LFS-13783; No of Pages 8 Life Sciences xxx (2013) xxx–xxx

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Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stress-mediated apoptosis Yongjun Zhu a, Ruoting Men a, Maoyao Wen a, Xiaolin Hu a,b, Xiaojing Liu c, Li Yang a,⁎ a b c

Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China Department of Internal Medicine, Hospital of Southwest University, Chongqing 400715, China Laboratory of Cardiovascular Diseases, West China Hospital, Sichuan University, Chengdu 610041, China

a r t i c l e

i n f o

Article history: Received 16 June 2013 Accepted 24 October 2013 Available online xxxx Keywords: Hepatic stellate cells Transient receptor potential melastatin-like 7 Apoptosis Endoplasmic reticulum stress

a b s t r a c t Aims: Proliferation is a ‘multiplier’ for extracellular matrix production and contraction of activated hepatic stellate cells (HSC) in fibrotic liver. Transient receptor potential melastatin-like 7 channels (TRPM7) are implicated in the survival and proliferation of several kinds of cells. This study was aimed to investigate the effect of TRPM7 blocker 2-APB on survival and proliferation of HSC and the underlying mechanisms. Main methods: Rat HSC were stimulated by 2-APB for 24 h and then collected for further use. Cell viability was detected by MTT, and apoptosis was determined by AnnexinV/PI staining and TUNEL assay. Gene expressions of TRPM7, α-SMA, bcl-2, bax, and endoplasmic reticulum (ER) stress key members CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin were evaluated with quantitative RT-PCR. Quantifications of α-SMA, TRPM7, CHOP and GRP78 proteins were carried out by Western blot. Transmission electron microscopy and Xbp1 mRNA splicing analysis were also used for detection of ER stress. Key findings: 2-APB decreased TRPM7 and α-SMA expressions in primary HSC, and inhibited proliferation of activated HSC in a dose-dependent manner. 2-APB also decreased total count of activated HSC and increased the number of apoptotic cells. 2-APB increased expressions of bax and ER stress key factors CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin. Meanwhile, ultra-structural ER changes and spliced Xbp1 mRNA were also observed in 2-APB treated HSC. Significance: Blockage of TRPM7 could inhibit activation and proliferation of primary HSC and induce apoptotic death of activated cells, in which ER stress was identified as one of possible underlying molecular bases. © 2013 Elsevier Inc. All rights reserved.

Introduction Hepatic fibrosis, the common pathway of chronic liver diseases regardless of the etiology, is a progressive pathological process which ultimately results in much mortality around the world (Chan et al., 2009). The pivotal cell event in this process is activation of hepatic stellate cells (HSC) (Friedman, 2008). Activated HSC synthesize and secrete pro-collagens which could accumulate as insoluble collagens and cause fibrosis (Jiao et al., 2009). They also aggregate and contract to raise intraluminal pressure of liver sinusoids, ultimately leading to disordered intrahepatic blood flow and increased portal pressure (Laleman et al., 2007; Iizuka et al., 2011). And cell proliferation, undoubtedly plays a role of ‘multiplier’ for these impairments. Considering that hepatic fibrosis could be reversed once noxious stimuli are removed and activated HSC are eliminated (Arthur, 2002; Issa et al., 2004; Sato et al., 2008), inhibition of quiescent HSC activation and

⁎ Corresponding author at: Wainan Guoxue Xiang 37#, Chengdu, Sichuan 610041, China. Tel. /fax: +86 28 85422383. E-mail address: [email protected] (L. Yang).

