1,25-Dihydroxyvitamin D3 up-regulates the renal vitamin D receptor through indirect gene activation and receptor stabilization

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Abstract

Expression of the vitamin D receptor (VDR) in the kidney and intestine plays a major role in calcium homeostasis and the metabolism of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Calcium and 1,25(OH)2D3-mediated regulation of renal and duodenal VDR expression has been analyzed in vivo and the mechanisms responsible for the renal regulation have been studied in mouse kidney TCMK-1 cells. Vitamin D-deficient mice were maintained on diets containing either 0.02 or 0.47% calcium, with or without 50 ng of 1,25(OH)2D3 per day. Renal VDR levels were significantly higher in the vitamin D-deficient mice fed the 0.47% calcium diet vs. the calcium-restricted diet, and were increased 5-fold by 1,25(OH)2D3 when dietary calcium was present. The renal VDR transcript was expressed at a basal level in the absence of calcium or 1,25(OH)2D3; 50 ng of 1,25(OH)2D3 elevated renal VDR mRNA levels approximately 10-fold in the presence of calcium. Neither calcium nor 1,25(OH)2D3 had any significant effect on duodenal VDR or VDR mRNA expression. In TCMK-1 cells, 1,25(OH)2D3 increased receptor and VDR mRNA content in both low and adequate calcium medium. The 1,25(OH)2D3-mediated increase in VDR mRNA did not result from increased stability of the transcript. Further, the increase in mRNA was blocked by cycloheximide, indicating a requirement for protein synthesis and an indirect regulation of VDR transcription. Thus, both dietary serum calcium and 1,25-(OH)2D3 are required for VDR expression in kidney but not in intestine where neither is required. The 1,25-(OH)2D3 requirement can also be shown in TCMK-1 cells in vitro, while the calcium requirement was not found.

Section snippets

Chemicals

1,25(OH)2D3 was purchased from Tetrionics (Madison, WI). Actinomycin D and cycloheximide were purchased from Sigma (St. Louis, MO).

Mice and diets

Breeding pairs of B6/CBA mice (The Jackson Laboratory) were maintained on chow LabDiet 5015 (PMI Nutrition International, St. Louis). Vitamin D-deficiency was generated by transferring visibly pregnant females into housing in which all fluorescent lighting was shielded, preventing the endogenous production of vitamin D3. These females were fed a 1.20% calcium diet

Dietary calcium and 1,25(OH)2D3 elevate renal VDR and VDR mRNA but have minimal impact on intestinal VDR expression

To study the effect of calcium and 1,25(OH)2D3 on renal and duodenal VDR expression, vitamin D-deficient mice were weaned onto purified diets that contained 0.02 or 0.47% calcium, with or without 50 ng of 1,25(OH)2D3 per day. As shown in Table 1, mice maintained on the 0.02% calcium diets suffered from severe hypocalcemia, even with 50 ng of 1,25(OH)2D3 per day in the diet. Vitamin D-deficient mice maintained on the 0.47% calcium diet were hypocalcemic, whereas mice on the 0.47% calcium diet

Discussion

We have analyzed the regulation of VDR expression by calcium and 1,25(OH)2D3 in mouse kidney and duodenum, and utilized a cell culture model to further our understanding of the basic mechanisms responsible for aspects of renal VDR regulation. Mice fed 50 ng of 1,25(OH)2D3 per day and 0.47% calcium had 5-fold higher levels of renal VDR than mice fed a calcium-restricted diet either with or without 50 ng of 1,25(OH)2D3. Initial reports of VDR up-regulation by 1,25(OH)2D3 were attributed to

Acknowledgments

We thank Jean Prahl for her assistance with the ELISA assays, Eric Danielson for his aid in genotyping, and Wendy Hellwig for her help in the quantification of serum calcium.

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    This work was supported by the Wisconsin Alumni Research Foundation.

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