KRT8 phosphorylation regulates the epithelial-mesenchymal transition in retinal pigment epithelial cells through autophagy modulation

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF-β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co-localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p-KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF-β2–induced EMT process, we found that p-KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p-KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p-KRT8 in the prevention and treatment of PVR.     

METTL3 attenuates proliferative vitreoretinopathy and epithelial-mesenchymal transition of retinal pigment epithelial cells via wnt/β-catenin pathway

Proliferative vitreoretinopathy (PVR) is a refractory vitreoretinal fibrosis disease, and epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is the key pathological mechanism of PVR. However, few studies focused on the role of METTL3, the dominating methyltransferase for m6A RNA modification in PVR pathogenesis. Immunofluorescence staining and qRT-PCR were used to determine the expression of METTL3 in human tissues. Lentiviral transfection was used to stably overexpress and knockdown METTL3 in ARPE-19 cells. MTT assay was employed to study the effects of METTL3 on cell proliferation. The impact of METTL3 on the EMT of ARPE-19 cells was assessed by migratory assay, morphological observation and expression of EMT markers. Intravitreal injection of cells overexpressing METTL3 was used to assess the impact of METTL3 on the establishment of the PVR model. We found that METTL3 expression was less in human PVR membranes than in the normal RPE layers. In ARPE-19 cells, total m6A abundance and the METTL3 expression were down-regulated after EMT. Additionally, METTL3 overexpression inhibited cell proliferation through inducing cell cycle arrest at G0/G1 phase. Furthermore, METTL3 overexpression weakened the capacity of TGFβ1 to trigger EMT by regulating wnt/β -catenin pathway. Oppositely, knockdown of METTL3 facilitated proliferation and EMT of ARPE-19 cells. In vivo, intravitreal injection of METTL3-overexpressing cells delayed the development of PVR compared with injection of control cells. In summary, this study suggested that METTL3 is involved in the PVR process, and METTL3 overexpression inhibits the EMT of ARPE-19 cells in vitro and suppresses the PVR process in vivo.     

The angiogenic effects of exosomes secreted from retinal pigment epithelial cells on endothelial cells

Exosomes are informative microvesicles associated with intercellular communication via the transfer of many molecular constituents such as proteins, lipids, and nucleic acids; environmental changes and the cellular status around cells greatly affect exosome components. Cells of the retinal pigment epithelium (RPE) are key players in retinal homeostasis. Transforming growth factor (TGF)-β and tumour necrosis factor (TNF)-α are increased in the vitreous and retina in several retinal diseases and activate and undergo epithelial-mesenchymal transition (EMT) in RPE cells. EMT is closely associated with mechanisms of wound healing, including fibrosis and related angiogenesis; however, whether exosome components depend on the cell status, epithelium or mesenchyme and whether these exosomes have pro- or anti-angiogenic roles in the retina are unknown. We performed this study to investigate whether these EMT inducers affect the kinds of components in exosomes secreted from RPE cells and to assess their angiogenic effects. Exosomes were collected from culture media supernatants of a human RPE cell line (ARPE-19) stimulated with or without 10 ng/ml TNF-α and/or 5 ng/ml TGF-β2. NanoSight tracking analysis and immunoblot analysis using exosome markers were used to qualify harvested vesicles. Angiogenic factor microarray analysis revealed that exosomes derived from ARPE-19 cells cultured with TNF-α alone (Exo-TNF) and co-stimulated with TNF-α and TGF-β2 (Exo-CO) contained more angiogenic factors than exosomes derived from control cells (Exo-CTL) or ARPE-19 cells cultured with TGF-β2 alone (Exo-TGF). To assess the effect on angiogenesis, we performed chemotaxis, tube formation, and proliferation assays of human umbilical vein endothelial cells (HUVECs) stimulated with or without exosomes. HUVECs migrated to RPE-derived exosomes, and exosomes derived from ARPE-19 cells accelerated HUVEC tube formation. In contrast, Exo-TNF and Exo-CO reduced HUVEC proliferation. Our findings provide insight into the mechanisms underlying the relation between angiogenesis and exosomes derived from RPE cells.     

TGF-β2 secretion from RPE decreases with polarization and becomes apically oriented

Retinal pigmented epithelium (RPE) secretes transforming growth factor beta 1 and 2 (TGF-β1 and -β2) cytokines involved in fibrosis, immune privilege, and proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be altered in various disease conditions including PVR and age-related macular degeneration, we determined levels of TGF-β from polarized human RPE (hRPE) and human stem cell derived RPE (hESC-RPE) as compared to nonpolarized cells. TGF-β2 was the predominant isoform in all cell culture conditions. Nonpolarized cells secreted significantly more TGF-β2 supporting the contention that loss of polarity of RPE in PVR leads to rise of intravitreal TGF-β2. Active TGF-β2, secreted mainly from apical side of polarized RPE, represented 6–10% of total TGF-β2. In conclusion, polarity is an important determinant of TGF-β2 secretion in RPE. Low levels of apically secreted active TGF-β2 may play a role in the normal physiology of the subretinal space. Comparable secretion of TGF-β from polarized hESC-RPE and hRPE supports the potential for hESC-RPE in RPE replacement therapies.     

