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Liu Y, Fu Z, Wang X, Yang Q, Liu S, Zhu D. Metformin attenuates diabetic osteoporosis by suppressing ferroptosis via the AMPK/Nrf2 pathway. Front Pharmacol 2025; 16:1527316. [PMID: 40206070 PMCID: PMC11979264 DOI: 10.3389/fphar.2025.1527316] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2024] [Accepted: 03/17/2025] [Indexed: 04/11/2025] Open
Abstract
Background Ferroptosis is a critical factor in the impairment of osteoblast function in osteoporosis. Metformin (Met), a biguanide antidiabetic drug, has demonstrated anti-osteoporotic effects and has been confirmed to exert therapeutic benefits in diabetic osteoporosis (DOP). Nevertheless, the underlying mechanisms through which Met affects bone metabolism remain ambiguous. Objective This study seeks to elucidate the function of Met in DOP and to explore the potential mechanisms through which it mediates treatment effects. Methods In vitro, we utilized osteoblasts to explore the impact of Met on osteoblast differentiation and anti-ferroptosis in a high glucose and palmitic acid (HGHF) environment. In vivo, we developed a DOP model utilizing a high-fat diet along with streptozocin injections and evaluated the bone-protective effects of Met through micro-CT and histomorphological analyses. Results Met inhibits HGHF-induced ferroptosis in osteoblasts, as indicated by the elevation of ferroptosis-protective proteins (GPX4, FTH1, and SLAC7A11), along with decreased lipid peroxidation and ferrous ion levels. Furthermore, Met augmented the levels of osteogenic markers (RUNX2 and COL1A1) and enhanced alkaline phosphatase activity in osteoblasts under HGHF conditions. Mechanistic investigations revealed that Met activates the AMPK/Nrf2 pathway, effectively preventing ferroptosis progression. Additionally, in vivo results demonstrated Met alleviates bone loss and microstructural deterioration in DOP rats. Conclusion Met can activate the AMPK/Nrf2 pathway to prevent ferroptosis, thereby protecting against DOP.
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Affiliation(s)
- Yanwei Liu
- Department of Orthopaedics, The First Hospital of Jilin University, Changchun, China
| | - Zhaoyu Fu
- Department of Emergency Surgery, The First Affiliated Hospital of Zhengzhou University, Henan Medical Key Laboratory of Emergency and Trauma Research, Zhengzhou, Henan, China
| | - Xinyu Wang
- Department of Orthopaedics, The First Hospital of Jilin University, Changchun, China
| | - Qifan Yang
- Department of Orthopaedics, The First Hospital of Jilin University, Changchun, China
| | - Shun Liu
- Department of Orthopaedics, The First Hospital of Jilin University, Changchun, China
| | - Dong Zhu
- Department of Orthopaedics, The First Hospital of Jilin University, Changchun, China
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Yu H, Yang S, Jiang T, Li T, Duan H, Li M. Repair mechanisms of bone system tissues based on comprehensive perspective of multi-omics. Cell Biol Toxicol 2025; 41:45. [PMID: 39966216 PMCID: PMC11836151 DOI: 10.1007/s10565-025-09995-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2024] [Accepted: 01/28/2025] [Indexed: 02/20/2025]
Abstract
Bone disorders affect more than half of the adult population worldwide who may have a poor quality of life and physical independence worldwide. Multi-omic techniques are increasingly adopted and applied to determine the molecular mechanisms of bone tissue repair, providing perspective towards personalized medical intervention. Data from genomics, epigenomics, transcriptomics, proteomics, glycomics, and lipidomics were combined to elucidate dynamic processes in bone repair. In this narrative review, the key role of genetic and epigenetic factors in regulating injured cellular responses is highlighted, and changes in RNA and protein expression during the healing phase, as well as glucolipid metabolism adaptation, are described in detail how the repair process is affected. In a word, the integration of multi-omic techniques in this review not only benefits the comprehensive identification of new biomarkers, but also facilitates the development of personalized treatment strategies of bone disorders to revolutionize regenerative medicine.
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Affiliation(s)
- Honghao Yu
- Departments of Spine Surgery, Shengjing Hospital of China Medical University, Shengyang, China
| | - Shize Yang
- Department of Thoracic Surgery, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Tianlong Jiang
- Department of Orthopedic Surgery, First Affiliated Hospital of China Medical University, Shenyang, 110001, China
| | - Tian Li
- Tianjin Key Laboratory of Acute Abdomen Disease-Associated Organ Injury and ITCWM Repair, Institute of Integrative Medicine of Acute Abdominal Diseases, Tianjin Nankai Hospital, Tianjin Medical University, 8 Changjiang Avenue, Tianjin, 300100, China.
| | - Hongmei Duan
- Department of Rheumatology and Immunology, First Affiliated Hospital of China Medical University, Shenyang, 110001, China.
| | - Minglei Li
- Department of Pediatric Orthopaedics, Shengjing Hospital of China Medical University, 36 Sanhao St, Shenyang, 110004, China.
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Wanionok NE, Molinuevo MS, Fernández JM, Lucas B, Cortizo AM, Castillo EJ, Jiron JM, Claudia S, Leon S, Aguirre JI, McCarthy AD. Skeletal Effects of a Prolonged Oral Metformin Treatment in Adult Wistar Rats. Exp Clin Endocrinol Diabetes 2024; 132:547-556. [PMID: 38740375 DOI: 10.1055/a-2324-8661] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 05/16/2024]
Abstract
INTRODUCTION We previously showed that a 3-week oral metformin (MET) treatment enhances the osteogenic potential of bone marrow stromal cells (BMSCs) and improves several bone histomorphometric parameters in Wistar rats with metabolic syndrome (MetS). However, the skeletal effects of extended periods of MET need to be completely elucidated. Hence, in this study, the impact of a prolonged (3-month) MET treatment was investigated on bone architecture, histomorphometric and biomechanics variables, and osteogenic potential of BMSCs in Wistar rats with or without MetS. MATERIALS AND METHODS Young male Wistar rats (n=36) were randomized into four groups (n=9) that received either 20% fructose (F), MET (MET), F plus MET treatments (FMET), or drinking water alone (Veh). Rats were euthanized, blood was collected, and bones were dissected and processed for peripheral quantitative computed tomography (pQCT) analysis, static and dynamic histomorphometry, and bone biomechanics. In addition, BMSCs were isolated to determine their osteogenic potential. RESULTS MET affected trabecular and cortical bone, altering bone architecture and biomechanics. Furthermore, MET increased the pro-resorptive profile of BMSCs. In addition, fructose-induced MetS practically did not affect the the structural or mechanical variables of the skeleton. CONCLUSION A 3-month treatment with MET (with or without MetS) affects bone architecture and biomechanical variables in Wistar rats.
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Affiliation(s)
- Nahuel E Wanionok
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - María S Molinuevo
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - Juan M Fernández
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - Besada Lucas
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - Ana M Cortizo
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - Evelyn J Castillo
- Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA
| | - Jessica M Jiron
- Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA
| | - Sedlinsky Claudia
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - Schurman Leon
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
| | - José I Aguirre
- Department of Physiological Sciences, University of Florida, Gainesville, Florida, USA
| | - Antonio D McCarthy
- Laboratorio de Investigaciones en Osteopatías y Metabolismo Mineral (LIOMM), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, Argentina
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Li F, Xie X, Xu X, Zou X. Water-soluble biopolymers calcium polymalate derived from fermentation broth of Aureobasidium pullulans markedly alleviates osteoporosis and fatigue. Int J Biol Macromol 2024; 268:132013. [PMID: 38697412 DOI: 10.1016/j.ijbiomac.2024.132013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2024] [Revised: 04/02/2024] [Accepted: 04/29/2024] [Indexed: 05/05/2024]
Abstract
Osteoporosis is a prevalent condition characterized by bone loss and decreased skeletal strength, resulting in an elevated risk of fractures. Calcium plays a crucial role in preventing and managing osteoporosis. However, traditional calcium supplements have limited bioavailability, poor solubility, and adverse effects. In this study, we isolated a natural soluble biopolymer, calcium polymalate (PMACa), from the fermentation broth of the fungus Aureobasidium pullulans, to investigate its potential as an anti-osteoporosis therapeutic agent. Characterization revealed that linear PMA-Ca chains juxtaposed to form a porous, rod-like state, in the presence of Ca2+. In vivo mouse models demonstrated that PMA-Ca significantly promoted the conversion of serum calcium into bone calcium, and stimulated bone growth and osteogenesis. Additionally, PMA-Ca alleviated exercise fatigue in mice by facilitating the removal of essential metabolites, such as serum lactate (BLA) and blood urea nitrogen (BUN), from their bloodstream. In vitro studies further showed that PMA-Ca strengthened osteoblast cell activity, proliferation, and mineralization. And PMA-Ca upregulated the expression of some genes involved in osteoblast differentiation, indicating a potential correlation between bone formation and PMACa. These findings indicate that soluble PMA-Ca has the potential to be a novel biopolymer-based calcium supplement with sustainable production sourced from the fermentation industry.
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Affiliation(s)
- Fulin Li
- College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Xin Xie
- College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Xingran Xu
- College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
| | - Xiang Zou
- College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China; Dongguan Juwei Biotechnology Co., Dongguan 523808, China.
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Egashira K, Kajiya H, Tsutsumi T, Taniguchi Y, Kakura K, Ohno J, Kido H. AMPK activation enhances osteoblast differentiation on a titanium disc via autophagy. Int J Implant Dent 2024; 10:2. [PMID: 38286943 PMCID: PMC10825085 DOI: 10.1186/s40729-024-00525-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2023] [Accepted: 01/18/2024] [Indexed: 01/31/2024] Open
Abstract
PURPOSE The acquisition of osseointegration during implant therapy is slower and poorer in patients with diabetes compared with healthy persons. The serum concentration of adiponectin in patients with type II diabetes is lower than that of healthy persons via the suppression of AMP-activated protein kinase (AMPK). Therefore, we hypothesized that the AMPK activation enhances bone formation around implants, resulting in the improved acquisition of osseointegration. The purpose of this study was to evaluate the impact of AMPK activation on osteoblast differentiation and its mechanism of downstream signaling on titanium disc (Ti). METHODS Confluent mouse pre-osteoblasts (MC3T3-E1) cells (1 × 105 cells/well) were cultured with BMP-2 for osteoblast differentiation, in the presence or absence AICAR, an AMPK activator. We examined the effects of AMPK activation on osteoblast differentiation and the underlying mechanism on a Ti using a CCK8 assay, a luciferase assay, quantitative RT-PCR, and western blotting. RESULTS Although the proliferation rate of osteoblasts was not different between a Ti and a tissue culture polystyrene dish, the addition of AICAR, AMPK activator slightly enhanced osteoblast proliferation on the Ti. AICAR enhanced the BMP-2-dependent transcriptional activity on the Ti, leading to upregulation in the expression of osteogenesis-associated molecules. AICAR simultaneously upregulated the expression of autophagy-associated molecules on the Ti, especially LC3-II. AdipoRon, an adiponectin receptor type1/type2 activator activated AMPK, and upregulated osteogenesis-associated molecules on Ti. CONCLUSIONS AMPK activation enhances osteoblast differentiation on a Ti via autophagy, suggesting that it promotes the acquisition of osseointegration during implant therapy.
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Affiliation(s)
- Kei Egashira
- Section of Oral Implantology, Department of Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan
- Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan
| | - Hiroshi Kajiya
- Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan.
- Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka, 814-0193, Japan.
| | - Takashi Tsutsumi
- Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan
- Department of General Dentistry, Fukuoka Dental College, Fukuoka, Japan
| | - Yusuke Taniguchi
- Section of Oral Implantology, Department of Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan
| | - Kae Kakura
- Section of Oral Implantology, Department of Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan
| | - Jun Ohno
- Oral Medicine Research Center, Fukuoka Dental College, Fukuoka, Japan
| | - Hirofumi Kido
- Section of Oral Implantology, Department of Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan
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Patel B, Koysombat K, Mills EG, Tsoutsouki J, Comninos AN, Abbara A, Dhillo WS. The Emerging Therapeutic Potential of Kisspeptin and Neurokinin B. Endocr Rev 2024; 45:30-68. [PMID: 37467734 PMCID: PMC10765167 DOI: 10.1210/endrev/bnad023] [Citation(s) in RCA: 18] [Impact Index Per Article: 18.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 06/13/2023] [Accepted: 07/11/2023] [Indexed: 07/21/2023]
Abstract
Kisspeptin (KP) and neurokinin B (NKB) are neuropeptides that govern the reproductive endocrine axis through regulating hypothalamic gonadotropin-releasing hormone (GnRH) neuronal activity and pulsatile GnRH secretion. Their critical role in reproductive health was first identified after inactivating variants in genes encoding for KP or NKB signaling were shown to result in congenital hypogonadotropic hypogonadism and a failure of pubertal development. Over the past 2 decades since their discovery, a wealth of evidence from both basic and translational research has laid the foundation for potential therapeutic applications. Beyond KP's function in the hypothalamus, it is also expressed in the placenta, liver, pancreas, adipose tissue, bone, and limbic regions, giving rise to several avenues of research for use in the diagnosis and treatment of pregnancy, metabolic, liver, bone, and behavioral disorders. The role played by NKB in stimulating the hypothalamic thermoregulatory center to mediate menopausal hot flashes has led to the development of medications that antagonize its action as a novel nonsteroidal therapeutic agent for this indication. Furthermore, the ability of NKB antagonism to partially suppress (but not abolish) the reproductive endocrine axis has supported its potential use for the treatment of various reproductive disorders including polycystic ovary syndrome, uterine fibroids, and endometriosis. This review will provide a comprehensive up-to-date overview of the preclinical and clinical data that have paved the way for the development of diagnostic and therapeutic applications of KP and NKB.
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Affiliation(s)
- Bijal Patel
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
| | - Kanyada Koysombat
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
- Department of Diabetes and Endocrinology, Imperial College Healthcare NHS Trust, 72 Du Cane Rd, London, W12 0HS, UK
| | - Edouard G Mills
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
- Department of Diabetes and Endocrinology, Imperial College Healthcare NHS Trust, 72 Du Cane Rd, London, W12 0HS, UK
| | - Jovanna Tsoutsouki
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
| | - Alexander N Comninos
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
- Department of Diabetes and Endocrinology, Imperial College Healthcare NHS Trust, 72 Du Cane Rd, London, W12 0HS, UK
| | - Ali Abbara
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
- Department of Diabetes and Endocrinology, Imperial College Healthcare NHS Trust, 72 Du Cane Rd, London, W12 0HS, UK
| | - Waljit S Dhillo
- Section of Investigative Medicine, Department of Metabolism, Digestion and Reproduction, Imperial College School of Medicine, Imperial College London, London, W12 0NN, UK
- Department of Diabetes and Endocrinology, Imperial College Healthcare NHS Trust, 72 Du Cane Rd, London, W12 0HS, UK
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Liu Z, Li Q, Wang X, Wu Y, Zhang Z, Mao J, Gong S. Proanthocyanidin enhances the endogenous regeneration of alveolar bone by elevating the autophagy of PDLSCs. J Periodontal Res 2023; 58:1300-1314. [PMID: 37715945 DOI: 10.1111/jre.13186] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 07/31/2023] [Accepted: 09/01/2023] [Indexed: 09/18/2023]
Abstract
OBJECTIVE This study aimed to investigate the effect of proanthocyanidin (PA) on osteogenesis mediated by periodontal ligament stem cells (PDLSCs) and endogenous alveolar bone regeneration. BACKGROUND Leveraging the osteogenic potential of resident stem cells is a promising strategy for alveolar bone regeneration. PA has been reported to be effective in osteogenesis. However, the effect and mechanism of PA on the osteogenic differentiation of PDLSCs remain elusive. METHODS Human PDLSCs were treated with various doses of PA to assess the cell proliferation using Cell Counting Kit-8. The osteogenic differentiation ability was detected by qRT-PCR analysis, western blot analysis, Alizarin red S staining, and Alkaline Phosphatase staining. The level of autophagy was evaluated by confocal laser scanning microscopy, transmission electron microscopy, and western blot analysis. RNA sequencing was utilized to screen the potential signaling pathway. The alveolar bone defect model of rats was created to observe endogenous bone regeneration. RESULTS PA activated intracellular autophagy in PDLSCs, resulting in enhanced osteogenic differentiation. Moreover, this effect could be abolished by the autophagy inhibitor 3-Methyladenine. Mechanistically, the PI3K/Akt/mTOR pathway was negatively correlated with PA-mediated autophagy activation. Lastly, PA promoted the alveolar bone regeneration in vivo, and this effect was reversed when the autophagy process was blocked. CONCLUSION PA may activate autophagy by inhibiting PI3K/Akt/mTOR signaling pathway to promote the osteogenesis of PDLSCs and enhance endogenous alveolar bone regeneration.
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Affiliation(s)
- Zhuo Liu
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Qilin Li
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Xiangyao Wang
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Yaxin Wu
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Zhixing Zhang
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Jing Mao
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
| | - Shiqiang Gong
- Center of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
- Hubei Province Key Laboratory of Oral and Maxillofacial Development and Regeneration, Wuhan, China
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Lee E, Moon JY, Ko JY, Park SY, Im GI. GSTT1 as a Predictive Marker and Enhancer for Osteogenic Potential of Human Adipose-Derived Stromal/Stem Cells. J Bone Miner Res 2023; 38:1480-1496. [PMID: 37537994 DOI: 10.1002/jbmr.4893] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2022] [Revised: 07/23/2023] [Accepted: 08/01/2023] [Indexed: 08/05/2023]
Abstract
Adipose-derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine for bone because of their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. On the other hand, the differentiation potential of ASCs is generally lower than that of bone marrow-derived stromal/stem cells and varies greatly depending on donors. In this study, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA-seq gene analysis, we discovered that GSTT1 (Glutathione S-transferase theta-1) was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation. On the other hand, GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species, and increased GSH/GSSG ratios were also detected in GSTT1-transfected ASCs. When the in vivo effect of GSTT1-transfected ASCs on bone regeneration was investigated with segmental long-bone defect model in rats, bone regeneration was significantly better after implantation of GSTT1-transfected ASCs compared with that of control vector-transfected ASCs. In conclusion, GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones. © 2023 American Society for Bone and Mineral Research (ASBMR).
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Affiliation(s)
- Eugene Lee
- Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University, Goyang, Republic of Korea
| | - Jae-Yeon Moon
- Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University, Goyang, Republic of Korea
| | - Ji-Yun Ko
- Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University, Goyang, Republic of Korea
| | - Seo-Young Park
- Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University, Goyang, Republic of Korea
| | - Gun-Il Im
- Research Institute for Integrative Regenerative Biomedical Engineering, Dongguk University, Goyang, Republic of Korea
- Department of Orthopedics, Dongguk University Ilsan Hospital, Goyang, Republic of Korea
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Kim KM, Son HE, Lim YJ, Jang WG. Topiramate promotes osteogenic differentiation through AMPK-dependent phosphorylation of Smad1/5/9. Acta Histochem 2023; 125:152095. [PMID: 37757516 DOI: 10.1016/j.acthis.2023.152095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2023] [Revised: 09/21/2023] [Accepted: 09/21/2023] [Indexed: 09/29/2023]
Abstract
Topiramate [2,3:4,5-bis-o-(1-methylethylidene) β-D-fructo-pyranose sulfamate; TPM] is one of the most used new-generation antiepileptic drugs. It has been reported to regulate the differentiation of human bone cells. However, the molecular mechanism of TPM in osteoblast differentiation is not fully elucidated. In the present study, we examined the effect of TPM on osteogenic differentiation of C3H10T1/2, MC3T3-E1, primary mouse calvarial cells, and primary bone marrow stem cells (BMSCs). Primary cells were isolated from mice calvaria and bone marrow respectively. Expression of the osteogenic gene was determined by RT-PCR. The osteogenic protein levels were measured by Western blot analysis. Alkaline phosphatase (ALP) staining experiment was performed to evaluate ALP activity. Alizarin red s (ARS) staining was performed to measure zebrafish caudal fin regeneration. Treatment of TPM up-regulated the osteogenic genes including distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, TPM also increased the Dlx5 and Runx2 protein levels, Smad1/5/9 phosphorylation, and alkaline phosphatase (ALP) activity. Furthermore, TPM activated AMPK, and inhibition of AMPK decreased TPM-induced osteogenic differentiation. In the zebrafish model, osteogenic effect of TPM was identified. TPM was increased amputated caudal fin rays of zebrafish. These results demonstrate that TPM enhances osteogenic differentiation via AMPK-mediated Smad1/5/9 phosphorylation.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea
| | - Hyo-Eun Son
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea
| | - Young-Ju Lim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
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Zhang Y, Jia S, Wen G, Xie S, Song Z, Qi M, Liang Y, Bi W, Dong W. Zoledronate Promotes Peri-Implant Osteogenesis in Diabetic Osteoporosis by the AMPK Pathway. Calcif Tissue Int 2023; 113:329-343. [PMID: 37392365 DOI: 10.1007/s00223-023-01112-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/25/2023] [Accepted: 06/23/2023] [Indexed: 07/03/2023]
Abstract
Together with diabetic osteoporosis (DOP), diabetes patients experience poor peri-implant osteogenesis following implantation for dentition defects. Zoledronate (ZOL) is widely used to treat osteoporosis clinically. To evaluate the mechanism of ZOL for the treatment of DOP, experiments with DOP rats and high glucose-grown MC3T3-E1 cells were used. The DOP rats treated with ZOL and/or ZOL implants underwent a 4-week implant-healing interval, and then microcomputed tomography, biomechanical testing, and immunohistochemical staining were performed to elucidate the mechanism. In addition, MC3T3-E1 cells were maintained in an osteogenic medium with or without ZOL to confirm the mechanism. The cell migration, cellular actin content, and osteogenic differentiation were evaluated by a cell activity assay, a cell migration assay, as well as alkaline phosphatase, alizarin red S, and immunofluorescence staining. The mRNA and protein expression of adenosine monophosphate-activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphogenetic protein 2 (BMP2), and collagen type I (Col-I) were detected using real-time quantitative PCRs and western blot assays, respectively. In the DOP rats, ZOL markedly improved osteogenesis, enhanced bone strength and increased the expression of AMPK, p-AMPK, and Col-I in peri-implant bones. The in vitro findings showed that ZOL reversed the high glucose-induced inhibition of osteogenesis via the AMPK signaling pathway. In conclusion, the ability of ZOL to promote osteogenesis in DOP by targeting AMPK signaling suggests that therapy with ZOL, particularly simultaneous local and systemic administration, may be a unique approach for future implant repair in diabetes patients.
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Affiliation(s)
- Yan Zhang
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Shunyi Jia
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Guochen Wen
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Shanen Xie
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Zhiqiang Song
- Oral and Maxillofacial Surgery, TangShan BoChuang Stomatology Hospital, Tangshan, 063000, Hebei, China
| | - Mengchun Qi
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Yongqiang Liang
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Wenjuan Bi
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China
| | - Wei Dong
- School of Stomatology, North China University of Science and Technology, Tangshan, 063210, Hebei, China.
- Institute of Stomatology, Chinese PLA General Hospital, Fuxing Lu 28#, Beijing, 100853, China.
