Correlations between Transmembrane 4 L6 Family Member 5 (TM4SF5), CD151, and CD63 in Liver Fibrotic Phenotypes and Hepatic Migration and Invasive Capacities

Transmembrane 4 L6 family member 5 (TM4SF5) is overexpressed during CCl₄-mediated murine liver fibrosis and in human hepatocellular carcinomas. The tetraspanins form tetraspanin-enriched microdomains (TEMs) consisting of large membrane protein complexes on the cell surface. Thus, TM4SF5 may be involved in the signal coordination that controls liver malignancy. We investigated the relationship between TM4SF5-positive TEMs with liver fibrosis and tumorigenesis, using normal Chang hepatocytes that lack TM4SF5 expression and chronically TGFβ1-treated Chang cells that express TM4SF5. TM4SF5 expression is positively correlated with tumorigenic CD151 expression, but is negatively correlated with tumor-suppressive CD63 expression in mouse fibrotic and human hepatic carcinoma tissues, indicating cooperative roles of the tetraspanins in liver malignancies. Although CD151 did not control the expression of TM4SF5, TM4SF5 appeared to control the expression levels of CD151 and CD63. TM4SF5 interacted with CD151, and caused the internalization of CD63 from the cell surface into late lysosomal membranes, presumably leading to terminating the tumor-suppressive functions of CD63. TM4SF5 could overcome the tumorigenic effects of CD151, especially cell migration and extracellular matrix (ECM)-degradation. Taken together, TM4SF5 appears to play a role in liver malignancy by controlling the levels of tetraspanins on the cell surface, and could provide a promising therapeutic target for the treatment of liver malignancies.     

Lefty antagonises TGF-β1 induced epithelial-mesenchymal transition in tubular epithelial cells

Lefty is a novel member of the transforming growth factor (TGF) supergene family which has the potential to antagonise actions of TGF-β1 - the main factor driving fibrotic disease in the kidney and in other organs. TGF-β1 can induce fibrosis through several mechanisms, including epithelial-mesenchymal transition (EMT) which contributes to myofibroblast accumulation in the renal interstitium. This study examined whether Lefty can antagonise TGF-β1 mediated EMT. A rat tubular epithelial cell line (NRK52E) was stably transfected with a Lefty expression plasmid (52E-Lefty) or control plasmid (52E-Control). 52E-Control cells underwent TGF-β1 induced EMT with up-regulation of α-smooth muscle actin (α-SMA), down-regulation of E-cadherin, and transition to an elongated fibroblast-like morphology. In contrast, 52E-Lefty cells were substantially protected from TGF-β1 induced EMT. Analysis of signalling pathways showed that 52E-Lefty cells had a marked reduction in TGF-β1 induced Smad activity and suppression of the secondary phase of JNK (but not p38) signalling. Treatment of NRK52E cells with a JNK inhibitor was shown to suppress TGF-β1 induced EMT. In conclusion, Lefty can antagonise TGF-β1 mediated EMT in renal tubular epithelial cells. Lefty may have potential as an anti-fibrotic molecule in the treatment of renal fibrosis.     

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl₄-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl(4) in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl(4)-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.     

Gefitinib resistance of cancer cells correlated with TM4SF5-mediated epithelial-mesenchymal transition

