Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun;23(6):3940-3950.
doi: 10.1111/jcmm.14275. Epub 2019 Apr 2.

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

Longquan Zuo  1 , Yueqin Zhu  2   3 , Lili Hu  2   3 , Yanyi Liu  2   3 , Yinghong Wang  4 , Yamin Hu  2   3 , Huan Wang  2   3 , Xuesheng Pan  2   3 , Kuayue Li  2   3 , Na Du  2   3 , Yan Huang  2   3
Affiliations

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

Longquan Zuo et al. J Cell Mol Med. 2019 Jun.

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.

Keywords: ERS; PDGF; ASIC1a; HSCs; PI3K/AKT; liver fibrosis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
ASIC1a was up‐regulated in liver tissues of patients with liver fibrosis. A, Photomicrographs showing the liver of patients with liver fibrosis stained with hematoxylin and eosin. B, Protein levels of α‐SMA, ASIC1a and GRP78 in liver fibrosis tissues (T) and normal tissues (NT). (original magnification × 200; n(T) = 20, n(NT) = 10)
Figure 2
Figure 2
ASIC1a and endoplasmic reticulum stress‐related proteins were up‐regulated in rat liver tissues and HSC‐T6 cells. A, Photomicrographs showing the liver of the experimental rats stained with hematoxylin and eosin (original magnification × 200; n = 15). B, The expression of ASIC1a, a‐SMA, GRP78 and C‐Caspase12 in the liver tissues of experimental rats. **P < 0.01 vs Control group (n = 3). C, The expression of ASIC1a, a‐SMA, GRP78 and C‐Caspase12 in HSC‐T6 cells induced by PDGF‐BB. **P < 0.01 vs Normal group (n = 3)
Figure 3
Figure 3
Silencing ASIC1a attenuated the activation and proliferation of HSC‐T6 cells induced by PDGF‐BB. A, B and C, Silencing ASIC1a inhibited the expression of a‐SMA and Collagen I in PDGF‐BB‐stimulated HSC‐T6 cells. **P < 0.01 vs Normal group; ## P < 0.01 vs PDGF group; && P<0.01 vs PDGF+NC‐ShRNA group. D and E, Silencing ASIC1a inhibited the expression of ERS marker proteins GRP78 and Caspase12, and IRE1‐XBP1 in PDGF‐BB‐stimulated HSC‐T6 cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group; && P<0.01 vs PDGF+NC‐ShRNA group. F, RT‐PCR analysis showing silencing ASIC1a attenuated the activation and proliferation of HSC‐T6 (n = 3)
Figure 4
Figure 4
PDGF activated membrane transport of ASIC1a in HSC by PI3K/AKT pathway. A, Toxicity of PI3K/AKT inhibitor (LY294002) on liver stellate cells at different concentrations. **P<0.01 vs Normal group. B, Effect of PI3K/AKT inhibitor (LY294002) on PDGF‐stimulated liver stellate cells at different concentrations. *P<0.05,** P<0.01 vs Normal group; ## P<0.01 vs PDGF group. C, Effect of PI3K/AKT inhibitor (LY294002) with different concentrations on expression of p‐AKT in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. D, LY294002 inhibited the expression of ASIC1a in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. E, Representative immunofluorescence analysis performed on HSCs using anti‐ASIC1a (green) antibody after PDGF treatment for 24 h. Nuclei were counterstained with DAPI. Merge contains the combined image of ASIC1a immunostaining and DAPI staining. Experiments were conducted in triplicate in three independent cell cultures (original magnification × 200) (n = 3)
Figure 5
Figure 5
PDGF induced intracellular Ca2 +influx activated by ASIC1a through PI3K/AKT pathway. Cellular confocal micrographs showing changes in [Ca2+]i concentration visualized by Fluo‐3‐AM in HSCs. A, PDGF‐induced elevation of [Ca2+]i in Ca2+‐free extracellular solution. B, PDGF‐induced elevation of [Ca2+]i in extracellular Ca2+ solution. C, PDGF‐induced elevation of [Ca2+]i in HSCs treated with Ca2+ inhibitor. D, PDGF‐induced elevation of [Ca2+]i in HSCs treated with PcTx1. E, PDGF‐induced elevation of [Ca2+]i in HSCs treated with PI3K/AKT inhibitor (LY294002). (n = 3)
Figure 6
Figure 6
The expression of ASIC1a is related to the GRP78‐IRE1‐XBP1 pathway under endoplasmic reticulum stress. A, Toxicity of ERS inhibitor 4‐PBA on liver stellate cells at different concentrations. **P<0.01 vs Normal group. B, Effect of ERS inhibitor 4‐PBA on PDGF‐stimulated liver stellate cells at different concentrations. *P<0.05, **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. C, Effect of ERS inhibitor 4‐PBA at different concentrations on ERS‐related protein expression in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. D, 4‐PBA inhibited the expression of ERS‐related proteins in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. E, 4‐PBA inhibited the expression of a‐SMA, Collagen I and ASIC1a in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. F, 4‐PBA inhibited the expression of p‐AKT in PDGF‐induced liver stellate cells. **P<0.01 vs Normal group; ## P<0.01 vs PDGF group. G, PcTx1 inhibited the expression of p‐AKT in PDGF‐induced liver stellate cells. ** P<0.01 vs Normal group; ## P<0.01 vs PDGF group. (n = 3)
Figure 7
Figure 7
Endoplasmic reticulum stress combined with ASIC1a via PI3K/AKT pathway contributes to HSC‐T6 activation and proliferation stimulated by PDGF
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M. A proton‐gated cation channel involved in acid‐sensing. Nature. 1997;386:173‐177. - PubMed
    1. Gautschi MX, van Bemmelen L. Schild, Proton and non‐proton activation of ASIC channels. PLoS ONE. 2017;12:e0175293. - PMC - PubMed
    1. Boscardin E, Alijevic O, Hummler E, Frateschi S, Kellenberger S. The function and regulation of acid‐sensing ion channels (ASICs) and the epithelial Na(+) channel (ENaC): IUPHAR Review 19. Br J Pharmacol. 2016;173:2671‐2701. - PMC - PubMed
    1. Li M, Inoue K, Branigan D, et al. Acid‐sensing ion channels in acidosis‐induced injury of human brain neurons. J Cereb Blood Flow Metab. 2010;30:1247‐1260. - PMC - PubMed
    1. Duan B, Wu LJ, Yu YQ, et al. Upregulation of acid‐sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity. J Neurosci. 2007;27:11139‐11148. - PMC - PubMed

Publication types

MeSH terms