Active nNOS Is Required for Grp94-Induced Antioxidant Cytoprotection: A Lesson from Myogenic to Cancer Cells.

The endoplasmic reticulum (ER) chaperone Grp94/gp96 appears to be involved in cytoprotection without being required for cell survival. This study compared the effects of Grp94 protein levels on Ca homeostasis, antioxidant cytoprotection and protein-protein interactions between two widely studied cell lines, the myogenic C2C12 and the epithelial HeLa, and two breast cancer cell lines, MDA-MB-231 and HS578T. In myogenic cells, but not in HeLa, Grp94 overexpression exerted cytoprotection by reducing ER Ca storage, due to an inhibitory effect on SERCA2. In C2C12 cells, but not in HeLa, Grp94 co-immunoprecipitated with non-client proteins, such as nNOS, SERCA2 and PMCA, which co-fractionated by sucrose gradient centrifugation in a distinct, medium density, ER vesicular compartment. Active nNOS was also required for Grp94-induced cytoprotection, since its inhibition by L-NNA disrupted the co-immunoprecipitation and co-fractionation of Grp94 with nNOS and SERCA2, and increased apoptosis. Comparably, only the breast cancer cell line MDA-MB-231, which showed Grp94 co-immunoprecipitation with nNOS, SERCA2 and PMCA, increased oxidant-induced apoptosis after nNOS inhibition or Grp94 silencing. These results identify the Grp94-driven multiprotein complex, including active nNOS as mechanistically involved in antioxidant cytoprotection by means of nNOS activity and improved Ca homeostasis.