PI3‐kinase/Akt pathway‐regulated membrane transportation of acid‐sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF‐induced HSC Activation

Abstract Acid‐sensing ion channel 1a (ASIC1a) allows Na+ and Ca2+ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non‐neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS‐related proteins were up‐regulated in carbon tetrachloride (CCl4)‐induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS‐related biomarkers GRP78, Caspase12 and IREI‐XBP1. And, ERS inhibition by 4‐PBA down‐regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF‐induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF‐activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.


ZUO et al.
ASIC1a is the most abundant isoform, playing important roles in a variety of physiological and pathological processes including learning, memory, fear, depression, pain, anxiety and ischaemic stroke. [5][6][7][8] Most ASIC1a research has focused on the peripheral nervous system and CNS neurons. Recent reports show ASIC1a is expressed in and affects non-neuronal cells. 9,10 Although ASICs subunits possess differing ion permeability, ASIC1a is solely permeable to Ca 2+ . 11-14. The endoplasmic reticulum (ER) is a complex and key organelle for protein folding, and lipid and carbohydrate metabolism. 15 It is also an important Ca 2+ storage station that regulates intracellular Ca 2+ signalling that affects many signalling pathways. [16][17][18][19][20] Intracellular calcium disorders affect ER functions including protein synthesis, folding and translation, which triggers endoplasmic reticulum stress (ERS) and activates the unfolded protein response (UPR). [21][22][23] UPR is mediated by three ER membrane-associated proteins, including protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring 1α (IRE1α) and activating transcription factor 6 (ATF6). [24][25][26][27] IRE1a is the most conservative branch, it regulates the activity of transcription factor X-box binding protein (Xbp1) through recruitment of signalling molecules, splicing, and production of an active transcription factor. 28 Liver fibrosis is a dynamic process triggered by chronic liver injury, chronic viral infections, alcoholic liver disease, autoimmune disease and hereditary metabolic disorders, which is the final common pathway in a variety of liver diseases. 29 Hepatic stellate cells (HSCs) are the primary fibre producing cells in the liver. During inflammation or mechanical stimulation, HSCs are activated, proliferate and increase the expression of a-smooth muscle actin (a-SMA) and extracellular matrix (ECM). 30,31 Our preliminary experimental results showed high ASIC1a expression in liver fibrosis tissue and PDGF-activated HSCs, and a strong correlation of increased ECM in HSCs. 32 Aissouni et al. and Bo Duan et al. showed that PI3K/Akt signalling affects ASIC1a expression and activity via membrane transduction in neurons. 33,34 This has not been reported in liver fibrosis or HSCs.
ERS is also involved in the pathogenesis of liver fibrosis, where it activates fat generation transcription factor, triggers inflammation and fibrosis and promotes fatty liver and liver fibrosis. 35,36 Given that ERS and ASIC1a both play roles in liver fibrosis pathogenesis, we analysed their effects on HSC activation and proliferation, and looked at possible signalling pathways associated with disease states.

| Animals
Adult male Sprague-Dawley (SD) rats (100-140 g) were provided by the Experimental Animal Center of Anhui Medical University (Hefei, China). All animal protocols were reviewed and approved by the University Animal Care and Use Committee of Anhui Medical University. Thirty SD rats were randomly divided into normal (n = 15) and liver fibrosis groups (n = 15). All animals were adapted 7 days prior to experiments. The liver fibrosis model was established by injecting carbon tetrachloride (CCl 4 , Shantou Xilong Chemistry Plant, China) diluted (3:2) in olive oil (4 ml of CCl 4 /kg bodyweight for the first dose, and 2 ml/kg for the remaining doses) twice weekly for 8 weeks. Control animals were treated with 2 ml/kg body weight olive oil. 35

| Patient tissue samples
Patient tissue samples (n =30) were collected from the First Affiliated Hospital of Anhui Medical University (HeFei, China). A total of 20 liver fibrosis samples were obtained from 20 patients with liver cancer with severe liver fibrosis, and 10 normal samples were obtained from liver tissue with biliary calculi. The study was approved by the Research Ethics Committee of the Anhui Medical University, and informed consent was obtained from all patients.
HSC-T6 cells were cultured in DMEM with 10% FBS and incubated in a humidified atmosphere 5% CO 2 at 37°C.

