The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNF?), and underlies a number of respiratory diseases. A fundamental question is why are some individuals more susceptible than others to the negative impact of airway inflammation. We will explore a novel homeostatic mechanism, which protects airway smooth muscle (hASM) cells from the negative impact of inflammation-induced reactive oxygen species (ROS) formation and protein unfolding (endoplasmic reticulum (ER) stress). We believe that a failure in this homeostatic mechanism leads to increased ROS formation thereby exacerbating oxidative and ER stress. Overall Hypothesis: TNF?-induced ROS formation and protein unfolding activates the pIRE1?/XBP1s ER stress pathway in hASM, which initiates a homeostatic response directed towards increasing mitochondrial biogenesis and mitochondrial volume density to reduce O2 consumption and ROS formation by individual mitochondrion, while still meeting the increase in ATP demand ? sharing the energetic load across mitochondria. Furthermore, reduced Mfn2 disrupts mitochondrial tethering to the ER, thereby decreasing mitochondrial Ca2+ influx and maximum respiratory capacity of mitochondria. Aim 1: TNF?-induced activation of pIRE1?/XBP1s ER stress pathway increases mitochondrial volume density and reduces O2 consumption and ROS formation per mitochondrion. In hASM cells, the downstream impact of TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be explored using transfection of a non-phosphorylatable IRE1? mutant plasmid (DP-IRE1?) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the effects of siRNA knockdown of PGC1? and Mfn2 overexpression on TNF?-induced changes in mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation. Aim 2: TNF?-induced reduction in Mfn2 disrupts mitochondrial tethering to ER, decreases mitochondrial Ca2+ influx and reduces maximum respiratory capacity of mitochondria. In hASM cells, we will examine the impact of DP-IRE1? or uXBP1 mRNA transfection and siRNA Mfn2 knockdown on TNF?-induced disruption of mitochondrial/ ER tethering, decreased mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria. Aim 3: The impact of TNF? on activation of the pIRE1?/XBP1s ER stress pathway and downstream effects are mitigated by ROS scavenging and chemical chaperone treatment. In hASM cells, the mitigating effects of ROS scavenging and chemical chaperone treatment on TNF?-induced activation of the pIRE1?/XBP1s ER stress pathway will be examined.
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