Intestinal resistance to 1,25 dihydroxyvitamin D in mice heterozygous for the vitamin D receptor knockout allele

We tested the hypothesis that low vitamin D receptor (VDR) level causes intestinal vitamin D resistance and intestinal calcium (Ca) malabsorption. To do so, we examined vitamin D regulated duodenal Ca absorption and gene expression [transient receptor potential channel, vallinoid subfamily member 6 (TRPV6), 24-hydroxylase, calbindin D(9k) (CaBP) mRNA, and CaBP protein] in wild-type mice and mice with reduced tissue VDR levels [i.e. heterozygotes for the VDR gene knockout (HT)]. Induction of 24-hydroxylase mRNA levels by 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] injection was significantly reduced in the duodenum and kidney of HT mice in both time-course and dose-response experiments. TRPV6 and CaBP mRNA levels in duodenum were significantly induced after 1,25(OH)(2) D(3) injection, but there was no difference in response between wild-type and HT mice. Feeding a low-calcium diet for 1 wk increased plasma PTH, renal 1alpha-hydroxylase (CYP27B1) mRNA level, and plasma 1,25(OH)(2) D(3), and this response was greater in HT mice (by 88, 55, and 37% higher, respectively). In contrast, duodenal TRPV6 and CaBP mRNA were not higher in HT mice fed the low-calcium diet. However, the response of duodenal Ca absorption and CaBP protein to increasing 1,25(OH)(2) D(3) levels was blunted by 40% in HT mice. Our data show that low VDR levels lead to resistance of intestinal Ca absorption to 1,25(OH)(2) D(3), and this resistance may be due to a role for the VDR (and VDR level) in the translation of CaBP.     

Identification of sequence elements in mouse calbindin-D28k gene that confer 1,25-dihydroxyvitamin D3- and butyrate-inducible responses.

We have examined the 5' flanking region of the mouse calbindin-D28k gene and identified a 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-responsive element by deletion mutant analysis of the native promoter as well as by studies with a heterologous thymidine kinase (TK) promoter. The segment between residues -200 and -169 was found to confer a dose-dependent 1,25-(OH)2D3 responsiveness through the TK promoter in Ros 17/2.8 cells as well as in CV-1 cells cotransfected with pAV-hVDR (human vitamin D receptor expression vector). This region contains sequences homologous to the rat osteocalcin vitamin D response element (VDRE). Incubation of this element with nuclear extracts from 1,25-(OH)2D3-treated Ros 17/2.8 cells or from 1,25-(OH)2D3-treated COS cells that had been transfected with pAV-hVDR resulted in a specific protein-DNA interaction. In addition to 1,25-(OH)2D3, sodium butyrate, a differentiating agent, has also been found to modulate expression of calbindin-D28k. Deletion analysis of the mouse calbindin-D28k promoter as well as studies with a heterologous TK promoter resulted in identification of a butyrate-responsive element between -180 and -150 that was found to bind specifically to nuclear factors from butyrate-treated Ros 17/2.8 cells. This butyrate-responsive element may represent a genetic element acted upon by enhancer binding proteins. In summary, the 5' flanking region of the mouse calbindin-D28k gene contains responsive elements that interact with nuclear factors and may mediate, at least in part, the enhanced expression of this gene by 1,25-(OH)2D3 and butyrate.     

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.     

Calbindin-D28k (CaBP28k) identification and regulation by 1,25-dihydroxyvitamin D3 in human choriocarcinoma cell line JEG-3

Calbindin-D28k (CaBP28k) is a cytosolic calcium (Ca2+)-binding protein expressed in tissues such as intestine, kidneys and placenta. This protein is thought to be involved in Ca2+ homeostasis. While it is well known that CaBP28k is influenced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the intestine and kidneys, nothing is known regarding the regulation of this protein in trophoblasts of human placenta. We used JEG-3 syncytiotrophoblast-like carcinoma cell line to study the regulation of CaBP28k in correlation with 1,25(OH)2D3 receptor (VDR) following 1,25(OH)2D3 treatments. Our data demonstrated for the first time that both CaBP28k mRNA and protein were highly induced by the addition of 1,25(OH)2D3 in dose-dependent manner. Moreover, the increase and subsequent decrease in the expression of CaBP28k and VDR mRNAs indicates the transient nature of the changes in gene expression in response to 1,25(OH)2D3. This is in contrast with the temporal pattern of increasing protein for CaBP28k and VDR. We also showed that new RNA and protein syntheses are required for 1,25(OH)2D3-induced upregulation of CaBP28k. Furthermore, a 25-carboxylic ester analogue of 1,25(OH)2D3, ZK159222, used as an antagonist of 1,25(OH)2D3 signaling confirmed that indeed 1,25(OH)2D3 was implicated in the induction of CaBP28k. These novel findings are a contribution to the processes that drive CaBP28k expression regulation in human placenta.     

Identification and characterization of a response element in the EGFR promoter that mediates transcriptional repression by 1,25-dihydroxyvitamin D3 in breast cancer cells

Reduction of epidermal growth factor receptor (EGFR) mRNA and protein by 1,25-dihydroxyvitamin D3 has been documented in MCF7, T47D, and BT549 breast cancer cells. In the present report, functional mapping of the EGFR promoter in BT549 cells has revealed a sequence of DNA between nucleotide positions -536 and -478 that resembles a consensus vitamin D response element (VDRE) and confers a vitamin D response upon both the homologous and a minimal heterologous promoter. In vitro footprinting and gel shift assays demonstrate the presence of an unidentified nuclear factor that is required for strong binding of the vitamin D receptor (VDR) to this putative VDRE. An Sp1 binding site was also identified in close proximity and shown to bind Sp1 from nuclear extract. Mutational analysis and functional studies using a minimal heterologous promoter provide evidence that the VDR in concert with an unknown nuclear partner mediates basal EGFR repression through displacement of Sp1 which is augmented in the presence of a ligand.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

A nuclear protein essential for binding of rat 1,25-dihydroxyvitamin D3 receptor to its response elements.

Recombinant 1,25-dihydroxyvitamin D3 receptor from a baculovirus expression system requires a mammalian-derived nuclear accessory protein for binding to a vitamin D response element (DRE). This was established by electrophoretic mobility shift analyses using radiolabeled DNA probes consisting of DREs from two vitamin D-responsive genes. Mammalian nuclear extract was also required for the binding of wild-type porcine vitamin D receptor to a DRE. Surprisingly, the accessory factor-dependent formation of receptor-DRE complex was independent of exogenous 1,25-dihydroxyvitamin D3. A 59- to 64-kDa accessory protein from porcine intestinal nuclear extract was identified by size-exclusion chromatography. Nuclear extracts from rat liver and kidney contained accessory factor, whereas smaller amounts were detected in heart muscle. Spleen and skeletal muscle contained no detectable accessory factor.