Original Paper Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
Received: December 2, 2013 Accepted: March 20, 2014 Published online: May 22, 2014
Endoplasmic Reticulum Stress Induces Epithelial-Mesenchymal Transition through Autophagy via Activation of c-Src Kinase Soo Young Moon Hyo Sang Kim Kyeong Woo Nho Young Joo Jang Sang Koo Lee Department of Internal Medicine, Asan Medical Center, University of Ulsan, Seoul, Korea
Key Words Autophagy · Epithelial-mesenchymal transition · Endoplasmic reticulum stress · c-Src kinase
Abstract Background: Endoplasmic reticulum (ER) stress has been implicated in inducing epithelial-mesenchymal transition (EMT). ER stress is also known to induce autophagy. However, it is unclear whether ER stress-induced autophagy contributes to EMT. We hypothesized that ER stress might induce EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. Method: All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Immunofluorescence and small interfering RNA (siRNA) experiments were performed. Results: Chemical ER stress inducers such as tunicamycin (TM, 0.2 μM) and thapsigargin (TG, 0.2 μM) induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ER stress inhibitors such as 4-PBA and salubrinal suppressed both TM- and TG-induced EMT. TM and TG also induced autophagy, as evidenced by upregulation of LC3-II and beclin-1, which were abolished by pretreatment with ER stress inhibitors. Transfection with siRNA targeting ER stress protein (IRE-1) blocked the TM- or TG-induced EMT and au-
© 2014 S. Karger AG, Basel 1660–2129/14/1263–0127$39.50/0 E-Mail [email protected] www.karger.com/nee
tophagy. Autophagy inhibitors such as 3-methyladenine and bafilomycin inhibited the TM- or TG-induced EMT. Transfection with siRNA targeting autophagy protein (beclin-1) also blocked the TM- or TG-induced EMT. Both TM and TG induced activation of c-Src kinase. Inhibitor of c-Src kinase (PP2) suppressed the TM- or TG-induced autophagy and EMT. Conclusion: ER stress by TM or TG induced EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. © 2014 S. Karger AG, Basel
Introduction
Endoplasmic reticulum (ER) stress refers to physiological or pathological states that result in accumulation of misfolded proteins in the ER. Cells respond to ER stress by activating a series of integrative stress pathways termed the unfolded protein response (UPR). The three arms of the UPR include processes designed to attenuate protein translation, increase the production of metabolism and redox proteins, enhance production of protein folding chaperones, and upregulate the production of protein degradation enzymes through the activation of the ER transmembrane sensors PERK, ATF6, and IRE-1. GRP78 is a central Sang Koo Lee, MD Division of Nephrology, Department of Internal Medicine Asan Medical Center, 88, Olympic-ro 43-gil Songpa-gu, Seoul 138-736 (Korea) E-Mail sklee2 @ amc.seoul.kr
regulator of ER homeostasis and phosphorylation of eIF2α leads to attenuate the translation initiation rate, both of which are biomarkers for ER stress. UPR may either promote cell survival, or if the ER stress is chronic or excessive may lead to apoptosis by inducing proapoptotic transcription factor CHOP [1]. In addition to effects on apoptosis, ER stress and UPR pathways have been suggested as important determinants of fibrotic remodeling in a number of internal organs, including the lungs, liver, gastrointestinal tract, heart and kidney [2–5] through induction of epithelial-mesenchymal transition (EMT) [6–9]. EMT defines a phenotypic conversion of primary epithelial cells into mesenchymal cells, leading to morphological changes to fibroblastoid morphology, downregulation of epithelial marker proteins such as E-cadherin, ZO-1 and cytokeratin, and finally upregulation of mesenchymal markers including α-smooth muscle actin (α-SMA), vimentin and fibroblast-specific protein-1 [10]. EMT is thought to contribute to fibrotic remodeling in a number of organs. In kidney, tubular epithelial cells have been implicated in tubulointerstitial fibrosis as a source of interstitial fibroblasts via EMT [11]. However, information about ER stress-induced EMT in renal tubular epithelial cells is still limited. Autophagy is a tightly regulated, programmed mechanism to eliminate damaged organelles and proteins to maintain cellular homeostasis. Autophagy involves sequestration of proteins and cell organelles in a double membrane-bound structure called autophagosome, which fuses with a lysosome, after which its contents and inner membrane are degraded and recycled. The formation of autophagosome is known to be dependent on the induction of several genes including LC3, beclin-1, phosphatidylinositide 3-kinase and Atgs [12]. During autophagosome formation, LC3 is converted from the unconjugated form (LC3-I) to the phosphatidylethanolamine (PE)-conjugated form (LC3-II), which is then incorporated into an autophagosomal membrane [13]. It has been suggested that autophagy plays an important role in a variety of biological processes, including cell death, development, etc. [14]. ER stress is also known to upregulate the autophagy machinery [13, 15, 16]. However, whether ER stress-induced autophagy contributes to EMT has not been explored so far. We hypothesized that ER stress might induce EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. In our study, ER stress was induced by two different chemical agents: tunicamycin (TM) and thapsigargin (TG). TM elicits ER stress by inhibiting protein N-linked glycosylation, leading to accumulation of unfolded pro128
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teins and consequent UPR activation [17]. TG inhibits sarco-endoplasmic reticulum Ca-ATPase and blocks calcium reuptake into the ER lumen, leading to disruption of protein folding and subsequent induction of ER stress [18]. We examined the effects of two different ER stress inhibitors (4-PBA and salubrinal) on the TM- or TG-induced autophagy and EMT. To confirm the role of ER stress on the autophagy and EMT, we investigated the effects of small interfering RNA (siRNA) targeting ER stress protein (IRE-1). We also examined the effects of autophagy inhibitors (3-MA, bafilomycin) and siRNA targeting autophagy protein (beclin-1) on the TM- or TG-induced EMT. c-Src is a member of the Src tyrosine kinase family, which plays a role in signal transduction in response to many external stimuli and its activity is under tight redox control [19]. Src activation in ER stress-induced EMT was reported in non-renal cells [7, 8]. However, it is unclear whether c-Src activation mediates ER stress-induced EMT in renal tubular epithelial cells. Furthermore, involvement of Src in the induction of ER stress-induced autophagy has not been reported so far. Therefore, we finally examined whether c-Src kinase was involved in both ER stress-induced autophagy and EMT. Methods Reagent TM, TG, 3-methyladenine (3-MA) and bafilomycin were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). 4-Phenylbutyric acid (4-PBA), salubrinal, PP2 and rapamycin were obtained from Calbiochem (San Diego, Calif., USA). Antibodies to α-SMA, E-cadherin and GRP78 were acquired from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Antibodies to CHOP, beclin-1, microtubule-associated protein-1 light chain-3 (LC3), IRE-1, total c-Src kinase, phosphospecific-c-Src kinase (Tyr416), and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, Mass., USA). Cell Culture and Conditioning All experiments were performed using HK-2 cells, a human proximal tubular cell line. HK-2 cells were obtained from the American Type Culture Collection and have been characterized previously [20]. The media were changed every 3 days until confluent. Cells were growth-arrested in serum-free medium for 24 h before being used in experiments. Cells were incubated with TM (0–0.2 μM) or TG (0–0.2 μM) for up to 24 h with or without 4-PBA (0.5 mM), salubrinal (10 μM), 3-MA (0.5 mM), bafilomycin (10 nM) and PP2 (10 μM). Western Blot Analysis An equal amount of protein from whole cell lysates was separated by a 10% SDS-polyacrylamide gels, transferred to nylon
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Fig. 1. Induction of GRP78 (a) and CHOP expression (b) by TM or TG and inhibition of TM- or TG-induced CHOP expression by both 4-PBA and salubrinal in HK-2 cells. Proximal tubular cells were incubated with TM (0–0.2 μM) or TG (0–0.2 μM) for up to 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression
of GRP78 and CHOP was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
membrane. Membranes were incubated for 2 h with primary antibody, followed by peroxidase-conjugated secondary antibody. Antibody-antigen complexes were detected with ECL plus chemiluminescence (Amersham Pharmacia Biotech, Arlington, Ill., USA). The band intensities were quantified using a GS-710 densitometer and QuantityOne software (Bio-Rad, Hercules, Calif., USA).