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11
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Kim KM, Lim YJ, Jang WG. Policosanol Stimulates Osteoblast Differentiation via Adenosine Monophosphate-Activated Protein Kinase-Mediated Expression of Insulin-Induced Genes 1 and 2. Cells 2023; 12:1863. [PMID: 37508527 PMCID: PMC10378419 DOI: 10.3390/cells12141863] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2023] [Revised: 07/12/2023] [Accepted: 07/13/2023] [Indexed: 07/30/2023] Open
Abstract
Policosanol is known as a hypocholesterolemic compound and is derived from plants such as sugar cane and corn. Policosanol can lower blood pressure or inhibit adipogenesis, but its effect on osteogenic differentiation and the molecular mechanism is unclear. This study aims to investigate the effect of policosanol on osteogenic differentiation in MC3T3-E1 cells and zebrafish models. Administration of policosanol into MC3T3-E1 induced the expression of the osteogenic genes such as distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). Alkaline phosphatase activity and extracellular mineralization also increased. Policosanol promoted activation of adenosine monophosphate-activated protein kinase (AMPK) and insulin-induced genes (INSIGs) expression and regulation of INSIGs modulated osteoblast differentiation. AMPK activation through transfection of the constitutively active form of AMPK (CA-AMPK) increased INSIGs expression, whereas policosanol-induced INSIGs expression was suppressed by inhibitor of AMPK (Com. C). Furthermore, the osteogenic effects of policosanol were verified in zebrafish. Amputated caudal fin rays were regenerated by policosanol treatment. Taken together, these results show that policosanol increases osteogenic differentiation and contributes to fin regeneration in zebrafish via AMPK-mediated INSIGs expression, suggesting that policosanol has potential as an osteogenic agent.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453, Republic of Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453, Republic of Korea
| | - Young-Ju Lim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453, Republic of Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453, Republic of Korea
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453, Republic of Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453, Republic of Korea
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12
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Yang C, Dong Z, Ling Z, Chen Y. The crucial mechanism and therapeutic implication of RNA methylation in bone pathophysiology. Ageing Res Rev 2022; 79:101641. [PMID: 35569786 DOI: 10.1016/j.arr.2022.101641] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/01/2022] [Revised: 04/19/2022] [Accepted: 05/09/2022] [Indexed: 12/12/2022]
Abstract
Methylation is the most common posttranscriptional modification in cellular RNAs, which has been reported to modulate the alteration of RNA structure for initiating relevant functions such as nuclear translocation and RNA degradation. Recent studies found that RNA methylation especially N6-methyladenosine (m6A) regulates the dynamic balance of bone matrix and forms a complicated network in bone metabolism. The modulation disorder of RNA methylation contributes to several pathological bone diseases including osteoporosis (OP), osteoarthritis (OA), rheumatoid arthritis (RA), and so on. In the review, we will discuss advanced technologies for detecting RNA methylation, summarize RNA methylation-related biological impacts on regulating bone homeostasis and pathological bone diseases. In addition, we focus on the promising roles of RNA methylation in early diagnosis and therapeutic implications for bone-related diseases. Then, we aim to establish a theoretical basis for further investigation in this meaningful field.
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13
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Guo X, Liang M. Metformin alleviates dexamethasone-induced apoptosis by regulating autophagy via AMPK/mTOR/p70S6K in osteoblasts. Exp Cell Res 2022; 415:113120. [PMID: 35341775 DOI: 10.1016/j.yexcr.2022.113120] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2021] [Revised: 03/21/2022] [Accepted: 03/23/2022] [Indexed: 01/07/2023]
Abstract
Glucocorticoid (GC)-induced osteoporosis (GIOP) is the most common type of secondary osteoporosis. Osteoblast apoptosis induced by GCs is now considered as a crucial factor for GIOP. Many clinical, in vivo, and in vitro studies have shown that metformin has a beneficial effect on bone metabolism and bone formation. To investigate whether metformin could be used to treat GIOP, we explored the influence of metformin on dexamethasone (Dex)-induced apoptosis of osteoblasts and its underlying mechanisms. In this study, the CCK8 assay was used to determine the optimal metformin concentration and processing time. The expression levels of target proteins were examined by Western blot and immunofluorescence; the expression levels of target genes were tested by quantitative PCR. Apoptotic cells were detected using flow cytometry. Characteristics of autophagy were observed by transmission electron microscopy. An autophagy inhibitor was administered to investigate whether autophagy decreases apoptosis. Sh-AMPK transfection and an mTOR activator were used to investigate the role of AMPK/mTOR signaling in metformin-induced autophagy. The results showed that metformin alleviated Dex-induced apoptosis of osteoblasts accompanied by increased autophagy. Treatment with the autophagy inhibitor 3-methyladenine (3-MA) attenuated the effect of metformin on apoptosis, autophagy, and the AMPK/mTOR/p70S6K signaling pathway. The anti-apoptotic effect of metformin on osteoblasts is associated with the promotion of autophagy. Furthermore, sh-AMPK transfection and the mTOR activator MHY1485 impaired metformin-mediated inhibition of osteoblast apoptosis and promotion of autophagy. The AMPK/mTOR/p70S6K signaling pathway plays a role in metformin-mediated apoptosis suppression and autophagy promotion. In conclusion, metformin can alleviate Dex-induced osteoblast apoptosis by inducing autophagy via the AMPK/mTOR/p70S6K pathway. This study highlights the potential value of metformin in the treatment of GIOP.
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Affiliation(s)
- Xintong Guo
- Department of Geriatric Endocrinology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Province, China
| | - Min Liang
- Department of Geriatric Endocrinology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi Province, China.
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14
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Wang X, Schepler H, Neufurth M, Wang S, Schröder HC, Müller WEG. Polyphosphate in Chronic Wound Healing: Restoration of Impaired Metabolic Energy State. PROGRESS IN MOLECULAR AND SUBCELLULAR BIOLOGY 2022; 61:51-82. [PMID: 35697937 DOI: 10.1007/978-3-031-01237-2_4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/15/2023]
Abstract
Many pathological conditions are characterized by a deficiency of metabolic energy. A prominent example is nonhealing or difficult-to-heal chronic wounds. Because of their unique ability to serve as a source of metabolic energy, inorganic polyphosphates (polyP) offer the opportunity to develop novel strategies to treat such wounds. The basis is the generation of ATP from the polymer through the joint action of two extracellular or plasma membrane-bound enzymes alkaline phosphatase and adenylate kinase, which enable the transfer of energy-rich phosphate from polyP to AMP with the formation of ADP and finally ATP. Building on these findings, it was possible to develop novel regeneratively active materials for wound therapy, which have already been successfully evaluated in first studies on patients.
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Affiliation(s)
- Xiaohong Wang
- ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Hadrian Schepler
- Department of Dermatology, University Clinic Mainz, Mainz, Germany
| | - Meik Neufurth
- ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Shunfeng Wang
- ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Heinz C Schröder
- ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
| | - Werner E G Müller
- ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany.
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15
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Kim KM, Jang WG. NXNL1 negatively regulates osteoblast differentiation via GDF15-induced PP2A Cα dependent manner in MC3T3-E1 cells. Biofactors 2022; 48:239-248. [PMID: 34932831 DOI: 10.1002/biof.1817] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/24/2021] [Accepted: 11/24/2021] [Indexed: 11/10/2022]
Abstract
Controlling the level of intracellular reactive oxygen species (ROS) is important for the survival and differentiation of osteoblasts. Intracellular ROS levels are controlled by antioxidant enzymes that modulate the redox state of the cell. Nucleoredoxin-like 1 (NXNL1) is an antioxidant enzyme that increases the viability of rod and cone cells by protecting them from oxidative stress, and is a potential pharmacological target for the treatment of retinitis pigmentosa. The present study investigated the role of NXNL on osteoblast differentiation of MC3T3-E1 preosteoblast cells. Results from qPCR experiments demonstrated that growth differentiation factor 15 (GDF15) increased NXNL1 expression, and that GDF15-induced NXNL1 decreased the expression of osteogenic genes such as distal-less homeobox 5 (Dlx5) and Runt-related transcription factor 2. Furthermore, NXNL1 also inhibits bone morphogenetic protein 2-induced phosphorylation of Smad1/5/9 and alkaline phosphatase activity. The inhibitory effects of NXNL1 on osteoblast differentiation were mediated by protein phosphatase 2A Cα (PP2A Cα). The expression of PP2A Cα was regulated by GDF15, and overexpression of PP2A Cα increased the expression of NXNL1. Taken together, our results demonstrate that NXNL1 inhibits osteoblast differentiation of MC3T3-E1 due to GDF15-induced expression of PP2A Cα.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, South Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, South Korea
- New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), Daegu, South Korea
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, South Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, South Korea
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16
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The effect of AMP kinase activation on differentiation and maturation of osteoblast cultured on titanium plate. J Dent Sci 2021; 17:1225-1231. [PMID: 35784162 PMCID: PMC9236888 DOI: 10.1016/j.jds.2021.12.003] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2021] [Revised: 12/02/2021] [Indexed: 12/01/2022] Open
Abstract
Background/purpose 5′ Adenosine monophosphate-activated protein kinase (AMPK) is known as an enzyme that maintains intracellular homeostasis and has various biological activity. The purpose of this study is evaluation effect of AMPK activation on implant prognosis. Materials & methods MC3T3-E1 osteoblast-like cells were cultured on titanium using a 24-well plate. The experimental group was divided into the following 3 groups: (1) the normal culture group (control group), (2) the osteogenic induction group, and (3) the osteogenic induction + AMPK activation group. The cell counts were measured; real-time PCR was used to assess the expression of ALP and Osterix as osteogenic related genes at Day 0,7,14 and 21 after experiments. Additionally, ALP activity and calcification were assessed. Results The results of the real-time PCR assessments revealed that the expression of ALP, which is a marker for the initial stages of calcification, was significantly increased by AMPK activation compared to the normal culture or osteogenic induction. A significant increase was also observed in the expression of Osterix, which is a marker for the later stages of calcification. Because significant increases were observed in ALP activity and calcification potential, this suggested that AMPK activation could elicit an increase in osteoblast calcification potential. Conclusion AMPK activation promotes implant peripheral osteoblast differentiation and maturation and enhances calcification. Our results suggest that AMPK activation may help to maintain implant stability.
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17
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Mills EG, Yang L, Nielsen MF, Kassem M, Dhillo WS, Comninos AN. The Relationship Between Bone and Reproductive Hormones Beyond Estrogens and Androgens. Endocr Rev 2021; 42:691-719. [PMID: 33901271 PMCID: PMC8599211 DOI: 10.1210/endrev/bnab015] [Citation(s) in RCA: 71] [Impact Index Per Article: 17.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Indexed: 12/20/2022]
Abstract
Reproductive hormones play a crucial role in the growth and maintenance of the mammalian skeleton. Indeed, the biological significance for this hormonal regulation of skeletal homeostasis is best illustrated by common clinical reproductive disorders, such as primary ovarian insufficiency, hypothalamic amenorrhea, congenital hypogonadotropic hypogonadism, and early menopause, which contribute to the clinical burden of low bone mineral density and increased risk for fragility fracture. Emerging evidence relating to traditional reproductive hormones and the recent discovery of newer reproductive neuropeptides and hormones has deepened our understanding of the interaction between bone and the reproductive system. In this review, we provide a contemporary summary of the literature examining the relationship between bone biology and reproductive signals that extend beyond estrogens and androgens, and include kisspeptin, gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, prolactin, progesterone, inhibin, activin, and relaxin. A comprehensive and up-to-date review of the recent basic and clinical research advances is essential given the prevalence of clinical reproductive disorders, the emerging roles of upstream reproductive hormones in bone physiology, as well as the urgent need to develop novel safe and effective therapies for bone fragility in a rapidly aging population.
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Affiliation(s)
- Edouard G Mills
- Division of Diabetes, Endocrinology and Metabolism, Imperial College London, London, UK
| | - Lisa Yang
- Division of Diabetes, Endocrinology and Metabolism, Imperial College London, London, UK
| | - Morten F Nielsen
- Department of Endocrinology, University Hospital of Odense & institute of Clinical Research, University of Southern Denmark, 5000 Odense C, Denmark
| | - Moustapha Kassem
- Department of Endocrinology, University Hospital of Odense & institute of Clinical Research, University of Southern Denmark, 5000 Odense C, Denmark.,Faculty of Health and Medical Sciences, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark
| | - Waljit S Dhillo
- Division of Diabetes, Endocrinology and Metabolism, Imperial College London, London, UK.,Department of Endocrinology, Imperial College Healthcare NHS Trust, London, UK
| | - Alexander N Comninos
- Division of Diabetes, Endocrinology and Metabolism, Imperial College London, London, UK.,Department of Endocrinology, Imperial College Healthcare NHS Trust, London, UK.,Endocrine Bone Unit, Imperial College Healthcare NHS Trust, London, UK
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18
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Self-Organization Provides Cell Fate Commitment in MSC Sheet Condensed Areas via ROCK-Dependent Mechanism. Biomedicines 2021; 9:biomedicines9091192. [PMID: 34572378 PMCID: PMC8470239 DOI: 10.3390/biomedicines9091192] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Revised: 08/30/2021] [Accepted: 09/07/2021] [Indexed: 12/13/2022] Open
Abstract
Multipotent mesenchymal stem/stromal cells (MSC) are one of the crucial regulators of regeneration and tissue repair and possess an intrinsic program from self-organization mediated by condensation, migration and self-patterning. The ability to self-organize has been successfully exploited in tissue engineering approaches using cell sheets (CS) and their modifications. In this study, we used CS as a model of human MSC spontaneous self-organization to demonstrate its structural, transcriptomic impact and multipotent stromal cell commitment. We used CS formation to visualize MSC self-organization and evaluated the role of the Rho-GTPase pathway in spontaneous condensation, resulting in a significant anisotropy of the cell density within the construct. Differentiation assays were carried out using conventional protocols, and microdissection and RNA-sequencing were applied to establish putative targets behind the observed phenomena. The differentiation of MSC to bone and cartilage, but not to adipocytes in CS, occurred more effectively than in the monolayer. RNA-sequencing indicated transcriptional shifts involving the activation of the Rho-GTPase pathway and repression of SREBP, which was concordant with the lack of adipogenesis in CS. Eventually, we used an inhibitory analysis to validate our findings and suggested a model where the self-organization of MSC defined their commitment and cell fate via ROCK1/2 and SREBP as major effectors under the putative switching control of AMP kinase.
