Mol Cell Biochem (2014) 394:1–12 DOI 10.1007/s11010-014-2073-8
Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways Yan Huang • Xiaohui Li • Yarui Wang Huan Wang • Cheng Huang • Jun Li
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Received: 31 December 2013 / Accepted: 18 April 2014 / Published online: 25 June 2014 Ó Springer Science+Business Media New York 2014
Abstract Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and
provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1a1 and a-smooth muscle actin (a-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis recovery. Keywords Liver fibrosis Endoplasmic reticulum stress Calpain Caspase MAPK Apoptosis
Y Huang and Xiaohui Li are co-first authors; they contributed equally to the work. Y. Huang X. Li Y. Wang H. Wang C. Huang J. Li (&) School of Pharmacy, Institute for Liver Diseases, Anhui key laboratory of bioactivity of natural products, Anhui Medical University, Hefei, China e-mail: [email protected] Y. Huang e-mail: [email protected] X. Li e-mail: [email protected]
Abbreviations a-SMA Alpha-smooth muscle actin CHOP C/EBP homologues protein ECM Extracellular matrix ER Endoplasmic reticulum ERK Extracellular signal-regulated protein kinase HF Hepatic fibrosis HSCs Hepatic stellate cells JNK C-jun N-terminal kinase MAPK Mitogen-activated protein kinase siRNA Small interfering RNA TG Thapsigargin UPR Unfolded protein response
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Introduction Liver fibrosis is the result of the wound-healing response of the liver to repeated injury [1]. It is caused by a variety of agents, including chronic viral hepatitis, alcohol toxicity, autoimmune disease, and hereditary metabolic disorders. In response to chronic liver injury, hepatic stellate cells (HSCs) develop into a myofibroblast-like phenotype with expression of a-smooth muscle actin (a-SMA) and secretion of excess extracellular matrix (ECM), predominantly collagen type I [2–4]. As HSCs play a crucial role in liver fibrosis, the elimination of activated HSCs through cell death is proposed as a mechanism to attenuate or reverse fibrogenesis [5, 6]. Therefore, it is necessary to accelerate the apoptosis of activated HSCs and it is a significant target in reversing hepatic fibrosis. Apoptosis is an essential mechanism for cell clearance. There are mainly three apoptotic signaling pathways that have been discovered: (1) the mitochondrial pathway; (2) the death receptor-associated pathway; (3) the ER-associated pathway [7–9]. Previous results revealed the effect of Kupffer cells releasing tumor necrosis factor related apoptosis-inducing ligand (TRAIL) on activated HSCs apoptosis in hepatic fibrosis regression and that the mechanism of it may correlate with the mitochondrial pathway and the ER-associated pathway [10–12]. In recent years, the study has found that the apoptosis mechanism of ER stress-mediated apoptosis is a new signaling pathway of apoptosis, but the exact mechanisms remain unclear. Endoplasmic reticulum (ER) plays pivotal roles in various cell processes, including synthesizing, sorting, assembling, modifying and trafficking proteins, and maintaining intracellular Ca2? homeostasis [13]. Accumulation of misfolded or unfolded proteins in ER, caused by impairment of Ca2? homeostasis or other noxious stimuli, would trigger an ER stress response [14, 15]. A short-term UPR functions as a pro-survival response via reducing deposition of unfolded proteins and restoring normal ER function [16]. However, if ER stress is prolonged, its down-stream signaling initiates a series of complex response networks to strengthen ER stress, activates proapoptosis signal pathways, and ultimately induces ER associated with CCAAT/enhancer-binding protein homologous protein (CHOP) and ER resident protein caspase-12 [17]. Previous reports showed that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER. Caspase-12 deficient mice are resistant to ER stress-induced apoptosis [18]. Calpain deficient mouse embryonic fibroblasts (MEFs) have been shown to decrease
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Fig. 1 Effect of TG on cell viability with MTT assay. The cell viability was assayed in PDGF-induced HSC stimulated with TG by 24 h at indicated concentrations (0, 0.25, 0.5, 1, and 2 lM). 1 control, 2 PDGF (20 ng/ml), 3 PDGF ? TG (0.25 lM), 4 PDGF ? TG (0.5 lM), 5 PDGF ? TG (1 lM), 6 PDGF ? TG (2 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \ 0.05 versus PDGF, #P \ 0.05 versus TG values
ER stress-induced activation of caspase-12 and be resistant to ER stress-induced apoptosis [19]. Both calpain and caspase have been proposed to mediate processing and activation of caspase-12 after induction of the ER stress [20]. Moreover, Ca2? released from the ER enters mitochondria leading to caspase-9, caspase-7, and caspase-3 [21, 22]. However, the role of these molecules in inducing HSCs apoptosis, their correlation with the Calpain/Caspase expression and the subsequent contribution of this process to hepatic fibrogenesis still remain unclear. Mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinases (JNK), and p38 MAPK, regulate signaling mechanisms for cell proliferation, differentiation, survival, death, and inflammation [23]. Indeed, previous studies showed that ERK, JNK, and p38 MAPK mediate HSCs activation in the pathogenesis of liver fibrosis [24]. However, our previous laboratory research showed that JNK may participate in mediating activated HSCs apoptosis though death receptors DR4/DR5 and pro-apoptosis factor CHOP [25]. Some other reports showed the engagement of activation of the cell death promoting kinase, MAPK in many kinds of cell death associated with ER stress [26, 27]. To further study the mechanism of its action, we monitored the effects of ER stress-associated factors, as well as those of MAPK signaling pathways. There are many signal conduction paths and mechanisms that may participate in the apoptosis reduced by ER stress. However, the mechanisms of ER stress effecting activated HSCs apoptosis have not been fully understood to date. The
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Fig. 2 Effect of EGTA and BAPTA/AM on TG-induced activated HSCs apoptosis. Apoptosis of HSCs were analyzed by flow cytometry with Annexin V-FITC and PI staining. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF?TG (1 lM); 4, PDGF?TG?EGTA (100 lM); 5, PDGF?TG?BAPTA/AM (50 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
aim of this study was therefore to explore the potential apoptotic process of activated HSCs associated with ER stress mechanisms and the resolution of liver fibrosis.
and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), and all other chemicals were purchased from Sigma Technology.
