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Li H, Su M, Lin H, Li J, Wang S, Ye L, Li X, Ge R. Patulin Stimulates Progenitor Leydig Cell Proliferation but Delays Its Differentiation in Male Rats during Prepuberty. Toxins (Basel) 2023; 15:581. [PMID: 37756007 PMCID: PMC10538017 DOI: 10.3390/toxins15090581] [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: 08/12/2023] [Revised: 09/14/2023] [Accepted: 09/15/2023] [Indexed: 09/28/2023] Open
Abstract
Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.
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Affiliation(s)
- Huitao Li
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
- Key Laboratory of Pediatric Anesthesiology, Ministry of Education, Wenzhou Medical University, Wenzhou 325027, China
- Key Laboratory of Environment and Male Reproductive Medicine of Wenzhou, Key Laboratory of Structural Malformations in Children of Zhejiang Province, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China
| | - Ming Su
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Hang Lin
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Jingjing Li
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Shaowei Wang
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Lei Ye
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Xingwang Li
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
| | - Renshan Ge
- Department of Anesthesiology and Perioperative Medicine, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China; (H.L.); (M.S.); (H.L.); (J.L.); (S.W.); (L.Y.); (X.L.)
- Key Laboratory of Pediatric Anesthesiology, Ministry of Education, Wenzhou Medical University, Wenzhou 325027, China
- Key Laboratory of Environment and Male Reproductive Medicine of Wenzhou, Key Laboratory of Structural Malformations in Children of Zhejiang Province, The Second Affiliated Hospital and Yuying Children’s Hospital, Wenzhou Medical University, Wenzhou 325027, China
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Baek SK, Jeon SB, Seo BG, Hwangbo C, Shin KC, Choi JW, An CS, Jeong MA, Kim TS, Lee JH. The Presence or Absence of Alkaline Phosphatase Activity to Discriminate Pluripotency Characteristics in Porcine Epiblast Stem Cell-Like Cells. Cell Reprogram 2021; 23:221-238. [PMID: 34227846 DOI: 10.1089/cell.2021.0014] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Porcine embryonic stem cells (pESCs) would provide potentials for agricultural- and biotechnological-related applications. However, authentic pESCs have not been established yet because standards for porcine stem cell-specific markers and culture conditions are not clear. Therefore, the present study reports attempts to derive pluripotent epiblast stem cells either from in vitro or in vivo derived porcine embryos. Nine epiblast cell lines (seven lines from Berkshire and two lines from Duroc) could only be isolated from day 9- to 9.5-old in vivo derived early conceptuses. Pluripotency features were analyzed in relation to the presence or absence of alkaline phosphatase (AP) activity. Interestingly, the mRNA expression of several marker genes for pluripotency or epiblast was different between putative epiblast stem cells of the two groups [AP-positive (+) pEpiSC-like cell 2 line and AP-negative (-) pEpiSC-like cell 8 line]. For example, expressions of OCT-3/4, NANOG, SOX2, c-MYC, FGF2, and NODAL in AP-negative (-) porcine epiblast stem cell (pEpiSC)-like cells were higher than those in AP-positive (+) pEpiSC-like cells. Expression of surface markers differed between the two groups to some extent. SSEA-1 was strongly expressed only in AP-negative (-) pEpiSC-like cells, whereas AP-positive (+) pEpiSC-like cells did not express. In addition, we report to have some differences in the in vitro differentiation capacity between AP-positive (+) and AP-negative (-) epiblast cell lines. Primary embryonic germ layer markers (cardiac actin, nestin, and GATA 6) and primordial germ cell markers (Dazl and Vasa) were strongly expressed in embryoid bodies (EBs) aggregated from AP-negative (-) pEpiSC-like cells, whereas EBs aggregated from AP-positive (+) pEpiSCs did not show expression of primary embryonic germ layers and primordial germ cell markers except GATA 6. These results indicate that pEpiSC-like cells display different pluripotency characteristics in relation to AP activity.
