Rho-dependent myosin contractility has emerged as a central regulator of AJ size and strength. In established AJ, increased contractility correlates with the weakening of the junctions and with increased endothelial permeability (Dudek and Garcia, 2001; Rolfe et al., 2005
). During sprouting, myosin activation must be tightly controlled because if it happens too fast or is unduly strong or prolonged, the junctions will not be plastic enough to allow sprouting (Abraham et al., 2009
). On the other hand, endogenous tugging forces generated locally by Rho/Rok-dependent myosin activation promote junction growth (Liu et al., 2010
); if these forces are not timely generated or they are too weak, the junction's growth and stabilization will be delayed. This dual role of myosin activation in junction plasticity is reflected by the effect of a Rok-inhibitor on sprouting f/f 3D cultures. Partial inhibition of Rok by low concentrations of the inhibitor, which reportedly increase Rac1 activity (Abraham et al., 2009
), enhances sprouting; higher concentrations, however, block AJ growth (Liu et al., 2010
) and inhibit sprouting (E). Similarly, both accelerating and delaying AJ maturation by activating or inhibiting Rap1 suppresses sprouting (Figure S5
). The latter treatments phenocopy the defects of ECs lacking Raf-1; in 3D sprouting assays, the most obvious consequence of Raf-1 ablation is the formation of cell-cell contacts that cannot be stabilized fast enough to withstand the pull of the tip cell, which breaks off the developing sprout and eventually dies (B; Movies S1 and S2
). Thus, in contrast to other cell types and tissues in which Raf-1's main role is that of an endogenous Rok-α inhibitor, and in which chemical or genetic Rok-α inhibition rescues the Raf-1 knock-out phenotypes (Ehrenreiter et al., 2009; Ehrenreiter et al., 2005; Niault et al., 2009; Piazzolla et al., 2005
), in ECs Raf-1 is a crucial component of the molecular machinery linking VEC to Rok-α signaling and myosin activation.
Raf-1 modulates myosin activation at VEC-containing contacts by bringing Rok-α to these structures. Both kinases are present in VEC immunoprecipitates from f/f cells treated with agents that modulate AJ formation, but much less Rok-α coprecipitates with VEC in Raf-1Δ/Δ
iMECs, resulting in a dramatic reduction of activated myosin selectively at AJ (). The hyperphosphorylation of Rok-α downstream targets observed in Raf-1 knock-out MECs (A), albeit subtle, suggests that Raf-1 might in addition function as an endogenous Rok-α inhibitor, as described in other cell types (Ehrenreiter et al., 2009; Ehrenreiter et al., 2005; Niault et al., 2009; Piazzolla et al., 2005
). On this basis, it is tempting to speculate that Raf-1 might both recruit Rok-α to VEC and dim its activity at the AJ. Such a mechanism would ensure tight regulation of Rok-α signaling at the AJ, preventing excessive actomyosin contractility and the generation of centripetal forces that might ultimately destabilize the AJ. The lack of phenotype in Raf-1Δ/Δ
MEC monolayers and in unperturbed c-raf-1Δ/ΔEC
adult mice compared with the defects in AJ development, sprouting and neovascularization, indicate that the Raf-1-mediated fine tuning of Rok-α is most important for AJ plasticity in the course of remodeling processes, rather than in the AJ maintenance.
