Intrinsic signaling functions of the beta4 integrin intracellular domain

A key issue regarding the role of alpha6beta4 in cancer biology is the mechanism by which this integrin exerts its profound effects on intracellular signaling, including growth factor-mediated signaling. One approach is to evaluate the intrinsic signaling capacity of the unique beta4 intracellular domain in the absence of contributions from the alpha6 subunit and tetraspanins and to assess the ability of growth factor receptor signaling to cooperate with this domain. Here, we generated a chimeric receptor composed of the TrkB extracellular domain and the beta4 transmembrane and intracellular domains. Expression of this chimeric receptor in beta4-null cancer cells enabled us to assess the signaling potential of the beta4 intracellular domain alone or in response to dimerization using brain-derived neurotrophic factor, the ligand for TrkB. Dimerization of the beta4 intracellular domain results in the binding and activation of the tyrosine phosphatase SHP-2 and the activation of Src, events that also occur upon ligation of intact alpha6beta4. In contrast to alpha6beta4 signaling, however, dimerization of the chimeric receptor does not activate either Akt or Erk1/2. Growth factor stimulation induces tyrosine phosphorylation of the chimeric receptor but does not enhance its binding to SHP-2. The chimeric receptor is unable to amplify growth factor-mediated activation of Akt and Erk1/2, and growth factor-stimulated migration. Collectively, these data indicate that the beta4 intracellular domain has some intrinsic signaling potential, but it cannot mimic the full signaling capacity of alpha6beta4. These data also question the putative role of the beta4 intracellular domain as an "adaptor" for growth factor receptor signaling.     

Injury and EGF mediate the expression of alpha6beta4 integrin subunits in corneal epithelium

Our goal was to evaluate the role of epidermal growth factor and injury on the expression of integrin subunits alpha6(alpha6) and beta4(beta4). An in vitro wound model was used to evaluate corneal wound repair and cellular migration. Primary rabbit corneal epithelial cell cultures were serum-starved and injured in the presence or absence of EGF or tyrphostin AG1478, an inhibitor of EGF receptor kinase activity. Repair was monitored morphologically and expression was analyzed using in situ hybridization and immunohistochemistry accompanied by confocal microscopy. The addition of EGF to cell cultures induced a dose-dependent increase in beta4 mRNA expression but the constitutive expression of alpha6 was several fold greater. In the wounded cultures there was a rapid change in expression at the edge of the wound that was enhanced with EGF. In our model there was an increase in beta4 and alpha6 protein in migrating cells. Changes in integrin expression were accompanied by a transient increase in activation of the EGF receptor. The addition of tyrphostin inhibited migration of cells and wound repair, the activation of the EGF receptor and phosphorylation of beta4 in the cytoplasm. These data indicate that the activation of the EGF receptor plays a critical role in the regulation of integrin receptors and the mediation of cellular migration.     

The integrin alpha 6 beta 4 is a laminin receptor

In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1.     

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes

In normal epithelial cells, integrin α(6)β(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)β(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)β(4). The binding of Necl-2 with integrin β(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)β(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)β(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.     

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.     

Dynamics of the alpha6beta4 integrin in keratinocytes

The integrin alpha6beta4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of alpha6beta4 in live cells two different beta4-chimeras were stably expressed in beta4-deficient PA-JEB keratinocytes. One chimera consisted of full-length beta4 fused to EGFP at its carboxy terminus (beta4-EGFP). In a second chimera the extracellular part of beta4 was replaced by EGFP (EGFP-beta4), thereby rendering it incapable of associating with alpha6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the beta4-EGFP and EGFP-beta4 hemidesmosomes disappear, and a proportion of the beta4-EGFP, but not of the EGFP-beta4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving "footprints" of the migrating cell. PA-JEB cells expressing beta4-EGFP migrate considerably more slowly than those that express EGFP-beta4. Studies with a beta4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (beta4(R1281W)-EGFP) suggest that the stabilization of the interaction between alpha6beta4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of beta4 with plectin renders the bond between alpha6beta4 and laminin-5 more stable, i.e., beta4-EGFP is less dynamic than beta4(R1281W)-EGFP. On the other hand, when alpha6beta4 is bound to laminin-5, the binding dynamics of beta4 to plectin are increased, i.e., beta4-EGFP is more dynamic than EGFP-beta4. We suggest that the stability of the interaction between alpha6beta4 and laminin-5 is influenced by the clustering of alpha6beta4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether alpha6beta4 will inhibit cell migration or not.     

