Zhang et al. TRPV4 Complex Regulates Tracheal Tension interaction of TRPV4 and IP 3 R 1 with NCX s. Taken together, these findings support a physical and functional interaction of TRPV4 and IP 3 R 1 with NCXs as a novel TRPV4mediated Ca 2+ signaling mechanism and suggest a potential target for regulation of ASM tension and treatment of respiratory diseases, especially tracheal spasm.
Background: Radiation-induced brain injury is a serious and treatment-limiting complication of brain radiation therapy. Although endothelial cell dysfunction plays a critical role in the development of this pathogenesis, the underlying molecular mechanisms remain elusive.Methods: Primary cultured rat brain microvascular endothelial cells (BMECs) were divided into five groups without or with exposure of x-rays delivered at 5 Gy or 20 Gy. For the irradiated groups, cells were continued to cultivate for 12 or 24 h after being irradiated. Then the mRNA libraries of each group were established and applied for next-generation sequencing. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were conducted to analyze the sequencing results. Quantitative polymerase chain reaction, western blotting, cck8 assay and intracellular calcium concentration assays were conducted to analyze the role of Orai2-associated SOCE in x-ray induced cellular injury.Results: In total, 3,005 transcripts in all the four x-ray–exposed groups of BMECs showed expression level changes compared with controls. With the dose of x-ray augment and the following cultured time extension, the numbers of differentially expressed genes (DEGs) increased significantly in BMECs. Venn diagrams identified 40 DEGs common to all four exposure groups. Functional pathway enrichment analyses indicated that those 40 DEGs were enriched in the calcium signaling pathway. Among those 40 DEGs, mRNA and protein expression levels of Orai2 were significantly upregulated for 24 h. Similarly, calcium influx via store-operated calcium entry, which is modulated by Orai2, was also significantly increased for 24 h in x-ray–exposed BMECs. Moreover, the change in SOCE was suppressed by btp-2, which is a non-selective inhibitor of Orai. Additionally, x-ray exposure induced a significant decrease of proliferation in BMECs in the dose- and time-dependent manner.Conclusion: These findings provide evidence for molecular mechanisms underlying BMECs dysfunction in development of radiation-induced brain injury and suggest new approaches for therapeutic targets.
Radiation-induced brain injury is a serious complication with complex pathogenesis that may accompany radiotherapy of head and neck tumors. Although studies have shown that calcium (Ca2+) signaling may be involved in the occurrence and development of radiation-induced brain injury, the underlying molecular mechanisms are not well understood. In this study, we used real-time quantitative polymerase chain reaction and Western blotting assays to verify our previous finding using next-generation sequencing that the mRNA and protein expression levels of Orai3 in rat brain microvascular endothelial cells (rBMECs) increased after X-ray irradiation. We next explored the role of Orai3 and Orai3-mediated store-operated Ca2+ entry (SOCE) in radiation-induced brain injury. Primary cultured rBMECs derived from wild-type and Orai3 knockout (Orai3(−/−)) Sprague–Dawley rats were used for in vitro experiments. Orai3-mediated SOCE was significantly increased in rBMECs after X-ray irradiation. However, X-ray irradiation-induced SOCE increase was markedly reduced in Orai3 knockout rBMECs, and the percentage of BTP2 (a nonselective inhibitor of Orai channels)-inhibited SOCE was significantly decreased in Orai3 knockout rBMECs. Functional studies indicated that X-ray irradiation decreased rBMEC proliferation, migration, and tube formation (a model for assessing angiogenesis) but increased rBMEC apoptosis, all of which were ameliorated by BTP2. In addition, occurrences of all four functional deficits were suppressed in X-ray irradiation-exposed rBMECs derived from Orai3(−/−) rats. Cerebrovascular damage caused by whole-brain X-ray irradiation was much less in Orai3(−/−) rats than in wild-type rats. These findings provide evidence that Orai3-mediated SOCE plays an important role in radiation-induced rBMEC damage and brain injury and suggest that Orai3 may warrant development as a potential therapeutic target for reducing or preventing radiation-induced brain injury.
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