Molecular mechanisms underlying TNFα-induced mitochondrial fragmentation in human airway smooth muscle cells

Tumor necrosis factor alpha (TNF alpha), 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 TNF alpha 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 (pDRP1(S616)) promotes its translocation and binding to the outer mitochondrial membrane (OMM) and mediates mitochondrial fragmentation. Previously, we reported that TNF alpha exposure triggers protein unfolding and triggers an endoplasmic reticulum (ER) stress response involving phosphorylation of inositol-requiring enzyme 1 alpha (pIRE1 alpha) at serine 724 (pIRE1 alpha(S724)) and subsequent splicing of X-box binding protein 1 (XBP1s) in hASM cells. We hypothesize that TNF alpha-mediated activation of the pIRE1 alpha(S724)/XBP1s ER stress pathway in hASM cells transcriptionally activates genes that encode kinases responsible for pDRP1(S616) phosphorylation. Using 3-D confocal imaging of MitoTracker green-labeled mitochondria, we found that TNF alpha treatment for 6 h induces mitochondrial fragmentation in hASM cells. We also confirmed that 6 h TNF alpha treatment activates the pIRE1 alpha/XBP1s ER stress pathway. Using in silico analysis and ChIP assay, we showed that CDK1 and CDK5, kinases involved in the phosphorylation of pDRP1(S616), are transcriptionally targeted by XBP1s. TNF alpha treatment increased the binding affinity of XBP1s on the promoter regions of CDK1 and CDK5, and this was associated with an increase in pDRP1(S616) and mitochondria fragmentation. This study reveals a new underlying molecular mechanism for TNF alpha-induced mitochondrial fragmentation in hASM cells.