nology, 20 nmol/ml) for 6 h in serum-free medium and then culture medium was changed to normal medium containing 10% FBS for 24 h. Non-specific siRNA was used as negative control. The efficiency of IRE-1 siRNA and beclin-1 siRNA was evaluated by Western blotting of IRE-1 and beclin-1 respectively. Transfected cells were incubated with TM or TG for 24 h. Western blotting for α-SMA, E-cadherin, LC3 and beclin-1 was performed.
Immunofluorescence Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 and then blocked with 2% BSA in PBS for 1 h. Cells were incubated overnight with primary antibody against α-SMA, E-cadherin, GRP78 and beclin-1. The specimens were then washed with PBS and incubated with FITC-conjugated secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI (4′,6-diamidino2-phenylindole). After washing with PBS, coverslips were mounted in 80% glycerol in PBS and photographed using a confocal microscope.
Statistical Analysis Data were expressed as mean ± SE. Kruskal-Wallis test was used for comparison of more than two groups, followed by a Mann-Whitney U-test for comparison using a microcomputerassisted program with SPSS for Windows 10.0 (SPSS, Inc., Chicago, Ill., USA). p < 0.05 was considered significant.
siRNA Transfection Transfection of siRNA was performed with Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen, Carlsbad, Calif., USA). Cells were transfected with siRNA against IRE-1 (Ambion, 100 pmol/ml) or beclin-1 (Cell Signaling Tech-
ER Stress Induces EMT via Autophagy
Results
Both TM and TG Induced ER Stress, Which Were Blocked by ER Stress Inhibitors Such as 4-PBA and Salubrinal in Our HK-2 Cells In initial studies, we sought to determine whether well-known chemical ER stress inducers (TM and TG) Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
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Color version available online
Fig. 2. Inhibition of TM- (a) or TG-induced (b) EMT by ER stress inhibitors such as 4-PBA and salubrinal. Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression of α-SMA and
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Fig. 3. Immunofluorescence study showing
that suppression of TM-induced upregulation of α-SMA and downregulation of Ecadherin by ER stress inhibitors. Proximal tubular cells were incubated with TM (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (10 μM). Immunofluorescence staining for α-SMA and Ecadherin was performed. Representative microscopic scans are shown.
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and ER stress inhibitors (4-PBA and salubrinal) could be used as ER stress inducers and inhibitors respectively in our HK-2 cells. To determine the effects of ER stress inducers and inhibitors on the ER stress, we examined the change of two ER stress biomarkers, upregulation of GRP78 and CHOP expression. As would be expected, both TM (0.2 μM) and TG (0.2 μM) induced the expression of GRP78 in a time-dependent manner (0–24 h) in HK-2 cells. They also increased the GRP78 expression in 130
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a dose-dependent manner (0–0.2 μM) at 24 h of incubation. 0.2 μM of TM and TG induced the expression of CHOP, which was attenuated by pretreatment with ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (10 μM) (fig. 1a, b). ER Stress by TM or TG Induced EMT To determine whether ER stress by TM or TG induced EMT in tubular epithelial cells, we examined the change Moon/Kim/Nho/Jang/Lee
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Fig. 4. Suppression of TM- (a) or TG-induced (b) EMT by siRNA targeting IRE-1 (c). Cells were transfected with non-specific (Con siRNA) or siRNA targeting IRE-1 (IRE-1 siRNA). The cells were then treated with TM (0.2 μM) or TG (0.2 μM) for 24 h. Expression of α-SMA and E-cadherin was examined by Western blot analysis. The efficiency of IRE-1 siRNA was evaluated by Western blotting of IRE-1. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
of two EMT biomarkers, upregulation of α-SMA and downregulation of E-cadherin. Exposure of tubular cells to TM or TG (0.2 μM) for 24 h induced EMT, as shown by upregulation of α-SMA and downregulation of E-cadherin, which was abolished by pretreatment with ER stress inhibitors such as 4-PBA and salubrinal (fig. 2a, b). In agreement with the Western blot data, immunofluorescence staining revealed that 4-PBA and salubrinal suppressed the TM-induced upregulation of α-SMA and downregulation of E-cadherin (fig. 3). To confirm the
role of ER stress in the induction of EMT, we knocked down the ER stress protein (IRE-1) expression by siRNA transfection. Transfection with siRNA targeting IRE-1 suppressed the TM or TG-induced EMT, but not nonspecific siRNA (fig. 4a, b). These data indicated that TMor TG-induced EMT was mediated by a process involving ER stress. The efficiency of siRNA targeting IRE-1 was also evaluated. As expected, siRNA targeting IRE-1 inhibited the TM- or TG-induced IRE-1 expression but not non-specific siRNA (fig. 4c).
ER Stress Induces EMT via Autophagy
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Fig. 5. Inhibition of TM- (a) and TG-induced (b) autophagy by ER stress inhibitors such as 4-PBA and salubrinal. Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression of LC3-ΙΙ and beclin-1 was examined by Western blot analysis. Representa-
tive blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG. Immunofluorescence staining for beclin-1 (c) was performed. Representative microscopic scans are shown.
ER Stress by TM or TG Induced Autophagy To determine whether ER stress by TM or TG induced autophagy in tubular epithelial cells, we examined the change of two autophagy biomarkers, upregulation of microtubule-associated protein-1 light chain3-II (LC3-II) and beclin-1. Exposure of tubular cells to TM or TG (0.2 μM) for 24 h induced autophagy, as evidenced by upregulation of LC3-II and beclin-1, which were abolished by pretreatment with ER stress inhibitors such as 4-PBA and salubrinal (fig. 5a, b). In line with the Western blot data, immunofluorescence staining demonstrated that 4-PBA and salubrinal suppressed the TM-induced beclin-1 expression (fig. 5c). Transfec-
tion with siRNA targeting the ER stress protein (IRE-1) also suppressed the TM or TG induced autophagy (fig. 6a, b).
