Lipid accumulation impairs adiponectin-mediated induction of activin A by increasing TGFbeta in primary human hepatocytes

Fatty liver is commonly detected in obesity and has been identified as a risk factor for the progression of hepatic fibrosis in a wide range of liver diseases. Transforming growth factor beta (TGFβ) and activin A, both members of the TGFβ superfamiliy, are central regulators in liver fibrosis and regeneration, and the effect of hepatocyte lipid accumulation on the release of these proteins was studied. Primary human hepatocytes (PHH) were incubated with palmitic acid or oleic acid to increase lipid storage. Whereas activin A and its natural inhibitor follistatin were not affected, TGFβ was 2-fold increased. The hepatoprotective adipokine adiponectin dose-dependently induced activin A while lowering follistatin but did not alter TGFβ. Activin A was markedly reduced in hepatocyte cell lines compared to PHH and was not induced upon adiponectin incubation demonstrating significant differences of primary and transformed cells. In free fatty acid (FFA)-incubated PHH adiponectin-mediated induction of activin A was impaired. Inhibition of TGFβ receptors ALK4/5 and blockage of SMAD3 phosphorylation rescued activin A synthesis in FFA and in TGFβ incubated cells suggesting that FFA inhibit adiponectin activity by inducing TGFβ. To evaluate whether serum levels of activin A and its antagonist are altered in patients with hepatic steatosis, both proteins were measured in the serum of patients with sonographically diagnosed fatty liver and age- and BMI-matched controls. Systemic adiponectin was significantly reduced in patients with fatty liver but activin A and follistatin were not altered. In summary the current data demonstrate that lipid accumulation in hepatocytes induces TGFβ which impairs adiponectin bioactivity, and thereby may contribute to liver injury.     

n-3 PUFAs inhibit TGFβ1-induced profibrogenic gene expression by ameliorating the repression of PPARγ in hepatic stellate cells

Activated hepatic stellate cells (HSCs) are primarily responsible for the accumulation of extracellular matrix substances during the development of liver fibrosis. It has been shown that n-3 polyunsaturated fatty acids (PUFAs) can prevent liver fibrosis development. However, the underlying mechanisms of action need further investigation. The objective of this study was to determine the regulatory roles of fatty acids (FAs) on the expression of profibrogenic genes in HSCs with the elucidation of mechanisms. LX-2 cells and primary human and mouse HSCs were treated with palmitic acid, oleic acid, linoleic acid, α-linolenic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) to determine their effect on profibrogenic gene expression upon the activation by transforming growth factor β1 (TGFβ1). PUFAs significantly suppressed TGFβ1-induced expression of profibrogenic genes in LX-2 and primary human HSCs with n-3 being more potent than n-6 PUFAs. However, PUFAs did not inhibit the phosphorylation and nuclear translocation of SMA- and MAD-related protein in primary human HSCs. Furthermore, PUFAs did not alter the profibrogenic gene expression in primary mouse HSCs. The inhibitory effect of EPA and DHA on TGFβ1-induced profibrogenic gene expression was diminished by peroxisome proliferator-activated receptor gamma (PPARG) knockdown, although chemical inhibition of PPARγ did not elicit a similar result. The results suggest that n-3 PUFAs possess the most potent protective effects against TGFβ1-induced profibrogenic gene expression, which is, at least in part, PPARγ-dependent in HSCs.     

Calcitonin gene-related peptide regulates mitogen-activated protein kinase pathway to decrease transforming growth factor β1-induced hepatic plasminogen activator inhibitor-1 mRNA expression in HepG2 cells

Transforming growth factor (TGF) β1-induced plasminogen activator inhibitor (PAI)-1 is one of factors associated with the development of hepatic fibrosis. Calcitonin gene-related peptide (CGRP) shows hepatoprotective effect during hepatic injuries, including fibrosis. However, the effects of CGRP on PAI-1 expression induced by TGFβ1 are unknown. In this study, we investigated the effect of CGRP on TGFβ1-induced PAI-1 expression and its regulatory mechanisms in HepG2 cells. CGRP inhibited TGFβ1-induced PAI-1 expression. H89, a protein kinase A inhibitor, abolished the inhibition of TGFβ1-induced PAI-1 expression by CGRP. TGFβ1 activated mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase, c-jun NH₂-terminal kinase, and p38, and this activation was abolished by CGRP. These results show that the CGRP-induced cAMP/PKA activation suppresses activation of MAPK induced by TGFβ1, leading to decreased PAI-1 expression in HepG2 cells.     

