|
1
|
Umetsu A, Watanabe M, Sato T, Higashide M, Nishikiori N, Furuhashi M, Ohguro H. TGF-β effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions. FEBS Open Bio 2024. [PMID: 39380256 DOI: 10.1002/2211-5463.13890] [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: 02/06/2024] [Revised: 04/04/2024] [Accepted: 08/23/2024] [Indexed: 10/10/2024] Open
Abstract
The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.
Collapse
Affiliation(s)
- Araya Umetsu
- Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan
| | - Megumi Watanabe
- Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan
| | - Tatsuya Sato
- Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Japan
- Department of Cellular Physiology and Signal Transduction, Sapporo Medical University School of Medicine, Japan
| | - Megumi Higashide
- Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan
| | - Nami Nishikiori
- Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan
| | - Masato Furuhashi
- Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Japan
| | - Hiroshi Ohguro
- Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan
| |
Collapse
|
|
2
|
Maharjan BR, McLennan SV, Twigg SM, Williams PF. The Effect of TGFβ1 in Adipocyte on Inflammatory and Fibrotic Markers at Different Stages of Adipocyte Differentiation. PATHOPHYSIOLOGY 2022; 29:640-649. [PMID: 36548206 PMCID: PMC9788619 DOI: 10.3390/pathophysiology29040050] [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: 10/20/2022] [Revised: 11/18/2022] [Accepted: 11/21/2022] [Indexed: 11/24/2022] Open
Abstract
Transforming growth factor beta (TGFβ) is a versatile cytokine. Although a profibrotic role of TGFβ is well established, its effect on tissue inhibitor of metalloproteinase (TIMPs) and inflammatory mediators are incompletely described. This study investigates the profibrotic and pro-inflammatory role of TGFβ1 during adipocyte differentiation. NIH3T3L1 cells were used for the in vitro study and were differentiated by adding a standard differentiation mix either with rosiglitazone (R-Diff) or without (S-Diff). Recombinant TGFβ1 (2 ng/mL) was added to the undifferentiated preadipocyte during the commitment stage and at the terminal differentiation stage. TGFβ1 treatment significantly decreased adiponectin mRNA at both early commitment (>300 fold) and terminal differentiated cells [S-Diff (~33%) or R-Diff (~20%)]. TGFβ1 upregulated collagen VI mRNA and its regulators connective tissue growth factor (CCN2/CTGF), TIMP1 and TIMP3 mRNA levels in undifferentiated preadipocytes and adipocytes at commitment stage. But in the terminal differentiated adipocytes, changes in mRNA and protein of collagen VI and TIMP3 mRNA were not observed despite an increase in CCN2/CTGF, TIMP1 mRNA. Although TGFβ1 upregulated interleukin-6 (IL6) and monocyte chemoattractant protein-1 (MCP1) mRNA at all stages of differentiation, decreased tumor necrosis factor-α (TNFα) mRNA was observed early in adipocyte differentiation. This study highlights the complex role of TGFβ1 on extracellular matrix (ECM) remodeling and inflammatory markers in stimulating both synthetic and inhibitory markers of fibrosis at different stages of adipocyte differentiation.
Collapse
Affiliation(s)
- Babu Raja Maharjan
- Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
- School of Medicine, Department of Biochemistry, Patan Academy of Health Sciences, Lalitpur 44700, Nepal
- Correspondence: (B.R.M.); (P.F.W.); Tel.: +61-2-8627-1889 (B.R.M. & P.F.W.)
| | - Susan V. McLennan
- Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
- New South Wales Health Pathology, Sydney, NSW 2050, Australia
| | - Stephen M. Twigg
- Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
- Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, NSW 2006, Australia
| | - Paul F. Williams
- Greg Brown Diabetes & Endocrinology Laboratory, Sydney Medical School, University of Sydney, Sydney, NSW 2006, Australia
- Correspondence: (B.R.M.); (P.F.W.); Tel.: +61-2-8627-1889 (B.R.M. & P.F.W.)
| |
Collapse
|
|
3
|
Rodríguez‐Barrueco R, Latorre J, Devis‐Jáuregui L, Lluch A, Bonifaci N, Llobet FJ, Olivan M, Coll‐Iglesias L, Gassner K, Davis ML, Moreno‐Navarrete JM, Castells‐Nobau A, Plata‐Peña L, Dalmau‐Pastor M, Höring M, Liebisch G, Olkkonen VM, Arnoriaga‐Rodríguez M, Ricart W, Fernández‐Real JM, Silva JM, Ortega FJ, Llobet‐Navas D. A microRNA Cluster Controls Fat Cell Differentiation and Adipose Tissue Expansion By Regulating SNCG. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2022; 9:e2104759. [PMID: 34898027 PMCID: PMC8811811 DOI: 10.1002/advs.202104759] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/22/2021] [Indexed: 05/08/2023]
Abstract
The H19X-encoded miR-424(322)/503 cluster regulates multiple cellular functions. Here, it is reported for the first time that it is also a critical linchpin of fat mass expansion. Deletion of this miRNA cluster in mice results in obesity, while increasing the pool of early adipocyte progenitors and hypertrophied adipocytes. Complementary loss and gain of function experiments and RNA sequencing demonstrate that miR-424(322)/503 regulates a conserved genetic program involved in the differentiation and commitment of white adipocytes. Mechanistically, it is demonstrated that miR-424(322)/503 targets γ-Synuclein (SNCG), a factor that mediates this program rearrangement by controlling metabolic functions in fat cells, allowing adipocyte differentiation and adipose tissue enlargement. Accordingly, diminished miR-424(322) in mice and obese humans co-segregate with increased SNCG in fat and peripheral blood as mutually exclusive features of obesity, being normalized upon weight loss. The data unveil a previously unknown regulatory mechanism of fat mass expansion tightly controlled by the miR-424(322)/503 through SNCG.