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ER Stress by TM or TG Induced EMT at Least in Part through Autophagy To determine whether ER stress by TM or TG induced EMT through autophagy, we examined the effects of autophagy inhibitors on the TM- or TG-induced EMT. Autophagy inhibitors such as 3-MA (0.5 mM) and bafilomycin (10 nM) inhibited the TG- or TM-induced EMT, as shown by inhibition of TG- or TM-induced upregulation of α-SMA and downregulation of E-cadherin Moon/Kim/Nho/Jang/Lee
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Fig. 6. Suppression of TM- (a) and TG-induced (b) autophagy by siRNA targeting IRE-1. Cells were transfected with non-specific (Con siRNA) or siRNA targeting IRE-1 (IRE-1 siRNA). The cells were then treated with TM (0.2 μM) or TG (0.2 μM) for 24 h. Expression of LC3-ΙΙ and beclin-1 was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
(fig. 7a, b). Consistent with the Western blot data, immunofluorescence staining showed that 3-MA and bafilomycin inhibited the TM-induced EMT (fig. 8a). To confirm the role of autophagy in the TM- or TGinduced EMT, transfection with siRNA targeting autophagy protein (beclin-1) was performed. Transfection with siRNA targeting beclin-1 suppressed the TM- or TG-induced upregulation of α-SMA and downregulation of E-cadherin, but not non-specific siRNA (fig. 9a, b). These data suggested that ER stress by TM or TG promoted EMT, at least in part, through induction of autophagy. To confirm whether autophagy per se was able to induce EMT, we examined the effect of autophagy inducer (rapamycin, 5 nM) on the EMT. Rapamycin was found to increase EMT as well, which was blocked by pretreatment with autophagy inhibitors and siRNA targeting beclin-1 (fig. 7–9). The efficiency of siRNA targeting beclin-1 was examined. As expected, siRNA tarER Stress Induces EMT via Autophagy
geting beclin-1 inhibited the TM-, TG- or rapamycininduced beclin-1 expression but not non-specific siRNA (fig. 10a, b). c-Src Tyrosine Kinase Was Involved in Both TM- or TG-Induced Autophagy and EMT We sought to determine whether TM- or TG-induced autophagy and EMT were mediated via activation of cSrc kinase. Western blots revealed that both TM and TG induced the activation of c-Src kinase (fig. 11a). To examine whether activation of c-Src kinase was directly involved in TM- or TG-induced autophagy and EMT, we examined the effect of c-Src kinase inhibitor (PP2, 10 μM). As expected, PP2 suppressed the TM- or TG-induced activation of c-Src kinase (fig. 11b). PP2 also inhibited the TM- or TG-induced autophagy (fig. 11c, d) and EMT (fig. 12a, b).
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mycin (Bafil) and 3-MA. Proximal tubular cells were incubated with TM (0.2 μM), TG (0.2 μM) or Ra (5 nM) for 24 h with or without pretreatment with autophagy inhibitors such as 3-MA (0.5 mM) and Bafil (10 nM). Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG, or Ra.
Discussion
The present study demonstrates that ER stress by TM or TG induces EMT, at least in part, through autophagy via activation of c-Src kinase in tubular epithelial cells. Tubulointerstitial fibrosis is a final common pathway to end-stage chronic kidney diseases and its severity correlates with renal prognosis. Studies suggest that tubular 134
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epithelial cells play an essential role in the development of tubulointerstitial fibrosis [11], but the specific mechanism underlying linking tubular cells to tubulointerstitial fibrosis has not been completely understood. Recently, ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in a number of internal organs [2–5] through induction of EMT [6–9]. In kidney, it has been reported that ER stress by Moon/Kim/Nho/Jang/Lee
Color version available online
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Fig. 8. Immunofluorescence study showing that suppression of TM- (a) or rapamycininduced (b) EMT by autophagy inhibitors.
Proximal tubular cells were incubated with TM (0.2 μM) or rapamycin (5 nM) for 24 h with or without pretreatment with autophagy inhibitors such as 3-MA (0.5 mM) and bafilomycin (10 nM). Immunofluorescence staining for α-SMA and E-cadherin was performed. Representative microscopic scans are shown.
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cyclosporine and TG in tubular epithelial cells induces EMT [21]. In an animal model of unilateral ureteral obstruction, tubular epithelial ER stress induced apotosis, leading to tubulointerstitial fibrosis [3]. Similarly, we found that ER stress by TM or TG induced EMT in tubular epithelial cells. However, the mechanisms linking ER stress to EMT are incompletely understood. Tanjore et al. [7] reported that siRNA targeting of IRE-1 was able to block TM-induced EMT in alveolar epithelial cells by attenuation of TM-induced Smad2 and Src phosphorylation, which were involved in ER stress-induced EMT. They suggested that the IRE-1 arm of the UPR appeared to be involved in activating these signaling pathways. In support of their results, we found that the IRE-1 arm was involved in TM- or TG-induced EMT in renal tubular epithelial cells as well. Our study, together with other reports, supported the notion that EMT might be a common response by epithelial cells to substantial or prolonged ER stress. Further work is needed to better define the mechanisms and to determine whether targeting ER stress could have thera-
peutic benefit. Autophagy is an intracellular self-digesting process to regenerate energy by removal of long-lived proteins and organelles to support cell survival during stress, such as starvation. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism [14]. Several studies have reported that ER stress also induces autophagy in mammalian cancer cell lines and mouse embryonic fibroblasts [15, 16]. In tubular epithelial cells, ER stress by chemical ER stress inducers such as TM and brefeldin A induces autophagy [13]. Similarly, we found that ER stress by TM and TG induced autophagy in tubular epithelial cells. We also found that activation of the IRE-1 arm of the UPR was able to regulate autophagy, which was consistent with the result of previous study showing that activation of the IRE1/TRAF2/JNK arm of ER stress might regulate autophagy through modulation of beclin-1 function and expression [22]. However, little is known yet whether ER stress-induced autophagy contributes to EMT in renal tubular epithelial cells. We found that au-
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transfected with non-specific (Con siRNA) or siRNA targeting beclin-1 (Beclin siRNA). The cells were then treated with TM (0.2 μM), TG (0.2 μM) or Ra (5 nM) for 24 h. Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG or Ra.
