Figure 1.
Expression of antimicrobial peptides in psoriatic plaques before and after treatment with vitamin D analogs.
4-mm punch biopsies from a marker psoriatic plaque were taken from patients (n = 8) before and after treatment with ointment containing the vitamin D analog calcipotriol (0.005%; applied twice daily for 5 to 7 days). Total mRNA was extracted and transcript levels of cathelicidin, HBD2, HBD3, psoriasin, IL-17A, IL-17F and IL-8 were analyzed by qPCR. Expression of antimicrobial peptides and markers of inflammation in untreated, lesional skin of psoriasis patients was normalized to the mean expression of target genes in skin of the healthy (non-psoriatic) controls (n = 7) (A). In (B) the relative changes of gene expression levels in calcipotriol treated vs. untreated lesional skin of psoriasis patients are displayed. The Y-axis in (A) and (B) is depicted in Log10 scale (n. s. not significant, *P<0.05, **P<0.01, *** P<0.001; Mann-Whitney test). (C) Punch biopsies from lesional and non-lesional psoriatic plaque were taken from patients (n = 6). Total mRNA was extracted and transcript levels of cathelicidin (left panel) and HBD2 (right panel) analyzed by qPCR. Statistical analysis of lesional or non-lesional, respectively, psoriatic skin vs. healthy (non-psoriatic) controls (n = 6) was performed with Mann-Whitney test, comparison of lesional vs. non-lesional biopsies was performed with Wilcoxon matched pairs test (n. s. not significant, *P<0.05, **P<0.01). In (D) Western blot analysis using an antibody which detects HBD2 was performed with total protein extracted from biopsies taken from lesional skin of one representative psoriasis patient before and after treatment with calcipotriol.
Figure 2.
Induction of HBD2 and IL-8 by IL-17A is decreased by calcipotriol through inhibition of NF-κB signaling.
(A) NHEK were stimulated with IL-17A (10 ng/ml) in the presence or absence of vitamin D analogs calcipotriol or ZK191784 (10−8 M). Cells were harvested after 24 h and HBD2 transcript levels were analyzed by qPCR. In (B) IL-8 transcript expression after stimulation of NHEK with IL-17A (10 ng/ml) in the presence or absence of calcipotriol (10−8 M) is displayed. Data are means±SD of a single experiment performed in triplicate and representative of 2 to 3 independent experiments (**P<0.01, ***P<0.001; Student's t test). (C) To study involvement of the Nf-κB pathway we performed reporter gene analyses with an Nf-κB reporter plasmid. 24 h after transfection of HaCaT keratinocytes cells were stimulated with IL-17A (10 ng/ml) in the presence or absence of calcipotriol or 1,25D3 (10−8 M). Luciferase activity was assayed (*P<0.05, **P<0.01; Student's t test). (D) To further confirm Nf-κB involvement cells were stimulated with IL-17A (10 ng/ml) in the presence or absence of calcipotriol (10−8 M) and harvested after 0, 15 or 30 min. Western blot analysis using antibodies against phospho-IκB-α and unphosphorylated IκB-α were performed to analyze activation of NF-κB. Staining for β-actin served as a loading control.
Figure 3.
Vitamin D analogs enhance cathelicidin promoter activity and induce expression in primary human epidermal keratinocytes.
(A) To analyze the effects of vitamin D analogs on the cathelicidin promoter a 5 kb fragment of the 5′ UTR of the cathelicidin gene CAMP was cloned into a luciferase reporter plasmid and transfected into HaCaT keratinocytes. Cells were stimulated with 1,25D3, ZK159222, ZK191784, ZK203278 and calcipotriol (all at 10−7 M) and luciferase activity was assayed (**P<0.01, ***P<0.001; Student's t test). In (B) primary human keratinocytes (NHEK) were stimulated with increasing concentrations of vitamin D analogs ZK159222, ZK191784, ZK203278 or calcipotriol (all: 10−10 M – 10−8 M). Cells were harvested after 24 h and cathelicidin transcript levels were analyzed by qPCR. (C) NHEK were stimulated with vitamin D analogs ZK191784 and calcipotriol (all at 10−8 M) for 0.5, 1 and 4 hours. Again, cathelicidin transcript abundance was analyzed by qPCR. All data are means±SD of a single experiment performed in triplicate and are representative of 2 to 3 independent experiments. (D) To evaluate cathelicidin peptide induction NHEK were treated with calcipotriol, 1,25D3, ZK191784, ZK203278 or ZK159222 (all at 10−8 M). Cathelicidin hCAP18 protein expression was analyzed in NHEK lysates by Western blot after 24 hours. Staining for β-actin served as loading control and hCAP18/β-actin ratios were analyzed using densitometry.
Figure 4.
VDR and the MEK/ERK signaling pathway are involved in cathelicidin induction by vitamin D analogs.
To characterize the role of the VDR in increased cathelicidin expression after treatment with vitamin D analogs, NHEK were transfected with siRNA to decrease VDR expression before stimulation with vitamin D analogs ZK191784, ZK203278 and calcipotriol (all at 10−8 M). Silencing of VDR was confirmed by qPCR (A; left panel) and Western blot (B; middle panel). siRNA suppression of VDR significantly reduced the induction of cathelicidin mRNA by all vitamin D analogs after 24 hours (A; right panel) (*P<0.05, **P<0.01; Student's t test). In (B; upper panel) the corresponding cathelicidin peptide hCAP18 expression levels are displayed as observed by Western blot. Staining for α-tubulin served as loading control and cathelicidin hCAP-18/α-tubulin ratios were analyzed using densitometry. (C) To analyze the role of MEK/ERK signaling in cathelicidin induction NHEK were transfected simultaneously with two different siRNAs (20 nM) to decrease ERK1 and ERK2 expression before stimulation with 1,25D3 and its analogs ZK191784, ZK203278 and calcipotriol (all at 10−8 M). Silencing of ERK1 and ERK2 was confirmed by qPCR (C; left panel). siRNA suppression of ERK1 and ERK2 resulted in reduced induction of cathelicidin mRNA by vitamin D analogs (C; right panel). Data are means±SD of a single experiment performed in triplicate and are representative of 2 to 3 independent experiments (*P<0.05, **P<0.01, ***P<0.001; Student's t test).
Figure 5.
HBD2 and HBD3 expression is induced by IL-17A via a MEK/ERK-dependent mechanism.
(A) To investigate the role of MEK/ERK signaling in HBD2 and HBD3 expression NHEK were treated with the specific MEK1 inhibitor PD98059 (20 µM) prior to stimulation with IL-17A (10 ng/ml). Cells were harvested after 24 h and transcript levels were analyzed by qPCR. Inhibition of MEK/ERK significantly blocked HBD2 and HBD3 induction by IL-17A. Data are means±SD of a single experiment performed in triplicate and are representative of 2 to 3 independent experiments (**P<0.01, ***P<0.001; Student's t test). The inhibitory effect of PD98059 on MEK/ERK was confirmed by Western blot analyses of phospho-p44/p42 (B, upper panel). Staining for α-tubulin served as loading control.
Table 1.
Target genes and corresponding primers for qPCR.