Collapse
Affiliation(s)
- Ruth Rodríguez‐Barrueco
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Anatomy UnitDepartment of Pathology and Experimental TherapySchool of MedicineUniversity of Barcelona (UB)L'Hospitalet de Llobregat08907Spain
| | - Jessica Latorre
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - Laura Devis‐Jáuregui
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
| | - Aina Lluch
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
| | - Nuria Bonifaci
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC)Instituto de Salud Carlos III, (ISCIII)Madrid28029Spain
| | - Francisco J. Llobet
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
| | - Mireia Olivan
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Anatomy UnitDepartment of Pathology and Experimental TherapySchool of MedicineUniversity of Barcelona (UB)L'Hospitalet de Llobregat08907Spain
| | - Laura Coll‐Iglesias
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
| | - Katja Gassner
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC)Instituto de Salud Carlos III, (ISCIII)Madrid28029Spain
| | - Meredith L. Davis
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Department of PathologyDuke University School of MedicineDurhamNC27710USA
| | - José M. Moreno‐Navarrete
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - Anna Castells‐Nobau
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
| | - Laura Plata‐Peña
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
| | - Miki Dalmau‐Pastor
- Anatomy UnitDepartment of Pathology and Experimental TherapySchool of MedicineUniversity of Barcelona (UB)L'Hospitalet de Llobregat08907Spain
- MIFAS by GRECMIP (Minimally Invasive Foot and Ankle Society)Merignac33700France
| | - Marcus Höring
- Institute of Clinical Chemistry and Laboratory MedicineRegensburg University HospitalRegensburg93053Germany
| | - Gerhard Liebisch
- Institute of Clinical Chemistry and Laboratory MedicineRegensburg University HospitalRegensburg93053Germany
| | - Vesa M. Olkkonen
- Minerva Foundation Institute for Medical Research (Biomedicum 2U)and Department of AnatomyFaculty of MedicineUniversity of HelsinkiHelsinki00290Finland
| | - Maria Arnoriaga‐Rodríguez
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - Wifredo Ricart
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - José M. Fernández‐Real
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - José M. Silva
- Department of PathologyIcahn School of Medicine at Mount SinaiNew YorkNY10029USA
| | - Francisco J. Ortega
- Department of DiabetesEndocrinology, and Nutrition (UDEN)Institut d'Investigació Biomèdica de Girona (IDIBGI)Salt17190Spain
- Centro de Investigación Biomédica en Red de la Fisiopatología de la Obesidad y la Nutrición (CIBEROBN)Instituto de Salud Carlos III (ISCIII)Madrid28029Spain
| | - David Llobet‐Navas
- Molecular Mechanisms and Experimental Therapy in Oncology‐Oncobell ProgramBellvitge Biomedical Research Institute (IDIBELL)L'Hospitalet de Llobregat08908Spain
- Centro de Investigación Biomédica en Red de Cáncer (CIBERONC)Instituto de Salud Carlos III, (ISCIII)Madrid28029Spain
| |
Collapse
|
|
4
|
Smad2/3 Activation Regulates Smad1/5/8 Signaling via a Negative Feedback Loop to Inhibit 3T3-L1 Adipogenesis. Int J Mol Sci 2021; 22:ijms22168472. [PMID: 34445177 PMCID: PMC8395197 DOI: 10.3390/ijms22168472] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 07/30/2021] [Accepted: 08/01/2021] [Indexed: 12/22/2022] Open
Abstract
Adipose tissues (AT) expand in response to energy surplus through adipocyte hypertrophy and hyperplasia. The latter, also known as adipogenesis, is a process by which multipotent precursors differentiate to form mature adipocytes. This process is directed by developmental cues that include members of the TGF-β family. Our goal here was to elucidate, using the 3T3-L1 adipogenesis model, how TGF-β family growth factors and inhibitors regulate adipocyte development. We show that ligands of the Activin and TGF-β families, several ligand traps, and the SMAD1/5/8 signaling inhibitor LDN-193189 profoundly suppressed 3T3-L1 adipogenesis. Strikingly, anti-adipogenic traps and ligands engaged the same mechanism of action involving the simultaneous activation of SMAD2/3 and inhibition of SMAD1/5/8 signaling. This effect was rescued by the SMAD2/3 signaling inhibitor SB-431542. By contrast, although LDN-193189 also suppressed SMAD1/5/8 signaling and adipogenesis, its effect could not be rescued by SB-431542. Collectively, these findings reveal the fundamental role of SMAD1/5/8 for 3T3-L1 adipogenesis, and potentially identify a negative feedback loop that links SMAD2/3 activation with SMAD1/5/8 inhibition in adipogenic precursors.
Collapse
|
|
5
|
Krambs JR, Abou Ezzi G, Yao JC, Link DC. Canonical signaling by TGF family members in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions. PLoS One 2020; 15:e0233751. [PMID: 32470079 PMCID: PMC7259882 DOI: 10.1371/journal.pone.0233751] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Accepted: 05/12/2020] [Indexed: 02/07/2023] Open
Abstract
Mesenchymal stromal cells are an important component of the bone marrow hematopoietic niche. Prior studies showed that signaling from members of the transforming growth factor (TGF) superfamily in mesenchymal stromal cells is required for normal niche development. Here, we assessed the impact of TGF family signaling on niche maintenance and stress responses by deleting Smad4 in mesenchymal stromal cells at birth, thereby abrogating canonical TGF signaling. No alteration in the number or spatial organization of CXCL12-abundant reticular (CAR) cells, osteoblasts, or adipocytes was observed in Osx-Cre, Smad4fl/fl mice, and expression of key niche factors was normal. Basal hematopoiesis and stress erythropoiesis responses to acute hemolytic anemia were normal. TGF-β potently inhibits stromal CXCL12 expression in vitro; however, G-CSF induced decreases in bone marrow CXCL12 expression and subsequent hematopoietic stem/progenitor cell mobilization were normal in Osx-Cre, Tgfbr2fl/fl mice, in which all TGF-β signaling in mesenchymal stromal is lost. Finally, although a prior study showed that TGF-β enhances recovery from myeloablative therapy, hematopoietic recovery following single or multiple doses of 5-flurauracil were normal in Osx-Cre, Tgfbr2fl/fl mice. Collectively, these data suggest that TGF family member signaling in mesenchymal stromal cells is dispensable for hematopoietic niche maintenance under basal and stress conditions.
Collapse
Affiliation(s)
- Joseph Ryan Krambs
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, United States of America
| | - Grazia Abou Ezzi
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, United States of America
| | - Juo-Chin Yao
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, United States of America
| | - Daniel C. Link
- Division of Oncology, Department of Medicine, Washington University School of Medicine, Saint Louis, MO, United States of America
- * E-mail:
| |
Collapse
|
|
6
|
Abou-Ezzi G, Supakorndej T, Zhang J, Anthony B, Krambs J, Celik H, Karpova D, Craft CS, Link DC. TGF-β Signaling Plays an Essential Role in the Lineage Specification of Mesenchymal Stem/Progenitor Cells in Fetal Bone Marrow. Stem Cell Reports 2019; 13:48-60. [PMID: 31204302 PMCID: PMC6626889 DOI: 10.1016/j.stemcr.2019.05.017] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2018] [Revised: 05/15/2019] [Accepted: 05/16/2019] [Indexed: 01/17/2023] Open
Abstract
Mesenchymal stromal cells are key components of hematopoietic niches in the bone marrow. Here we abrogated transforming growth factor β (TGF-β) signaling in mesenchymal stem/progenitor cells (MSPCs) by deleting Tgfbr2 in mesenchymal cells using a doxycycline-repressible Sp7 (osterix)-Cre transgene. We show that loss of TGF-β signaling during fetal development results in a marked expansion of CXCL12-abundant reticular (CAR) cells and adipocytes in the bone marrow, while osteoblasts are significantly reduced. These stromal alterations are associated with significant defects in hematopoiesis, including a shift from lymphopoiesis to myelopoiesis. However, hematopoietic stem cell function is preserved. Interestingly, TGF-β signaling is dispensable for the maintenance of mesenchymal cells in the bone marrow after birth under steady-state conditions. Collectively, these data show that TGF-β plays an essential role in the lineage specification of fetal but not definitive MSPCs and is required for the establishment of normal hematopoietic niches in fetal and perinatal bone marrow.
