Abstract 525: Bone Morphogenetic Protein 2 (BMP 2) and Wingless 3a (Wnt 3a) Stimulate Human Pulmonary Artery Smooth Muscle Cell (hPASMC) Motility via Activation of a Rho and Rac Dependent Non-Canonical Wnt Pathway
Increased PASMC motility has been related to the neointimal thickening observed in patients with systemic vascular disease as well as pulmonary arterial hypertension (PAH). Changes in activity of monomeric GTPases Rho and Rac are necessary to relay cytoskeletal changes that result in cell movement. We previously showed that BMP 2 and Wnt 3a induce hPASMC migration similar to that of the mitogen, PDGF-BB, and now investigate how this might be coordinated through the Rho/Rac pathway.
Methods: Recombinant BMP 2 and Wnt 3a were used to stimulate hPASMC motility in a Boyden chamber assay. Whole cell lysates were obtained at various time points and exposed to Rho-binding (RBD) or Rac-binding (RABP) protein in glutathione beads to precipitate active Rho and Rac, respectively. Levels of active Rho and Rac and those of phosphorylated LIM domain kinase 1 (LIMK1) and C-Jun N terminal Kinase (JNK), known targets of Rho and Rac respectively, were measured by immunoblotting. These features were related to the presence of stress fibers assessed by Alexa 488-labeled phalloidin. The functional significance of our findings was assessed by the ability of Y-27623, a ROCK I/II inhibitor, to block hPASMC motility.
Results: Both BMP 2 (10ng/ml) and Wnt 3a (100ng/ml) induce activation of Rho and Rac in hPASMC. This is associated with phosphory-lation of LIMK and JNK, with the appearance of stress fibers, and with increased formation of lamellipodia and fillopodia. BMP 2 and Wnt 3a induced hPASMC migration, and this is significantly reduced in the presence of the ROCK I/II inhibitor Y-27632 (10, 50 and 100uM). These observations correlate with the fall in active beta catenin levels in response to both BMP 2 and Wnt 3a and implicate induction of a non-canonical planar cell polarity (PCP) pathway.
Conclusions: Activation of Rho- and Rac-dependent pathways may result from BMP 2 and Wnt 3a cross-talk: the fall in beta catenin suggests that sequestration of Disheveled and inhibition of canonical Wnt activity may be necessary for PASMC motility.