Actin Can Reorganize into Podosomes in Aortic Endothelial Cells, a Process Controlled by Cdc42 and RhoA

Abstract

Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

Sodium fluoride induces podosome formation in endothelial cells

“…Also in contrast with other models (Berdeaux et al, 2004;Tatin et al, 2006), RhoA is not required for podosome formation upon NaF treatment and Rho protein inhibition favours podosome formation in endothelial cells. These results are consistent with our previous study describing the role of RhoA in V12Cdc42-driven endothelial podosome assembly (Moreau et al, 2003). These results confirm that Cdc42 plays a master role in the general process of podosome formation and that podosome assembly and possibly podosome arrangement are controlled by the integration of several GTPase activities.…”

“…PAE cells, clone p23, and PAE cell lines expressing V12Cdc42 under the control of an IPTG (isopropyl β-D-thiogalactoside)-inducible promoter were described previously (Moreau et al, 2003). HAECs and HUAECs were purchased from Promocell and cultured in the endothelial cell basal medium supplemented with the 'supplement pack' recommended by the manufacturer (EGM-MV).…”

“…Anti-RhoA antibodies were from Santa Cruz and anti-p85α antibody was from Serotec (U5 clone). Constructs encoding GST-RBD-rhotekin, GST-CRIB (Cdc42/Rac-interacting binding)-PAK, active mutants of RhoA or Cdc42, GFP (green fluorescent protein)-N-WASP and GFP-WIP were described previously (Moreau et al, 2003). Rho inhibitor C3 transferase was purchased from Cytoskeleton (Tebu).…”

“…Cells plated onto glass coverslips were prepared for immunofluorescence microscopy as previously described (Moreau et al, 2003). Fluorescent images were recorded on an Eclipse Nikon microscope using a 63× oil immersion lens.…”

“…Podosomes form spontaneously in monocyte-derived cells such as macrophages, osteoclasts and immature dentritic cells (Linder, 2009). Previously, we discovered that endothelial cells are also capable of assembling podosomes but only in response to specific inducers such as a constitutively active form of Cdc42 (Moreau et al, 2003), an oncogenic form of Src , phorbol esters or transforming growth factor-β .…”