Materials and methods
Cell culture and activation
Regent
A cell line of rat HSC was purchased from Chinese Academy of Sciences (Shanghai, China). HSCs were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10 % fetal bovine serum and 1 % antibiotic mixture comprising penicillin–streptomycin, and maintained at 37 °C in a humidified incubator containing 5 % CO2. HSCs were activated by treating with platelet derived growth factor (PDGF) (20 ng/ml) for 24 h.
Antibodies directed against caspase-3, p-p38 MAPK, p-ERK, p-JNK, p38 MAPK, ERK, and JNK were purchased from Cell Signaling Technology. Caspase-12 was purchased from Biovision Technology. CHOP was purchased from Bioworld Technology. a-SMA was purchased from Proteintech Technology. Calpain and calpeptin were purchased from Santa Cruz Technology. Thapsigargin, dimethylsulfoxide (DMSO),
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a
Calpain
α-SMA C-Caspase-12 Caspase-9
Col1α1
Caspase-3
C-Caspase-3
β-actin β-actin
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Fig. 3 Effect of TG on a-SMA and Col1a1 expression in activated HSCs. The protein level of a-SMA and Col1a1 was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM). Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
MTT assay Cell viability assay was determined by MTT. HSCs were seeded into each well of 96-well culture plates and then cultured in DMEM for 24 h. After culture, 150 ll of MTT
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b
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Mol Cell Biochem (2014) 394:1–12 b Fig. 4 Effect of EGTA and BAPTA/AM on calpain/Caspases
expression in TG-induced activated HSCs apoptosis. TG-induced activated HSCs apoptosis is Ca2? dependent involving the activation of calpain, C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 were detected using western blotting. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, PDGF ? TG ? EGTA (100 lM); 5, PDGF ? TG ? BAPTA/AM (50 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
solution (5 mg/ml) was added to each well, and the plates were then incubated at 37 °C for another 4 h. The optical density (OD) was measured at 490 nm after removing the supernatant and shaking with 150 ll DMSO by Thermomax microplate reader (Bio-TekEL, USA). All experiments were performed in triplicate and repeated at least three times. Flow cytometric analysis Since apoptosis analysis cells were performed with Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen, USA), samples from different groups were collected by trypsinization, and washed twice with cold phosphate buffered saline (PBS) buffer. Before analyses were performed on BD LSR flow cytometer (BD Biosciences), cells were resuspended in 400 ll Annexin-V binding buffer, added with 5 ll Annexin-V-FITC cultured at 2–8 °C for 15 min, and then added with 10 ll propidium iodide (PI) cultured at 2–8 °C for 5 min in dark. All experiments were performed in triplicate and repeated at least three times. siRNA transfection To stably knock down CHOP expression, RNAi (GenePharma, China) experiments in HSCs were performed by forward transfection in day 2 cultured HSCs using LipofectamineTM2000 (Invitrogen, USA) according to the manufacturer’s protocol. A negative control RNAi was purchased from Genepharma. The sense sequence for CHOP was showed as follows: SiCHOP (rat): 50 -CCAGUGCCAAGCAUGUAAUTT-30 ; 50 -AUUACAUGCUUGGCACUGGTT-30 . Western blotting analysis Proteins were isolated and quantified using the BCA protein assay kit (Boster, China). Total protein (30 or 50 mg) from samples was separated by SDS-PAGE and blotted onto a PVDF membrane (Millipore Corp, Billerica, MA, USA). The membranes were blocked with 5 % non-fat milk 3 h, and then incubated with the primary antibody overnight at 4 °C. Horseradish peroxidase-conjugated antirabbit
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antibodies were used as secondary antibodies correspondingly. After extensive washing, protein was visualized with ECL assay kit (ECL plus, Thermo-Scientific). b-actin (Santa Cruz, USA) was used as a sample loading control. Statistical analysis Data are presented as mean and standard error (SE). Comparison among groups was performed using Student’s t test or one-way analysis of variance (ANOVA). P \ 0.05 was considered statistically significant.
Results TG-induced activated HSCs apoptosis correlation with increased intracellular Ca2? To assess the effect of TG on activated HSCs, cell viability was determined by the MTT assay. Activated HSCs were seeded in 96-well plates and treated with different concentrations (0.25, 0.5, 1, and 2 lM) of TG for 24 h. As shown in Fig. 1, lower doses of TG (0.25 and 0.5 lM) had no significant effect on cell viability, while higher concentrations (1 and 2 lM) decreased cell viability, compared to that of the control. Moreover, to further evaluate the effect of activated HSCs apoptosis, activated HSCs were pretreated with 25 lM EGTA or 50 lM BAPTA/AM, and then exposed to 1 lM TG for 24 h. Cells apoptosis was determined by the flow cytometric analysis. As shown in Fig. 2, pretreatment of activated HSCs with EGTA or BAPTA/AM decreased apoptosis compared to TG-induced HSCs apoptosis. These data suggest that TG may represent a crucial target to trigger cell apoptosis in activated HSCs, correlation with increased intracellular Ca2?. TG attenuates the expression of a-SMA and Col1a1 in activated HSCs Exposure of HSC to PDGF resulted in liver fibrosis associated with expression of ECM proteins, collagen I, and aSMA. In this study, we found that the protein level of collagen I and a-SMA were increased in response to PDGF in HSCs for 24 h. As shown in Fig. 3, exposure of HSCs to 1 lM TG resulted in dramatic expression levels of a-SMA and Col1a1 in activated HSCs. The results showed that TG significantly attenuates the expression of a-SMA and Col1a1 in activated HSCs. Calpain/Caspases involved in ER stress-induced activated HSCs apoptosis To examine whether ER stress-induced apoptosis may have an effect on calpain and caspases expression in activated
