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The Role of TRPV6 and Calbindin-D9k
in Calcium Homeostasis

Bryan S. Benn
MD/Ph.D. Program

B.A. 2003 Johns Hopkins University

Thesis Advisor: Sylva Christakos, Ph.D.
Biochemistry and Molecular Biology

Biochemistry and Molecular Biology


Tuesday, May 27, 2008
12:00 p.m.



To study the role of the epithelial calcium channel TRPV6 and the calcium binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knock out (KO), calbindin-D9k KO, and TRPV6/calbindin-D9k double knock out (DKO) mice were generated. Null mutation was verified by RT-PCR and Western blot analyses for intestinal and renal expression of TRPV6 and calbindin-D9k. When fed a standard rodent chow diet, TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild type (WT) mice (~10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8 fold increase in serum PTH levels (p<0.05 compared to WT). Levels of calbindin-D9k mRNA and protein in intestine were not significantly different in TRPV6 KO mice compared to WT nor were levels of intestinal TRPV6 mRNA significantly different in calbindin-D9k KO mice compared to WT.
Active intestinal calcium absorption was measured using the everted gut sac method and the first 5 cm of the duodenum of WT or null mutant mice. Under low dietary calcium conditions [mice were fed a low calcium (0.02%) diet from weaning for 4 weeks] there was a 4.1, 2.9, and 3.9 fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n=8-22/group; p>0.1 WT vs. calbindin-D9k KO and p<0.05 WT vs. TRPV6 KO on the low calcium diet). Duodenal calcium transport was increased 2.1 fold in the TRPV6/calbindin-D9k DKO mice fed the low calcium diet (p<0.05 WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or null mutant mice. In addition to low dietary calcium, 1,25(OH)2D3 administration (three injections of 1,25(OH)2D3 100ng/100g body weight; 48, 24 and 12h prior to sacrifice) to vitamin D deficient mice also resulted in a significant increase in duodenal calcium transport (1.4 V 2.0 fold, p<0.05 compared to vitamin D deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are needed for vitamin D induced active intestinal calcium transport.
The goal of the second part of this study was to examine the regulation of the human TRPV6 (hTRPV6) promoter by 1,25(OH)2D3, which is known to increase the rate of calcium entry into the intestinal cell. Recently the apical calcium channel TRPV6, which is co-localized with calbindin and is induced by 1,25(OH)2D3 in intestine, has been identified, suggesting a calcium entry mechanism. Previous studies have shown that vitamin D response elements (VDREs) located at -1.2, -2.1 and -4.3kb in the hTRPV6 promoter are required for the full transcriptional response to 1,25(OH)2D3 and that the VDREs at -2.1 and -4.3kb appear to be most significant for 1,25(OH)2D3 mediated transcriptional activation. C/EBP has been shown previously to enhance 1,25(OH)2D3 mediated 24(OH)ase promoter activation. To determine if the C/EBP isoforms LIP and LAP are involved in 1,25(OH)2D3 mediated hTRPV6 transcription, Caco-2 cells were transfected with the hTRPV6 promoter (-7000/+160) and increasing concentrations of LIP or LAP in the presence or absence of 10-8M 1,25(OH)2D3. Increasing concentrations of LIP, which has been reported to repress LAP induced transcription, was consistently found to result in a significant potentiation of 1,25(OH)2D3 induced hTRPV6 transcription (2-4 fold; p<0.05 compared to 1,25(OH)2D3 treatment alone; maximal 1,25(OH)2D3 induced transcription in the presence of LIP was 14-17 fold). LIP alone had no effect on hTRPV6 transcription. Increasing concentrations of LAP, C/EBP or C/EBP in the presence or absence of 1,25(OH)2D3 also had no effect on hTRPV6 transcription. In COS-7 cells LIP enhancement of VDR mediated hTRPV6 transcription was not observed, suggesting cell type specificity.
LIP enhancement of 1,25(OH)2D3 induced transcription was also observed using a hTRPV6 promoter construct with a mutation in the -2.1 VDRE but with a functional -4.3 VDRE. No effect of LIP was observed using a hTRPV6 promoter construct with the -4.3 and -2.1 VDREs mutated. Caco-2 cells were transfected with an ~200bp construct containing the -4.3 VDRE upstream of the minimal thymidine kinase (tk) promoter. Results of these experiments yielded a similar enhancement with LIP. Further, co-immunoprecipitation experiments indicated that VDR and LIP interact. ChIP/re-ChIP experiments indicated that VDR and LIP can bind simultaneously to the hTRPV6 promoter at the -4.3 VDRE. These findings suggest a novel function for LIP where LIP acts as a transcriptional activator in the 1,25(OH)2D3 mediated regulation of TRPV6.

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