RGS3 inhibits TGF‐β1/Smad signalling in adventitial fibroblasts

Recent evidence suggests that adventitial fibroblasts (AFs) are crucially implicated in atherosclerosis. However, the mechanisms by which AFs are dysfunctional and contribute to atherosclerosis remain unclear. This study aimed to investigate the role of regulator of G‐protein signalling 3 (RGS3) in the regulation of AFs using apoE knockout mouse as the model. Pathological changes in aortic arteries of apoE knockout mice fed with hyperlipid diet were examined by Movat staining. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in the adventitia was detected by immunohistochemistry. Adventitial fibroblasts were isolated from aortic arteries of apoE knockout mice and infected with RGS3 overexpression lentivirus or empty lentivirus. The expression of RGS3, α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs was detected by real‐time polymerase chain reaction and Western blot analysis. We found that hyperlipidic diet caused significant aortic intima thickening and atherosclerotic plaques in 15‐week‐old apoE knockout mice. Compared to wild‐type mice, RGS3 expression was lower while α‐SMA, TGF‐β1, Smad2, and Smad3 expression was higher in the adventitia of apoE knockout mice. In addition, lentivirus mediated overexpression of RGS3 caused decreased expression of α‐SMA, TGF‐β1, Smad2, and Smad3 in AFs derived from apoE(?/?) mice. In conclusion, these results suggest that RGS3 may provide protection against pathological changes of AFs and the development of atherosclerosis by inhibiting TGF‐β1/Smad signalling. RGS3 may be a potential therapeutic target for atherosclerosis.     

Periostin down‐regulation attenuates the pro‐fibrogenic response of hepatic stellate cells induced by TGF‐β1

Liver fibrosis is characterized by an exacerbated accumulation of deposition of the extracellular matrix (ECM), and the activation of hepatic stellate cells (HSC) plays a pivotal role in the development of liver fibrosis. Periostin has been shown to regulate cell adhesion, proliferation, migration and apoptosis; however, the involvement of periostin and its role in transforming growth factor (TGF)‐β1‐induced HSC activation remains unclear. We used RT‐PCR and Western blot to evaluate the expression level of periostin in hepatic fibrosis tissues and HSCs, respectively. Cell proliferation was determined using the Cell Proliferation ELISA BrdU kit, cell cycle was analysed by flow cytometry. The expression of α‐smooth muscle actin (α‐SMA), collagen I, TGF‐β1, p‐Smad2 and p‐Smad3 were determined by western blot. Our study found that periostin was up‐regulated in liver fibrotic tissues and activated HSCs. In addition, siRNA‐periostin suppressed TGF‐β1‐induced HSC proliferation. The HSC transfected with siRNA‐periostin significantly inhibited TGF‐β1‐induced expression levels of α‐SMA and collagen I. Furthermore, TGF‐β1 stimulated the expression of periostin, and siRNA‐periostin attenuated TGF‐β1‐induced Smad2/3 activation in HSCs. These results suggest that periostin may function as a novel regulator to modulate HSC activation, potentially by promoting the TGF‐β1/Smad signalling pathway, and propose a strategy to target periostin for the treatment of liver fibrosis.     

Lipopolysaccharide enhances TGF‐β1 signalling pathway and rat pancreatic fibrosis

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor‐beta1 (TGF‐β1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF‐β1 signalling and pancreatic fibrosis. Sprague‐Dawley rats fed with a Lieber‐DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP‐2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF‐β1 which was paralleled by an increased number of TLR4‐positive or TGF‐β1‐positive Mφs or PSCs in ALC‐fed rats. In vitro, TLR4 or TGF‐β1 production in Mφs or RP‐2 cells was up‐regulated by LPS. LPS alone or in combination with TGF‐β1 significantly increased type I collagen and α‐SMA production and Smad2 and 3 phosphorylation in serum‐starved RP‐2 cells. TGF‐β pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si‐TLR4 RNA or by inhibitors of MyD88/NF‐kB. Additionally, knockdown of Bambi with Si‐Bambi RNA significantly increased TGF‐β1 signalling in RP‐2 cells. These findings indicate that LPS increases TGF‐β1 production through paracrine and autocrine mechanisms and that LPS enhances TGF‐β1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF‐kB activation.     

