Podosome rosettes induced by TGF-β in aortic endothelial cells mediate matrix degradation events which could be involved in either basement membrane maintenance of large vessels or in the initiation of an invasive process (29
). Here, we demonstrate a crucial role for Fgd1 in podosome formation and its importance in endothelial cell functions.
Fgd1 is an important actor in skeletal formation because mutations in this gene result in the disease faciogenital dysplasia or Aarskog-Scott syndrome (AAS), an X-linked developmental disorder that adversely affects the formation of multiple skeletal structures (27
). Consequently, most studies have thus far focused on osteoblasts and osteoblast-like cell lines. Only recently, Fgd1 was shown to be expressed in certain tumor-derived cell lines and human tumors (3
). By demonstrating Fgd1 expression in aortic endothelial cells, we establish here that Fgd1 is more widely expressed than initially thought in normal tissues. Furthermore, we show that Fgd1 might play an important role in vascular physiology and responses to inflammation. In support of this view, a human gene array technology study evaluating differences in gene expression in abdominal aortic aneurysm and arterial occlusive disease versus control aortic tissue revealed downregulation of Fgd1 gene expression in both diseases (1
Fgd1 is the founding member of a family of highly related Fgd1-like genes. Fgd1 family members are modular proteins that contain in sequence an N-terminal domain, a Dbl homology (DH) domain, an adjacent pleckstrin homology (PH) domain, a FYVE-finger domain, and a second C-terminal PH domain (PH2). Each member contains a different N-terminal domain, suggesting interactions with distinct sets of regulatory molecules. The Fgd1 N-terminal domain contains a proline-rich region (PRD) involved in cortactin binding (16
). In cultured primary mouse osteoblasts, Fgd1 was found in the cytosol, as well as in the Golgi and plasma membranes (10
). In contrast, we found that in unstimulated BAE cells, the amount of Fgd1 at the subcortical actin cytoskeleton was low but increased upon TGF-β stimulation. We therefore anticipated that the activity of Fgd1 was turned on at an appropriate intracellular compartment through the interaction with upstream adaptor modules activated in a temporally and spatially regulated manner.
Our study shows that one such adaptor could be cortactin. Fgd1 is expected to regulate podosome formation through its exchange activity toward Cdc42, as supported by the consequences of Fgd1 knock down on TGF-β-induced Cdc42 activation. Indeed, the results obtained with the constitutively active Fgd1-RKB3 mutant show that this mutant behaves like V12Cdc42, suggesting that the large deletion in the PRD results in a loss of at least some of Fgd1 specificities. Constitutively active Fgd1-2DBDEL carrying a smaller deletion of the PRD but still lacking the cortactin binding domain retained some features of Fgd1, a fraction of the mutated protein localized at the cell cortex, possibly through a contribution of the PRD. However, this mutant failed to induce podosomes, indicating that, an as-yet-undefined membrane targeting function and/or the cortactin binding domain are critically involved in podosome formation. Moreover, unlike, FL-Fgd1, a mutant deleted of the sole cortactin binding region (Fgd1Δ156-165) was unable to support TGF-β-induced podosome formation. It therefore seems that Fgd1Δ156-165 cannot be recruited in TGF-β signaling pathways and competes with the endogenous molecule, thus preventing podosome assembly. Finally, TGF-β promoted Fgd1-cortactin association. Collectively, our results show that Fgd1 cortactin binding domain is essential for TGF-β-induced podosome formation.
