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.     

The interaction of LFA-1 on mononuclear cells and ICAM-1 on tubular epithelial cells accelerates TGF-β1-induced renal epithelial-mesenchymal transition

The epithelial-mesenchymal transition (EMT) of renal epithelial cells (RTECs) has pivotal roles in the development of renal fibrosis. Although the interaction of lymphocyte function-associated antigen 1 (LFA-1) on leukocytes and its ligand, intracellular adhesion molecule 1 (ICAM-1), plays essential roles in most inflammatory reactions, its pathogenetic role in the EMT of RTECs remains to be clarified. In the present study, we investigated the effect of the interaction of LFA-1 on peripheral blood mononuclear cells (PBMCs) and ICAM-1 on HK-2 cells after stimulation with TGF-β(1) on the EMT of RTECs. ICAM-1 was highly expressed in HK-2 cells. After TGF-β(1) stimulation, the chemokines CCL3 and CXCL12 increased on HK-2 cells. After co-culture of PBMCs and HK-2 cells pre-stimulated with TGF-β(1) (0.1 ng/ml) (HK-2-TGF-β(1) (0.1)), the expression of the active form of LFA-1 increased on PBMCs; however, total LFA-1 expression did not change. The expression of the active form of LFA-1 on PBMCs did not increase after co-culture with not CCL3 but CXCL12 knockdown HK-2-TGF-β(1) (0.1). The expression of epithelial cell junction markers (E-cadherin and occludin) further decreased and that of mesenchymal markers (vimentin and fibronectin) further increased in HK-2-TGF-β(1) (0.1) after co-culture with PBMCs for 24 hrs (HK-2-TGF-β(1) (0.1)-PBMCs). The phosphorylation of ERK 1/2 but not smad2 and smad3 increased in HK-2-TGF-β(1) (0.1)-PBMCs. The snail and slug signaling did not increase HK-2-TGF-β(1) (0.1)-PBMCs. Although the migration and invasion of HK-2 cells induced full EMT by a high dose (10.0 ng/ml) and long-term (72-96 hrs) TGF-β(1) stimulation increased, that of HK-2-TGF-β(1) (0.1)-PBMCs did not increase. These results suggested that HK-2 cells stimulated with TGF-β(1) induced conformational activation of LFA-1 on PBMCs by increased CXCL12. Then, the direct interaction of LFA-1 on PBMCs and ICAM-1 on HK-2 cells activated ERK1/2 signaling to accelerate the part of EMT of HK-2 cells induced by TGF-β(1).     

Lefty A attenuates the TGF-β1-induced epithelial to mesenchymal transition of human renal proximal epithelial tubular cells

The epithelial to mesenchymal transition (EMT) is a crucial event for renal fibrosis that can be elicited by TGF-β1/Smads signaling and its downstream mediator connective tissue growth factor (CTGF). As a distinct member of the TGF-β superfamily, Lefty A has been shown to be significantly downregulated in the kidneys of patients with severe ureteral obstruction, suggesting its role in renal fibrosis induced by obstructive nephropathy. In order to determine whether Lefty A prevents TGF-β1-induced EMT, human proximal tubule epithelial cells (HK-2) were stably transfected with Lefty A or control vectors and stimulated with 10 ng/ml TGF-β1 for 48 h. The results show that stimulation with TGF-β1 led to EMT including cell morphology changes, Smad2/3 signaling pathway activation, increased α-SMA, collagen type I, and CTGF expression, and decreased E-cadherin expression in mock-transfected HK-2 cells. Overexpression of Lefty A efficiently blocked p-Smad2/3 activation and attenuated all these EMT changes induced by TGF-β1. This finding suggests that Lefty A may serve as a potential new therapeutic target to inhibit or even reverse EMT during the process of renal fibrosis.     

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.     