Distribution of TGF‐β isoforms and signaling intermediates in corneal fibrotic wound repair

In this study, temporal and spatial distribution of three TGF‐β isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF‐β1, ‐2, and ‐3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF‐β1 was primarily deposited in the fibrin clot and the unwounded BL. TGF‐β2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF‐β3 was mainly detected in the unwound region of basal epithelial cells. α‐Smooth muscle actin (α‐SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, α‐SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF‐β2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF‐β2 were released into the posterior region of healing stroma on day 14. High levels of α‐SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF‐β2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF‐β2‐mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo. J. Cell. Biochem. 108: 476–488, 2009. © 2009 Wiley‐Liss, Inc.     

Acetyl‐11‐keto‐β‐boswellic acid ameliorates renal interstitial fibrosis via Klotho/TGF‐β/Smad signalling pathway

Acetyl‐11‐keto‐β‐boswellic acid (AKBA), an active triterpenoid compound from the extract of Boswellia serrate, has been reported previously in our group to alleviate fibrosis in vascular remodelling. This study aimed to elucidate the in vivo and in vitro efficacy and mechanism of AKBA in renal interstitial fibrosis. The experimental renal fibrosis was produced in C57BL/6 mice via unilateral ureteral obstruction (UUO). Hypoxia‐induced HK‐2 cells were used to imitate the pathological process of renal fibrosis in vitro. Results showed that the treatment of AKBA significantly alleviated UUO‐induced impairment of renal function and improved the renal fibrosis by decreasing the expression of TGF‐β1, α‐SMA, collagen I and collagen IV in UUO kidneys. In hypoxia‐induced HK‐2 cells, AKBA displayed remarkable cell protective effects and anti‐fibrotic properties by increasing the cell viability, decreasing the lactate dehydrogenase (LDH) release and inhibiting fibrotic factor expression. Moreover, in obstructed kidneys and HK‐2 cells, AKBA markedly down‐regulated the expression of TGFβ‐RI, TGFβ‐RII, phosphorylated‐Smad2/3 (p‐Smad2/3) and Smad4 in a dose‐dependent fashion while up‐regulated the expression of Klotho and Smad7 in the same manner. In addition, the effects of AKBA on the Klotho/TGF‐β/Smad signalling were reversed by transfecting with siRNA‐Klotho in HK‐2 cells. In conclusion, our findings provide evidence that AKBA can effectively protect kidney against interstitial fibrosis, and this renoprotective effect involves the Klotho/TGF‐β/Smad signalling pathway. Therefore, AKBA could be considered as a promising candidate drug for renal interstitial fibrosis.     

MeCP2‐421‐mediated RPE epithelial‐mesenchymal transition and its relevance to the pathogenesis of proliferative vitreoretinopathy

Proliferative vitreoretinopathy (PVR) is a blinding eye disease. Epithelial‐mesenchymal transition (EMT) of RPE cells plays an important role in the pathogenesis of PVR. In the current study, we sought to investigate the role of the methyl‐CpG‐binding protein 2 (MeCP2), especially P‐MeCP2‐421 in the pathogenesis of PVR. The expressions of P‐MeCP2‐421, P‐MeCP2‐80, PPAR‐γ and the double labelling of P‐MeCP2‐421 with α‐SMA, cytokeratin, TGF‐β and PPAR‐γ in human PVR membranes were analysed by immunohistochemistry. The effect of knocking down MeCP2 using siRNA on the expressions of α‐SMA, phospho‐Smad2/3, collagen I, fibronectin and PPAR‐γ; the expression of α‐SMA stimulated by recombinant MeCP2 in ARPE‐19; and the effect of TGF‐β and 5‐AZA treatment on PPAR‐γ expression were analysed by Western blot. Chromatin immunoprecipitation was used to determine the binding of MeCP2 to TGF‐β. Our results showed that P‐MeCP2‐421 was highly expressed in PVR membranes and was double labelled with α‐SMA, cytokeratin and TGF‐β, knocking down MeCP2 inhibited the activation of Smad2/3 and the expression of collagen I and fibronectin induced by TGF‐β. TGF‐β inhibited the expression of PPAR‐γ, silence of MeCP2 by siRNA or using MeCP2 inhibitor (5‐AZA) increased the expression of PPAR‐γ. α‐SMA was up‐regulated by the treatment of recombinant MeCP2. Importantly, we found that MeCP2 bound to TGF‐β as demonstrated by Chip assay. The results suggest that MeCP2 especially P‐MeCP2‐421 may play a significant role in the pathogenesis of PVR and targeting MeCP2 may be a potential therapeutic approach for the treatment of PVR.     