C G A A C G T G T C A C G T T C T C T T A A A C G T G A C A C G T T C
GGAGAAC-3' (reverse).

| Hematoxylin and eosin staining and immunohistochemistry
Tissue samples were fixed in 10% buffered formalin for 48 hours and then embedded in paraffin; 5-μm slides were cut and dried overnight.

| Quantitative real-time reverse transcriptase polymerase chain reaction
RD SYBR ® qPCR Mix (Toyobo Co. Ltd, Osaka, Japan) was used according to the manufacturer's instructions. β-actin was used as the endogenous control. The primers were designed and synthesized by Shanghai Biology Engineering Corporation according to the serial number from GenBank, shown in Table 1. Similar results were obtained from three independent samples.

| Western blot analysis
Rat liver tissues and HSC-T6 cells were lysed with RIPA lysis buffer (Beyotime, China). After centrifugation at 12 000 g at 4°C for 30 minutes, the supernatants were collected. Proteins were mixed with Laemmli sample buffer and boiled at 100°C for 10 minutes. Extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE, 10%, 80 V for 30 minutes and then 120 V for 60 minutes), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), blocked in TBS/Tween-20 containing 5% nonfat dry milk at 37°C for 3 hours, washed in TBS/Tween-20 three times, then incubated with primary antibodies against ASIC1a

| Statistics
Results are represented as mean ± SD. The comparison among the different treatment groups was performed with analysis of variance (ANOVA) and unpaired Student's t test. P < 0.05 is regarded as statistically significant.

| ASIC1a was up-regulated in liver tissues of patients with liver fibrosis
To investigate the potential function of ASIC1a in human liver fibrosis, we examined ASIC1a expression in liver tissues and normal tissues by immunohistochemical staining. We found ASIC1a was significantly up-regulated in disease tissue compared with control ( Figure 1A). In addition, we found the liver fibrosis protein marker α-SMA and PI3K/AKT pathway proteins GRP78 and p-AKT were significantly up-regulated in liver tissues compared with controls ( Figure 1B). These results suggest ASIC1a is important in the regulation of liver fibrosis by affecting ERS and the PI3K/AKT pathway.

| ASIC1a and ERS-related proteins were upregulated in rat liver tissues and PDGF-induced HSC cells
We activated HSCs with PDGF-BB(10 ng/ml, Protech, USA)and measured the effects. We found that liver tissue and liver lobule were completely intact with the normal structure and healthy arrangement of cells in the control group (Figure 2A

| Silencing ASIC1a attenuated the activation and proliferation of HSC-T6 cells induced by PDGF-BB
To investigate ASIC1a's effect on HSC-T6 cells induced by PDGF-BB, we silenced ASIC1a with a nonspecific inhibitor (Amiloride), a specific inhibitor (PcTx1) and ASIC1a-ShRNA. Results show levels of α-SMA and Collagen I significantly reduced after Amiloride, PcTx1 or ASIC1a-ShRNA administration ( Figure 3A-C respectively). This suggests that ASIC1a participates in the activation and proliferation of HSCs.
To confirm whether ASIC1a affects ERS in HSC-T6 cells stimulated by PDGF-BB, we measured ERS protein markers GRP78, Caspase12 and downstream pathway marker IRE1-XBP1 after silencing ASIC1a. We found that GRP78, Caspase12 and IRE1-XBP1 significantly reduced after PcTx1 or ASIC1a-ShRNA administration ( Figure 3D, E respectively). Consistently, q-PCR results align with Western blot data ( Figure 3F). These results suggest that ASIC1a plays an important role in the pathogenesis of liver fibrosis through ERS related pathway.

