Syndecan-4 independently regulates multiple small GTPases to promote fibroblast migration during wound healing

Abstract

Upon wounding, syndecan-4 detects the appearance of fibronectin in the wound bed and mediates regulation of the small GTPases, Rac1, RhoA and RhoG. Cohesive regulation of these molecules results in cycles of membrane protrusion and cytoskeletal contraction, and triggers the endocytosis of α 5β 1-integrin, which collectively lead to immigration of fibroblasts into the wound bed. In this manuscript we identify the regulation of a fourth GTPase, Arf6 that is responsible for α 5β 1-integrin recycling and thereby completes the cycle of syndecan-4-regulated integrin trafficking. We demonstrate that each of the GTPase signals can be regulated by syndecan-4, but that they are independent of one another. By doing so we identify syndecan-4 as the coordinating center of pro-migratory signals.

Figure 1. Syndecan-4 regulates both endocytic and recycling phases of integrin trafficking. Contacts were made sequentially with 50K, then Fn, then 50K again to follow α₅β₁-integrin endocytosis upon syndecan-4-engagement and recovery upon removal of syndecan-4 ligand. (A) Control fibroblasts exhibit reduction in unbinding energy upon syndecan-4 engagement, and recover high unbinding energy upon return to 50K. (B) Reduction of Arf6 expression by RNAi has no effect on integrin endocytosis, but blocks recovery upon return to 50K. (C) Arf6 expression of fibroblasts transfected with control or Arf6-targeted siRNA. (D) Engagement of syndecan-4 triggers activation of Arf6 in fibroblasts prespread on 50K. (E) Redistribution of β₁-integrin between plasma membrane and vesicle fractions when fibroblasts, prespread on 50K, were stimulated with H/0 over a time course. Segregation of membrane was confirmed by blotting fractions for Na⁺K⁺ATPase and Rab4. Energy values represent at least 80 measurements per condition from four experiments, western blot data represent eight experiments. Error bars represent s.e.m., significance was tested by ANOVA.

Figure 2. Syndecan-4-regulated integrin trafficking is independent of Rho kinase activity. Energy required to detach fibroblasts from 50K or Fn was measured by atomic force microscopy. (A) Energy measurements of untreated and Y27632-inhibited fibroblasts reveal that Rho kinase activity affects cell avidity itself, but is not required for syndecan-4-induced integrin endocytosis. (B and C) Contacts were made sequentially with 50K, then Fn, then 50K again to follow α₅β₁-integrin endocytosis upon syndecan-4-engagement and recovery upon removal of syndecan-4 ligand. (B) Untreated fibroblasts exhibit reduction in unbinding energy upon syndecan-4 engagement, and recover high unbinding energy upon return to 50K. (C) Treatment with Y27632 blocks neither the endocytic nor recovery phases caused by contact with fibronectin. Values represent at least 80 measurements per condition from 4 experiments. Error bars represent s.e.m., significance was tested by ANOVA.

Figure 3. Relationships between integrin and GTPase signaling. (A–C) Engagement of syndecan-4 triggers Rac1 activation in cells spread on 50K (A and C, dotted line) but not cells in suspension (B and C, solid line), demonstrating that Rac1 activation is integrin dependent. (D) Engagement of syndecan-4 still triggers loss of α₅β₁-integrin from the plasma membrane fraction when fibroblasts are in suspension. (E) Kinetics of regulation of the multiple GTPases regulated by syndecan-4. Data represent eight experiments. Error bars represent s.e.m.

Figure 4. Parallel regulation of multiple GTPases by syndecan-4. Engagement of syndecan-4 triggers early signals (bold): RhoG, Rac1 and RhoA inhibition, followed by late signals: Arf6 and RhoA activation.