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19
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Kim KM, Kim CH, Cho KH, Jang WG. Policosanol attenuates Pi-induced calcification via AMPK-mediated INSIGs expression in rat VSMCs. Clin Exp Pharmacol Physiol 2021; 48:1336-1345. [PMID: 34053129 DOI: 10.1111/1440-1681.13530] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2020] [Revised: 05/25/2021] [Accepted: 05/26/2021] [Indexed: 12/25/2022]
Abstract
Policosanol is a hypocholesterolemic derived from sugar cane and corn that downregulates blood cholesterol levels. It can further lower blood pressure and reduce liver inflammation. Policosanol can also affect vascular calcification, however, its molecular mechanisms are not well understood. This study investigated the effect of policosanol on vascular calcification and its molecular mechanism. Policosanol decreased the expression of inorganic phosphate (Pi)-induced osteogenic genes such as distal-less homeobox 5 (Dlx5) and runt-related transcription factor 2 (Runx2). In addition, following policosanol treatment, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation increased in a time-dependent manner. The constitutively active form of AMPK (CA-AMPK) dramatically suppressed Pi-induced Dlx5 and Runx2 protein levels. Inactivation of AMPK using compound C (Com. C; AMPK inhibitor) recovered policosanol-suppressed Alizarin Red S staining levels. Insulin-induced genes (INSIGs) were induced by CA-AMPK, their overexpression suppressed Pi-induced Dlx5 and Runx2 expression. Taken together, the results demonstrate that policosanol inhibits Pi-induced vascular calcification by regulating AMPK-induced INSIG expression in vascular smooth muscle cells.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk, Republic of Korea.,Research institute of Anti-Aging, Daegu University, Gyeongbuk, Republic of Korea
| | | | - Kyung-Hyun Cho
- LipoLab, Yeungnam University, Gyeongsan, Republic of Korea
| | - Won-Gu Jang
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk, Republic of Korea.,Research institute of Anti-Aging, Daegu University, Gyeongbuk, Republic of Korea
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20
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Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress. Int J Mol Sci 2021; 22:ijms22094312. [PMID: 33919218 PMCID: PMC8122383 DOI: 10.3390/ijms22094312] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Revised: 04/17/2021] [Accepted: 04/19/2021] [Indexed: 02/06/2023] Open
Abstract
Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research.
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21
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Lim YJ, Kim KM, Jang WG. Chrysophanol increases osteoblast differentiation via AMPK/Smad1/5/9 phosphorylation in vitro and in vivo. Clin Exp Pharmacol Physiol 2021; 48:515-523. [PMID: 33300218 DOI: 10.1111/1440-1681.13443] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2020] [Accepted: 11/05/2020] [Indexed: 01/20/2023]
Abstract
Chrysophanol (Chrysophanic acid; CA) is a natural anthraquinone found in Senna tora and rhubarb that has various characteristic features, including the ability to suppress adipogenesis. However, its effects on osteoblast differentiation have not been investigated. Herein, this study aimed to demonstrate the mechanism by which CA induces the osteoblast differentiation. CA increased the expression of osteogenic genes. The staining levels Alkaline phosphatase (ALP) and Alizarin Red S (ARS) were increased by chrysophanol. CA induced osteoblast differentiation through AMP-activated protein kinase (AMPK)/Small mothers against decapentaplegic (Smad1/5/9) activation in MC3T3-E1 cells. In addition, compound C, AMPK inhibitor (Comp. C)-induced cells suppressed osteogenic genes expression and AMPK/Smad1/5/9 activation. Interestingly, AMPK in the CA-induced AMPK/Smad1/5/9 signalling pathway was an upstream regulator of Smad1/5/9. In order to further dissect in bone development, we used a zebrafish model to investigate the effect of CA on bone development. These results suggest that CA stimulated bone development via AMPK/Smad1/5/9. Overall, our results demonstrate that CA promotes osteoblast differentiation via AMPK/Smad1/5/9 expression in vitro and in vivo.
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Affiliation(s)
- Young-Ju Lim
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk, Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, Korea
| | - Kyeong-Min Kim
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk, Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, Korea
| | - Won-Gu Jang
- Department of Biotechnology, College of Engineering, Daegu University, Gyeongbuk, Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, Korea
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22
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Sun R, Liang C, Sun Y, Xu Y, Geng W, Li J. Effects of metformin on the osteogenesis of alveolar BMSCs from diabetic patients and implant osseointegration in rats. Oral Dis 2021; 28:1170-1180. [PMID: 33606350 DOI: 10.1111/odi.13808] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 02/09/2021] [Accepted: 02/15/2021] [Indexed: 12/15/2022]
Abstract
OBJECTIVES This study aimed to explore the effects of metformin on osteogenic differentiation of alveolar bone marrow mesenchymal stem cells (BMSCs) from type-2 diabetes mellitus (T2DM) patients (DM-BMSCs) and implant osseointegration in rats, screen the optimal concentration, and investigate whether metformin could protect against the adverse impact of T2DM on BMSC osteogenic capacity. SUBJECTS AND METHODS Different concentrations of metformin were administered to human-derived BMSCs and Wistar rats receiving implants. ALP detection, alizarin red staining, real-time RT-PCR and Western blotting were performed to detect osteogenesis and investigate the mechanism. Toluidine blue staining was performed to analyse bone-implant contact in rats. RESULTS Metformin increased implant osseointegration in a rat model and promoted the osteogenic capacity of DM-BMSCs via the AMPK/BMP/Smad signalling pathway, and 125 μM was the optimal concentration; however, concentrations over 200 µM, metformin showed an inhibitory effect on DM-BMSCs. Additionally, metformin at the optimal concentration (125 µM) identified in this study could compensate for the negative impacts of T2DM on the osteogenic differentiation of BMSCs. CONCLUSIONS Metformin can promote the osteogenesis of BMSCs from T2DM patients and osseointegration in rats, and it might be an effective drug for increasing the success rate of T2DM-associated implants.
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Affiliation(s)
- Rongxin Sun
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China
| | - Chao Liang
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China
| | - Yujie Sun
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China
| | - Yifan Xu
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China
| | - Wei Geng
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China
| | - Jun Li
- Department of Dental Implant Center, Beijing Stomatological Hospital, School of Stomatology, Capital Medical University, Beijing, China.,Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, School of Stomatology, Capital Medical University, Beijing, China
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23
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Abstract
INTRODUCTION Preclinical, clinical, and population-based studies have provided evidence that anti-diabetic drugs affect bone metabolism and may affect the risk of fracture in diabetic patients. AREAS COVERED An overview of the skeletal effects of anti-diabetic drugs used in type 2 diabetes is provided. Searches on AdisInsight, PubMed, and Medline databases were conducted up to 1st July 2020. The latest evidence from randomized clinical trials and population-based studies on the skeletal safety of the most recent drugs (DPP-4i, GLP-1RA, and SGLT-2i) is provided. EXPERT OPINION Diabetic patients present with a higher risk of fracture for a given bone mineral density suggesting a role of bone quality in the etiology of diabetic fracture. Bone quality is difficult to assess in human clinical practice and the use of preclinical models provides valuable information on diabetic bone alterations. As several links have been established between bone and energy homeostasis, it is interesting to study the safety of anti-diabetic drugs on the skeleton. So far, evidence for the newest molecules suggests a neutral fracture risk, but further studies, especially in different types of patient populations (patients at risk or with history of cardiovascular disease, renal impairment, neuropathy) are required to fully appreciate this matter.
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Affiliation(s)
- Guillaume Mabilleau
- Groupe Etude Remodelage Osseux et biomatériaux, GEROM, UPRES EA 4658, UNIV Angers, SFR ICAT 4208, Institut de Biologie en Santé , Angers, France
- Service Commun d'Imagerie et Analyses Microscopiques, SCIAM, UNIV Angers, SFR ICAT 4208, Institut de Biologie en Santé , Angers, France
- Bone pathology unit, Angers University hospital , Angers Cedex, France
| | - Béatrice Bouvard
- Groupe Etude Remodelage Osseux et biomatériaux, GEROM, UPRES EA 4658, UNIV Angers, SFR ICAT 4208, Institut de Biologie en Santé , Angers, France
- Rheumatology department, Angers University Hospital , Angers Cedex, France
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Chen X, Wang Y, Chen R, Qu N, Zhang B, Xia C. Suppressing PLCγ1 enhances osteogenic and chondrogenic potential of BMSCs. Biochem Biophys Res Commun 2020; 532:292-299. [PMID: 32868075 DOI: 10.1016/j.bbrc.2020.08.049] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Accepted: 08/14/2020] [Indexed: 11/28/2022]
Abstract
Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-β3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.
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Affiliation(s)
- Xiaolei Chen
- Bone & Joint Research Institute, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, 361004, China
| | - Yue Wang
- Bone & Joint Research Institute, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, 361004, China
| | - Ri Chen
- School of Medicine, Xiamen University, Xiamen, Fujian, 361102, China
| | - Ning Qu
- School of Medicine, Xiamen University, Xiamen, Fujian, 361102, China
| | - Bing Zhang
- School of Medicine, Xiamen University, Xiamen, Fujian, 361102, China.
| | - Chun Xia
- Bone & Joint Research Institute, Zhongshan Hospital, Xiamen University, Xiamen, Fujian, 361004, China.
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Biomimetic bone regeneration using angle-ply collagen membrane-supported cell sheets subjected to mechanical conditioning. Acta Biomater 2020; 112:75-86. [PMID: 32505802 DOI: 10.1016/j.actbio.2020.05.041] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2020] [Revised: 05/28/2020] [Accepted: 05/28/2020] [Indexed: 02/07/2023]
Abstract
Bone injuries are common and new strategies are desired for achieving ideal bone regeneration for bone defect repair. Scaffolds with bone-mimicking characteristics may provide an appropriate microenvironment to promote bone regeneration. Meanwhile, mechanical stimulation effectively regulates a wide range of cellular behaviors such as cell proliferation and differentiation. In this study, biomimetic multi-layer cell-collagen constructs with angle-ply structural feature were prepared by assembling micropatterned collagen membranes on which aligned MC3T3-E1 cells were cultured. The anisotropic microgrooved collagen membranes effectively guided the alignment of cells and promoted the osteogenic differentiation of them. To further promote cell differentiation and extracellular matrix production, the multi-layer cell-collagen constructs were cultured under mechanical conditioning through cyclic stretching. It was found that the constructs with both cell alignment and mechanical conditioning resulted in better osteogenic potential than those with cell alignment or mechanical conditioning alone. Upon implantation into the critical-sized calvarial defects of mice, the constructs with both cell alignment and mechanical conditioning achieved best new bone formation efficacy. Together, findings from this study reveal that synergized use of microstructural and mechanical cues may provide an effective new approach toward bone regeneration. STATEMENT OF SIGNIFICANCE: Biomimicking is an effective strategy to promote bone regeneration for repairing bone defects. Although numerous studies which micro-structurally mimicked native bone using various scaffolds, far less studies have paid attention to the mechanical environment of bone. In this study, angle-ply collagen membrane-supported cell sheets were prepared and pre-conditioned using mechanical loading prior to implantation at bone defects. The constructs with cell alignment and subjected to mechanical conditioning resulted in better osteogenic differentiation of cells in vitro and new bone formation in vivo than those with cell alignment or mechanical conditioning alone. Therefore, recapitulation of both microstructural and mechanical features of native bone may result in a synergistic effect and provides an effective approach toward bone regeneration.
