Zona occludens proteins modulate podosome formation and function.

Podosomes are highly dynamic structures that are involved in cell adhesion and extracellular matrix remodeling. They present as intracellular columns composed of an actin-rich core region and a surrounding ring-like structure containing focal adhesion proteins, actin binders as well as cell signaling molecules. A key player in podosome biogenesis is the scaffolding protein cortactin, which is thought to control actin assembly at the core region. We show that the zona occludens protein 1 (ZO-1), a pivotal tight junction protein and known binding partner of cortactin, is a component of podosomes. In the smooth muscle cell line A7r5, phorbol ester treatment induced a rapid relocation of ZO-1 from the cell cortex and cytosolic pools toward newly formed podosomes. Podosomal localization was also observed for the known ZO-1-binding proteins l-afadin, α-catenin, and phospho-connexin 43. Truncation studies revealed that the actin-binding domain but not the association with cortactin is necessary for ZO-1 recruitment to podosomes. Moreover, impaired ZO-1 expression leads to significantly reduced podosome formation and concomitant decreased matrix degradation at podosomes. Our findings demonstrate that besides their known function in tight junction assembly and intercellular communication, zona occludens proteins and their binding partners may play a novel role in podosome formation and associated function, thus regulating cell adhesion and matrix remodeling.

TGFbeta-induced endothelial podosomes mediate basement membrane collagen degradation in arterial vessels.

Podosomes are specialized plasma-membrane actin-based microdomains that combine adhesive and proteolytic activities to spatially restrict sites of matrix degradation in in vitro assays, but the physiological relevance of these observations remain unknown. Inducible rings of podosomes (podosome rosettes) form in cultured aortic cells exposed to the inflammatory cytokine TGFbeta. In an attempt to prove the existence of podosomes in living tissues, we developed an ex vivo endothelium observation model. This system enabled us to visualize podosome rosettes in the endothelium of native arterial vessel exposed to biologically active TGFbeta. Podosomes induced in the vessel appear similar to those formed in cultured cells in terms of molecular composition, but in contrast to the latter, arrange in a protruding structure that is similar to invadopodia. Local degradation of the basement membrane scaffold protein collagen-IV, is observed underneath the structures. Our results reveal for the first time the presence of podosome rosettes in the native endothelium and provide evidence for their capacity to degrade the basement membrane, opening up new avenues to study their role in vascular pathophysiology. We propose that podosome rosettes are involved in arterial vessel remodeling.

TGFbeta1 regulates endothelial cell spreading and hypertrophy through a Rac-p38-mediated pathway.

BACKGROUND INFORMATION: TGFbeta (transforming growth factor beta) is a multifunctional cytokine and a potent regulator of cell growth, migration and differentiation in many cell types. In the vascular system, TGFbeta plays crucial roles in vascular remodelling, but the signalling pathways involved remain poorly characterized. RESULTS: Using the model of porcine aortic endothelial cells, we demonstrated that TGFbeta stimulates cellular spreading when cells are on collagen I. TGFbeta-stimulated Rac1-GTP accumulation, which was associated with increased MAPK (mitogen-activated protein kinase) p38 phosphorylation. Furthermore, ectopic expression of a dominant-negative Rac mutant, or treatment of the cells with the p38 pharmacological inhibitor SB203580, abrogated TGFbeta-induced cell spreading. Our results demonstrate for the first time that prolonged exposure to TGFbeta stimulates endothelial cell hypertrophy and flattening. Collectively, these data indicate that TGFbeta-induced cell spreading and increase in cell surface areas occurs via a Rac-p38-dependent pathway. CONCLUSIONS: The Rac-p38 pathway may have conceptual implications in pathophysiological endothelial cell responses to TGFbeta, such as wound healing or development of atherosclerotic lesions.

Regulatory signals for endothelial podosome formation.

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.

ALK5 and ALK1 play antagonistic roles in transforming growth factor ß-induced podosome formation in aortic endothelial cells.

Transforming growth factor ß (TGF-ß) and related cytokines play a central role in the vascular system. In vitro, TGF-ß induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-ß-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-ß receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-ß drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-ß-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-ß-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-ß-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.