How is the complex recruited to AJ? The N-terminal regulatory domain of Raf-1 is sufficient to rescue the sprouting defects of Raf-1Δ/Δ
pMECs, but mutation of either the RBD or the CRD, required for the binding of Raf-1 to Rok-α (Niault et al., 2009
), nullifies this effect (). Thus, binding to Rok-α and to an activated G protein is essential for the function of Raf-1 in EC sprouting. With the exception of K-Ras, which contributes to the formation of Oncostatin M-induced E-cadherin cell-cell contacts in hepatocytes (Matsui et al., 2002
), Ras activation is mostly associated with junction disassembly (Popoff and Geny, 2009
). We show that another Ras-like G protein, Rap1, which is activated at newly formed VEC-containing cell-cell contacts (Sakurai et al., 2006; Wittchen et al., 2005
) and plays an important role in the extension of nascent contacts in ECs () (Kooistra et al., 2005; Noda et al., 2010
) and epithelial cells (Dubé et al., 2008
), but is dispensable for AJ maintenance (Dubé et al., 2008; Hogan et al., 2004
), is the upstream regulator of Raf-1 at AJ. Rap1 activation is necessary and sufficient to recruit Raf-1 and Rok-α to VEC, and Rap1-induced AJ maturation is blunted in Raf-1Δ/Δ
iMECs (). Unlike Ras activation in epithelial cells and in the epidermis (Ehrenreiter et al., 2009; Niault et al., 2009
), Rap1 activation does not promote the association between Raf-1 and Rok-α. Collectively, therefore, the data are consistent with a model in which Rap1 activation recruits the Raf-1/Rok-α complex to VEC to locally regulate MLC2 activity and AJ dynamics during sprouting.
In contrast to Raf-1, endothelial B-Raf is not required for sprouting and does not coimmunoprecipitate with VEC under any of the conditions tested (data not shown). In good correlation with our model, Rap1 binds more efficiently to Raf-1 than to B-Raf (Hu et al., 1997; Okada et al., 1999
) because of its higher affinity to the Raf-1 CRD (Okada et al., 1999
). The Raf-1 CRD is also required for Rok-α binding and inhibition (Niault et al., 2009
), and an exhaustive two-hybrid screen for A-Raf and Raf-1 interactors has identified the CRD as the discriminator between these two proteins (Yuryev and Wennogle, 2003
). Thus, the CRD has a prominent role in conferring specificity to Raf isoforms.
EPAC and Rap1 are also crucial regulators of endothelial permeability (Cullere et al., 2005; Kooistra et al., 2005; Wittchen et al., 2005; Yan et al., 2008
), and the Rap1 effector KRIT1 (CCM1; a member of the cerebral cavernous malformation family), as well as its interacting protein CCM2, are particularly important in this context (Glading et al., 2007; Stockton et al., 2010
). Intriguingly, KRIT1 or CCM2-depleted cells show a dramatic increase in active RhoA and in MLC2 phosphorylation, particularly at the junctions. This correlates with the destabilization of mature junctions and with vascular leakage, which can be alleviated by treatment with a Rok inhibitor (Stockton et al., 2010
). On the contrary, Raf-1Δ/Δ
cells show a selective lack of MLC2 activation at nascent AJ. This correlates with defects in junction formation and sprouting, which are phenocopied by treatment of f/f cells with a Rok inhibitor or with siRNA against Rok-α (). Whether the depletion of KRIT1/CCM2 has any effect on nascent AJ dynamics has not been investigated; similarly, we currently do not know whether Raf-1 ablation has an effect on endothelial permeability. However, the existence of these two pathways, impacting Rho/Rok signaling at the AJ in opposite ways, would allow for the fine regulation of AJ plasticity by EPAC/Rap1.
The control of cadherin-containing AJ by EPAC/Rap1 and myosin is not unique to ECs; epithelial cell junctions are regulated in a similar way (Dubé et al., 2008; Pannekoek et al., 2009; Papusheva and Heisenberg, 2010
). Preliminary results indicate that Raf-1 is recruited to E-cadherin and regulates its distribution to the membrane in the epidermis (R.W. and M.B., unpublished data). We are currently determining whether this function of Raf-1 has an impact on epidermal barrier function.
In keratinocytes, Raf-1 inhibition of Rok-α is required for the development and maintenance of Ras-dependent tumors (Ehrenreiter et al., 2009
). This is related to Rok-α's ability to promote keratinocyte differentiation by inducing EDC expression (McMullan et al., 2003
) and cell cycle exit through the phosphorylation of other downstream targets, such as cofilin (Honma et al., 2006
). Thus, the consequences of the interaction of Rok-α with Raf-1 are cell- and context-dependent, but their net effect is to promote tumorigenesis, either in a cell autonomous manner (inhibition of cell differentiation) or by supporting tumor-driven angiogenesis. Accordingly, approaches targeting the Raf-1/Rok-α complex may be considered attractive in the (co)therapy of cancer.