Blocking core fucosylation of TGF-β1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells

Posttranslational modification of proteins could regulate their multiple biological functions. Transforming growth factor-β receptor I and II (ALK5 and TGF-βRII), which are glycoproteins, play important roles in the renal tubular epithelial-mesenchymal transition (EMT). In the present study, we examined the role of core fucosylation of TGF-βRII and ALK5, which is regulated by α-1,6 fucosyltransferase (Fut8), in the process of EMT of cultured human renal proximal tubular epithelial (HK-2) cells. The typical cell model of EMT induced by TGF-β1 was constructed to address the role of core fucosylation in EMT. Core fucosylation was found to be essential for both TGF-βRII and ALK5 to fulfill their functions, and blocking it with Fut8 small interfering RNA greatly reduced the phosphorylation of Smad2/3 protein, caused the inactivation of TGF-β/Smad2/3 signaling, and resulted in remission of EMT. More importantly, even with high levels of expressions of TGF-β1, TGF-βRII, and ALK5, blocking core fucosylation also could attenuate the EMT of HK-2 cells. Thus blocking core fucosylation of TGF-βRII and ALK5 may attenuate EMT independently of the expression of these proteins. This study may provide new insight into the role of glycosylation in renal interstitial fibrosis. Furthermore, core fucosylation may be a novel potential therapeutic target for treatment of renal tubular EMT.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.     

Ectopic expression of Ligand-of-Numb protein X promoted TGF-β induced epithelial to mesenchymal transition of proximal tubular epithelial cells

Ligand-of-Numb protein X (LNX) was initially characterized as a RING finger type E3 ubiquitin ligase that targeted the intrinsic cell fate determinant Numb for ubiquitination dependent degradation. However, the physiological function of LNX remains largely unknown. In the present study, we demonstrate that ectopic expression of LNX in human proximal tubular epithelial cells (HK-2 cells) significantly enhanced TGF-β1 induced epithelial to mesenchymal transition (EMT). The EMT-promoting effect of LNX manifested as strong inhibition of E-cadherin expression, enhanced expression of vimentin, fibronectin or PAI-1, and increased cell migration. This function of LNX was shown to be independent of its ligase activity because ectopic expression of a mutant form of LNX (C48ALNX) that lacks E3 ligase activity had the similar effect as the wild-type LNX. Overexpression of E-cadherin could inhibit LNX augmented EMT. This study suggests a potential role for LNX in promoting EMT in human proximal tubular epithelial cells.     

Role of Smad2/3 and p38 MAP kinase in TGF-β1-induced epithelial-mesenchymal transition of pulmonary epithelial cells

Idiopathic pulmonary fibrosis is characterized by myofibroblast accumulation, extracellular matrix (ECM) remodeling, and excessive collagen deposition. ECM-producing myofibroblasts may originate from epithelial cells through epithelial to mesenchymal transition (EMT). TGF-β1 is an inducer of EMT in pulmonary epithelial cells in vitro and in vivo, though the mechanisms are unclear. We hypothesized that TGF-β1 induced EMT through Smad-dependent and -independent processes. To test this hypothesis, we studied the roles and mechanisms of TGF-β1-induced Smad and p38 mitogen-activated protein kinase (MAPK) signaling in EMT-related changes in pulmonary epithelial cells. Exposure of pulmonary epithelial 1HAEo(-) cells to TGF-β1 resulted in morphological and molecular changes of EMT over a 96-h period; loss of cell-cell contact, cell elongation, down-regulation of E-cadherin, up-regulation of fibronectin, and up-regulation of collagen I. Both Smad2/3 and p38 MAPK signaling pathways were activated by TGF-β1. However, neither Smad2/3 nor p38 MAPK were required for the down-regulation of E-cadherin, yet p38 MAPK was associated with fibronectin up-regulation. Both Smad2/3 and p38 MAPK had a role in regulation of TGF-β1-induced collagen expression. Furthermore, these data demonstrate that Smads and p38 MAPK differentially regulate EMT-related changes in pulmonary epithelial cells.     