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Affiliation(s)
- Sang-Ki Baek
- Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Soo-Been Jeon
- Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Bo-Gyeong Seo
- Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Cheol Hwangbo
- Division of Life Science, College of Natural Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Keum-Chul Shin
- Institute of Agriculture & Life Science, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea.,Department of Forest Environmental Resources, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Jung-Woo Choi
- College of Animal Life Science, Kangwon National University, Chuncheon, Republic of Korea
| | - Chang-Seop An
- Gyeongsangnamdo Livestock Experiment Station, Sancheong, Republic of Korea
| | - Mi-Ae Jeong
- Gyeongsangnamdo Livestock Experiment Station, Sancheong, Republic of Korea
| | - Tae-Suk Kim
- Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
| | - Joon-Hee Lee
- Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea.,Institute of Agriculture & Life Science, College of Agriculture and Life Sciences, Gyeongsang National University, Jinju, Republic of Korea
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Urbach A, Witte OW. Divide or Commit - Revisiting the Role of Cell Cycle Regulators in Adult Hippocampal Neurogenesis. Front Cell Dev Biol 2019; 7:55. [PMID: 31069222 PMCID: PMC6491688 DOI: 10.3389/fcell.2019.00055] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2019] [Accepted: 03/28/2019] [Indexed: 12/21/2022] Open
Abstract
The adult dentate gyrus continuously generates new neurons that endow the brain with increased plasticity, helping to cope with changing environmental and cognitive demands. The process leading to the birth of new neurons spans several precursor stages and is the result of a coordinated series of fate decisions, which are tightly controlled by extrinsic signals. Many of these signals act through modulation of cell cycle (CC) components, not only to drive proliferation, but also for linage commitment and differentiation. In this review, we provide a comprehensive overview on key CC components and regulators, with emphasis on G1 phase, and analyze their specific functions in precursor cells of the adult hippocampus. We explore their role for balancing quiescence versus self-renewal, which is essential to maintain a lifelong pool of neural stem cells while producing new neurons “on demand.” Finally, we discuss available evidence and controversies on the impact of CC/G1 length on proliferation versus differentiation decisions.
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Affiliation(s)
- Anja Urbach
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Germany
| | - Otto W Witte
- Hans Berger Department of Neurology, Jena University Hospital, Jena, Germany
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Caron N, Genin EC, Marlier Q, Verteneuil S, Beukelaers P, Morel L, Hu MG, Hinds PW, Nguyen L, Vandenbosch R, Malgrange B. Proliferation of hippocampal progenitors relies on p27-dependent regulation of Cdk6 kinase activity. Cell Mol Life Sci 2018; 75:3817-3827. [PMID: 29728713 PMCID: PMC11105564 DOI: 10.1007/s00018-018-2832-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Revised: 03/02/2018] [Accepted: 05/02/2018] [Indexed: 10/17/2022]
Abstract
Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.
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Affiliation(s)
- Nicolas Caron
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Emmanuelle C Genin
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Quentin Marlier
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Sébastien Verteneuil
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Pierre Beukelaers
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Laurence Morel
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida, Gainesville, FL, USA
| | - Miaofen G Hu
- Molecular Oncology Research Institute, Tufts Medical Center, 75 Kneeland Street, Boston, MA, USA
| | - Philip W Hinds
- Molecular Oncology Research Institute, Tufts Medical Center, 75 Kneeland Street, Boston, MA, USA
- Department of Developmental, Molecular, and Chemical Biology, Tufts University School of Medicine, Boston, MA, USA
| | - Laurent Nguyen
- Molecular Regulation of Neurogenesis, GIGA-Neurosciences, University of Liège, Liège, Belgium
| | - Renaud Vandenbosch
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium
| | - Brigitte Malgrange
- Laboratory of Developmental Neurobiology, GIGA-Neurosciences, Quartier Hôpital, University of Liège, Avenue Hippocrate 15, B36 +1, 4000, Liège, Belgium.
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Kuppers DA, Schmitt TM, Hwang HC, Samraj L, Clurman BE, Fero ML. The miR-106a~363 Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27 Kip1 cell cycle inhibitor. Oncotarget 2017; 8:50680-50691. [PMID: 28881594 PMCID: PMC5584189 DOI: 10.18632/oncotarget.16932] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2016] [Accepted: 03/22/2017] [Indexed: 12/19/2022] Open
Abstract
The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.
