Expression of 25-Hydroxyvitamin D3-24-Hydroxylase mRNA in Cultured Human Keratinocytes

Abstract

It is well documented that 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25[OH]2D3), the most active vitamin D metabolite, inhibits epidermal keratinocyte proliferation and promotes differentiation. 1 alpha,25(OH)2D3 can be produced in keratinocytes from 25-hydroxyvitamin D3 by the enzyme 25-hydroxyvitamin D3-1 alpha-hydroxylase (1-OHase). Hydroxylation of 1 alpha,25(OH)2D3 by 25-hydroxyvitamin D3-24-hydroxylase (24-OHase), the first step in the catabolic pathway of 1 alpha,25(OH)2D3 could significantly reduce the intracellular concentration of 1 alpha,25(OH)2D3. Therefore, the expression of 24-OHase could have a critical regulatory role in 1 alpha,25(OH)2D3-dependent gene expression. As a first step to examine this possibility, the steady state level of 24-OHase mRNA in cultured human keratinocytes (CHK) was investigated. 24-OHase mRNA was not detected in control CHK. 1 alpha,25(OH)2D3 caused a dose- and time-dependent increase in 24-OHase mRNA level. The highest accumulation of 24-OHase mRNA was observed in CHK treated with 0.1-1 microM 1 alpha,25(OH)2D3. The level of 24-OHase mRNA reached a plateau 12-24 hr after 1 alpha,25(OH)2D3 treatment. 1 beta,25-dihydroxyvitamin D3, the stereoisomer of 1 alpha,25(OH)2D3, failed to induce 24-OHase mRNA expression significantly. In addition to 24-OHase mRNA, a 1.0-kb mRNA hybridized strongly with both rat and human 24-OHase cDNA probes. The origin of this 1.0-kb message is unknown at present, however, it was regulated by 1 alpha,25(OH)2D3. These results demonstrate that 1 alpha,25(OH)2D3 up-regulates the expression of 24-OHase mRNA, and this may be an important first step in the initiation of catabolism of 1 alpha,25(OH)2D3 in human keratinocytes.

UVB-induced production of 1,25-dihydroxyvitamin D3 and vitamin D activity in human keratinocytes pretreated with a sterol Δ7-reductase inhibitor

“…Transfection experiments in which a VDRE containing luciferase reporter construct was introduced in epidermal keratinocytes revealed the involvement of the VDR and therefore, UVB-induced 24-hydroxylase reflects UVB-induced vitamin D activity. Indeed, 24-hydroxylase induction represents a negative feedback mechanism in 1,25(OH) 2 D 3 -dependent gene expression, reducing the availability of VDR ligand [Chen et al, 1994]. The UVB-induced 24-hydroxylase mRNA expression started from 8 h after UVB irradiation, which is completely in line with the production of active vitamin D metabolites in keratinocytes upon UVB irradiation.…”

“…In analogy, significant quantities of 1,25(OH) 2 D 3 are found between 5 and 10 h after irradiation of 7-DHC-supplemented keratinocytes [Lehmann et al, 2000]. Furthermore, keratinocytes were shown to induce 24-hydroxylase mRNA as early as 1 h after addition of 1,25(OH) 2 D 3 [Chen et al, 1994]. Together with the enhancing effect of ketoconazole on 24-hydroxylase induction from 5 h after irradiation, these data fit with the production of active vitamin D metabolites already 5-7 h following Fig.…”

“…We demonstrate that epidermal keratinocytes pretreated with the specific sterol D 7 -reductase inhibitor BM15766 induce 24-hydroxylase, one of the most sensitive 1,25 (OH) 2 D 3 -responsive genes [Makin et al, 1989;Chen et al, 1994] upon irradiation with a physiological UVB-dose. Transfection experiments in which a VDRE containing luciferase reporter construct was introduced in epidermal keratinocytes revealed the involvement of the VDR and therefore, UVB-induced 24-hydroxylase reflects UVB-induced vitamin D activity.…”

“…Vitamin D-dependent gene activation usually, although not exclusively, reflects the presence of active vitamin D metabolites acting as a VDR-ligand [Chen et al, 1994;Pascussi et al, 2005]. As the enzymes responsible for vitamin D activation and catabolism are cytochrome P450 [CYP450] enzymes [Bikle et al, 1986a;Lehmann et al, 1999Lehmann et al, , 2001Schuessler et al, 2001], we used ketoconazole as a universal CYP450 inhibitor in our experiments.…”

UVB‐induced 1,25(OH)2D3 production and vitamin D activity in intestinal CaCo‐2 cells and in THP‐1 macrophages pretreated with a sterol Δ7‐reductase inhibitor

“…Transfection experiments in preconfluent CaCo‐2 cells in which a VDRE‐containing luciferase reporter construct was introduced revealed the involvement of the VDR and therefore, UVB‐induced CYP24 reflects UVB‐induced vitamin D activity. Indeed, CYP24 is one of the most sensitive 1,25(OH) 2 D 3 responsive genes [Makin et al, 1989; Chen et al, 1994, and its induction represents a negative feedback mechanism in 1,25(OH) 2 D 3 ‐dependent gene expression reducing the availability of VDR ligand [Chen et al, 1994. Furthermore, the modulating effect of ketoconazole supports the contention that VDR transactivation took place by the generation of photoproducts (like vitamin D 3 ) that need activation in order to act as a VDR ligand.…”