Collapse
Affiliation(s)
- Grazia Abou-Ezzi
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Teerawit Supakorndej
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Jingzhu Zhang
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Bryan Anthony
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Joseph Krambs
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Hamza Celik
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Darja Karpova
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA
| | - Clarissa S Craft
- Division of Bone and Mineral Diseases, Washington University, St. Louis, MO, USA
| | - Daniel C Link
- Division of Oncology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8007, St. Louis, MO 63110, USA.
| |
Collapse
|
|
7
|
Wang MK, Sun HQ, Xiang YC, Jiang F, Su YP, Zou ZM. Different roles of TGF-β in the multi-lineage differentiation of stem cells. World J Stem Cells 2012; 4:28-34. [PMID: 22993659 PMCID: PMC3443709 DOI: 10.4252/wjsc.v4.i5.28] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2011] [Revised: 03/10/2012] [Accepted: 03/25/2012] [Indexed: 02/06/2023] Open
Abstract
Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.
Collapse
Affiliation(s)
- Ming-Ke Wang
- Ming-Ke Wang, Fan Jiang, Zhong-Min Zou, Department of Chemical Defense and Toxicology, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China
| | | | | | | | | | | |
Collapse
|
|
8
|
Zhu J, Pang D, Zhou Y, Tang X, Huang Y, Xie W, Gao F, Lai L, Zhang M, Ouyang H. Direct conversion of porcine embryonic fibroblasts into adipocytes by chemical molecules. Cell Reprogram 2012; 14:99-105. [PMID: 22372576 DOI: 10.1089/cell.2011.0074] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
Direct reprogramming of terminally differentiated cells to specify different cell types may allow somatic cells to be reprogrammed to an alternative, differentiated fate without intervening stem or progenitor cells. Recent studies have shown that the conversion of fibroblasts to other cell lines can be accomplished by the introduction of master regulator transcription factors. These findings have raised the question as to whether chemical molecules could replace transcription factor cocktails to directly alter defined somatic cell fate. Here, we demonstrate the generation of adipocytes directly from porcine embryonic fibroblasts (PEFs) using defined chemical molecules. Treatment with SB431542 and Thiazovivin, which are transforming growth factor-beta (TGF-β) and ROCK signaling pathway inhibitors, respectively, allowed PEFs to directly convert to fat-laden adipocytes. These induced adipocytes expressed multiple fat marker genes. We believe that these findings demonstrate that committed adipocytes can be directly reprogrammed from differentiated somatic cells using defined chemical molecules. The generation of adipocytes from nonadipogenic lineages has important implications for studies of adipogenesis, obesity modeling, and regenerative medicine. Additionally, these findings may enlighten a new method that direct reprogramming committed cell lines to other somatic cells using defined chemical molecules.
Collapse
Affiliation(s)
- Jianguo Zhu
- Jilin Provincial Key Laboratory of Animal Embryo Engineering, College of Animal Science and Veterinary Medicine, Jilin University, Changchun, Jilin 130062, People's Republic of China
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
9
|
Hausman GJ, Dodson MV, Ajuwon K, Azain M, Barnes KM, Guan LL, Jiang Z, Poulos SP, Sainz RD, Smith S, Spurlock M, Novakofski J, Fernyhough ME, Bergen WG. Board-invited review: the biology and regulation of preadipocytes and adipocytes in meat animals. J Anim Sci 2008; 87:1218-46. [PMID: 18849378 DOI: 10.2527/jas.2008-1427] [Citation(s) in RCA: 242] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
The quality and value of the carcass in domestic meat animals are reflected in its protein and fat content. Preadipocytes and adipocytes are important in establishing the overall fatness of a carcass, as well as being the main contributors to the marbling component needed for consumer preference of meat products. Although some fat accumulation is essential, any excess fat that is deposited into adipose depots other than the marbling fraction is energetically unfavorable and reduces efficiency of production. Hence, this review is focused on current knowledge about the biology and regulation of the important cells of adipose tissue: preadipocytes and adipocytes.
Collapse
Affiliation(s)
- G J Hausman
- USDA-ARS, Richard B. Russell Agricultural Research Station, Athens, GA 30604, USA
| | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Role of gender and anatomical region on induction of osteogenic differentiation of human adipose-derived stem cells. Ann Plast Surg 2008; 60:306-22. [PMID: 18443514 DOI: 10.1097/sap.0b013e3180621ff0] [Citation(s) in RCA: 139] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Adipose-derived stem cells (ASCs) display multilineage plasticity and, under appropriate conditions, can mineralize their extracellular matrix and undergo osteogenesis. The aims of this study are to examine in vitro osteogenic differentiation properties of ASCs to assess the role of gender, fat depot, and optimal duration as variables for differentiation. Human ASCs were isolated from superficial and deep adipose layers of the abdominoplasty specimens obtained from patients undergoing elective surgeries. ASCs were cultured in osteogenic media (OM). After 1, 2, and 4 weeks of differentiation, cultures were assessed for markers of osteogenesis. Alkaline phosphatase (AP), alizarin red (AR) and Masson trichrome (MT) stainings for osteoblastic transformation, matrix mineralization, and collagen production; enzyme-linked immunosorbent assay (ELISA) for Gla-osteocalcin; and Western blot analysis for osteonectin protein expression were performed. Osteogenic differentiation began as early as 1 week. Cells exhibited a vertical growth pattern, lacunae formed in the cultures, matrix volume increased, and mineralization was observed. Differences in AP staining were most evident during the first week. AR activity progressively increased over 4 weeks, and collagen was secreted only by differentiated ASCs. There was no significant difference in the degree of osteogenic differentiation between the ASCs from both depots in the female. In the male, the superficial depot ASCs differentiated faster and more efficiently than those of the deep depot. Male ASCs from both depots differentiated more effectively than female ASCs from both depots. We describe a hierarchy of osteogenic differentiation potential based on gender and anatomic harvest site by layering adipose tissues of the abdominal wall. ASCs derived from male superficial layer were most efficient in achieving osteogenesis. In future clinical applications using stem cells for osseous healing, these gender and depot differences will guide our clinical methods.
Collapse
|
|
11
|
Hemmrich K, Thomas GPL, Abberton KM, Thompson EW, Rophael JA, Penington AJ, Morrison WA. Monocyte chemoattractant protein-1 and nitric oxide promote adipogenesis in a model that mimics obesity. Obesity (Silver Spring) 2007; 15:2951-7. [PMID: 18198303 DOI: 10.1038/oby.2007.352] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
OBJECTIVE An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low-level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute-phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)-1 and zymosan-A (Zy) in a murine tissue engineering model. RESEARCH METHODS AND PROCEDURES MCP-1 and Zy were added to chambers filled with Matrigel and fibroblast growth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. RESULTS Our results show that MCP-1 generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. DISCUSSION Our findings demonstrate that low-grade inflammation and iNOS expression are important factors in adipogenesis. Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage-derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.
Collapse
Affiliation(s)
- Karsten Hemmrich
- Bernard O'Brien Institute of Microsurgery, Fitzroy, Victoria, Australia.
| | | | | | | | | | | | | |
Collapse
|
|
12
|
Taha MF, Valojerdi MR, Mowla SJ. Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells. Anat Histol Embryol 2006; 35:271-8. [PMID: 16836593 DOI: 10.1111/j.1439-0264.2006.00680.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.