The molecular association of Fgd1 and cortactin may be one aspect of the mechanism regulating the initial clustering of cortactin which act as nucleation platforms onto which the actin polymerization machinery assemble. Cortactin is composed of several protein interaction domains, an N-terminal acidic (NTA) region contains a conserved DDW region responsible for interaction with Arp3 and subsequent activation of the Arp2/3 complex, an F-actin binding region, a helical region, and a region rich in Pro, Ser, Thr, and Tyr that harbor sites of phosphorylation. Cortactin is therefore involved in subcellular regions engaged in de novo
actin assembly, as typified by podosomes. Phosphorylation of cortactin by Src does not explain cortactin translocation at podosomes (39
). Previous studies in vascular smooth muscle cells have shown that the SH3 domain of cortactin is required for cortactin clustering at the onset of podosome formation, while the actin binding repeat region is required for the translocation of cortactin to the actin-rich core of mature podosomes (39
). These results suggest that the SH3 domain and the actin-binding repeat region are involved, respectively, in the early and late stages of podosome formation process (34
). Fgd1 also binds Mabp1 (mammalian actin-binding protein 1) (16
), a protein that structurally and functionally resembles cortactin (17
) and which could therefore also contributes to rendering Fgd1 fully active. Although Mabp1 contains a helical, proline-rich, and SH3 domain at its carboxyl terminus, with sequence similarity with the cognate domains in cortactin, it does not contain an NTA domain or cortactin repeats. In our model, overexpression of FL-Fgd1 did not promote filopodia, a finding that is consistent with the fact that V12Cdc42 does not induce these structures in endothelial cells (21
), nor did it promote podosome formation. It is likely that overexpression of Fgd1 leads to formation of unproductive Fgd1-cortactin (and Mabp1) complexes in the absence of concomitant Cdc42 activation. Our results provide the basis of a molecular mechanism involving Fgd1 and cortactin at the onset of podosome assembly. It is worth pointing out that Fgd1 has been shown is important for export from the Golgi bodies in some cells (9
). Therefore, we cannot exclude that Fgd1 depletion also affects protein export from the Golgi bodies to the cell surface. However, the fact that the neoexpression of a plasmid encoding constitutively active Fgd1 mutant drives podosome formation supports our conclusions.
The induction of endothelial podosomes by TGF-β requires protein synthesis with a peak at 24 h after stimulation (32
). At this stage, 20% of podosome rosettes stained for Fgd1, whereas cortactin was always present. This situation is similar to that found for invadopodia (3
). The reasons for this are unclear, but it seems plausible that this observation reflects a dynamic turnover of Fgd1. One may envision that Fgd1 is recruited locally to initiate/drive actin polymerization and thus trigger podosome formation. Fgd1 then dissociates from this prepodosome and is recycled, thus becoming available for a new round of podosome rosette formation.
There is no published indication that Fgd1 may be tyrosine phosphorylated upon activation. Fgd1 protein domain organization, however, is similar to other GEFs already reported to be tyrosine kinase substrates. Also, putative phosphorylation sites in Fgd1 amino acid sequence point at Fgd1 as a plausible substrate for Src family kinases (data not shown). Finally, previous data obtained in this model show Src regulation upon exposure to TGF-β (32
). Our data show that Fgd1 becomes tyrosine phosphorylated shortly after TGF-β addition, and this phosphorylation was found sensitive to Src family kinase inhibitors.
Considering these findings, we suggest a model whereby a signaling cascade triggered by TGF-β activates Fgd1. Activation is mediated by Src family kinases and promotes its association to binding partners, possibly cortactin (and/or Mabp1), to efficiently stimulate Cdc42 at the appropriate time and place. As far as cortactin is concerned, the mechanism may involve cortactin dimerization (18
). Cdc42 in turn recruits the actin polymerization machinery, including actin related protein 2/3 complex (Arp2/3) and neuronal Wiskott-Aldrich syndrome protein (N-WASp), leading to actin polymerization at the core of the podosome and subsequent recruitment of adhesion proteins.
Our previous studies established that Smad signaling is required for TGF-β-induced podosome assembly. The present study brings evidence for the contribution of a parallel signaling pathway leading to Cdc42 activation, in the process of podosome formation. The description of this novel noncanonical pathway may also shed light on mechanisms such as ossification and skeletal development, where Fgd1 signaling plays a critical role. Because TGF-β is an important regulator of bone biogenesis, it is tempting to speculate that the defects observed in faciogenital dysplasia may result, at least in part, from altered TGF-β signaling. Likewise, our findings predict a perturbation in the vascular system in AAS. In this respect, arterial dysplasia, malformation, and aneurysms have recently been reported in this disease (7
). It is thus likely that future studies will further elucidate the involvement of Fgd1 in vascular diseases.
The present findings contribute to our understanding of the basic mechanisms of podosome formation and function, by shedding light on the mechanism by which Fgd1 in combination with cortactin mediates the clustering of podosome components and initiate actin polymerization which gives rise to podosomal structures. They not only clarify a novel mechanism for podosome formation but also provide clues to understanding the role of podosomes in TGF-β-enriched tissue environments. For the first time, Fgd1 emerges as an important regulator in the vascular system. These studies also establish that Fgd1 is a regulatory element of signaling cascades triggered by TGF-β and thereby provide the basis of a novel noncanonical pathway. Such pathway is involved in the process of podosome formation but could also contribute to other TGF-β-mediated processes in cell physiology and pathology.