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Affiliation(s)
| | | | | | | | - Bruce E. Clurman
- Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
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Lin H, Huang Y, Su Z, Zhu Q, Ge Y, Wang G, Wang CQF, Mukai M, Holsberger DR, Cooke PS, Lian QQ, Ge RS. Deficiency of CDKN1A or both CDKN1A and CDKN1B affects the pubertal development of mouse Leydig cells. Biol Reprod 2015; 92:77. [PMID: 25609837 DOI: 10.1095/biolreprod.114.118463] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Cyclin-dependent kinase inhibitors p21(Cip1) (CDKN1A) and p27(Kip1) (CDKN1B) are expressed in Leydig cells. Previously, we reported that Cdkn1b knockout in the mouse led to increased Leydig cell proliferative capacity and lower steroidogenesis. However, the relative importance of CDKN1A and CDKN1B in these regulations was unclear. In the present study, we examined the relative importance of CDKN1A and CDKN1B in regulation of Leydig cell proliferation and steroidogenesis by whole-body knockout of CDKN1A (Cdkn1a(-/-)) and CDKN1A/CDKN1B double knockout (DBKO). The cell number, 5-bromo-2-deoxyuridine incorporation rate, steroidogenesis, and steroidogenic enzyme mRNA levels and activities of Leydig cells were compared among wild-type (WT), Cdkn1a(-/-), and DBKO mice. Relative to WT mice, Leydig cell number per testis was doubled in the DBKO and unchanged in the Cdkn1a(-/-) mice. Testicular testosterone levels and mRNA levels for luteinizing hormone receptor (Lhcgr), steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), 17alpha-hydroxylase/17,20-lyase (Cyp17a1), and 17beta-hydroxysteroid dehydrogenase 3 (Hsd17b3) and their respective proteins were significantly lower in the DBKO mice. However, testicular testosterone level was unchanged in the Cdkn1a(-/-) mice, although Lhcgr mRNA levels were significantly lower relative to those in the WT control. We conclude that Cdkn1a(-/-) did not increase Leydig cell numbers (although a defect of Leydig cell function was noted), whereas DBKO caused a significant increase of Leydig cell numbers but a decrease of steroidogenesis.
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Affiliation(s)
- Han Lin
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Yadong Huang
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, Guangdong, P.R. China
| | - Zhijian Su
- Guangdong Provincial Key Laboratory of Bioengineering Medicine, Jinan University, Guangzhou, Guangdong, P.R. China
| | - Qiqi Zhu
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Yufei Ge
- Population Council & Rockefeller University, New York, New York
| | - Guimin Wang
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Claire Q F Wang
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Motoko Mukai
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois
| | - Denise R Holsberger
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois
| | - Paul S Cooke
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois Department of Physiological Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida
| | - Qing-Quan Lian
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China
| | - Ren-Shan Ge
- The 2nd Affiliated Hospital & Yuying Children's Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China Population Council & Rockefeller University, New York, New York
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Zhu DH, Niu BL, Du HM, Ren K, Sun JM, Gong JP. Hath1 inhibits proliferation of colon cancer cells probably through up-regulating expression of Muc2 and p27 and down-regulating expression of cyclin D1. Asian Pac J Cancer Prev 2014; 13:6349-55. [PMID: 23464457 DOI: 10.7314/apjcp.2012.13.12.6349] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.
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Affiliation(s)
- Dai-Hua Zhu
- Department of General Surgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing, China.
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Kríz V, Pospíchalová V, Masek J, Kilander MBC, Slavík J, Tanneberger K, Schulte G, Machala M, Kozubík A, Behrens J, Bryja V. β-arrestin promotes Wnt-induced low density lipoprotein receptor-related protein 6 (Lrp6) phosphorylation via increased membrane recruitment of Amer1 protein. J Biol Chem 2013; 289:1128-41. [PMID: 24265322 PMCID: PMC3887180 DOI: 10.1074/jbc.m113.498444] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.
