To study this, we took advantage of the finding that the cell surface expression levels of CD38 on activated T cells are dependent on the concen tration of 1,25 2D3 present during T cell activation. Consequently, we activated CD4 T cells in serum free medium in the presence MG132 133407-82-6 of increasing con centrations of 25 D3 or 1,25 2D3 and then mea sured CD38 expression at day 3. In accordance with previous studies, we found that 1,25 2D3 signifi cantly up regulated CD38 expression in activated T cells pression in T cells cultured in medium supplemented with serum. In contrast, the presence of serum did not signifi cantly shift the concentration of 1,25 2D3 required to induce CD38 expression.

To separately analyse the role of DBP and albumin in this serum mediated inhibition of the effect of 25 D3 on T cells, we activated CD4 T cells in the presence of 100 nM 25 D3 in serum free medium supplemented with increasing concentrations of either DBP or albumin and Inhibitors,Modulators,Libraries measured CD38 expression Inhibitors,Modulators,Libraries at day 3. We found that DBP at concentrations above 250 500 nM completely abolished the effect of 25 D3 on CD38 expression, whereas albumin did not affect the ef fect of 25 D3. Although T cells converted 25 D3 to 1,25 2D3 when activated in serum free medium, we could not exclude the possibility that the observed 25 D3 induced up regulation of CD38 was caused directly by 25 D3 and not by 1,25 2D3. To exclude this possibility, we determined the effect of the CYP27B1 inhibitor ketoconazole on CD38 up regulation. We ac tivated T cells in the absence or presence of ketocona zole and increasing concentrations of 25 D3.

We found that ketoconazole efficiently inhibited 25 D3 in duced Inhibitors,Modulators,Libraries CD38 up regulation. These observations Inhibitors,Modulators,Libraries indicated that conversion is required for 25 D3 induced CD38 up regulation. If this is the Inhibitors,Modulators,Libraries case and ketoconazole does not in itself influ ence CD38 expression then ketoconazole should not affect 1,25 2D3 induced CD38 up regulation. Consequently, we activated T cells in the absence or presence of ketoco nazole and increasing concentrations of 1,25 2D3. We found that ketoconazole did not affect 1,25 2D3 in duced CD38 up regulation. These experiments indicated that it is not 25 D3 itself that induces CD38 up regulation, but that the conversion of 25 D3 to 1,25 2D3 is required for Thus, we could conclude that activated T cells have the capacity to produce sufficient 1,25 2D3 to affect vitamin D responsive genes as measured by CD38 expres sion when cultured with physiological concentrations of selleck DAPT secretase 25 D3 in serum free medium. However, addition of DBP to the medium inhibited the effect of 25 D3. In contrast, DBP did not seem to affect the concentration of 1,25 2D3 required to regulate vitamin D responsive genes.