We have shown previously that T1α/podoplanin is required for capillary tube formation by human lung microvascular lymphatic endothelial cells (HMVEC-LLy) and that cells with decreased podoplanin expression fail to properly activate the
small GTPase RhoA shortly after the beginning of the lymphangiogenic process. The objective of this study was to determine whether podoplanin regulates HMVEC-LLy migration and whether this regulation is via modulation of
small GTPase activation. In analysis of scratch
wound assays, we found that
small interfering RNA (
siRNA) depletion of podoplanin expression in HMVEC-LLy inhibits
VEGF-induced microtubule-organizing center (MTOC) and Golgi polarization and causes a dramatic reduction in directional migration compared with control
siRNA-transfected cells. In addition, a striking redistribution of cortical actin to fiber networks across the cell body is observed in these cells, and, remarkably, it returns to control levels if the cells are cotransfected with a dominant-negative mutant of Cdc42. Moreover, cotransfection of a dominant-negative construct of Cdc42 into podoplanin knockdown HMVEC-LLy completely abrogated the effect of podoplanin deficiency, rescuing MTOC and Golgi polarization and cell migration to control level. Importantly, expression of constitutively active Cdc42 construct, like podoplanin knockdown, decreased RhoA-
GTP level in HMVEC-LLy, demonstrating cross talk between both
GTPases. Taken together, the results indicate that polarized migration of lymphatic endothelial cells in response to
VEGF is mediated via a pathway of podoplanin regulation of
small GTPase activities, in particular Cdc42.