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beclin-1. Cells were transfected with non-specific (Con siRNA) or siRNA targeting beclin-1 (Beclin siRNA). The cells were then treated with TM (0.2 μM), TG (0.2 μM) and Ra (5 nM) for 24 h. The efficiency of beclin-1 siRNA was evaluated by Western blotting of beclin-1. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG or Ra.
tophagy inhibitors (3-MA and bafilomycin) blocked the ER stress-induced EMT in tubular epithelial cells. siRNA targeting autophagy protein (beclin-1) also suppressed the ER stress-induced EMT. In addition, rapamycin, an autophagy inducer, increased EMT, which was blocked by autophagy inhibitors and siRNA targeting beclin-1 as well. These results indicated that induction of autophagy per se was able to promote EMT in tubular epithelial cells. In support of our observation, a similar finding was reported in a cancer cell. Li et al. [23] reported that starvation-induced autophagy played a crucial role in the invasion of hepatocellular carcinoma cells through activation of EMT. They also demonstrated that inhibition of autophagy by siRNA-Atgs (3 or 7) suppressed both Smad3 and TGF-β1, which were known to mediate EMT. We found that beclin-1 (Agt 6) also played an important role in autophagy-induced EMT. The underlying
mechanism linking beclin-1 to EMT is unclear. However, it has been reported that siRNA targeting beclin-1 inhibits TNFα-driven NF-κB activation [24]. TNFα-driven NF-κB activation is known to stabilize the transcription factor Snail which is involved in inducing EMT [25], suggesting a possible linkage between beclin-1 and EMT. Numerous signaling molecules are involved in inducing autophagy and EMT. We found that both TM and TG induced c-Src phosphorylation and PP2, a c-Src kinase inhibitor attenuated effects of TM- or TG-induced EMT, suggesting that activated c-Src kinase mediated ER stressinduced EMT in tubular epithelial cells. These results were compatible with previous reports in alveolar epithelial cells and thyroid cells [7, 8]. We also found that c-Src kinase was involved in ER stress-induced autophagy, which was compatible with a previous report that Src was involved in zVAD-induced autophagic cell death in murine fibrosarcoma cells [26].
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Fig. 11. Suppression of TM- or TG-induced autophagy by c-Srckinase inhibitor (PP2) (a, b). Proximal tubular cells were incubated with TM (0.2 μM) (c) or TG (0.2 μM) (d) for 24 h with or without pretreatment with c-Src kinase inhibitor (PP2, 10 μM). Expression of p-c-Src kinase, LC3-ΙΙ and beclin-1 was examined by Western
blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
Our study demonstrated that induction of ER stress led to autophagy, which resulted in EMT in renal tubular epithelial cells via activation of c-Src kinase, indicating possible links among ER stress, autophagy and EMT. Although our studies provide in vitro evidence of a link among ER stress, autophagy and EMT, in vivo models will be required to further dissect these pathways and their contributions in tubulointerstitial fibrosis. EMT is one of several proposed mechanisms through which myofibroblasts are generated. However, there still exists a controversy about the role of EMT in vivo kidney fibro-
sis. Iwano et al. [11] had reported that up to one third of fibroblasts could originate from tubular epithelia through EMT. Recent lineage-tracing studies suggest that the majority of interstitial myofibroblasts are probably derived from pericyte instead of EMT [27]. However, it has been suggested that results of lineage tracing may be very different in different injury/fibrogenic models [28]. Therefore, additional studies are necessary to determine to what extent EMT contributes to in vivo renal fibrosis in a variety of animal models.
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kinase inhibitor (PP2). Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with c-Src kinase inhibitor (PP2, 10 μM). Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative
In conclusion, ER stress by TM or TG induces EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. Identifying the pathways involved in ER stress-induced EMT may lead to new therapeutic strategies to limit tubulointerstitial fibrosis.
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Disclosure Statement The authors have no conflicts of interest to disclose.
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ER Stress Induces EMT via Autophagy
5 Lawson WE, Crossno PF, Polosukhin VV, Roldan J, Cheng DS, Lane KB, Blackwell TR, Xu C, Markin C, Ware LB, Miller GG, Loyd JE, Blackwell TS: Endoplasmic reticulum stress in alveolar epithelial cells is prominent in IPF: association with altered surfactant protein processing and herpesvirus infection. Am J Physiol Lung Cell Mol Physiol 2008; 294:L1119–L1126. 6 Lawson WE, Cheng DS, Degryse AL, Tanjore H, Polosukhin VV, Xu XC, Newcomb DC, Jones BR, Roldan J, Lane KB, Morrisey EE, Beers MF, Yull FE, Blackwell TS: Endoplasmic reticulum stress enhances fibrotic remodeling in the lungs. Proc Natl Acad Sci USA 2011;108:10562–10567. 7 Tanjore H, Cheng DS, Degryse AL, Zoz DF, Abdolrasulnia R, Lawson WE, Blackwell TS: Alveolar epithelial cells undergo epithelial to mesenchymal transition in response to endoplasmic reticulum stress. J Biol Chem 2011; 286:30972–30980.
8 Ulianich L, Garbi C, Treglia AS, Punzi D, Miele C, Raciti GA, Beguinot F, Consiglio E, Di Jeso B: ER stress is associated with dedifferentiation and an epithelial-to-mesenchymal transition-like phenotype in PC Cl3 thyroid cells. J Cell Sci 2008;121:477–486. 9 Yang L, Carlson SG, McBurney D, Horton WE Jr: Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis. J Biol Chem 2005;280:31156–31165. 10 Liu Y: Epithelial to mesenchymal transition in renal fibrogenesis: pathologic significance, molecular mechanism, and therapeutic intervention. J Am Soc Nephrol 2004;15:1–12. 11 Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG: Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 2002;110:341–350.
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12 Xie Z, Klionsky DJ: Autophagosome formation: core machinery and adaptations. Nat Cell Biol 2007;9:1102–1109. 13 Kawakami T, Inagi R, Takano H, Sato S, Ingelfinger JR, Fujita T, Nangaku M: Endoplasmic reticulum stress induces autophagy in renal proximal tubular cells. Nephrol Dial Transplant 2009;24:2665–2672. 14 Boya P, González-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, Métivier D, Meley D, Souquere S, Yoshimori T, Pierron G, Codogno P, Kroemer G: Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol 2005;25:1025–1040. 15 Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, Ogawa S, Kaufman RJ, Kominami E, Momoi T: ER stress (PERK/eIFα phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death Differ 2007;14:230–239. 16 Ding WX, Ni HM, Gao W, Hou YF, Melan MA, Chen X, Stolz DB, Shao ZM, Yin XM: Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem 2007;282:4702–4710.
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17 Kozutsumi Y, Segal M, Normington K, Gething MJ, Sambrook J: The presence of malfolded proteins in the endoplasmic reticulum signals the induction of glucose-regulated proteins. Nature 1988;332:462–464. 18 Treiman M, Caspersen C, Christensen SB: A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+ATPases. Trends Pharmacol Sci 1998;19:131– 135. 19 Wang YH, Li F, Schwartz JH, Flint PJ, Borkan SC: c-Src and HSP72 interact in ATP-depleted renal epithelial cells. Am J Physiol Cell Physiol 2001;281:C1667–C1675. 20 Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA, Torok-Storb B: HK-2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int 1994;45:48–57. 21 Pallet N, Bouvier N, Bendjallabah A, Rabant M, Flinois JP, Hertig A, Legendre C, Beaune P, Thervet E, Anglicheau D: Cyclosporine-induced endoplasmic reticulum stress triggers tubular phenotypic changes and death. Am J Transplant 2008;8:2283–2296. 22 Verfaillie T, Salazar M, Velasco G, Agostinis P: Linking ER stress to autophagy: potential implications for cancer therapy. Int J Cell Biol 2010;2010:930509.
