Figure 8.

Integrin α1β1–dependent Rac1/CDC42 activation mediates dynamic remodeling of the actin cytoskeleton in cultured primary mesangial cells (MES). A: Migration of primary cultured mesangial cells is significantly reduced under conditions of integrin α1 deletion, integrin-linked kinase inhibition, Rac1 inhibition, but not AKT inhibition. Migration was measured by Boyden chamber assay in the presence or absence of the ILK inhibitor QLT-0267 (QLT), the Rac1 inhibitor NSC 23766, or the pan-AKT inhibitor GSK 690693, and for integrin α1–null mesangial cells (Iα1 Null MES). Multiple replicate experiments were performed on multiple independent derivations of mesangial cells and the data analyzed by Student’s t-test. ^∗P < 0.05 versus 10% FCS alone. BSA, bovine serum albumin. B–E: Treatment of cultured mesangial cells with LPS induced cytoskeletal rearrangement with numerous actin spikes (asterisks in C), as determined by phalloidin staining (untreated cells, B; LPS-treated cells, C), and these morphological changes were blocked by treatment of cells with either Rac1 inhibitors (D) or CDC42 inhibitors (E). Untreated integrin α1–null cells did not respond to LPS treatment (data not shown). F–I: Treatment of cultured mesangial cells with LPS results in polarized localization of CDC42 (red) and is associated with filopodia (phalloidin in green) (G, inset), compared to Golgi and cytosolic localization of CDC42 in wild-type cells (F). Pretreatment of cells with the Rac1 inhibitor NSC 23766 abolished LPS-activated polarized localization of CDC42 (H), indicating crosstalk between Rac1 and CDC42. I: GTP-Rac1 pull-down assay confirms LPS-mediated activation of Rac1 in cultured mesangial cells is blocked by pretreatment with Rac1 inhibitors, but not by CDC42 inhibitor. Scale bars: 12 μm (B–H); 6 μm (inset, G). Cont, control; inh, inhibitor; lys, lysates.