Abstract
Endothelial cell migration is a key step during angiogenesis and its dysregulation contributes to aberrant neovascularization. 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. Using microarray analyses, 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. 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. Likewise, cellular alignment induced by BMP6 is Myo10-dependent. Using Dunn chamber assay, MEC transfected with control siRNA and pretreated with BMP6 for 4 hours before application of the BMP6 gradient demonstrated increased alignment towards the BMP6 gradient (from 32±10 to 95±5 cells). However, cells transfected with Myo10 siRNA failed to align in the proper direction (from 28±7 to 27±9 cells). Boyden chamber assays indicate that the knockdown of Myo10 with specific small interfering RNA (siRNA) inhibits BMP6-directed 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 directed by BMP6. Surprisingly, we found that Myo10 and BMP6 receptor ALK6 co-localized in a BMP6-dependent fashion. ALK6 was translocated into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possessed intrafilopodial motility. Additionally, Myo10 was required for BMP6-dependent Smad activation, indicating that-in addition to its role in filopodial assembly-Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 plays a critical role in the guidance of endothelial migration towards BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.
Published Version
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