The 1,25D3-MARRS protein: contribution to steroid stimulated calcium uptake in chicks and rats

There are currently two main candidates for the membrane receptor for 1,25(OH)2D3: the 1,25D3-MARRS protein/ERp57; and the classical VDR. The 1,25D3-MARRS protein is essential for hormone-stimulated phosphate and calcium uptake in chick intestinal cells, whereas the VDR is not. The 1,25D3-MARRS protein also shows a high degree of correlation with growth periods in which bone is rapidly formed, whereas the VDR does not. However, in rat enterocytes, both the 1,25D3-MARRS protein and the VDR play a role in the rapid, steroid-mediated uptake of phosphate or calcium. Therefore, the theory that alternate binding sites on the VDR for various analogs account for all membrane-initiated phenomena, is incorrect.     

Phosphate uptake in chick kidney cells: effects of 1,25(OH)2D3 and 24,25(OH)2D3

Phosphate homeostasis is controlled in part by absorption from the intestine, and reabsorption in the kidney. While the effect of Vitamin D metabolites on enterocytes is well documented, in the current study we assess selected responses in primary cultures of kidney cells. Time course studies revealed a rapid stimulation of phosphate uptake in cells treated with 1,25(OH)(2)D(3), relative to controls. Dose-response studies indicated a biphasic curve with optimal stimulation at 300 pM 1,25(OH)(2)D(3) and inhibition at 600 pM seco-steroid. Antibody 099--against the 1,25D(3)-MARRS receptor - abolished stimulation by the steroid hormone. Moreover, phosphate uptake was mediated by the protein kinase C pathway. The metabolite 24,25(OH)(2)D(3), which was found to inhibit the rapid stimulation of phosphate uptake in intestinal cells, had a parallel effect in cultured kidney cells. Finally, the 24,25(OH)(2)D(3) binding protein, catalase, was assessed for longer term down regulation. In both intestinal epithelial cells and kidney cells incubated with 24,25(OH)(2)D(3) for 5-24h, both the specific activity of the enzyme and protein levels were decreased relative to controls, while 1,25(OH)(2)D(3) increased both parameters over the same time periods. We conclude that the Vitamin D metabolites have similar effects in both kidney and intestine, and that 24,25(OH)(2)D(3) may have effects at the level of gene expression.     

Involvement of 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH)₂D₃ traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH)₂D₃ called 1,25D₃-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D₃-MARRS expression modulates 1,25(OH)₂D₃ activity in breast cancer cells.Relative levels of 1,25D₃-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D₃-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH)₂D₃ in MCF-7 cells, a ribozyme construct designed to knock down 1,25D₃-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D₃-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH)₂D₃ ( IC₅₀ 56 ± 24 nM) compared to controls (319 ± 181 nM; P < 0.05). Reduction in 1,25D₃-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH)₂D₃. Knockdown of 1,25D₃-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D₃-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH)₂D₃ in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D₃-MARRS expression or activity as anticancer agents.     

Differential effects of 1,25-dihydroxyvitamin D3 upon intestinal vitamin D3-dependent calbindin (a 28,000-dalton calcium binding protein) and its mRNA in D-replete and D-deficient chickens

The effect of vitamin D3 status upon the responsiveness of chick intestinal epithelium to exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was studied. Intestinal calbindin [A recent consensus decision was made to redesignate the vitamin D-dependent calcium binding protein as "calbindin-D28K" (R.H. Wasserman (1985) in Vitamin D: Chemical, Biochemical, and Clinical Update (Norman, A.W., Schaefer, K., Grigoleit, H.-G., and Herrath, D.V., Eds.), pp. 321-322, de Gruyter, Berlin/New York).] protein and intestinal calbindin mRNA were quantitated in birds which had been raised on a vitamin D3-deplete (-D) or on a vitamin D3-replete (+D) diet. 1,25(OH)2D3 stimulated intestinal calbindin mRNA levels in -D chickens in a proportional dose-dependent manner, when measured at both 12 and 48 h after administration of the hormone. A first increase was observed with 1,25(OH)2D3 concentrations between 0.065 and 0.65 nmol. The maximal stimulation achieved by 1,25(OH)2D3 (6.5-18 nmol) in -D tissue was approximately 10-fold over the calbindin mRNA levels present in vehicle-treated birds. The increase of calbindin mRNA in -D birds was associated with a similar dose-dependent increase in calbindin protein in 1,25(OH)2D3-treated -D birds after 12 or 48 h. In +D intestine, while exogenous 1,25(OH)2D3 also increased calbindin mRNA levels in a dose-dependent fashion, the maximal stimulation observed after 5 h (1.2- to 2-fold) was clearly less than that observed in -D intestine. In contrast to -D birds, intestinal calbindin levels in +D birds were decreased by administration of exogenous 1,25(OH)2D3. Administration of 32.5 to 65 nmol 1,25(OH)2D3 resulted in an approximately 1.8-fold repression compared to vehicle-treated birds. This differential responsiveness between +D and -D intestines with respect to 1,25(OH)2D3 was not explained either by differences in the uptake in the chromatin fractions of these tissues or by metabolism of radiolabeled 1,25(OH)2D3. Dietary withdrawal of vitamin D3 led to a gradual decline in ambient intestinal calbindin levels, while intestinal sensitivity to 1,25(OH)2D3 was restored. These findings suggest that vitamin D3 status regulates intestinal responsiveness to the seco-steroid 1,25(OH)2D3.     

