We have found previously that the integrin αvβ8 inhibits epithelial cell growth (Cambier et al., 2000
). Furthermore, αvβ8 is expressed in either quiescent cells or cells with a low rate of turnover (Nishimura et al., 1998
; Cambier et al., 2000
) and is lost in the process of neoplastic transformation (Cambier et al., 2000
). These findings led us to hypothesize that β8 plays a role in the homeostatic control of normal tissues.
In support of this hypothesis, we now demonstrate that the integrin αvβ8 mediates growth inhibition through a novel mechanism of activation of TGF-β1, a cytokine with a central role in homeostatic cellular processes (Blobe et al., 2000
Two molecular mechanisms have been proposed that may lead to the activation of TGF-β1: conformational change leading to activation of the SLC complex (Crawford et al., 1998
; Munger et al., 1999
) or proteolysis of LAP-β1 leading to the release of active TGF-β1 (Munger et al., 1997
; Yu and Stamenkovic, 2000
). Our data demonstrate that a mechanism of conformational change leading to activation of TGF-β, as proposed for the αvβ6 integrin (Munger et al., 1999
) or TSP-1 (Crawford et al., 1998
), is not responsible for αvβ8-mediated activation of TGF-β1. Specifically, αvβ8-mediated activation of SLC does not require the β8 cytoplasmic domain in contrast to the mechanism of αvβ6-mediated activation of TGF-β, which requires the β6-cytoplasmic domain (Munger et al., 1999
). Furthermore, αvβ8 is unlikely to bind directly or indirectly to LAP-β1 through a TSP-1–dependent mechanism because αvβ8 lacks the defined TSP-1 binding site for LAP-β1 (Crawford et al., 1998
) and αvβ8 does not bind to TSP-1 (unpublished data). Moreover, unlike secreted TSP-1 (Crawford et al., 1998
) secreted αvβ8 cannot activate TGF-β1. Thus, the mechanism by which αvβ8 activates TGF-β1 is not dependent on conformational changes, resulting from “inside-out” signal transduction as mediated by the β6 cytoplasmic domain (Munger et al., 1999
) or direct physical interaction as mediated by TSP-1(Crawford et al., 1998
Our findings support a biologically relevant mechanism whereby SLC binds with high affinity to αvβ8 on the cell surface, which results in the metalloprotease-dependent release of active TGF-β. Evidence to support this mechanism follows: (a) secreted αvβ8 binds to LAP-β1 with a high affinity with a dissociation constant similar to other TGF-β receptors (Tucker et al., 1984
); (b) both synthetic and endogenous MMP inhibitors block αvβ8-mediated activation of TGF-β1; (c) reconstitution of MT1-MMP into the H1264 MT1-MMP–deficient cell line rescues αvβ8-mediated TGF-β activation; (d) αvβ8 and MT1-MMP specifically colocalize in LAP-β1 substrate contacts; (e) consistent with a proteolytic event, active TGF-β is liberated by an αvβ8-dependent mechanism into the supernatants of tumor cell lines and into the aqueous phase of lung cancer xenografts; (f) the proteolytic substrate of αvβ8-, MT1-MMP–dependent activation of TGF-β1 is likely to be LAP-β1, since β8-overexpressing, MT1-MMP–expressing H1264 cells cleave and inactivate LAP-β1, whereas β8-overexpressing, MT1-MMP–deficient H1264 cells do not; (g) cleavage of LAP-β1 requires the concomitant activity of both β8 and MT1-MMP, since β8-specific RGD inhibitors and metalloprotease inhibitors both block cleavage. Precedent for such a proteolytic mechanism is that plasmin (Lyons et al., 1990
) and MMP-9 (Yu and Stamenkovic, 2000
) have each been shown to activate TGF-β1 and TGF-β2, respectively, by cleavage of LAP.
It is also possible that MT1-MMP acts indirectly by proteolytically modifying the activity of αvβ8 as suggested recently for the MT1-MMP–dependent modification of the integrin αvβ3 (Deryugina et al., 2000
). However, this is unlikely because of the following: (a) cell lines expressing αvβ8 attach to LAP-β1 equally well whether or not they express MT1-MMP (unpublished data), suggesting that coexpression of MT1-MMP does not modify the activity of αvβ8; (b) flow cytometry of H1264 cells overexpressing both β8 and MT1-MMP using two different anti-β8 monoclonal antibodies shows no alteration in surface expression of αvβ8, indicating that antibody epitopes are preserved along with adhesive capability; (c) immunoprecipitations or Western blots of cells coexpressing αvβ8 and MT1-MMP, using polyclonal antibodies against the cytoplasmic domain of β8, show no electrophoretic shift or proteolytic degradation products. Therefore, we have no evidence of modification of αvβ8 by MT1-MMP.