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Affiliation(s)
- Vítezslav Kríz
- From the Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic
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Lin H, Hu GX, Dong L, Dong Q, Mukai M, Chen BB, Holsberger DR, Sottas CM, Cooke PS, Lian QQ, Li XK, Ge RS. Increased proliferation but decreased steroidogenic capacity in Leydig cells from mice lacking cyclin-dependent kinase inhibitor 1B. Biol Reprod 2009; 80:1232-8. [PMID: 19211806 PMCID: PMC2804804 DOI: 10.1095/biolreprod.108.074229] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2008] [Revised: 11/04/2008] [Accepted: 01/23/2009] [Indexed: 11/01/2022] Open
Abstract
Proliferating cells express cyclins, cell cycle regulatory proteins that regulate the activity of cyclin-dependent kinases (CDKs). The actions of CDKs are regulated by specific inhibitors, the CDK inhibitors (CDKIs), which are comprised of the Cip/Kip and INK4 families. Expression of the Cip/Kip CDKI 1B (Cdkn1b, encoding protein CDKN1B, also called p27(kip1)) in developing Leydig cells (LCs) has been reported, but the function of CDKN1B in LCs is unclear. The goal of the present study was to determine the effects of CDKN1B on LC proliferation and steroidogenesis by examining these parameters in Cdkn1b knockout (Cdkn1b(-/-)) mice. LC proliferation was measured by bromodeoxyuridine incorporation. Testicular testosterone levels, mRNA levels, and enzyme activities of steroidogenic enzymes were compared in Cdkn1b(-/-) and Cdkn1b(+/+) mice. The labeling index of LCs in Cdkn1b(-/-) mice was 1.5% +/- 0.2%, almost 7-fold higher than 0.2% +/- 0.08% (P < 0.001) in the Cdkn1b(+/+) control mice. LC number per testis in Cdkn1b(-/-) mice was 2-fold that seen in the Cdkn1b(+/+) control mice. However, testicular testosterone levels, mRNA levels of steroidogenic acute regulatory protein (Star), cholesterol side-chain cleavage enzyme (Cyp11a1), and 3beta-hydroxtsteroid dehydrogenase 6 (Hsd3b6), and their respective proteins, were significantly lower in Cdkn1b(-/-) mice. We conclude that deficiency of CDKN1B increased LC proliferation, but decreased steroidogenesis. Thus, CDKN1B is an important regulator of LC development and function.
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Affiliation(s)
- Han Lin
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | - Guo-Xin Hu
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | - Lei Dong
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | - Qiang Dong
- Department of Urology, Huaxi Hospital, Sichuan University, Chendu, Sichuan, China
| | - Motoko Mukai
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois
| | - Bing-Bing Chen
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | | | - Chantal M. Sottas
- Population Council and the Rockefeller University, New York, New York
| | - Paul S. Cooke
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois
| | - Qing-Quan Lian
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | - Xiao-Kun Li
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
| | - Ren-Shan Ge
- Population Council and the Rockefeller University, New York, New York
- The Second Affiliated Hospital, School of Pharmacy, and the First Affiliated Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, China
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Bryja V, Pacherník J, Vondráček J, Souček K, Čajánek L, Horvath V, Holubcová Z, Dvořák P, Hampl A. Lineage specific composition of cyclin D-CDK4/CDK6-p27 complexes reveals distinct functions of CDK4, CDK6 and individual D-type cyclins in differentiating cells of embryonic origin. Cell Prolif 2008; 41:875-893. [PMID: 19040567 PMCID: PMC2659368 DOI: 10.1111/j.1365-2184.2008.00556.x] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Abstract. Objectives: This article is to study the role of G1/S regulators in differentiation of pluripotent embryonic cells. Materials and methods: We established a P19 embryonal carcinoma cell‐based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G1/S regulators were analysed with respect to growth and differentiation parameters of the cells. Results and Conclusions: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E–CDK2 but not of cyclin D–CDK4/6–p27 complexes. In an exponentially growing P19 cell population, the cyclin D1–CDK4 complex is detected, which is replaced by cyclin D2/3–CDK4/6–p27 complex following density arrest. During endodermal differentiation kinase‐inactive cyclin D2/D3–CDK4–p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2–CDK4–p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D–CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G1/S transition‐regulating machinery in early embryonic cells.
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Affiliation(s)
- V Bryja
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - J Pacherník
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - J Vondráček
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - K Souček
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - L Čajánek
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - V Horvath
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - Z Holubcová
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - P Dvořák
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
| | - A Hampl
- Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic,Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic,Center for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic,Department of Molecular Embryology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic, andDepartment of Biology, Faculty of Medicine, Masaryk University, Brno, Czech Republic
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11
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Zheng Y, Bie W, Yang R, Perekatt AO, Poole AJ, Tyner AL. Functions of p21 and p27 in the regenerating epithelial linings of the mouse small and large intestine. Cancer Biol Ther 2008; 7:873-9. [PMID: 18344686 PMCID: PMC3005242 DOI: 10.4161/cbt.7.6.5868] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
The epithelial linings of the small and large intestine are rapidly turned over and provide an ideal system for exploring links between differentiation and regulation of cell cycle exit. We utilized wild type, p21-/-, p27-/- and p21/p27-/- mice to address contributions of the Cdk inhibitors p21 and p27 to proliferation and differentiation in the mouse gastrointestinal tract. We did not detect any significant differences in proliferation, and all differentiated epithelial cell lineages were represented in all four genotypes. These data indicate that p21 and p27 do not play essential roles in the regulation of normal epithelial renewal in the intestine. These Cdk inhibitors are not needed in vivo for either assembly of Cdk/Cyclin complexes that drive active proliferation, or inhibition of Cdk/Cyclin complexes during cell cycle exit. However, expression of Cyclin D2 and to a lesser degree Cyclin D3 was reduced in p27-/- and p21/p27-/- mice, indicating a unique role for p27 in the regulation of these specific D-type Cyclins in vivo. In the absence of p27, reduced levels of Cyclin D2 and D3 may help to counteract increased proproliferative signals in the intestine.
