Functional effects of Tribbles homolog 2 in bovine ovarian granulosa cells†

Abstract

Tribbles homologs (TRIB) 1, 2, and 3 represent atypical members of the serine/threonine kinase superfamily. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GCs) of bovine preovulatory follicles. The current study aimed to further investigate TRIB2 regulation and study its function in the ovary. GCs were collected from follicles at different developmental stages: small antral follicles (SF), dominant follicles (DF) at day 5 of the estrous cycle, and hCG-induced ovulatory follicles (OFs). RT-qPCR analyses showed greater expression of TRIB2 in GC of DF as compared to OF and a significant downregulation of TRIB2 steady-state mRNA amounts by hCG/LH, starting at 6 h through 24 h post-hCG as compared to 0 h. Specific anti-TRIB2 polyclonal antibodies were generated and western blot analysis confirmed TRIB2 downregulation by hCG at the protein level. In vitro studies showed that FSH stimulates TRIB2 expression in GC. Inhibition of TRIB2 using CRISPR/Cas9 resulted in a significant increase in PCNA expression and an increase in steroidogenic enzyme CYP19A1 expression, while TRIB2 overexpression tended to decrease GC proliferation. TRIB2 inhibition also resulted in a decrease in transcription factors connective tissue growth factor (CTGF) and ankyrin repeat domain-containing protein 1 (ANKRD1) expression, while TRIB2 overexpression increased CTGF and ANKRD1. Additionally, western blot analyses showed reduction in ERK1/2 (MAPK3/1) and p38MAPK (MAPK14) phosphorylation levels following TRIB2 inhibition, while TRIB2 overexpression increased p-ERK1/2 and p-p38MAPK. These results provide evidence that TRIB2 modulates MAPK signaling in GC and that TRIB2 could act as a regulator of GC proliferation and function, which could affect steroidogenesis during follicular development.

Tribbles Pseudokinase 2 (TRIB2) Regulates Expression of Binding Partners in Bovine Granulosa Cells

“…The results confirmed TRIB2 downregulation via CRISPR/Cas9 ( Figure 3A; p < 0.01), while TRIB2 was overexpressed using the pQE system ( Figure 3B; p < 0.01). These data confirming TRIB2 inhibition and overexpression in our model were previously published [14]. Subsequent analyses demonstrated that TRIB2 inhibition did not change CALM1 abundance while TRIB2 overexpression resulted in significant increase in CALM1 abundance ( Figure 4A; p < 0.01).…”

“…Finally, RAB14 displayed a surprising result as both inhibition and overexpression of TRIB2 resulted in increased expression of RAB14 ( Figure 4G). [14]. TRIB2 knockdown was confirmed by RT-qPCR showing that TRIB2 was significantly reduced in GCs via CRISPR/Cas9 as compared to the control and empty vector.…”

“…We previously reported the regulation of the expression of Tribbles genes in granulosa cells of ovarian follicles during follicular development [14]. Overall, the expression of genes in the bovine preovulatory follicle assures its growth but abrogates the development of other potentially ovulatory follicles through atresia.…”

“…TRIB2 knockdown was confirmed by RT-qPCR showing that TRIB2 was significantly reduced in GCs via CRISPR/Cas9 as compared to the control and empty vector. (B) The pQE-Trisystem was used to overexpress TRIB2 in GCs as described in Materials and Methods and previously reported [14]. TRIB2 overexpression in GCs was confirmed by RT-qPCR as compared to control and empty vector.…”

“…Specifically, TRIB3 inhibits ubiquitylation and degradation of promyelocytic leukemia protein (PML) in acute promyelocytic leukemia progression by repressing proteasome recruitment and interaction with PML and by inhibiting the ubiquitin proteasome system activity [13]. As for TRIB2 in particular, it was also associated with ERK signaling pathway in ovarian granulosa cells as increased ERK1/2 phosphorylation was observed in TRIB2-overexpressed cells [14].…”