The results of the present study show that confluent airway epithelial cells, despite secreting two TGF-β isoforms and expressing two members of the integrin family that can activate TGF-β, do not activate either isoform under resting conditions. In response to mechanical wounding, however, there is rapid induction of integrin-dependent activation of TGF-β, which slows the degree of wound closure. TGF-β activation following wounding appears to be mediated by both the αvβ6 and αvβ8 integrins, but αvβ8-mediated activation is principally responsible for the reduction in the degree of wound closure, since blocking antibodies to αvβ8 substantially accelerated wound closure, whereas antibodies to αvβ6 had no effect on their own. This difference is explained in part by a TGF-β–independent contribution of αvβ6 to the degree of wound closure, as demonstrated by inhibition of the degree of closure by antibodies to αvβ6 after TGF-β activity was inhibited by antibodies to either TGF-β or αvβ8. Airway epithelial cells make substantial amounts of TGF-β2 and smaller amounts of TGF-β1, but the concentrations of each TGF-β isoform are not affected by wounding, at least during the time course of our experiments. The αvβ8-mediated decrease in the degree of wound closure is mediated by activation of TGF-β1, but TGF-β2 does not appear to contribute, consistent with the identification of the arginine-glycine–aspartic acid site in TGF-β1 LAP as the site of binding of this integrin. The RGD site is present in TGF-β1 and -β3 LAP, but absent from TGF-β2 LAP (25
Numerous previous reports have examined effects of TGF-β on individual migration of a variety of cell types. Based on these studies, it is clear that TGF-β can exert dramatically different effects on different cell types and under different circumstances. TGF-β isoforms have been shown to be chemotactic for macrophages, mast cells, and fibroblasts, enhancing the migration of each cell type toward gradients of diffusible TGF-β (26
). TGF-β has also been reported to enhance the migration of epithelial tumor cells and immortalized epithelial cells (18
). Finally, TGF-β is a potent inducer of epithelial-to-mesenchymal transformation (EMT), inducing primary epithelial cells to take on mesenchymal cell characteristics, including enhanced detachment from epithelial sheets and enhanced speed of migration (31
). However, EMT occurs over a time course of several days, much longer than the time course of primary epithelial wound closure (33
Primary epithelial wounds, like the scratch wounds studied here, close by a process of continuous sheet migration, which appears to be distinct from migration of isolated individual cells. The one previous study that looked at the effects of exogenous TGF-β on the rate of closure of airway epithelial wounds found significant slowing of the rate of closure, just as we did in the current study (21
). Furthermore, this effect is consistent with results reported for the rate of cutaneous wound closure in vivo
in mice with impaired responsiveness to TGF-β as a consequence of a null mutation of the TGF-β signaling molecule, SMAD3. These mice have dramatically accelerated wound closure, as expected if endogenously activated TGF-β normally slows the rate of wound closure (20
Many of the previous studies that examined the potential roles of TGF-β on migration of epithelial cells focused primarily on exogenously administered TGF-β. In the current study, we demonstrated that TGF-β is activated by airway epithelial cells themselves in response to mechanical wounding, and that locally activated TGF-β significantly slows the degree of wound closure. Based on studies with blocking antibodies, it is clear that TGF-β1 is endogenously activated and contributes to this response. In response to mechanical wounding, TGF-β1 is activated by the actions of at least two members of the integrin family, αvβ6 and αvβ8. Airway epithelial cells express one other integrin, αvβ5, that has also recently been reported to be capable of activating TGF-β on the surface of fibroblasts from patients with sceleroderma (14
). Blockade of αvβ5 had no effect on either TGF-β activity or on the degree of wound closure in our assay. These results are consistent with our own inability to demonstrate αvβ5-mediated TGF-β activation by any primary cells or cell lines we have examined (data not shown). We are uncertain why our results in this regard are different from those reported by Asano and coworkers (14
), but perhaps this function of αvβ5 uniquely occurs on fibroblasts from patients with scleroderma. An unanswered question from our experiments is how wounding stimulates αvβ6- and αvβ8-mediated TGF-β activation. This effect does not appear to be due to either a change in the level of expression of either integrin or a change in expression of any TGF-β isoforms, since the levels of each of these proteins was unaffected by wounding over the time course of our experiments.
Our finding that airway epithelial cells make large amounts of TGF-β2, but do not appear to activate it in response to injury raises the questions of how TGF-β2 becomes activated and whether or not it might contribute to the slowing of wound closure or other TGF-β-dependent effects in wounded airways in vivo
. In addition to integrins, TGF-β isoforms can be activated by a variety of proteases and by interaction with the secreted protein, thrombospondin and it is certainly possible that other cells that are present in or recruited to injured airways could secrete these other activators and lead to an important contribution of TGF-β2 to in vivo
Interestingly, although we found that both αvβ6 and αvβ8 contributed to TGF-β activation after wounding, and the contribution of αvβ6 was at least as large as that of αvβ8, we could not demonstrate any contribution of αvβ6 to the decrease in the degree of wound closure we observed. As noted above, one possible explanation for this confusing result might be that, in addition to activating TGF-β (an effect that would be expected to slow the degree of wound closure), αvβ6 itself enhances the degree of wound closure through a TGF-β–independent mechanism. This possibility is supported by our finding that blockade of αvβ6 actually slowed the degree of wound closure under conditions in which the effects of endogenously activated TGF-β were prevented by addition of TGF-β–blocking antibodies. However, there may be additional explanations for these results. Although αvβ6 and αvβ8 both bind to the same RGD sites in TGF-β1 and -β3, the mechanisms of TGF-β activation by these two integrins are actually quite different. αvβ6 induces activation of latent complexes through a mechanism that depends on actin polymerization and appears to induce a conformational change in latent TGF-β but does not release any free TGF-β from the latent complex. Rather, this pathway is completely dependent of direct contact between the cell expressing the activated integrin and an adjacent target cell expressing TGF-β receptors (12
). In contrast, αvβ8-mediated TGF-β activation involves presentation of the latent complex to transmembrane proteases, with resultant proteolytic cleavage of the LAP and release of freely diffusible active TGF-β from the cell surface (13
). It is thus conceivable that TGF-β locally activated by wounding of the epithelial cells at the wound edge might not be sufficient to globally affect the migration of a continuous sheet of epithelial cells, whereas the diffusible TGF-β released by αvβ8-mediated activation could have more long-range effects on the entire epithelial sheet. However, we have not been able to demonstrate any difference in TGF-β activity between the wound edge and the rest of the wounded epithelial sheet, so we have no data to support this possibility.
In summary, we have found that airway epithelial cells respond to injuring by inducing TGF-β activation mediated by two different integrins, αvβ6 and αvβ8. Although these cells make large amounts of TGF-β2 and small amounts of TGF-β1, only TGF-β1 is activated by this pathway. At least in the case of αvβ8, activation of endogenous TGF-β1 slows the degree of wound closure, an effect that can be prevented by blocking either TGF-β or the αvβ8 integrin. These observations could have important implications for understanding the mechanisms underlying a variety of lung and airway diseases that are characterized by abnormal airway repair.