5 cells to produce calcitriol,

we tested the expression level of the gene CYP27B1 encoding for 1α-hydroxylase, the enzyme responsible for the synthesis of calcitriol. Real-time RT-PCR analysis showed that these cells express CYP27B1, the level of which increased following 24 hours incubation with vitamin D3 (Fig. 2A). Calcitriol binds to VDR to induce the expression of the first enzyme in the LY2606368 price pathway leading to its catabolism, 24-hydroxylase, in most of its target cells. In fact, induction of this enzyme serves as an indicator of the transcriptional activation of VDR. Therefore, we tested the expression level of the VDR-regulated gene CYP24A1, encoding for 24-hydroxylase, in response to vitamin D by real-time RT-PCR. Treatment with vitamin D3 (5 μM) markedly up-regulated the CYP24A1 expression level (Fig. 2B). These data demonstrate that treatment with vitamin D3 leads to transactivation of VDR, presumably as a consequence of its conversion to the VDR ligand, calcitriol. Furthermore, the addition of vitamin D3 up-regulated the mRNA level of VDR (Fig. 2C), which may increase the responsiveness to calcitriol in these cells. Next we directly examined the potential DAPT mw of Huh7.5 cells to convert vitamin D into its active form, calcitriol. The cells were treated with vitamin D3 (5 μM) and the level of calcitriol in the supernatant at 5 and 24 hours posttreatment was measured by specific ELISA. The

results presented in Aldehyde dehydrogenase Fig. 2D demonstrate that Huh7.5 cells convert vitamin D3 into calcitriol. Production of calcitriol was detected as early as 5 hours after treatment and was markedly higher at 24 hours posttreatment. Treatment for 48 hours did not further increase

calcitriol production (data not shown). Thus, calcitriol can be constitutively produced in these cells. Taken together, these results indicate that the hepatoma Huh7.5 cell system contains: the complete enzymatic machinery needed for the conversion of the parent compound, vitamin D, to its hormonal metabolite, a functional vitamin D response system, and the enzyme responsible for the first step of calcitriol catabolism. Next we examined whether infection with HCV affects vitamin D metabolism in Huh7.5 cells. First, we evaluated the expression level of CYP27B1 and CYP24A1 genes in vitamin D3-treated cells in response to HCV infection. Infection with the virus did not significantly affect the expression of CYP27B1 (Fig. 3A), while significantly reducing CYP24A1 expression (Fig. 3B). These findings may suggest that vitamin D conversion to calcitriol may not be enhanced by HCV but rather calcitriol catabolism may be decreased, which may result in higher levels of calciriol in the infected cells. This was confirmed by comparing the levels of calcitriol in the supernatant of infected and uninfected Huh7.5 cells after treatment with vitamin D3 (5 μM) as measured by ELISA. The level of calcitriol was significantly higher in the supernatant of infected cells (Fig. 3C).