Project description:Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β-catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt-mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor-β (TGF-β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α-SMA and collagen I) and the TGF-β signalling pathway that include smad2/3 and its phosphorylated form p-smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β-catenin revealed epithelial-mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β-catenin in regulation of the signalling network, which acts to counteract autocrine TGF-β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1-smad2/3 signalling through Wnt/β-catenin contribute to lung fibrosis.
Project description:Subretinal fibrosis remains a major obstacle to the management of neovascular age-related macular degeneration. Choroidal pericytes were found to be a significant source of subretinal fibrosis, but the underlying mechanisms of pericyte-myofibroblast transition (PMT) remain largely unknown. The goal of this study was to explore the role and potential mechanisms by which PMT contributes to subretinal fibrosis. Choroidal neovascularization (CNV) was induced by laser photocoagulation in transgenic mice with the collagen1α1-green fluorescent protein (Col1α1-GFP) reporter, and recombinant adeno-associated virus 2 (rAAV2)-mediated TGF-β2 (rAAV2-TGF-β2) was administered intravitreally to further induce PMT. Primary mouse choroidal GFP-positive pericytes were treated with TGF-β2 in combination with siRNAs targeting Smad2/3, the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin to examine cell proliferation, migration, and differentiation into myofibroblasts. The involvement of the Akt/mTOR pathway in PMT in subretinal fibrosis was further investigated in vivo. Intraocular TGF-β2 overexpression induced GFP-positive pericyte infiltration and PMT in subretinal fibrosis, which was mimicked in vitro. Knockdown of Smad2/3 or inhibition of Akt/mTOR decreased cell proliferation, PMT and migration in primary mouse pericytes. Combined inhibition of Smad2/3 and mTOR showed synergistic effects on attenuating α-smooth muscle actin (α-SMA) expression and cell proliferation. In mice with laser-induced CNV, the administration of the Akt/mTOR inhibitors suppressed pericyte proliferation and alleviated the severity of subretinal fibrosis. Our results showed that PMT plays a pivotal role in subretinal fibrosis, which was induced by TGF-β2 through the Smad2/3 and Akt/mTOR pathways. Thus, inhibiting PMT may be a novel strategy for the treatment of subretinal fibrosis.
Project description:Pulmonary fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc) with no effective medication. Polyporus polysaccharide (PPS), extracted from Chinese herbs, has immune regulation, anticancer, antioxidant and antiinflammatory activities. This study aims to investigate antifibrotic effects of PPS. We show that PPS markedly ameliorates bleomycin-induced lung fibrosis in mice. Myofibroblasts are the effector cells responsible for excessive deposition of extracellular matrix (ECM) proteins in fibrotic diseases. In vitro evidence reveals that PPS exerts potent antifibrotic effects by inhibiting fibroblast-to-myofibroblast transition, suppressing ECM deposition, and repressing lung fibroblast proliferation and migration. We also find that PPS inhibits TGF-β1-induced Smad2/3 activating. This study is the first to demonstrate an antifibrotic role of PPS in lungs, thus warranting further therapeutic evaluation.
Project description:Airway remodelling with subepithelial fibrosis, which abolishes the physiological functions of the bronchial wall, is a major issue in bronchial asthma. Human bronchial fibroblasts (HBFs) derived from patients diagnosed with asthma display in vitro predestination towards TGF-β1-induced fibroblast-to-myofibroblast transition (FMT), a key event in subepithelial fibrosis. As commonly used anti-asthmatic drugs do not reverse the structural changes of the airways, and the molecular mechanism of enhanced asthma-related TGF-β1-induced FMT is poorly understood, we investigated the balance between the profibrotic TGF-β/Smad2/3 and the antifibrotic TGF-β/Smad1/5/9 signalling pathways and its role in the myofibroblast formation of HBF populations derived from asthmatic and non-asthmatic donors. Our findings showed for the first time that TGF-β-induced activation of the profibrotic Smad2/3 signalling pathway was enhanced, but the activation of the antifibrotic Smad1/5/(8)9 pathway by TGF-β1 was significantly diminished in fibroblasts from asthmatic donors compared to those from their healthy counterparts. The impairment of the antifibrotic TGF-β/Smad1/5/(8)9 pathway in HBFs derived from asthmatic donors was correlated with enhanced FMT. Furthermore, we showed that Smad1 silencing in HBFs from non-asthmatic donors increased the FMT potential in these cells. Additionally, we demonstrated that activation of antifibrotic Smad signalling via BMP7 or isoliquiritigenin [a small-molecule activator of the TGF-β/Smad1/5/(8)9 pathway] administration prevents FMT in HBFs from asthmatic donors through downregulation of profibrotic genes, e.g., α-SMA and fibronectin. Our data suggest that influencing the balance between the antifibrotic and profibrotic TGF-β/Smad signalling pathways using BMP7-mimetic compounds presents an unprecedented opportunity to inhibit subepithelial fibrosis during airway remodelling in asthma.
