β-Catenin regulates ovarian granulosa cell cycle and proliferation in laying hens by interacting with TCF4

Abstract

Ovarian follicle development depends on the proliferation and differentiation of granulosa cells and is a complex biological process. The Wnt/β-catenin signaling pathway can regulate ovarian follicle development, and β-catenin, encoded by catenin beta 1 (CTNNB1), is the core component of this pathway. Although several studies of the mechanisms by which the Wnt/β-catenin pathway regulates cell proliferation in humans and mammals have reported, it remains unclear how β-catenin functions in poultry. To investigate the function of β-catenin in laying hens' follicle development, we evaluated the effect of CTNNB1 on cell cycle, proliferation, and apoptosis in ovarian granulosa cells (GCs) isolated from laying hens. We demonstrated that CTNNB1 significantly affected the expression of cyclin D1 (CCND1) and v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) (P < 0.01 and P < 0.05), key genes related to cell cycle and proliferation, to promote cell cycle progression from G1 to S phase, and thus accelerate granulosa cell proliferation. CTNNB1 did not however affect apoptosis or the expression of related genes baculoviral IAP repeat containing 5 (BIRC5) and BCL2 apoptosis regulator (Bcl-2). Overexpression of transcription factor 7-like 2 (TCF4) resulted in increased expression of CCND1, accelerated cell cycle progression, and granulosa cell proliferation. Direct physical interaction between β-catenin and TCF4 was demonstrated by immunofluorescence and coimmunoprecipitation. The proliferation of granulosa cells was inhibited by silencing CCND1; overexpression of TCF4 in CCND1-silenced cells restored their proliferation rate to normal levels. These results indicate that the interaction of TCF4 and β-catenin promotes CCND1 expression which in turn accelerates the cell cycle process of laying hen hierarchical follicular granulosa cells.