Protein kinase C isotypes in signal transduction for the 1,25D3-MARRS receptor (ERp57/PDIA3) in steroid hormone-stimulated phosphate uptake.

We undertook studies to determine which isotype(s) of protein kinase C (PKC) is/are activated by ligand binding to the 1,25D(3)-MARRS receptor (ERp57/PDIA3) and subsequent stimulation of phosphate uptake. Isolated intestinal epithelial cells from vitamin D-replete chicks were exposed to 1,25(OH)(2)D(3) for 1, 3, or 5min, thoroughly chilled, homogenized, and P(2) fractions (20,000xg post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P(2) membranes 1min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCalpha exhibited redistribution to membranes in a biphasic dose-response curve: slightly stimulated at the lowest dose, maximal at 300pM 1,25(OH)(2)D(3), and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCbeta also revealed hormone-mediated differences, while those with anti PKCgamma did not. RNAi studies were then performed with siRNA against PKCalpha or PKCbeta. Untransfected cells treated with hormone for 7min exhibited enhanced (32)P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased (32)P uptake in both controls (relative to untransfected controls), and hormone treated cells. However, control and transfected cells treated with hormone had equivalent levels of uptake. Western blot analyses confirmed decreased immunoreactivity in transfected cells. Chemical PKCalpha (safingol) and PKCbeta ([3-(1-(3-Imidazol-1-ylpropyl)-1H-indol-3-yl)-4-anilino-1H-pyrrole-2,5-dione] blockers also confirmed the results from siRNA and demonstrated decreased (32)P uptake in cells treated with 1,25(OH)(2)D(3) plus blockers in comparison with cells treated with 1,25(OH)(2)D(3) alone. Thus, PKCalpha and PKCbeta are both involved in steroid-stimulated phosphate uptake.