Collapse
Affiliation(s)
- M F Taha
- Department of Anatomy, School of Medical Sciences, Tarbiat Modarres University, Tehran, Iran
| | | | | |
Collapse
|
|
13
|
Shin SM, Kim KY, Kim JK, Yoon SR, Choi I, Yang Y. Dexamethasone reverses TGF-beta-mediated inhibition of primary rat preadipocyte differentiation. FEBS Lett 2003; 543:25-30. [PMID: 12753899 DOI: 10.1016/s0014-5793(03)00371-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Dexamethasone and transforming growth factor-beta (TGF-beta) show contrary effects on differentiation of adipocytes. Dexamethasone stimulates adipocyte differentiation whereas TGF-beta inhibits it. In the present study, we investigated whether dexamethasone could reverse the TGF-beta-mediated inhibition of preadipocyte differentiation. Primary rat preadipocytes, obtained from Sprague-Dawley rats, were pretreated with dexamethasone in the presence or absence of TGF-beta, prior to the induction of differentiation. Co-treatment of dexamethasone and TGF-beta before inducing differentiation reversed the TGF-beta-mediated inhibition of preadipocyte differentiation. In order to elucidate the mechanism by which dexamethasone reversed the effect of TGF-beta on the inhibition of preadipocyte differentiation, the expression of CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) was examined. Dexamethasone increased C/EBPalpha and PPARgamma expression in the absence of TGF-beta and also recovered the TGF-beta-mediated suppression of C/EBPalpha expression in preadipocytes. Its effect was sustained in differentiated adipocytes as well. However, those effects were not observed in 3T3-L1 preadipocytes or differentiated adipocytes. These results indicate that dexamethasone reverses the TGF-beta-mediated suppression of adipocyte differentiation by regulating the expression of C/EBPalpha and PPARgamma, which is dependent on the cellular context.
Collapse
Affiliation(s)
- Sun Mi Shin
- Laboratory of Immunology, Korea Research Institute of Bioscience and Biotechnology, 305-333, Daejon, South Korea
| | | | | | | | | | | |
Collapse
|
|
14
|
Choy L, Derynck R. Transforming growth factor-beta inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function. J Biol Chem 2003; 278:9609-19. [PMID: 12524424 DOI: 10.1074/jbc.m212259200] [Citation(s) in RCA: 274] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Transforming growth factor (TGF)-beta is a potent inhibitor of adipocyte differentiation. To identify which adipocyte transcription factors might be targeted by TGF-beta, we overexpressed key adipogenic transcription factors, C/EBPbeta, C/EBPdelta, or peroxisome proliferator-activated receptor (PPAR) gamma in NIH3T3 cells and tested the ability of TGF-beta to block adipogenesis. We show that TGF-beta inhibits adipocyte differentiation driven by either C/EBPbeta or C/EBPdelta without affecting C/EBP protein expression levels, suggesting that these C/EBPs are a direct target of TGF-beta action. Because TGF-beta inhibits adipogenesis by signaling through Smad3, we examined physical and functional interactions of Smad3 and Smad4 with C/EBPbeta, C/EBPdelta, and PPARgamma2. C/EBPbeta and C/EBPdelta were found to physically interact with Smad3 and Smad4, and Smad3 cooperated with Smad4 and TGF-beta signaling to repress the transcriptional activity of C/EBPs. Thus, repression of the activity of C/EBPs by Smad3/4 at C/EBP binding sites inhibited transcription from the PPARgamma2 and leptin promoters. In contrast, PPARgamma interacted only very weakly with Smad3 and its transcriptional activity was not repressed by Smad3/4 or in response to TGF-beta. Smad3/4 did not reduce the ability of C/EBP to bind to its cognate DNA sequence, but repressed transcription by inhibiting the transactivation function of C/EBP.
Collapse
Affiliation(s)
- Lisa Choy
- Department of Growth and Development, Programs in Cell Biology and Developmental Biology, University of California, San Francisco, California 94143-0640, USA
| | | |
Collapse
|
|
15
|
Albrektsen T, Frederiksen KS, Holmes WE, Boel E, Taylor K, Fleckner J. Novel genes regulated by the insulin sensitizer rosiglitazone during adipocyte differentiation. Diabetes 2002; 51:1042-51. [PMID: 11916924 DOI: 10.2337/diabetes.51.4.1042] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Thiazolidinediones (TZDs) are a new class of compounds that improve insulin sensitivity in type 2 diabetic patients as well as in rodent models of this disease. These compounds act as ligands for a member of the nuclear hormone receptor superfamily, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), which is highly expressed in adipose tissue and, moreover, has been shown to play an important role in adipocyte differentiation. The strong correlation between the antidiabetic activity of TZDs and their ability to activate PPAR-gamma suggests that PPAR-gamma, through downstream-regulated genes, mediates the effects of TZDs. In this report, we present the isolation and characterization of 81 genes, encoding proteins of known function, differentially expressed during TZD-stimulated differentiation of 3T3-L1 cells. By the use of different reverse- Northern blot techniques, the differential expression of 50 of these genes could be verified, and 21 genes were specifically regulated by a potent TZD during the course of adipocyte differentiation, whereas no effect of a PPAR-gamma antagonist could be observed in mature adipocytes. The differential expression of a large fraction of the isolated genes was also shown to occur in white adipose tissue of ob/ob mice treated with rosiglitazone; combined, our results suggest that an important effect of rosiglitazone in adipose tissue is based on activation of PPAR-gamma in preexisting preadipocytes found among the mature adipocytes, resulting in subsequent adipocyte differentiation.
Collapse
|
|
16
|
Ahdjoudj S, Lasmoles F, Holy X, Zerath E, Marie PJ. Transforming growth factor beta2 inhibits adipocyte differentiation induced by skeletal unloading in rat bone marrow stroma. J Bone Miner Res 2002; 17:668-77. [PMID: 11918224 DOI: 10.1359/jbmr.2002.17.4.668] [Citation(s) in RCA: 103] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
Skeletal unloading induced by hindlimb suspension in rats reduces bone formation and induces osteopenia, but its effect on adipogenesis is unknown. We assessed the effects of unloading and transforming growth factor (TGF) beta2 on bone marrow stromal cell adipocyte differentiation in relation with osteoblast differentiation. Skeletal unloading rapidly (4-7 days) decreased osteoblast transcription factor Runx2, osteocalcin (OC), and type I collagen messenger RNA (mRNA) levels and reduced bone formation in the long bone metaphysis. Conversely, unloading increased expression of the adipocyte transcription factor peroxisome proliferator-activated receptor gamma2 (PPARgamma2) at 4 days and increased expression of the adipocyte differentiation genes lipoprotein lipase (LPL) and aP2 in the bone marrow stroma at 7 days. Consistently, unloading increased the number and volume of adipocytes in the bone marrow stroma. Continuous (0-7 days) and late (4-7 days) treatments with TGF-beta2 corrected the abnormal expression of Cbfa1/Runx2, OC, and type I collagen mRNAs and normalized bone formation in unloaded metaphyseal bone. Moreover, both TGF-beta2 treatments decreased PPARy2 and C/EBPalpha mRNA levels at 4 days and normalized aP2 and LPL expression and adipocyte number and volume at 7 days. These results show that skeletal unloading increases adipocyte differentiation concomitantly with inhibition of osteoblast differentiation. These abnormalities are prevented and reversed by TGF-beta2, suggesting a role for TGF-beta in the control of adipogenic differentiation in the bone marrow stroma.