Although cancers can be initially treated with the epidermal growth factor receptor (EGFR) inhibitor, gefitinib, continued gefitinib therapy does not benefit the survival of patients due to acquired resistance through EGFR mutations, c-MET amplification, or epithelial-mesenchymal transition (EMT). It is of further interest to determine whether mesenchymal-like, but not epithelial-like, cancer cells can become resistant to gefitinib by bypassing EGFR signaling and acquiring alternative routes of proliferative and survival signaling. Here we examined whether gefitinib resistance of cancer cells can be caused by transmembrane 4 L six family member 5 (TM4SF5), which has been shown to induce EMT via cytosolic p27Kip1 stabilization. Gefitinib-resistant cells exhibited higher and/or sustained TM4SF5 expression, cytosolic p27Kip1 stabilization, and mesenchymal phenotypes, compared with gefitinib-sensitive cells. Conversion of gefitinib-sensitive to -resistant cells by introduction of the T790M EGFR mutation caused enhanced and sustained expression of TM4SF5, phosphorylation of p27Kip1 Ser10 (responsible for cytosolic location), loss of E-cadherin from cell-cell contacts, and gefitinib-resistant EGFR and survival signaling activities. Additionally, TM4SF5 overexpression lessened the sensitivity of NSCLC cells to gefitinib. Suppression of TM4SF5 or p27Kip1 in gefitinib-resistant cells via the T790M EGFR mutation or TM4SF5 expression rendered them gefitinib-sensitive, displaying more epithelial-like and less mesenchymal-like characteristics. Together, these results indicate that TM4SF5-mediated EMT may have an important function in the gefitinib resistance of cancer cells.     

Proteasome inhibition causes epithelial-mesenchymal transition upon TM4SF5 expression

Transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in hepatocarcinoma and causes epithelial-mesenchymal transition (EMT) of hepatocytes. We found that TM4SF5-expressing cells showed lower mRNA levels but maintained normal protein levels in certain gene cases, indicating that TM4SF5 mediates stabilization of proteins. In this study, we explored whether regulation of proteasome activity and TM4SF5 expression led to EMT. We observed that TM4SF5 expression caused inhibition of proteasome activity and proteasome subunit expression, causing morphological changes and loss of cell-cell contacts. shRNA against TM4SF5 recovered proteasome expression, with leading to blockade of proteasome inactivation and EMT. Altogether, TM4SF5 expression appeared to cause loss of cell-cell adhesions via proteasome suppression and thereby proteasome inhibition, leading to repression of cell-cell adhesion molecules, such as E-cadherin.     

Adiponectin induces the transforming growth factor decoy receptor BAMBI in human hepatocytes

Transforming growth factor (TGF) β is the central cytokine in fibrotic liver diseases. We analyzed whether hepatoprotective adiponectin directly interferes with TGFβ1 signaling in primary human hepatocytes (PHH). Adiponectin induces the TGFβ decoy receptor BMP-and activin-membrane-bound inhibitor (BAMBI) in PHH. Overexpression of BAMBI in hepatoma cells impairs TGFβ-mediated phosphorylation of SMAD2 and induction of connective tissue growth factor. BAMBI is lower in human fatty liver with a higher susceptibility to liver fibrosis and negatively correlates with BMI of the donors. Hepatic BAMBI is reduced in rodent models of liver inflammation and fibrosis. In summary, the current data show that hepatoprotective effects of adiponectin include induction of BAMBI which is reduced in human fatty liver and rodent models of metabolic liver injury.     

V-ATPase promotes transforming growth factor-β-induced epithelial-mesenchymal transition of rat proximal tubular epithelial cells

The ubiquitous vacuolar H(+)-ATPase (V-ATPase), a multisubunit proton pump, is essential for intraorganellar acidification. Here, we hypothesized that V-ATPase is involved in the pathogenesis of kidney tubulointerstitial fibrosis. We first examined its expression in the rat unilateral ureteral obstruction (UUO) model of kidney fibrosis and transforming growth factor (TGF)-β1-mediated epithelial-to-mesenchymal transition (EMT) in rat proximal tubular epithelial cells (NRK52E). Immunofluorescence experiments showed that UUO resulted in significant upregulation of V-ATPase subunits (B2, E, and c) and α-smooth muscle actin (α-SMA) in areas of tubulointerstitial injury. We further observed that TGF-β1 (10 ng/ml) treatment resulted in EMT of NRK52E (upregulation of α-SMA and downregulation of E-cadherin) in a time-dependent manner and significant upregulation of V-ATPase B2 and c subunits after 48 h and the E subunit after 24 h, by real-time PCR and immunoblot analyses. The ATP hydrolysis activity tested by an ATP/NADH-coupled assay was increased after 48-h TGF-β1 treatment. Using intracellular pH measurements with the SNARF-4F indicator, Na(+)-independent pH recovery was significantly faster after an NH(4)Cl pulse in 48-h TGF-β1-treated cells than controls. Furthermore, the V-ATPase inhibitor bafilomycin A1 partially protected the cells from EMT. TGF-β1 induced an increase in the cell surface expression of the B2 subunit, and small interfering RNA-mediated B2 subunit knockdown partially reduced the V-ATPase activity and attenuated EMT induced by TGF-β1. Together, these findings show that V-ATPase may promote EMT and chronic tubulointerstitial fibrosis due to increasing its activity by either overexpression or redistribution of its subunits.     