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Fig. 5 Effect of EGTA and BAPTA/AM on MAPK expression in TG-induced activated HSCs apoptosis. The protein level of P-p-38 MAPK, P38 MAPK, P-JNK, JNK, ERK and P-ERK was measured by Western blot assay. a The protein level of P-p-38 MAPK, P38 MAPK, P-JNK and JNK was measured by Western blot assay. b The protein level of ERK and P-ERK was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, PDGF ? TG ? EGTA (100 lM); 5, PDGF ? TG ? BAPTA/AM (50 lM). Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
a P-p-38
P38
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b P-ERK ERK
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HSCs, immunoblots were performed with lysates from thapsigargin-treated HSCs. As shown in Fig. 4, exposure of HSCs to 1 lM TG resulted in dramatic expression levels of the active form of calpain and 35-kD active caspase-12 and caspase-9, and significantly caused the increase in 17-kD active caspase-3 activity . These results indicated that an intrinsic ER stress-dependent apoptotic pathway plays an important role in activated HSCs apoptosis. To further investigate the relationship between TG-induced activated HSCs apoptosis and the increased intracellular Ca2? concentration, activated HSCs were pre-treated with the EGTA or BAPTA/AM and then exposed to TG (1 lM) for 24 h. These results suggest that treatment of HSCs with EGTA
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or BAPTA/AM had a profound inhibitory effect on TG induced apoptosis of HSCs. MAPK involved in ER stress-induced activated HSCs apoptosis To explore the role of the MAPK pathway in the TG-induced activated HSCs apoptosis, the next experiments were performed in the presence or absence of EGTA or BAPTA/AM. As shown in Fig. 5, the protein phosphorylation levels of JNK and p38 MAPK except for those of ERK, were significantly increased after treatment of HSCs with TG for 24 h. The effects induced by TG could be abrogated by pre-
Mol Cell Biochem (2014) 394:1–12 Fig. 6 Effect of z-VAD-FKM, Calpeptin, SB202190 and SP600125 on C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 expression in TGinduced activated HSCs apoptosis. The protein level of C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 was measured by Western blot assay. 1, PDGF (20 ng/ml); 2, PDGF ? TG (2 lM); 3, TG ? PDGF ? z-VAD-FKM (25 lM); 4, TG ? PDGF ? Calpeptin (50 lM); 5, TG ? PDGF ? SB202190 (25 lM); 6, TG ? PDGF ? SP600125 (30 lM). Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
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C-Caspase-12
Caspase-9
Caspase-3 C-Caspase-3
β-actin
treatment with EGTA or BAPTA/AM, indicating that intracellular Ca2? concentration plays a critical role in TGinduced JNK and p38 MAPK activation. These results suggested that the activation of the JNK/p38 MAPK signaling pathway lead to activated HSCs apoptosis induced by TG. Connecting Calpain/Caspases and JNK/p38 MAPK on ER stress-induced activated HSCs apoptosis To further investigate the relationship between the activation of Calpain/Caspases and the phosphorylation of JNK/p38 MAPK, activated HSCs were pre-treated with the specific JNK inhibitor (SP600125, 25 lM), p38 MAPK inhibitor (SB202190, 25 lM), TG ? PDGF ?
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z-VAD-FKM (25 lM), or TG ? PDGF ? Calpeptin (50 lM) and then exposed to TG (1 lM) for 24 h. The result showed that pretreatment with Calpeptin, z-VADFKM, SP600125, or SB202190, significantly reduced the activation of caspase-12, caspase-9, and caspase-3 (Fig. 6). However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain (Fig. 7). Moreover, flow cytometric analysis showed that pre-treatment with the Calpeptin, z-VAD-FKM, SP600125, or SB202190 also significantly attenuated TG-induced activated HSCs apoptosis (Fig. 8). Taken together, our findings suggested that the activation of the JNK/p38 MAPK signaling pathway and Calpain/Caspases lead to activated HSCs apoptosis induced by TG.
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Calpain
β-actin 1
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Fig. 7 Effect of pan-caspase inhibitor z-VAD-FMK on calpain expression in TG-induced activated HSCs apoptosis. The protein level of calpain was measured by Western blot assay. 1, PDGF (20ng/ ml); 2, PDGF?TG (1lM); 3, PDGF?TG?z-VAD-FKM (25lM); 4, PDGF?TG?Calpeptin (50lM). Data were representative of at least three separate experiments and presented as mean ± SE. *P\0.05 versus PDGF values, #P\0.05 versus PDGF?TG values
CHOP is essential for ER stress-induced apoptosis To investigate cell apoptosis in response to ER stress, expression of CHOP, a UPR marker implicated in apoptosis, was evaluated after treatment of activated HSCs with TG (1 lM) for 24 h. As shown in Fig. 9, activated HSCs were transfected with the control or CHOP-siRNA, CHOP-siRNA effectively suppressed TGinduced CHOP protein. To further determine the underlying mechanism of CHOP on ER stress-induced activated HSCs apoptosis. The effects induced by TG could be abrogated by pretreatment with SP600125 or SB202190, indicating that the phosphorylation of JNK/p38 MAPK plays a critical role in TG-induced CHOP activation (Fig. 10). Taken together, our findings indicated that CHOP plays an important role in ER stress-induced activated HSCs apoptosis and JNK/P38 MAPK participated in CHOP expression.
