The IL-6 trans-signaling-STAT3 pathway mediates ECM and cellular proliferation in fibroblasts from hypertrophic scar

Abstract

The molecular mechanisms behind the pathogenesis of postburn hypertrophic scar (HS) remain unclear. Here, we investigate the role of the IL-6 trans-signaling-signal transducer and activator of transcription (STAT)3 pathway in HS fibroblasts (HSFs) derived from post-burn HS skin. HSF showed increased Tyr 705 STAT3 phosphorylation compared with normal fibroblast (NF) after IL-6•IL-6Rα stimulation by immunoassays. The endogenous STAT3 target gene, SOCS3, was upregulated in HSFs and showed increased STAT3 binding on its promoter relative to NFs in a chromatin immunoprecipitation assay. We observed that the cell-surface signaling transducer glycoprotein 130 is upregulated in HSFs by quantitative real-time reverse-transcriptase-PCR and flow cytometry. The production of excessive extracellular matrix (ECM), including the expression of alpha2 (1) procollagen (Col1A2) and fibronectin 1 (FN), was seen in HSFs. A STAT3 peptide inhibitor abrogated FN and Col1A2 gene expression in HSFs indicating involvement of STAT3 in ECM production. The cellular proliferation markers Cyclin D1, Bcl-Xl, and c-Myc were also upregulated in HSF, and knockdown of STAT3 by small interfering RNA attenuated c-Myc expression indicating the essential role of STAT3 in fibroblast proliferation. Taken together, our results suggest that the IL-6 trans-signaling-STAT3 pathway may have an integral role in HS pathogenesis, and disruption of this pathway could be a potential therapeutic strategy for the treatment of post-burn HS.

Trial registration: ClinicalTrials.gov NCT00675714.

Figure 1

(a) Immunohistochemical analysis of phospho-STAT3 (Tyr 705) expression in burned HSskin. ‘A-F’ and G are HS skin stained with anti-phosphoTyr705-STAT3 Ab and control IgG respectively. Panel H-I are normal skin stained with anti-phosphoTyr705-STAT3 Ab. Arrowheads (D-F) show the strong nuclear staining for activated STAT3. A-C, G-H and D-F, I are X10 and X20 magnifications respectively, scale bar: 10µm. (b) Intensity of nuclear staining in the dermis of HS (n=10) and normal skin (n=5) (MetaMorph 7.3). *, p<0.01. (c) Western Immunoblot analysis of phospho-STAT3 (Tyr 705) in HSFs. WCE of NF and HSF were fractionated and Western immunoblot analysis with phospho-STAT3 (Tyr 705) Ab is shown. The numbers at the bottom of the lane indicate the fold induction. β-actin blot (lower panel) shows loading control.

Figure 1

(a) Immunohistochemical analysis of phospho-STAT3 (Tyr 705) expression in burned HSskin. ‘A-F’ and G are HS skin stained with anti-phosphoTyr705-STAT3 Ab and control IgG respectively. Panel H-I are normal skin stained with anti-phosphoTyr705-STAT3 Ab. Arrowheads (D-F) show the strong nuclear staining for activated STAT3. A-C, G-H and D-F, I are X10 and X20 magnifications respectively, scale bar: 10µm. (b) Intensity of nuclear staining in the dermis of HS (n=10) and normal skin (n=5) (MetaMorph 7.3). *, p<0.01. (c) Western Immunoblot analysis of phospho-STAT3 (Tyr 705) in HSFs. WCE of NF and HSF were fractionated and Western immunoblot analysis with phospho-STAT3 (Tyr 705) Ab is shown. The numbers at the bottom of the lane indicate the fold induction. β-actin blot (lower panel) shows loading control.

Figure 1

(a) Immunohistochemical analysis of phospho-STAT3 (Tyr 705) expression in burned HSskin. ‘A-F’ and G are HS skin stained with anti-phosphoTyr705-STAT3 Ab and control IgG respectively. Panel H-I are normal skin stained with anti-phosphoTyr705-STAT3 Ab. Arrowheads (D-F) show the strong nuclear staining for activated STAT3. A-C, G-H and D-F, I are X10 and X20 magnifications respectively, scale bar: 10µm. (b) Intensity of nuclear staining in the dermis of HS (n=10) and normal skin (n=5) (MetaMorph 7.3). *, p<0.01. (c) Western Immunoblot analysis of phospho-STAT3 (Tyr 705) in HSFs. WCE of NF and HSF were fractionated and Western immunoblot analysis with phospho-STAT3 (Tyr 705) Ab is shown. The numbers at the bottom of the lane indicate the fold induction. β-actin blot (lower panel) shows loading control.

