Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line

Abstract

S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein β (C/EBPβ). Mutated C/EBPβ binding sequences or C/EBPβ-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPβ-dependent transcriptional activity.

Keywords: C/EBPβ; ERK; IL-1 receptor; IL-1R1; IL-1α; JNK; Keratinocytes; S100A9; TLR5; Toll-like receptor 5; c-JUN N-terminal kinase; extracellular-regulated kinase; p38; p38 kinase; siRNA; small interfering RNA.

Figure 1. Effect of IL-1α, IL-1α antibody and IL-1R1 antagonist on the expression of IL-1R1 and S100A9 mRNA in HaCaT cells

(A) HaCaT cells were cultured for 5 days. To some cells, IL-1α (20 ng/ml) was added and all cultures continued for another 24 h. After RNA isolation from the cultured cells, IL-1R1-specific mRNA expression was analyzed by RT-PCR. After culture for 5 days, HaCaT cells were pre-cultured with anti-IL-1α antibody (1/100 dilution) or IL-1α receptor antagonist (100 ng/ml) for 1 h and further cultured with IL-1α (20 ng/ml) for 24 h. The expression of S100A9 mRNA was analyzed by northern blot analysis (B) and quantitative PCR (C). The RT-PCR and northern blot analyses were repeated three times and a typical result is shown. S100A9 mRNA expression was normalized to GAPDH mRNA. Data are expressed as fold change relative to the vehicle treatment. NS = not significant. The vehicle only control reflected addition of DMEM into the culture medium. Bars represent the mean ± S.D. of three independent RNA samples. *P < 0.05.

Figure 2. Effect of MAPK inhibitors on S100A9 expression and phosphorylation of MAPK in HaCaT cells

HaCaT cells were cultured for 5 days and pre-cultured with SB203580 (30 μM), U0126 (10 μM) or SP600125 (10 μM) for 1 h, and further cultured with IL-1α (20 ng/ml) for 30 min (Western blotting) or 24 h (Northern blotting). (A) MAPK phosphorylation was analyzed by a western blot analysis of the cell protein fraction using anti-p38 and phospho-p38, anti-ERK and phospho-ERK, anti-JNK and phospho-JNK antibodies. (B) RNA was isolated from the cultured cells and S100A9 mRNA expression was quantified by a northern blot analysis. The vehicle only control reflected addition of DMSO (0.05% concentration) into the culture medium. (C) Cells were grown to approximately 60-80% confluency and transfected with control siRNA or p38 siRNA (25 nM) for 24 h. The medium was then removed, and cells were treated with fresh medium containing IL-1α (20 ng/ml) or vehicle (DMEM medium) for another 24 h before harvesting for (C) Western blot analysis or (D) quantitative PCR analysis. Data are expressed as fold change relative to the control siRNA transfection and vehicle treatment. These results were confirmed by three independent experiments. *P < 0.05. **P<0.01.

Figure 3. Effect of IL-1α on S100A9 transcription in HaCaT cells

HaCaT cells were transiently co-transfected with deletion constructs in the S100A9 promoter region (A: -2574 to +1; B: -500 to +1) and an internal control Renilla vector (phRL-TK), and cultured with or without IL-1α (100 ng/ml) for 12 h. The transcriptional start site was set as +1 (57). Luciferase activity was measured using Dual-Luciferase Reporter Assay System. Relative luciferase activity was normalized to Renilla luciferase expression using the phRL-TK Vector control and expressed as the percentage activity of the pGL3(-500/+430) deletion construct in the absence of IL-1α as described in the Experimental Procedures. Cell samples from IL-1α-stimulated (black bar) and non-stimulated cells (white bar; vehicle) were analyzed and the data expressed as the mean ± S.D. of cell samples from six separate experiments. The vehicle only control reflected an addition of Opti-MEM into the culture medium. *P < 0.01 **P < 0.05.

Figure 4. C/EBPβ associated with IL-αa-stimulated S100A9 expression

(A) Location of oligonucleotide sequence for EMSA is boxed and asterisks (*) show mutated nucleotide positions. S100A9 promoter (-203/+50) sequences amplified for ChIP assay primers are underlined. (B) C/EBPβ binding to the S100A9 promoter region as analyzed by EMSA. HaCaT cells were cultured for 5 days followed by culture for 6 h with or without IL-1α (10 ng/ml). The nuclear protein (10 μg) extracted from the cultured cells was incubated with DIG-labeled double-stranded oligonucleotide and used for EMSA as described in the Experimental Procedures. Oligonucleotide competition was performed using 100-fold excess of each unlabeled oligonucleotide or mutated oligonucleotide. A supershift assay was performed by preincubation with anti-C/EBPα or anti-C/EBPβ antibody. An asterisk identifies the supershift band. (C) C/EBPβ binding to the S100A9 promoter in vivo. The S100A9 promoter region (−203/+50) containing the putative C/EBPβ binding site was amplified as described in Experimental Procedures. The S100A9 upstream fragment (-5360/-5130) was amplified and served as control for the absence of C/EBPβ binding sites. The vehicle only control consisted of addition of DMEM into the culture medium. β-actin antibody and mouse IgG were used as negative controls, whereas “Input” was sheared DNA prior to immunoprecipitation. Detailed methods can be seen in Experimental Procedures. (D) Dose-response increase in C/EBPβ-binding after treatment with IL-1α. Amplification of C/EBPβ immunoprecipitates after treatment with IL-1α (2.5 and 10 ng/ml) shows increasing recovery of S100A9 promoter fragments when compared with vehicle control. The transcriptional start site is set as +1. The analyses reported in panels B, C, and D were repeated three separate times and a typical result is shown for each.

Figure 5. C/EBPβ binding site at -89/-76 critical for IL-1α-stimulated S100A9 expression

(A, B) Downregulation of C/EBPβ by siRNA reduces S100A9 promoter activity. Cells were grown to approximately 60-80% confluency and transfected with pGL3(−500/+430) and pRL-TK for 24 h. Cells were then were transfected with 25 nM C/EBPβ siRNA or non-specific control siRNA, medium removed, and growth continued for an additional 24 h before (A) Western blot analysis and (B) luciferase activity assay. (C) Mutational analysis of the C/EBPβ binding site in the S100A9 promoter region using a luciferase reporter as described in the Experimental Procedures. HaCaT cells were transiently transfected with the luciferase reporter construct with a mutation (M1) or deletion (M2) of the C/EBPβ binding site and cultured with or without IL-1α (100 ng/ml) for 12 h. Luciferase activity from IL-1α-stimulated (black bar) and non-stimulated cells (white bar; vehicle) is expressed as the mean ± S.D. of cell samples from six separate experiments. The vehicle only control reflected an addition of Opti-MEM into the culture medium. *P < 0.01.