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First published online 31 January 2006
doi: 10.1242/jcs.02787

Journal of Cell Science 119, 769-781 (2006)
Published by The Company of Biologists 2006
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Research Article

A signalling cascade involving PKC, Src and Cdc42 regulates podosome assembly in cultured endothelial cells in response to phorbol ester

Florence Tatin1,2, Christine Varon1,2, Elisabeth Génot1,2 and Violaine Moreau1,2,*

1 Institut Européen de Chimie-Biologie, 2 rue Robert Escarpit, 33600 Pessac, France
2 INSERM Unité 441 / Université Victor Segalen Bordeaux 2, Avenue du Haut-Lévêque, 33600 Pessac, France

* Author for correspondence (e-mail: v.moreau{at}iecb.u-bordeaux.fr)

Accepted 9 November 2005

The involvement of Src, Cdc42, RhoA and PKC in the regulation of podosome assembly has been identified in various cell models. In endothelial cells, the ectopic expression of constitutively active mutants of Src or Cdc42, but not RhoA, induced the formation of podosomes. Short-term exposure to phorbol-12-myristate-13-acetate (PMA) induced the appearance of podosomes and rosettes after initial disruption of stress fibres. Molecular analysis of PMA-induced podosomes and rosettes revealed that their composition was identical to that of podosomes described in other models. Pharmacological inhibition and siRNA knock-down experiments revealed that both PKC{alpha} and PKC{delta} isotypes were necessary for podosome assembly. However, only constitutively active PKC{alpha} could mimic PMA in podosome formation. Src, Cdc42 and RhoA were required downstream of PKCs in this process. Src could be positioned between PKC and Cdc42 in a linear cascade leading to podosome assembly. Using in vitro matrix degradation assays, we demonstrated that PMA-induced podosomes are endowed with proteolytic activities involving MT1-MMP-mediated activation of MMP2. Endothelial podosomes may be involved in subendothelial matrix degradation during endothelium remodelling in pathophysiological processes.

Key words: Podosomes, PKC, Src, RhoGTPases, ECM, Endothelial cells


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