Activation of Smad‐mediated TGF‐β signaling triggers epithelial–mesenchymal transitions in murine cloned corneal progenitor cells

Epithelial–mesenchymal transition (EMT), via activation of Wnt signaling, is prevailing in embryogenesis, but postnatally it only occurs in pathological processes, such as in tissue fibrosis and tumor metastasis. Our prior studies led us to speculate that EMT might be involved in the loss of limbal epithelial stem cells in explant cultures. To examine this hypothesis, we successfully grew murine corneal/limbal epithelial progenitors by prolonging the culture time and by seeding at a low density in a serum‐free medium. Single cell‐derived clonal growth was accompanied by a gradient of Wnt signaling activity, from the center to the periphery, marked by a centrifugal loss of E‐cadherin and β‐catenin from intercellular junctions, coupled with nuclear translocation of β‐catenin and LEF‐1. Large‐colony‐forming efficiency at central location of colony was higher than peripheral location. Importantly, there was also progressive centrifugal differentiation, with positive K14 keratin expression and the loss of p63 and PCNA nuclear staining, and irreversible EMT, evidenced by cytoplasmic expression of α‐SMA and nuclear localization of S100A4; and by nuclear translocation of Smad4. Furthermore, cytoplasmic expression of α‐SMA was promoted by high‐density cultures and their conditioned media, which contained cell density‐dependent levels of TGF‐β1, TGF‐β2, GM‐CSF, and IL‐1α. Exogenous TGF‐β1 induced α‐SMA positive cells in a low‐density culture, while TGF‐β1 neutralizing antibody partially inhibited α‐SMA expression in a high‐density culture. Collectively, these results indicate that irreversible EMT emerges in the periphery of clonal expansion where differentiation and senescence of murine corneal/limbal epithelial progenitors occurs as a result of Smad‐mediated TGF‐β‐signaling. J. Cell. Physiol. 228: 225–234, 2013. © 2012 Wiley Periodicals, Inc.     

MicroRNA‐144‐3p suppressed TGF‐β1‐induced lung cancer cell invasion and adhesion by regulating the Src–Akt–Erk pathway

Lung cancer remains a leading cause to cancer‐related death worldwide. The anti‐cancer ability of microRNA‐144‐3p has been reported in many cancer types. This study focused on the mechanisms underlying miR‐144‐3p in inhibiting lung cancer. The expression levels of miR‐144‐3p and steroid receptor coactivator (Src) in different lung cancer cell lines and those in bronchial epithelial cells (16HBE) were compared. miR‐144‐3p mimic and siSrc were transfected into A549 cells. Under the conditions of transforming growth factor‐β1 (TGF‐β1). Small interfering transfection or TGF‐β1 treatment, cell invasive and adhesive abilities were analyzed by Transwell and cell adhesion assays. miR‐144‐3p inhibitor and siSrc were co‐transfected into A549 cells and the changes in cell invasion and adhesion were detected. The activation of Src–protein kinase B–extracellular‐regulated protein kinases (Src–Akt–Erk) pathway was determined using Western blot. The downregulated miR‐144‐3p and upregulated Src were generally detected in lung cancer cell lines and were the most significant genes in A549 cells. Both miR‐144‐3p overexpression and Src inhibition could obviously inhibit the invasion and adhesion abilities of A549 cells in the presence or absence of the effects of TGF‐β1. The inhibition of Src could block the promotive effects of miR‐144‐3p inhibitor and TGF‐β1 on cell invasion and adhesion. Furthermore, we found that miR‐144‐3p could negatively regulate the phosphorylation levels of Akt and Erk. Our data indicated the essential role of Src in the mechanisms underlying TGF‐β1‐induced cell invasion and adhesion of lung cancer, and that miR‐144‐3p could effectively suppress TGF‐β1‐induced aggressive lung cancer cells by regulating Src expression.     