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23 Li J, Yang B, Zhou Q, Wu Y, Shang D, Guo Y, Song Z, Zheng Q, Xiong J: Autophagy promotes hepatocellular carcinoma cell invasion through activation of epithelial-mesenchymal transition. Carcinogenesis 2013; 34: 1343– 1351. 24 Criollo A, Chereau F, Malik SA, Niso-Santano M, Mariño G, Galluzzi L, Maiuri MC, Baud V, Kroemer G: Autophagy is required for the activation of NF-κB. Cell Cycle 2012; 11: 194– 199. 25 Wu Y, Deng J, Rychahou PG, Qiu S, Evers BM, Zhou BP: Stabilization of snail by NF-κB is required for inflammation-induced cell migration and invasion. Cancer Cell 2009; 15: 416–428. 26 Chen SY, Chiu LY, Maa MC, Wang JS, Chien CL, Lin WW: zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation. Autophagy 2011;7:217–228. 27 Grgic I, Duffield JS, Humphreys BD: The origin of interstitial myofibroblasts in chronic kidney disease. Pediatr Nephrol 2012;27:183– 193. 28 Kage H, Borok Z: EMT and interstitial lung disease: a mysterious relationship. Curr Opin Pulm Med 2012;18:517–523.
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Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
Received: December 2, 2013 Accepted: March 20, 2014 Published online: May 22, 2014
Endoplasmic Reticulum Stress Induces Epithelial-Mesenchymal Transition through Autophagy via Activation of c-Src Kinase Soo Young Moon Hyo Sang Kim Kyeong Woo Nho Young Joo Jang Sang Koo Lee Department of Internal Medicine, Asan Medical Center, University of Ulsan, Seoul, Korea
Key Words Autophagy · Epithelial-mesenchymal transition · Endoplasmic reticulum stress · c-Src kinase
Abstract Background: Endoplasmic reticulum (ER) stress has been implicated in inducing epithelial-mesenchymal transition (EMT). ER stress is also known to induce autophagy. However, it is unclear whether ER stress-induced autophagy contributes to EMT. We hypothesized that ER stress might induce EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. Method: All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Immunofluorescence and small interfering RNA (siRNA) experiments were performed. Results: Chemical ER stress inducers such as tunicamycin (TM, 0.2 μM) and thapsigargin (TG, 0.2 μM) induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ER stress inhibitors such as 4-PBA and salubrinal suppressed both TM- and TG-induced EMT. TM and TG also induced autophagy, as evidenced by upregulation of LC3-II and beclin-1, which were abolished by pretreatment with ER stress inhibitors. Transfection with siRNA targeting ER stress protein (IRE-1) blocked the TM- or TG-induced EMT and au-
© 2014 S. Karger AG, Basel 1660–2129/14/1263–0127$39.50/0 E-Mail [email protected] www.karger.com/nee
tophagy. Autophagy inhibitors such as 3-methyladenine and bafilomycin inhibited the TM- or TG-induced EMT. Transfection with siRNA targeting autophagy protein (beclin-1) also blocked the TM- or TG-induced EMT. Both TM and TG induced activation of c-Src kinase. Inhibitor of c-Src kinase (PP2) suppressed the TM- or TG-induced autophagy and EMT. Conclusion: ER stress by TM or TG induced EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. © 2014 S. Karger AG, Basel
Introduction
Endoplasmic reticulum (ER) stress refers to physiological or pathological states that result in accumulation of misfolded proteins in the ER. Cells respond to ER stress by activating a series of integrative stress pathways termed the unfolded protein response (UPR). The three arms of the UPR include processes designed to attenuate protein translation, increase the production of metabolism and redox proteins, enhance production of protein folding chaperones, and upregulate the production of protein degradation enzymes through the activation of the ER transmembrane sensors PERK, ATF6, and IRE-1. GRP78 is a central Sang Koo Lee, MD Division of Nephrology, Department of Internal Medicine Asan Medical Center, 88, Olympic-ro 43-gil Songpa-gu, Seoul 138-736 (Korea) E-Mail sklee2 @ amc.seoul.kr
regulator of ER homeostasis and phosphorylation of eIF2α leads to attenuate the translation initiation rate, both of which are biomarkers for ER stress. UPR may either promote cell survival, or if the ER stress is chronic or excessive may lead to apoptosis by inducing proapoptotic transcription factor CHOP [1]. In addition to effects on apoptosis, ER stress and UPR pathways have been suggested as important determinants of fibrotic remodeling in a number of internal organs, including the lungs, liver, gastrointestinal tract, heart and kidney [2–5] through induction of epithelial-mesenchymal transition (EMT) [6–9]. EMT defines a phenotypic conversion of primary epithelial cells into mesenchymal cells, leading to morphological changes to fibroblastoid morphology, downregulation of epithelial marker proteins such as E-cadherin, ZO-1 and cytokeratin, and finally upregulation of mesenchymal markers including α-smooth muscle actin (α-SMA), vimentin and fibroblast-specific protein-1 [10]. EMT is thought to contribute to fibrotic remodeling in a number of organs. In kidney, tubular epithelial cells have been implicated in tubulointerstitial fibrosis as a source of interstitial fibroblasts via EMT [11]. However, information about ER stress-induced EMT in renal tubular epithelial cells is still limited. Autophagy is a tightly regulated, programmed mechanism to eliminate damaged organelles and proteins to maintain cellular homeostasis. Autophagy involves sequestration of proteins and cell organelles in a double membrane-bound structure called autophagosome, which fuses with a lysosome, after which its contents and inner membrane are degraded and recycled. The formation of autophagosome is known to be dependent on the induction of several genes including LC3, beclin-1, phosphatidylinositide 3-kinase and Atgs [12]. During autophagosome formation, LC3 is converted from the unconjugated form (LC3-I) to the phosphatidylethanolamine (PE)-conjugated form (LC3-II), which is then incorporated into an autophagosomal membrane [13]. It has been suggested that autophagy plays an important role in a variety of biological processes, including cell death, development, etc. [14]. ER stress is also known to upregulate the autophagy machinery [13, 15, 16]. However, whether ER stress-induced autophagy contributes to EMT has not been explored so far. We hypothesized that ER stress might induce EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. In our study, ER stress was induced by two different chemical agents: tunicamycin (TM) and thapsigargin (TG). TM elicits ER stress by inhibiting protein N-linked glycosylation, leading to accumulation of unfolded pro128