Discussion In the present study, we demonstrated that ER stress could promote HSCs apoptosis, consequently leading to fibrosis
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resolution and reversibility. Four significant findings were presented in this study: (1) ER stress leads to activated HSCs apoptosis correlation with increased intracellular Ca2?, (2) activated multiple molecular mechanisms involving JNK/p38 MAPK and Calpain/Caspase for apoptosis, (3) CHOP is essential for ER stress-induced apoptosis, and (4) TG attenuates the expression of hepatic a-SMA and Col1a1 in activated HSCs. The ER stress response is an important homeostatic device for the liver during the onset and progression of chronic liver disease. Indeed, previous studies demonstrated the presence of ER stress during fibrosis, the consequently expected hepatocytes apoptosis and induced an inflammatory response in vivo [28]. In our study, we aimed to reveal that ER stress could promote activated HSCs apoptosis, leading to fibrosis resolution. ER stress-induced cells death pathway could be induced by a variety of chemicals inducer such as brefeldin A (ER to Golgi transport inhibitor), tunicamycin (asparagine-linked protein glycosylation inhibitor, TNC), calcium ionophore A23187, and thapsigargin (ER Ca2? ATPase inhibitor, TG). Disruption of Ca2? homeostasis in the ER induced by TG is also considered important to trigger ER stress and ERSinduced apoptosis [29, 30]. However, previous study showed that an increased intracellular Ca2? was more likely to have an adverse effect on HSCs activation and proliferation by PDGF in vitro [31, 32]. Similarly, Herna´ndez-Gea et al. [33] found that TNC was sufficient to induced HSCs activation 6 h, and a strong increase in mRNA expression of Col1a1, Col1a2, b-PDGF, and aSMA. This discrepancy might be explained by the state of ER stress model system. The mechanisms for the transition between adaptation to ER stress and ER stress-induced cell death may be explained by diverse disease stages, and the mechanism underlying liver fibrosis in vivo still remains to be determined. Therefore, in the present study, we aimed to reveal whether TG plays an essential role in PDGF-induced activation of HSCs in vitro. Results indicated that pretreatment with EGTA and BAPTA/AM significantly inhibited activated HSCs apoptosis. Noteworthily, we found that persistent and massive overload Ca2? induced by TG was more likely to have an adverse effect on apoptosis cells. We found that TG significantly decreased a-SMA and Col1a1, which markers for HSCs activation and elevated in PDGF-induced HSCs. Furthermore, in the present study, we showed that TG significantly decreased PDGF-induced upregulation of a-SMA and Col1a1. These findings suggested that ER stress may represent a crucial target to trigger cell apoptosis in activated HSCs and promote fibrosis resolution. To further determine the underlying mechanism of ER stress trigger activated HSCs apoptosis. For instance, De
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Fig. 8 Effect of z-VAD-FKM, Calpeptin, SB202190 and SP600125 on TG-induced activated HSCs apoptosis. Apoptosis of HSCs were analyzed by flow cytometry with Annexin V-FITC and PI staining. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1lM); 4, TG ? PDGF ? z-VAD-FKM (25 lM); 5, TG ? PDGF ? Calpeptin (50 lM); 6, TG ? PDGF ? SB202190 (25 lM); 7, TG ? PDGF ? SP600125 (30
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lM). Control: the cells without treatment. Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
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10 Fig. 9 Cells were transfected with specific small interfering RNA against CHOP or with nontargeting control siRNA as mentioned above for 72 h. The protein level of CHOP was measured by Western blot assay. a Western blotting shows CHOP protein was inhibited by CHOP-siRNA. b Representative photos show cells at 72 h following transfection with control (left) or CHOP-siRNA (right). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
Mol Cell Biochem (2014) 394:1–12
a Bright field image
Fluorescent image
b CHOP
β-actin
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Minicis et al. [34] showed that fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), led to fibrosis resolution through a mechanism of HSCs apoptosis which is potentially associated with ER stress. Recent studies have suggested that calpain and caspase were activated by
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elevated intracellular calcium and both may play important roles in mediating ER stress-induced apoptotic pathway [35, 36]. We examined the possible interaction between these two cysteine proteases in activated HSCs. In present study, we found that pre-treatment with EGTA and BAPTA/AM significantly inhibited the increasing of
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induced apoptosis, and while being normally undetectable in proliferating cells, it becomes highly synthesized in cells exposed to conditions that perturb the homeostasis of ER and is linked to the development of apoptosis. In summary, these data suggested that ER stress-mediated apoptosis may represent a key target to trigger cell apoptosis in activated HSCs and promote fibrosis resolution. Although the mechanism of this ER stress-mediated liver fibrosis recovery remains largely unknown, through the increase of intracellular calcium concentration releasing from ER, we showed that the relationship between Calpain/Caspase activation and JNK/p38 MAPK phosphorylation might involve ER stress-induced apoptosis during the regression of liver fibrosis. Our observation may open new opportunities for therapeutic strategies to reverse liver fibrosis. Acknowledgments This study is supported by the Chinese National Natural Science Foundation Project (No. 81102493, 81273526, 81072686) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20103420120001).
Fig. 10 Effect of SP600125 and SB202190 on CHOP expression in TG-induced activated HSCs apoptosis. The protein level of CHOP was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, TG ? PDGF ? SB202190 (25 lM); 5, TG ? PDGF ? SP600125 (30 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P\0.05 versus PDGF values, #P\0.05 versus PDGF ? TG values
intracellular Ca2? induced by TG. Furthermore, our study determined that pre-treatment with EGTA and BAPTA/ AM, also significantly inhibited the TG-induced activation and cleavage of apoptosis-associated proteins of calpain, caspase-3, caspase-9, and caspase-12. According to several reports from different laboratories, the phosphorylations of ERK, JNK, and p38 MAPK were essential components in CCl4-induced liver fibrosis model and its spontaneous recovery [37, 38]. Similarly, previous study showed that MAPK cascades play an important role in the mitogenic response and the induction of apoptosis in response to various stresses, including ER stress [39, 40]. However, the identification of numerous points of crosstalk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress. In present data, compared with PDGF-induced HSCs, the increased of JNK and p38 MAPK phosphorylation in TG-induced activated HSCs were measured by Western blot. As a result, our data suggested that JNK/p38 MAPK might be involved in TG-induced apoptosis in activated HSCs. Furthermore, p38 MAPK inhibition SB202190 and JNK inhibition SP600125 inhibited the TGinduced activation of caspase-12, -9, -3, and CHOP. Indeed, the expression of CHOP is involved in ER stress-
Conflict of interest of interest.
