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CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway.

ABSTRACT: Collaborator of ARF (CARF) regulates cell proliferative fate through both p53-dependent and -independent mechanisms. Recently, we reported a new function of CARF as a positive regulator of Wnt signaling. Despite these findings, the physiological function of CARF has not been well studied. Here, we generated CARF knockout mice and found that male CARF^-/- mice exhibited significantly impaired fertility and Sertoli-cell-only (SCO) syndrome phenotypes. Further studies revealed that loss of CARF in Sertoli cells led to decreased GDNF expression, which hindered spermatogonial stem cells (SSCs) self-renewal. Meanwhile, CARF loss in undifferentiated spermatogonia impaired their proliferation. These two mechanisms together led to SCO syndrome phenotypes, which could be functionally rescued by pharmacological or genetic reactivation of Wnt signaling. Finally, we identified CARF^S351F as a potential pathogenic mutation in an SCO patient. Overall, our findings reveal important roles of CARF in spermatogonial self-renewal and proliferation through the Wnt signaling pathway.

SUBMITTER: Cui W

PROVIDER: S-EPMC7674451 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway.

Cui Wenhao W He Xiaoli X Zhai Xiaohong X Zhang Huan H Zhang Yuanwei Y Jin Fei F Song Xiaomin X Wu Dianqing D Shi Qinghua Q Li Lin L

Cell discovery 20201117 1

Collaborator of ARF (CARF) regulates cell proliferative fate through both p53-dependent and -independent mechanisms. Recently, we reported a new function of CARF as a positive regulator of Wnt signaling. Despite these findings, the physiological function of CARF has not been well studied. Here, we generated CARF knockout mice and found that male CARF<sup>-/-</sup> mice exhibited significantly impaired fertility and Sertoli-cell-only (SCO) syndrome phenotypes. Further studies revealed that loss o ...[more]

PMID: 33298864

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Project description:BackgroundStem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells.MethodsIn the present study, we used the previously constructed luciferase reporters driven by the promoter and 3'-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways.ResultsWe screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways.ConclusionsThese findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

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CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway.

Publications

CARF promotes spermatogonial self-renewal and proliferation through Wnt signaling pathway.

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