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teins and consequent UPR activation [17]. TG inhibits sarco-endoplasmic reticulum Ca-ATPase and blocks calcium reuptake into the ER lumen, leading to disruption of protein folding and subsequent induction of ER stress [18]. We examined the effects of two different ER stress inhibitors (4-PBA and salubrinal) on the TM- or TG-induced autophagy and EMT. To confirm the role of ER stress on the autophagy and EMT, we investigated the effects of small interfering RNA (siRNA) targeting ER stress protein (IRE-1). We also examined the effects of autophagy inhibitors (3-MA, bafilomycin) and siRNA targeting autophagy protein (beclin-1) on the TM- or TG-induced EMT. c-Src is a member of the Src tyrosine kinase family, which plays a role in signal transduction in response to many external stimuli and its activity is under tight redox control [19]. Src activation in ER stress-induced EMT was reported in non-renal cells [7, 8]. However, it is unclear whether c-Src activation mediates ER stress-induced EMT in renal tubular epithelial cells. Furthermore, involvement of Src in the induction of ER stress-induced autophagy has not been reported so far. Therefore, we finally examined whether c-Src kinase was involved in both ER stress-induced autophagy and EMT. Methods Reagent TM, TG, 3-methyladenine (3-MA) and bafilomycin were obtained from Sigma Chemical Co. (St. Louis, Mo., USA). 4-Phenylbutyric acid (4-PBA), salubrinal, PP2 and rapamycin were obtained from Calbiochem (San Diego, Calif., USA). Antibodies to α-SMA, E-cadherin and GRP78 were acquired from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Antibodies to CHOP, beclin-1, microtubule-associated protein-1 light chain-3 (LC3), IRE-1, total c-Src kinase, phosphospecific-c-Src kinase (Tyr416), and horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Beverly, Mass., USA). Cell Culture and Conditioning All experiments were performed using HK-2 cells, a human proximal tubular cell line. HK-2 cells were obtained from the American Type Culture Collection and have been characterized previously [20]. The media were changed every 3 days until confluent. Cells were growth-arrested in serum-free medium for 24 h before being used in experiments. Cells were incubated with TM (0–0.2 μM) or TG (0–0.2 μM) for up to 24 h with or without 4-PBA (0.5 mM), salubrinal (10 μM), 3-MA (0.5 mM), bafilomycin (10 nM) and PP2 (10 μM). Western Blot Analysis An equal amount of protein from whole cell lysates was separated by a 10% SDS-polyacrylamide gels, transferred to nylon
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Fig. 1. Induction of GRP78 (a) and CHOP expression (b) by TM or TG and inhibition of TM- or TG-induced CHOP expression by both 4-PBA and salubrinal in HK-2 cells. Proximal tubular cells were incubated with TM (0–0.2 μM) or TG (0–0.2 μM) for up to 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression
of GRP78 and CHOP was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
membrane. Membranes were incubated for 2 h with primary antibody, followed by peroxidase-conjugated secondary antibody. Antibody-antigen complexes were detected with ECL plus chemiluminescence (Amersham Pharmacia Biotech, Arlington, Ill., USA). The band intensities were quantified using a GS-710 densitometer and QuantityOne software (Bio-Rad, Hercules, Calif., USA).
nology, 20 nmol/ml) for 6 h in serum-free medium and then culture medium was changed to normal medium containing 10% FBS for 24 h. Non-specific siRNA was used as negative control. The efficiency of IRE-1 siRNA and beclin-1 siRNA was evaluated by Western blotting of IRE-1 and beclin-1 respectively. Transfected cells were incubated with TM or TG for 24 h. Western blotting for α-SMA, E-cadherin, LC3 and beclin-1 was performed.
Immunofluorescence Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2% Triton X-100 and then blocked with 2% BSA in PBS for 1 h. Cells were incubated overnight with primary antibody against α-SMA, E-cadherin, GRP78 and beclin-1. The specimens were then washed with PBS and incubated with FITC-conjugated secondary antibody for 1 h at room temperature. Nuclei were stained with DAPI (4′,6-diamidino2-phenylindole). After washing with PBS, coverslips were mounted in 80% glycerol in PBS and photographed using a confocal microscope.
Statistical Analysis Data were expressed as mean ± SE. Kruskal-Wallis test was used for comparison of more than two groups, followed by a Mann-Whitney U-test for comparison using a microcomputerassisted program with SPSS for Windows 10.0 (SPSS, Inc., Chicago, Ill., USA). p < 0.05 was considered significant.
siRNA Transfection Transfection of siRNA was performed with Lipofectamine 2000, according to the manufacturer’s instructions (Invitrogen, Carlsbad, Calif., USA). Cells were transfected with siRNA against IRE-1 (Ambion, 100 pmol/ml) or beclin-1 (Cell Signaling Tech-
ER Stress Induces EMT via Autophagy
Results
Both TM and TG Induced ER Stress, Which Were Blocked by ER Stress Inhibitors Such as 4-PBA and Salubrinal in Our HK-2 Cells In initial studies, we sought to determine whether well-known chemical ER stress inducers (TM and TG) Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
129
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E-cadherin was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
Color version available online
Fig. 2. Inhibition of TM- (a) or TG-induced (b) EMT by ER stress inhibitors such as 4-PBA and salubrinal. Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression of α-SMA and
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Fig. 3. Immunofluorescence study showing
that suppression of TM-induced upregulation of α-SMA and downregulation of Ecadherin by ER stress inhibitors. Proximal tubular cells were incubated with TM (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (10 μM). Immunofluorescence staining for α-SMA and Ecadherin was performed. Representative microscopic scans are shown.
(FDGKHULQ
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and ER stress inhibitors (4-PBA and salubrinal) could be used as ER stress inducers and inhibitors respectively in our HK-2 cells. To determine the effects of ER stress inducers and inhibitors on the ER stress, we examined the change of two ER stress biomarkers, upregulation of GRP78 and CHOP expression. As would be expected, both TM (0.2 μM) and TG (0.2 μM) induced the expression of GRP78 in a time-dependent manner (0–24 h) in HK-2 cells. They also increased the GRP78 expression in 130
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
70
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a dose-dependent manner (0–0.2 μM) at 24 h of incubation. 0.2 μM of TM and TG induced the expression of CHOP, which was attenuated by pretreatment with ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (10 μM) (fig. 1a, b). ER Stress by TM or TG Induced EMT To determine whether ER stress by TM or TG induced EMT in tubular epithelial cells, we examined the change Moon/Kim/Nho/Jang/Lee
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Fig. 4. Suppression of TM- (a) or TG-induced (b) EMT by siRNA targeting IRE-1 (c). Cells were transfected with non-specific (Con siRNA) or siRNA targeting IRE-1 (IRE-1 siRNA). The cells were then treated with TM (0.2 μM) or TG (0.2 μM) for 24 h. Expression of α-SMA and E-cadherin was examined by Western blot analysis. The efficiency of IRE-1 siRNA was evaluated by Western blotting of IRE-1. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
of two EMT biomarkers, upregulation of α-SMA and downregulation of E-cadherin. Exposure of tubular cells to TM or TG (0.2 μM) for 24 h induced EMT, as shown by upregulation of α-SMA and downregulation of E-cadherin, which was abolished by pretreatment with ER stress inhibitors such as 4-PBA and salubrinal (fig. 2a, b). In agreement with the Western blot data, immunofluorescence staining revealed that 4-PBA and salubrinal suppressed the TM-induced upregulation of α-SMA and downregulation of E-cadherin (fig. 3). To confirm the
role of ER stress in the induction of EMT, we knocked down the ER stress protein (IRE-1) expression by siRNA transfection. Transfection with siRNA targeting IRE-1 suppressed the TM or TG-induced EMT, but not nonspecific siRNA (fig. 4a, b). These data indicated that TMor TG-induced EMT was mediated by a process involving ER stress. The efficiency of siRNA targeting IRE-1 was also evaluated. As expected, siRNA targeting IRE-1 inhibited the TM- or TG-induced IRE-1 expression but not non-specific siRNA (fig. 4c).
