20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes.
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-kappaB (NF-kappaB) 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-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol) as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha 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 IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB 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-kappaB. Since NF-kappaB 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.
Janjetovic
Zorica
Z
Department of Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
Zmijewski
Michal A
MA
Tuckey
Robert C
RC
DeLeon
Damon A
DA
Nguyen
Minh N
MN
Pfeffer
Lawrence M
LM
Slominski
Andrzej T
AT
D049109:Cell Proliferation; D002762:Cholecalciferol / Q000737:chemistry*; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006887:Hydroxycholecalciferols / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism*; D015151:Immunoblotting / Q000379:methods; D007249:Inflammation; D053582:Interleukin-1alpha / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D006887:Hydroxycholecalciferols; D020672:I-kappa B Proteins; D053582:Interleukin-1alpha; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D014157:Transcription Factors; D000072000:NF-KappaB Inhibitor alpha; D002762:Cholecalciferol; D006899:Mixed Function Oxygenases; D002786:Cholesterol Side-Chain Cleavage Enzyme