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A5732 - Syndecan-1 Mediates Lung Endothelial Cell Inflammatory Signaling by Integrin β4
Author Block: Y. Epshtein1, M. Anis1, W. Chen2, T. Wang3, A. E. Cress3, J. R. Jacobson4; 1Division of Pulmonary, Critical Care, Sleep and Allergy, Department of medicine., University of Illinois at Chicago, Chicago, IL, United States, 2Division of Pulmonary, Critical Care, Sleep and Allergy, Department of medicine., University of Illinois at Chicago, chicago, IL, United States, 3The University of Arizona, Tucson, AZ, United States, 4University of Illinois at Chicago, Chicago, IL, United States.
Rationale: We previously reported lung endothelial cell (EC) inflammatory responses are mediated by integrin β4 (ITGB4). In addition, we identified decreased ITGB4 phosphorylation as a key vascular-protective effect of simvastatin, an HMG CoA-reductase inhibitor. To explore the mechanisms involved in regulating ITGB4 phosphorylation we investigated the role of syndecan-1 (SDC1), a cell-surface proteoglycan that binds the ITGB4 cytoplasmic domain, is expressed by EC, and has been implicated as a mediator of ALI. Accordingly, we hypothesized that syndecan-1 mediates ITGB4 signaling associated with vascular inflammation. Methods: Human pulmonary artery EC were treated with simvastatin (5 µM, 16 h) and lysates were used for Western blotting for ITGB4 and SDC1 or for immunoprecipitation with an ITGB4 antibody followed by Western blotting for SDC1. To investigate a functional link between SDC1 and inflammatory responses regulated by ITGB4 EC were transfected with siRNA specific for SDC1 (siSDC1, 100 mM, 3 d) or non-specific siRNA prior to excessive cyclic stretch (18% elongation, 2 h) followed by immunoprecipitation of ITGB4 and Western blotting with a phospho-tyrosine antibody as well as measurements of inflammatory cytokines, IL-6 and IL-8, in the media. Finally, we utilized SDC1 C-terminal peptide, a competitive inhibitor of ITGB4-SDC1 interaction to assess effects of SDC1 inhibition on LPS-induced ITGB4 phosphorylation and EC barrier function. EC were pretreated with the competitive peptide (30 µM) or control peptide 30 min prior to LPS (1 µg/ml, 1 h) followed by Western blotting for total and p-ITGB4. Transendothelial electrical resistance (TER) measurements of EC under similar conditions were performed to assess barrier effects. Results: Simvastatin treatment of EC was associated with dramatically increased ITGB4 and markedly decreased SDC1 expression levels. Simvastatin treatment also abrogated the strong association of SDC-1 with ITGB4 that was present under basal conditions. Moreover, SDC1 silencing significantly decreased ITGB4 phosphorylation after cyclic stretch and attenuated CS-induced expression of IL-6 and IL-8. Finally, LPS-induced ITGB4 phosphorylation and EC barrier disruption measured by TER were both significantly attenuated by inhibition of the ITGB4-SDC1 interaction with the SDC-1 competitive inhibitor. Conclusions: These studies implicate SDC1 as a critical mediator of ITGB4 signaling in lung EC inflammatory responses. Inhibition or knockdown of SDC1 is associated with decreased ITGB4 phosphorylation and an attenuation of agonist-induced inflammatory cytokine expression levels and EC barrier disruption. Our findings suggest SDC1 may serve as a novel target to mitigate lung vascular inflammatory responses in a variety of clinical contexts.