Molecular Mechanisms Underlying TNFα-Induced Mitochondrial Biogenesis in Human Airway Smooth Muscle

Proinflammatory cytokines such as TNFa mediate airway inflammation. Previously, we showed that TNFa increases mitochondrial biogenesis in human ASM (hASM) cells, which is associated with increased PGC1a expression. We hypothesized that TNFa induces CREB and ATF1 phosphorylation (pCREB(S133) and pATF1(S63)), which transcriptionally co-activate PGC1a expression. Primary hASM cells were dissociated from bronchiolar tissue obtained from patients undergoing lung resection, cultured (one-three passages), and then differentiated by serum deprivation (48 h). hASM cells from the same patient were divided into two groups: TNFa (20 ng/mL) treated for 6 h and untreated controls. Mitochondria were labeled using MitoTracker green and imaged using 3D confocal microscopy to determine mitochondrial volume density. Mitochondrial biogenesis was assessed based on relative mitochondrial DNA (mtDNA) copy number determined by quantitative real-time PCR (qPCR). Gene and/or protein expression of pCREB(S133), pATF1(S63), PCG1a, and downstream signaling molecules (NRFs, TFAM) that regulate transcription and replication of the mitochondrial genome, were determined by qPCR and/or Western blot. TNFa increased mitochondrial volume density and mitochondrial biogenesis in hASM cells, which was associated with an increase in pCREB(S133), pATF1(S63) and PCG1a expression, with downstream transcriptional activation of NRF1, NRF2, and TFAM. We conclude that TNFa increases mitochondrial volume density in hASM cells via a pCREB(S133)/pATF1(S63)/PCG1a-mediated pathway.

Automated summary

Inflammatory cytokines like TNFa promote airway inflammation. Our study explored the mechanism by which TNFa increases mitochondrial biogenesis in human ASM cells, finding that it involves the phosphorylation of CREB and ATF1, which in turn co-activate the expression of PGC1a. We demonstrated that TNFa treatment of hASM cells resulted in increased mitochondrial volume density, biogenesis, and the activation of downstream transcription factors NRF1, NRF2, and TFAM via the pCREB(S133)/pATF1(S63)/PCG1a pathway.