Molecular Mechanisms Underlying TNFα Induced Mitochondrial Fragmentation in Human Airway Smooth Muscle Cells

Abstract

Tumor necrosis factor α (TNFa), a proinflammatory cytokine, plays a significant role in mediating the effects of acute inflammation in response to allergens, pollutants, and respiratory infections. Previously, we showed that acute exposure to TNFa induces mitochondrial fragmentation in human airway smooth muscle (hASM) cells, which is associated with increased expression of dynamin-related protein 1 (DRP1). Phosphorylation of DRP1 at serine 616 (pDRP1S616) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNFa exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1α (pIRE1a) at serine 724 (pIRE1aS724) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNFa-mediated activation of the pIRE1aS724/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1S616 phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNFa treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNFa treatment activates the pIRE1a/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1S616, are transcriptionally targeted by XBP1s. TNFa treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1S616 and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNFa-induced mitochondrial fragmentation in hASM cells.