Monoclonal antibodies to the porcine intestinal receptor for 1,25-dihydroxyvitamin D3: interaction with distinct receptor domains

Monoclonal antibodies to different domains of the porcine intestinal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor have been produced. A nuclear extract enriched in the 1,25-(OH)2D3 receptor was prepared from small intestinal mucosa of young pigs. The receptor was purified an additional 6600-fold by chromatography on DNA-cellulose, ammonium sulfate precipitation, gel filtration high-performance liquid chromatography, and DEAE-Sepharose chromatography, with an overall yield of 23% and an average purity of 24%. A BALB/c mouse immunized with this material developed serum polyclonal antibodies to the 1,25-(OH)2D3 receptor, as demonstrated by a change in sedimentation of the porcine receptor on sucrose gradients. Spleen cells from this animal were fused with mouse myeloma cells (P3-NSI/1-Ag4-1, SP2/0-Ag14), and 24 hybridomas secreting antibodies to the 1,25-(OH)2D3 receptor were identified by both a radiometric immunosorbent assay and an immunoprecipitation assay. Twenty-one hybridoma lines were cloned by limiting dilution and further characterized as subclass IgG1 antibodies with the exception of one which is an IgA. All but two of the antibodies cross-react with the 1,25-(OH)2D3 receptor from both mammalian (human, monkey, and rat) and avian (chicken) intestine; two antibodies recognize only porcine intestinal receptor. All antibodies are unreactive to the vitamin D serum transport protein. Eight of the antibodies bind denatured receptor on an immunoblot. A solid-phase competition assay was used to identify four groups of antibodies that bind to distinct epitopes on the 1,25-(OH)2D3 receptor. One antibody from each of the four groups was used to examine the effect of antibody binding on the DNA-binding activity of the receptor-hormone complex. One antibody completely inhibited the binding of the 1,25-(OH)2D3 receptor complex to DNA-cellulose, suggesting that the epitope for this antibody may be located in the polynucleotide binding domain of the protein. Antibodies from two additional groups only slightly perturbed DNA binding, while one had no effect, suggesting that these antibodies bind to receptor epitopes distant from the region of the polypeptide directly involved in polynucleotide binding. These antibodies that are directed to several different binding sites on the 1,25-(OH)2D3 receptor provide important new tools to probe the biochemistry and topology of the 1,25-(OH)2D3 receptor and to investigate its role in mediating target tissue response to hormone.     

Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D3-II: calcium transport, receptor kinetics, and signal transduction in intestine of female chickens

We recently reported (Larsson and Nemere [2003]: Endocrinology 144:1726) the effects of growth and maturation on 1,25(OH)2D3-membrane initiated effects in the intestine of male chickens. Here we extend our observations to studies on females with two stages of high calcium demand: growth (7-14 weeks) and egg laying (28-58 weeks). The rapid stimulatory effect of 130 pM 1,25(OH)2D3 on calcium transport was assessed as a physiological response in perfused duodena of 7-, 14-, 28-, and 58-week-old chickens, and determined to be 308%, 184%, 170%, and 153%, respectively, of corresponding controls after 40 min. Saturation analyses of [3H]1,25(OH)2D3 binding to nuclear vitamin D receptor (VDR) indicated an absence of cooperative binding, no changes in dissociation constant (Kd) with age, and an increase in maximum binding capacity (Bmax) between 7-week birds and older age groups. Analyses of saturable binding of [3H]1,25(OH)2D3 to the membrane associated rapid response steroid binding protein (1,25D3-MARRS bp) in basal lateral membranes (BLM), indicated cooperative binding, and an increase in both Bmax and Kd with age. No changes in the age-related expression of 1,25D3-MARRS bp were found, as judged by Western analyses, suggesting that a shift in ligand binding to lower affinity membrane components accounted for the increase in calculated Bmax. Basal levels of protein kinase C (PKC) activity decreased with age, as did hormone enhancement of activity. Basal levels of protein kinase A (PKA) activity remained constant with age, while the magnitude of hormone stimulation increased. Comparison of dose-response curves for ion transport and kinase activities in 7-week chicks suggested that PKC mediates phosphate transport while PKA mediates calcium transport. Thus, the age-related loss of calcium transport is most likely related to loss of PKC-mediated phosphate transport.     

