20,23-dihydroxyvitamin D3, novel P450scc product, stimulates differentiation and inhibits proliferation and NF-kappaB activity in human keratinocytes

Abstract

We have examined effects of the 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), on differentiation and proliferation of human keratinocytes and the anti-inflammatory potential of 20,23(OH)2D3 from its action on nuclear factor-kappaB (NF-kappaB). 20,23(OH)2D3 inhibited growth of keratinocytes with a potency comparable to that for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell cycle analysis showed that this inhibition was associated with G1/G0 and G2/M arrests. 20,23(OH)2D3 stimulated production of involucrin mRNA and inhibited production of cytokeratin 14 mRNA in a manner similar to that seen for 1,25(OH)2D3. Flow cytometry showed that these effects were accompanied by increased involucrin protein expression, and an increase in the cell size and granularity. Silencing of the vitamin D receptor (VDR) by corresponding siRNA abolished the stimulatory effect on involucrin gene expression demonstrating an involvement of VDR in 20,23(OH)2D3 action. This mode of action was further substantiated by stimulation of CYP24 gene expression and stimulation of the CYP24 promoter-driven reporter gene activity. 20,23(OH)2D3 displayed several fold lower potency for induction of CYP24 gene expression than 1,25(OH)2D3. Finally, 20,23(OH)2D3 inhibited the transcriptional activity of NF-kappaB in keratinocytes as demonstrated by EMSA, NF-kappaB-driven reporter gene activity assays and measurements of translocation of p65 from the cytoplasm to the nucleus. These inhibitory effects were connected with stimulation of the expression of IkappaBalpha with subsequent sequestration of NF-kappaB in the cytoplasm and consequent attenuation of transcriptional activity. In summary, we have characterized 20,23(OH)2D3 as a novel secosteroidal regulator of keratinocytes proliferation and differentiation and a modifier of their immune activity.

Fig. 1

20,23(OH)2D3 inhibits proliferation and arrests keratinocytes at G0/G1 and G2/M phases of the cell cycle. HaCaT keratinocytes were treated with 20,23(OH)2D3, 1,25(OH)2D3or 20(OH)D3 for 48 h, at concentrations ranging from 0.1to 100 nM (A) orat a 100 nM concentration at different time points (24 h and 48 h) (B), and [³H]-thymidine incorporation into the DNA of cells was measured. Differences to control (ethanol treated cells) and between every dose are significant(*P < 0.05; **P < 0.01, ***P < 0.001). HaCaTcells were also treated with 10 nM 20,23 (OH)2D3 or 1,25(OH)2D3 for 24 h, cells harvested, fixed, stained with PI and read with a flow cytometer (C). Data are presented as mean ± SD (n = 3, *P < 0.05 between control and treatment).

Fig. 2

20,23(OH)2D3 stimulates keratinocytes differentiation on mRNA level. HEKn or HaCaT cells were treated for 6 h with 100 nM 20,23(OH)2D3, 1,25(OH)2D3 or ethanol as a vehicle. The mRNA was isolated and RTPCR performed using specific primers for involucrin (A) and CK14 genes (B). Data are presented as mean ± SD (n = 3, *P < 0.05; ***P < 0.001).

Fig. 3

20,23(OH)2D3 stimulates keratinocytes differentiation on protein level. HaCaT cells were treated with 10 nM 20,23(OH)2D3 or ethanol as a vehicle for 24 h. The cells were harvested, fixed in 2% PFA, stained with anti-involucrin antibody and read with a flow cytometer. Data are presented as mean ± SD (n = 3, *P < 0.05 between control and treatment). IgG is used as a control. For immunofluorescent analysis, HaCaT cells were plated onto cover glass in 6-well plates and treated for 24 h with 10 nM 20,23(OH)2D3 or ethanol, fixed, stained with anti-involucrin antibody (green) and nuclei with PI (red). Cells were photographed with a fluorescent microscope using 40× magnification.

Fig. 4

20,23(OH)2D3 does not inhibit expression of the 25-hydroxyvitamin D 1 α-hydroxylase (CYP27B1) in keratinocytes. HEKn (A) or HaCaT (B) keratinocytes were treated with 20,23(OH)2D3 or1,25(OH)2D3 at a100 nM concentration for 0, 6, and 24 h. Total RNA was extracted and reverse transcribed. CYP27B1 mRNA levels were measured according to manufacturer’s protocol (Roche). Data are presented as mean ± SEM (n = 3).