induction of activated HSC death selectively could be a part of the therapeutic strategy to attenuate or even reverse liver fibrosis. Transient receptor potential (TRP) channels are an unselective and non-voltage-gated cation ion channel superfamily, which constitutively express on the membrane of various kinds of cells (Fonfria et al., 2006). TRP melastatin-like 7(TRPM7) is responsible mainly for Ca2+ entry and implicated in sustaining intracellular Ca2 + homeostasis (Aarts et al., 2003; Jin et al., 2008), which was reported to be associated with proliferation of several types of cells, such as retinoblastoma cells, breast cancer cells, and human head and neck squamous carcinoma cells (Hanano et al., 2004; Jiang et al., 2007; Guilbert et al., 2009). However, whether TRPM7 is implicated in activation and proliferation of HSC was seldom reported, and the underlying mechanism still remains elusive. Endoplasmic reticulum (ER) plays pivotal roles in various cell processes, including synthesizing, sorting, assembling, modifying and trafficking proteins, and maintaining intracellular Ca2 + homeostasis. Accumulation of misfolded or unfolded proteins in ER, caused by impairment of Ca2+ homeostasis or other noxious stimuli, would trigger an ER stress response (Szegezdi et al., 2006; Fu et al., 2011). In the initial part of this response signaling, at least three resident ER transmembrane proteins, also called ‘sensors’, are activated: PERK, ATF6 and

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Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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IRE1. In resting cells, these sensors remain physiologically in an inactive state combining with chaperones, like GRP78. Upon ER stress, accumulation of unfolded proteins leads to activation of sensors via dissociating from GRP78 and triggers unfolded protein response (UPR) (Lim et al., 2011; Gorman et al., 2012). A short-term UPR functions as a prosurvival response via reducing deposition of unfolded proteins and restoring normal ER function. However, if UPR is prolonged, its downstream signaling initiates a series of complex response networks to strengthen ER stress, activates pro-apoptosis signal pathways and ultimately induces cell death (Rao et al., 2002b; Mollica et al., 2011). ER in HSC, especially in activated ones, are often overburdened with work and sensitive to ER stress stimuli. And ER stress was reported as one of the mechanisms of rat HSC apoptotic death (Svegliati-Baroni et al., 2006; Lim et al., 2011; Minicis et al., 2012). Therefore, we hypothesized that blockage of TRPM7 channel could induce ER stress in HSC, and finally cause apoptotic cell death. Herein, we used TRPM7 inhibitor 2-Aminoethoxydiphenyl borate (2-APB) to stimulate primary rat HSC and HSC line T6 cells to investigate evidences for this hypothesis.

Table 1 Primers

Sequence (5′-3′)

Size (bp)

TRPM7

Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense

120

α-SMA bmf Beclin-1 Caspase-3 Caspase-12 CHOP GRP78 ATF6 bax

Materials and methods

bcl-2

Rat HSC and culture

ATF4 Xbp1

Rat primary HSC were isolated from the livers of normal male Sprague-Dawley rats with an optimal body weight of 400–550 g (Animal Center of Sichuan University, China), by in situ perfusion of pronase and collagenase and single-step Nycodenz gradient centrifugation as we previously reported (Pan et al., 2010). With Trypan blue dye exclusion test, total cell number was counted about 4.15–8.67 × 107/rat, and purity and viability of the cells exceeded 90% and 95%, respectively. Cells were seeded at a density of 1.5 × 106/ml on 50-ml culture flasks or 60-mm or 100-mm dishes and maintained in DMEM supplemented with 20% fetal calf serum (FBS, Hyclone Labs, USA). All culture flasks and dishes were kept in a sterile humidified incubator (SANYO, Japan) with 5% CO2 at 37 °C and 100% humidity. This study was approved by the Medical Ethics Committee of the West China Hospital, Sichuan University. Immortalized rat HSC line T6 cells were passaged in Dulbecco's modified Eagle's medium (DMEM, Gibico, USA) supplemented with 10% newborn calf serum (MinhaiBio, China). Experiments were carried out while the cells were in exponential growth phase. Unless otherwise stated, T6 cells in this study were starved with serum-free medium for 16 h and primary HSC for 6 h before they were treated with 200 μmol/L of 2-APB, because primary HSC were more sensitive to serum-free starvation than immortalized T6 cells. Cell viability assay Cell viability was assessed by MTT test as reported previously (Zhu et al., 2012). Briefly, T6 cells were seeded in a 96-well plate. After 24 h, the cells were treated with control (0.1% DMSO) or 2-APB (50, 100, 150, 200, 500 μmol/L) for 24, 48 and 72 h, and then incubated in 5 g/L MTT substrate (200 μL/well) for 4 h at 37 °C. The resulting violet formazan precipitate was solubilized by 200 μL DMSO. After gently shaking at 37 °C for 10 min, the absorbance of the dissolved formazan grains within the cells was measured at 570 nm using VARIOSKAN (Thermo Electron Co., USA). Experiments were carried out three times. Cell viability was calculated by the formula: (test OD570 − blank OD570) / (control OD570 − blank OD570) × 100%. RNA isolation and quantitative RT-PCR (qRT-PCR) Total RNA from HSC was harvested using Trizol reagent (Invitrogen, USA), and extracted, quantified and the quality evaluated as described (Pan et al., 2010). The cDNA was prepared from 1.0 μg of total RNA using ReverTra Ace qPCR RT Kit (TOYOBO, Japan) according to the

