Our observations are of interest for several reasons. First, the data indicate that one origin of the capacity for integrins to mediate both matrix engagement and cross-talk with other adhesion pathways arises from the integrin β-propeller itself, implying that these signaling pathways can diverge at the cell surface. Most current thinking about integrin cross-talk focuses on “spillover” between intracellular signaling cascades (Schwartz and Ginsberg, 2002
). Second, the experiments elucidate a previously unrecognized, Src-dependent signaling pathway linking integrins, at least α3β1, with expression of the SLUG family of transcription factors, key elements in epithelial–mesenchymal development. And finally, the data reveal a mechanistic connection between α3β1, uPAR, and E-cadherin that may be pertinent to the observation that uPAR and α3β1 levels are commonly enhanced and E-cadherin function suppressed in invasive human carcinomas. These observations were empowered by the reported crystal structure of αvβ3, the crystal revealing the presence of a β-propeller structure at the NH2
-terminal region of the α chain and its relationship to the I-like domain of the integrin β chain (). This allowed selection of point mutants of known location in the assembled integrin to probe for distinct functions in different regions of the α chain propeller. Our data indicate that this is the case. Matrix engagement by α3β1 leads to the expected transient wave of FAK activation, likely reflecting transient Src activation, and supports formation of stable E-cadherin connections, whereas engagement of uPAR by α3β1 leads to a low but sustained level of Src activation with eventual loss of cell–cell borders and frank mesenchymal transition as reflected by up-regulation of SLUG. Single amino acid mutations in α3β1 that abrogate effective laminin-5 or uPAR engagement, or both, block Src activation and its downstream consequences.
A possible caveat to these findings is that while uPAR expression in kidney epithelial cells regulates E-cadherin function in an integrin-specific manner, the levels of uPAR expression in this experimental system are high and could be judged as nonphysiological. There is only a very low level of endogenous mouse uPAR, as judged by immunoblotting, in R10 cells (unpublished data). However, TGFβ1 and other growth factors implicated in tumor progression induce expression of uPAR in these cells. We have recently also observed that TGFβ1, known to promote dissolution of cell–cell contacts and mesenchymal transition of kidney epithelial cells, promotes dissociation of R10 cells in culture and does so in an α3β1-dependent manner. Cells bearing the H245A mutation do not lose stable E-cadherin junctions in response to TGFβ1 (unpublished data). Whether the TGFβ1 response is critically dependent on uPAR interacting with α3β1 is under study. In any case, these findings indicate that the mechanism linking α3β1 and E-cadherin demonstrated here may also apply to epithelial cells responding to physiological stimulation accompanied by enhanced uPAR expression and altered integrin function.
An important unanswered question is why Src activation appears sustained upon uPAR engagement, whereas Src activation is transient after matrix attachment. Classical ligand-induced integrin signaling is thought to lead to Src activation by altered conformation of the integrin cytoplasmic tails and the clustering of integrins. The latter leads to the concentration and cross-activation of signaling molecules, including Src kinases, at the site of attachment (Schwartz and Ginsberg, 2002
). Tyrosine phosphatases, such as SHP-1, rapidly accumulate at attachment sites and limit or reverse protein phosphorylation (Rock et al., 1997
). Engagement of uPAR by α3β1 could be expected to be different in at least two respects. There is little evidence to indicate clustering of integrins by uPAR, at least when cells are plated on laminin-5 or fibronectin. This alone may alter the accumulation of kinases and phosphatases at the site of integrin engagement. Second, as a cis-acting integrin ligand, this glycosylphosphorylinositol (GPI)-anchored membrane protein could be expected to bring its own set of associated membrane proteins to the site of integrin interaction. Because uPAR associates with lipid rafts, containing an array of signaling molecules (Sargiacomo et al., 1993
), we presume that the juxtaposition of rafts and integrins leads to the distinct signaling patterns and observed downstream consequences. This speculation is supported by the pattern of enhanced tyrosine-phosphorylated proteins observed in R10/U cells. Whereas classical downstream targets of integrin signals, such as p130CAS and FAK, did not show sustained phosphorylation, the raft-localized protein caveolin-1 did ( C). Tyrosine-phosphorylated caveolin-1 has been reported to bind the SH2 domain of the adaptor protein Grb7, implying that this caveolin-1 phosphorylation itself could be a mediator of integrin cross-talk (Lee et al., 2000
). The fact that DnSrc–GFP, which alters association of Src with its binding partners, causes reversion to a clustered phenotype ( C) further highlights the importance of Src localization. Exactly how raft components might regulate Src kinase activity in an integrin-specific manner, and vice versa, remains an important goal of future studies.