Project description:Smad2 and Smad3 (Smad2/3) are structurally similar proteins that primarily mediate the transforming growth factor-β (TGF-β) signaling responsible for driving cell proliferation, differentiation, and migration. The dynamics of the Smad2/3 phosphorylation provide the key mechanism for regulating the TGF-β signaling pathway, but the details surrounding this phosphorylation remain unclear. Here, using in vitro kinase assay coupled with mass spectrometry, we identified for the first time that nemo-like kinase (NLK) regulates TGF-β signaling via modulation of Smad2/3 phosphorylation in the linker region. TGF-β-mediated transcriptional and cellular responses are suppressed by NLK overexpression, whereas NLK depletion exerts opposite effects. Specifically, we discovered that NLK associates with Smad3 and phosphorylates the designated serine residues located in the linker region of Smad2 and Smad3, which inhibits phosphorylation at the C terminus, thereby decreasing the duration of TGF-β signaling. Overall, this work demonstrates that phosphorylation on the linker region of Smad2/3 by NLK counteracts the canonical phosphorylation in response to TGF-β signals, thus providing new insight into the mechanisms governing TGF-β signaling transduction.
Project description:Sphingosine kinase 1 (SPHK1), the enzyme that produces sphingosine 1 phosphate (S1P), is known to be highly expressed in many cancers. However, the role of SPHK1 in cells of the tumor stroma remains unclear. Here, we show that SPHK1 is highly expressed in the tumor stroma of high-grade serous ovarian cancer (HGSC), and is required for the differentiation and tumor promoting function of cancer-associated fibroblasts (CAFs). Knockout or pharmacological inhibition of SPHK1 in ovarian fibroblasts attenuated TGF-β-induced expression of CAF markers, and reduced their ability to promote ovarian cancer cell migration and invasion in a coculture system. Mechanistically, we determined that SPHK1 mediates TGF-β signaling via the transactivation of S1P receptors (S1PR2 and S1PR3), leading to p38 MAPK phosphorylation. The importance of stromal SPHK1 in tumorigenesis was confirmed in vivo, by demonstrating a significant reduction of tumor growth and metastasis in SPHK1 knockout mice. Collectively, these findings demonstrate the potential of SPHK1 inhibition as a novel stroma-targeted therapy in HGSC.
Project description:Corneal scarring, whether caused by trauma, laser refractive surgery, or infection, remains a significant problem for humans. Certain ligands for peroxisome proliferator-activated receptor gamma (PPARγ) have shown promise as antiscarring agents in a variety of body tissues. In the cornea, their relative effectiveness and mechanisms of action are still poorly understood. Here, we contrasted the antifibrotic effects of three different PPARγ ligands (15-deoxy-Δ12,14-prostaglandin J2, troglitazone, and rosiglitazone) in cat corneal fibroblasts. Western blot analyses revealed that all three compounds reduced transforming growth factor (TGF)-β1-driven myofibroblast differentiation and up-regulation of α-smooth muscle actin, type I collagen, and fibronectin expression. Because these effects were independent of PPARγ, we ascertained whether they occurred by altering phosphorylation of Smads 2/3, p38 mitogen-activated protein kinase, stress-activated protein kinase, protein kinase B, extracellular signal-regulated kinase, and/or myosin light chain 2. Only p38 mitogen-activated protein kinase phosphorylation was significantly inhibited by all three PPARγ ligands. Finally, we tested the antifibrotic potential of troglitazone in a cat model of photorefractive keratectomy-induced corneal injury. Topical application of troglitazone significantly reduced α-smooth muscle actin expression and haze in the stromal ablation zone. Thus, the PPARγ ligands tested here showed great promise as antifibrotics, both in vitro and in vivo. Our results also provided new evidence for the signaling pathways that may underlie these antifibrotic actions in corneal fibroblasts.