Calnexin β-Actin Xbp1r*

ACC CTC ACA GAT GTC TTC CAG CAT CTG AGT ATT TTG TGG CAA G CCG AGA TCT CAC CGA CTA CC TCC AGA GCG ACA TAG CAC AG GAG CCA AGT AGT CAG TTA CCA C GTC CAA TCC AGT CCA GTC AT CAG GAG GAA GCT CAG TAC CA CAT AGC GCA TCT GGT TCT CT TGG AGC ACT GTA GCA CAC AT CCA CCA CTG AAG GAT GGT AG GAA GGA ATC TGT GGG GTG AA TCC CTT TGC TTG TGG ATA CC AGC AGA GGT CAC AAG CAC CT CTC CTT CAT GCG CTG TTT CC CCC CAG ATT GAA GTC ACC TTT GAG CAG GCG GTT TTG GTC ATT G GCA GGT GTA TTA CGC TTC GC TGT GGT CTT GTT ATG GGT GG AGC TGC AGA GGA TGA TTG CT GAT CAG CTC GGG CAC TTT AG GGT GGT GGA GGA ACT CTT CA ATG CCG GTT CAG GTA CTC AG TCC TGA ACA GCG AAG TGT TG CAT CCA TAG CCA GCC ATT CT ATT CTG ACG CTG TTG CCT CT CTC TGG GGA AGG ACA TTT GA GAT GCT GTC AAG CCA GAT GA TTA GGG TTG GCA ATC TGA GG ACT ATC GGC AAT GAG CGG TTC ATG CCA CAG GAT TCC ATA CCC AAA CAG AGT AGC AGC GCA GAC TGC GGA TCT CTA AAA CTA GAG GCT TGG TG

120 100 100 176 181 157 117 136 176 157 174 101 188 77 601

Summary of oligonucleotide sequences used for PCR.

manufacturer's instructions. qPCR amplification conditions were as described (Pan et al., 2010), and the primer sequences are listed in Table 1. Results were calculated with the Livak Method (2−ΔΔCt) and expressed as the ratio of Ct value of cDNA concentrations of target genes relative to that of β-actin.

Western blot analysis Protein from cell extracts was quantified by VARIOSKAN (Thermo, USA) using a BCA protein assay kit (Thermo/Pierce, USA). Equivalent amount (25 μg) of total protein were separated by 10% SDS-PAGE and transferred to a 0.45 μm nitrocellulose membrane (Roche, Germany) which was subsequently blocked with 10% skim milk in TBST solution for 2 h and incubated with primary antibodies (see Table 2) at 4 °C overnight. Horseradish peroxidase conjugated goat anti-mouse IgG was used as secondary antibody. The antigen-antibody complexes were detected by Pierce ECL substrate kit (Thermo/Pierce, USA). Specific bands were scanned and quantified by Gel Doc 2000 software, β-actin as loading control. The results were reported as the mean of triplicate assays.