Inositol-requiring protein 1 – X-box-binding protein 1 pathway promotes epithelial–mesenchymal transition via mediating snail expression in pulmonary fibrosis

Epithelial–mesenchymal transition (EMT) is a complex biological program during which cells loss epithelial phenotype and acquire mesenchymal features. EMT is thought to be involved in the pathogenesis of various fibrotic diseases including pulmonary fibrosis (PF). Recent studies suggest that endoplasmic reticulum (ER) stress is associated with EMT in the progression of PF. However, the exact mechanism is unclear. Here, we developed a PF model with bleomycin (BLM) administration in rats and conducted several simulation experiments in alveolar epithelial cell (AECs) RLE-6TN to unravel the role of inositol-requiring protein 1 (IRE1) – X-box-binding protein 1 (XBP1) signal pathway in ER stress-induced EMT in PF. First, we observed that ER stress was occurred in type II AECs accompanied by EMT in BLM-induced PF. Then we explored the role of IRE1-XBP1-snail pathway in transforming growth factor (TGF)-β1/tunicamycin (TM)-induced EMT. When TGF-β1/TM was treated on AECs, IRE1 and XBP1 were overexpressed, meanwhile, snail expression was upregulated accompanied with EMT. However, when IRE1 or XBP1 was knockdown, TGF-β1/TM-induced EMT were blocked while the expression of snail was inhibited. Then we silenced snail and found that TGF-β1/TM-induced EMT were also suppressed, but it had no effect on the up-regulated expression of IRE1 and XBP1. Thus, we concluded that IRE1-XBP1 pathway promotes EMT via mediating snail expression in PF.     

MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-β1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-β1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl(4)-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-β1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-β-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-β1 induction by targeting SMAD4.     

Axin pathway activity regulates in vivo pY654-β-catenin accumulation and pulmonary fibrosis

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.     

Inhibition of activated factor X; a new pathway in ameliorating carbon tetrachloride–induced liver fibrosis in rats

Activated factor X has a central role in the coagulation activation and also contributes to chronic inflammation and tissue fibrosis. In this study, rivaroxaban, a direct factor X inhibitor, attenuates liver fibrosis induced by carbon tetrachloride (CCl₄). Male rats were randomly allocated into three groups: a control group, CCl ₄ fibrotic group, and CCl ₄+rivaroxaban (5 mg/kg) group. Liver fibrosis was induced by subcutaneous injection of CCl ₄ twice a week for 6 weeks. Rivaroxaban significantly restored the biochemical parameter including inflammatory and fibrosis markers with histopathological evidence using routine and Masson trichrome staining. It reduced also the expression of tissue factor, fibrin, transforming growth factor and α‐smooth muscle actin in the liver tissues. This concludes that rivaroxaban attenuates liver injury caused by CCl ₄, at least in part by inhibiting coagulation and proinflammatory activation. In conclusion, rivaroxaban may be used for the management of liver fibrosis.     

The antifibrotic effect of TGF-beta1 siRNAs in murine model of liver cirrhosis

Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-beta1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-beta1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-beta1 siRNAs reduced the expression of TGF-beta1 in the livers of CCl(4) injection compared with those of control group, and the expression of type I collagen and alpha-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-beta1 siRNAs inhibit TGF-beta1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human.     

