The Integrin Pathway Partially Mediates Stretch-Induced Deficits in Primary Rat Microglia

Stretch-injured microglia display significantly altered morphology, function and inflammatory-associated gene expression when cultured on a synthetic fibronectin substrate. However, the mechanism by which stretch induces these changes is unknown. Integrins, such as α5β1, mediate microglial attachment to fibronectin via the RGD binding peptide; following integrin ligation the integrin-associated signaling enzyme, focal adhesion kinase (FAK), autophosphorylates tyrosine residue 397 and mediates multiple downstream cellular processes. We therefore hypothesize that blocking the RGD binding/integrin pathway with a commercially available RGD peptide will mimic the stretch-induced morphological alterations and functional deficits in microglia. Further, we hypothesize that upregulation of stretch-inhibited downstream integrin signaling will reverse these effects. Using primary rat microglia, we tested the effects of RGD binding peptide and a FAK activator on cellular function and structure and response to stretch-injury. Similar to injured cells, RGD peptide administration significantly decreases media nitric oxide (NO) levels and iNOS expression and induced morphological alterations and migratory deficits. While stretch-injury and RGD peptide administration decreased phosphorylation of the tyrosine 397 residue on FAK, 20 nM of ZINC 40099027, an activator specific to the tyrosine 397 residue, rescued the stretch-induced decrease in FAK phosphorylation and ameliorated the injury-induced decrease in media NO levels, iNOS expression and inflammatory associated gene expression. Additionally, treatment alleviated morphological changes observed after stretch-injury and restored normal migratory behavior to control levels. Taken together, these data suggest that the integrin/FAK pathway partially mediates the stretch-injured phenotype in microglia, and may serve as a pathway to modulate microglial responses.