Antagonistic regulation of transmembrane 4 L6 family member 5 attenuates fibrotic phenotypes in CCl(4) -treated mice

The development of liver fibrosis from chronic inflammation can involve epithelial-mesenchymal transition (EMT). Severe liver fibrosis can progress to cirrhosis, and further to hepatocellular carcinoma. Because the tetraspanin transmembrane 4 L6 family member 5 (TM4SF5) induces EMT and is highly expressed in hepatocellular carcinoma, it is of interest to investigate whether TM4SF5 expression is correlated with EMT processes during the development of fibrotic liver features. Using hepatic cells in vitro and a CCl(4) -mediated mouse liver in?vivo model, we examined whether TM4SF5 is expressed during liver fibrosis mediated by CCl(4) administration and whether treatment with anti-TM4SF5 reagent blocks the fibrotic liver features. Here, we found that TM4SF5 expression was induced by the transforming growth factor (TGF)β1 and epidermal growth factor signaling pathways in hepatocytes in vitro. In the CCl(4) -mediated mouse liver model, TM4SF5 was expressed during the liver fibrosis mediated by CCl(4) administration and correlated with α-smooth muscle actin expression, collagen I deposition, and TGFβ1 and epidermal growth factor receptor signaling activation in fibrotic septa regions. Interestingly, treatment with anti-TM4SF5 reagent blocked the TM4SF5-mediated liver fibrotic features: the formation of fibrotic septa with α-smooth muscle actin expression and collagen I deposition was attenuated by treatment with anti-TM4SF5 reagent. These results suggest that TM4SF5 expression mediated by TGFβ1 and growth factor can facilitate fibrotic processes during chronic liver injuries. TM4SF5 is thus a candidate target for prevention of liver fibrosis following chronic liver injury.     

The role and mechanism of transforming growth factor beta 3 in human myocardial infarction‐induced myocardial fibrosis

Transforming growth factor beta (TGFβ) plays a crucial role in tissue fibrosis. A number of studies have shown that TGFβ3 significantly attenuated tissue fibrosis. However, the mechanism involved in this effect is poorly understood. In this study we found that the expression level of TGFβ3 was higher in human myocardial infarction (MI) tissues than in normal tissues, and interestingly, it increased with the development of fibrosis post‐myocardial infarction (post‐MI). In vitro, human cardiac fibroblasts (CFs) were incubated with angiotensin II (Ang II) to mimic the ischaemic myocardium microenvironment and used to investigate the anti‐fibrotic mechanism of TGFβ3. Then, fibrosis‐related proteins were detected by Western blot. It was revealed that TGFβ3 up‐regulation attenuated the proliferation, migration of human CFs and the expression of collagens, which are the main contributors to fibrosis, promoted the phenotype shift and the cross‐linking of collagens. Importantly, the expression of collagens was higher in the si‐smad7 groups than in the control groups, while silencing smad7 increased the phosphorylation level of the TGFβ/smad signalling pathway. Collectively, these results indicated that TGFβ3 inhibited fibrosis via the TGFβ/smad signalling pathway, possibly attributable to the regulation of smad7, and that TGFβ3 might serve as a potential therapeutic target for myocardial fibrosis post‐MI.     

Bromodomain protein 4 is a key molecular driver of TGFβ1-induced hepatic stellate cell activation

Liver fibrosis is characterized by the excessive deposition of extracellular matrix in liver. Chronic liver injury induces the activation of hepatic stellate cell (HSCs), a key step in liver fibrogenesis. The activated HSC is the primary source of ECM and contributes significantly to liver fibrosis. TGFβ1 is the most potent pro-fibrotic cytokine. Bromodomain protein 4 (BrD4), an epigenetic reader of histone acetylation marks, was crucial for profibrotic gene expression in HSCs. The present study aimed to investigate the roles of BRD4 in TGFβ1-dependent HSC activation and liver fibrosis, focusing on TGFβ1-induced alterations of the levels of the fibrotic-related important proteins in HSCs by employing the heterozygous TGFβ1 knockout mice and BrD4 knockdown in vivo and in vitro. Results revealed that BrD4 protein level was significantly upregulated by TGFβ1 and BrD4 knockdown reduced TGFβ1-induced HSC activation and liver fibrosis. BrD4 was required for the influences of TGFβ1 on PDGFβ receptor and on the pathways of Smad3, Stat3, and Akt. BrD4 also mediated TGFβ1-induced increases in histone acetyltransferase p300, the pivotal pro-inflammatory NFkB p65, and tissue inhibitor of metalloproteinase 1 whereas BrD4 reduced Caspase-3 protein levels in HSCs during liver injury, independent of TGFβ1. Further experiments indicated the interaction between TGFβ1-induced BrD4 and NFkB p65 in HSCs and in liver of TAA-induced liver injury. Human cirrhotic livers were demonstrated a parallel increase in the protein levels of BrD4 and NFkB p65 in HSCs. This study revealed that BrD4 was a key molecular driver of TGFβ1-induced HSC activation and liver fibrosis.     

