Investigation of the Role of Cytokeratin 8/18 Intermediate Filaments in Granulosa Cell Fate.

Abstract

Apoptosis of granulosa cells is an early indication of follicular atresia; but the cellular mechanisms that drive this process, especially its cell specificity, are relatively unknown. Recent evidence suggests cytoskeletal stress filaments, such as cytokeratin 8/18 (KRT8/18) intermediate filaments, intrinsically protect epithelial cells from apoptosis by influencing intracellular signaling and diminishing death receptor expression (e.g., FAS) on the cell surface. The objective of this study was to determine the relative expression of KRT8/18 filaments in a human granulosa tumor cell line (KGN cells) and evaluate their role in the context of FAS-mediated apoptosis. Flow cytometric analysis following culture revealed ~ 92% of cells stained positively for KRT8/18 expression. Caspase-Glo 3/7 and MTS assays were used to monitor cell apoptosis after 8- and 24-hour treatment of KGN cells with anti-FAS antibody, CH11 (1 μg/mL) to activate FAS. CH11 treatment caused a modest increase in caspase activity (2 fold) but had no effect on cell viability (P<0.05; n= 6 expts). However, CH11 combined with a non-toxic concentration of the protein synthesis inhibitor cycloheximide (0.25 μg/mL) provoked extensive apoptosis (~60% cells; P<0.05; n=6 expts) characterized by 10 fold increases in caspase 3/7 activity and reduced cell viability. Under these conditions, inhibition of ERK1/2 signaling with the MEK1/2 inhibitor PD98059 (30 μM) had no effect (P>0.05; n=3 expts), but inhibition of the PI3K/Akt pathway with Wortmannin (100 nM) provided modest protection from FAS-mediated apoptosis (P<0.05; n=3 expts). Initial experiments to genetically knock-down KRT8/18 expression with short interfering RNA (siRNA; 10 nM) sensitized KGN cells to FAS-induced apoptosis (P<0.05) compared to cells exposed to a scrambled form of siRNA (n=3 expts). The present study provides initial evidence that human granulosa cells express KRT8/18, and that KRT8/18 filaments are cellular determinants that prevent FAS-mediated apoptosis. Moreover, the results suggest that the synthesis of labile protein(s) and the regulation of the PI3K/Akt pathway provide granulosa cells with additional mechanisms to avoid cell death and ultimately, follicular atresia. This project was supported by National Research Initiative Competitive Grant no. 2007-35203-18074 from the USDA Cooperative State Research, Education, and Extension Service.