The authors declare that there are no conflicts
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Endoplasmic reticulum stress-induced hepatic stellate cell apoptosis through calcium-mediated JNK/P38 MAPK and Calpain/Caspase-12 pathways Yan Huang • Xiaohui Li • Yarui Wang Huan Wang • Cheng Huang • Jun Li
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Received: 31 December 2013 / Accepted: 18 April 2014 / Published online: 25 June 2014 Ó Springer Science+Business Media New York 2014
Abstract Recent reports considered that it was the disturbance of calcium homeostasis and the accumulation of misfolded proteins in the endoplasmic reticulum (ER) that activated hepatic stellate cells (HSCs) apoptosis and promoted fibrosis resolution. However, the signal-transducing events that are activated by ER stress after HSCs activation were incompletely understood. In this study, we induced ER stress with thapsigargin (TG), and determined the activation of calpain and the cleavage of caspase by analyzing the protein levels and the correspondingly increased intracellular calcium levels and the induction of the proapoptotic transcription factor CHOP. Moreover, the phosphorylation of JNK and p38 MAPK were followed by the activation of the executioner caspases, caspase-3. As expected, preventing an increase in intracellular calcium levels using intracellular calcium chelators, EGTA, and BAPTA/AM, could substantially inhibit the phosphorylation of JNK and p38 MAPK, abolish the activation of calpains, namely caspase-12, caspase-9, and caspase-3, and
provide significant protection for TG-treated activated HSCs. Interestingly, pretreatment with p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, the pan-caspase inhibitor z-VAD-FMK, or calpain inhibitors calpeptin, significantly reduced the cell apoptosis and the cleavage of caspase-12 and caspase-3. However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain. Additionally, pretreatment with SB202190 and SP600125 also decreased the expression of CHOP. Importantly, PDGF-induced collagen Col1a1 and a-smooth muscle actin (a-SMA), markers for the perpetuation phase of HSCs activation, were inhibited in TG-treated activated HSCs. These findings showed that the Calpain/Caspase-12 activation induced by ER stress and the JNK/p38 MAPK phosphorylation induced by the increase of intracellular calcium concentration releasing from ER are the novel signaling pathway underlying the molecular mechanism of fibrosis recovery. Keywords Liver fibrosis Endoplasmic reticulum stress Calpain Caspase MAPK Apoptosis
Y Huang and Xiaohui Li are co-first authors; they contributed equally to the work. Y. Huang X. Li Y. Wang H. Wang C. Huang J. Li (&) School of Pharmacy, Institute for Liver Diseases, Anhui key laboratory of bioactivity of natural products, Anhui Medical University, Hefei, China e-mail: [email protected] Y. Huang e-mail: [email protected] X. Li e-mail: [email protected]
Abbreviations a-SMA Alpha-smooth muscle actin CHOP C/EBP homologues protein ECM Extracellular matrix ER Endoplasmic reticulum ERK Extracellular signal-regulated protein kinase HF Hepatic fibrosis HSCs Hepatic stellate cells JNK C-jun N-terminal kinase MAPK Mitogen-activated protein kinase siRNA Small interfering RNA TG Thapsigargin UPR Unfolded protein response
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Introduction Liver fibrosis is the result of the wound-healing response of the liver to repeated injury [1]. It is caused by a variety of agents, including chronic viral hepatitis, alcohol toxicity, autoimmune disease, and hereditary metabolic disorders. In response to chronic liver injury, hepatic stellate cells (HSCs) develop into a myofibroblast-like phenotype with expression of a-smooth muscle actin (a-SMA) and secretion of excess extracellular matrix (ECM), predominantly collagen type I [2–4]. As HSCs play a crucial role in liver fibrosis, the elimination of activated HSCs through cell death is proposed as a mechanism to attenuate or reverse fibrogenesis [5, 6]. Therefore, it is necessary to accelerate the apoptosis of activated HSCs and it is a significant target in reversing hepatic fibrosis. Apoptosis is an essential mechanism for cell clearance. There are mainly three apoptotic signaling pathways that have been discovered: (1) the mitochondrial pathway; (2) the death receptor-associated pathway; (3) the ER-associated pathway [7–9]. Previous results revealed the effect of Kupffer cells releasing tumor necrosis factor related apoptosis-inducing ligand (TRAIL) on activated HSCs apoptosis in hepatic fibrosis regression and that the mechanism of it may correlate with the mitochondrial pathway and the ER-associated pathway [10–12]. In recent years, the study has found that the apoptosis mechanism of ER stress-mediated apoptosis is a new signaling pathway of apoptosis, but the exact mechanisms remain unclear. Endoplasmic reticulum (ER) plays pivotal roles in various cell processes, including synthesizing, sorting, assembling, modifying and trafficking proteins, and maintaining intracellular Ca2? homeostasis [13]. Accumulation of misfolded or unfolded proteins in ER, caused by impairment of Ca2? homeostasis or other noxious stimuli, would trigger an ER stress response [14, 15]. A short-term UPR functions as a pro-survival response via reducing deposition of unfolded proteins and restoring normal ER function [16]. However, if ER stress is prolonged, its down-stream signaling initiates a series of complex response networks to strengthen ER stress, activates proapoptosis signal pathways, and ultimately induces ER associated with CCAAT/enhancer-binding protein homologous protein (CHOP) and ER resident protein caspase-12 [17]. Previous reports showed that caspase-12 is localized to the ER and activated by ER stress, including disruption of ER calcium homeostasis and accumulation of excess proteins in ER. Caspase-12 deficient mice are resistant to ER stress-induced apoptosis [18]. Calpain deficient mouse embryonic fibroblasts (MEFs) have been shown to decrease
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Fig. 1 Effect of TG on cell viability with MTT assay. The cell viability was assayed in PDGF-induced HSC stimulated with TG by 24 h at indicated concentrations (0, 0.25, 0.5, 1, and 2 lM). 1 control, 2 PDGF (20 ng/ml), 3 PDGF ? TG (0.25 lM), 4 PDGF ? TG (0.5 lM), 5 PDGF ? TG (1 lM), 6 PDGF ? TG (2 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \ 0.05 versus PDGF, #P \ 0.05 versus TG values
ER stress-induced activation of caspase-12 and be resistant to ER stress-induced apoptosis [19]. Both calpain and caspase have been proposed to mediate processing and activation of caspase-12 after induction of the ER stress [20]. Moreover, Ca2? released from the ER enters mitochondria leading to caspase-9, caspase-7, and caspase-3 [21, 22]. However, the role of these molecules in inducing HSCs apoptosis, their correlation with the Calpain/Caspase expression and the subsequent contribution of this process to hepatic fibrogenesis still remain unclear. Mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinases (JNK), and p38 MAPK, regulate signaling mechanisms for cell proliferation, differentiation, survival, death, and inflammation [23]. Indeed, previous studies showed that ERK, JNK, and p38 MAPK mediate HSCs activation in the pathogenesis of liver fibrosis [24]. However, our previous laboratory research showed that JNK may participate in mediating activated HSCs apoptosis though death receptors DR4/DR5 and pro-apoptosis factor CHOP [25]. Some other reports showed the engagement of activation of the cell death promoting kinase, MAPK in many kinds of cell death associated with ER stress [26, 27]. To further study the mechanism of its action, we monitored the effects of ER stress-associated factors, as well as those of MAPK signaling pathways. There are many signal conduction paths and mechanisms that may participate in the apoptosis reduced by ER stress. However, the mechanisms of ER stress effecting activated HSCs apoptosis have not been fully understood to date. The
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Fig. 2 Effect of EGTA and BAPTA/AM on TG-induced activated HSCs apoptosis. Apoptosis of HSCs were analyzed by flow cytometry with Annexin V-FITC and PI staining. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF?TG (1 lM); 4, PDGF?TG?EGTA (100 lM); 5, PDGF?TG?BAPTA/AM (50 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
aim of this study was therefore to explore the potential apoptotic process of activated HSCs associated with ER stress mechanisms and the resolution of liver fibrosis.
and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide), and all other chemicals were purchased from Sigma Technology.
