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Suppression of store-operated Ca²⁺ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion.

ABSTRACT: Store-operated Ca²⁺ entry (SOCE) plays an important role in regulating Ca²⁺ influx, which participates in tumor cell survival and motility. We aim to elucidate the role of SOCE in the behavior of C6 glioma cells. Lentiviral vector inserted with the Orai1-targeting shRNA was used to inhibit SOCE in C6 glioma cells. The down-regulation of Orai1 was confirmed by western blot. The ability of shOrai1 or SOCE inhibitor (SKF96365) in regulating SOCE inhibition was evaluated by measuring Ca²⁺ concentration. Additionally, its effect on cell behavior was assessed using methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell assay, and adhesion assay. Focal adhesions were visualized by immunofluorescence assay. Further, the expression of proline-rich tyrosine kinase 2 (Pyk2) and phosphorylated Pyk2 (p-Pyk2) was analyzed using western blot. Both, SKF96365 treatment and the Orai1 down-regulation inhibited SOCE by perturbing Ca²⁺ influx. The inhibitory effects of shOrai1 on C6 cell proliferation, migration, and invasion were similar to that of SKF96365. Moreover, Orai1 inhibition enhanced C6 cell adhesion by increasing the size of focal adhesion plaques. The down-regulation of Pyk2 was observed in both SKF96365-treated and Orai1-silenced C6 cells. Additionally, Orai1 inhibition blocked AKT/mTOR, NFAT, and NF-κB pathways. The silencing of Orai1 inhibited the C6 glioma cell migration, invasion and contributed to focal adhesion.

SUBMITTER: Zhu M

PROVIDER: S-EPMC7781592 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

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Suppression of store-operated Ca²⁺ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion.

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Suppression of store-operated Ca2+ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion.

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OmicsDI is part of the ELIXIR infrastructure

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Suppression of store-operated Ca²⁺ entry regulated by silencing Orai1 inhibits C6 glioma cell motility via decreasing Pyk2 activity and promoting focal adhesion.