Table 2 Summary of antibodies used for western blot. Antibodies

Source

Optimal dilution

Primary antibodies Monoclonal TRPM7 S74-25 Monoclonal α-SMA 1A4 Monoclonal CHOP (H-5) Monoclonal GRP78 (E-4) Monoclonal β-actin 1E9A3

Abcam ab85016 Abcam ab7817 Santa Cruz sc-166682 Santa Cruz sc-166490 ZSBio TA-09

1/500 1/200 1/500 1/400 1/1000

Secondary antibody Goat anti-mouse IgG(H + L)

ZSBio ZB-2305

1/5000

Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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Transmission electron microscopy (TEM) T6 cells seeded in 60 mm plate were washed with PBS, trypsinized, fixed with 2% glutaraldehyde at 4 °C for 1 h, and then washed with 0.2 mol/L sodium cacodylate buffer (pH 7.4). The resulting pellet was embedded in 1% agarose at 4 °C overnight and then dehydrated with increasing serial concentrations (60%, 70%, 80% and 100%) of acetone for 30 min each. Low viscosity Spurr's epoxy group resin was then mixed with increasing serial concentrations of acetone. After infiltration with pure resin, the samples were transferred to silicone sample embedding molds and kept at 70 °C overnight. Before choosing areas for ultrastructural studies, semi-thick sections of 3 μm were cut with a Reichert Ultracut R microtome (Leica, Germany) and examined with a microscope. Ultrathin sections of 80 nm size were prepared and stained with 2% uranyl acetate and 1% lead citrate. Ultrathin sections were analyzed with a transmission electron microscope (Philips, Netherlands). Measurements of apoptosis The following methods were applied for evaluation of apoptosis: (1) AnnexinV-FITC/propidium iodide (AnnexinV/PI) staining. After 2APB treatment, T6 cells seeded on 60 mm plate were stained with prediluted mixture of 20 μl AnnexinV-FITC, 20 μl PI and 100 μl binding buffer (Roche, Germany), and incubated for 15 min at room temperature in the dark before being evaluated by fluorescence microscopy. AnnexinV-FITC and PI were excited at 488 and 546 nm, respectively. The cells showing visible AnnexinV (green) without or with PI (red) staining were categorized as early or late apoptotic cells, respectively. (2) TUNEL assay: T6 cells seeded on coverslips were fixed with 4% paraformaldehyde for 30 min after treatment. The TUNEL assay was performed as previously described (Baskin-Bey et al., 2005). Xbp1 mRNA splicing analysis ER stress-induced processing of Xbp1 mRNA was evaluated by RTPCR and restriction site analysis as described (Paschen et al., 2003; Williams and Lipkin, 2006). Briefly, the fragments of 601 bp PCR product, encompassing the IRE1 cleavage site of Xbp1, were amplified by regular PCR from cDNA of HSC using the Xbp1r primer listed in Table 1. PCR products were separated by a 2% agarose gel for 1.5 h before or after incubation with the restriction enzyme Pst I at 37 °C for 5 h. The gels were then steeped in ethidium bromide for about 40 min, and specific bands were scanned using Gel Doc 2000 software. Statistical analysis Data were presented as means ± SD. Multiple comparisons for different groups were carried out using unpaired t-test or one-way analysis of variance (ANOVA) followed by S.N.K. test as post-hoc analysis with SPSS 18.0 software. Statistical significance was acceptable when p b 0.05. Results Effect of TRPM7 blockage on activation and proliferation of HSC Primary HSC isolated freshly (0-day), cultivated for 3 days (3-day) or 10 days (10-day) were used as quiescent, half- and completelyactivated cells in this study respectively, based on marked morphological changes (Fig. 1A) and gradually increased mRNA and protein levels of α-SMA with self-activation of HSC when cultivated in vitro (Fig. 1B, C). Small amount of TRPM7 and almost no α-SMA were detected in 0day HSC on both mRNA and protein profiles, which were both increased with cell activation, and decreased by 2-APB treatment (Fig. 1B, C), suggesting that TRPM7 blockage could postpone HSC activation.

