Paxillin mechanisms control epithelial tissue remodeling under inflammatory conditions

Objective: We aim to investigate bacterial liposaccharide (LPS) induced wound healing mechanisms via deconvoluting cell-matrix signaling pathways Hypothesis: Normal wounds typically heal at a standard rate, whereas infectious wounds experience delayed recovery, posing significant clinical challenges such as chronic wound formation and an increased risk of infections. These wounds are particularly difficult to treat, highlighting the need to identify new therapeutic targets by unravelling early mechanistic events. Although bacterial roles in infectious diseases have been extensively studied, the mechanisms of cell-cell and cell-matrix interactions in host tissues remain insufficiently explored. Therefore, this study investigates bacterial lipopolysaccharide (LPS)-induced wound healing mechanisms by deconvoluting cell-matrix signaling pathways. To achieve this, keratinocytes were exposed to E. coli LPS, with a focus on paxillin—a pivotal regulator of cell-matrix interactions through integrin signaling. Paxillin plays a crucial role in cell adhesion, migration, and differentiation during epithelial restoration. By unraveling the cellular dynamics and signaling pathways involved in inflamed wounds, this research aims to contribute to the development of targeted therapies for improving healing in infection-induced wound scenarios. A brief statement of methods: 1) Cell culture: In all experiments, Human immortalized gingival keratinocyte cells (HIGKs) were cultured in serum-free media. Subsequently, the cells were exposed to lipopolysaccharide (LPS) derived from E. coli at a concentration of 100 ng/ml. 2) Scratch assay: Scratch assays were performed to monitor wound closure and cell migration respectively. 3) Live migration of keratinocytes: We performed a time-lapse study to evaluate the cell migration under LPS conditions. 4) Immunofluorescence staining was used to visualize paxillin. 5) Western blotting assessed paxillin and phospho-paxillin expression, while Rac activation was analyzed using Rac pulldown assays with PAK-PBD beads. Summary of Results: Our study uncovered the complex mechanisms underlying LPS-mediated effects on wound healing, providing insights into cellular dynamics under inflammatory conditions. Scratch assays demonstrated delayed wound closure after 18 hours of LPS exposure compared to controls. Live migration assays revealed increased protrusion formation and reduced migration upon LPS treatment. Immunofluorescence analysis highlighted elevated Paxillin expression with its localization predominantly on the distal side of cells, indicating its role in adhesion and migration processes. Investigation of phospho-paxillin sites identified Ser273 as a critical phosphorylation site linked to migration signaling, activated via PAK enzymes triggered by Rac activation under LPS exposure. Rac inhibitors (NSC23766) reduced Rac expression and paxillin phosphorylation at Ser273, and their addition during scratch assays improved wound closure by 2-fold under LPS conditions. Conclusion: Collectively, these findings suggest that LPS-induced wound impairment is mediated by Rac-dependent phosphorylation of Paxillin at Ser273, positioning these pathways as promising therapeutic targets for enhancing wound recovery in infection scenarios. Overall, the results emphasize the complex interplay between inflammatory signals, cellular signaling cascades, and cytoskeletal dynamics in regulating wound healing processes. Furthermore, ongoing studies in animal models aim to validate these findings and provide critical insights for developing targeted interventions to accelerate wound healing under infectious conditions. • Georgetown University Medical School (Georgetown Startup Funds, Assignee: 91252)•National Institutes of Health (R01DE031046, R21AR076497, and R21CA294025) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.