PLK1 regulates hepatic stellate cell activation and liver fibrosis through Wnt/β‐catenin signalling pathway

As an outcome of chronic liver disease, liver fibrosis involves the activation of hepatic stellate cells (HSCs) caused by a variety of chronic liver injuries. It is important to explore approaches to inhibit the activation and proliferation of HSCs for the treatment of liver fibrosis. PLK1 is overexpressed in many human tumour cells and has become a popular drug target in tumour therapy. Therefore, further study of the function of PLK1 in the cell cycle is valid. In the present study, we found that PLK1 expression was elevated in primary HSCs isolated from CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. Knockdown of PLK1 inhibited α‐SMA and Col1α1 expression and reduced the activation of HSCs in CCl₄‐induced liver fibrosis mice and LX‐2 cells stimulated with TGF‐β1. We further showed that inhibiting the expression of PLK1 reduced the proliferation of HSCs and promoted HSCs apoptosis in vivo and in vitro. Furthermore, we found that the Wnt/β‐catenin signalling pathway may be essential for PLK1‐mediated HSCs activation. Together, blocking PLK1 effectively suppressed liver fibrosis by inhibiting HSC activation, which may provide a new treatment strategy for liver fibrosis.     

HYDAMTIQ,a selective PARP‐1 inhibitor,improves bleomycin‐induced lung fibrosis by dampening the TGF‐β/SMAD signalling pathway

Idiopathic pulmonary fibrosis is a severe disease characterized by excessive myofibroblast proliferation, extracellular matrix and fibrils deposition, remodelling of lung parenchyma and pulmonary insufficiency. Drugs able to reduce disease progression are available, but therapeutic results are unsatisfactory; new and safe treatments are urgently needed. Poly(ADP‐ribose) polymerases‐1 (PARP‐1) is an abundant nuclear enzyme involved in key biological processes: DNA repair, gene expression control, and cell survival or death. In liver and heart, PARP‐1 activity facilitates oxidative damage, collagen deposition and fibrosis development. In this study, we investigated the effects of HYDAMTIQ, a potent PARP‐1 inhibitor, in a murine model of lung fibrosis. We evaluated the role of PARP on transforming growth factor‐β (TGF‐β) expression and TGF‐β/SMAD signalling pathway in lungs. Mice were intratracheally injected with bleomycin and then treated with either vehicle or different doses of HYDAMTIQ for 21 days. Airway resistance to inflation and lung static compliance, markers of lung stiffness, were assayed. Histochemical and biochemical parameters to evaluate TGF‐β/SMAD signalling pathway with alpha‐smooth muscle actin (αSMA) deposition and the levels of a number of inflammatory markers (tumour necrosis factor‐α, interleukin‐1β, iNOS and COX‐2) were performed. Bleomycin administration increased lung stiffness. It also increased lung PARP activity, TGF‐β levels, pSMAD3 expression, αSMA deposition and content of inflammatory markers. HYDAMTIQ attenuated all the above‐mentioned physiological, biochemical and histopathological markers. Our findings support the proposal that PARP inhibitors could have a therapeutic potential in reducing the progression of signs and symptoms of the disease by decreasing TGF‐β expression and the TGF‐β/SMAD transduction pathway.     

MMP‐13 deletion decreases profibrogenic molecules and attenuates N‐nitrosodimethylamine‐induced liver injury and fibrosis in mice

Connective tissue growth factor (CTGF) is involved in inflammation, pathogenesis and progression of liver fibrosis. Matrix metalloproteinase‐13 (MMP‐13) cleaves CTGF and releases several fragments, which are more potent than the parent molecule to induce fibrosis. The current study was aimed to elucidate the significance of MMP‐13 and CTGF and their downstream effects in liver injury and fibrosis. Hepatic fibrosis was induced using intraperitoneal injections of N‐nitrosodimethylamine (NDMA) in doses of 10 μg/g body weight on three consecutive days of each week over a period of 4 weeks in both wild‐type (WT) and MMP‐13 knockout mice. Administration of NDMA resulted in marked elevation of AST, ALT, TGF‐β1 and hyaluronic acid in the serum and activation of stellate cells, massive necrosis, deposition of collagen fibres and increase in total collagen in the liver of WT mice with a significant decrease in MMP‐13 knockout mice. Protein and mRNA levels of CTGF, TGF‐β1, α‐SMA and type I collagen and the levels of MMP‐2, MMP‐9 and cleaved products of CTGF were markedly increased in NDMA‐treated WT mice compared to the MMP‐13 knockout mice. Blocking of MMP‐13 with CL‐82198 in hepatic stellate cell cultures resulted in marked decrease of the staining intensity of CTGF as well as protein levels of full‐length CTGF and its C‐terminal fragments and active TGF‐β1. The data demonstrate that MMP‐13 and CTGF play a crucial role in modulation of fibrogenic mediators and promote hepatic fibrogenesis. Furthermore, the study suggests that blocking of MMP‐13 and CTGF has potential therapeutic implications to arrest liver fibrosis.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

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.