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Kim KM, Kim EJ, Jang WG. Carbohydrate responsive element binding protein (ChREBP) negatively regulates osteoblast differentiation via protein phosphatase 2A Cα dependent manner. Int J Biochem Cell Biol 2020; 124:105766. [PMID: 32416328 DOI: 10.1016/j.biocel.2020.105766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2019] [Revised: 05/06/2020] [Accepted: 05/09/2020] [Indexed: 11/29/2022]
Abstract
Carbohydrate responsive element binding protein (ChREBP) is a major transcription factor of lipogenesis regulated by glucose status in the liver. However, the function of ChREBP in osteogenic differentiation is unclear. The present study examined the role of ChREBP in osteoblast differentiation in MC3T3-E1 preosteoblast cell line. The mRNA expression of ChREBP, protein phosphatase 2A catalytic subunit-α (PP2A Cα) and the osteogenic genes such as, DNA-binding protein inhibitor (Id1), runt-related transcription factor-2 (Runx2), and alkaline phosphatase (ALP) was measured by qPCR and RT-PCR. Runx2, ChREBP, and PP2A Cα, protein levels were evaluated by Western blotting. ALP staining experiment was carried out to evaluate ALP enzyme activity, and a luciferase reporter assay was performed to analyze Runx2 transcriptional activity. Expression of ChREBP and PP2A Cα did not change during bone morphogenetic protein-2 (BMP2)-induced osteoblast differentiation. Overexpression of ChREBP reduced the osteogenic genes (Runx2 and ALP) expression and ALP activity, while knockdown of ChREBP had the opposite effects. Overexpression of PP2A Cα increased ChREBP expression, while inhibition of PP2A Cα using okadaic acid not only inhibited the expression of ChREBP, but also restored the mRNA and protein expression of Runx2 and activity of ALP enzyme. These results demonstrate that ChREBP inhibits BMP2-induced osteoblast differentiation in a PP2A Cα- dependent manner.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
| | - Eun-Jung Kim
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea; Department of Immunology, Kyungpook National University School of Medicine, Daegu 41944, Republic of Korea.
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
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Son HE, Min HY, Kim EJ, Jang WG. Fat Mass and Obesity-Associated (FTO) Stimulates Osteogenic Differentiation of C3H10T1/2 Cells by Inducing Mild Endoplasmic Reticulum Stress via a Positive Feedback Loop with p-AMPK. Mol Cells 2020; 43:58-65. [PMID: 31940720 PMCID: PMC6999711 DOI: 10.14348/molcells.2019.0136] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2019] [Revised: 09/29/2019] [Accepted: 12/05/2019] [Indexed: 12/14/2022] Open
Abstract
Fat mass and obesity-associated (FTO) gene helps to regulate energy homeostasis in mammals by controlling energy expenditure. In addition, FTO functions in the regulation of obesity and adipogenic differentiation; however, a role in osteogenic differentiation is unknown. This study investigated the effects of FTO on osteogenic differentiation of C3H10T1/2 cells and the underlying mechanism. Expression of osteogenic and endoplasmic reticulum (ER) stress markers were characterized by reverse-transcriptase polymerase chain reaction and western blotting. Alkaline phosphatase (ALP) staining was performed to assess ALP activity. BMP2 treatment increased mRNA expression of osteogenic genes and FTO. Overexpression of FTO increased expression of the osteogenic genes distal-less homeobox5 (Dlx5) and runt-related transcription factor 2 (Runx2). Activation of adenosine monophosphate-activated protein kinase (AMPK) increased FTO expression, and there was a positive feedback loop between FTO and p-AMPK. p-AMPK and FTO induced mild ER stress; however, tunicamycin-induced severe ER stress suppressed FTO expression and AMPK activation. In summary, FTO induces osteogenic differentiation of C3H10T1/2 cells upon BMP2 treatment by inducing mild ER stress via a positive feedback loop with p-AMPK. FTO expression and AMPK activation induce mild ER stress. By contrast, severe ER stress inhibits osteogenic differentiation by suppressing FTO expression and AMPK activation.
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Affiliation(s)
- Hyo-Eun Son
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453,
Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453,
Korea
| | - Hyeon-Young Min
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453,
Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453,
Korea
| | - Eun-Jung Kim
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453,
Korea
- Department of Immunology, Kyungpook National University School of Medicine, Daegu 41944,
Korea
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan 38453,
Korea
- Research Institute of Anti-Aging, Daegu University, Gyeongsan 38453,
Korea
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Zhu J, Ye H, Deng D, Li J, Wu Y. Electrospun metformin-loaded polycaprolactone/chitosan nanofibrous membranes as promoting guided bone regeneration membranes: Preparation and characterization of fibers, drug release, and osteogenic activity in vitro. J Biomater Appl 2020; 34:1282-1293. [PMID: 31964207 DOI: 10.1177/0885328220901807] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Affiliation(s)
- Junjin Zhu
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Huilin Ye
- State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
| | - Dan Deng
- Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, Chengdu, China
| | - Jidong Li
- Research Center for Nano-Biomaterials, Analytical and Testing Center, Sichuan University, Chengdu, China
| | - Yingying Wu
- Department of Implantology, State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China
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Chai X, Zhang W, Chang B, Feng X, Song J, Li L, Yu C, Zhao J, Si H. GPR39 agonist TC-G 1008 promotes osteoblast differentiation and mineralization in MC3T3-E1 cells. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2020; 47:3569-3576. [PMID: 31448639 DOI: 10.1080/21691401.2019.1649270] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/09/2023]
Abstract
Osteoporosis-related bone fracture and falls have a severe impact on patients' daily lives. Osteoblasts are bone-building cells that play a vital role in bone formation and remodeling. Imbalanced osteoblast differentiation could lead to osteoporosis. GPR39 is an orphan G protein-coupled receptor that mediates metabolic pathways. In this study, we show that GPR39 is expressed in MC3T3-E1 cells. Osteoblast differentiation culture media induces GPR39, suggesting that GPR39 is a differentiation-responsive factor. Activation of GPR39 using its selective agonist TC-G 1008 induces alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen (Col-I) expression, and increases cellular ALP activity and calcium deposition, implying that GPR activation promotes cells toward osteoblast differentiation. Treatment with TC-G 1008 also increases Runx-2 expression and AMPK activation. However, the inhibition of AMPK by Compound C abolished TC-G 1008-mediated ALP, OCN, and Col-I induction, and reduces ALP activity and cellular calcium deposition as well as Runx-2 induction. These data indicate that TC-G 1008-mediated GPR39 activation involves AMPK-mediated Runx-2 induction. In summary, our study uncovers a new role of GPR39 activation in osteoblast differentiation, implying that GPR39 could be a promising therapeutic target for osteoporosis.
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Affiliation(s)
- Xingyu Chai
- School of Medicine, Shandong University , Ji'nan , China
| | - Wencan Zhang
- Department of Orthopedics, Qilu Hospital, Shandong University , Ji'nan , China
| | - Bingying Chang
- Department of Orthopedics, East Hospital of Shouguang People's Hospital , Weifang , China
| | - Xianli Feng
- Department of Orthopedics, Shandong Tai'an Coal Mine Hospital , Tai'an , China
| | - Jiang Song
- Department of Spine Surgery, Tengzhou Central People's Hospital , Tengzhou , China
| | - Le Li
- Department of Orthopedics, Qilu Hospital, Shandong University , Ji'nan , China
| | - Chenxiao Yu
- Department of Orthopedics, Qilu Hospital, Shandong University , Ji'nan , China
| | - Junyong Zhao
- College of Physics and Electronic Sciences, Shandong Normal University , Ji'nan , China
| | - Haipeng Si
- Department of Orthopedics, Qilu Hospital, Shandong University , Ji'nan , China
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Kim YH, Jang WG, Oh SH, Kim JW, Lee MN, Song JH, Yang JW, Zang Y, Koh JT. Fenofibrate induces PPARα and BMP2 expression to stimulate osteoblast differentiation. Biochem Biophys Res Commun 2019; 520:459-465. [PMID: 31607484 DOI: 10.1016/j.bbrc.2019.10.048] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2019] [Accepted: 10/03/2019] [Indexed: 10/25/2022]
Abstract
The peroxisome proliferator-activated receptor (PPAR)-α agonist fenofibrate is used as a lipid-lowering agent to reduce cholesterol and triglyceride in blood. In this study, we investigated whether fenofibrate affects osteoblast differentiation of osteogenic precursor cells. Quantitative real-time PCR and alkaline phosphatase (ALP) staining assays revealed that fenofibrate can enhance the osteoblast differentiation of C3H10T1/2 and MC3T3-E1 cells. In contrast with fenofibrate, the PPARγ agonist rosiglitazone decreased or did not affect the expression of osteogenic genes in these cells. Fenofibrate dose- and time-dependently increased PPARα expression, and concomitantly increased the expression of bone morphogenetic protein 2 (BMP2). Knockdown of PPARα abolished fenofibrate-induced BMP2 expression, activity of the BMP2 promoter gene, and calcium deposition. The chromatin immunoprecipitation assay demonstrated that fenofibrate increased BMP2 expression by inducing direct binding of PPARα to the BMP2 promoter region. Taken together, we suggest that fenofibrate has a stimulatory effect on osteoblast differentiation via the elevation of PPARα levels and the PPARα-mediated BMP2 expression. Our findings provide fenofibrate as a useful agent for controlling hypercholesterolemic patients with osteoporosis.
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Affiliation(s)
- Yu-Ha Kim
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongsan, Republic of Korea
| | - Sin-Hye Oh
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Jung-Woo Kim
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Mi Nam Lee
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Ju Han Song
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Jin-Woo Yang
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Yaran Zang
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Jeong-Tae Koh
- Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea; Department of Pharmacology and Dental Therapeutics, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea.
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Bai J, Xu Y, Dieo Y, Sun G. Combined low-dose LiCl and LY294002 for the treatment of osteoporosis in ovariectomized rats. J Orthop Surg Res 2019; 14:177. [PMID: 31196133 PMCID: PMC6567919 DOI: 10.1186/s13018-019-1210-1] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2018] [Accepted: 05/20/2019] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND To provide a low-toxicity and high-efficacy clinical treatment for osteoporosis via a novel combination of LiCl and LY294002. METHODS The protein levels of p-AKT, AKT, p-GSK3β, GSK3β, β-catenin, p-β-catenin, and NFATC1 were measured in osteoblasts and osteoclasts by Western blot. ALP activity and TRACP activity were measured using the corresponding kit. The levels of BALP, PINP, CTX, and TRACP-5b were determined in accordance with the requirements of the ELISA kits. Microstructural analysis was performed on the left distal femur using microcomputed tomography. RESULTS Treatment with the combination of LiCl and LY294002 led to a markedly increased osteoblast activity but significantly decreased osteoclast differentiation and bone absorption capacity compared with the treatment with LiCl or LY294002 alone (P < 0.01). In serum, the low-dose combination of LiCl and LY294002 significantly enhanced BALP levels (P < 0.01) and significantly decreased PINP, TRACP-5b, and CTX levels (P < 0.01) compared with the application of either drug alone. CONCLUSIONS This study indicates that drug combinations directed at multiple targets could be used for osteoporosis treatment by promoting osteoblast proliferation and inhibiting differentiation with high efficiency.
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Affiliation(s)
- Jianhai Bai
- Department of Ophthalmology, Taizhou Central Hospital (Taizhou University Hospital), 999 Donghaidadao St, Jiaojiang District, Taizhou, 318000, Zhejiang Province, China
| | - Yier Xu
- The Fourth Affiliated Hospital of Nanchang University, 133 Guangchangnan St., Xihu District, Nanchang, China
| | - Yan Dieo
- School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin, China
| | - Guicai Sun
- The Fourth Affiliated Hospital of Nanchang University, 133 Guangchangnan St., Xihu District, Nanchang, China.
