Abstract
We have crossed ERp57(flx/flx) mice with commercially available mice expressing villin-driven cre-recombinase. Lysates of intestinal epithelial cells were prepared from knock-out (KO) mice and littermates (LM) and used in Western blot analyses with Ab099 against the N terminus of the 1,25D(3)-MARRS (membrane-associated, rapid response steroid-binding) receptor: LM mice exhibited one positive band, which was absent in preparations from KO mice. Saturation analyses of cell lysates with [(3)H]1,25D(3) revealed negligible binding in preparations from 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 1,25D(3) elicited enhanced calcium uptake in females and males 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 vitamin D receptors. However, antibodies to the vitamin D receptor failed to block 1,25D(3)-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 and for LM females reached 250% of controls within 5 min, and 150% of controls in cells prepared from LM males. Enterocytes from female or male KO mice failed to exhibit steroid hormone-stimulated PKA activity, but did respond to forskolin with enhanced calcium uptake. We conclude that the 1,25D(3)-MARRS receptor is of central importance to steroid hormone-stimulated calcium uptake in mammalian intestinal cells.
Highlights
Genomic responses have been documented for other steroid hormones [8, 9]
In the studies described in this report, genetically engineered mice are used to produce a targeted knock-out of the 1,25D3MARRS receptor in intestinal epithelial cells and are tested, along with littermates, for their response to the steroid hormone 1,25(OH)2D3
The present work demonstrates that the targeted disruption of the 1,25D3-MARRS receptor gene in intestinal epithelial cells eliminates the rapid response to 1,25(OH)2D3 with respect to rapidly enhanced calcium uptake and PKA signaling
Summary
Genomic responses have been documented for other steroid hormones [8, 9]. It is well accepted that steroid hormones are capable of acting through membrane-localized receptors to initiate pre-genomic effects as well as through regulation of gene expression [9]. For protein kinase A activity determination, isolated intestinal epithelial cells were resuspended in GBSS, incubated at 23 °C for 10 min, and a 100-l basal aliquot was removed at t ϭ Ϫ1 min. Calcium Uptake in Female Mice—Intestinal epithelial cells isolated from female LM mice responded to 300 pM 1,25(OH)2D3 with a significant increase in calcium uptake within 1 min, reaching 175% of corresponding controls by 5 min of incubation with steroid (Fig. 6).
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