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Meanwhile, decreased gene and protein expressions of TRPM7 were also detected in 2-APB-treated HSC T6 cells (Figs. 1D and 2D). Cell proliferation was detected in activated HSC T6 cells with MTT assay. We found out that stimulation of a series of concentration of 2APB for 24 h decreased cell viability in a dose-dependent manner (Fig. 1E). Based on this, 200 μmol/L of 2-APB was chosen for further experiments. Apoptosis was involved in activated HSC death caused by TRPM7 blockage Using fluorescence microscopy with AnnexinV/PI staining, we found that apoptotic HSC T6 cells in early or late stage were both markedly increased whereas total cell count was decreased by 2-APB in a dosedependent manner (Fig. 2A). TUNEL assay also demonstrated similar results (Fig. 2B), suggesting that apoptosis was involved in decreased cell proliferation caused by 2-APB treatment. Additionally, the cells incubated with 200 μmol/L of 2-APB revealed markedly shrunken morphological changes (Fig. 2A and B). Our qRT-PCR results showed that, with HSC activation, gene expressions of both bax and bcl-2 decreased gradually, and the ratio bax/bcl-2 showed no difference, implying that pro- and anti-apoptotic gene families were getting a balance. 2-APB increased mRNA levels of bax (9.35 fold) and bcl-2 (2.53 fold) and the bax/bcl-2 ratio (3.99 fold) in 10day HSC (Fig. 2C). Similar result was also detected in HSC T6 cells (Fig. 2D). These results further demonstrated that apoptosis was involved in TRPM7 blockage caused activated HSC death. We also detected increased beclin-1 (autophagy specific) and decreased bmf (necroptosis specific) gene expression in 2-APB treated HSC T6 cells (Fig. 2D), suggesting that autophagy might be activated whereas necroptosis was inhibited during TRPM7 blockage-induced cell death. Taken together, these results demonstrated that apoptosis was involved, at least partially in HSC death caused by TRPM7 blockage. TRPM7 blockage caused apoptotic death of activated HSC was mediated by ER stress With TEM, we observed markedly morphological changes of HSC T6 cells (Fig. 3A), suggesting an ER stress injury from 2-APB. This conclusion was drawn mainly from transition of uniform ERs scattered in the cytoplasm to excessive swollen and remarkably elongated vacuolelike structures around the nucleus. Meanwhile, pyknosis, margination of nuclear chromatin and disappearance of glycoproteins on the cell surface were also observed, suggesting apoptotic cell damage from TRPM7 blockage. Furthermore, a couple of autophagosomes were found in cytoplasm of 2-APB treated cells, further supportive for activation of autophagy. Casepase-12 is considered as one pivotal factor in the ER stress signal pathway. Our qRT-PCR results showed that gene expression of caspase12 was higher in 10-day HSC than those in 0-day and 3-day cells, which means higher ER stress level in activated cells, maybe caused by increasing heavy work during the process of HSC activation (Fig. 3B). With 2APB treatment, gene expressions of caspase-12 in 3-day and 10-day cells were all markedly increased. CHOP is an important potentiator of pro-apoptotic signaling following ER stress, and in this study we found that gene (Fig. 3B) and protein (Fig. 3C) expressions of CHOP did not show any difference with HSC activation, which were both increased markedly by 2-APB treatment in 3-day and 10-day HSC. We also detected increased gene and/or protein expression of caspase-12 (Fig. 3D) and CHOP (Fig. 3D, E) in 2-APB treated HSC T6 cells, whereas change of caspase-3 mRNA level did not show any significance (1.30 ± 0.29 vs. 1.19 ± 0.13, P = 0.22, Fig. 3D). Taken together, these results indicated that ER stress might be one of the underlying mechanisms through which TRPM7 blockage caused apoptotic death of activated HSC. To further verify the involvement of ER stress in 2-APB induced HSC apoptosis, we investigated expressions of downstream effectors of ER

Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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Fig. 2. Apoptosis was involved in activated HSC death caused by TRPM7 blockage. (A) Representative morphology of apoptotic HSC T6 cells detected by Annexin-V/PI staining with fluorescence microscope (×200 magnification, n = 3). Cells in early (green) and late (red) stages of apoptosis were markedly increased by 2-APB treatment in a dose-dependent manner. (B) Representative morphology of apoptotic HSC T6 cells assessed by TUNEL assay (×400 magnification, n = 3). The number of total cells was reduced with increase of 2-APB concentration (numbers in the picture, μmol/L), and TUNEL staining positive apoptotic cells showed dark brown and shrunken appearance. (C) Gene expressions of apoptosis associated factors during the process of HSC activation detected by qRT-PCR (n = 3). β-Actin was used as a housekeeping gene. ##P b 0.01 vs. 10 DC. (D) Gene expressions of TRPM7 and cell deathassociated factors in HSC T6 cells detected by qRT-PCR (n = 3). β-Actin was used as a housekeeping gene. *P b 0.05 and **P b 0.01 vs. control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

stress ATF4, ATF6 and Xbp1, and chaperones GRP78 and calnexin. We found out that, mRNA levels of these genes did not show any significant difference with HSC activation, which were all increased markedly in 2APB treated 3-day and 10-day primary rat HSC and HSC T6 cells (Fig. 3B, F and D), indicating that ER stress-mediated apoptosis was induced not by HSC activation, but by 2-APB stimulation. Similar results were found in protein expression of GRP78 detected by Western blot (Fig. 3C, E).

We also examined the effect of 2-APB on production of spliced form of Xbp1 mRNA (Xbp1s) using regular RT-PCR with specific primers. The result showed that 2-APB created Xbp1s (575 bp) in 3-day and 10-day HSC and decreased 289 and 312 bp fragments produced by Pst I (Fig. 3G), suggesting activation of ER stress sensor IRE1. Nevertheless, the 575 bp band from 10-day cells was much more prominent than that from 3-day cells. Besides, 601 bp, 289 bp and 312 bp bands could

Fig. 1. Effect of TRPM7 blockage on the activation and proliferation of HSC. (A) Representative morphology of primary rat HSC (×100 magnification). 0 DC, 0-day HSC; 3 DC and 3 DA, 3day HSC without or with 200 μmol/L of 2-APB treatment for 24 h; 10 DC and 10 DA, 10-day HSC without or with 2-APB treatment. (B) Gene expressions of TRPM7 and α-SMA during HSC activation detected by quantitative RT-PCR (n = 3). β-Actin was used as a housekeeping gene. (C) Representative Western blot analysis of samples prepared from primary rat HSC using antibodies specific against TRPM7 and α-SMA (n = 3). β-Actin was used as loading control. *P b 0.05 and **P b 0.01 vs. 3 DC, #P b 0.05, ##P b 0.01 vs. 10 DC. (D) Representative Western blot analysis of samples prepared from HSC T6 cells using antibodies specific against TRPM7 (n = 3). β-Actin was used as loading control. (E) HSC T6 cell viability was assessed by MTT assay at the indicated time points following exposure to different concentrations of 2-APB (n = 6 for each group). *P b 0.05 and **P b 0.01 vs. control.

Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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hardly be detected before or after Pst I processing respectively in sample of the 10-day HSC, which were observed obviously in that of the 3-day cells. These results indicated that IRE1 was activated by 2-APB treatment, and the activation level is associated with level of HSC activation. Above all, these results demonstrated that TRPM7 blockage led to induction of all three signaling arms of ER stress response. Along with our observation that 2-APB caused apoptosis of HSC, this provided further evidences for ER stress induced apoptosis as one of the underlying mechanisms mediating cell death caused by TRPM7 blockage. TRPM7 blockage-induced ER stress-mediated apoptosis was associated with level of cell activation With further calculation, we found out that, increases of gene expressions of CHOP, caspase-12, GRP78, ATF6, bax, and the bax/bcl-2 ratio in 10-day HSC caused by 2-APB were more than those in 3-day cells. The increase in protein profile of CHOP and GRP78 showed similar

results (Fig. 3H). This conclusion was further supported by Xbp1 mRNA processing analysis (Fig. 3G). Taken together, these results indicated that level of HSC activation might be an underlying factor, at least in part, determining cell sensitivity to 2-APB and apoptosis mediated by ER stress. Discussion In the present study, we made a couple of significant findings: 1) Blockage of TRPM7 channels inhibited activation and proliferation of HSC; 2) TRPM7 blockage induced apoptotic cell death of activated HSC; and 3) ER stress was identified as the molecular basis underlying this process, which was associated with level of cell activation. Currently there is no approved therapeutic strategy against hepatic fibrosis. Inhibition of HSC activation and proliferation, and induction of activated HSC removal were always hot topics of discussion trying to delay or reverse this process (Lim et al., 2011; Yang et al., 2012). Recent

Fig. 3. TRPM7 blockage caused apoptotic death of activated HSC was mediated by ER stress. (A) Representative ultrastructure of 2-APB-treated T6 cells assessed by TEM (×10,000 magnification, n = 3). Arrow (→) was for excessive swollen and remarkably elongated vacuole-like ER, # for pyknotic and margined nuclear chromatin, and Δ for autophagosomes. (B, F) Gene expressions of ER stress associated factors during HSC activation detected by qRT-PCR (n = 3). β-Actin was used as a housekeeping gene. *P b 0.05 and **P b 0.01 vs. 3 DC, ## P b 0.01 vs. 10 DC. (C) Representative Western blot analysis of samples prepared from primary HSC using antibodies specific against CHOP and GRP78 (n = 3). β-Actin was used as loading control. **P b 0.01 vs. 3 DC, ##P b 0.01 vs. 10 DC. (D) Gene expressions of CHOP, caspase-12 and caspase-3 and ER stress key factors in HSC T6 cells detected by qRT-PCR (n = 3). β-Actin was used as a housekeeping gene. *P b 0.05 and **P b 0.01 vs. control. (E) Representative Western blot analysis of samples prepared from T6 cells using antibodies specific against CHOP and GRP78 (n = 3). β-Actin was used as loading control. *P b 0.05 as compared with control. (G) Analysis of Xbp1 mRNA processing in samples prepared from primary rat HSC. PCR products were illustrated before (a) and after (b) restriction enzyme Pst I incubation. In control HSC, PCR yielded the expected amplification product of 601 bp (a, →), which was completely cut into 289 bp and 312 bp fragments (b, b) by Pst I, indicating the absence of detectable levels of processed Xbp1 mRNA. In 2-APB-treated cells, however, a new band at 575 bp (a and b, Δ) and less (3DA) or even no (10DA) 289 and 312 bp fragments appeared. Numbers indicate size of bands of the ladder (bp). (H) Association of ER stress mediated apoptosis with level of HSC activation. Increase amount of gene expressions of CHOP, caspase-12, GRP78 and bax, protein expressions of CHOP and GRP78, and the bax/bcl-2 ratio in 10-day HSC caused by 2-APB were more than those in 3-day cells.

Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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Fig. 3 (continued).

studies have shown that TRPM7 is implicated in cell survival and proliferation. TRPM7 was considered as the major channel responsible for Ca2+ entry in human atrial fibroblasts (Du et al., 2010), and critical for proliferation, differentiation, and ECM production of fibroblasts (Yue et al., 2011). Besides, TRPM7 was involved in the proliferative potentiality of breast cancer cells (Guilbert et al. 2009) and human retinoblastoma cells (Hanano et al., 2004) by regulating Ca2 + influx. Blockage of TRPM7 channels with Gd3+ or 2-APB or suppression of TRPM7 expression with specific siRNA inhibited growth and proliferation of human head and neck carcinoma cells (Jiang et al., 2007). The present study provided direct evidences that quiescent HSC expressed a small amount of TRPM7, which increased with HSC activation. With 2-APB administration, not only the function of TRPM7, but also expressions of TRPM7 and α-SMA were inhibited, indicating postponement of HSC activation. MTT analysis showed that 2-APB decreased cell viability and inhibited cell proliferation, suggesting induction of cell death. How does TRPM7 inhibition induce HSC death? To answer this question, we investigated various cell death manners. AnnexinV/PI staining and TUNEL assay showed that apoptotic cells in early and late stages were both markedly increased whereas total cells were decreased by

2-APB treatment. Bax and bcl-2 are key members of pro- and antiapoptotic factors respectively, and the ratio of Bax-to-Bcl-2 can determine susceptibility of the cell to apoptosis (Zhou et al., 2011; Lee et al., 2012). In this study, mRNA level of bax and the bax/bcl-2 ratio were increased in 2-APB treated activated primary HSC and T6 cells. These results demonstrated that apoptosis was, at least partially, involved in TRPM7 blockage-induced cell death. We also detected increased beclin-1 and decreased bmf mRNA levels in 2-APB treated cells, implying activation of autophagy and inhibition of necroptosis. Autophagy was also supported by a couple of autophagosomes observed with TEM. Autophagy could be considered as a protective response to ER stress resulting from short-term stimulus (Coates et al., 2010). And necroptosis, a novel cell death manner different from necrosis and apoptosis, does not commonly co-exist with apoptosis, as our result demonstrated. Taken together, these data indicated that apoptosis was one of the main causes of 2-APB induced HSC death. Multiple complex signaling pathways were involved in apoptosis, including the classic (caspase-3) (Huang et al., 2010) and ER stress (Rao et al., 2004; Puthalakath et al., 2007). Casepase-12 is considered as the initial and indispensable factor in ER stress-induced apoptotic

Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030

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cascade (Morishima et al., 2004; Szegezdi et al., 2006). Our results showed that 2-APB increased gene expression of caspase-12 (not caspase-3), suggesting that 2-APB induced apoptosis was mediated, at least partially by ER stress pathway, which agreed with the work of Rao et al. (2002a). This conclusion was further confirmed by highly increased gene and protein expressions of CHOP and GRP78 in 2-APB treated cells. Upon ER stress, the sensors PERK, ATF6 and IRE1 dissociate from chaperones and initiate three arms of signaling events, resulting in increased expression of genes critical for overcoming ER stress, including transcription factors and molecular chaperones (Fig. 2D). So we detected key factors on PERK and ATF6 arms: ATF4, ATF6, Xbp1 and calnexin, and found that mRNA expressions of these genes were all increased significantly by 2-APB. IRE1 is a transmembrane Ser/Thr protein kinase with site-specific endoribonuclease activity that removes a 26-nucleotide intron from Xbp1 mRNA (Lim et al., 2011). Our results showed that Xbp1 mRNA was spliced by 2-APB treatment, indicating phosphorylation of IRE1. Taken together, TRPM7 blockage caused HSC death was induced by ER stress-mediated apoptosis. Interestingly, our results showed that expression of TRPM7 increased with HSC activation, and TRPM7 blockage-induced apoptosis might impact more on activated HSC phenotype via a currently unexplored mechanism of ER stress, which implied that cells producing ECM and promoting fibrosis could be removed with less injury of unactivated HSC in fibrotic liver. This study identified TRPM7 as a novel potential therapeutic target for liver fibrosis, and provided a molecular basis for further investigations in vitro or in vivo. Conflict of interest statement The authors declare that there are no conflicts of interest.

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Please cite this article as: Zhu Y, et al, Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stressmediated apoptosis, Life Sci (2013), http://dx.doi.org/10.1016/j.lfs.2013.10.030