How does MT1-MMP interact with the αvβ8–TGF-β1 complex? Our data suggest that upon ligation of αvβ8 with SLC, αvβ8 and MT1-MMP become closely associated to form a complex on the cell surface. The cell surface appears to be required for productive interactions, since the secreted forms of αvβ8 and MT1-MMP do not mediate activation of TGF-β. Evidence for a physical association on the cell surface is that αvβ8 and MT1-MMP colocalize in substrate contacts specifically on LAP-β1. The nature of the MT1-MMP–β8 interaction awaits elucidation by coimmunoprecipitation and domain interaction studies. Because the localization of MT1-MMP in LAP-β1 substrate contacts is dependent on the presence of β8, it is likely that αvβ8–SLC interactions are required to initiate the recruitment of MT1-MMP. The dynamic recruitment of MT1-MMP to αvβ8–TGF-β complexes could provide a basis for the homeostatic regulation of TGF-β activity in cellular microenvironments.
Although reconstitution of wild-type MT1-MMP is sufficient to support αvβ8-mediated activation, other metalloproteases could potentially be involved. For instance, MT1-MMP binds to and is potently inhibited by TIMP-2 (Brew et al., 2000
), but MT1-MMP–TIMP-2 complexes also serve as a cell surface receptor for MMP-2, and the function of this complex is activation of MMP-2 (Strongin et al., 1995
). As such, it is not inconceivable that MMP-2 could also be involved in αvβ8-mediated activation of TGF-β. However, in H1264s cells MMP-2 is unlikely to be involved, since TIMP-1, a potent inhibitor of MMP-2 and weak inhibitor of MT1-MMP (Brew et al., 2000
), has no effect on αvβ8-mediated activation of TGF-β. In contrast, β8-mediated TGF-β activation is inhibited by TIMP-2, suggesting that MT1-MMP may alone be sufficient to support β8-mediated activation of TGF-β. Although formally we cannot exclude additional roles for other MMPs or related metalloproteases such as ADAMs or ADAMTS, family members in αvβ8 mediated activation of TGF-β in other systems or cell types.
The β8 subunit appears to be the only integrin subunit capable of coordinating metalloprotease activity with SLC bound to the cell surface because the other LAP-β1 binding integrins are either incapable of activating TGF-β (Munger et al., 1998
) or, in the case of αvβ6, activating TGF-β via a metalloprotease-independent pathway (Munger et al., 1999
). Furthermore, αvβ8-mediated TGF-β activation is solely dependent on metalloproteases and not other proteases because inhibitors of aspartyl, serine, and cysteine proteases do not inhibit activation. Thus, αvβ8-mediated activation of TGF-β1 is not dependent on other proteases that have been implicated in SLC activation, including plasmin (Lyons et al., 1990
), calpain (Abe et al., 1998
), and cathepsin (Lyons et al., 1988
Integrins (Brooks et al., 1996
) and other cell surface molecules (Yu and Stamenkovic, 1999
) have also been shown to localize MMP activity to the cell surface. For instance, the integrin αvβ3 has been shown to form an SDS stable cell surface complex with MMP-2 (Brooks et al., 1996
) and to colocalize with MT1-MMP (Deryugina et al., 2001
), whereas CD44 has been shown to mediate localization of MMP-9 (Yu and Stamenkovic, 2000
) to the cell surface. However, αvβ3 and CD44 are unlikely to be required for αvβ8-mediated activation of TGF-β because αvβ3 is not expressed in multiple cell lines that support αvβ8-mediated activation of TGF-β () and because anti-CD44 antibodies do not inhibit αvβ8-mediated activation of TGF-β (unpublished data).
The selective MMP dependence of αvβ8- but not αvβ6-mediated activation of TGF-β1 clearly demonstrates that the mechanisms of αvβ8- and αvβ6-mediated activation of TGF-β1 are different. A structural basis for these different mechanisms may be the striking difference in the predicted secondary structure of the extracellular domains of the β8 and β6 subunits (Moyle et al., 1991
). Different integrin-mediated mechanisms of TGF-β activation may have evolved to support distinct biologic functions. For instance, in the airway epithelium, a site where β8 is normally expressed (Cambier et al., 2000
), a mechanism to support a low and persistent level of activation of TGF-β1 is necessary for homeostasis (Crawford et al., 1998
). We speculate that αvβ8 could sequester SLC to the cell surface where, in response to an environmental cue, changes in the local balance of MMP/TIMP activity could lead to αvβ8-dependent liberation of active TGF-β1. Thus, αvβ8-mediated activation of TGF-β1 might liberate the low levels of active TGF-β1 sufficient to promote local paracrine effects but insufficient for undesirable local and systemic fibrogenic effects of TGF-β1 (Border and Noble, 1994
). Conversely, if αvβ6 were to liberate TGF-β by an MMP-dependent mechanism undesirable pathologic levels of TGF-β might be released locally and into the systemic circulation because after injury expression of αvβ6 (Breuss et al., 1993
; Pilewski et al., 1997
) and MMPs (Holgate et al., 1999
) are both strongly and rapidly induced.
In summary, abundant evidence implicates the cytokine TGF-β1, integrins, and MMPs as important mediators of homeostatic cell behaviors. This article provides the first evidence of the coordination of activity of members of these three major multigene families in the maintenance of homeostasis.