Abstract 817: Myo10 is a Sensor for Endothelial Migration Induced by BMP6 via Regulation of Filopodia
Endothelial cell migration is a key step during angiogenesis and dysregulation of this process is held responsible for many aspects of cardiovascular and other diseases. The bone morphogenetic proteins (BMP) are potent stimulators of cell migration and angiogenesis. However, the signaling pathways necessary for migration induced by BMPs are still poorly understood. To search for novel genes contributing to BMP-induced endothelial migration, we performed gene expression profiling experiments using microarray analysis technology. We found that Myosin X (Myo10), which is known to regulate filopodial integrity, is potently upregulated by BMP2 or BMP6. RT-PCR and western blotting analysis indicate more than 10-fold increase of Myo10 mRNA and protein levels. The upregulation of Myo10 is regulated through the Smad pathway since Smad inhibitors Smad6 and Smad7 significantly inhibit Myo10 upregulation induced by BMP6. In endothelial cells, BMP6-induced Myo10 localizes in filopodia and filopodia number increases from 20±11 to 54±17 per cell as determined by scanning electron microscopy. This finding is consistent with our observation that increased filopodial extensions are induced by overexpression of Myo10. Co-immunoprecipitation experiments demonstrates that endogenous Myo10 and BMP6 type I receptor ALK6 are associated and that BMP6 treatment increases their binding. Immunofluorescence data demonstrates that with BMP6 treatment ALK6 translocates into filopodia, co-localizes with Myo10 and undergoes intrafilopodia motility. Boyden chamber assays indicate that the knockdown of Myo10 with specific small hairpin- (sh−) RNAs inhibits BMP6-induced cell migration (from 53±5 to 4±2 cells per field), surporting the model that Myo10 located in filopodia plays a key role in endothelial migration induced by BMP6. However, overexpression of Myo10 alone is not sufficient to increase endothelial migration compared to control (from 4±1 to 7±1 cells per field), and that a BMP6 gradient is needed to increase cell migration significantly (from 7±1 to 29±5 cells per field). Therefore, our data strongly suggest that Myo10 plays a critical role as a sensor in the guidance of endothelial migration towards BMP6 gradient via the regulation of filopodia function.