20-hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-κB activity by increasing IκBα levels in human keratinocytes

Zorica Janjetovic, Michal A. Zmijewski, Robert C. Tuckey, Damon A. DeLeon, Minh N. Nguyen, Lawrence Pfeffer, Andrzej T. Slominski

Research output: Contribution to journalArticle

81 Citations (Scopus)

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.

Original languageEnglish (US)
Article numbere5988
JournalPloS one
Volume4
Issue number6
DOIs
StatePublished - Jun 19 2009

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hydroxycholecalciferols
Hydroxylation
Hydroxycholecalciferols
cholecalciferol
Cholecalciferol
keratinocytes
hydroxylation
Keratinocytes
Cholesterol Side-Chain Cleavage Enzyme
calcitriol
Calcitriol Receptors
Calcitriol
vitamin D
Messenger RNA
receptors
Proteins
proteins
extracts
Cell proliferation
skin diseases

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

20-hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-κB activity by increasing IκBα levels in human keratinocytes. / Janjetovic, Zorica; Zmijewski, Michal A.; Tuckey, Robert C.; DeLeon, Damon A.; Nguyen, Minh N.; Pfeffer, Lawrence; Slominski, Andrzej T.

In: PloS one, Vol. 4, No. 6, e5988, 19.06.2009.

Research output: Contribution to journalArticle

Janjetovic, Zorica ; Zmijewski, Michal A. ; Tuckey, Robert C. ; DeLeon, Damon A. ; Nguyen, Minh N. ; Pfeffer, Lawrence ; Slominski, Andrzej T. / 20-hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-κB activity by increasing IκBα levels in human keratinocytes. In: PloS one. 2009 ; Vol. 4, No. 6.
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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.",
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AU - Janjetovic, Zorica

AU - Zmijewski, Michal A.

AU - Tuckey, Robert C.

AU - DeLeon, Damon A.

AU - Nguyen, Minh N.

AU - Pfeffer, Lawrence

AU - Slominski, Andrzej T.

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AB - 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.

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