| PDGF activated membrane transport of ASIC1a in HSC by PI3K/AKT pathway
To further determine the role of ASIC1a activation in liver fibrosis, we inhibited the PI3K/AKT pathway with LY294002 in PDGF-BB induced HSC-T6 cells. MTT assay results show that LY294002 reduced the number of HSCs ( Figure 4A, B), and WB results show that LY294002 reduced the expression of p-AKT ( Figure 4C). We found LY294002 inhibited PI3K/AKT most effectively at 15 μmol/L. Therefore, this concentration was used in further experiments. We found that ASIC1a expression significantly decreased after silencing PI3K/AKT, suggesting this pathway up-regulates the activity of the ASIC1a membrane channel ( Figure 4D).
We measured the surface population of ASIC1a channels in cultured HSC-T6 cells by immunofluorescence and found significantly
In all experiments, voltage-gated Ca 2+ channels were blocked with 10 μmol/L nimodipine to remove the possibility of secondary channel activation and release of internal Ca 2+ stores. Confocal micrograph results show PDGF significantly increased [Ca 2+ ] i in HSCs ( Figure 5A, B). And, silencing ASIC1a with PcTx1 significantly reduced intracellular Ca 2+ compared with PDGF group ( Figure 5C). Consistently, intracellular Ca 2+ was also reduced after PI3K/AKT inhibition with LY294002. This increased PDGF increased cytosolic Ca 2+ concentration in HSCs by activating ASIC1a through PI3K/AKT pathway.

| Effect of GRP78-IRE1-XBP1 pathway on ASIC1a expression in PDGF-induced HSCs
To further investigate the role of ASIC1a, we inhibited ERS with 4-  Figure 6D). In addition, 4-PBA abolished PDGF-induced expression of ASIC1a, α-SMA and Collagen I ( Figure 6E) and reduced p-AKT levels ( Figure 6F). Finally, PcTx1 treatment also inhibited the PI3K/AKT pathway ( Figure 6G). These results demonstrate a correlation between ERS and ASIC1a via PI3K/AKT signalling, where ASIC1a affects ERS-related pathways by regulating intracellular calcium. Our previous studies showed that ERS is involved in liver fibrosis and associated with liver inflammation. ERS-mediated apoptosis may be a key step in activating HSCs that trigger apoptosis and promote fibrosis. 40 We know that ERS enhances fibrosis through IRE1a-XBP1. 36,41 Caspase-12 is expressed in the ER, where cleavage is important for stress-induced apoptosis. 42 We found increased expression of ERS protein markers GRP78 and Caspase12 and IREI-XBP1 in HSCs, indicating that IREI-XBP1 is involved in liver fibrosis.

| D ISCUSS I ON
However, we do not know how this is related to ASIC1a.
To this end, we examined the expression of ERS-related protein after inhibiting ASIC1a. WB showed that the expressions of GRP78, c-Caspase12 and IRE1-XBP1 were inhibited by PcTx1 and ShRNA-ASIC1a; q-PCR results confirm these findings. In order to investigate whether ERS modulates ASIC1a, we used 4-PBA as an ERS inhibitor, given its broad and effective usage. We found that GRP78, c-Caspase12 and IRE1-XBP1 were significantly inhibited, and α-SMA and Collagen1 significantly decreased. Notably, ASIC1a was also reduced, which may be associated with 4-PBA inhibition of PI3K/AKT pathway in ERS. Results showed that PcTx1 and 4-PBA reduced the expression of p-AKT.
In conclusion, our findings suggest ASIC1a is involved in the activation and proliferation of liver fibrosis and ERS through the PI3K/ AKT pathway.

ACK N OWLED G EM ENTS
The authors thank Z.S. WU for the help with HE and immumohistochemical staining in patients with liver fibrosis, and thank Dr. Austin Cape at ASJ Editors for careful reading and helpful suggestions.
F I G U R E 7 Endoplasmic reticulum stress combined with ASIC1a via PI3K/AKT pathway contributes to HSC-T6 activation and proliferation stimulated by PDGF

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