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Peroxiredoxin II negatively regulates BMP2-induced osteoblast differentiation and bone formation via PP2A Cα-mediated Smad1/5/9 dephosphorylation. Exp Mol Med 2019; 51:1-11. [PMID: 31160554 PMCID: PMC6546700 DOI: 10.1038/s12276-019-0263-x] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2018] [Revised: 02/08/2019] [Accepted: 02/18/2019] [Indexed: 12/12/2022] Open
Abstract
Peroxiredoxin II (Prx II), an antioxidant enzyme in the Prx family, reduces oxidative stress by decreasing the intracellular ROS levels. Osteoblast differentiation is promoted by bone morphogenetic protein 2 (BMP2), which upregulates the expression of osteoblast differentiation marker genes, through Smad1/5/9 phosphorylation. We found that Prx II expression was increased by a high dose of lipopolysaccharide (LPS) but was not increased by a low dose of LPS. Prx II itself caused a decrease in the osteogenic gene expression, alkaline phosphatase (ALP) activity, and Smad1/5/9 phosphorylation induced by BMP2. In addition, BMP2-induced osteogenic gene expression and ALP activity were higher in Prx II knockout (KO) cells than they were in wild-type (WT) cells. These inhibitory effects were mediated by protein phosphatase 2A Cα (PP2A Cα), which was increased and is known to induce the dephosphorylation of Smad1/5/9. The overexpression of Prx II increased the expression of PP2A Cα, and PP2A Cα was not expressed in Prx II KO cells. Moreover, PP2A Cα reduced the level of BMP2-induced osteogenic gene expression and Smad1/5/9 phosphorylation. LPS inhibited BMP2-induced Smad1/5/9 phosphorylation and the suppressed phosphorylation was restored by the PP2A inhibitor okadaic acid (OA). Bone phenotype analyses using microcomputed tomography (μCT) revealed that the Prx II KO mice had higher levels of bone mass than the levels of the WT mice. We hypothesize that Prx II has a negative role in osteoblast differentiation through the PP2A-dependent dephosphorylation of Smad1/5/9. An antioxidant enzyme actively works to reduce bone synthesis under oxidative stress conditions in order to protect bone cells from damage and cell death. Bone is generated by cells called osteoblasts, which differentiate from stem cells. In osteoporosis and diabetes, excessive reactive oxygen species (ROS) within cells can disrupt osteoblast differentiation. South Korean researchers led by Eun-jung Kim at Kyungpook National University, Daegu, and Won-Gu Jang at Daegu University, Gyeongbuk, have shown that an antioxidant enzyme, peroxiredoxin II (PrxII), helps regulate bone formation under oxidative stress. The team generated PrxII-deficient mice and compared them with healthy normal mice. Under oxidative stress conditions, the mice had higher bone mass and higher expression of genes related to bone formation than the normal mice. PrxII limits osteoblast differentiation during elevated ROS by influencing associated protein activity and signalling pathways.
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Cheng Y, Huang L, Wang Y, Huo Q, Shao Y, Bao H, Li Z, Liu Y, Li X. Strontium promotes osteogenic differentiation by activating autophagy via the the AMPK/mTOR signaling pathway in MC3T3‑E1 cells. Int J Mol Med 2019; 44:652-660. [PMID: 31173178 PMCID: PMC6605659 DOI: 10.3892/ijmm.2019.4216] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2019] [Accepted: 05/24/2019] [Indexed: 12/23/2022] Open
Abstract
Strontium (Sr) is an alkaline earth metal that exerts the dual effect of improving bone formation and suppressing bone resorption, resulting in increased bone apposition rates and bone mineral density. However, the mechanisms through which Sr exerts these beneficial effects on bone have yet to be fully elucidated. The present study aimed to reveal the underlying molecular mechanisms associated with Sr‑induced osteogenic differentiation. The effects of Sr on cell proliferation and osteogenic differentiation were analyzed by MTT assay, RT‑qPCR, western blot analysis, alkaline phosphatase (ALP) and Alizarin red staining assays. The extent of autophagy was determined by monodansylcadaverine (MDC) staining and western blot analysis of two markers of cellular autophagic activity, the steatosis‑associated protein, sequestosome‑1 (SQSTM1/p62), and the two isoforms of microtubule‑associated protein 1 light chain 3 (LC3), LC‑3‑I/II. The expression levels of AMP‑activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were also detected by western blot analysis. Sr at a concentration of 3 mM exerted the most pronounced effect on osteogenic differentiation, without any apparent cell toxicity. At the same time, cellular autophagy was active during this process. Subsequently, autophagy was blocked by 3‑methyladenine, and the enhancement of osteogenic differentiation in response to Sr was abrogated. Additionally, the phosphorylation level of AMPK was significantly increased, whereas that of mTOR was significantly decreased, in the Sr‑treated group. Taken together, the findings of the present study demonstrate that Sr stimulates AMPK‑activated autophagy to induce the osteogenic differentiation of MC3T3‑E1 cells.
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Affiliation(s)
- You Cheng
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Lunhui Huang
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Yichao Wang
- Department of Clinical Laboratory Medicine, Taizhou University Hospital, Taizhou, Zhejiang 318000, P.R. China
| | - Qianyu Huo
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Yanhong Shao
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Huijing Bao
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Zhaoyang Li
- School of Materials Science and Engineering, Tianjin University, Tianjin 300350, P.R. China
| | - Yunde Liu
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
| | - Xue Li
- School of Medical Laboratory, Tianjin Medical University, Tianjin 300070, P.R. China
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MacKenzie RK, Sankar PR, Bendall AJ. Dlx5 and Dlx6 can antagonize cell division at the G 1/S checkpoint. BMC Mol Cell Biol 2019; 20:8. [PMID: 31041891 PMCID: PMC6460778 DOI: 10.1186/s12860-019-0191-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2018] [Accepted: 04/02/2019] [Indexed: 11/17/2022] Open
Abstract
Background Dlx5 and Dlx6 stimulate differentiation of diverse progenitors during embryonic development. Their actions as pro-differentiation transcription factors includes the up-regulation of differentiation markers but the extent to which differentiation may also be stimulated by regulation of the cell cycle has not been addressed. Results We document that expression of Dlx5 and Dlx6 antagonizes cell proliferation in a variety of cell types without inducing apoptosis or promoting cell cycle exit. Rather, a variety of evidence indicates that elevated Dlx5 and Dlx6 expression reduces the proportion of cells in S phase and affects the length of the cell cycle. Conclusions Antagonism of S-phase entry by Dlx5 and Dlx6 proteins likely represents a lineage-independent function to effect Dlx-mediated differentiation in multiple progenitor cell types.
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Affiliation(s)
- Rachel K MacKenzie
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd East, Guelph, Ontario, N1G 2W1, Canada
| | - Parvathy Ravi Sankar
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd East, Guelph, Ontario, N1G 2W1, Canada
| | - Andrew J Bendall
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Rd East, Guelph, Ontario, N1G 2W1, Canada.
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35
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Kalaitzoglou E, Fowlkes JL, Popescu I, Thrailkill KM. Diabetes pharmacotherapy and effects on the musculoskeletal system. Diabetes Metab Res Rev 2019; 35:e3100. [PMID: 30467957 PMCID: PMC6358500 DOI: 10.1002/dmrr.3100] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/04/2018] [Revised: 11/14/2018] [Accepted: 11/19/2018] [Indexed: 12/13/2022]
Abstract
Persons with type 1 or type 2 diabetes have a significantly higher fracture risk than age-matched persons without diabetes, attributed to disease-specific deficits in the microarchitecture and material properties of bone tissue. Therefore, independent effects of diabetes drugs on skeletal integrity are vitally important. Studies of incretin-based therapies have shown divergent effects of different agents on fracture risk, including detrimental, beneficial, and neutral effects. The sulfonylurea class of drugs, owing to its hypoglycemic potential, is thought to amplify the risk of fall-related fractures, particularly in the elderly. Other agents such as the biguanides may, in fact, be osteo-anabolic. In contrast, despite similarly expected anabolic properties of insulin, data suggests that insulin pharmacotherapy itself, particularly in type 2 diabetes, may be a risk factor for fracture, negatively associated with determinants of bone quality and bone strength. Finally, sodium-dependent glucose co-transporter 2 inhibitors have been associated with an increased risk of atypical fractures in select populations, and possibly with an increase in lower extremity amputation with specific SGLT2I drugs. The role of skeletal muscle, as a potential mediator and determinant of bone quality, is also a relevant area of exploration. Currently, data regarding the impact of glucose lowering medications on diabetes-related muscle atrophy is more limited, although preclinical studies suggest that various hypoglycemic agents may have either aggravating (sulfonylureas, glinides) or repairing (thiazolidinediones, biguanides, incretins) effects on skeletal muscle atrophy, thereby influencing bone quality. Hence, the therapeutic efficacy of each hypoglycemic agent must also be evaluated in light of its impact, alone or in combination, on musculoskeletal health, when determining an individualized treatment approach. Moreover, the effect of newer medications (potentially seeking expanded clinical indication into the pediatric age range) on the growing skeleton is largely unknown. Herein, we review the available literature regarding effects of diabetes pharmacotherapy, by drug class and/or by clinical indication, on the musculoskeletal health of persons with diabetes.
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Affiliation(s)
- Evangelia Kalaitzoglou
- University of Kentucky Barnstable Brown Diabetes Center Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA
| | - John L Fowlkes
- University of Kentucky Barnstable Brown Diabetes Center Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA
| | - Iuliana Popescu
- University of Kentucky Barnstable Brown Diabetes Center Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA
| | - Kathryn M Thrailkill
- University of Kentucky Barnstable Brown Diabetes Center Department of Pediatrics, University of Kentucky College of Medicine, Lexington, KY, USA
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36
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Ma WQ, Sun XJ, Wang Y, Zhu Y, Han XQ, Liu NF. Restoring mitochondrial biogenesis with metformin attenuates β-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of PDK4/oxidative stress-mediated apoptosis. Mol Cell Endocrinol 2019; 479:39-53. [PMID: 30170182 DOI: 10.1016/j.mce.2018.08.012] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Revised: 08/27/2018] [Accepted: 08/27/2018] [Indexed: 12/19/2022]
Abstract
Mitochondrial abnormalities have long been observed in the development of vascular calcification. Metformin, a member of the biguanide class of antidiabetic drugs, has recently received attention owing to new findings regarding its protective role in cardiovascular disease. Since the precise control of mitochondrial quantity and quality is critical for the survival and function of vascular smooth muscle cells (VSMCs), maintaining mitochondrial homeostasis may be a potential protective factor for VSMCs against osteoblast-like phenotypic transition. However, limited studies have been reported in this area. Here, we investigated the role of metformin in the phenotypic transformation of VSMCs, as well as its intracellular signal transduction pathways. We demonstrated that supplementation with metformin restored the β-glycerophosphate (β-GP)-mediated impairment of mitochondrial biogenesis in VSMCs, as evidenced by an increased mitochondrial DNA copy number, a restored mitochondrial membrane potential (MMP), and upregulated mitochondrial biogenesis-related gene expression, whereas the AMP-activated protein kinase (AMPK) inhibitor compound C suppressed these effects. We also observed that overexpression of pyruvate dehydrogenase kinase 4 (PDK4), an important mitochondrial matrix enzyme in cellular energy metabolism, exacerbated β-GP-induced oxidative stress and subsequent apoptosis in VSMCs but that these effects were suppressed by dichloroacetate, a widely reported PDK4 inhibitor. More importantly, enhanced mitochondrial biogenesis attenuated the β-GP-induced phenotypic transformation of VSMCs into an osteogenic phenotype through inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, whereas disruption of mitochondrial biogenesis by zidovudine aggravated β-GP-induced apoptosis in VSMCs. In addition, inhibition of autophagy by small interfering RNA targeting Atg5 reduced mitochondrial biogenesis in VSMCs. In summary, we uncovered a novel mechanism by which metformin attenuates the phenotypic transformation of VSMCs into an osteogenic phenotype via inhibition of the PDK4/oxidative stress-mediated apoptosis pathway, and mitochondrial homeostasis is involved in this process.
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Affiliation(s)
- Wen-Qi Ma
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China
| | - Xue-Jiao Sun
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China
| | - Ying Wang
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China
| | - Yi Zhu
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China
| | - Xi-Qiong Han
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China
| | - Nai-Feng Liu
- Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, PR China.
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Zheng L, Shen X, Ye J, Xie Y, Yan S. Metformin alleviates hyperglycemia-induced apoptosis and differentiation suppression in osteoblasts through inhibiting the TLR4 signaling pathway. Life Sci 2018; 216:29-38. [PMID: 30414431 DOI: 10.1016/j.lfs.2018.11.008] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2018] [Revised: 10/27/2018] [Accepted: 11/05/2018] [Indexed: 02/07/2023]
Abstract
AIMS Metformin was found to protect against hyperglycemia-induced injury in osteoblasts, but the cellular mechanisms involved remain unclear. Therefore, the aim of this study was to determine the effect of metformin on hyperglycemia-induced apoptosis and differentiation suppression in osteoblasts and to explore its relationships with the TLR4 signaling pathway. MAIN METHODS A mouse osteoblast cell line, MC3T3-E1, and a diabetic rat model were used to survey the protective effects of metformin on hyperglycemia-induced injury. TLR4 expression was altered using small interfering (si)RNA and lentivirus-mediated TLR4 overexpression. LPS was used as a specific TLR4 activator, and CLI-095 was used as a TLR4 inhibitor. KEY FINDINGS Metformin improved osteoblast differentiation, reduced apoptosis in hyperglycemic osteoblasts, and inhibited TLR4, MyD88 and NF-κB expression in a dose-dependent manner. Down-regulating the expression or inhibiting the activity of TLR4 enhanced these protective effects of metformin on osteoblast differentiation, cell viability and cell apoptosis in hyperglycemic conditions, whereas up-regulating the expression or activating the activity of TLR4 had the opposite effects. Activating NF-κB suppressed the protective effects of metformin, while inhibiting NF-κB activity had the opposite effects. Metformin increased ALP and OCN secretion, enhanced BMP-2 expression, improved bone mineral density (BMD), and decreased TLR4, MyD88 and NF-κB levels in the femur tissues of diabetic rats. SIGNIFICANCE Taken together our experimentation support the hypothesis that metformin may alleviate hyperglycemia-induced apoptosis and differentiation suppression in osteoblasts by inhibiting the TLR4/MyD88/NF-κB signaling pathway.
