Fig. 4. Tropoelastin regulates migration and actin polymerization via a non-integrin, G-protein-coupled signaling pathway. (A) Eln^-/- vascular smooth muscle cells migrate to recombinant tropoelastin in an EDTA insensitive manner. By comparison, we show that vascular smooth muscle cell migration to collagen, which is mediated by integrins, is EDTA sensitive. (B) Tropoelastin mediated actin polymerization of Eln^-/- cells is EDTA insensitive. (C) Migration of Eln^-/- vascular smooth muscle cells towards tropoelastin is pertussis toxin sensitive. Control experiments with the B protomer of pertussis toxin alone demonstrate that the effect of pertussis toxin is specific to its ability to inhibit G-protein signaling. The migration of Eln^-/- cells to PDGF (30 ng/ml) is insensitive to pertussis toxin. This control experiment demonstrates that Eln^-/- cells treated with pertussis toxin can respond normally to stimuli other than tropoelastin. (D) Tropoelastin-mediated actin polymerization of Eln^-/- vascular smooth muscle cells is pertussis toxin sensitive. Control experiments demonstrate that the B protomer of pertussis toxin does not inhibit actin polymerization. Together, these experiments indicate that tropoelastin regulates vascular smooth muscle cells through a pertussis toxin-sensitive G-protein signaling pathway. (E) Cholera toxin (ctxn) and forskolin (frskln) increase the baseline levels of cAMP in Eln^-/- vascular smooth muscle cells by constitutive activation of the G_s pathway. In the presence of tropoelastin and forskolin, there is a marked decrease in cAMP. A similar decrease in cAMP levels is observed when tropoelastin is added to cholera toxin pretreated cells. The reduction in cAMP was blocked by pertussis toxin and not B-protomer indicating a G_i specific pathway. These experiments indicate that tropoelastin activates a receptor that signals through G_i to inhibit adenylate cyclase, and reduce cAMP levels. (F) Tropoelastin activates RhoA in a pertussis toxin-sensitive manner. Co-immunoprecipitation experiments demonstrate that tropoelastin treatment of Eln^-/- cells results in a marked elevation of activated RhoA. This response is pertussis toxin sensitive and B-protomer insensitive indicating that tropoelastin activation of the Rho pathway requires G_i activity. (G) Proposed molecular mechanism for tropoelastin-mediated actin polymerization in vascular smooth muscle cells.

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