Phosphorylation-stabilized CRTC1 cooperates with CBP and androgen receptor to transactivate AMH expression and drive polycystic ovary syndrome.

Abstract

Background: Anti-Müllerian hormone (AMH) levels are frequently elevated in women with polycystic ovary syndrome (PCOS) and serve as a valuable diagnostic biomarker. However, the molecular mechanisms driving AMH overexpression in PCOS remain poorly understood.

Methods: A PCOS mouse model was generated by subcutaneously administering dehydroepiandrosterone (DHEA). Gene and protein expression levels were measured using RT-qPCR and Western blot analysis, respectively. Isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis was performed to identify differentially expressed proteins. Protein interactions were assessed via immunoprecipitation and co-immunoprecipitation experiments. Additionally, both in vitro and in vivo experiments were conducted to evaluate phosphorylation and ubiquitination processes.

Results: Conducting proteomic profiling of polycystic ovaries, we identified 417 differentially expressed proteins, including upregulated CRTC1 (CREB-regulated transcription coactivator 1), AR (androgen receptor), HIPK2 protein kinase, and AMH. We demonstrated that CRTC1 interacted with the histone acetyltransferase CBP (CREB binding protein) and AR to form a transcriptional complex that activates AMH expression. Importantly, the stability of CRTC1 was regulated by HIPK2-mediated phosphorylation. Specifically, HIPK2 phosphorylated CRTC1 at Ser36, which prevented its ubiquitination and subsequent proteasomal degradation. Inhibition or knockdown of HIPK2 disrupted this protective mechanism, leading to RNF121 (Ring finger protein 121) E3 ubiquitin ligase-mediated degradation of CRTC1. This resulted in the dissociation of the CRTC1-CBP-AR complex and a significant reduction in AMH expression. Furthermore, in DHEA-induced PCOS mice, administration of HIPK2 inhibitors effectively suppressed AMH expression and attenuated PCOS progression.

Conclusion: These findings provide novel insights into the molecular mechanisms underlying AMH upregulation in PCOS. They highlight the CRTC1-CBP-AR transcriptional complex and HIPK2-mediated phosphorylation as critical regulatory nodes in PCOS pathogenesis.

Clinical trial number: Not applicable.