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Affiliation(s)
| | | | | | | | | | - Angela L. Tyner
- Correspondence should be addressed to ALT, University of Illinois College of Medicine, Department of Biochemistry and Molecular Genetics, M/C 669, 900 S. Ashland Ave., Chicago, Illinois 60607, (312) 996-7964 (Telephone), (312) 413-0353 (FAX),
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12
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Zhu JS, Song MQ, Chen GQ, Li Q, Sun Q, Zhang Q. Molecular mechanisms of paclitaxel and NM-3 on human gastric cancer in a severe combined immune deficiency mice orthotopic implantation model. World J Gastroenterol 2007; 13:4131-5. [PMID: 17696236 PMCID: PMC4205319 DOI: 10.3748/wjg.v13.i30.4131] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To explore the molecular mechanisms of action of paclitaxel and NM-3 on human gastric cancer in severe combined immune deficiency (SCID) mice.
METHODS: Human gastric cancer cells SGC-7901 were implanted into SCID mice and mice were treated with paclitaxel and NM-3. The effects of paclitaxel and NM-3 on apoptosis of human gastric cancer cells were analyzed using flow cytometry, TUNEL assays, and DNA fragment analyses.
RESULTS: Apoptosis of SGC-7901 cells was successfully induced by paclitaxel, NM-3, and the combination of paclitaxel and NM-3 24 h after injection as shown by the presence of apoptotic hypodiploid peaks on the flow cytometer before G1-S and a characteristic apoptotic band pattern in the DNA electrophoresis. The apoptotic rate detected by TUNEL assay was found to be significantly higher in the paclitaxel/NM-3 compared to the control group (38.5% ± 5.14% vs 13.2% ± 1.75%, P < 0.01).
CONCLUSION: Paclitaxel in combination with NM-3 is able to induce apoptosis of the human gastric cancer cells in SCID mice effectively and synergistically.
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Affiliation(s)
- Jin-Shui Zhu
- Department of Gastroenterology, Affiliated Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China.
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13
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Ishihara K, Takahashi A, Kaneko M, Sugeno H, Hirasawa N, Hong J, Zee O, Ohuchi K. Differentiation of eosinophilic leukemia EoL-1 cells into eosinophils induced by histone deacetylase inhibitors. Life Sci 2007; 80:1213-20. [PMID: 17258775 DOI: 10.1016/j.lfs.2006.12.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2006] [Revised: 11/16/2006] [Accepted: 12/13/2006] [Indexed: 01/29/2023]
Abstract
EoL-1 cells differentiate into eosinophils in the presence of n-butyrate, but the mechanism has remained to be elucidated. Because n-butyrate can inhibit histone deacetylases, we hypothesized that the inhibition of histone deacetylases induces the differentiation of EoL-1 cells into eosinophils. In this study, using n-butyrate and two other histone deacetylase inhibitors, apicidin and trichostatin A, we have analyzed the relationship between the inhibition of histone deacetylases and the differentiation into eosinophils in EoL-1 cells. It was demonstrated that apicidin and n-butyrate induced a continuous acetylation of histones H4 and H3, inhibited the proliferation of EoL-1 cells without attenuating the level of FIP1L1-PDGFRA mRNA, and induced the expression of markers for mature eosinophils such as integrin beta7, CCR1, and CCR3 on EoL-1 cells, while trichostatin A evoked a transient acetylation of histones and induced no differentiation into eosinophils. These findings suggest that the continuous inhibition of histone deacetylases in EoL-1 cells induces the differentiation into mature eosinophils.
