Intestinal cell calcium uptake and the targeted knockout of the 1,25D3-MARRS (membrane-associated, rapid response steroid-binding) receptor/PDIA3/Erp57.
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.
Nemere
Ilka
I
Department of Nutrition, Utah State University, Logan, Utah 84322, USA. ilka.nemere@usu.edu
Garbi
Natalio
N
Hämmerling
Gunter J
GJ
Khanal
Ramesh C
RC
D000818:Animals; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005260:Female; D012739:Gonadal Steroid Hormones / Q000378:metabolism / Q000494:pharmacology; D007408:Intestinal Absorption / Q000502:physiology*; D007421:Intestine, Small / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D018345:Mice, Knockout; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D014815:Vitamins / Q000378:metabolism / Q000494:pharmacology
D012739:Gonadal Steroid Hormones; D014815:Vitamins; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; C513195:Pdia3 protein, mouse; D019704:Protein Disulfide-Isomerases; D002117:Calcitriol; D002118:Calcium