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TGF-β1-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts.

ABSTRACT: HSP47 is required for the production of collagen and serves an important role in tissue remodeling, a pathophysiologic mechanism of chronic rhinosinusitis (CRS). We investigated the relationship between HSP47 expression and tissue remodeling in CRS. We also determined the underlying molecular mechanisms of TGF-β1-induced HSP47 and extracellular matrix (ECM) production in nasal fibroblasts. HSP47, α-SMA, fibronectin, and collagen type I expression levels were measured using real-time PCR, western blotting, and immunofluorescence staining. Fibroblast migration was analyzed using scratch and transwell migration assays. Contractile activity was measured with a collagen gel contraction assay. HSP47 is increased in patients with CRS without nasal polyps. TGF-β1 induced HSP47 expression in nasal fibroblasts. Myofibroblast differentiation and ECM production, which are induced by TGF-β1, were inhibited by siHSP47. We also confirmed that the Smad2/3 signaling pathway is involved in TGF-β1-induced HSP47 expression in nasal fibroblasts. In a functional assay, TGF-β1-enhanced migration and contraction ability were inhibited by HSP47 knockout. Glucocorticoid reversed the stimulatory effects of TGF-β1 on HSP47 expression and ECM production in nasal fibroblasts and ex vivo organ cultures. HSP47 expression is involved in TGF-β1-induced myofibroblast differentiation and ECM production through the Smad2/3 signaling pathway, which might contribute to tissue remodeling in chronic rhinosinusitis.

SUBMITTER: Kim HJ

PROVIDER: S-EPMC6820875 | biostudies-other | 2019 Oct

REPOSITORIES: biostudies-other

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TGF-β1-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts.

Kim Hae-Ji HJ Park Joo-Hoo JH Shin Jae-Min JM Yang Hyun-Woo HW Lee Heung-Man HM Park Il-Ho IH

Scientific reports 20191029 1

HSP47 is required for the production of collagen and serves an important role in tissue remodeling, a pathophysiologic mechanism of chronic rhinosinusitis (CRS). We investigated the relationship between HSP47 expression and tissue remodeling in CRS. We also determined the underlying molecular mechanisms of TGF-β1-induced HSP47 and extracellular matrix (ECM) production in nasal fibroblasts. HSP47, α-SMA, fibronectin, and collagen type I expression levels were measured using real-time PCR, western ...[more]

PMID: 31664133

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Project description:Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growth factor beta 1, and longitudinally quantified cell phenotype by measuring expression of α-smooth muscle actin, the most robust indicator of myofibroblasts. Our data indicate that, although extracellular matrix rigidity influenced differentiation after one day of transforming growth factor beta 1 treatment, ultimately transforming growth factor beta 1 superseded extracellular matrix rigidity as the primary regulator of myofibroblast differentiation. We also measured expression of POSTN, FAP, and FSP1, proposed secondary indicators of fibroblast/myofibroblast phenotypes. Although these genes partially trended with α-smooth muscle actin expression, they were relatively inconsistent. Finally, we demonstrated that activated myofibroblasts incompletely revert to a fibroblast phenotype after they are re-plated onto new surfaces without transforming growth factor beta 1, suggesting differentiation is partially reversible. Our results provide new insights into how microenvironmental cues affect human cardiac fibroblast differentiation in the context of myocardial pathology, which is important for identifying effective therapeutic targets and dictating supporting cell phenotypes for engineered human cardiac disease models. Impact statement Heart disease is the leading cause of death worldwide. Many forms of heart disease are associated with fibrosis, which increases extracellular matrix (ECM) rigidity and compromises cardiac output. Fibrotic tissue is synthesized primarily by myofibroblasts differentiated from fibroblasts. Thus, defining the cues that regulate myofibroblast differentiation is important for understanding the mechanisms of fibrosis. However, previous studies have focused on non-human cardiac fibroblasts and have not tested combinations of chemical and mechanical cues. We tested the effects of TGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidity on the differentiation of human cardiac fibroblasts to myofibroblasts. Our results indicate that differentiation is initially influenced by ECM rigidity, but is ultimately superseded by TGF-β1. This suggests that targeting TGF-β signaling pathways in cardiac fibroblasts may have therapeutic potential for attenuating fibrosis, even in rigid microenvironments. Additionally, our approach can be leveraged to engineer more precise multi-cellular human cardiac tissue models.

Project description:BackgroundExcessive cardiac fibrosis due to maladaptive remodeling leads to progression of cardiac dysfunction and is modulated by TGF-β1-activated intracellular phospho-SMAD signaling effectors and transcription regulators. SMAD2/3 phosphorylation, regulated by protein-phosphatases, has been studied in different cell types, but its role in human ventricular fibroblasts (hVFs) is not defined as a target to reduce cytokine-mediated excessive fibrotic response and adverse cardiac remodeling. Statins are a class of drugs reported to reduce cardiac fibrosis, although underlying mechanisms are not completely understood. We aimed to assess whether simvastatin-mediated reduction in TGF-β1-augmented profibrotic response involves reduction in phospho-SMAD2/3 owing to activation of protein-phosphatase in hVFs.Methods and resultsCultures of hVFs were used. Effect of simvastatin on TGF-β1-treated hVF proliferation, cytotoxicity, myofibroblast differentiation/activation, profibrotic gene expression and protein-phosphatase activity was assessed. Simvastatin (1 μM) reduced effect of TGF-β1 (5 ng/mL) on hVF proliferation, myofibroblast differentiation (reduced α-smooth muscle actin [α-SMA-expression]) and activation (decreased procollagen-peptide release). Simvastatin also reduced TGF-β1-stimulated time-dependent increases in SMAD2/3 phosphorylation and nuclear translocation, mediated through catalytic activation of protein-phosphatases PPM1A and PP2A, which physically interact with SMAD2/3, thereby promoting their dephosphorylation. Effect of simvastatin on TGF-β1-induced fibroblast activation was annulled by okadaic acid, an inhibitor of protein-phosphatase.ConclusionsThis proof-of-concept study using an in vitro experimental cell culture model identifies the protective role of simvastatin against TGF-β1-induced hVF transformation into activated myofibroblasts through activation of protein phosphatase, a novel target that can be therapeutically modulated to curb excessive cardiac fibrosis associated with maladaptive cardiac remodeling.

Dataset Information

TGF-β1-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts.

Publications

TGF-β1-induced HSP47 regulates extracellular matrix accumulation via Smad2/3 signaling pathways in nasal fibroblasts.

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