Cdc42-interacting protein-4 promotes TGF-Β1-induced epithelial-mesenchymal transition and extracellular matrix deposition in renal proximal tubular epithelial cells

Cdc42-interacting protein-4 (CIP4) is an F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family member that regulates membrane deformation and endocytosis, playing a key role in extracellular matrix (ECM) deposition and invasion of cancer cells. These processes are analogous to those observed during the initial epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. The role of CIP4 in renal tubular EMT and renal tubulointerstitial fibrosis was investigated over the course of the current study, demonstrating that the expression of CIP4 increased in the tubular epithelia of 5/6-nephrectomized rats and TGF-β1 treated HK-2 cells. Endogenous CIP4 evidenced punctate localization throughout the cytosol, with elevated levels observed in the perinuclear region of HK-2 cells. Subsequent to TGF-β1 treatment, CIP4 expression increased, forming clusters at the cell periphery that gradually redistributed into the cytoplasm. Simultaneously, EMT induction in cells was confirmed by the prevalence of morphological changes, loss of E-cadherin, increase in α-SMA expression, and secretion of fibronectin. Overexpression of CIP4 promoted characteristics similar to those commonly observed in EMT, and small interfering RNA (siRNA) molecules capable of CIP4 knockdown were used to demonstrate reversed EMT. Cumulatively, results of the current study suggest that CIP4 promotes TGF-β1-induced EMT in tubular epithelial cells. Through this mechanism, CIP4 is capable of inducing ECM deposition and exacerbating progressive fibrosis in chronic renal failure.     

Evidence of TGF-β1 mediated epithelial-mesenchymal transition in immortalized benign prostatic hyperplasia cells

Expression of epithelial-mesenchymal transition (EMT) markers has been detected clinically in benign prostatic hyperplasia (BPH) tissues. To understand the molecular basis, we investigated the role of stromal microenvironment in the progression of EMT in BPH cells. First, we used cell culture supernatant from normal prostate stromal WPMY-1 cells to provide supernatant-conditioned medium (WSCM) to culture the BPH-1 cell line. Then, the morphological changes and migratory capacity were detected in BPH-1 cells. The expression of EMT markers was examined in BPH-1 cells by Western blot and immunofluorescent analysis. Finally, to investigate the role of transforming growth factor beta 1 (TGF-β1) in this process, the WSCM-cultured cells were treated with monoclonal antibody against TGF-β1 to study its effect on EMT. We found that the morphology of BPH-1 cells changed to a spindle-like shape after cultured in WSCM, and the levels of E-cadherin and cytokeratin 5/8 (CK5/8) were significantly lower than the cells cultured in ordinary medium. These BPH-1 cells were also tested positive for mesenchymal markers vimentin and a-smooth muscle actin (SMA) as well as Snail. We also found WSCM can increase the migratory capacity of BPH-1 cells. In addition, when they were treated with anti-TGF-β1, upregulation of E-cadherin and CK5/8 levels was observed but no expression of vimentin, alpha-SMA or Snail was detected. Furthermore, phosphorylated-Smad3 expression in WSCM-cultured BPH-1 cells was also suppressed by anti-TGF-β1 treatment. Our results demonstrated that stromal cell supernatant was able to induce EMT in BPH-1 cells, possibly through secreting TGF-β1 to activate Smad signaling. Our results suggest novel molecular targets for clinical treatment of BPH by modification of stromal microenvironment through inhibiting TGF-β1/Smad expression.     

Dioscin suppresses TGF-β1-induced epithelial-mesenchymal transition and suppresses A549 lung cancer migration and invasion

Epithelial-to-mesenchymal transition (EMT), an important cellular process, occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Dioscin is a polyphenolic component isolated from Phyllanthus amarus, which exhibits a wide range of pharmacological and physiological activities, such as anti-tumor, anti-inflammatory, anti-obesity, anti-fungal, and anti-viral activities. However, the possible role of dioscin in the EMT is unclear. We investigated the suppressive effect of dioscin on the EMT. Transforming growth factor-beta 1 (TGF-β1) is known to induce EMT in a number of cancer cell types and promote lung adenocarcinoma migration and invasion. To verify the inhibitory role of dioscin in lung cancer migration and invasion, we investigated the use of dioscin as inhibitors of TGF-β1-induced EMT in A549 lung cancer cells in vitro. Here, we found that dioscin prominently increased expression of the epithelial marker E-cadherin and expression of the mesenchymal marker N-cadherin and Snail during the TGF-β1-induced EMT. In addition, dioscin inhibited the TGF-β1-induced increase in cell migration and invasion of A549 lung cancer cells. Also, dioscin remarkably inhibited TGF-β1-regulated activation of MMP-2/9, Smad2, and p38. Taken together, our findings provide new evidence that dioscin suppresses lung cancer migration, and invasion in vitro by inhibiting the TGF-β1-induced EMT.     

