Targeted deletion of the C-terminal segment of the β4 tail does not affect adhesion to laminin-5 and assembly of hemidesmosomes, allowing a genetic analysis of α6β4 signaling in the absence of loss of adhesion. The results of this analysis indicate that the β4 substrate domain controls laminin-5-dependent nuclear translocation of P-ERK, P-JNK, and NF-κB and, through this mechanism, plays a role in epidermal growth and wound healing. Indeed, although both β1 and αv integrins can support epidermal proliferation and migration (15
), the epidermis of β4 mutant mice displays a significant proliferative defect and it undergoes reepithelialization at a reduced rate, presumably because of decreased epidermal migration. In addition, the β4 mutant mice display defective postnatal angiogenesis (37
). Thus, whereas the adhesive function of α6β4 is essential for epidermal stability, and its loss is incompatible with life (10
), signaling by the β4 substrate domain plays a role in epidermal homeostasis and repair and in angiogenesis. The observation that the β4 mutant mice are viable and fertile and do not display evident abnormalities in the absence of stress suggests that the novel signaling mechanism identified here does not contribute to embryonic development and normal adult life. Alternatively, other integrins may play redundant or compensatory roles.
We find that the β4 substrate domain performs key functions in epidermal cells. Upon plating on laminin-5, the mutant keratinocytes display poor mitogenic response to EGF, and they rapidly undergo apoptosis upon removal of the growth factor. These defects are not observed in cells plated on fibronectin, indicating that the β4 substrate domain is necessary for cell proliferation and survival on laminin-5 but not other permissive matrix substrates. In addition, keratinocytes lacking the β4 substrate domain migrate inefficiently in response to EGF, providing evidence that β4 signaling controls keratinocyte migration. Although prior studies had suggested that α6β4 activates promigratory signaling pathways in epidermal cells (43
), our results are the first to provide genetic evidence that this is the case. Finally, the mutant keratinocytes show enhanced activation of Rac, spreading, and assembly of α3β1-containing focal contacts on laminin-5, suggesting that loss of the β4 substrate domain enhances the adhesive and signaling function of α3β1, which also binds to laminin-5. These results suggest that the transdominant-negative effect that α6β4 exerts on α3β1 (36
) is mediated by the β4 substrate domain. In addition, they imply that the α3β1 integrin is unable to compensate for the effects of loss of β4 signaling on cell proliferation, survival, and migration. In fact, it is remarkable that the β4 mutant keratinocytes are unable to migrate efficiently, despite upregulation of α3β1, as this latter integrin clearly promotes migration on laminin-5 (6
). Taken together, our observations suggest that α6β4 activates potent promigratory signals able to overcome the partial suppression of α3β1-dependent spreading.
Our conclusions stand in stark contrast with those reached recently by Sonnenberg and colleagues (42
). These authors have used a K14-Cre transgene to delete β4 from the basal layer of the skin of β4 floxed mice but have obtained only a mosaic pattern of expression of Cre and, thereby, limited excision of floxed β4. They report that the β4-negative areas of the skin of these mice do not contain a decreased number of proliferating keratinocytes (42
). We note, however, that it may have been very difficult to identify continuous sections of the epidermis of these mice totally lacking β4 and, hence, to perform a statistically significant analysis. In addition, it is possible that the β4-positive keratinocytes rescued the proliferation of their β4-negative neighboring cells through a paracrine mechanism. Sonnenberg and colleagues have also failed to detect a proliferation defect in immortalized, p53-negative keratinocytes lacking β4 under standard culture conditions. By contrast, we have documented a significant proliferation defect in early-passage primary keratinocytes lacking the β4 signaling domain. Notably, this defect was evident when the cells were plated on laminin-5 but not when they were plated on fibronectin. We suspect that serum-derived factors, such as fibronectin or vitronectin, or loss of p53-mediated control of the cell cycle may have allowed the β4-null immortalized keratinocytes to proliferate despite loss of α6β4 signaling. Finally, Sonnenberg and colleagues also report that their p53-negative, β4−/−
keratinocytes migrate more efficiently—not less efficiently—than the β4+/+
). The apparent contrast between the migratory behavior of keratinocytes lacking α6β4 and those lacking only the β4 signaling domain is fully consistent with the model that α6β4-mediated assembly of hemidesmosomes opposes cell migration, whereas α6β4 signaling promotes it. In line with this conclusion, prior studies have indicated that the EGF-R and other RTKs enhance phosphorylation of β4, causing disruption of hemidesmosomes and increased keratinocyte migration (7
What are the molecular mechanisms by which the β4 substrate domain exerts its biological function? Our biochemical studies indicate that this segment of β4 mediates activation of PI-3K-to-Akt and Ras-to-ERK signaling independently of the EGF-R, as anticipated from prior studies (30
). Since inhibition of PI-3K, but not MEK, induces apoptosis of keratinocytes plated on laminin-5, it is likely that the β4 substrate domain protects these cells from apoptosis through PI-3K-to-Akt signaling (Fig. ). Thus, although α6β4-mediated assembly of hemidesmosomes may impart polarity to epithelial cells and protect them from apoptotic insults (54
), α6β4 promotes cell survival also by a direct signaling mechanism. We note that the β4 substrate domain is not necessary for efficient activation and nuclear translocation of P-Akt in the presence of EGF, suggesting that the antiapoptotic effect of α6β4 signaling through PI-3K may be important only when the amounts of trophic factors available to the cell are limiting. In accordance with this model, we have not detected increased apoptosis in the epidermis of mutant mice in vivo.