Selective activation of p120ctn-Kaiso signaling to unlock contact inhibition of ARPE-19 cells without epithelial-mesenchymal transition

Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues.     

Interleukin-2 induces extracellular matrix synthesis and TGF-β2 expression in retinal pigment epithelial cells

Macular fibrosis is a vital obstacle of vision acuity improvement of age-related macular degeneration patients. This study was to investigate the effects of interleukin 2 (IL-2) on epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) synthesis and transforming growth factor β2 (TGF-β2) expression in retinal pigment epithelial (RPE) cells. 10 μg/L IL-2 was used to induce fibrosis in RPE cells for various times. Western blot was used to detect the EMT marker α-smooth muscle actin (α-SMA), ECM markers fibronectin (Fn) and type 1 collagen (COL-1), TGF-β2, and the activation of the JAK/STAT3 and NF-κB signaling pathway. Furthermore, JAK/STAT3 and NF-κB signaling pathways were specifically blocked by WP1066 or BAY11-7082, respectively, and the expression of α-SMA, COL-1, Fn and TGF-β2 protein were detected. Wound healing and Transwell assays were used to measure cell migration ability of IL-2 with or without WP1066 or BAY11-7082. After induction of IL-2, the expressions of Fn, COL-1, TGF-β2 protein were significantly increased, and this effect was correlated with IL-2 treatment duration, while α-SMA protein expression did not change significantly. Both WP1066 and BAY11-7082 could effectively downregulate the expression of Fn, COL-1 and TGF-β2 induced by IL-2. What's more, both NF-κB and JAK/STAT3 inhibitors could suppress the activation of the other signaling pathway. Additionally, JAK/STAT3 inhibitor WP1066 and NF-κB inhibitor BAY 11-7082 could obviously decrease RPE cells migration capability induced by IL-2. IL-2 promotes cell migration, ECM synthesis and TGF-β2 expression in RPE cells via JAK/STAT3 and NF-κB signaling pathways, which may play an important role in proliferative vitreoretinopathy.     

TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Autophagy links β-catenin and Smad signaling to promote epithelial-mesenchymal transition via upregulation of integrin linked kinase

TGF-β1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-β1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-β1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-β1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-β1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of β-catenin at Y-654 leading to pY654-β-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-β-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-β1-induced autophagy links β-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-β-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/β-catenin-mediated cell–cell contact to induction of EMT via upregulation of ILK.     

Metastasis-associated protein 1 promotes epithelial-mesenchymal transition in idiopathic pulmonary fibrosis by up-regulating Snail expression

Idiopathic pulmonary fibrosis (IPF) is a progressive and usually fatal lung disease that lacking effective interventions. It is well known that aberrant activation of transforming growth factor-beta1 (TGF-β1) frequently promotes epithelial-mesenchymal transition (EMT) in IPF. Metastasis-associated gene 1 (MTA1) has identified as an oncogene in several human tumours, and aberrant MTA1 expression has been related to the EMT regulation. However, its expression and function in IPF remain largely unexplored. Using a combination of in vitro and in vivo studies, we found that MTA1 was significantly up-regulated in bleomycin-induced fibrosis rats and TGF-β1-treated alveolar type Ⅱ epithelial (RLE-6TN) cells. Overexpression of MTA1 induced EMT of RLE-6TN cells, as well as facilitates cell proliferation and migration. In contrast, knockdown of MTA1 reversed TGF-β1-induced EMT of RLE-6TN cells. The pro-fibrotic action of MTA1 was mediated by increasing Snail expression through up-regulating Snail promoter activity. Moreover, inhibition of MTA1 effectively attenuated bleomycin-induced fibrosis in rats. Additionally, we preliminarily found astragaloside IV (ASV), which was previously validated having inhibitory effects on TGF-β1-induced EMT, could inhibit MTA1 expression in TGF-β1-treated RLE-6TN cells. These findings highlight the role of MTA1 in TGF-β1-mediated EMT that offer novel strategies for the prevention and treatment of IPF.     

Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells

Epithelial to mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both high glucose and transforming growth factor-β (TGF-β) are able to induce EMT in cell culture. In this study, we examined the role of the thioredoxin-interacting protein (TXNIP) on EMT induced by high glucose or TGF-β1 in HK-2 cells. EMT was assessed by the expression of α-smooth muscle actin (α-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. High glucose (30 mM) was shown to induce EMT at 72 h. This was blocked by knockdown of TXNIP or antioxidant NAC. Meanwhile, we also found that knockdown of TXNIP or antioxidant NAC inhibited high glucose-induced generation of reactive oxygen species (ROS), phosphorylation of p38 MAPK and ERK1/2 and expression of TGF-β1. HK-2 cells that were exposed to TGF-β1 (4 ng/ml) also underwent EMT. The expression of TXNIP gene and protein was increased in HK-2 cells treated with TGF-β1. Transfection with TXNIP shRNA was able to attenuate TGF-β1 induced-EMT. These results suggested that knockdown of TXNIP antagonized high glucose-induced EMT by inhibiting ROS production, activation of p38 MAPK and ERK1/2, and expression of TGF-β1, highlighting TXNIP as a potential therapy target for diabetic nephropathy.     

