Zinc deficiency in bovine mural granulosa cells results in hippo pathway-mediated apoptosis†

Zinc is essential for the proper functioning of a variety of cells and tissues. Due to the limited understanding of the role of zinc in the bovine ovary, our objective was to investigate the role of zinc in bovine mural granulosa cell (mGC) viability and identify cell signaling pathways regulated by zinc in this cell type. Key indicators of cell behavior were measured after cultured bovine mGCs were incubated with increasing levels of a zinc chelator, TPEN. Cell viability decreased in a dose dependent manner, with a 91% loss of cell viability seen at 10 μM TPEN (p = 1.02 × 10-9), which corresponded to a 49% increase in caspase 3/7 activity (p = 0.006) and an upregulation of p-YAP at Ser127 (p = 0.006). TRULI, a potent LATS1/2 inhibitor, in combination with TPEN treatments, partially rescued the decrease in cell viability seen at 3.5 μM TPEN (p = 2.54 × 10-7) and the increase in caspase 3/7 activity seen at 10 μM TPEN (p = 0.0003). Surprisingly, TPEN treatment for 6 h showed a dose dependent increase in relative fold change in expression of the known YAP gene targets, CCN1 (p = 3.85 × 10-10) and CCN2 (p = 1.95 × 10-7). While the mechanism by which increased Hippo activation mediates TPEN-stimulated apoptosis is still unknown, these results demonstrate that zinc is an essential micronutrient for bovine mGC viability, and that zinc deficiency promotes Hippo signaling leading to increased apoptosis in bovine mGCs.