Model of integrin α6β4 signaling.
Prior studies have led to the hypothesis that the β4 substrate domain promotes carcinoma cell invasion through recruitment of PI-3K and consequent activation of Rac (48
). By contrast, we find that, at least in primary keratinocytes, α6β4 activates Rac and induces signaling to JNK and phosphorylation and degradation of IκB independently of the β4 substrate domain, possibly through the integrin-associated Src family kinase (32
). This apparent discrepancy may reflect the existence of partially redundant signaling mechanisms activated in a cell-type-specific manner. In addition, we had proposed that the β4 substrate domain promotes epithelial cell proliferation by cooperating with the EGF-R to activate Ras-to ERK signaling (30
). Yet, we now find that deletion of the β4 substrate domain does not impair activation of ERK in mutant keratinocytes treated with optimal concentrations of EGF. Thus, our genetic analysis clearly indicates that the β4 substrate domain is required for keratinocyte migration and proliferation, but it also reveals that it does not exert its function exclusively through activation of the signaling pathways previously identified through cell biological studies.
What is then the mechanism by which α6β4 promotes keratinocyte migration and proliferation? Our studies indicate that the EGF-R is unable to induce nuclear accumulation of P-ERK, P-JNK, and NF-κB, and hence presumably transcription of target genes, in mutant keratinocytes adhering to laminin-5. This striking result indicates that the β4 substrate domain is necessary for nuclear translocation of three distinct transcriptional regulators (Fig. ). Prior studies have indicated that α5β1-mediated adhesion to fibronectin promotes nuclear accumulation of P-ERK, but not P-JNK and P-p38, and this occurs through activation of Rac (1
). The effect of α6β4 signaling is more general, as it involves three distinct nuclear effectors, and it does not appear to be mediated by Rac, as the levels of activation of Rac are enhanced, not diminished, in β4 mutant cells. In addition, whereas the effect of fibronectin-mediated adhesion on nuclear translocation of ERK requires changes in the actin cytoskeleton consequent to cell spreading (2
), the β4 mutant keratinocytes display defective nuclear accumulation of P-ERK, P-JNK, and NF-κB in spite of increased spreading on laminin-5. Also, it has been reported that the leukocyte integrin αLβ2 promotes nuclear translocation of the c-Jun coactivator JAB1 (3
). However, this effect is mediated by cytohesin-1, which binds selectively to the integrin β2 cytotail (39
). These observations suggest that the mechanism by which the β4 substrate domain promotes nuclear translocation of mitogen-activated protein kinases and NF-κB is novel. Future studies will be required to elucidate it.
Our results provide evidence that α6β4-dependent activation of NF-κB and JNK is necessary for keratinocyte proliferation and migration (Fig. ). In agreement with this hypothesis, genetic studies of dorsal closure in Drosophila melanogaster
and wound healing in mice have indicated that JNK signaling and AP-1-dependent transcription are crucial for epidermal migration and proliferation (26
). In addition, Ron cooperates with α6β4 to promote NF-κB signaling and keratinocyte migration (44
). In apparent contrast to our results, prior transgenic studies have indicated that overexpression of dominant-negative IκBα promotes proliferation and inhibits differentiation of basal keratinocytes, whereas overexpression of the NF-κB subunit p50 exerts the opposite effects (47
). We note, however, that epidermis-specific deletion of the IκB kinase IKKβ, which mediates NF-κB signaling in the skin, does not cause cell-autonomous hyperproliferation, or impaired differentiation, but rather inhibits proliferation of keratinocytes (38
). Because the EGF-R activates NF-κB (52
) and NF-κB participates in the transcriptional control of cyclin D (16
), transient activation of NF-κB may be required for progression of keratinocytes through the G1
phase of the cell cycle. By contrast, persistent activation of NF-κB may lead to the upregulation of genes encoding cytokines, such as tumor necrosis factor alpha and interleukin-1, and extracellular factors, such as growth inhibitory factor, which inhibit keratinocyte growth (19
). In this model, the apparently contradictory effects of NF-κB signaling in the skin are explained by two temporally and kinetically distinct roles of the transcription factor. Similar considerations may apply also to the apparent discrepancy between the reported role of Ras-ERK signaling in the control of epidermal proliferation (5
) and our observation that inhibition of MEK with two distinct compounds inhibits keratinocyte proliferation on laminin-5 only modestly.
In conclusion, our results provide genetic evidence that α6β4 signaling promotes epidermal growth and wound healing by controlling nuclear translocation of NF-κB and P-JNK. These findings identify a novel integrin signaling mechanism and highlight its physiological function.