Tumor necrosis factor α-induced endoplasmic reticulum stress promotes airway smooth muscle cell proliferation

Proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) induce airway smooth muscle (ASM) cell proliferation. Previously, we showed that TNFα induces an endoplasmic reticulum (ER) stress response involving autophosphorylation of inositol requiring enzyme 1α at serine 724 (pIRE1α^S724) and alternative splicing of X-box binding protein 1 (XBP1s). XBP1s transcriptionally activates the expression of cyclin-dependent kinases 1 and 5 (CDK1 and CDK5). In the present study, we hypothesized that TNFα induced activation of the pIRE1α^S724/XBP1s ER stress pathway mediates transcriptional activation of cyclin B1 and ASM cell proliferation. Human ASM (hASM) cells were dissociated from bronchiolar tissue samples obtained from female and male patients with no history of respiratory disease. Isolated hASM cells were either treated or untreated with TNFα (20 ng/mL) for 6 h. For loss of function experiments, hASM cells were either treated with 4µ8C, a pharmacological inhibitor of IRE1α endoribonuclease activity, or transfected with a nonspliceable XBP1 mutant (δXBP1). The binding site sequences of XBP1s to the <i>CCNB1</i> (cyclin B1 gene) promoter were identified by bioinformatic analysis and confirmed by chromatin immunoprecipitation (ChIP) assay. hASM cell proliferation was measured using a CyQuant cell proliferation assay. TNFα induced pIRE1α^S724 phosphorylation and XBP1s splicing in hASM cells. XBP1 transcriptionally activates expression of cyclin B1 mRNA and protein. Nuclear localization of cyclin B1 increased significantly in TNFα-treated hASM cells, consistent with the formation of cyclin B1/CDKs complexes, and increased cell proliferation. Inhibition of pIRE1α^S724/XBP1s pathway mitigated TNFα-induced cyclin B1 and CDKs expression and hASM cell proliferation.<b>NEW & NOTEWORTHY</b> Airway hyperreactivity and hASM cell proliferation (hyperplasia) are hallmark characteristics of asthma. The results of the present study showed that short-term exposure of nonasthmatic hASM to TNFα (a proinflammatory cytokine) can also induce hASM cell proliferation via activation of an ER stress pathway. Identification of the ER stress signaling pathway as important in mediating hASM hyperplasia may provide an important therapeutic target for treating asthma or acute inflammation.