FGF2 antagonizes aberrant TGFβ regulation of tropomyosin: role for posterior capsule opacity

Transforming growth factor (TGF) β2 and fibroblast growth factor (FGF) 2 are involved in regulation of posterior capsule opacification (PCO) and other processes of epithelial–mesenchymal transition (EMT) such as cancer progression, wound healing and tissue fibrosis as well as normal embryonic development. We previously used an in vivo rodent PCO model to show the expression of tropomyosin (Tpm) 1/2 was aberrantly up‐regulated in remodelling the actin cytoskeleton during EMT. In this in vitro study, we show the Tpms family of cytoskeleton proteins are involved in regulating and stabilizing actin microfilaments (F‐actin) and are induced by TGFβ2 during EMT in lens epithelial cells (LECs). Importantly, we found TGFβ2 and FGF2 played contrasting roles. Stress fibre formation and up‐regulation of α‐smooth muscle actin (αSMA) induced by TGFβ2 could be reversed by Tpm1/2 knock‐down by siRNA. Expression of Tpm1/2 and stress fibre formation induced by TGFβ2 could be reversed by FGF2. Furthermore, FGF2 delivery to TGFβ‐treated LECs perturbed EMT by reactivating the mitogen‐activated protein kinase (MAPK)/ extracellular signal‐regulated kinase (ERK) pathway and subsequently enhanced EMT. Conversely, MEK inhibitor (PD98059) abated the FGF2‐mediated Tpm1/2 and αSMA suppression. However, we found that normal LECs which underwent EMT showed enhanced migration in response to combined TGFβ and FGF2 stimulation. These findings may help clarify the mechanism reprogramming the actin cytoskeleton during morphogenetic EMT cell proliferation and fibre regeneration in PCO. We propose that understanding the physiological link between levels of FGF2, Tpm1/2 expression and TGFβs‐driven EMT orchestration may provide clue(s) to develop therapeutic strategies to treat PCO based on Tpm1/2.     

RAR‐Related Orphan Receptor Gamma (ROR‐γ) Mediates Epithelial‐Mesenchymal Transition Of Hepatocytes During Hepatic Fibrosis

The epithelial‐mesenchymal transition (EMT) is involved in many different types of cellular behavior, including liver fibrosis. In this report, we studied a novel function of RAR‐related orphan receptor gamma (ROR‐γ) in hepatocyte EMT during liver fibrosis. To induce EMT in vitro, primary hepatocytes and FL83B cells were treated with TGF‐β1. Expression of ROR‐γ was analyzed by Western blot in the fibrotic mouse livers and human livers with cirrhosis. To verify the role of ROR‐γ in hepatocyte EMT, we silenced ROR‐γ in FL83B cells using a lentiviral short hairpin RNA (shRNA) vector. The therapeutic effect of ROR‐γ silencing was investigated in a mouse model of TAA‐induced fibrosis by hydrodynamic injection of plasmids. ROR‐γ expression was elevated in hepatocyte cells treated with TGF‐β1, and ROR‐γ protein levels were elevated in the fibrotic mouse livers and human livers with cirrhosis. Knockdown of ROR‐γ resulted in the attenuation of TGF‐β1‐induced EMT in hepatocytes. Strikingly, ROR‐γ bound to ROR‐specific DNA response elements (ROREs) in the promoter region of TGF‐β type I receptor (Tgfbr1) and Smad2, resulting in the downregulation of Tgfbr1 and Smad2 after silencing of ROR‐γ. Therapeutic delivery of shRNA against ROR‐γ attenuated hepatocyte EMT and ameliorated liver fibrosis in a mouse model of TAA‐induced liver fibrosis. Overall, our results suggest that ROR‐γ regulates TGF‐β‐induced EMT in hepatocytes during liver fibrosis. We suggest that ROR‐γ may become a potential therapeutic target in treating liver fibrosis. J. Cell. Biochem. 118: 2026–2036, 2017. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals Inc.     

Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.