B29-10 AKAPs Restrain PDGF-PDGFR-PI3k-Mediated Migration by Compartmentalizing Adhesion Proteins in Human Airway Smooth Muscle

Rationale A-kinase Anchoring Proteins (AKAPs) are scaffolding and signaling proteins that regulate multiple cellular functions such as adhesion, migration, and signaling cascades. We recently demonstrated that AKAPs regulate cAMP/PKA signaling by G protein-coupled receptors (GPCRs) in a compartmentalized manner and regulate both contraction and migration of human airway smooth muscle (HASM) cells. Phosphatidylinositol 3-kinase (PI3K) signaling by growth factors such as platelet-derived growth factor (PDGF) plays a pivotal role in HASM migration and polarity. In this study, our aim is to delineate the role of AKAPs Ezrin- and Gravin- in modulating the spatiotemporal compartmentalization of PDGF-PI3K signaling. We hypothesize that AKAPs regulate the spatial and temporal distribution of essential adhesion proteins at adhesion sites on the leading edge of the ASM cell inducing a migratory phenotype thereby influencing migratory function of ASM cells. Material & Methods Ezrin and Gravin expression in HASM cells was knocked down using Dharmacon smartpool siRNA in serum free F12 media and subsequently plated in media with serum in MatTek™ dishes, 12-well plates, or E-plates. xCELLigence and immunoblot were used to assess cell adhesion and changes in total and phosphorylated (p) protein expression, respectively. Biosensors for F-actin, CDC-42, and FMNL-1 were used to assess the changes in expression of adhesion proteins in different compartments and leading-edge dynamics in migrating HASM cells using confocal microscopy followed by image analysis using multiple tools. Results Ezrin-, Gravin- or double- knockdown (KD) in HASM cells resulted in significantly decreased cell adhesion with an associated increase in PI3K activity as assessed using immunoblot analysis of p-110α and p-AKT. Image analysis showed differential expression of adhesion proteins; integrin alpha 8 and Talin-1 in different compartments of cells. Live imaging of PDGF-treated HASM cells showed; (i) initiation of the formation of the leading edge sooner (<1 h) in DKD cells compared to scrambled cells (>4 h); (ii) significantly narrowed leading edge in DKD cells compared to the wide leading edge in scrambled cells; (iii) decreased polarity, allowing the DKD cells to change direction during migration, synonymous to epithelial-mesenchymal transition (EMT). Conclusion Data suggest knockdown of AKAPs significantly disrupts the spatial-temporal organization of adhesion proteins, causing decreased -adhesion and -polarity, as AKAPs are essential modulators of the PDGF/PI3K pathway that regulate cell adhesion and migration. Therefore, disrupting AKAP interactions with cellular proteins is a potential therapeutic approach for modulating aberrant signaling in ASM cells.