β-Casomorphin-7 attenuates the development of nephropathy in type I diabetes via inhibition of epithelial-mesenchymal transition of renal tubular epithelial cells

This study was designed to investigate the putative protective effect of β-casomorphin-7 on diabetic nephropathy in a rat model, and to explore the possible mechanism of this effect. SD rats were randomly divided into the following three groups: control group, diabetes group and β-casomorphin-7-treatment group. All rats were euthanized after 30 days with or without β-casomorphin-7 treatment. Biochemical parameters including blood glucose and renal function were quantified. The concentration of plasma TGF-β1 was measured by ELISA. Histopathological changes to the kidney were studied by Masson and Sirius red staining. Expressions of α-smooth muscle actin (α-SMA), E-cadherin, vimentin, cytokeratin19 and TGF-β1 mRNA in rat renal cortices were analyzed by real-time PCR. Changes in α-SMA and E-cadherin protein expression in rat renal cortices were quantified by Western blot. β-Casomorphin-7 treatment of diabetic rats reduced urinary glucose, urinary protein, serum creatinine, blood urinary nitrogen, plasma TGF-β1 and the ratio of kidney: body weight. Masson and Sirius red staining showed that β-casomorphin-7 treatment attenuated renal interstitial fibrosis in diabetic rats. Compared to the control rats, diabetic rats had elevated expressions of α-SMA, vimentin and TGF-β1 mRNA and α -SMA protein and decreased expression of E-cadherin and cytokeratin19 mRNA, and E-cadherin protein. β-Casomorphin-7 treatment of diabetic rats partially normalized these changes. Our results suggest that administration of β-casomorphin-7 attenuates renal interstitial fibrosis caused by diabetes. This protective effect may be associated, in part, with down regulation of epithelial-mesenchymal transition of renal tubular epithelial cells.     

Starvation-induced autophagy promotes the invasion and migration of human bladder cancer cells via TGF-β1/Smad3-mediated epithelial-mesenchymal transition activation

The biological characteristics of bladder cancer include enhanced invasion and migration, which are the main causes of death in patients. Starvation is a typical feature of the bladder cancer microenvironment and can induce autophagy. Autophagy has an important relationship with the invasion and migration of tumors. However, the role of autophagy in the invasion and migration of bladder cancer cells remains unclear. Hence, the aim of the current study was to clarify this role and underlying mechanism. In this study, we found that starvation enhanced the epithelial-mesenchymal transition (EMT)-mediated invasion and migration of T24 and 5637 cells while inducing autophagy. The inhibition of autophagy with chloroquine (CQ) or 3-methyladenine (3MA) decreased EMT-mediated invasion and migration. In addition, the expression of transforming growth factor 1 (TGF-β1) and phosphorylated Smad3 (p-Smad3) increased after starvation. The inhibition of autophagy with CQ or 3MA also decreased the expression of TGF-β1 and p-Smad3. The inhibitor of TGF-β receptor sb431542 also inhibited the invasion, migration, and EMT of T24 and 5637 cells during starvation. Furthermore, recombinant TGF-β1 induced autophagy and inhibition of the TGF-β/Smad signaling pathway with sb431542 suppressed autophagy. In summary, our results suggested that autophagy promotes the invasion and migration of bladder cancer cells by inducing EMT through the TGF-β1/Smad3 signaling pathway. Moreover, autophagy and TGF-β1 can form a positive feedback loop to synergistically promote invasion and migration. Thus, our findings may provide a theoretical basis for the prevention of invasion and migration in bladder cancer.