Collapse
Affiliation(s)
- Souhila Ahdjoudj
- Unit 349 INSERM, CNRS, Laboratory of Osteoblast Biology and Pathology, Lariboisière Hospital, Paris, France
| | | | | | | | | |
Collapse
|
|
17
|
Abstract
The recently discovered protein, leptin, which is secreted by fat cells in response to changes in body weight or energy, has been implicated in regulation of feed intake, energy expenditure and the neuroendocrine axis in rodents and humans. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. Administration of leptin to ob/ob mice led to improved reproduction as well as reduced feed intake and weight loss. The porcine leptin receptor has been cloned and is a member of the class 1 cytokine family of receptors. Leptin has been implicated in the regulation of immune function and the anorexia associated with disease. The leptin receptor is localized in the brain and pituitary of the pig. The leptin response to acute inflammation is uncoupled from anorexia and is differentially regulated among swine genotypes. In vitro studies demonstrated that the leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig. Expression of CCAAT-enhancer binding proteinalpha (C/EBPalpha) mediates insulin dependent preadipocyte leptin gene expression during lipid accretion. In contrast, insulin independent leptin gene expression may be maintained by C/EBPalpha auto-activation and phosphorylation/dephosphorylation. Adipogenic hormones may increase adipose tissue leptin gene expression in the fetus indirectly by inducing preadipocyte recruitment and subsequent differentiation. Central administration of leptin to pigs suppressed feed intake and stimulated growth hormone (GH) secretion. Serum leptin concentrations increased with age and estradiol-induced leptin mRNA expression in fat was age and weight dependent in prepuberal gilts. This occurred at the time of expected puberty in intact contemporaries and was associated with greater LH secretion. Further work demonstrated that leptin acts directly on pituitary cells to enhance LH and GH secretion, and brain tissue to stimulate gonadotropin releasing hormone secretion. Thus, development of nutritional schemes and (or) gene therapy to manipulate leptin secretion will lead to practical methods of controlling appetite, growth and reproduction in farm animals, thereby increasing efficiency of lean meat production.
Collapse
Affiliation(s)
- C R Barb
- USDA-ARS, Animal Physiology Unit, Russell Research Center, P. O. Box 5677, Athens, GA 30604-5677, USA.
| | | | | |
Collapse
|
|
18
|
Abstract
Adipogenesis, or the development of fat cells from preadipocytes, has been one of the most intensely studied models of cellular differentiation. In part this has been because of the availability of in vitro models that faithfully recapitulate most of the critical aspects of fat cell formation in vivo. More recently, studies of adipogenesis have proceeded with the hope that manipulation of this process in humans might one day lead to a reduction in the burden of obesity and diabetes. This review explores some of the highlights of a large and burgeoning literature devoted to understanding adipogenesis at the molecular level. The hormonal and transcriptional control of adipogenesis is reviewed, as well as studies on a less well known type of fat cell, the brown adipocyte. Emphasis is placed, where possible, on in vivo studies with the hope that the results discussed may one day shed light on basic questions of cellular growth and differentiation in addition to possible benefits in human health.
Collapse
Affiliation(s)
- E D Rosen
- Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA.
| | | |
Collapse
|
|
19
|
Kim HS, Liang L, Dean RG, Hausman DB, Hartzell DL, Baile CA. Inhibition of preadipocyte differentiation by myostatin treatment in 3T3-L1 cultures. Biochem Biophys Res Commun 2001; 281:902-6. [PMID: 11237745 DOI: 10.1006/bbrc.2001.4435] [Citation(s) in RCA: 92] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Myostatin, a new TGF-beta family member, is known as a muscle growth inhibitor, but its role in adipocyte development has not been studied. To test the role of Myostatin in 3T3-L1 preadipocyte differentiation, we treated cultured 3T3-L1 preadipocytes with Myostatin dissolved in 0.1% trifluoroacetic acid (TFA) during differentiation after they had become confluent. Myostatin treatment significantly decreased glycerol-3-phosphate dehydrogenase (GPDH) activity and oil Red-O staining compared to controls that did not receive Myostatin. Western blot analysis showed that the expression levels of CCAAT/enhancer binding protein alpha (C/EBP alpha) and peroxisome proliferator-activated receptor gamma (PPAR gamma) were significantly decreased by Myostatin treatment (P < 0.05). However, the expression of C/EBP beta was not significantly changed by the treatment (P > 0.05). From RT-PCR result, the relative level of leptin mRNA in Myostatin-treated cells was not significantly different (P > 0.1) from the level in cells without Myostatin treatment. Our data show that Myostatin, a secreted protein from muscle, inhibits preadipocyte differentiation in 3T3-L1 cells, which is mediated, in part, by altered regulation of C/EBP alpha and PPAR gamma.
Collapse
Affiliation(s)
- H S Kim
- Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602, USA
| | | | | | | | | | | |
Collapse
|
|
20
|
Choy L, Skillington J, Derynck R. Roles of autocrine TGF-beta receptor and Smad signaling in adipocyte differentiation. J Cell Biol 2000; 149:667-82. [PMID: 10791980 PMCID: PMC2174852 DOI: 10.1083/jcb.149.3.667] [Citation(s) in RCA: 253] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
TGF-beta inhibits adipocyte differentiation, yet is expressed by adipocytes. The function of TGF-beta in adipogenesis, and its mechanism of action, is unknown. To address the role of TGF-beta signaling in adipocyte differentiation, we characterized the expression of the TGF-beta receptors, and the Smads which transmit or inhibit TGF-beta signals, during adipogenesis in 3T3-F442A cells. We found that the cell-surface availability of TGF-beta receptors strongly decreased as adipogenesis proceeds. Whereas mRNA levels for Smads 2, 3, and 4 were unchanged during differentiation, mRNA levels for Smads 6 and 7, which are known to inhibit TGF-beta responses, decreased severely. Dominant negative interference with TGF-beta receptor signaling, by stably expressing a truncated type II TGF-beta receptor, enhanced differentiation and decreased growth. Stable overexpression of Smad2 or Smad3 inhibited differentiation and dominant negative inhibition of Smad3 function, but not Smad2 function, enhanced adipogenesis. Increased Smad6 and Smad7 levels blocked differentiation and enhanced TGF-beta-induced responses. The inhibitory effect of Smad7 on adipocyte differentiation and its cooperation with TGF-beta was associated with the C-domain of Smad7. Our results indicate that endogenous TGF-beta signaling regulates the rate of adipogenesis, and that Smad2 and Smad3 have distinct functions in this endogenous control of differentiation. Smad6 and Smad7 act as negative regulators of adipogenesis and, even though known to inhibit TGF-beta responses, enhance the effects of TGF-beta on these cells.