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Affiliation(s)
- Lifeng Zheng
- Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China
| | - Ximei Shen
- Department of Endocrinology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China; Diabetes Research Institute of Fujian Province, Fuzhou 350005, Fujian, China
| | - Junjian Ye
- Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China
| | - Yun Xie
- Department of Orthopedics, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China
| | - Sunjie Yan
- Department of Endocrinology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, China; Diabetes Research Institute of Fujian Province, Fuzhou 350005, Fujian, China.
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Kim KM, Jeon WJ, Kim EJ, Jang WG. CRTC2 suppresses BMP2-induced osteoblastic differentiation via Smurf1 expression in MC3T3-E1 cells. Life Sci 2018; 214:70-76. [PMID: 30449452 DOI: 10.1016/j.lfs.2018.10.052] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2018] [Revised: 10/18/2018] [Accepted: 10/25/2018] [Indexed: 12/14/2022]
Abstract
AIMS CREB (cAMP response element-binding protein)-regulated transcription coactivator (CRTC2) has been reported to act as a coactivator of CREB during gluconeogenesis. The role of CRTC2 in osteoblastic differentiation has not yet been elucidated. The aim of this study is to identify the mechanism of CRTC2 in osteoblast differentiation. MAIN METHODS The mRNA expression was determined by RT-PCR and qPCR. Protein levels were measured using Western blot assay. Alkaline phosphatase (ALP) staining was performed to evaluate ALP activity. Alizarin red S (ARS) staining was performed to measure extracellular mineralization. Transcriptional activity was detected using a luciferase assay. KEY FINDINGS In the present study, TNF-α was found to stimulate CRTC2 expression. However, TNF-α did not increase the gene expression of osteoblast differentiation markers and inhibited BMP2-induced osteoblastic differentiation. Overexpression of CRTC2 decreased the expression of osteogenic genes, ALP activity and extracellular matrix mineralization. Knockdown of CRTC2 restored BMP2-induced osteogenic gene expression and ALP activity. CRTC2 increased Smurf1 mRNA expression, Smurf 1 promoter activity, and protein level. Furthermore, Smurf 1 decreased Smad 1/5/9 protein levels. These results suggest that CRTC2 decreased BMP2-induced osteoblastic differentiation via Smurf 1 expression. SIGNIFICANCE Our results indicate that CRTC2 regulates the expression of Smurf1 in osteoblast differentiation.
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Affiliation(s)
- Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea
| | - Wan-Jin Jeon
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea
| | - Eun-Jung Kim
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea; Department of Immunology, Kyungpook National University School of Medicine, Daegu 41944, Republic of Korea.
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk 38453, Republic of Korea; Research Institute of Anti-Aging, Daegu University, Gyeongbuk 38453, Republic of Korea.
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Kanazawa I, Takeno A, Tanaka KI, Notsu M, Sugimoto T. Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice. Biochem Biophys Res Commun 2018; 503:1955-1961. [DOI: 10.1016/j.bbrc.2018.07.141] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2018] [Accepted: 07/27/2018] [Indexed: 01/07/2023]
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40
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A novel phosphorylation by AMP-activated kinase regulates RUNX2 from ubiquitination in osteogenesis over adipogenesis. Cell Death Dis 2018; 9:754. [PMID: 29988028 PMCID: PMC6037667 DOI: 10.1038/s41419-018-0791-7] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 05/10/2018] [Accepted: 06/11/2018] [Indexed: 12/13/2022]
Abstract
Mesenchymal stem cells (MSCs) function as progenitors to a variety of cell types. The reported association between osteogenic and adipogenic commitment during differentiation is due to the regulation of key transcription factors in the signaling pathways. However, the process of adipogenesis at the expense of osteogenic phenotype during metabolic stress is still unclear. In this study, we showed for the first time that RUNX2 is a novel substrate of AMP-activated kinase (AMPK), which directly phosphorylates at serine 118 residue in the DNA-binding domain of RUNX2. Our results in in vitro MSC lineage differentiation models confirmed that active AMPK and RUNX2-S118 phosphorylation are preferentially associated with osteogenic commitment, whereas the lack of this phosphorylation leads to adipogenesis. This interplay is regulated by the ubiquitination of non-phosphorylated RUNX2-S118, which is evident in the dominant mutant RUNX2-S118D. Pharmacological activation of AMPK by metformin significantly abrogated the loss of RUNX2-S118 phosphorylation and protected from tunicamycin-induced endoplasmic reticulum stress, high glucose-induced in vitro adipogenesis and streptozotocin-induced in vivo bone adiposity and bone phenotype. In conclusion, results from this study demonstrated that RUNX2 is a direct target of AMPK which simplified the outlook towards several complex mechanisms that are currently established concerning cellular metabolism and pathogenesis.
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41
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Lee W, Ko KR, Kim HK, Lee DS, Nam IJ, Lim S, Kim S. Dehydrodiconiferyl Alcohol Inhibits Osteoclast Differentiation and Ovariectomy-Induced Bone Loss through Acting as an Estrogen Receptor Agonist. JOURNAL OF NATURAL PRODUCTS 2018; 81:1343-1356. [PMID: 29869503 DOI: 10.1021/acs.jnatprod.7b00927] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Estrogen deficiency after menopause increases bone loss by activating RANKL-induced osteoclast differentiation. Dehydrodiconiferyl alcohol (DHCA), a lignan originally isolated from Cucurbita moschata, has been thought to be a phytoestrogen based on its structure. In this study, we tested whether DHCA could affect RANKL-induced osteoclastogenesis in vitro and ovariectomy-induced bone loss in vivo. In RAW264.7 cells, DHCA inhibited RANKL-induced differentiation of osteoclasts. Consistently, expression of the six osteoclastogenic genes induced by RANKL was down-regulated. DHCA was also shown to suppress the NF-κB and p38 MAPK signaling pathways by activating AMPK. Data from transient transfection assays suggested that DHCA might activate the estrogen receptor signaling pathway. Effects of DHCA on RANKL-induced osteoclastogenesis were reduced when cells were treated with specific siRNA to ERα, but not to ERβ. Interestingly, DHCA was predicted from molecular docking simulation to bind to both ERα and ERβ. Indeed, data from an estrogen receptor competition assay revealed that DHCA acted as an agonist on both estrogen receptors. In the ovariectomized (Ovx) mouse model, DHCA prevented Ovx-induced bone loss by inhibiting osteoclastogenesis. Taken together, our results suggest that DHCA may be developed as an efficient therapeutic for osteoporosis by regulating osteoclastogenesis through its estrogenic effects.
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Affiliation(s)
- Wonwoo Lee
- Department of Biological Sciences , Seoul National University , Seoul 151-742 , Korea
- ViroMed Co., Ltd. , Seoul 151-747 , Korea
| | - Kyeong Ryang Ko
- Department of Biological Sciences , Seoul National University , Seoul 151-742 , Korea
- ViroMed Co., Ltd. , Seoul 151-747 , Korea
| | - Hyun-Keun Kim
- Department of Biological Sciences , Seoul National University , Seoul 151-742 , Korea
| | | | | | - Seonung Lim
- Department of Biological Sciences , Seoul National University , Seoul 151-742 , Korea
| | - Sunyoung Kim
- Department of Biological Sciences , Seoul National University , Seoul 151-742 , Korea
- ViroMed Co., Ltd. , Seoul 151-747 , Korea
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Sun X, Fu X, Du M, Zhu MJ. Ex vivo gut culture for studying differentiation and migration of small intestinal epithelial cells. Open Biol 2018; 8:170256. [PMID: 29643147 PMCID: PMC5936714 DOI: 10.1098/rsob.170256] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2017] [Accepted: 03/09/2018] [Indexed: 12/16/2022] Open
Abstract
Epithelial cultures are commonly used for studying gut health. However, due to the absence of mesenchymal cells and gut structure, epithelial culture systems including recently developed three-dimensional organoid culture cannot accurately represent in vivo gut development, which requires intense cross-regulation of the epithelial layer with the underlying mesenchymal tissue. In addition, organoid culture is costly. To overcome this, a new culture system was developed using mouse embryonic small intestine. Cultured intestine showed spontaneous peristalsis, indicating the maintenance of the normal gut physiological structure. During 10 days of ex vivo culture, epithelial cells moved along the gut surface and differentiated into different epithelial cell types, including enterocytes, Paneth cells, goblet cells and enteroendocrine cells. We further used the established ex vivo system to examine the role of AMP-activated protein kinase (AMPK) on gut epithelial health. Tamoxifen-induced AMPKα1 knockout vastly impaired epithelial migration and differentiation of the developing ex vivo gut, showing the crucial regulatory function of AMPK α1 in intestinal health.
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Affiliation(s)
- Xiaofei Sun
- School of Food Science, Washington State University, Pullman, WA 99164, USA
- School of Food Science, University of Idaho, Moscow, ID 83844, USA
| | - Xing Fu
- Department of Animal Science, Washington State University, Pullman, WA 99164, USA
| | - Min Du
- Department of Animal Science, Washington State University, Pullman, WA 99164, USA
| | - Mei-Jun Zhu
- School of Food Science, Washington State University, Pullman, WA 99164, USA
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Takeno A, Kanazawa I, Notsu M, Tanaka KI, Sugimoto T. Inhibition of adenosine monophosphate-activated protein kinase suppresses bone morphogenetic protein-2-induced mineralization of osteoblasts via Smad-independent mechanisms. Endocr J 2018; 65:291-298. [PMID: 29249772 DOI: 10.1507/endocrj.ej17-0229] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Previous studies showed that adenosine monophosphate-activated protein kinase (AMPK), which plays as an intracellular energy sensor, promotes the differentiation and mineralization of osteoblasts via enhancing expression of bone morphogenetic protein (BMP)-2, which is a potent inducer of osteoblastogenesis. Thus, the aim of this study was to examine the roles of AMPK in BMP-2-induced osteoblastogenesis. We used a murine osteoblastic cell line MC3T3-E1 and a murine marrow stromal cell line ST2. BMP-2 (50 and 100 ng/mL) stimulated alkaline phosphatase (ALP) activity and enhanced mineralization of MC3T3-E1 cells, while the effects of BMP-2 were partly abolished by an inhibitor of AMPK, ara-A (0.1 mM). Real-time PCR showed that BMP-2 significantly increased the mRNA expressions of Alp, osteocalcin (Ocn), Runx2, Osterix and Dlx-5 in MC3T3-E1 cells, while co-incubation of ara-A significantly decreased the BMP-2-stimulated expression of Alp, Ocn, and Runx2. Moreover, co-incubation of ara-A suppressed the BMP-2-induced upregulation of Alp and Ocn in ST2 cells. Western blot analysis showed that BMP-2 phosphorylated Smad1/5 although it did not affect AMPK phosphorylation in MC3T3-E1 cells. Furthermore, a BMP receptor inhibitor LDN-193189 inhibited the phosphorylation of Smad1/5, but did not affect AMPK. In addition, co-incubation of ara-A did not affect BMP-2-induced phosphorylation of Smad1/5. These findings suggest that the inhibition of AMPK activation reduces the osteo-inductive effects of BMP-2 by decreasing the expression of Alp, Ocn, and Runx2 through Smad-independent mechanisms in osteoblastic cells.