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.     

Long Non-Coding RNA MALAT1 Mediates Transforming Growth Factor Beta1-Induced Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells

Purpose

To study the role of long non-coding RNA (lncRNA) MALAT1 in transforming growth factor beta 1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells.

Methods

ARPE-19 cells were cultured and exposed to TGF-β1. The EMT of APRE-19 cells is confirmed by morphological change, as well as the increased expression of alpha-smooth muscle actin (αSMA) and fibronectin, and the down-regulation of E-cadherin and Zona occludin-1(ZO-1) at both mRNA and protein levels. The expression of lncRNA MALAT1 in RPE cells were detected by quantitative real-time PCR. Knockdown of MALAT1 was achieved by transfecting a small interfering RNA (SiRNA). The effect of inhibition of MALAT1 on EMT, migration, proliferation, and TGFβ signalings were observed. MALAT1 expression was also detected in primary RPE cells incubated with proliferative vitreoretinopathy (PVR) vitreous samples.

Results

The expression of MALAT1 is significantly increased in RPE cells incubated with TGFβ1. MALAT1 silencing attenuates TGFβ1-induced EMT, migration, and proliferation of RPE cells, at least partially through activating Smad2/3 signaling. MALAT1 is also significantly increased in primary RPE cells incubated with PVR vitreous samples.

Conclusion

LncRNA MALAT1 is involved in TGFβ1-induced EMT of human RPE cells and provides new understandings for the pathogenesis of PVR.     

Calreticulin regulates TGF-β1-induced epithelial mesenchymal transition through modulating Smad signaling and calcium signaling

As a Ca²⁺ binding protein, calreticulin (CRT) has many functions and plays an important role in a variety of tumors. The role of CRT in TGF-β1-induced EMT is unknown. In this study, we demonstrated in vitro that TGF-β1-induced EMT elevated the expression of CRT in A549 lung cancer cells. Subsequently, we confirmed that overexpression CRT had no capacity to induce A549 cells EMT alone, but successfully enhanced TGF-β1-induced-EMT. Furthermore, knockdown of CRT in A549 cells significantly suppressed changes of EMT marks expression induced by TGF-β1. On treatment with TGF-β1, overexpression of CRT could enhance the phosphorylation of both Smad2 and Smad3. Consistently, the knockdown of CRT by siRNA-CRT could inhibit Smad signaling pathway activated by TGF-β1. These results indicated that CRT regulates EMT induced by TGF-β1 through Smad signaling pathway. Finally, TGF-β1-induced-EMT enhanced store-operated Ca²⁺ influx in A549 cells. CRT knockdown was able to abolish the effect of TGF-β1 on thapsigargin (TG) −induced Ca²⁺ release, but had failed to reduce store-operated Ca²⁺ influx. The alteration of intracellular Ca²⁺ concentration by TG or BAPTA-AM was able to regulate EMT induced by TGF-β1 through Smad signaling pathway. Together, these data identify that CRT regulates TGF-β1-induced-EMT through modulating Smad signaling. Furthermore, TGF-β1-induced-EMT is highly calcium-dependent, CRT was partly involved in it.     

Cigarette smoke-induced alveolar epithelial–mesenchymal transition is mediated by Rac1 activation

Background

Epithelial–mesenchymal transition (EMT) is the major pathophysiological process in lung fibrosis observed in chronic obstructive pulmonary disease (COPD) and lung cancer. Smoking is a risk factor for developing EMT, yet the mechanism remains largely unknown. In this study, we investigated the role of Rac1 in cigarette smoke (CS) induced EMT.

Methods

EMT was induced in mice and pulmonary epithelial cells by exposure of CS and cigarette smoke extract (CSE) respectively.

Results

Treatment of pulmonary epithelial cells with CSE elevated Rac1 expression associated with increased TGF-β1 release. Blocking TGF-β pathway restrained CSE-induced changes in EMT-related markers. Pharmacological inhibition or knockdown of Rac1 decreased the CSE exposure induced TGF-β1 release and ameliorated CSE-induced EMT. In CS-exposed mice, pharmacological inhibition of Rac1 reduced TGF-β1 release and prevented aberrations in expression of EMT markers, suggesting that Rac1 is a critical signaling molecule for induction of CS-stimulated EMT. Furthermore, Rac1 inhibition or knockdown abrogated CSE-induced Smad2 and Akt (PKB, protein kinase B) activation in pulmonary epithelial cells. Inhibition of Smad2, PI3K (phosphatidylinositol 3-kinase) or Akt suppressed CSE-induced changes in epithelial and mesenchymal marker expression.

Conclusions and general significance

Altogether, these data suggest that CS initiates EMT through Rac1/Smad2 and Rac1/PI3K/Akt signaling pathway. Our data provide new insights into the fundamental basis of EMT and suggest a possible new course of therapy for COPD and lung cancer.     

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.