Collapse
Affiliation(s)
- Lisa Choy
- Department of Growth and Development, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
- Department of Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
| | - Jeremy Skillington
- Department of Growth and Development, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
- Department of Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
| | - Rik Derynck
- Department of Growth and Development, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
- Department of Anatomy, Programs in Cell Biology and Developmental Biology, University of California at San Francisco, San Francisco, California 94143-0640
| |
Collapse
|
|
21
|
Abstract
The adipocyte plays a critical role in energy balance. Adipose tissue growth involves an increase in adipocyte size and the formation of new adipocytes from precursor cells. For the last 20 years, the cellular and molecular mechanisms of adipocyte differentiation have been extensively studied using preadipocyte culture systems. Committed preadipocytes undergo growth arrest and subsequent terminal differentiation into adipocytes. This is accompanied by a dramatic increase in expression of adipocyte genes including adipocyte fatty acid binding protein and lipid-metabolizing enzymes. Characterization of regulatory regions of adipose-specific genes has led to the identification of the transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein (C/EBP), which play a key role in the complex transcriptional cascade during adipocyte differentiation. Growth and differentiation of preadipocytes is controlled by communication between individual cells or between cells and the extracellular environment. Various hormones and growth factors that affect adipocyte differentiation in a positive or negative manner have been identified. In addition, components involved in cell-cell or cell-matrix interactions such as preadipocyte factor-1 and extracellular matrix proteins are also pivotal in regulating the differentiation process. Identification of these molecules has yielded clues to the biochemical pathways that ultimately result in transcriptional activation via PPAR-gamma and C/EBP. Studies on the regulation of the these transcription factors and the mode of action of various agents that influence adipocyte differentiation will reveal the physiological and pathophysiological mechanisms underlying adipose tissue development.
Collapse
Affiliation(s)
- F M Gregoire
- Department of Nutritional Sciences, University of California, Berkeley, USA
| | | | | |
Collapse
|
|
22
|
Chen XL, Hausman DB, Dean RG, Hausman GJ. Hormonal regulation of leptin mRNA expression and preadipocyte recruitment and differentiation in porcine primary cultures of S-V cells. OBESITY RESEARCH 1998; 6:164-72. [PMID: 9545024 DOI: 10.1002/j.1550-8528.1998.tb00331.x] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The hormonal regulation of leptin mRNA expression and the association between leptin expression and adipocyte differentiation were examined in primary cultures of porcine S-V cells with Northern blot and immunocytochemical analysis. Seeding for 3 days with fetal bovine serum (FBS) with varying levels of dexamethasone (Dex) increased levels of leptin mRNA in a dose-dependent manner in parallel with increases in the proportion of preadipocytes (AD-3 positive cells; AD-3, a preadipocyte marker). Six-day treatment with 10 or 850 nM insulin after FBS+Dex treatment resulted in a similar increase in leptin mRNA expression and morphological differentiation. However, significantly lower levels of leptin mRNA and smaller fat cells were observed in cultures treated with 1 nM insulin or 10 nM insulin-like growth factor-I (IGF-I). Dex-induced increases in leptin mRNA levels and AD-3 cell numbers were blocked completely by the addition of transforming growth factor-beta (TGF-beta) to FBS+Dex-treated cultures. However TGF-beta significantly increased fat cell size and leptin mRNA expression when added to ITS (insulin, 850 nM; transferrin, 5 microg/ml; and selenium, 5 microg/mL) treated cultures during the lipid-filling stage. When added with FBS+DEX for the first 3 days, growth hormone (GH) did not influence the Dex-induced increase in AD-3 cells and leptin mRNA expression, but GH reduced leptin mRNA levels when added with insulin for 6 days after FBS+Dex. These results demonstrated that regulation of leptin mRNA expression by Dex, insulin, IGF-I, TGF-beta, and GH may be associated with changes in preadipocyte number and fat cell size.
Collapse
Affiliation(s)
- X L Chen
- Department of Foods and Nutrition, University of Georgia, Athens
| | | | | | | |
Collapse
|
|
23
|
Nishimura N, Harada-Shiba M, Tajima S, Sugano R, Yamamura T, Qiang QZ, Yamamoto A. Acquisition of secretion of transforming growth factor-beta 1 leads to autonomous suppression of scavenger receptor activity in a monocyte-macrophage cell line, THP-1. J Biol Chem 1998; 273:1562-7. [PMID: 9430696 DOI: 10.1074/jbc.273.3.1562] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Macrophage cells derived from the human monocytic leukemia cell line, THP-1, accumulate esterified cholesterol when cultivated in the presence of acetylated low density lipoprotein (Ac-LDL) through scavenger receptors (ScR). In the present study, we isolated a subtype of THP-1 cells that failed to accumulate esterified cholesterol when cultivated in the presence of Ac-LDL. The cells had negligible amounts of cell association and degradation of Ac-LDL compared with the parent THP-1 cells. The subtype THP-1 cells did not express ScR mRNA as well as that of lipoprotein lipase. In contrast, the expression of apolipoprotein E mRNA was greater than that found in parent THP-1 cells. The culture medium of subtype THP-1 cells treated with 12-O-tetradecanoylphorbol-13-acetate inhibited the uptake of Ac-LDL and the expression of ScR in parent THP-1 cells. After a 48-h incubation in the culture medium containing 12-O-tetradecanoylphorbol-13-acetate, the culture medium of differentiated subtype THP-1 cells contained 6.9 ng/ml transforming growth factor (TGF)-beta 1, while that of parent THP-1 cells secreted below detection level, which was less than 3 ng/ml. This inhibitory effect of the conditioned medium on the expression of ScR in parent THP-1 cells was abolished by pretreatment of the culture medium with anti-TGF-beta 1 antibodies. Parent THP-1 cells expressed as much TGF-beta 1 mRNA as sTHP-1 cells after stimulation of differentiation. Although the precursor forms of TGF-beta 1 that were synthesized in both parent and subtype THP-1 cells were of similar size and were expressed at similar levels, latent TGF-beta 1-binding protein, which is necessary for the secretion of TGF-beta 1, could only be co-immunoprecipitated with anti-TGF-beta 1 antibody from subtype THP-1 cells. This suggests that subtype THP-1 cells secrete TGF-beta 1 into the medium by forming a functional complex with the latent TGF-beta 1-binding protein. We conclude that subtype THP-1 cells could not take up Ac-LDL because ScR was inhibited (leading to a loss of function) caused by the secreted TGF-beta 1.
Collapse
Affiliation(s)
- N Nishimura
- Department of Etiology and Pathophysiology, National Cardiovascular Center Research Institute, Osaka, Japan
| | | | | | | | | | | | | |
Collapse
|
|
24
|
Clouthier DE, Comerford SA, Hammer RE. Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-beta1 transgenic mice. J Clin Invest 1997; 100:2697-713. [PMID: 9389733 PMCID: PMC508473 DOI: 10.1172/jci119815] [Citation(s) in RCA: 226] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
Transgenic mice overexpressing a constitutively active human TGF-beta1 under control of the rat phosphoenolpyruvate carboxykinase regulatory sequences developed fibrosis of the liver, kidney, and adipose tissue, and exhibited a severe reduction in body fat. Expression of the transgene in hepatocytes resulted in increased collagen deposition, altered lobular organization, increased hepatocyte turnover, and in extreme cases, hemorrhage and thrombosis. Renal expression of the transgene was localized to the proximal tubule epithelium, and was associated with tubulointerstitial fibrosis, characterized by excessive collagen deposition and increased fibronectin and plasminogen activator inhibitor-1 immunoreactivity. Pronounced glomerulosclerosis was evident, and hydronephrosis developed with low penetrance. Expression of TGF-beta1 in white and brown adipose tissue resulted in a lipodystrophy-like syndrome. All white fat depots and brown fat pads were severely reduced in size, and exhibited prominent fibroplasia. This reduction in WAT was due to impaired adipose accretion. Introduction of the transgene into the ob/ob background suppressed the obesity characteristic of this mutation; however, transgenic mutant mice developed severe hepato- and splenomegaly. These studies strengthen the link between TGF-beta1 expression and fibrotic disease, and demonstrate the potency of TGF-beta1 in modulating mesenchymal cell differentiation in vivo.