Materials and methods
Cell culture and activation
Regent
A cell line of rat HSC was purchased from Chinese Academy of Sciences (Shanghai, China). HSCs were propagated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) supplemented with 10 % fetal bovine serum and 1 % antibiotic mixture comprising penicillin–streptomycin, and maintained at 37 °C in a humidified incubator containing 5 % CO2. HSCs were activated by treating with platelet derived growth factor (PDGF) (20 ng/ml) for 24 h.
Antibodies directed against caspase-3, p-p38 MAPK, p-ERK, p-JNK, p38 MAPK, ERK, and JNK were purchased from Cell Signaling Technology. Caspase-12 was purchased from Biovision Technology. CHOP was purchased from Bioworld Technology. a-SMA was purchased from Proteintech Technology. Calpain and calpeptin were purchased from Santa Cruz Technology. Thapsigargin, dimethylsulfoxide (DMSO),
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a
Calpain
α-SMA C-Caspase-12 Caspase-9
Col1α1
Caspase-3
C-Caspase-3
β-actin β-actin
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Fig. 3 Effect of TG on a-SMA and Col1a1 expression in activated HSCs. The protein level of a-SMA and Col1a1 was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM). Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
MTT assay Cell viability assay was determined by MTT. HSCs were seeded into each well of 96-well culture plates and then cultured in DMEM for 24 h. After culture, 150 ll of MTT
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Mol Cell Biochem (2014) 394:1–12 b Fig. 4 Effect of EGTA and BAPTA/AM on calpain/Caspases
expression in TG-induced activated HSCs apoptosis. TG-induced activated HSCs apoptosis is Ca2? dependent involving the activation of calpain, C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 were detected using western blotting. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, PDGF ? TG ? EGTA (100 lM); 5, PDGF ? TG ? BAPTA/AM (50 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
solution (5 mg/ml) was added to each well, and the plates were then incubated at 37 °C for another 4 h. The optical density (OD) was measured at 490 nm after removing the supernatant and shaking with 150 ll DMSO by Thermomax microplate reader (Bio-TekEL, USA). All experiments were performed in triplicate and repeated at least three times. Flow cytometric analysis Since apoptosis analysis cells were performed with Annexin-V-FITC Apoptosis Detection Kit (BD Pharmingen, USA), samples from different groups were collected by trypsinization, and washed twice with cold phosphate buffered saline (PBS) buffer. Before analyses were performed on BD LSR flow cytometer (BD Biosciences), cells were resuspended in 400 ll Annexin-V binding buffer, added with 5 ll Annexin-V-FITC cultured at 2–8 °C for 15 min, and then added with 10 ll propidium iodide (PI) cultured at 2–8 °C for 5 min in dark. All experiments were performed in triplicate and repeated at least three times. siRNA transfection To stably knock down CHOP expression, RNAi (GenePharma, China) experiments in HSCs were performed by forward transfection in day 2 cultured HSCs using LipofectamineTM2000 (Invitrogen, USA) according to the manufacturer’s protocol. A negative control RNAi was purchased from Genepharma. The sense sequence for CHOP was showed as follows: SiCHOP (rat): 50 -CCAGUGCCAAGCAUGUAAUTT-30 ; 50 -AUUACAUGCUUGGCACUGGTT-30 . Western blotting analysis Proteins were isolated and quantified using the BCA protein assay kit (Boster, China). Total protein (30 or 50 mg) from samples was separated by SDS-PAGE and blotted onto a PVDF membrane (Millipore Corp, Billerica, MA, USA). The membranes were blocked with 5 % non-fat milk 3 h, and then incubated with the primary antibody overnight at 4 °C. Horseradish peroxidase-conjugated antirabbit
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antibodies were used as secondary antibodies correspondingly. After extensive washing, protein was visualized with ECL assay kit (ECL plus, Thermo-Scientific). b-actin (Santa Cruz, USA) was used as a sample loading control. Statistical analysis Data are presented as mean and standard error (SE). Comparison among groups was performed using Student’s t test or one-way analysis of variance (ANOVA). P \ 0.05 was considered statistically significant.
