TNFa regulates mitochondrial biogenesis via CREB phosphorylation in human airway smooth muscle
Authors: Dasgupta Debanjali , Price Alexis , Mahadev Bhat Sanjana , Delmotte Philippe , Sieck Gary 
Published: May 2023

Airway inflammation is the central driver behind the pathogenesis of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and the very recent pandemic outbreak of COVID-19. Previous studies reported that proinflammatory cytokine TNFa plays a significant role in airway inflammation by inducing hyper-reactivity of airway smooth muscle (ASM) and airway remodeling. TNFa also increases ASM force generation and ATP hydrolysis that requires energy. The excess energy demand is met by an increase in the level of PGC1a protein, mitochondrial biogenesis and subsequently increased mitochondrial volume density. Based on these previous findings, we hypothesized that TNFa promotes CREB phosphorylation at a specific serine 133 residue (pCREBS133) thereby transcriptionally activating PGC1a expression leading to mitochondrial biogenesis and increased mitochondrial volume density. In the study, human ASM cells were dissociated from bronchiolar tissue samples collected during bronchiolar surgery from female and male donors (34 to 71 years of age) with no history of respiratory diseases or current smoking. The dissociated ASM cells were serum-deprived for 48 h before experimentation. The ASM cells from each donor were separated into two groups: 1) time-matched untreated controls, and 2) TNFa treated (20 ng/ml for 6 h). Expression of CREB, pCREBS133, PCG1a, VDAC (an indicator of mitochondrial volume), and downstream signaling molecules (NRFs, TFAM), which regulate transcription and replication of the mitochondrial genome, were determined by quantitative real-time PCR (qPCR) and/or Western blot analysis. The binding of pCREBS133 to the PGC1a promoter was confirmed by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. Mitochondria within ASM cells were labeled using MitoTracker green and imaged by 3D confocal microscopy for the assessment of mitochondrial volume density. Mitochondrial biogenesis was quantified by mitochondrial DNA (mtDNA) copy number relative to nuclear DNA using qPCR. Results were analyzed by one-way analysis of variance (ANOVA). Human ASM cells treated with TNFa showed increased pCREBS133 phosphorylation without any significant change in total CREB. The ChIP assay using a CREB-specific antibody revealed that pCREBS133 binds to the promoter of PGC1a and TNFa treatment further increased the binding affinity. Consistent with these findings, TNFa treatment increased PCG1a expression in ASM cells, and subsequently activated its downstream signaling pathway revealed by increased expression of its target genes, NRF1, NRF2 and TFAM. TNFa-induced activation of the PCG1a signaling pathway in ASM cells resulted in increased mitochondrial biogenesis as indicated by increased mitochondrial DNA copy number and increased mitochondrial volume density. Supported by NIH grant HL157984 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.