Collapse
Affiliation(s)
- D E Clouthier
- Department of Biochemistry and the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9050, USA
| | | | | |
Collapse
|
|
25
|
Wu X, Robinson CE, Fong HW, Gimble JM. Analysis of the native murine bone morphogenetic protein serine threonine kinase type I receptor (ALK-3). J Cell Physiol 1996; 168:453-61. [PMID: 8707881 DOI: 10.1002/(sici)1097-4652(199608)168:2<453::aid-jcp24>3.0.co;2-2] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
The bone morphogenetic proteins, members of the transforming growth factor-beta cytokine family, induce the osteoblast phenotype and promote osteogenesis in the bone marrow stroma. Simultaneously, these cytokines inhibit other mesodermal differentiation pathways, such as adipogenesis and myogenesis. The receptors for the bone morphogenetic proteins belong to a family of transmembrane serine/ threonine kinase TGF beta type I and type II receptor proteins. In man, these include the activin receptor like kinase-3 (ALK-3), a type I receptor protein. We have used a polyclonal antibody to examine the expression of the native murine ALK-3 protein in murine tissues and bone morphogenetic protein-responsive cell lines. On Western blot analyses, we found that the native 85 kDa native ALK-3 protein was expressed in a number of murine tissues; protein and mRNA levels did not necessarily correlate. Two bone morphogenetic protein-responsive cell lines, BMS2 bone marrow stromal cells and C2C12 myoblasts, expressed the ALK-3 protein constitutively. Cell differentiation was accompanied by modest changes in ALK-3 protein levels. Immunoprecipitation of the ALK-3 protein cross linked to [125I] BMP-4 revealed two major receptor complexes of approximately 90 kDa and 170 kDa in size. Biotin surface-labeling experiments revealed that the 85 kDa ALK-3 protein was constitutively associated with a novel 140 kDa surface glycoprotein. Deglycosylation reduced the protein's size to 116 kDa, comparable in size to that of the recently described BMP type II receptor. These findings support the current model that BMP interacts with a pre-existing complex consisting of a type I and type II receptor protein.
Collapse
Affiliation(s)
- X Wu
- Immunobiology and Cancer Program, Oklahoma Medical Research Foundation Oklahoma City 73104, USA
| | | | | | | |
Collapse
|
|
26
|
Fontaine RN, Gossett RE, Schroeder F, O'Toole BA, Doetschman T, Kier AB. Liver and intestinal fatty acid binding proteins in control and TGF beta 1 gene targeted deficient mice. Mol Cell Biochem 1996; 159:149-53. [PMID: 8858565 DOI: 10.1007/bf00420917] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023]
Abstract
The effect of transforming growth factor beta-1 (TGF beta 1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF beta 1-deficient mice. Homozygous TGF beta 1-deficient 129 x CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF beta 1-deficient/immunodeficient C3H mice on a SCID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (I-FABP) which decreased 3-fold in the TGF beta 1-deficient/immunodeficient C3H mice only. Thus, TGF beta 1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.
Collapse
Affiliation(s)
- R N Fontaine
- Department of Veterinary Pathobiology, Texas A & M University, College Station 77843-4467, USA
| | | | | | | | | | | |
Collapse
|
|
27
|
Gimble JM, Morgan C, Kelly K, Wu X, Dandapani V, Wang CS, Rosen V. Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells. J Cell Biochem 1995; 58:393-402. [PMID: 7593260 DOI: 10.1002/jcb.240580312] [Citation(s) in RCA: 142] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-beta gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.
Collapse
Affiliation(s)
- J M Gimble
- Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City 73104, USA
| | | | | | | | | | | | | |
Collapse
|
|
28
|
Teruel T, Valverde AM, Benito M, Lorenzo M. Transforming growth factor beta 1 induces differentiation-specific gene expression in fetal rat brown adipocytes. FEBS Lett 1995; 364:193-7. [PMID: 7750569 DOI: 10.1016/0014-5793(95)00385-m] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Fetal rat brown adipocytes show a low number of transforming growth factor beta 1 (TGF-beta 1) binding sites of high affinity, revealing the presence of type I, II and III TGF-beta 1 receptors and a minor-labeled species of approximately 140 kDa. The culture of cells in the presence of TGF-beta 1 induced the expression of the tissue-specific gene uncoupling protein in a dose- and time-dependent manner. In addition, TGF-beta 1 up-regulates the expression of genes involved in adipogenesis such as fatty acid synthase, glycerol 3-phosphate dehydrogenase, malic enzyme and glucose 6-phosphate dehydrogenase, as well as induces the expression of fibronectin (specific target gene for TGF-beta 1). Our results suggest that TGF-beta 1 is a major signal involved in initiating and/or maintaining the thermogenic and adipogenic differentiation of rat fetal brown adipocytes.
Collapse
Affiliation(s)
- T Teruel
- Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Madrid, Spain
| | | | | | | |
Collapse
|
|
29
|
Friedman G, Ben-Yehuda A, Ben-Naim M, Matsa D, Stein O, Stein Y. Effect of transforming growth factor-beta on lipoprotein lipase in rat mesenchymal heart cell cultures. BIOCHIMICA ET BIOPHYSICA ACTA 1995; 1254:140-6. [PMID: 7827118 DOI: 10.1016/0005-2760(94)00175-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/27/2023]
Abstract
The effect of recombinant transforming growth factor-beta 2 (rTGF-beta 2) on lipoprotein lipase (LPL) synthesis was studied in mesenchymal rat heart cell cultures. Addition of rTGF-beta 2 to culture medium containing 20% serum resulted in a time-dependent decrease in LPL activity. With 10 ng/ml a 30% fall occurred after 12 h and only 20% of enzyme activity remained after 24 h with 5 or 10 ng/ml. The minimal effective dose of rTGF-beta 2 was 0.1 ng/ml and a 20% decrease occurred after exposure for 24 h. Antibodies specific to TGF-beta 2 blocked this effect. The decrease in enzymic activity was accompanied by a decrease in enzyme mass and LPL mRNA. Addition of rTGF-beta 2 was effective only during the first week in culture, when enzyme activity was increasing but not after 12 days when the cultures were overconfluent, and the enzyme activity was high.