Project description:The master cytokine TGF-β mediates tissue fibrosis associated with inflammation and tissue injury. TGF-β induces fibroblast activation and differentiation into myofibroblasts that secrete extracellular matrix proteins. Canonical TGF-β signaling mobilizes Smad2 and Smad3 transcription factors that control fibrosis by promoting gene expression. However, the importance of TGF-β-Smad2/3 signaling in fibroblast-mediated cardiac fibrosis has not been directly evaluated in vivo. Here, we examined pressure overload-induced cardiac fibrosis in fibroblast- and myofibroblast-specific inducible Cre-expressing mouse lines with selective deletion of the TGF-β receptors Tgfbr1/2, Smad2, or Smad3. Fibroblast-specific deletion of Tgfbr1/2 or Smad3, but not Smad2, markedly reduced the pressure overload-induced fibrotic response as well as fibrosis mediated by a heart-specific, latency-resistant TGF-β mutant transgene. Interestingly, cardiac fibroblast-specific deletion of Tgfbr1/2, but not Smad2/3, attenuated the cardiac hypertrophic response to pressure overload stimulation. Mechanistically, loss of Smad2/3 from tissue-resident fibroblasts attenuated injury-induced cellular expansion within the heart and the expression of fibrosis-mediating genes. Deletion of Smad2/3 or Tgfbr1/2 from cardiac fibroblasts similarly inhibited the gene program for fibrosis and extracellular matrix remodeling, although deletion of Tgfbr1/2 uniquely altered expression of an array of regulatory genes involved in cardiomyocyte homeostasis and disease compensation. These findings implicate TGF-β-Smad2/3 signaling in activated tissue-resident cardiac fibroblasts as principal mediators of the fibrotic response.
Project description:TGFβ is the quintessential cytokine of T cell homeostasis. TGFβ signaling is required for the efficient differentiation and maintenance of CD4(+)FOXP3(+) T cells that inhibit immune responses. Conversely, in conjunction with the inflammatory cytokine IL-6, TGFβ promotes Th17 cell differentiation. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ-signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. Although mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-deficient T cells are impaired in their response to TGFβ in vitro and in vivo, and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is uniquely essential for TGFβ signaling in CD4(+) T effector cell differentiation.
Project description:Drosophila melted encodes a pleckstrin homology (PH) domain-containing protein that enables normal tissue growth, metabolism, and photoreceptor differentiation by modulating Forkhead box O (FOXO), target of rapamycin, and Hippo signaling pathways. Ventricular zone expressed PH domain-containing 1 (VEPH1) is the mammalian ortholog of melted, and although it exhibits tissue-restricted expression during mouse development and is potentially amplified in several human cancers, little is known of its function. Here we explore the impact of VEPH1 expression in ovarian cancer cells by gene-expression profiling. In cells with elevated VEPH1 expression, transcriptional programs associated with metabolism and FOXO and Hippo signaling were affected, analogous to what has been reported for Melted. We also observed altered regulation of multiple transforming growth factor-β (TGF-β) target genes. Global profiling revealed that elevated VEPH1 expression suppressed TGF-β-induced transcriptional responses. This inhibitory effect was verified on selected TGF-β target genes and by reporter gene assays in multiple cell lines. We further demonstrated that VEPH1 interacts with TGF-β receptor I (TβRI) and inhibits nuclear accumulation of activated Sma- and Mad-related protein 2 (SMAD2). We identified two TβRI-interacting regions (TIRs) with opposing effects on TGF-β signaling. TIR1, located at the N terminus, inhibits canonical TGF-β signaling and promotes SMAD2 retention at TβRI, similar to full-length VEPH1. In contrast, TIR2, located at the C-terminal region encompassing the PH domain, decreases SMAD2 retention at TβRI and enhances TGF-β signaling. Our studies indicate that VEPH1 inhibits TGF-β signaling by impeding the release of activated SMAD2 from TβRI and may modulate TGF-β signaling during development and cancer initiation or progression.