Results TG-induced activated HSCs apoptosis correlation with increased intracellular Ca2? To assess the effect of TG on activated HSCs, cell viability was determined by the MTT assay. Activated HSCs were seeded in 96-well plates and treated with different concentrations (0.25, 0.5, 1, and 2 lM) of TG for 24 h. As shown in Fig. 1, lower doses of TG (0.25 and 0.5 lM) had no significant effect on cell viability, while higher concentrations (1 and 2 lM) decreased cell viability, compared to that of the control. Moreover, to further evaluate the effect of activated HSCs apoptosis, activated HSCs were pretreated with 25 lM EGTA or 50 lM BAPTA/AM, and then exposed to 1 lM TG for 24 h. Cells apoptosis was determined by the flow cytometric analysis. As shown in Fig. 2, pretreatment of activated HSCs with EGTA or BAPTA/AM decreased apoptosis compared to TG-induced HSCs apoptosis. These data suggest that TG may represent a crucial target to trigger cell apoptosis in activated HSCs, correlation with increased intracellular Ca2?. TG attenuates the expression of a-SMA and Col1a1 in activated HSCs Exposure of HSC to PDGF resulted in liver fibrosis associated with expression of ECM proteins, collagen I, and aSMA. In this study, we found that the protein level of collagen I and a-SMA were increased in response to PDGF in HSCs for 24 h. As shown in Fig. 3, exposure of HSCs to 1 lM TG resulted in dramatic expression levels of a-SMA and Col1a1 in activated HSCs. The results showed that TG significantly attenuates the expression of a-SMA and Col1a1 in activated HSCs. Calpain/Caspases involved in ER stress-induced activated HSCs apoptosis To examine whether ER stress-induced apoptosis may have an effect on calpain and caspases expression in activated
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Fig. 5 Effect of EGTA and BAPTA/AM on MAPK expression in TG-induced activated HSCs apoptosis. The protein level of P-p-38 MAPK, P38 MAPK, P-JNK, JNK, ERK and P-ERK was measured by Western blot assay. a The protein level of P-p-38 MAPK, P38 MAPK, P-JNK and JNK was measured by Western blot assay. b The protein level of ERK and P-ERK was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, PDGF ? TG ? EGTA (100 lM); 5, PDGF ? TG ? BAPTA/AM (50 lM). Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
a P-p-38
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HSCs, immunoblots were performed with lysates from thapsigargin-treated HSCs. As shown in Fig. 4, exposure of HSCs to 1 lM TG resulted in dramatic expression levels of the active form of calpain and 35-kD active caspase-12 and caspase-9, and significantly caused the increase in 17-kD active caspase-3 activity . These results indicated that an intrinsic ER stress-dependent apoptotic pathway plays an important role in activated HSCs apoptosis. To further investigate the relationship between TG-induced activated HSCs apoptosis and the increased intracellular Ca2? concentration, activated HSCs were pre-treated with the EGTA or BAPTA/AM and then exposed to TG (1 lM) for 24 h. These results suggest that treatment of HSCs with EGTA
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or BAPTA/AM had a profound inhibitory effect on TG induced apoptosis of HSCs. MAPK involved in ER stress-induced activated HSCs apoptosis To explore the role of the MAPK pathway in the TG-induced activated HSCs apoptosis, the next experiments were performed in the presence or absence of EGTA or BAPTA/AM. As shown in Fig. 5, the protein phosphorylation levels of JNK and p38 MAPK except for those of ERK, were significantly increased after treatment of HSCs with TG for 24 h. The effects induced by TG could be abrogated by pre-
Mol Cell Biochem (2014) 394:1–12 Fig. 6 Effect of z-VAD-FKM, Calpeptin, SB202190 and SP600125 on C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 expression in TGinduced activated HSCs apoptosis. The protein level of C-Caspase-12, Caspase-9, Caspase-3 and C-Caspase-3 was measured by Western blot assay. 1, PDGF (20 ng/ml); 2, PDGF ? TG (2 lM); 3, TG ? PDGF ? z-VAD-FKM (25 lM); 4, TG ? PDGF ? Calpeptin (50 lM); 5, TG ? PDGF ? SB202190 (25 lM); 6, TG ? PDGF ? SP600125 (30 lM). Data were representative of at least three separate experiments and presented as mean ± SE. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
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C-Caspase-12
Caspase-9
Caspase-3 C-Caspase-3
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treatment with EGTA or BAPTA/AM, indicating that intracellular Ca2? concentration plays a critical role in TGinduced JNK and p38 MAPK activation. These results suggested that the activation of the JNK/p38 MAPK signaling pathway lead to activated HSCs apoptosis induced by TG. Connecting Calpain/Caspases and JNK/p38 MAPK on ER stress-induced activated HSCs apoptosis To further investigate the relationship between the activation of Calpain/Caspases and the phosphorylation of JNK/p38 MAPK, activated HSCs were pre-treated with the specific JNK inhibitor (SP600125, 25 lM), p38 MAPK inhibitor (SB202190, 25 lM), TG ? PDGF ?
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z-VAD-FKM (25 lM), or TG ? PDGF ? Calpeptin (50 lM) and then exposed to TG (1 lM) for 24 h. The result showed that pretreatment with Calpeptin, z-VADFKM, SP600125, or SB202190, significantly reduced the activation of caspase-12, caspase-9, and caspase-3 (Fig. 6). However, pretreatment with z-VAD-FMK failed to reduce the activation of calpain (Fig. 7). Moreover, flow cytometric analysis showed that pre-treatment with the Calpeptin, z-VAD-FKM, SP600125, or SB202190 also significantly attenuated TG-induced activated HSCs apoptosis (Fig. 8). Taken together, our findings suggested that the activation of the JNK/p38 MAPK signaling pathway and Calpain/Caspases lead to activated HSCs apoptosis induced by TG.
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β-actin 1
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Fig. 7 Effect of pan-caspase inhibitor z-VAD-FMK on calpain expression in TG-induced activated HSCs apoptosis. The protein level of calpain was measured by Western blot assay. 1, PDGF (20ng/ ml); 2, PDGF?TG (1lM); 3, PDGF?TG?z-VAD-FKM (25lM); 4, PDGF?TG?Calpeptin (50lM). Data were representative of at least three separate experiments and presented as mean ± SE. *P\0.05 versus PDGF values, #P\0.05 versus PDGF?TG values
CHOP is essential for ER stress-induced apoptosis To investigate cell apoptosis in response to ER stress, expression of CHOP, a UPR marker implicated in apoptosis, was evaluated after treatment of activated HSCs with TG (1 lM) for 24 h. As shown in Fig. 9, activated HSCs were transfected with the control or CHOP-siRNA, CHOP-siRNA effectively suppressed TGinduced CHOP protein. To further determine the underlying mechanism of CHOP on ER stress-induced activated HSCs apoptosis. The effects induced by TG could be abrogated by pretreatment with SP600125 or SB202190, indicating that the phosphorylation of JNK/p38 MAPK plays a critical role in TG-induced CHOP activation (Fig. 10). Taken together, our findings indicated that CHOP plays an important role in ER stress-induced activated HSCs apoptosis and JNK/P38 MAPK participated in CHOP expression.