Collapse
Affiliation(s)
- G Friedman
- Lipid Research Laboratory, Hadassah University Hospital, Jerusalem, Israel
| | | | | | | | | | | |
Collapse
|
|
30
|
Sparks RL, Strauss EE, Manga AV. Regulation of differentiation and protein kinase C expression in 3T3 T proadipocytes: effects of TGF-beta and transformation. Cell Prolif 1994; 27:139-51. [PMID: 10465005 DOI: 10.1111/j.1365-2184.1994.tb01412.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/02/2023] Open
Abstract
We are studying the mechanisms that regulate proliferation and differentiation of normal 3T3 T proadipocytes and neoplastically transformed clones which have lost the ability to differentiate. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and transforming growth factor beta (TGF-beta) are known inhibitors of the same step of the differentiation process in 3T3 T cells. Here, we examined the expression of the phorbol ester receptor/protein kinase C (PKC) during adipocytic differentiation of 3T3 T cells and its modulation by the differentiation inhibitor TGF-beta. PKC receptor assays were performed using a tritiated analogue of TPA and it was found that PKC receptor levels decreased approximately threefold during differentiation. Northern blot analyses revealed an even greater decrease of PKC transcripts during differentiation. TGF-beta inhibited not only differentiation, but the differentiation-dependent decrease in PKC levels as well. Transformed 3T3 T cells which have lost the ability to differentiate were found to express aberrant levels of PKC. The data suggest that TGF-beta may inhibit differentiation via a PKC-dependent pathway and that disruption of normal PKC levels or its regulation may be involved in the loss of differentiation control in transformed 3T3 T cells.
Collapse
Affiliation(s)
- R L Sparks
- Department of Anatomy, Tulane Medical School, New Orleans, Louisiana 70112, USA
| | | | | |
Collapse
|
|
31
|
Gimble JM, Wanker F, Wang CS, Bass H, Wu X, Kelly K, Yancopoulos GD, Hill MR. Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 protein. J Cell Biochem 1994; 54:122-33. [PMID: 8126083 DOI: 10.1002/jcb.240540113] [Citation(s) in RCA: 84] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone-induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor alpha protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.
Collapse
Affiliation(s)
- J M Gimble
- Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, Oklahoma City 73104
| | | | | | | | | | | | | | | |
Collapse
|
|
32
|
Abstract
Adipocyte hyperplasia occurs by the proliferation and differentiation of adipocyte precursor cells or preadipocytes. Although the process of commitment to the adipocyte lineage is poorly understood, a great deal of information has accumulated about the processes and regulatory mechanisms involved in preadipocyte differentiation. The differentiation of preadipocytes is known to be characterized by increased transcription of a number of specific genes. AP-1 and C/EBP binding sites within these genes have been identified as important regulatory sequences. In addition, a specific enhancer sequence has been shown to confer adipose tissue specificity. This article will review the changes in gene transcription that occur during preadipocyte differentiation and how these are regulated. The potential role of autocrine/paracrine acting factors in the proliferation and differentiation of the preadipocyte is also discussed.
Collapse
Affiliation(s)
- S C Butterwith
- Agricultural and Food Research Council, Roslin Institute (Edinburgh), Department of Cellular and Molecular Biology, Midlothian, U.K
| |
Collapse
|
|
33
|
|
|
34
|
Smyth MJ, Sparks RL, Wharton W. Proadipocyte cell lines: models of cellular proliferation and differentiation. J Cell Sci 1993; 106 ( Pt 1):1-9. [PMID: 8270617 DOI: 10.1242/jcs.106.1.1] [Citation(s) in RCA: 27] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Affiliation(s)
- M J Smyth
- Cell Growth, Damage and Repair Group, Los Alamos National Laboratory, New Mexico 87545
| | | | | |
Collapse
|
|
35
|
Enerbäck S, Gimble JM. Lipoprotein lipase gene expression: physiological regulators at the transcriptional and post-transcriptional level. BIOCHIMICA ET BIOPHYSICA ACTA 1993; 1169:107-25. [PMID: 8343535 DOI: 10.1016/0005-2760(93)90196-g] [Citation(s) in RCA: 133] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Affiliation(s)
- S Enerbäck
- Department of Molecular Biology, University of Göteborg, Sweden
| | | |
Collapse
|
|
36
|
Smyth MJ, Wharton W. Multiparameter flow cytometric analysis of the effects of indomethacin on adipocyte differentiation in A31T6 cells. Cell Prolif 1993; 26:103-14. [PMID: 8471668 DOI: 10.1111/j.1365-2184.1993.tb00011.x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
A31T6 proadipocytes, derived from BALB/c-3T3 clone A31, develop responsiveness to differentiation-promoting agents at density-arrest and differentiate into adipocytes, as determined by the accumulation of cytoplasmic lipid droplets. A flow cytometric assay is being employed to monitor the acquisition of aspects of the differentiated phenotype. In this study, the assay is used to monitor both the rate of differentiation, as defined by the appearance of cells containing lipid droplets and the rate of adipocyte maturation, which involves measurement of increases in cytoplasmic lipid in cells already committed to the differentiation programme. Specifically, we show that: 1) treatment with a combination of indomethacin and dexamethasone causes the maximum percentage differentiation in the population, 2) addition of indomethacin in combination with either dexamethasone or insulin increases the rate of differentiation, and 3) indomethacin selectively increases the maturation of adipocytes, measured as an increase in the amount of lipid per cell. The cytometric assay used in these experiments has allowed determination of the effects of indomethacin on aspects of the adipocyte phenotype that cannot be measured by standard techniques.
Collapse
Affiliation(s)
- M J Smyth
- Life Sciences Division, Los Alamos National Laboratory, New Mexico 87545
| | | |
Collapse
|
|
37
|
Wang H, Scott RE. Inhibition of distinct steps in the adipocyte differentiation pathway in 3T3 T mesenchymal stem cells by dimethyl sulphoxide (DMSO). Cell Prolif 1993; 26:55-66. [PMID: 8439589 DOI: 10.1111/j.1365-2184.1993.tb00006.x] [Citation(s) in RCA: 26] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
The process of adipocyte differentiation in murine 3T3 T mesenchymal stem cells involves three well-defined steps: 1 predifferentiation growth arrest; 2 non-terminal (reversible) differentiation and 3 terminal differentiation associated with the irreversible loss of proliferative potential. To further investigate these processes, the effects of dimethyl sulphoxide (DMSO), an agent that affects differentiation in several other cell systems, was tested. The results show that DMSO modulates two distinct steps of adipocyte differentiation. The first effect is evident when growing 3T3 T cells are cultured in differentiation-inducing medium in the presence of DMSO. Therein the expression of adipocyte phenotype is inhibited because the cells fail to growth-arrest at the predifferentiation growth arrest state. Instead in the presence of DMSO, cells growth-arrest at a biological state that does not support differentiation. The second effect is evident if nonterminally differentiated adipocytes are cultured in terminal differentiation-inducing medium containing DMSO. Therein the terminal step in differentiation is inhibited. These inhibitory effects occur in a dosage-dependent manner; maximum inhibition of differentiation requires 2% DMSO. Therefore, whereas DMSO typically promotes differentiation in other cell systems, DMSO inhibits multiple steps in the process of adipocyte differentiation. These observations support the conclusion that a single pharmacological agent can have markedly different effects on specific cell types. Even more important, the data establish that DMSO can now be used as a tool to study the molecular mechanisms involved in the multistep process of adipocyte differentiation.
Collapse
Affiliation(s)
- H Wang
- Department of Pathology, University of Tennessee Medical Center, Memphis 38163
| | | |
Collapse
|