Rapamycin inhibits transforming growth factor β-induced peritoneal angiogenesis by blocking the secondary hypoxic response

Patients with end-stage kidney disease on peritoneal dialysis often develop progressive scarring of the peritoneal tissues. This manifests as submesothelial thickening and is associated with increased vascularization that leads to ultrafiltration dysfunction. Hypoxia induces a characteristic series of responses including angiogenesis and fibrosis. We investigated the role of hypoxia in peritoneal membrane damage. An adenovirus expressing transforming growth factor (TGF) β was used to induce peritoneal fibrosis. We evaluated the effect of the mTOR inhibitor rapamycin, which has been previously shown to block hypoxia-inducible factor (HIF) 1α. We also assessed the effect of HIF1α independently using an adenovirus expressing active HIF1α. To identify the TGFβ1-independent effects of HIF1α, we expressed HIF1α in the peritoneum of mice lacking the TGFβ signalling molecule Smad3. We demonstrate that TGFβ-induced fibroproliferative tissue is hypoxic. Rapamycin did not affect the early angiogenic response, but inhibited angiogenesis and submesothelial thickening 21 days after induction of fibrosis. In primary mesothelial cell culture, rapamycin had no effect on TGFβ-induced vascular endothelial growth factor (VEGF) but did suppress hypoxia-induced VEGF. HIF1α induced submesothelial thickening and angiogenesis in peritoneal tissue. The fibrogenic effects of HIF1α were Smad3 dependent. In summary, submesothelial hypoxia may be an important secondary factor, which augments TGFβ-induced peritoneal injury. The hypoxic response is mediated partly through HIF1α and the mTOR inhibitor rapamycin blocks the hypoxic-induced angiogenic effects but does not affect the direct TGFβ-mediated fibrosis and angiogenesis.     

Syndecan-2 is a novel target of insulin-like growth factor binding protein-3 and is over-expressed in fibrosis

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3.     

Transglutaminase 2: a novel therapeutic target for idiopathic pulmonary fibrosis using selective small molecule inhibitors

This study investigates the effects of a site-directed TG2-selective inhibitor on the lung myofibroblast phenotype and ECM deposition to elucidate TG2 as a novel therapeutic target in idiopathic pulmonary fibrosis (IPF)—an incurable progressive fibrotic disease. IPF fibroblasts showed increased expression of TG2, α smooth muscle actin (αSMA) and fibronectin (FN) with increased extracellular TG2 and transforming growth factor β1 (TGFβ1) compared to normal human lung fibroblasts (NHLFs) which do not express αSMA and express lower levels of FN. The myofibroblast phenotype shown by IPF fibroblasts could be reversed by selective TG2 inhibition with a reduction in matrix FN and TGFβ1 deposition. TG2 transduction or TGFβ1 treatment of NHLFs led to a comparable phenotype to that of IPF fibroblasts which was reversible following selective TG2 inhibition. Addition of exogenous TG2 to NHLFs also induced the myofibroblast phenotype by a mechanism involving TGFβ1 activation which could be ameliorated by selective TG2 inhibition. SMAD3-deleted IPF fibroblasts via CRISPR-cas9 genome editing, showed reduced TG2 protein levels following TGFβ1 stimulation. This study demonstrates a key role for TG2 in the induction of the myofibroblast phenotype and shows the potential for TG2-selective inhibitors as therapeutic agents for the treatment of fibrotic lung diseases like IPF.

     

Decursin attenuates hepatic fibrogenesis through interrupting TGF-beta-mediated NAD(P)H oxidase activation and Smad signaling in vivo and in vitro

Aims

We studied that a potent antifibrotic effect of decursin on in vivo liver damage model and the mechanism in inhibiting which transforming growth factor (TGF)-β1-induced human hepatic stellate cells (HSCs) activation.

Main methods

Liver injury was induced in vivo by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without decursin for 4 weeks in mice. Human hepatic stellate cell line, an immortalized human HSC line, was used in in vitro assay system. The effects of decursin on HSC activation were measured by analyzing the expression of α-smooth muscle actin (α-SMA) and collagen I in liver tissue and human HSCs.

Key findings

Decursin treatment significantly reduced the ratio of liver/body weight, α-SMA activation, and type I collagen overexpression in CCl₄ treated mice liver. The elevated serum levels, including ALT, AST, and ALP, were also decreased by decursin treatment. Treatment of decursin markedly proved the generation of reactive oxygen species, NAD(P)H oxidase (NOX) protein (1, 2, and 4) upregulation, NOX activity, and superoxide anion production in HSCs by TGF-β1. It also significantly reduced TGF-β1-induced Smad 2/3 phosphorylation, nuclear translocation of Smad 4, and association of Smad 2/3–Smad 4 complex. Consistent with in vitro results, decursin treatment effectively blocked the levels of NOX protein, and Smad 2/3 phosphorylation in injured mice liver.

