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.     

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.     

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 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.     

Knockdown of circular RNA VANGL1 inhibits TGF-β-induced epithelial-mesenchymal transition in melanoma cells by sponging miR-150-5p

Melanoma is one of the most aggressive and life-threatening skin cancers, and in this research, we aimed to explore the functional role of circular RNA VANGL1 (circVANGL1) in melanoma progression. The expression levels of circVANGL1 were observed to be significantly increased in clinical melanoma tissues and cell lines. Moreover, circVANGL1 knockdown suppressed, while circVANGL1 overexpression promoted the proliferation, migration and invasion abilities of melanoma cells. Further investigations confirmed the direct binding relation between circVANGL1 and miR-150-5p in melanoma, and restoration of miR-150-5p blocked the effects of circVANGL1 overexpression in melanoma cells. We further found that circVANGL1 was up-regulated by TGF-β treatment, and the enhanced EMT of TGF-β-treated melanoma cells was blocked by circVANGL1 knockdown. In conclusion, these results indicated that circVANGL1 might serve as a promising therapeutic target for melanoma.     

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.     

Formin mDia1 contributes to migration and epithelial-mesenchymal transition of tubular epithelial cells exposed to TGF-β1

Renal tubular epithelial cells may undergo epithelial-mesenchymal transition (EMT) in response to stimuli, such as transforming growth factor (TGF)-β1, leading to myofibroblast activation and renal fibrosis. The formin mDia1 is required for nucleation and polymerization of actin and the microtubule cytoskeleton. The present study sought to explore the role of mDia1 in EMT of tubular epithelial cells. A rat model of unilateral ureteral obstruction (UUO) was established. The expression of TGF-β1, collagen I, collagen III, and mDia1 in the kidneys was examined at day 7 after surgery. The effect of mDia1 on EMT was explored in NRK-52E cells by exposing them to TGF-β1. Increased expression of TGF-β1, collagen I, collagen III, and mDia1 was found in obstructive kidneys of UUO model rats. Exposing rat tubular epithelial cells to TGF-β1 promoted collagen I and collagen III expression but had no effect on mDia1 expression. Silencing mDia1 expression impeded epithelial cell migration as well as reduced TGF-β1, collagen, and Profilin1 expression, whereas mDia1 overexpression exerted an opposite effect. Furthermore, mDia1 regulated the expression of vimentin, α-smooth muscle actin, and E-cadherin and focal adhesion-kinase (FAK)/Src activation through Profilin1. Inhibition of the mDia1 activator RhoA by fasudil reversed EMT, and FAK/Src activation induced by mDia1. In conclusion, mDia1 regulated tubular epithelial cell migration, collagen expression, and EMT in NRK-52E cells exposed to TGF-β1. Thus, suppression of mDia1 activation might be a strategy to counteract renal fibrosis.     

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.     

Novel TGF-β1 inhibitor antagonizes TGF-β1-induced epithelial-mesenchymal transition in human A549 lung cancer cells

Transforming growth factor β1 (TGF-β1), a multifunctional cytokine, is known to promote tumor invasion and metastasis and induce epithelial-mesenchymal transition (EMT) in various cancer cells. Inhibition of TGF-β1 signaling is a new strategy for cancer therapy. Most cancer cells display altered or nonfunctional TGF-β1 signaling; hence, TGF-β1 inhibitors exert limited effects on these cells. Recent studies have suggested that developing a TGF-β1 inhibitor from natural compounds is a key step to create novel therapeutic agents. This study aimed to develop a new anti-TGF-β1 therapy for cancer. We found an improved analog of chalcones, compound 67, and investigated its effects in vitro. We demonstrated the inhibitory role of compound 67 through migration and invasion assays on TGF-β1-induced EMT of human A549 lung cancer cells. Compound 67 inhibited TGF-β1-induced smad2 phosphorylation, suppressed TGF-β1-induced EMT markers, matrix metalloproteinase-2 (MMP-2) and MMP-9, and inhibited migration and invasion of A549 cells. The study results showed that compound 67 is useful to prevent tumor growth and metastasis.     

Glucocorticoid induces mesenchymal-to-epithelial transition and inhibits TGF-β1-induced epithelial-to-mesenchymal transition and cell migration

Epithelial-to-mesenchymal transition (EMT) has been implicated in various physiological and pathological events. In this study, we found that the synthetic glucocorticoid dexamethasone (Dex) can inhibit transforming growth factor-beta1-induced EMT and cell migration. We also demonstrated that Dex inhibits EMT through a mechanism involving the suppression of ROS generation. Surprisingly, Dex alone induced mesenchymal-to-epithelial transition (MET). Dexamethasone treatment abolished Snail1 binding to the E-cadherin promoter, suggesting that suppression of Snail1 contributes to the above roles of Dex. Our findings demonstrate that Dex functions as both a suppressor of EMT and as an inducer of MET and therefore may be implicated in certain pathophysiological events.     

miR-29b inhibits TGF-β1-induced cell proliferation in articular chondrocytes

Transforming growth factor β1 (TGF-β1) is a known regulator of chondrocyte proliferation and promotes cartilage repair in osteoarthritis (OA). microRNA-29b-3p (miR-29b-3p) is downregulated by TGF-β1 and overexpressed in OA cartilage. However, the ability of miR-29b-3p to mediate the chondrocyte pro-proliferative effects of TGF-β1 is not yet understood. This current study aimed to investigate the effect of miR-29b-3p on TGF-β1-induced cell proliferation in murine articular chondrocytes. The stimulation of chondrocytes by TGF-β1 for 24 h resulted in the downregulation of miR-29b-3p expression. The ratio of G0/G1 phase cells decreased in response to TGF-β1 whereas the ratio of S phase cells was increased. Consistent with this observation, miR-29b-3p overexpression inhibited TGF-β1’s ability to promote the ratio of S phase cells and downregulate the ratio of G0/G1 phase cells. These findings suggest that the downregulation of miR-29b-3p is a likely requirement for TGF-β1-mediated proliferation of murine articular chondrocytes. Furthermore, implying that miR-29b-3p expression may be involved in reduced chondrocyte proliferation in OA.     

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.     

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.     

TGF-β1-induced epithelial–mesenchymal transition in lung cancer cells involves upregulation of miR-9 and downregulation of its target,E-cadherin

Background

TGF-β1 plays an important role in the epithelial–mesenchymal transition (EMT) of epithelial cancers, including non-small cell lung cancer (NSCLC). While the full underlying mechanism remains unclear, miR-9 is known to play a critical role in the regulation of NSCLC cell invasion. We tested whether miR-9 targets E-cadherin and thus affects TGF-β1-induced EMT in NSCLC cells by assessing the expression levels of miR-9 and E-cadherin for NSCLC patients and then verifying the targeting of E-cadherin by miR-9 using the dual luciferase reporter system.

Results

MiR-9 was significantly upregulated in NSCLC tissues compared with its level in adjacent normal tissues. The expression of E-cadherin in NSCLC tissues was significantly decreased. In addition, we found that TGF-β1 significantly upregulated the expression of miR-9 and downregulated the expression of E-cadherin. E-cadherin was confirmed as a direct target gene of miR-9. Using an miR-9 inhibitor reversed the TGF-β1-mediated inhibition of E-cadherin expression and upregulation of the mesenchymal marker α-SMA. TGF-β1 significantly induced cell invasion, and this effect was significantly inhibited by miR-9 inhibitors.

Conclusions

TGF-β1 induced EMT in NSCLC cells by upregulating miR-9 and downregulating miR-9’s target, E-cadherin.