The major finding of this study is that GIT1 is a key mediator of VEGF stimulated podosome formation in endothelial cells. Specifically, we define a novel role for GIT1 to mediate EC migration and ECM degradation by activating PLCγ, and regulating podosome formation. GIT1 has been previously shown to regulate migration of SMC and HEK293 cells.32-34
We propose that VEGF stimulates GIT1-PLCγ translocation to the site where podosomes will form, and then recruits Src, which phosphorylates and activates PLCγ. Functionally, GIT1 mediates cell invasion and migration by increasing podosome formation and ECM degradation. Evidence to support this concept includes: 1) GIT1 knockdown inhibits EC podosome formation induced by VEGF (); 2) GIT1 colocalizes with podosomes induced by VEGF (); 3) PLCγ colocalizes with podosomes induced by VEGF (); 4) Inhibiting PLCγ with U73122 blocks podosome formation induced by VEGF (); 5) VEGF-induced podosome formation is also dependent on Src and small GTPase (); 6) Downregulation of GIT1 by siRNA significantly inhibited VEGF-induced MMP activation and ECM degradation ( and ); and 7) GIT1 mediated VEGF-induced cell migration correlated with podosome formation ().
PLCγ has been reported to associate with GIT1-βPIX complex (p21-activated kinase interacting exchange factor), which requires both GIT1 and β-PIX.35
Tyrosine phosphorylation of the βPIX/GIT1 complex is essential for the interaction with PLCγ, the subsequent activation of PLCγ, and the progression to an elongated cell morphology.35
Depletion of βPIX, GIT1 or PLCγ shows that all three components of the complex are necessary to promote cell spreading, and overexpression of individual components is not sufficient to replace deficiencies in the other components of the PLCγ/GIT1/βPIX complex.35
However, constitutively active forms of Cdc42 or Rac1 were able to rescue the elongation of these cells, suggesting that PLCγ, with complexes containing GIT1 and βPIX, is essential for cell spreading and motility by activating Cdc42 and Rac1.35
Data from several labs, including ours, demonstrate important roles for at least three small GTPases in podosome formation (). The apparent redundancy in effects of inhibiting a single GTPase () limits our ability to comment on the specific and hierarchical nature of their roles in podosome formation. We can only suggest that there are likely interdependent pathways for the small GTPases. In contrast, we show that both GIT1 and PLCγ localize in podosomes ( and ) and are required for VEGF-induced podosome formation ( and ). Therefore we propose that PLCγ is a critical component of a PLCγ-GIT1-PIX complex that mediates activation of small GTPases and is required for podosome formation.35
The present study demonstrates that VEGF increases PLCγ activation, which is GIT1 dependent. We previously showed that GIT1 mediated activation of PLCγ by angiotensin II and epidermal growth factor in SMC, which is dependent on GIT1 tyrosine phosphorylation via c-Src.14
Therefore, we propose that VEGF stimulated, Src-dependent, tyrosine phosphorylation of GIT1 creates a scaffold to mediate localization and activation of PLCγ, regulation of podosome formation, and cell migration by stimulating small GTPases.
Several small GTPases have been reported to mediate podosome formation in different cell types.19, 28, 36, 37
Our data show that Rac1, RhoA and CDC42 are involved in EC podosome formation in response to VEGF (), but Rac1 had the greatest effect (). It is possible that the prominent role for Rac1 depends on PLCγ-mediated calcium dependent events, because PLCγ has been reported to regulate cell spreading by increasing intracellular calcium.38
Specifically, calcium-activated proteases, calpains, associate with βPIX, and cleavage of βPIX by calpains is an early event required for Rac activation in some cellular systems.39
Therefore, GIT1 may act as a scaffold to link PLCγ with βPIX, which is small GTPase GEF. The role of PLCγ in the complex could be to increase local calcium concentration which causes calpain activation and subsequent Rac1 stimulation by the GEF function of βPIX.40
PIX has been shown to play a central role in podosome formation, dependent in part, on its GEF activity.41
A PIX-PAK complex was shown to function both upstream and downstream of small GTPases and generate localized feedback loops that could regulate podosome formation.41-43
Our lab previously showed that GIT1 interacted with MEK1 directly, and acted as a scaffold to mediate ERK1/2 activation in focal adhesions.13
Recently, a MEK1 - ERK1/2 - caldesmon signaling cascade was shown to regulate PKC-mediated podosome dynamics in A7r5 cells.44
Based on these data we suggest that GIT1 serves as a scaffold to facilitate the localization and activation of PLCγ, small GTPases, PIX and ERK1/2, thereby promoting podosome formation.
Besides podosomes, filopodia and lamellipodia are also important structures for endothelial cell migration and angiogenesis.45
Recently, bone morphogenetic protein-6 was shown to be a potent stimulator of angiogenesis by regulating filopodial assembly in endothelial tip cells.46
Specifically these authors found that myosin-X translocated into filopodia, stimulated filopodial motility, and increased EC migration and angiogenesis.46
GIT1 may also regulate lamellipodia and filopodia formation by mediating Rac activation due to the Arf-GAP activity of GIT1.47
Our data demonstrate that GIT1 is essential for podosome formation by mediating PLCγ activation. These data suggest that GIT1 may be involved in functions of several macromolecular structures that regulate EC migration and angiogenesis.
In summary, we showed that a c-Src - GIT1 - PLCγ signaling pathway is required for VEGF-mediated podosome formation and cell migration. Because podosomes play an important role in cell invasion and migration, these findings suggest a novel function for GIT1 in EC as a mediator of angiogenesis and tissue remodeling.