Abstract
The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1) to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-κB (NF-κB) plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-κB, using 1,25-dihydroxycholecalciferol (calcitriol) as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFκB DNA binding activity as well as NF-κB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-κB inhibitor protein, IκBα, in a time dependent manner, while no changes in total NF-κB-p65 mRNA or protein levels were observed. Another measure of NF-κB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IκBα was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR), 20-hydroxycholecalciferol did not affect IκBα mRNA levels, indicating that it requires VDR for its action on NF-κB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-κB. Since NF-κB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.
Highlights
Human keratinocytes have the ability to metabolize vitamin D3 autonomously [1,2,3]
We have recently identified new pathway for metabolism of vitamin D and pro-vitamin D that is catalyzed by cytochrome P450scc (CYP11A1) [6,7,8,9,10,11,12], the enzyme catalyzing the conversion of cholesterol to pregnenolone for steroid hormone synthesis [13]. 20-Hydroxyvitamin D3 (20(OH)D3) is the major product of P450scc activation of vitamin D3 as well as an intermediate in the sequential synthesis of other hydroxylated derivatives including 20,23-dihydroxyvitamin D3 (20,23(OH)2D3 and 17,20,23-trihydroxyvitamin D3 (17,20,23(OH)3D3) [7,10,11]
The inhibitory effect of 20(OH)D3 on nuclear factor-kB (NF-kB) activity in keratinocytes was shown by several complementary approaches including NF-kB dependent DNA binding assays, NFkB-driven reporter gene activity assays, as well as western blotting and immunofluorescence analysis of the translocation of p65 NF-kB subunit from cytoplasm into the nucleus
Summary
Human keratinocytes have the ability to metabolize vitamin D3 autonomously [1,2,3] Are they the site of conversion of 7-dehydrocholesterol (7DHC) to vitamin D3 following UVBradiation [2,4,5], but they express enzymes to hydroxylate vitamin D3 to the hormonally active form known as calcitriol or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). 1,25(OH)2D3 reduces NFkB DNA binding activity by increasing IkBa protein levels, which inhibits IL-8 production [55]. The effects of 1,25(OH)2D3 on both expression of genes involved in its metabolism and the biological activity of the encoded proteins have previously been studied in these cells [26,60]. Action of 20(OH)D3 on NF-kB activity would suggest a role for a novel endogenous secosteroidogenic metabolic pathway [7,8,9,10,11] in the regulation of the systemic and cutaneous immune activity
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