CITED4 mediates proliferation, apoptosis and steroidogenesis of Hu sheep granulosa cells in vitro.

Being a novel target of luteinizing hormone (LH), the effect of CREB-binding protein/P300-interacting trans-activator with ED-rich tail member 4 (CITED4) gene on the proliferation, apoptosis, and steroidogenesis of ovarian granulosa cells (GCs) in Hu sheep was investigated. The presence of CITED4, CREB-binding protein (CBP), CCAAT/enhancer-binding protein alpha (C/EBPα) and -beta (C/EBPβ) proteins was demonstrated in GCs and luteal cells. CITED4 protein in GCs was induced by LH, and CITED4 overexpression moderately increased GC responses to LH. In contrast, CITED4 knockdown in GCs decreased prostaglandin (PGE2)-induced LH target gene levels. Moreover, PGE2-stimulated CITED4 mRNA expression was blocked by ERK1/2 inhibition (U0126), suggesting that CITED4 is a downstream target of the ERK1/2 pathway in sheep GCs. In contrast to CITED4 knockdown, CITED4 overexpression promoted GC proliferation, inhibited apoptosis, upregulated cell cycle-related genes, and downregulated apoptosis-related genes. Additionally, CITED4 overexpression induced cell cycle transition from S to G2/M phase. No effect was observed with CITED4 knockdown. CITED4 overexpression increased progesterone (P4) production levels and STAR mRNA expression, whereas CITED4 knockdown decreased P4 production and STAR and 3β-HSD mRNA expression levels. Thus, our results suggest that CITED4 is involved in regulating the expression of LH-induced genes and the ERK1/2 pathway and the proliferation, apoptosis, and steroidogenesis in Hu sheep GCs by modulating the expression of related genes. These findings will help understand the role of CITED4 in follicular development and ovulation of pre-ovulatory follicles.