Significance

Decursin blocked CCl₄-induced liver fibrosis and inhibited TGF-β1-mediated HSC activation in vitro. These data demonstrated that decursin exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-β1 induced NOX activation and Smad signaling.     

A novel reciprocal loop between microRNA-21 and TGFβRIII is involved in cardiac fibrosis

Cardiac fibrosis is characterized by aberrant proliferation of cardiac fibroblasts and exaggerated deposition of extracellular matrix (ECM) in the myocardial interstitial, and ultimately impairs cardiac function. It is still controversial whether microRNA-21 (miR-21) participates in the process of cardiac fibrosis. Our previous study confirmed that transforming growth factor beta receptor III (TGFβRIII) is a negative regulator of TGF-β pathway. Here, we aimed to decipher the relationship between miR-21 and TGFβRIII in the pathogenic process of myocardial fibrosis. We found that TGF-β1 and miR-21 were up-regulated, whereas TGFβRIII was down-regulated in the border zone of mouse hearts in response to myocardial infarction. After transfection of miR-21 into cardiac fibroblasts, TGFβRIII expression was markedly reduced and collagen content was increased. And, luciferase results confirmed that TGFβRIII was a target of miR-21. It suggests that up-regulation of miR-21 could increase the collagen content and at least in part through inhibiting TGFβRIII. Conversely, we also confirmed that overexpression of TGFβRIII could inhibit the expression of miR-21 and reduce collagen production in fibroblasts. Further studies showed that overexpression of TGFβRIII could also deactivate TGF-β1 pathway by decreasing the expression of TGF-β1 and phosphorylated-Smad3 (p-Smad3). TGF-β1 has been proven as a positive regulator of miR-21. Taken together, we found a novel reciprocal loop between miR-21 and TGFβRIII in cardiac fibrosis caused by myocardial infarction in mice, and targeting this pathway could be a new strategy for the prevention and treatment of myocardial remodeling.     

Myocardial expression of transforming growth factor beta family and endothelin-1 in the progression from heart failure to ascites in broilers with cold-induced pulmonary hypertension

We determined mRNA expression of genes of endothelin-1 (ET-1), and of the transforming growth factor beta ligands (TGFβ1, TGFβ2 and TGFβ3), their receptors (TβRI and TβRII) and their pseudoreceptor BAMBI in the heart of broilers raised under cold temperature conditions and affected by pulmonary hypertension. Gene expression was determined by RT-qPCR in right myocardial ventricle samples from 4-week-old chickens (n?=?48) raised either under normal (control) or cold temperature conditions (22?°C versus 14?°C). We do not find differences among healthy birds, birds with cardiac failure and ascitic birds in the mRNA levels of TGFβ2, TGFβ3 and BAMBI. In the control group, ET-1 mRNA level was increased in the ascitic birds as compared with healthy birds and birds with cardiac failure (p?<?0.05) whereas in the cold treated group, no increase was observed (p?>?0.05); yet, ascitic birds in the cold group showed lower mean than ascitic birds in the control group (p?<?0.05). TβRII mRNA expression was higher in ascitic than in healthy birds (p?<?0.05) in both control and cold treated groups; however, in the ascitic birds of the cold treated group TβRII expression was lower than in ascitic birds from the control group (p?<?0.05). Thus, the higher ET-1 and TβRII levels observed in ascitic birds seem to be attenuated by cold.     

A novel molecular mechanism of microRNA‐21 inducing pulmonary fibrosis and human pulmonary fibroblast extracellular matrix through transforming growth factor β1–mediated SMADs activation

Pulmonary fibrosis (PF), characterized by the destruction of lung tissue architecture and the abnormal deposition of extracellular matrix (ECM) proteins, currently has no satisfactory treatment. The role of microRNA (miR)‐21 in PF has been reported; the current study attempted to investigate a novel molecular mechanism by which miR‐21 exerted its function. Consistent with previous studies, miR‐21 inhibition reduced ECM protein levels in bleomycin (BLM)‐induced mouse model of PF. In human pulmonary fibroblast (IMR‐90), miR‐21 inhibition reduced transforming growth factor β1 (TGFβ1)–induced ECM protein expression. Regarding a novel molecular mechanism, TGFβ1 combined with TGFβ1 receptor 1 (TGFβ1RI) to activate SMAD2/3, promote SMAD4 nucleus transformation, and thus regulate miR‐21 expression and ECM. SMAD3 and SMADs complex could bind to the promoter region of miR‐21 to promote miR‐21 expression. In conclusion, miR‐21 exerts promotive effects on BLM‐induced PF and TGFβ1‐induced ECM in IMR‐90; TGFβ1 combines with TGFβ1RI to activate SMAD2/3, promote SMAD4 nucleus transformation, promote miR‐21 expression, and thus to promote BLM‐induced PF and TGFβ1‐induced ECM in IMR‐90 cells.