20-Hydroxycholecalciferol, Product of Vitamin D3 Hydroxylation by P450scc, Decreases NF-κB Activity by Increasing IκBα 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-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

20(OH)D3 increases mRNA levels of IκBα in keratinocytes.

<p>HEKn and HaCaT keratinocytes were treated with 100 nM 20(OH)D3 or 1,25(OH)₂D3 or vehicle for the indicated period of time. Cells were then lysed and total RNA extracted. mRNA levels for IκBα were measured using reagents for RTPCR according to the manufacturer's protocol (Roche Applied Science, Manheim, Germany) and normalized relative to Cyclophylin B RNA. Data are presented as mean±STD (n = 3) *p<0.05 versus control, or **p<0.01 versus control. Time response in the expression of mRNA was shown for NFκBI (IκBα) in both cells.</p

A schematic representation of the effects of 20(OH)D3 on NFκB signal transduction in keratinocytes.

<p>A schematic representation of the effects of 20(OH)D3 on NFκB signal transduction in keratinocytes.</p

20(OH)D3 treatment inhibits the activation of NFκB-dependant activity in keratinocytes.

<p>Keratinocytes were transiently transfected with a NFκB-Luc construct for 24 h then treated with 100 nM 20(OH)D3, 1,25(OH)₂D3 or ethanol as a vehicle for the indicated time periods (A), or additionally stimulated with LPS (1 µg/ml) or IL-1α (10 ng/ml) for 30 min (B and C, respectively). Cell lysates prepared from HaCaT and normal human keratinocytes were assayed for luciferase activity. The data from six experiments performed in quadruplicate are presented as means±STDEV. *p<0.05 and **p<0.01 between control (non treated cells) and treated cells.</p

20(OH)D3 increases IκBα protein concentration in keratinocytes and has no effect on NFκB-p65.

<p>Keratinocytes, HEKn and HaCaT were stimulated for the indicated times with 100 nM 20(OH)D3, and 100 nM 1,25(OH)₂D3 (HEKn keratinocytes). Cells were lysed, whole cell extracts prepared, and equivalent amounts of protein were loaded onto polyacrylamide gels. Membranes were incubated with either anti-IκBα, anti-NFκB-p65 or anti β-actin (internal control) (A, C). Protein concentration expressed relative to β-actin was significantly different to the zero-time control for IκBα (p<0.05)(B, D). Results from three separate experiments are expressed as mean±STDEV.</p

20(OH)D3 inhibits the translocation of NFκB-p65 complex into the nucleus and increases the expression of IκBα in the cytosol of keratinocytes.

<p>Primary human keratinocytes, third passage, were incubated for 4 h in KBM medium containing KGF with 100 nM 20(OH)D3 or ethanol vehicle, stimulated with IL-1α for 30 min and then fixed. Cells were stained with anti IκBα or NFκB-p65 antibody, followed by secondary antibody linked to FITC. Nuclei were stained red with propidium iodide. Cells were analyzed using fluorescent microscope at 40× magnification.</p

P450scc (CYP11A1) can hydroxylate vitamin D3 to 20-hydroxycholecalciferol with following sequential metabolism to other hydroxyderivatives [10].

<p>P450scc (CYP11A1) can hydroxylate vitamin D3 to 20-hydroxycholecalciferol with following sequential metabolism to other hydroxyderivatives <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005988#pone.0005988-Tuckey2" target="_blank">[10]</a>.</p

20(OH)D3 increases IκBα protein concentration in cytosolic extracts in keratinocytes.

<p>Normal keratinocytes were treated with 100 nM 20(OH)D3 for 1 h and 4 h and then stimulated with or without IL-1α (10 ng/ml) for 30 min. Cells were lysed in lysis buffer and cytosolic extracts prepared. An equal amount of proteins was loaded onto the polyacrylamide gel. Membranes were incubated with antibodies: anti-IκBα and anti β-actin (internal control) (A). Protein concentration expressed relative to the concentration of β-actin is shown in (B). Results are expressed as mean±STDEV.</p