The impact of acute airway inflammation is mediated by pro-inflammatory cytokines (e.g., TNFa),
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: TNFa-induced ROS formation and protein unfolding activates the
pIRE1a/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: TNFa-induced activation of pIRE1a/XBP1s ER stress pathway increases mitochondrial
volume density and reduces O2 consumption and ROS formation per mitochondrion.
In hASM cells, the downstream impact of TNFa-induced activation of the pIRE1a/XBP1s ER
stress pathway will be explored using transfection of a non-phosphorylatable IRE1a mutant
plasmid (DP-IRE1a) or an unspliceable XBP1 (uXBP1) mRNA. In addition, we will examine the
effects of siRNA knockdown of PGC1a and Mfn2 overexpression on TNFa-induced changes in
mitochondrial biogenesis, mitochondrial volume density, O2 consumption and ROS formation.
Aim 2: TNFa-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-IRE1a or uXBP1 mRNA transfection and
siRNA Mfn2 knockdown on TNFa-induced disruption of mitochondrial/ ER tethering, decreased
mitochondrial Ca2+ influx and reduced maximum respiratory capacity of mitochondria.
Aim 3: The impact of TNFa on activation of the pIRE1a/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 TNFa-induced activation of the pIRE1a/XBP1s ER stress pathway will be examined.