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 1,25D3-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)2D3 for 1, 3, or 5 min, thoroughly chilled, homogenized, and P2 fractions (20,000 × g post-nuclear pellet) prepared. Western analyses with anti-pan PKC revealed steroid-stimulated redistribution to P2 membranes 1 min after hormone. Using this time point, cells were treated with vehicle, 130-, 300- or 650-pM hormone. Western blots with anti-PKCα exhibited redistribution to membranes in a biphasic dose–response curve: slightly stimulated at the lowest dose, maximal at 300 pM 1,25(OH)2D3, and equivalent to control levels at the highest dose, paralleling hormone-mediated phosphate uptake. Westerns with anti PKCβ also revealed hormone-mediated differences, while those with anti PKCγ did not. RNAi studies were then performed with siRNA against PKCα or PKCβ. Untransfected cells treated with hormone for 7 min exhibited enhanced 32P uptake relative to vehicle controls. Cells transfected with either active siRNA revealed decreased 32P 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 PKCα (safingol) and PKCβ ([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 32P uptake in cells treated with 1,25(OH)2D3 plus blockers in comparison with cells treated with 1,25(OH)2D3 alone. Thus, PKCα and PKCβ are both involved in steroid-stimulated phosphate uptake.