Pulmonary infections caused by Pseudomonas aeruginosa remain a significant clinical challenge, particularly in patients with cystic fibrosis and diffuse panbronchiolitis. This opportunistic pathogen exhibits remarkable resistance to antibiotics and produces multiple virulence factors, including proteases such as elastase (PE), which contribute to tissue damage and systemic infection. PE is known to disrupt epithelial barriers and promote inflammation, but its role in modulating inflammatory responses in lung fibroblasts has not been fully elucidated. This study investigates the molecular mechanism by which PE induces inflammatory signaling in human lung fibroblasts, focusing on the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway and its downstream effects on interleukin-8 (IL-8) production.
We utilized IMR-90 human lung fibroblasts to examine the effects of PE on intracellular signaling pathways. Treatment with enzymatically active PE led to rapid phosphorylation of EGFR at Tyr1068 and subsequent activation of ERK1/2, a key component of the MAPK cascade. This activation was time- and concentration-dependent, peaking at 10 minutes after exposure. Western blot analysis confirmed that PE-induced ERK1/2 phosphorylation was significantly attenuated by pretreatment with U0126, a specific MEK inhibitor, indicating dependence on the MEK-ERK axis. Furthermore, neutralizing antibodies against EGFR or the tyrosine kinase inhibitor AG1478 effectively blocked both EGFR phosphorylation and downstream ERK activation, demonstrating that PE signals through EGFR to initiate the MAPK pathway.SLC34A2 ProteinBiological Activity
In addition to MAPK activation, PE induced nuclear translocation of NF-κB, a master regulator of proinflammatory gene expression. This was observed within 10 minutes of treatment and persisted for at least one hour. Nuclear fractionation followed by Western blotting revealed increased levels of the p65 subunit of NF-κB in PE-treated cells compared to controls. The involvement of NF-κB in IL-8 regulation was further supported by experiments using BAY 11-7085, a specific NF-κB inhibitor, which significantly reduced PE-induced IL-8 secretion.
To assess functional outcomes, we measured IL-8 mRNA and protein levels using Northern blotting, real-time PCR, and ELISA. PE treatment resulted in a robust increase in IL-8 mRNA expression, reaching peak levels at 2 hours post-exposure and declining thereafter.1,5-Diiodopentane Data Sheet This induction was dose-dependent and critically dependent on EGFR, MEK, and NF-κB activity.PMID:34626678 Pretreatment with inhibitors of any of these components dramatically suppressed both IL-8 gene expression and protein secretion. Notably, heat-inactivated or phosphoramidon-treated PE failed to induce either signaling or IL-8 production, confirming that enzymatic activity is essential.
These findings demonstrate that Pseudomonas aeruginosa elastase activates lung fibroblasts via an EGFR/MAPK/NF-κB signaling cascade, leading to enhanced IL-8 production. This mechanism contributes to the amplification of local inflammation by recruiting neutrophils and exacerbating tissue injury. Given the central role of IL-8 in chronic lung diseases like cystic fibrosis, targeting this pathway may offer therapeutic potential for mitigating inflammation during P. aeruginosa infection.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com