Tumor necrosis factor α-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor κB - PubMed (original) (raw)
Tumor necrosis factor α-mediated induction of interleukin 17C in human keratinocytes is controlled by nuclear factor κB
Claus Johansen et al. J Biol Chem. 2011.
Abstract
IL-17C is a member of the IL-17 family of cytokines. The expression of IL-17C has been demonstrated to be strongly induced by TNFα in human keratinocytes, and recently the level of IL-17C was found to be increased in the inflammatory skin disease psoriasis. However, little is known about the molecular mechanisms involved in the regulation of IL-17C. Here, we show that pretreatment of cultured human keratinocytes with the inhibitor of κB kinase 2 inhibitor, SC-514, resulted in a significant reduction in both IL-17C mRNA and protein expression, indicating the significance of this pathway in the regulation of IL-17C. NF-κB binding sites were identified upstream from the IL-17C gene, and by electrophoretic mobility shift assay NF-κB was shown to bind to all three identified binding sites. Moreover, NF-κB binding to these sites was inducible by TNFα. Supershift analysis revealed binding of the NF-κB subunits p65 and p50 to all three NF-κB binding sites. To determine the contribution of NF-κB in IL-17C expression, we conducted luciferase gene reporter experiments and demonstrated that a 3204-bp promoter fragment of IL-17C containing three putative NF-κB binding sites was strongly activated by TNFα. Interestingly, mutations of the three NF-κB binding sites revealed that one specific NF-κB binding site was crucial for the TNFα-mediated IL-17C induction because mutation of this specific site completely abolished TNFα-induced IL-17C promoter activation. We conclude that the activation of NF-κB (p65/p50) is crucial for the TNFα-induced stimulation of IL-17C expression in human keratinocytes.
Figures
FIGURE 1.
IL-17C expression in cultured normal human keratinocytes is increased in response to TNFα. Cultured normal human keratinocytes were stimulated with TNFα (10 ng/ml) for the indicated time points. A, total RNA was isolated, and the mRNA expression of IL-17C was analyzed by qPCR. B, extracellular medium from cultured keratinocytes was isolated, and the IL-17C protein level was analyzed by ELISA. Four separate experiments were conducted, and all results are expressed as mean ± S.D. *, p < 0.05 compared with controls. Error bars, S.D.
FIGURE 2.
IL-17C is regulated by a NF-κB-dependent mechanism in human keratinocytes. A, cultured normal human keratinocytes were preincubated with an IKK2 inhibitor (SC-514, 50 μ
m
) for 45 min before stimulated with TNFα (10 ng/ml) for 2 h. Total RNA was isolated and analyzed for the expression of IL-17C by qPCR. B, cultured human keratinocytes were preincubated with SC-514 as in A before stimulated with TNFα for 6 h. The extracellular medium was isolated and the IL-17C protein level examined by ELISA. Four separate experiments were conducted, and all results are expressed as mean ± S.D. (error bars). *, p < 0.05 compared with nonstimulated cells; **, p < 0.05 compared with keratinocytes stimulated with TNFα. C, cultured human keratinocytes were preincubated with SC-514 (50 μ
m
) or BMS-345541 (50 μ
m
) for 45 min before stimulated with TNFα for 15 min. Protein extracts were isolated, and Western blotting analysis used to measure the phosphorylated level of p38 MAPK and MK2 as well as the total protein level of IκBα. Equal loading was confirmed by incubation with an anti-β-actin antibody. Data from one representative experiment of three are shown.
FIGURE 3.
Identification and characterization of three NF-κB DNA binding sites in the promoter region of the IL-17C gene. A, cultured normal human keratinocytes were stimulated with TNFα (10 ng/ml) for the indicated time points before the DNA binding activity to the three putative NF-κB binding sites (oligo1–3) located upstream from the start codon of the IL-17C gene was analyzed by EMSA. B, supershift analysis was carried out. Antibodies directed against p50 and p65 were added to the incubation mixture after which the NF-κB DNA binding activity to the three different oligonucleotides was analyzed. C, oligo1–3 were mutated as described under “Experimental Procedures,” and the NF-κB DNA binding activity was examined by EMSA. Representative gels from four different experiments are shown.
FIGURE 4.
TNFα-induced NF-κB DNA binding activity is inhibited by SC-514. Cultured normal human keratinocytes were preincubated with SC-514 (50 μ
m
) before stimulated with TNFα (10 ng/ml) for 1 h. The nuclear extract from the cells was isolated, and the NF-κB DNA binding activity to the three putative NF-κB binding sites (oligo1–3) was analyzed by EMSA. Representative gels from four different experiments are shown.
FIGURE 5.
IL-17C promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type (IL-17C-2-3204-luc2) IL-17C-promoter-luciferase plasmids together with an internal control (Renilla luciferase expression plasmid). After transfection, cells were stimulated with TNFα (10 ng/ml) for the indicated time points (A) or preincubated with SC-514 (50 μ
m
) for 1 h (B) before being stimulated with TNFα (10 ng/ml) for 24 h. The activity was determined as a ratio between firefly and Renilla luciferase activity. Results represent mean ± S.D. (error bars) from four different experiments. All measurements were performed in doublets. *, p < 0.05 compared with vehicle-treated cells; **, p < 0.05 compared with TNFα-stimulated cells.
FIGURE 6.
Mutation of a specific NF-κB binding site inhibits TNFα-mediated IL-17C promoter activation. A, IL-17C promoter constructs. Nucleotide positions are marked relative to the IL-17C transcription start. Three NF-κB binding sites in the IL-17C promoter (−3204 to −1 bp) linked to the luciferase gene were mutated in different combinations. B, cultured normal human keratinocytes were transfected with wild-type (IL-17C-2–3204-luc2) or mutated IL-17C-promoter-luciferase plasmids together with an internal control (Renilla luciferase expression plasmid). After transfection with the indicated plasmids the keratinocytes were stimulated with TNFα (10 ng/ml) for 24 h. The activity was determined as a ratio between firefly and Renilla luciferase activity. Results represent mean ± S.D. (error bars) from four different experiments. All measurements were performed in doublets. *, p < 0.05 compared with IL-17C-2–3204-luc2 reporter plasmid-transfected cells stimulated with TNFα.
FIGURE 7.
Transfection of keratinocytes with decoy oligonucleotides containing the indentified NF-κB DNA binding sites inhibits TNFα-induced IL-17C expression. Cultured normal human keratinocytes were transfected with a pool of hIL-17C(oligo1–3) or with a pool of hIL-17C(MUT1–3) for 6 h before being stimulated with TNFα (10 ng/ml) for 2 h. Total RNA from the cells was isolated, and the mRNA expression of IL-17C was analyzed by qPCR and normalized with RPLP0. Four separate experiments were conducted, and all results are expressed as mean ± S.D. (error bars). *, p < 0.05 compared with nonstimulated cells; **, p < 0.05 compared with TNFα-stimulated cells.
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