TGF-β/SMAD4 signaling pathway activates the HAS2-HA system to regulate granulosa cell state.

Both TGF-β/SMAD4 signaling pathway and HAS2-HA system have been shown to control granulosa cell (GC) state in mammalian ovary. However, the regulatory relationship between TGF-β/SMAD4 signaling pathway and HA system in GCs is not well known. Here, we report that the TGF-β/SMAD4 signaling pathway activates the HAS2-HA system by binding directly to the HAS2 promoter, ultimately controlling the GC state via the CD44-Caspase3 axis. SMAD4-induced HAS2 expression, HAS2-driven HA secretion, and HAS2-mediated GC state (proliferation and apoptosis) by interacting directly with the promoter region of the HAS2 gene. The CD44-Caspase3 axis, located downstream of the HAS2-HA system, was also activated by SMAD4 and the TGF-β/SMAD4 signaling pathway. However, there was no feedback regulation of the TGF-β/SMAD4 signaling pathway by the HAS2-HA system in GCs. In addition, we found that miRNA-26b attenuated HAS2 expression via SMAD4-dependent and -independent mechanisms. Our findings provide compelling evidence that HAS2 is a direct transcriptional target of SMAD4. They also reveal a novel mechanism by which the TGF-β/SMAD4 signaling pathway controls the GC state and alters the structural components of GCs in porcine ovaries.