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Calbindin (CaBP), the vitamin D-dependent calcium-binding protein, is believed to play an important role in intracellular calcium transport. The aim of this study was to investigate the effect of high dietary calcium on the expression of CaBP-D9k and CaBP-D28k in the presence and absence of a functional vitamin D receptor (VDR). Treatment with the HCa-Lac diet containing 2% calcium, 1.5% phosphorus and 20% lactose reversed the hypocalcemia seen in adult VDR-null mice in 3 weeks but did not significantly change the blood ionized calcium in wild-type mice. This dietary treatment dramatically suppressed both the duodenal and the renal CaBP-D9k expression in wild-type mice at both mRNA and protein levels but had little effect on the expression of the same gene in VDR-null mice. Removal of this diet gradually restored the expression of CaBP-D9k to the untreated level in wild-type mice. Only moderate or little change in CaBP-D28k expression was seen in wild-type and VDR-null mice fed with the HCa-Lac diet. The VDR content in the duodenum or kidney of wild-type mice was not altered by the dietary treatment. These results suggest that calcium regulates CaBP-D9k expression by modulating the circulating 1,25-dihydrxyvitamin D(3) level and that VDR is thus required for the dietary calcium-induced suppression of CaBP-D9k expression. Calcium regulation of the CaBP-D9k level may represent an important mechanism by which animals maintain their calcium balance.

As the fourth most abundant anion in the body, sulfate plays an essential role in numerous physiological processes. One key protein involved in transcellular transport of sulfate is the sodium-sulfate cotransporter NaSi-1, and previous studies suggest that vitamin D modulates sulfate homeostasis by regulating NaSi-1 expression. In the present study, we found that, in mice lacking the vitamin D receptor (VDR), NaSi-1 expression in the kidney was reduced by 72% but intestinal NaSi-1 levels remained unchanged. In connection with these findings, urinary sulfate excretion was increased by 42% whereas serum sulfate concentration was reduced by 50% in VDR knockout mice. Moreover, levels of hepatic glutathione and skeletal sulfated proteoglycans were also reduced by 18 and 45%, respectively, in the mutant mice. Similar results were observed in VDR knockout mice after their blood ionized calcium levels and rachitic bone phenotype were normalized by dietary means, indicating that vitamin D regulation of NaSi-1 expression and sulfate metabolism is independent of its role in calcium metabolism. Treatment of wild-type mice with 1,25-dihydroxyvitamin D3 or vitamin D analog markedly stimulated renal NaSi-1 mRNA expression. These data provide strong in vivo evidence that vitamin D plays a critical role in sulfate homeostasis. However, the observation that serum sulfate and skeletal proteoglycan levels in normocalcemic VDR knockout mice remained low in the absence of rickets and osteomalacia suggests that the contribution of sulfate deficiency to development of rickets and osteomalacia is minimal.

Calbindin (CaBP)-D9k is a major vitamin D target gene involved in calcium homeostasis. However, studies on the molecular mechanisms of CaBP-D9k gene regulation have been hampered by the lack of an appropriate cell culture system. In the present study, we used mouse primary renal tubular cell (PRTC) cultures to investigate the regulation of CaBP-D9k expression by 1,25(OH)(2)D(3). Both CaBP-D9k mRNA and protein were highly induced by 1,25(OH)(2)D(3) in a time- and dose-dependent manner in PRTCs, and new RNA and protein synthesis was required for the induction. Transfection of VDR(-/-) cells derived from VDR null mice with human VDR restored the induction of CaBP-D9k expression by 1,25(OH)(2)D(3), confirming the requirement of VDR for CaBP-D9k expression. Treatment of the PRTCs with 1,25(OH)(2)D(3) also increased VDR protein abundance, suggesting that enhanced VDR transactivation is involved in the CaBP-D9k up-regulation. Moreover, PTH had a synergistic effect on the 1,25(OH)(2)D(3) induction of CaBP-D9k. These data demonstrate that CaBP-D9k is highly regulated by 1,25(OH)(2)D(3) and PTH in mouse PRTCs, which provides a suitable in vitro system for further investigating the molecular mechanisms involved in CaBP-D9k gene regulation.