Figure 2

(a) Activation of IL-6 trans-signaling in HSF. NF and HSF cells were treated with IL-6•sIL-6Rα (8ng/ml •25ng/ml) for 30 minutes, or left untreated. Total cellular RNA was subjected to Q-RT-PCR for hSOCS3 transcript. Shown is fold change mRNA expression relative to GAPDH. Data represents mean±SD, *, p<0.01. (b) Inhibition of phospho-STAT3 with STAT3 siRNA. NFs and HSFs were transfected with either control or STAT3 siRNA. 72 hours after transfection, cells were stimulated as above and WCE were measured for phosphoTry705-STAT3 by immunoassay (Bio-Rad). Shown is the representative fluorescence intensity of phospho-STAT3 relative to total protein. (c) Inhibition of hSOCS3 gene with STAT3 silencing. Cells were treated as in (b) and total cellular RNA was subjected to Q-RT-PCR for hSOCS3 expression relative to GAPDH. *, p<0.05.

Figure 2

(a) Activation of IL-6 trans-signaling in HSF. NF and HSF cells were treated with IL-6•sIL-6Rα (8ng/ml •25ng/ml) for 30 minutes, or left untreated. Total cellular RNA was subjected to Q-RT-PCR for hSOCS3 transcript. Shown is fold change mRNA expression relative to GAPDH. Data represents mean±SD, *, p<0.01. (b) Inhibition of phospho-STAT3 with STAT3 siRNA. NFs and HSFs were transfected with either control or STAT3 siRNA. 72 hours after transfection, cells were stimulated as above and WCE were measured for phosphoTry705-STAT3 by immunoassay (Bio-Rad). Shown is the representative fluorescence intensity of phospho-STAT3 relative to total protein. (c) Inhibition of hSOCS3 gene with STAT3 silencing. Cells were treated as in (b) and total cellular RNA was subjected to Q-RT-PCR for hSOCS3 expression relative to GAPDH. *, p<0.05.

Figure 2

(a) Activation of IL-6 trans-signaling in HSF. NF and HSF cells were treated with IL-6•sIL-6Rα (8ng/ml •25ng/ml) for 30 minutes, or left untreated. Total cellular RNA was subjected to Q-RT-PCR for hSOCS3 transcript. Shown is fold change mRNA expression relative to GAPDH. Data represents mean±SD, *, p<0.01. (b) Inhibition of phospho-STAT3 with STAT3 siRNA. NFs and HSFs were transfected with either control or STAT3 siRNA. 72 hours after transfection, cells were stimulated as above and WCE were measured for phosphoTry705-STAT3 by immunoassay (Bio-Rad). Shown is the representative fluorescence intensity of phospho-STAT3 relative to total protein. (c) Inhibition of hSOCS3 gene with STAT3 silencing. Cells were treated as in (b) and total cellular RNA was subjected to Q-RT-PCR for hSOCS3 expression relative to GAPDH. *, p<0.05.

Figure 3

hSOCS3 promoter occupancy of STAT3 in HSF and NF. Protein-DNA crosslinked extracts of IL-6•sIL-6Rα (8ng/ml •25ng/ml) stimulated NF and HSF cells were immunoprecipitated with IgG or anti-STAT3 Ab. SOCS3 promoter occupancy of STAT3 were detected by two step ChIP assays as described in the method. Shown is the fold change in quantitative-genomic PCR (Q-gPCR) normalized to input DNA. *, p<0.01, students t- test.

Figure 4

(a) Activation of gp130 in HSF. NFs and HSFs were treated with IL-6•sIL-6Rα for 30 min. Total RNA was subjected to Q-RT-PCR for human gp130 mRNA expression, carried out in triplicate. Shown is fold change mRNA expression relative to GAPDH as internal control. Data represents mean±SD *, p<0.01, students t test. (b) Cell surface activation of gp130 in HSF. Cultured NF and HSF cells were left untreated or IL-6•sIL-6Rα (8ng/ml •25ng/ml) stimulated for 30 min. The expression of cell surface gp130 was analyzed by flow cytometry after staining with anti-gp130-PE. Events were plotted as a function of fluorescence intensity (x-axis). Shaded histograms represent isotype antibody control and open histograms represents either unstimulated anti-gp130-PE stained cells (dotted line) or IL-6•sIL-6Rα stimulated anti-gp130-PE (solid line) as indicated.

Figure 4

(a) Activation of gp130 in HSF. NFs and HSFs were treated with IL-6•sIL-6Rα for 30 min. Total RNA was subjected to Q-RT-PCR for human gp130 mRNA expression, carried out in triplicate. Shown is fold change mRNA expression relative to GAPDH as internal control. Data represents mean±SD *, p<0.01, students t test. (b) Cell surface activation of gp130 in HSF. Cultured NF and HSF cells were left untreated or IL-6•sIL-6Rα (8ng/ml •25ng/ml) stimulated for 30 min. The expression of cell surface gp130 was analyzed by flow cytometry after staining with anti-gp130-PE. Events were plotted as a function of fluorescence intensity (x-axis). Shaded histograms represent isotype antibody control and open histograms represents either unstimulated anti-gp130-PE stained cells (dotted line) or IL-6•sIL-6Rα stimulated anti-gp130-PE (solid line) as indicated.