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Affiliation(s)
- Kenji Ishihara
- Laboratory of Pathophysiological Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi 980-8578, Japan
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14
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Wang L, Zhu JS, Song MQ, Chen GQ, Chen JL. Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray. World J Gastroenterol 2006; 12:6949-54. [PMID: 17109515 PMCID: PMC4087337 DOI: 10.3748/wjg.v12.i43.6949] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection (LMD) in combination with cDNA microarray.
METHODS: Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients (group 1) with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients (group 2) were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer.
RESULTS: The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes (OPCML, RNASE1, YES1 and ACK1) could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes (OPCML, RNASE1 and YES1) was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer.
CONCLUSION: LMD in combination with cDNA microarray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes (OPCML, RNASE1 and YES1) associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.
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Affiliation(s)
- Long Wang
- Department of Gastroenterology, Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200233, China
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15
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Glorioso C, Sabatini M, Unger T, Hashimoto T, Monteggia LM, Lewis DA, Mirnics K. Specificity and timing of neocortical transcriptome changes in response to BDNF gene ablation during embryogenesis or adulthood. Mol Psychiatry 2006; 11:633-48. [PMID: 16702976 DOI: 10.1038/sj.mp.4001835] [Citation(s) in RCA: 74] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Brain-derived neurotrophic factor (BDNF) has been reported to be critical for the development of cortical inhibitory neurons. However, the effect of BDNF on the expression of transcripts whose protein products are involved in gamma amino butric acid (GABA) neurotransmission has not been assessed. In this study, gene expression profiling using oligonucleotide microarrays was performed in prefrontal cortical tissue from mice with inducible deletions of BDNF. Both embryonic and adulthood ablation of BDNF gave rise to many shared transcriptome changes. BDNF appeared to be required to maintain gene expression in the SST-NPY-TAC1 subclass of GABA neurons, although the absence of BDNF did not alter their general phenotype as inhibitory neurons. Furthermore, we observed expression alterations in genes encoding early-immediate genes (ARC, EGR1, EGR2, FOS, DUSP1, DUSP6) and critical cellular signaling systems (CDKN1c, CCND2, CAMK1g, RGS4). These BDNF-dependent gene expression changes may illuminate the biological basis for transcriptome changes observed in certain human brain disorders.
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Affiliation(s)
- C Glorioso
- Department of Psychiatry, University of Pittsburgh School of Medicine, PA 15261, USA
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16
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Zhu JS, Wang L, Cheng GQ, Li Q, Zhu ZM, Zhu L. Apoptosis mechanisms of human gastric cancer cell line MKN-45 infected with human mutant p27. World J Gastroenterol 2006; 11:7536-40. [PMID: 16437730 PMCID: PMC4725169 DOI: 10.3748/wjg.v11.i47.7536] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4+/-3.12%) compared to the control group (3.12+/-0.13%, P < 0.01). CONCLUSION Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.
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Affiliation(s)
- Jin-Shui Zhu
- Department of Gastroenterology, Affiliated Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China.
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17
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Nadella KS, Kirschner LS. Disruption of protein kinase a regulation causes immortalization and dysregulation of D-type cyclins. Cancer Res 2006; 65:10307-15. [PMID: 16288019 DOI: 10.1158/0008-5472.can-05-3183] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Phosphorylation is a key event in cell cycle control, and dysregulation of this process is observed in many tumors, including those associated with specific inherited neoplasia syndromes. We have shown previously that patients with the autosomal dominant tumor predisposition Carney complex carry inactivating mutations in the PRKAR1A gene, which encodes the type 1A regulatory subunit of protein kinase A (PKA), the cyclic AMP-dependent protein kinase. This defect was associated with dysregulation of PKA signaling, and genetic analysis has suggested that complete loss of the gene may be required for tumorigenesis. To determine the mechanism by which dysregulation of PKA causes tumor formation, we generated in vitro primary mouse cells lacking the Prkar1a protein. We report that this genetic disruption of PKA regulation causes constitutive PKA activation and immortalization of primary mouse embryonic fibroblasts (MEFs). At the molecular level, knockout of Prkar1a leads to up-regulation of D-type cyclins, and this increase occurs independently of other pathways known to increase cyclin D levels. Despite the immortalized phenotype, known mediators of cellular senescence (e.g., p53 and p19ARF) seem to remain intact in Prkar1a-/- MEFs. Mechanistically, cyclin D1 mRNA levels are not altered in the knockout cells, but protein half-life is markedly increased. Using this model, we provide the first direct genetic evidence that dysregulation of PKA promotes important steps in tumorigenesis, and that cyclin D1 is an essential target of PKA.
