Intestinal Cell Calcium Uptake and the Targeted Knockout Of the 1,25D3‐MARRS Receptor/PDIA3/Erp57

Abstract

We have crossed ERp57lfx/flx mice with mice expressing villin‐driven cre‐recombinase. Lysates of enterocytes from knockout (KO) mice and littermates (LM) were used in Western analyses with Ab099 against the N‐terminus of the 1,25D3‐MARRS receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [3H]1,25D3 revealed negligible binding in either female or male KOs. Lysates from female and male LM mice had similar affinities but different numbers of binding sites. Isolated enterocytes were tested for steroid stimulated calcium uptake. Treatment of cells from female or male LM mice with 1,25D3 elicited enhanced within 5 min. Intestinal cells from KO mice exhibited a severely blunted or completely absent response to hormone. Confocal microscopy of intestinal cells revealed the presence of cell‐surface VDR. However, antibodies to the VDR failed to block 1,25D3‐ stimulated calcium uptake. In chick enterocytes we have found that the PKA pathway mediates calcium uptake. The time course for activation of PKA in mouse enterocytes paralleled that for enhanced calcium uptake. Enterocytes from female or male KO mice failed to exhibit 1,25D3 stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D3‐MARRS receptor is of central importance to steroid hormone stimulated calcium uptake in mammalian intestinal cells.