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Anti-Müllerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB

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Abstract

Anti-Müllerian hormone (AMH) downregulates the level of stem cell factor (SCF) via the cAMP/PKA signaling pathway in human granulosa cells (GCs). Little information is available on the molecular mechanism underlying the interaction. This study is aimed at determining whether AMH regulates expression of SCF via the cAMP-PKA-CREB signaling pathway in human GCs. In the present study, we verified the binding of cAMP-response element-binding protein (CREB) to promoter of SCF in human GCs. Furthermore, the effect of CREB was tested on the SCF promoter, and the site of CREB binding to SCF promoter was identified using truncations as well as assays of SCF-promoted mutation and CREB mutation. To investigate the correlation among AMH, SCF promoter, and CREB, pGL-Basic-SCF+CREB was transfected into overexpressed AMH GCs (AMH-high GCs), low expressed AMH GCs (AMH-low GCs), and normal GCs (GCs), respectively. Finally, immunofluorescence, double immunostaining, and Western blot were carried out in AMH-high and AMH-low GCs to confirm the AMH-mediated regulation of SCF expression by inhibiting the phosphorylation of CREB (pCREB) in GCs. Results indicated CREB interacted with SCF promoter and significantly enhanced the transcription level of SCF. The CREB binding site was localized at 318–321 bp of SCF gene promote. AMH inhibits the expression of SCF by phosphorylation of CREB via the PKA signaling pathway in GCs. These findings provide an in-depth understanding of the molecular mechanism underlying AMH suppressing the follicle growth, which would aid in the development of a novel therapy.

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Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgments

We thank the Laboratory of Fertility Maintenance of Ningxia Medical University and the Clinical Pathogen Microbiology Laboratory of the General Hospital of Ningxia Medical University for providing an experimental platform. And we thank professor Hai Wang for modifying the article.

Funding

This work was supported by the Natural Science Foundation of China (No. 81660257), Ningxia Natural Science Foundation (NZ16125, NZ16143), Personnel Agency Overseas talent Project, 2017 Autonomous Region Leader in Science and Technology Innovation, First-Class Discipline Construction Founded Project of Ningxia Medical University and the School of Clinical Medicine (NXYLXK2017A05), and 2018 Advantageous Subjects Project of Ningxia Medical University (XY201807). Key Research and Development program of Ningxia Hui Autonomous Region (2019BFG02005).

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Authors and Affiliations

  1. Ningxia Medical University, General Hospital of Ningxia Medical University, Yinchuan, 750004, Ningxia, China

    Yun-Xing Fu, Hui-Min Yang, Ting Hu, Ya-Fei Wang, Yan-Fei Wang & Hui Wang

  2. Reproductive Medicine Center, General Hospital of Ningxia Medical University, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, 750004, Ningxia, China

    Fei-Miao Wang & Rong Hu

  3. Taihe Hospital, Shiyan, 442000, Hubei Province, China

    Xiao-E Ou-yang

Authors
  1. Yun-Xing Fu
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  2. Fei-Miao Wang
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  3. Xiao-E Ou-yang
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  4. Hui-Min Yang
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  5. Ting Hu
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  6. Ya-Fei Wang
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  7. Yan-Fei Wang
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  8. Hui Wang
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  9. Rong Hu
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Contributions

YXF and FMW conceived and designed the study with inputs from RH. YXF and FMW have the same contribution to this article. FMW and RH were responsible for the supervision and coordination of the project. YXF and XE performed most of the in vitro experiments. HMY collected the FF. TH and HW led the data analysis with inputs from YXF, Yanfei-Wang, XE, Yafei-Wang, and HMY. The first draft of the manuscript was written by YXF and FMW; then, RH, XE, Yafei-Wang, and Yanfei-Wang contributed to revise and review the manuscript. All authors read and approved the final manuscript before submission.

Corresponding author

Correspondence to Rong Hu.

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Ethics Approval and Consent to Participate

The use of human follicular fluid was reviewed and approved by the Ethics Committee of Ningxia Medical University and was performed in accordance with the approved guidelines. Written informed consent was obtained from each participating patient.

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The authors declare that they have no conflict of interest.

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Fu, YX., Wang, FM., Ou-yang, XE. et al. Anti-Müllerian Hormone Regulates Stem Cell Factor via cAMP/PKA Signaling Pathway in Human Granulosa Cells by Inhibiting the Phosphorylation of CREB. Reprod. Sci. 27, 325–333 (2020). https://doi.org/10.1007/s43032-019-00033-4

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  • DOI: https://doi.org/10.1007/s43032-019-00033-4

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