(R1826) Tribbles homolog 2 regulates binding partners expression in bovine granulosa cells

Aly Warma (Centre de recherche en reproduction et fertilité (CRRF), Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal)| Jacques Lussier (Centre de recherche en reproduction et fertilité (CRRF), Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal)| Kalidou Ndiaye (Centre de recherche en reproduction et fertilité (CRRF), Département de biomédecine vétérinaire, Faculté de médecine vétérinaire, Université de Montréal)

The Tribbles (TRIB) family including TRIB2 are serine/threonine pseudokinases, which function as signaling mediators and scaffolding proteins regulating numerous cellular events such as proliferation, differentiation and cell death through protein stability and activity. However, the signaling pathways affected by TRIB2 in granulosa cells (GC) during the ovarian follicular development is not fully defined. We previously identified and reported TRIB2 as differentially expressed in growing dominant follicles (DF) while downregulated by the luteinizing hormone (LH) surge in ovulatory follicles. In the present study, we used the yeast two-hybrid screening and in vitro co-immunoprecipitation to identify and confirm TRIB2 interactions in GC. We identified CALM1, INHBA, INPPL1, NT5E, SCD, SDHB and RAB14 as TRIB2 binding partners. RT-PCR analyses showed all TRIB2 binding partners are present in GC of DF but differently regulated throughout the different stages of follicular development. TRIB2 inhibition and overexpression using, respectively, CRISPR/Cas9 and pQE systems revealed TRIB2 differently regulates the expression of its binding partners. INPPL1 and INHBA were decreased in TRIB2-inhibited GC and increased in TRIB2-overexpressed GC (P <0.05) while NT5E and SDHB were increased following TRIB2 inhibition (P <0.05). CALM1 was increased following TRIB2 overexpression and both inhibition and overexpression of TRIB2 resulted in increased RAB14 expression (P <0.05). The regulation of these partners by TRIB2 reveal a crucial role of TRIB2 in a number of biological processes in GC, and provide a larger view of potential TRIB2-regulated signal transduction pathways and a strong evidence that TRIB2 may act as a regulator of target effectors during ovarian follicular development.