Figure 5

(a) ECM expression in HSF. NFs and HSFs were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to Q-RT-PCR for Col1A2 and FN expression. Shown is fold change mRNA expression relative to GAPDH. *, p<0.01. (b) STAT3 peptide inhibitor attenuates hSOCS3 expression. NF and HSFs were treated with 500 nM STAT3 inhibitor peptide for 6 hoursrs or left untreated. Total cellular RNA (stimulated) was subjected to Q-RT-PCR for hSOCS3 transcript. *, p<0.01. (c) STAT3 peptide inhibitor abrogates FN production in HSF. Cells were treated as in (b) and FN gene expression was measured relative to GAPDH.*, p<0.05. (d) STAT3 peptide inhibitor abrogates Col1A2 production in HSF. Cells were treated as in (b) and Col1A2 gene expression was measured relative to GAPDH. *, p<0.05.

Figure 5

(a) ECM expression in HSF. NFs and HSFs were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to Q-RT-PCR for Col1A2 and FN expression. Shown is fold change mRNA expression relative to GAPDH. *, p<0.01. (b) STAT3 peptide inhibitor attenuates hSOCS3 expression. NF and HSFs were treated with 500 nM STAT3 inhibitor peptide for 6 hoursrs or left untreated. Total cellular RNA (stimulated) was subjected to Q-RT-PCR for hSOCS3 transcript. *, p<0.01. (c) STAT3 peptide inhibitor abrogates FN production in HSF. Cells were treated as in (b) and FN gene expression was measured relative to GAPDH.*, p<0.05. (d) STAT3 peptide inhibitor abrogates Col1A2 production in HSF. Cells were treated as in (b) and Col1A2 gene expression was measured relative to GAPDH. *, p<0.05.

Figure 5

(a) ECM expression in HSF. NFs and HSFs were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to Q-RT-PCR for Col1A2 and FN expression. Shown is fold change mRNA expression relative to GAPDH. *, p<0.01. (b) STAT3 peptide inhibitor attenuates hSOCS3 expression. NF and HSFs were treated with 500 nM STAT3 inhibitor peptide for 6 hoursrs or left untreated. Total cellular RNA (stimulated) was subjected to Q-RT-PCR for hSOCS3 transcript. *, p<0.01. (c) STAT3 peptide inhibitor abrogates FN production in HSF. Cells were treated as in (b) and FN gene expression was measured relative to GAPDH.*, p<0.05. (d) STAT3 peptide inhibitor abrogates Col1A2 production in HSF. Cells were treated as in (b) and Col1A2 gene expression was measured relative to GAPDH. *, p<0.05.

Figure 5

(a) ECM expression in HSF. NFs and HSFs were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to Q-RT-PCR for Col1A2 and FN expression. Shown is fold change mRNA expression relative to GAPDH. *, p<0.01. (b) STAT3 peptide inhibitor attenuates hSOCS3 expression. NF and HSFs were treated with 500 nM STAT3 inhibitor peptide for 6 hoursrs or left untreated. Total cellular RNA (stimulated) was subjected to Q-RT-PCR for hSOCS3 transcript. *, p<0.01. (c) STAT3 peptide inhibitor abrogates FN production in HSF. Cells were treated as in (b) and FN gene expression was measured relative to GAPDH.*, p<0.05. (d) STAT3 peptide inhibitor abrogates Col1A2 production in HSF. Cells were treated as in (b) and Col1A2 gene expression was measured relative to GAPDH. *, p<0.05.

Figure 6

(a) IL-6 trans-signaling increases expression of cellular proliferation markers in HSF. NF and HSF cells were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to QPCR for Cyclin D1, c-Myc and Bcl-XL gene expression. Shown is fold change mRNA expression relative to GAPDH as internal control performed in triplicate. Data represents mean ± SD.*, p<0.05, students t test. (b) STAT3 siRNA inhibits expression of cellular proliferation markers in HSF. NF and HSF cells were transfected with either STAT3 siRNA or control siRNA. 72 h after transfection cells were stimulated with IL-6•sIL-6Rα for 30 min and total cellular RNA was subjected to Q-PCR for c-Myc, Bcl-XL and Cyclin D1 gene expression. *, p<0.01, students t test.

Figure 6

(a) IL-6 trans-signaling increases expression of cellular proliferation markers in HSF. NF and HSF cells were stimulated with IL-6•sIL-6Rα and total cellular RNA was subjected to QPCR for Cyclin D1, c-Myc and Bcl-XL gene expression. Shown is fold change mRNA expression relative to GAPDH as internal control performed in triplicate. Data represents mean ± SD.*, p<0.05, students t test. (b) STAT3 siRNA inhibits expression of cellular proliferation markers in HSF. NF and HSF cells were transfected with either STAT3 siRNA or control siRNA. 72 h after transfection cells were stimulated with IL-6•sIL-6Rα for 30 min and total cellular RNA was subjected to Q-PCR for c-Myc, Bcl-XL and Cyclin D1 gene expression. *, p<0.01, students t test.