Discussion In the present study, we demonstrated that ER stress could promote HSCs apoptosis, consequently leading to fibrosis
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resolution and reversibility. Four significant findings were presented in this study: (1) ER stress leads to activated HSCs apoptosis correlation with increased intracellular Ca2?, (2) activated multiple molecular mechanisms involving JNK/p38 MAPK and Calpain/Caspase for apoptosis, (3) CHOP is essential for ER stress-induced apoptosis, and (4) TG attenuates the expression of hepatic a-SMA and Col1a1 in activated HSCs. The ER stress response is an important homeostatic device for the liver during the onset and progression of chronic liver disease. Indeed, previous studies demonstrated the presence of ER stress during fibrosis, the consequently expected hepatocytes apoptosis and induced an inflammatory response in vivo [28]. In our study, we aimed to reveal that ER stress could promote activated HSCs apoptosis, leading to fibrosis resolution. ER stress-induced cells death pathway could be induced by a variety of chemicals inducer such as brefeldin A (ER to Golgi transport inhibitor), tunicamycin (asparagine-linked protein glycosylation inhibitor, TNC), calcium ionophore A23187, and thapsigargin (ER Ca2? ATPase inhibitor, TG). Disruption of Ca2? homeostasis in the ER induced by TG is also considered important to trigger ER stress and ERSinduced apoptosis [29, 30]. However, previous study showed that an increased intracellular Ca2? was more likely to have an adverse effect on HSCs activation and proliferation by PDGF in vitro [31, 32]. Similarly, Herna´ndez-Gea et al. [33] found that TNC was sufficient to induced HSCs activation 6 h, and a strong increase in mRNA expression of Col1a1, Col1a2, b-PDGF, and aSMA. This discrepancy might be explained by the state of ER stress model system. The mechanisms for the transition between adaptation to ER stress and ER stress-induced cell death may be explained by diverse disease stages, and the mechanism underlying liver fibrosis in vivo still remains to be determined. Therefore, in the present study, we aimed to reveal whether TG plays an essential role in PDGF-induced activation of HSCs in vitro. Results indicated that pretreatment with EGTA and BAPTA/AM significantly inhibited activated HSCs apoptosis. Noteworthily, we found that persistent and massive overload Ca2? induced by TG was more likely to have an adverse effect on apoptosis cells. We found that TG significantly decreased a-SMA and Col1a1, which markers for HSCs activation and elevated in PDGF-induced HSCs. Furthermore, in the present study, we showed that TG significantly decreased PDGF-induced upregulation of a-SMA and Col1a1. These findings suggested that ER stress may represent a crucial target to trigger cell apoptosis in activated HSCs and promote fibrosis resolution. To further determine the underlying mechanism of ER stress trigger activated HSCs apoptosis. For instance, De
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Fig. 8 Effect of z-VAD-FKM, Calpeptin, SB202190 and SP600125 on TG-induced activated HSCs apoptosis. Apoptosis of HSCs were analyzed by flow cytometry with Annexin V-FITC and PI staining. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1lM); 4, TG ? PDGF ? z-VAD-FKM (25 lM); 5, TG ? PDGF ? Calpeptin (50 lM); 6, TG ? PDGF ? SB202190 (25 lM); 7, TG ? PDGF ? SP600125 (30
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lM). Control: the cells without treatment. Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
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10 Fig. 9 Cells were transfected with specific small interfering RNA against CHOP or with nontargeting control siRNA as mentioned above for 72 h. The protein level of CHOP was measured by Western blot assay. a Western blotting shows CHOP protein was inhibited by CHOP-siRNA. b Representative photos show cells at 72 h following transfection with control (left) or CHOP-siRNA (right). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P \0.05 versus PDGF values, #P \0.05 versus PDGF ? TG values
Mol Cell Biochem (2014) 394:1–12
a Bright field image
Fluorescent image
b CHOP
β-actin
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Minicis et al. [34] showed that fibrosis resolution observed in rat livers subjected to bile duct ligation (BDL) and subsequent bile duct diversion (BDD), led to fibrosis resolution through a mechanism of HSCs apoptosis which is potentially associated with ER stress. Recent studies have suggested that calpain and caspase were activated by
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elevated intracellular calcium and both may play important roles in mediating ER stress-induced apoptotic pathway [35, 36]. We examined the possible interaction between these two cysteine proteases in activated HSCs. In present study, we found that pre-treatment with EGTA and BAPTA/AM significantly inhibited the increasing of
Mol Cell Biochem (2014) 394:1–12
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CHOP
β-actin 1
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induced apoptosis, and while being normally undetectable in proliferating cells, it becomes highly synthesized in cells exposed to conditions that perturb the homeostasis of ER and is linked to the development of apoptosis. In summary, these data suggested that ER stress-mediated apoptosis may represent a key target to trigger cell apoptosis in activated HSCs and promote fibrosis resolution. Although the mechanism of this ER stress-mediated liver fibrosis recovery remains largely unknown, through the increase of intracellular calcium concentration releasing from ER, we showed that the relationship between Calpain/Caspase activation and JNK/p38 MAPK phosphorylation might involve ER stress-induced apoptosis during the regression of liver fibrosis. Our observation may open new opportunities for therapeutic strategies to reverse liver fibrosis. Acknowledgments This study is supported by the Chinese National Natural Science Foundation Project (No. 81102493, 81273526, 81072686) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20103420120001).
Fig. 10 Effect of SP600125 and SB202190 on CHOP expression in TG-induced activated HSCs apoptosis. The protein level of CHOP was measured by Western blot assay. 1, Control; 2, PDGF (20 ng/ml); 3, PDGF ? TG (1 lM); 4, TG ? PDGF ? SB202190 (25 lM); 5, TG ? PDGF ? SP600125 (30 lM). Data were representative of at least three separate experiments and presented as mean ± SE. Control: the cells were cultured in DMEM containing 10 % FBS without PDGF stimulation. *P\0.05 versus PDGF values, #P\0.05 versus PDGF ? TG values
intracellular Ca2? induced by TG. Furthermore, our study determined that pre-treatment with EGTA and BAPTA/ AM, also significantly inhibited the TG-induced activation and cleavage of apoptosis-associated proteins of calpain, caspase-3, caspase-9, and caspase-12. According to several reports from different laboratories, the phosphorylations of ERK, JNK, and p38 MAPK were essential components in CCl4-induced liver fibrosis model and its spontaneous recovery [37, 38]. Similarly, previous study showed that MAPK cascades play an important role in the mitogenic response and the induction of apoptosis in response to various stresses, including ER stress [39, 40]. However, the identification of numerous points of crosstalk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress. In present data, compared with PDGF-induced HSCs, the increased of JNK and p38 MAPK phosphorylation in TG-induced activated HSCs were measured by Western blot. As a result, our data suggested that JNK/p38 MAPK might be involved in TG-induced apoptosis in activated HSCs. Furthermore, p38 MAPK inhibition SB202190 and JNK inhibition SP600125 inhibited the TGinduced activation of caspase-12, -9, -3, and CHOP. Indeed, the expression of CHOP is involved in ER stress-
Conflict of interest of interest.
The authors declare that there are no conflicts
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