ER Stress Induces EMT via Autophagy
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
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Color version available online
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Fig. 5. Inhibition of TM- (a) and TG-induced (b) autophagy by ER stress inhibitors such as 4-PBA and salubrinal. Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with the ER stress inhibitors such as 4-PBA (0.5 mM) and salubrinal (Salu, 10 μM). Expression of LC3-ΙΙ and beclin-1 was examined by Western blot analysis. Representa-
tive blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG. Immunofluorescence staining for beclin-1 (c) was performed. Representative microscopic scans are shown.
ER Stress by TM or TG Induced Autophagy To determine whether ER stress by TM or TG induced autophagy in tubular epithelial cells, we examined the change of two autophagy biomarkers, upregulation of microtubule-associated protein-1 light chain3-II (LC3-II) and beclin-1. Exposure of tubular cells to TM or TG (0.2 μM) for 24 h induced autophagy, as evidenced by upregulation of LC3-II and beclin-1, which were abolished by pretreatment with ER stress inhibitors such as 4-PBA and salubrinal (fig. 5a, b). In line with the Western blot data, immunofluorescence staining demonstrated that 4-PBA and salubrinal suppressed the TM-induced beclin-1 expression (fig. 5c). Transfec-
tion with siRNA targeting the ER stress protein (IRE-1) also suppressed the TM or TG induced autophagy (fig. 6a, b).
132
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
ER Stress by TM or TG Induced EMT at Least in Part through Autophagy To determine whether ER stress by TM or TG induced EMT through autophagy, we examined the effects of autophagy inhibitors on the TM- or TG-induced EMT. Autophagy inhibitors such as 3-MA (0.5 mM) and bafilomycin (10 nM) inhibited the TG- or TM-induced EMT, as shown by inhibition of TG- or TM-induced upregulation of α-SMA and downregulation of E-cadherin Moon/Kim/Nho/Jang/Lee
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Fig. 6. Suppression of TM- (a) and TG-induced (b) autophagy by siRNA targeting IRE-1. Cells were transfected with non-specific (Con siRNA) or siRNA targeting IRE-1 (IRE-1 siRNA). The cells were then treated with TM (0.2 μM) or TG (0.2 μM) for 24 h. Expression of LC3-ΙΙ and beclin-1 was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
(fig. 7a, b). Consistent with the Western blot data, immunofluorescence staining showed that 3-MA and bafilomycin inhibited the TM-induced EMT (fig. 8a). To confirm the role of autophagy in the TM- or TGinduced EMT, transfection with siRNA targeting autophagy protein (beclin-1) was performed. Transfection with siRNA targeting beclin-1 suppressed the TM- or TG-induced upregulation of α-SMA and downregulation of E-cadherin, but not non-specific siRNA (fig. 9a, b). These data suggested that ER stress by TM or TG promoted EMT, at least in part, through induction of autophagy. To confirm whether autophagy per se was able to induce EMT, we examined the effect of autophagy inducer (rapamycin, 5 nM) on the EMT. Rapamycin was found to increase EMT as well, which was blocked by pretreatment with autophagy inhibitors and siRNA targeting beclin-1 (fig. 7–9). The efficiency of siRNA targeting beclin-1 was examined. As expected, siRNA tarER Stress Induces EMT via Autophagy
geting beclin-1 inhibited the TM-, TG- or rapamycininduced beclin-1 expression but not non-specific siRNA (fig. 10a, b). c-Src Tyrosine Kinase Was Involved in Both TM- or TG-Induced Autophagy and EMT We sought to determine whether TM- or TG-induced autophagy and EMT were mediated via activation of cSrc kinase. Western blots revealed that both TM and TG induced the activation of c-Src kinase (fig. 11a). To examine whether activation of c-Src kinase was directly involved in TM- or TG-induced autophagy and EMT, we examined the effect of c-Src kinase inhibitor (PP2, 10 μM). As expected, PP2 suppressed the TM- or TG-induced activation of c-Src kinase (fig. 11b). PP2 also inhibited the TM- or TG-induced autophagy (fig. 11c, d) and EMT (fig. 12a, b).
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
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Fig. 7. Inhibition of TM- (a), TG- (b) or rapamycin (Ra)-induced EMT by autophagy inhibitors such as bafilo-
mycin (Bafil) and 3-MA. Proximal tubular cells were incubated with TM (0.2 μM), TG (0.2 μM) or Ra (5 nM) for 24 h with or without pretreatment with autophagy inhibitors such as 3-MA (0.5 mM) and Bafil (10 nM). Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG, or Ra.
Discussion
The present study demonstrates that ER stress by TM or TG induces EMT, at least in part, through autophagy via activation of c-Src kinase in tubular epithelial cells. Tubulointerstitial fibrosis is a final common pathway to end-stage chronic kidney diseases and its severity correlates with renal prognosis. Studies suggest that tubular 134
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
epithelial cells play an essential role in the development of tubulointerstitial fibrosis [11], but the specific mechanism underlying linking tubular cells to tubulointerstitial fibrosis has not been completely understood. Recently, ER stress and UPR pathways are emerging as important determinants of fibrotic remodeling in a number of internal organs [2–5] through induction of EMT [6–9]. In kidney, it has been reported that ER stress by Moon/Kim/Nho/Jang/Lee
Color version available online
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Fig. 8. Immunofluorescence study showing that suppression of TM- (a) or rapamycininduced (b) EMT by autophagy inhibitors.
Proximal tubular cells were incubated with TM (0.2 μM) or rapamycin (5 nM) for 24 h with or without pretreatment with autophagy inhibitors such as 3-MA (0.5 mM) and bafilomycin (10 nM). Immunofluorescence staining for α-SMA and E-cadherin was performed. Representative microscopic scans are shown.