Fig. 5

20,23(OH)2D3 is less potent at stimulating CYP24 gene expression in keratinocytes than 1,25(OH)2D3. A: HEKn and HaCaT cells were treated with 100 nM 20,23(OH)2D3, 1,25(OH)2D3 or ethanol as a vehicle for 24 h, mRNA was isolated and submitted for RTPCR. Data are presented as mean ± SD(n = 3, *P < 0.05, **P < 0.01, ***P < 0.001). B:HaCaT cells were transfected with luciferase constructs: CYP24 alone or with human VDR receptor and empty vector pLuc, using lipofectamine. 24 h post-transfection cells were treated with 10 nM, 20,23(OH)2D3, 1,25(OH)2D3 or ethanol as a vehicle for 24 h. Cells were lysed and luciferase activity measured with a luminometer. Data are presented as mean ± SD (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001).

Fig. 6

20,23(OH)2D3 requires VDR for its action on keratinocytes. HaCaT keratinocytes were transfected with 20 nM scrambled or VDR small interfering RNA (siRNA). Cells were incubated with 100 nM 20,23(OH)2D3 (A–C) or 1,25(OH)2D3 (D–F) or the vehicle control for 24 h. mRNA levels were measured for VDR (A, D), involucrin (B, E), and CYP24 (C, F), using reagents for RTPCR according to manufacturer’s protocol (Roche Applied Science, Manheim, Germany) and normalized relative to Cyclophilin B mRNA. Data are presented as mean ± SD (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control). Levels of VDR and β-actin proteins were assessed 24 h after transfection with VDR or scrambled siRNA by Western blotting of whole cell extracts (A).

Fig. 7

20,23(OH)2D3 attenuates NF-κB binding activity in HaCaT cells. A: Nuclear extracts were prepared from HaCaT cells treated with 100 nM 20,23(OH)2D3 for 0 h (vehicle control), or times indicated, and subjected to an electrophoresis mobility shift assay using 2.5 μg of proteins per lane. “Cold” represents extract incubated with an excess of unlabeled NF-κB oligonucleotide. Normal keratinocytes (B) and HaCaT (C) were transfected with the NF-κB luciferase construct (NF-κB-Luc) using lipofectamine. 24 h post-transfection cells were treated for the indicated periods with 100 nM 20,23(OH)2D3 or 1,25(OH)2D3 (or ethanol vehicle). Cells were then lysed and luciferase activity was measured with a luminometer. Data are presented as mean ± SD (n = 4).

Fig. 8

20,23(OH)2D3 inhibits translocation of NF-κ B-p65 proteinin keratinocytes. Primary human keratinocytes, third passage, were incubated for 4 h in KBM medium containing KGF and 100 nM 20,23(OH)2D3, 1,25(OH)2D3 or ethanol as a vehicle, and additionally treated without (A) or with IL-1α (B) for 30 min and then fixed. Cells were stained with anti IκBα or NF-κB-p65 antibody followed by secondary antibody linked to FITC. Nuclei were stained red with PI. Cells were photographed using a fluorescent microscope at 40× magnification.

Fig. 9

20,23(OH)2D3 increases IκBα protein levels in keratinocytes. HaCaT keratinocytes were stimulated with 100 nM 20,23(OH)2D3 (A) or 1,25(OH)2D3 (B) for the indicated times. Cells were lysed and whole cell extracts prepared. An equal amount of protein for each cell treatment was loaded onto the polyacrylamide gel. Membranes were incubated with antibodies: anti-IκBα, anti-NF-κB-p65, anti-IκBβ and anti-β-actin (internal control).

Fig. 10

20,23(OH)2D3 stimulates IκBα mRNA expression. HEKn and HaCaT keratinocytes were treated with 100 nM 20,23(OH)2D3, 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 by quantitative RTPCR and normalized relative to Cyclophilin B mRNA. Data are presented as mean ± SD (n = 3) (*P < 0.05 vs. control, or **P < 0.01 vs. control).

Fig. 11

20,23(OH)2D3 requires VDR expression for its action on the NF- κB pathway in keratinocytes. HaCaT keratinocytes were transfected with 20 nM scrambled or VDR siRNA and incubated with 100 nM20,23(OH)2D3 (A), 1,25(OH)2D3 (B) or vehicle (ethanol) for 4 h, cells lysed and total RNA extracted. I κBα mRNA levels were measured by quantitative RTPCR and normalized relative to Cyclophilin B mRNA. Data are presented as mean ± SD (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control).