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Affiliation(s)
- Kiran S Nadella
- Human Cancer Genetics Program, The Ohio State University, Columbus, Ohio 43210, USA
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18
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Mukai M, Dong Q, Hardy MP, Kiyokawa H, Peterson RE, Cooke PS. Altered Prostatic Epithelial Proliferation and Apoptosis, Prostatic Development, and Serum Testosterone in Mice Lacking Cyclin-Dependent Kinase Inhibitors1. Biol Reprod 2005; 73:951-8. [PMID: 16014817 DOI: 10.1095/biolreprod.105.040980] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2022] Open
Abstract
Normal prostatic development and some prostatic diseases involve altered expression of the cell-cycle regulators p27 and p21 (also known as CDKN1B and CDKN1A, respectively). To determine the role of these proteins in the prostate, we examined prostatic phenotype and development in mice lacking p27 and/or p21. In p27-knockout (p27KO) mice, epithelial proliferation was increased 2- and 3.8-fold in the ventral and dorsolateral prostate, respectively, versus wild-type (WT) mice, although prostatic weights were not different. Epithelial apoptosis was increased in p27KO mice and may account for the lack of a concurrent increase in weight. Testosterone deficiency observed in this group was not the cause of this increase, because vehicle- and testosterone-treated p27KO mice had similar percentages of apoptotic cells. Also observed was a trend toward a decreased functional epithelial cytodifferentiation, indicating a potential role of p27 in this process. Conversely, dorsolateral prostate and seminal vesicle (SV) of p21-knockout (p21KO) mice, and all prostatic lobes and SV of p21/p27 double-knockout mice, weighed significantly less compared to the WT mice, and their epithelial proliferation was normal. Decreased testosterone concentrations may contribute to the decreased prostatic weights. However, other factors may be involved, because testosterone replacement only partially restored prostatic weights. We conclude that loss of p27 increases prostatic epithelial proliferation and alters differentiation but does not result in prostatic hyperplasia because of increased epithelial cell loss. The p21KO mice showed phenotypes distinctly different from those of p27KO mice, suggesting nonredundant roles of p21 and p27 in prostatic development. Loss of p27 or of both p21 and p27 results in serum testosterone deficiency, complicating analysis of the prostatic effects of these cell-cycle regulators.
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Affiliation(s)
- Motoko Mukai
- Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802, USA
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Bryja V, Cajánek L, Pacherník J, Hall AC, Horváth V, Dvorák P, Hampl A. Abnormal development of mouse embryoid bodies lacking p27Kip1 cell cycle regulator. Stem Cells 2005; 23:965-74. [PMID: 15941856 DOI: 10.1634/stemcells.2004-0174] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Cultures of three-dimensional aggregates of embryonic stem cells (ESCs) called embryoid bodies (EBs) provide a valuable system for analyzing molecular mechanisms that regulate differentiation of this unique cell type. Cyclin-dependent kinase inhibitor p27Kip1 (p27) becomes elevated during the differentiation of mouse ESCs (mESCs). In this study, various aspects of differentiation of EBs produced from normal and p27-deficient mESCs were analyzed to address the biological significance of this elevation. It was found that EBs lacking p27 grew significantly bigger, but this was not accompanied by detect-able abnormalities in the activities of cyclin-dependent kinases (CDKs). In most EB cells, downregulation of activating cyclins rather than upregulation of inhibiting p27 is probably responsible for lowering the activity of their CDKs. Abnormalities in the development of specific cell lineages were also observed in p27-deficient EBs. These included elimination of cells positive for cytokeratin endo-A (TROMA-I) and increased proliferation and formation of cavities originating from cells positive for Lewis-X. Our data also suggest that although two different pools of Lewis-X-expressing cells, cluster forming (ESC-like) and cavity forming (neural progenitors), normally exist in EBs, the absence of p27 leads to the enhancement of only the neural pool. No failure was found when the neurogenic capacity of p27-deficient mESCs was tested using various protein markers. Together, our data point to a dual role of p27 in mESCs, with one role being in the regulation of proliferation and the other role in establishing some other aspects of a differentiated phenotype.
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Affiliation(s)
- Vítezslav Bryja
- Laboratory of Molecular Embryology, Mendel University Brno, Zemedelská1, 61300 Brno, Czech Republic
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