E-cadherin
b
cyclosporine and TG in tubular epithelial cells induces EMT [21]. In an animal model of unilateral ureteral obstruction, tubular epithelial ER stress induced apotosis, leading to tubulointerstitial fibrosis [3]. Similarly, we found that ER stress by TM or TG induced EMT in tubular epithelial cells. However, the mechanisms linking ER stress to EMT are incompletely understood. Tanjore et al. [7] reported that siRNA targeting of IRE-1 was able to block TM-induced EMT in alveolar epithelial cells by attenuation of TM-induced Smad2 and Src phosphorylation, which were involved in ER stress-induced EMT. They suggested that the IRE-1 arm of the UPR appeared to be involved in activating these signaling pathways. In support of their results, we found that the IRE-1 arm was involved in TM- or TG-induced EMT in renal tubular epithelial cells as well. Our study, together with other reports, supported the notion that EMT might be a common response by epithelial cells to substantial or prolonged ER stress. Further work is needed to better define the mechanisms and to determine whether targeting ER stress could have thera-
peutic benefit. Autophagy is an intracellular self-digesting process to regenerate energy by removal of long-lived proteins and organelles to support cell survival during stress, such as starvation. In response to stress, autophagy is induced and may either contribute to cell death or serve as a cell survival mechanism [14]. Several studies have reported that ER stress also induces autophagy in mammalian cancer cell lines and mouse embryonic fibroblasts [15, 16]. In tubular epithelial cells, ER stress by chemical ER stress inducers such as TM and brefeldin A induces autophagy [13]. Similarly, we found that ER stress by TM and TG induced autophagy in tubular epithelial cells. We also found that activation of the IRE-1 arm of the UPR was able to regulate autophagy, which was consistent with the result of previous study showing that activation of the IRE1/TRAF2/JNK arm of ER stress might regulate autophagy through modulation of beclin-1 function and expression [22]. However, little is known yet whether ER stress-induced autophagy contributes to EMT in renal tubular epithelial cells. We found that au-
ER Stress Induces EMT via Autophagy
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
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transfected with non-specific (Con siRNA) or siRNA targeting beclin-1 (Beclin siRNA). The cells were then treated with TM (0.2 μM), TG (0.2 μM) or Ra (5 nM) for 24 h. Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG or Ra.
136
Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
Moon/Kim/Nho/Jang/Lee
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beclin-1. Cells were transfected with non-specific (Con siRNA) or siRNA targeting beclin-1 (Beclin siRNA). The cells were then treated with TM (0.2 μM), TG (0.2 μM) and Ra (5 nM) for 24 h. The efficiency of beclin-1 siRNA was evaluated by Western blotting of beclin-1. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM, TG or Ra.
tophagy inhibitors (3-MA and bafilomycin) blocked the ER stress-induced EMT in tubular epithelial cells. siRNA targeting autophagy protein (beclin-1) also suppressed the ER stress-induced EMT. In addition, rapamycin, an autophagy inducer, increased EMT, which was blocked by autophagy inhibitors and siRNA targeting beclin-1 as well. These results indicated that induction of autophagy per se was able to promote EMT in tubular epithelial cells. In support of our observation, a similar finding was reported in a cancer cell. Li et al. [23] reported that starvation-induced autophagy played a crucial role in the invasion of hepatocellular carcinoma cells through activation of EMT. They also demonstrated that inhibition of autophagy by siRNA-Atgs (3 or 7) suppressed both Smad3 and TGF-β1, which were known to mediate EMT. We found that beclin-1 (Agt 6) also played an important role in autophagy-induced EMT. The underlying
mechanism linking beclin-1 to EMT is unclear. However, it has been reported that siRNA targeting beclin-1 inhibits TNFα-driven NF-κB activation [24]. TNFα-driven NF-κB activation is known to stabilize the transcription factor Snail which is involved in inducing EMT [25], suggesting a possible linkage between beclin-1 and EMT. Numerous signaling molecules are involved in inducing autophagy and EMT. We found that both TM and TG induced c-Src phosphorylation and PP2, a c-Src kinase inhibitor attenuated effects of TM- or TG-induced EMT, suggesting that activated c-Src kinase mediated ER stressinduced EMT in tubular epithelial cells. These results were compatible with previous reports in alveolar epithelial cells and thyroid cells [7, 8]. We also found that c-Src kinase was involved in ER stress-induced autophagy, which was compatible with a previous report that Src was involved in zVAD-induced autophagic cell death in murine fibrosarcoma cells [26].
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Fig. 11. Suppression of TM- or TG-induced autophagy by c-Srckinase inhibitor (PP2) (a, b). Proximal tubular cells were incubated with TM (0.2 μM) (c) or TG (0.2 μM) (d) for 24 h with or without pretreatment with c-Src kinase inhibitor (PP2, 10 μM). Expression of p-c-Src kinase, LC3-ΙΙ and beclin-1 was examined by Western
blot analysis. Representative blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
Our study demonstrated that induction of ER stress led to autophagy, which resulted in EMT in renal tubular epithelial cells via activation of c-Src kinase, indicating possible links among ER stress, autophagy and EMT. Although our studies provide in vitro evidence of a link among ER stress, autophagy and EMT, in vivo models will be required to further dissect these pathways and their contributions in tubulointerstitial fibrosis. EMT is one of several proposed mechanisms through which myofibroblasts are generated. However, there still exists a controversy about the role of EMT in vivo kidney fibro-
sis. Iwano et al. [11] had reported that up to one third of fibroblasts could originate from tubular epithelia through EMT. Recent lineage-tracing studies suggest that the majority of interstitial myofibroblasts are probably derived from pericyte instead of EMT [27]. However, it has been suggested that results of lineage tracing may be very different in different injury/fibrogenic models [28]. Therefore, additional studies are necessary to determine to what extent EMT contributes to in vivo renal fibrosis in a variety of animal models.
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Nephron Exp Nephrol 2014;126:127–140 DOI: 10.1159/000362457
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Fig. 12. Suppression of TM- (a) or TG-induced (b) EMT by c-Src
kinase inhibitor (PP2). Proximal tubular cells were incubated with TM (0.2 μM) or TG (0.2 μM) for 24 h with or without pretreatment with c-Src kinase inhibitor (PP2, 10 μM). Expression of α-SMA and E-cadherin was examined by Western blot analysis. Representative
In conclusion, ER stress by TM or TG induces EMT through autophagy via activation of c-Src kinase in tubular epithelial cells. Identifying the pathways involved in ER stress-induced EMT may lead to new therapeutic strategies to limit tubulointerstitial fibrosis.
TG
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blots and quantitative analysis from three independent experiments are shown. Results were expressed as n-fold increase over control as mean ± SE. # p < 0.05 vs. control (Con); ## p < 0.05 vs. TM or TG.
Disclosure Statement The authors have no conflicts of interest to disclose.
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Copyright: S. Karger AG, Basel 2014. Reproduced with the permission of S. Karger AG, Basel. Further reproduction or distribution (electronic or otherwise) is prohibited without permission from the copyright holder.
