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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-6R? 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.

SUBMITTER: Ray S

PROVIDER: S-EPMC3626764 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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

Ray Sutapa S Ju Xiaoxi X Sun Hong H Finnerty Celeste C CC Herndon David N DN Brasier Allan R AR

The Journal of investigative dermatology 20130110 5

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 ...[more]

PMID: 23303450

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Project description:Background: As a fibrotic disease with a high incidence, the pathogenesis of hypertrophic scarring is still not fully understood, and the treatment of this disease is also challenging. In recent years, human adipose-derived mesenchymal stem cells (AD-MSCs) have been considered an effective treatment for hypertrophic scars. This study mainly explored whether the therapeutic effect of AD-MSCs on hypertrophic scars is associated with oxidative-stress-related proteins. Methods: AD-MSCs were isolated from adipose tissues and characterized through flow cytometry and a differentiation test. Afterwards, coculture, cell proliferation, apoptosis, and migration were detected. Western blotting and a quantitative real-time polymerase chain reaction (qRT-PCR) were used to detect oxidative stress-related genes and protein expression in hypertrophic scar fibroblasts (HSFs). Flow cytometry was used to detect reactive oxygen species (ROS). A nude mouse animal model was established; the effect of AD-MSCs on hypertrophic scars was observed; and hematoxylin and eosin staining, Masson's staining, and immunofluorescence staining were performed. Furthermore, the content of oxidative-stress-related proteins, including nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), B-cell lymphoma 2(Bcl2), Bcl2-associated X(BAX) and caspase 3, was detected. Results: Our results showed that AD-MSCs inhibited HSFs' proliferation and migration and promoted apoptosis. Moreover, after coculture, the expression of antioxidant enzymes, including HO-1, in HSFs decreased; the content of reactive oxygen species increased; and the expression of Nrf2 decreased significantly. In animal experiments, we found that, at 14 days after injection of AD-MSCs into human hypertrophic scar tissue blocks that were transplanted onto the dorsum of nude mice, the weight of the tissue blocks decreased significantly. Hematoxylin and eosin staining and Masson's staining demonstrated a rearrangement of collagen fibers. We also found that Nrf2 and antioxidant enzymes decreased significantly, while apoptotic cells increased after AD-MSC treatment. Conclusions: Our results demonstrated that AD-MSCs efficiently cured hypertrophic scars by promoting the apoptosis of HSFs and by inhibiting their proliferation and migration, which may be related to the inhibition of Nrf2 expression in HSFs, suggesting that AD-MSCs may provide an alternative therapeutic approach for the treatment of hypertrophic scars.

Project description:BackgroundThe hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-β1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions.ObjectiveTo explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10.MethodsExpression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins.ResultsHS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs.ConclusionIL-10 apparently inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.

Project description:BackgroundHypertrophic-scar (HS) is the most common pathological healing phenomenon after trauma, especially after deep burns. We aimed to investigate the expression and role of microRNA-211-5p (miR-211-5p) in HS and explore its underlying mechanism.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-211-5p in 15 cases of HS tissues and normal skin tissues, as well as its expression in human hypertrophic scar fibroblasts (hHSFs) and normal fibroblasts. At the same time, the cell counting kit-8 (CCK-8), scratch test, cell invasion test, and flow cytometry were used to determine cell proliferation, migration, invasion, and apoptosis, respectively. Western blot assay was used to determine the expression of proteins. TargetScan was performed to predict the potential binding sites between miR-211-5p and TGFβR2, which was then verified by western blotting and luciferase reporter gene experiments. Also, co-transfection of plasmids that overexpress miR-211-5p and TGFβR2 were used to observe the reversal effect of miR-211-5p.ResultsThe level of miR-211-5p in HS tissues and hHSFs cells was significantly down-regulated (both P<0.05). The TGFβR2/Smad3 signaling pathway was activated (both P<0.05). Furthermore, the overexpression of miR-211-5p could inhibit the proliferation (P<0.05), migration (P<0.05), and invasion (P<0.05) of hHSFs cells, and induce their apoptosis (P<0.05), and could also regulate the expression of related proteins (all P<0.05). Moreover, the overexpression of miR-211-5p could also inhibit the accumulation of ECM and the activation of the TGF-βR2/Smad3 pathway (all P<0.05), while the opposite effect (all P<0.05) was observed when the level of miR-211-5p was interfered with. Finally, it was confirmed that miR-211-5p could target TGFβR2 (all P<0.05), and when hHSFs cells simultaneously overexpressed miR-211-5p and TGFβR2, the promotion effect of TGFβR2 on cells was reversed by miR-211-5p (all P<0.05).ConclusionsmiR-211-5p can inhibit the activation of the TGF-βR2/Smad3 signaling pathway by targeting TGFβR2, thereby suppressing the proliferation, migration, invasion, and ECM production of hHSFs, and inducing their apoptosis, suggesting that miR-211-5p can become a potential target for the treatment of HS.

Dataset Information

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

Publications

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

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