Abstract 3569: Bone Morphogenetic Protein 2 Promotes Pulmonary Smooth Muscle Cell Motility by Activation of the Wnt/RhoA-Rac1 Pathway via Recruitment of Disheveled by Integrin Linked Kinase 1
Background: Migration of pulmonary artery smooth muscle cells (PASMCs) to areas of ongoing vascular injury may contribute to the abnormal vascular remodeling seen in idiopathic pulmonary artery hypertension (IPAH). While mutations in bone morphogenetic protein (BMP) signaling promote PASMC growth, their impact on motility is unknown. We have previously reported that BMP-2 recruits Wnt/RhoA-Rac1 signaling in pulmonary artery endothelial cell (PAEC) to promote motility while simultaneously activating Wnt/β catenin (β C) to induce proliferation and survival. Based on this work, we hypothesized that BMP-2 promotes PASMC motility via Wnt/RhoA-Rac1 signaling independent of β C activation.
Results: BMP-2 (10ng/ml) stimulated PASMCs exhibited higher motility compared to unstimulated cells (P<0.001). In contrast to PAECs, transient elevation in βC was only observed during the first hour of BMP-2 stimulation (P<0.001) followed by steady decline after 4 hours (P<0.0001). While βC levels were being suppressed, RhoA-Rac1 activation was observed, suggesting preferential recruitment of Wnt/RhoA-Rac1 signaling. To determine the role of early βC activation, motility assays were performed using PASMC treated with nonspecific or βC specific RNAi. Knockdown of βC abrogated BMP-2 induced motility and RhoA-Rac1 activation, suggesting that βC may be necessary for induction of genes responsible for Wnt/RhoA-Rac1 activation. We found that synthesis and release of fibronectin (FN), a known βC target, is increased by BMP-2 in a βC dependent fashion and correlates with activation of Integrin linked kinase 1 (ILK1). Knockdown of ILK1 was associated with reduced motility and blunted RhoA-Rac1 activation despite BMP-2 stimulation. Confocal and time lapse video microscopy showed that, under BMP-2, ILK1 and Disheveled (Dvl) strongly colocalize in migrating PASMCs and when cells were transfected with either dominant negative (DN) dvl or ILK1, both BMP-2 mediated Dvl-ILK interaction and cell motility were lost.
Conclusions: BMP induced hPASMC motility is dependent on cross-talk with both Wnt/βC and Wnt/RhoA-Rac1 pathways. Abnormalities in both pathways may predispose to abnormal cellular motility responses and contribute to development of IPAH.