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IRE1A/ XBP1s Axis of ER Stress Promotes DRP1 Phosphorylation at Specific Serine Residue (S616) and Induces Mitochondrial Fragmentation During Airway Inflammation
American Journal of Respiratory and Critical Care Medicine ; 205(1), 2022.
Article in English | EMBASE | ID: covidwho-1927865
ABSTRACT
RATIONALE Airway inflammation plays a role in airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID-19 that affect millions of people worldwide. Previously, we showed that acute (24-h) exposure to the pro-inflammatory cytokine tumor necrosis factor α (TNFα) triggers an endoplasmic reticulum (ER) stress response in human airway smooth muscle (hASM) cells. In hASM cells, TNFα selectively activates the inositol requiring enzyme 1α (IRE1α) ER stress pathway with downstream splicing of X-box binding protein 1 (XBP1s), which transcriptionally activates expression of target genes that include proteins mediating phosphorylation of dynamin-related protein 1 (pDRP1) at the Ser616 (S616) residue. Increased pDRP1 at S616 is associated with mitochondrial fission (fragmentation);however, DRP1 is also phosphorylated at Ser637 (S637) residue, and the balance between phosphorylation at S616 and S637 regulates the translocation of DRP1 from cytosol to mitochondria and subsequent fragmentation of mitochondria. In the present study, we hypothesized that TNFα induces ER stress leading to XBP1s mediated increase in the expression of specific kinases that phosphorylate DRP1 at S616 and promote mitochondrial fragmentation.

METHODS:

hASM cells, dissociated from bronchial tissue obtained from patients with no history of respiratory diseases, were exposed to TNFα (20 ng/ ml for 6-h). As an inhibitor of fragmentation, cells were treated with Mdivi1 (50 μM for 6-h), GTPase inhibitor of DRP1. The expression and phosphorylation status of IRE1α, DRP1, XBP1, cyclin dependent kinases (CDK1, CDK5) and cyclin B1 were quantified by Western blot and immunohistochemistry. Mitochondrial morphology was assessed by 3D confocal microscopy using MitoTracker. XBP1-targets were confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR.

RESULTS:

Bioinformatics analysis predicted putative binding sites of XBP1 in the promoter region of CDK1, CDK5 and cyclin B1 genes that are reported to phosphorylate DRP1 at S616. Consistent with our previous findings, we found that TNFα increases IRE1α phosphorylation and XBP1 splicing. The TNFα induced increase in XBP1s transcriptionally activated expression of CDK1, CDK5 and cyclin B1 and leads to subsequent phosphorylation of DRP1 at S616 with no change in S637 phosphorylation. As a result, TNFα mediated increase in the ratio of S616/ S637 phosphorylation, which promoted translocation of DRP1 from cytosol to mitochondria and mitochondrial fragmentation. We also showed that Mdivi1 mediated inhibition of DRP1-GTPase activity ameliorated phosphorylation at S616 residue and significantly reduced mitochondrial fragmentation.

CONCLUSIONS:

The present study elucidates the mechanism underlying TNFα induced ER stress and mitochondrial fragmentation.
Keywords

Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: American Journal of Respiratory and Critical Care Medicine Year: 2022 Document Type: Article

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Full text: Available Collection: Databases of international organizations Database: EMBASE Language: English Journal: American Journal of Respiratory and Critical Care Medicine Year: 2022 Document Type: Article