1,25–Dihydroxyvitamin D and 25–hydroxyvitamin D—mediated regulation of TRPV6 (a putative epithelial calcium channel) mRNA expression in Caco–2 cells
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TRPV6 is a member of the vanilloid subfamily of transient receptor potential (TRP) proteins and likely functions as an epithelial calcium channel in calcium–transporting organs, such as the intestine, kidney, and placenta. TRPV6 mRNA expression is strongly regulated by 1,25–dihydroxyvitamin D (1,25VD), the active hormonal form of vitamin D, in intestine and in Caco–2 cells, a human colon cancer cell line.
Aim of the study
The aim of the present study was to characterise the mode of regulation of the 1,25VD–mediated TRPV6 mRNA expression and to test the effect of the precursor of 1,25VD namely 25 hydroxyvitamin D (25 VD) on TRPV6 mRNA expression in Caco–2 cells.
Caco–2 cells were treated in a 2 x 2 format with 1,25VD and the transcriptional inhibitor actinomycin D (AD, 4μg/ml), and also with translational inhibitor cycloheximide (CHX, 10 μg/ml) after 14 days in culture and TRPV6 mRNA levels were determined using reverse transcription–real time PCR.TRPV6 mRNA half life studies were performed by inhibiting transcription followed by sampling at various time points for TRPV6 mRNA. Varying concentrations of 25 VD were used to test their effect on TRPV6 mRNA in the presence of 5% FBS and also in the absence of serum (but containing insulin–transferrin–selenium mixture) for 24 h.
Treatment with 10–7 M 1,25VD for 8 h resulted in a 60–fold increase in TRPV6 mRNA and this increase could be completely blocked with AD. Treatment with CHX to inhibit de novo protein synthesis did not prevent the initiation of 1,25VD–induced TRPV6 expression, although it did reduce the extent of TRPV6 mRNA accumulation. We found that TRPV6 mRNA half–life was 8 h in Caco–2 cells and was not altered by 1,25VD treatment. Finally, we observed that treatment with 10–6 M of the pro–hormone 25 VD for 24 h resulted in a significant increase in TRPV6 expression in Caco–2 cells, which is consistent with the presence of 1α–hydroxylase (CYP27B1) expression in Caco–2 cells and a possible autocrine vitamin D signaling pathway in colon cells.
1,25 dihydroxyvitamin D regulates TRPV6 expression by a process that requires new mRNA and protein synthesis and the point of regulation lies likely at the transcriptional level especially since vitamin D did not increase the half life of TRPV6 mRNA. In addition, the prohormone form of 1,25 dihydroxyvitamin D, i. e. the 25 hydroxyvitamin D, induced TRPV6 mRNA expression in Caco–2 cells.
Key wordsCaT1 TRPV6 gene expression vitamin D intestine
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