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Affiliation(s)
- Ayumu Takeno
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan
| | - Ippei Kanazawa
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan
| | - Masakazu Notsu
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan
| | - Ken-Ichiro Tanaka
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan
| | - Toshitsugu Sugimoto
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane 693-8501, Japan
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Li Y, Su J, Sun W, Cai L, Deng Z. AMP-activated protein kinase stimulates osteoblast differentiation and mineralization through autophagy induction. Int J Mol Med 2018; 41:2535-2544. [PMID: 29484369 PMCID: PMC5846672 DOI: 10.3892/ijmm.2018.3498] [Citation(s) in RCA: 38] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2016] [Accepted: 01/18/2018] [Indexed: 12/25/2022] Open
Abstract
Previous studies have reported that adenosine monophosphate‑activated protein kinase (AMPK) activation can enhance osteoblast differentiation and mineralization; however, the underlying mechanism is not fully understood. Autophagy also serves an important role in osteoblast mineralization and bone homeostasis. The present study aimed to explore whether activation of AMPK could enhance osteoblast differentiation and mineralization via the induction of autophagy. The fracture healing and nonunion animal models were established and verified by X-ray imaging. Bone maturation was measured by Masson staining and the expression of AMPK, p-AMPK, microtubule-associated proteins 1A/1B light chain 3B II, and p62 in the fracture ends were detected by immunohistochemical staining. The mRNA expression levels of alkaline phosphatase (ALP), osteocalcin ,runt-related transcription factor 2 and BCN1 were determined by reverse transcription-quantitative polymerase chain reaction. 5-Bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium staining was used to determine ALP activity and alizarin red staining was adopted to examine mineralization. Western blot analysis was performed to detect protein expression. Autophagosome was observed by Transmission electron microscopy. Small interfering (si)RNA was used to knock down the expression of target gene. In vivo experiments demonstrated that new bone mineralization and maturation was markedly restrained in the nonunion group, alongside decreased AMPK activation and autophagic activity, compared with in the fracture healing group. The results of an in vitro study indicated that AMPK activation stimulated the osteogenic differentiation of MC3T3‑E1 cells, with increases in ALP activity, mineralization, and the mRNA expression levels of ALP, osteocalcin and runt-related transcription factor 2. Furthermore, AMPK activation induced autophagy, as determined by upregulation of microtubule‑associated proteins 1A/1B light chain 3B, increased autophagosome density and downregulation of p62. In addition, inhibition of autophagy reversed the effects of AMPK activation on osteoblast differentiation. These results suggested that AMPK activation may stimulate osteoblast differentiation and mineralization via the induction of autophagy, and provides evidence to suggest that enhancing AMPK activation and autophagic activity may be a potential novel approach to promote fracture healing.
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Affiliation(s)
- Yi Li
- Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China
| | - Jiajia Su
- Department of Radiology, Hubei Cancer Hospital, Wuhan, Hubei 430000, P.R. China
| | - Wenchao Sun
- Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China
| | - Lin Cai
- Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China
| | - Zhouming Deng
- Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, P.R. China
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Son HE, Kim KM, Kim EJ, Jang WG. Kisspeptin-10 (KP-10) stimulates osteoblast differentiation through GPR54-mediated regulation of BMP2 expression and activation. Sci Rep 2018; 8:2134. [PMID: 29391507 PMCID: PMC5794871 DOI: 10.1038/s41598-018-20571-2] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2017] [Accepted: 01/15/2018] [Indexed: 11/09/2022] Open
Abstract
Kisspeptin-10 (KP-10) acts as a tumor metastasis suppressor via its receptor, G-protein-coupled receptor 54 (GPR54). The KP-10-GPR54 system plays an important role in embryonic kidney development. However, its function in osteoblast differentiation is unknown. Osteoblast differentiation is controlled by a range of hormones and cytokines, such as bone morphogenetic protein (BMPs), and multiple transcription factors, such as Runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and Distal-less homeobox 5 (Dlx5). In the present study, KP-10-treatment significantly increased the expression of osteogenic genes, including mRNA and protein levels of BMP2, in C3H10T1/2 cells. Moreover, KP-10 induced BMP2-luc activity and increased phosphorylation of Smad1/5/9. In addition, NFATc4 specifically mediated KP-10-induced BMP2 gene expression. However, KP-10 treatment did not induce expression of the BMP2 and Runx2 genes in GPR54-/- cells. To examine whether KP-10 induced secretion of BMP2 to the culture medium, we used the conditioned-medium (C.M) of KP-10 treated medium on C3H10T1/2 cells. Dlx5 and Runx2 expressions were higher in GPR54-/- cells treated with C.M than in those treated with KP-10. These results demonstrate that BMP2 protein has an autocrine effect upon KP-10 treatment. Taken together, these findings suggest that KP-10/GPR54 signaling induces osteoblast differentiation via NFATc4-mediated BMP2 expression.
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Affiliation(s)
- Hyo-Eun Son
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, 38453, Republic of Korea.,Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea
| | - Kyeong-Min Kim
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, 38453, Republic of Korea.,Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea
| | - Eun-Jung Kim
- Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea. .,Department of Immunology, Kyungpook National University School of Medicine, Daegu, 41944, Republic of Korea.
| | - Won-Gu Jang
- Department of Biotechnology, School of Engineering, Daegu University, Gyeongbuk, 38453, Republic of Korea. .,Research Institute of Anti-Aging, Daegu University, Gyeongbuk, 38453, Republic of Korea.
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Kanazawa I, Takeno A, Tanaka KI, Notsu M, Sugimoto T. Osteoblast AMP-Activated Protein Kinase Regulates Postnatal Skeletal Development in Male Mice. Endocrinology 2018; 159:597-608. [PMID: 29126229 DOI: 10.1210/en.2017-00357] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/13/2017] [Accepted: 10/30/2017] [Indexed: 11/19/2022]
Abstract
Studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in bone development. Thus, to investigate in vivo roles of osteoblast AMPK, we conditionally inactivated Ampk in osterix (Osx)-expressing cells by crossing Osx-Cre mice with floxed AMPKα1 to generate mice lacking AMPKα1 in osteoblasts (Ampk-/- mice). Compared with wild-type and Ampk+/- mice, Ampk-/- mice displayed retardation of postnatal bone development, although bone deformity was not observed at birth. Microcomputed tomography showed significant reductions in trabecular bone volume, cortical bone length, and density, as well as increased cortical porosity in femur as well as development defects of skull in 8-week-old Ampk-/- mice. Surprisingly, histomorphometric analysis demonstrated that the number of osteoclasts was significantly increased, although bone formation rate was not altered. Loss of trabecular network connections and mass, as well as shortened growth plates and reduced thickness of cartilage adjacent to the growth plate, was observed in Ampk-/- mice. In primary cultured osteoblasts from calvaria, the expressions of alkaline phosphatase, type 1 collagen, osteocalcin, bone morphogenetic protein 2, Runx2, and osterix were significantly inhibited in Ampk-/- osteoblasts, whereas the expression of receptor activator of nuclear κB ligand (RANKL) and the RANKL/osteoprotegerin ratio were significantly increased. These findings indicate that osteoblastic AMPK plays important roles in bone development in vivo and that deletion of AMPK in osteoblasts decreases osteoblastic differentiation and enhances bone turnover by increasing RANKL expression.
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Affiliation(s)
- Ippei Kanazawa
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane, Japan
| | - Ayumu Takeno
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane, Japan
| | - Ken-Ichiro Tanaka
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane, Japan
| | - Masakazu Notsu
- Internal Medicine 1, Shimane University Faculty of Medicine, Shimane, Japan
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Zhang F, Xie J, Wang G, Zhang G, Yang H. Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model. J Cell Physiol 2018; 233:4626-4633. [PMID: 28926099 DOI: 10.1002/jcp.26187] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2017] [Accepted: 09/14/2017] [Indexed: 01/01/2023]
Affiliation(s)
- Fuzhan Zhang
- Department of Orthopaedic Surgery; The First Affiliated Hospital of Soochow University; Suzhou P.R. China
| | - Jile Xie
- Department of Orthopaedic Surgery; The First Affiliated Hospital of Soochow University; Suzhou P.R. China
| | - Genlin Wang
- Department of Orthopaedic Surgery; The First Affiliated Hospital of Soochow University; Suzhou P.R. China
| | - Ge Zhang
- Department of Orthopaedic Surgery; The First Affiliated Hospital of Soochow University; Suzhou P.R. China
| | - Huilin Yang
- Department of Orthopaedic Surgery; The First Affiliated Hospital of Soochow University; Suzhou P.R. China
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Kim DY, Kim EJ, Jang WG. Piperine induces osteoblast differentiation through AMPK-dependent Runx2 expression. Biochem Biophys Res Commun 2018; 495:1497-1502. [DOI: 10.1016/j.bbrc.2017.11.200] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2017] [Accepted: 11/29/2017] [Indexed: 12/29/2022]
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Lee W, Ko KR, Kim HK, Lim S, Kim S. Dehydrodiconiferyl alcohol promotes BMP-2-induced osteoblastogenesis through its agonistic effects on estrogen receptor. Biochem Biophys Res Commun 2017; 495:2242-2248. [PMID: 29253565 DOI: 10.1016/j.bbrc.2017.12.079] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Accepted: 12/14/2017] [Indexed: 12/11/2022]
Abstract
Estrogen deficiency results in an imbalance between the levels of bone-resorping osteoclasts and bone-forming osteoblasts, eventually leading to overall bone loss. Dehydrodiconiferyl alcohol (DHCA), a lignan compound originally isolated from Cucurbita moschata, has been shown to bind to estrogen receptor, and indeed exhibits various activities of estrogen, such as anti-inflammatory and anti-oxidative stress effects. In this study, we tested whether synthetic DHCA could affect the BMP-2-induced osteoblastogenesis in vitro. In MC3T3-E1 cells, DHCA promoted BMP-2-induced differentiation of osteoblasts. Consistently, the expression of three osteoblastogenic genes known to be induced by BMP-2, ALP, osteocalcin and OPG, was up-regulated by DHCA treatment. DHCA was also shown to activate the production of RUNX2 by activating Smad1/5/9 and AMPK. Data from transient transfection assays suggested that DHCA might activate the estrogen receptor signaling pathway. Effects of DHCA on BMP-2-induced osteoblastogenesis were reduced when cells were treated with a specific siRNA to ERα or ERβ. Taken together, our results suggest that DHCA may be developed as an efficient therapeutic for osteoporosis by regulating osteoblastogenesis through its estrogenic effects.
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Affiliation(s)
- Wonwoo Lee
- Department of Biological Sciences, Seoul National University, Seoul 151-742, South Korea; ViroMed Co., Ltd., Seoul 151-747, South Korea
| | - Kyeong Ryang Ko
- Department of Biological Sciences, Seoul National University, Seoul 151-742, South Korea; ViroMed Co., Ltd., Seoul 151-747, South Korea
| | - Hyun-Keun Kim
- Department of Biological Sciences, Seoul National University, Seoul 151-742, South Korea
| | - Seonung Lim
- Department of Biological Sciences, Seoul National University, Seoul 151-742, South Korea
| | - Sunyoung Kim
- Department of Biological Sciences, Seoul National University, Seoul 151-742, South Korea; ViroMed Co., Ltd., Seoul 151-747, South Korea.
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Abstract
Accumulating evidence has shown that bone and glucose metabolism are closely associated with each other. Since the risk of osteoporotic fractures is increased in patients with diabetes mellitus (DM), osteoporosis is recently recognized as one of diabetic complications, called DM-induced bone fragility. Previous studies showed that collagen cross-links of advanced glycation end products (AGEs) and dysfunctions of osteoblast and osteocyte are involved in DM-induced bone fragility. Circulating levels of AGEs and homocysteine are increased in patients with DM, and they directly impair the functions of osteoblast and osteocyte, resulting in decreased bone formation and bone remodeling. On the other hand, bone is recently recognized as an endocrine organ. Previous studies based on in vitro and animal studies showed that osteocalcin, which is specifically expressed in osteoblasts and secreted into the circulation, may regulate glucose homeostasis. Although several clinical studies reported the relationship between osteocalcin and glucose metabolism, further large-scale and intervention studies are necessary to confirm the beneficial effects of osteocalcin on glucose metabolism in human. It has been shown that adenosine monophosphate-activated protein kinase (AMPK), an intracellular energy sensor, is involved in bone metabolism. Adiponectin and metformin stimulate osteocalcin expression and the differentiation of osteoblasts via AMPK activation. Also, AMPK activation protects against oxidative stress-induced apoptosis of osteocytes. These findings suggest that AMPK in osteoblasts and osteocytes may be a therapeutic target for DM-induced bone fragility.
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Affiliation(s)
- Ippei Kanazawa
- Internal Medicine 1, Shimane University Faculty of Medicine, Izumo 693-8501, Japan
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