The observation that uPAR expression in epithelial cells can regulate E-cadherin expression and function has not been previously recognized. However, the capacity of uPAR to influence E-cadherin function specifically through α3β1 is not a complete surprise. This integrin localizes closely with adherens junctions as well as with focal adhesion sites (Wang et al., 1999
). Indeed plating epithelial cells on laminin-5 reportedly promotes E-cadherin–dependent cell–cell adhesion (Dogic et al., 1998
), consistent with our observations ( B). The expression of uPAR on the other hand has been linked to enhanced migratory capacity of epithelial cells both in vitro and in vivo (Ossowski and Aguirre-Ghiso, 2000
). Migration of epithelial cells depends on both the disruption of cell–cell contacts and reversible matrix engagement. Our observations shed further light on how this occurs. Sustained expression of uPAR led to loss of cell–cell clustering and markedly increased, integrin-dependent motility ( C; Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200304065/DC1
). This was associated with decreases in E-cadherin and γ-catenin, the latter being a key component of both adherens junctions and desmosomes (Kofron et al., 2002
), and was dependent on sustained Src-kinase activation. What is the connection between activated Src and altered expression of E-cadherin and γ-catenin? Overexpression of active Src kinases in either colon carcinoma cells or keratinocytes has been reported to phosphorylate components of the adherens junction complex, leading to its dissolution and a redistribution of E-cadherin similar to our observations ( A) (Owens et al., 2000
; Avizienyte et al., 2002
). However, in contrast to these prior transfection approaches, which lead to high levels of active Src, the level of sustained Src activation as a consequence of α3β1–uPAR engagement appears much lower with no evident change in the phosphorylation states of many of the known Src substrates ( C). Instead, our data point to a different mechanism for loss of cell–cell borders, up-regulation of SLUG. Expression of SLUG strictly correlated with the phenotype of the kidney epithelial cells with elevated SLUG in dispersed R10/U cells and falling SLUG levels after inhibition of either Src kinase or proper Src localization (DnSrc–GFP) in parallel with reversion to an epithelial phenotype ( A and C). In addition, uPAR had no effect on SLUG levels in H245A mutant cells, which also failed to scatter with uPAR expression or alter cadherin or catenin levels. Although we cannot be sure that the observed changes in SLUG levels alone cause reversion to epithelial levels of E-cadherin and γ-catenin and an epithelial phenotype, these observations are consonant with prior observations in several other cell systems that SLUG regulates cadherin and catenin transcription and cell–cell contact (Vallin et al., 2001
; Bolos et al., 2003
). To our knowledge, these are the first data directly linking integrin function with SLUG levels and further suggest that a specific integrin, α3β1, and how it is engaged by ligands, is an important determinant of SLUG expression. How low levels of active Src ultimately lead to SLUG transcription remains to be defined.
High levels of SLUG repress E-cadherin promoter function, and levels of SLUG correlate inversely with E-cadherin levels in various breast cancer cell lines (Hajra et al., 2002
). Although we cannot predict to what extent in human tumors uPAR expression promotes SLUG expression with its consequent negative effects on cadherin function, many carcinomas exhibit both up-regulated uPAR and SLUG and suppressed E-cadherin protein levels. Up-regulation of uPAR and down-regulation of E-cadherin are both considered risk factors for tumor progression and poor prognosis. Our data raise the possibility that one explanation for why both observations are commonly found in more invasive tumors is that they are mechanistically linked.