Role of the 1,25D3-MARRS receptor in the 1,25(OH)2D3-stimulated uptake of calcium and phosphate in intestinal cells

We have used mice with a targeted knockout (KO) of the 1,25D(3)-MARRS receptor (ERp57/PDIA3) in intestine to study rapid responses to 1,25-dihydroxyvitamin D(3) [1,25D(3)] with regards to calcium or phosphate uptake. Western analyses indicated the presence of the 1,25D(3)-MARRS receptor in littermate (LM) mice, but not KO mice. Saturation analyses for [(3)H]1,25D(3) binding revealed comparable affinities for the hormone in lysates from female and male LM, but a reduced B(max) in females. Binding in lysates from KO mice was absent or severely reduced. Enterocytes from KO mice failed to respond to hormone with regard to either ion uptake, while cells from LM mice exhibited an increase in uptake. For calcium uptake, the protein kinase (PK) A pathway mediated the response to 1,25D(3). Enterocytes from LM mice responded to 1,25D(3) with enhanced PKA activity, while cells from KO mice did not, although both cell types responded to forskolin. Calcium transport in LM mice in vivo was greater than in KO mice. Cells from LM and KO mice had cell surface VDR; however, anti-VDR antibodies had no effect on ion uptake. Unlike chicks, the PKC pathway was not involved in phosphate uptake. As in chicks and rats, intestinal cells from adult male mice lost the ability to respond to 1,25D(3) with enhanced phosphate uptake, whereas in female mice, uptake in cells from adults was greater than that observed in young mice. Finally, when we tested phosphate uptake in vivo, we found that young female mice had a much greater rate of transport than young male mice.     

Analysis of the mRNA coding for the chick vitamin D-induced calbindin and its regulation by 1,25-dihydroxyvitamin D3

We have used specific cloned cDNA probes generated from the mRNA coding for the vitamin D-induced 28,000-Da chick intestinal calcium binding protein (calbindin) to study the hormonal regulation of the expression of this mRNA by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The calbindin-mRNA has been analyzed in chicken intestinal poly(A)+ mRNA samples as well as other chicken tissues by "Northern" blot analysis. There exists a predominant mRNA species of approximately 2000 nucleotides and two minor cross-hybridizing species that are nearly equivalent in proportion; their sizes are approximately 2600 and 3100 nucleotides. All three mRNA species are nonexistent in the chick intestine in the absence of vitamin D3 intake. However, all three mRNA species begin to accumulate at the same time in the chick intestine following the administration of the hormonally active metabolite of vitamin D3, 1,25-(OH)2D3. This response in the intestine is very similar to other steroid hormone-regulated gene products. All three mRNA species exist in the cell cytoplasm and are present on soluble polysome complexes, suggesting that all three are engaged in protein synthesis. Examination of other chick tissues (both vitamin D-deficient and -replete) reveals a close association between mRNA expression and previously observed calbindin expression. Each tissue is unique in the steady-state level of expression of the calbindin-mRNAs.     

Contributions of pro-oxidant and anti-oxidant conditions to the actions of 24,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on phosphate uptake in intestinal cells

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against the 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in the presence of the catalase inhibitor 3-amino-1,2,4-triazole, or with 1,25(OH)2D3 alone. Cells exposed to hormone alone again showed an increased accumulation of 32P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for 32P uptake, occurred. Incubation of cells with phorbol-13-myristate (PMA) increased 32P uptake, while cells pretreated with 50 microM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.     

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

C-terminal proteolysis of the avian 1,25-dihydroxyvitamin D3 receptor

Exposure of the 60 kDa chick intestinal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) receptor to carboxypeptidase A resulted in a time dependent decrease in receptor hormone-binding; after 2 h, there was no detectable macro-molecular-bound 1,25(OH)2[3H]D3. Upon DNA-cellulose chromatography of this preparation, a 56 kDa protein adsorbed to the column and eluted as a function of para-chloromercuribenzene sulfonate (a sulfhydryl blocking reagent). The 56 kDa fragment was detected by anti-receptor monoclonal antibodies via immunoblot technology. The 1,25(OH)2[3H]D3 eluted in the fall through fractions of the column. Thus, cleavage of up to 40 amino acids from the carboxy-terminus of the 1,25(OH)2D3 receptor results in a protein which no longer binds to hormone, but retains its capacity to interact with DNA-cellulose and monoclonal antibody. These results represent novel biochemical evidence that allows us to orient the 1,25(OH)2D3 binding domain near the C-terminus of the receptor.     

Computer analysis of 1,25-dihydroxyvitamin D3-receptor regulated promoters: identification of a candidate D3-response element

The receptor for the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] belongs to a superfamily of steroid and triidothyronine (T3) receptors. As yet no 1,25(OH)2D3 response element (DRE) has been identified. Since the T3 and 1,25(OH)2D3 receptors are structurally homologous, we have used the nucleotide sequence of the T3 response element to carry out a computer search of the promoter region of several 1,25(OH)2D3 regulated genes. We report here one candidate DRE from the chick calbindin-D28K gene (AGCCCAATGGCTGAACA) and two candidate DRE's from the rat osteocalcin gene (TCCCCACTGGATGAGCG and CCTGCACTGGGTGAATGA).