RhoA function in lamellae formation and migration is regulated by the alpha6beta4 integrin and cAMP metabolism

Clone A colon carcinoma cells develop fan-shaped lamellae and exhibit random migration when plated on laminin, processes that depend on the ligation of the alpha6beta4 integrin. Here, we report that expression of a dominant negative RhoA (N19RhoA) in clone A cells inhibited alpha6beta4-dependent membrane ruffling, lamellae formation, and migration. In contrast, expression of a dominant negative Rac (N17Rac1) had no effect on these processes. Using the Rhotekin binding assay to assess RhoA activation, we observed that engagement of alpha6beta4 by either antibody-mediated clustering or laminin attachment resulted in a two- to threefold increase in RhoA activation, compared with cells maintained in suspension or plated on collagen. Antibody-mediated clustering of beta1 integrins, however, actually suppressed Rho A activation. The alpha6beta4-mediated interaction of clone A cells with laminin promoted the translocation of RhoA from the cytosol to membrane ruffles at the edges of lamellae and promoted its colocalization with beta1 integrins, as assessed by immunofluorescence microscopy. In addition, RhoA translocation was blocked by inhibiting phosphodiesterase activity and enhanced by inhibiting the activity of cAMP-dependent protein kinase. Together, these results establish a specific integrin-mediated pathway of RhoA activation that is regulated by cAMP and that functions in lamellae formation and migration.     

Compartmentalization of integrin alpha6beta4 signaling in lipid rafts

Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.     

Evidence that ligand and metal ion binding to integrin alpha 4beta 1 are regulated through a coupled equilibrium

We have used the highly selective alpha(4)beta(1) inhibitor 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino)-pentanoylamino]-butyric acid (BIO7662) as a model ligand to study alpha(4)beta(1) integrin-ligand interactions on Jurkat cells. Binding of [(35)S]BIO7662 to Jurkat cells was dependent on the presence of divalent cations and could be blocked by treatment with an excess of unlabeled inhibitor or with EDTA. K(D) values for the binding of BIO7662 to Mn(2+)-activated alpha(4)beta(1) and to the nonactivated state of the integrin that exists in 1 mm Mg(2+), 1 mm Ca(2+) were <10 pm, indicating that it has a high affinity for both activated and nonactivated integrin. No binding was observed on alpha(4)beta(1) negative cells. Through an analysis of the metal ion dependences of ligand binding, several unexpected findings about alpha(4)beta(1) function were made. First, we observed that Ca(2+) binding to alpha(4)beta(1) was stimulated by the addition of BIO7662. From solution binding studies on purified alpha(4)beta(1), two types of Ca(2+)-binding sites were identified, one dependent upon and the other independent of BIO7662 binding. Second, we observed that the metal ion dependence of ligand binding was affected by the affinity of the ligand for alpha(4)beta(1). ED(50) values for the metal ion dependence of the binding of BIO7762 and the binding of a lower affinity ligand, BIO1211, differed by 2-fold for Mn(2+), 30-fold for Mg(2+), and >1000-fold for Ca(2+). Low Ca(2+) (ED(50) = 5-10 microm) stimulated the binding of BIO7662 to alpha(4)beta(1). The effects of microm Ca(2+) closely resembled the effects of Mn(2+) on alpha(4)beta(1) function. Third, we observed that the rate of BIO7662 binding was dependent on the metal ion concentration and that the ED(50) for the metal ion dependence of BIO7662 binding was affected by the concentration of the BIO7662. These studies point to an even more complex interplay between metal ion and ligand binding than previously appreciated and provide evidence for a three-component coupled equilibrium model for metal ion-dependent binding of ligands to alpha(4)beta(1).     

Endothelial cell migration on fibronectin is regulated by syntaxin 6-mediated alpha5beta1 integrin recycling

The α5β1 integrin heterodimer regulates many processes that contribute to embryonic development and angiogenesis, in both physiological and pathological contexts. As one of the major adhesion complexes on endothelial cells, it plays a vital role in adhesion and migration along the extracellular matrix. We recently showed that angiogenesis is modulated by syntaxin 6, a Golgi- and endosome-localized t-SNARE, and that it does so by regulating the post-Golgi trafficking of VEGFR2. Here we show that syntaxin 6 is also required for α5β1 integrin-mediated adhesion of endothelial cells to, and migration along, fibronectin. We demonstrate that syntaxin 6 and α5β1 integrin colocalize in EEA1-containing early endosomes, and that functional inhibition of syntaxin 6 leads to misrouting of β1 integrin to the degradation pathway (late endosomes and lysosomes) rather transport along recycling pathway from early endosomes; an increase in the pool of ubiquitinylated α5 integrin and its lysosome-dependent degradation; reduced cell spreading on fibronectin; decreased Rac1 activation; and altered Rac1 localization. Collectively, our data show that functional syntaxin 6 is required for the regulation of α5β1-mediated endothelial cell movement on fibronectin. These syntaxin 6-regulated membrane trafficking events control outside-in signaling via haptotactic and chemotactic mechanisms.     

Differential regulation of alpha6beta4 integrin by PKC isoforms in murine skin keratinocytes

In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.     

A regulated interaction between alpha5beta1 integrin and osteopontin

The extracellular matrix protein osteopontin (OPN) interacts with a number of integrins, namely alphavbeta1, alphavbeta3, alphavbeta5, alpha9beta1, alpha8beta1, and alpha4beta1. We have investigated the interaction of alpha5beta1 integrin with OPN using K562 cells, which only express alpha5beta1. alpha5beta1 is in a low activation state in this cell line, but can be stimulated to a higher activation state by the phorbol ester TPA. Treating K562 wild-type cells (K562-WT) with TPA stimulated an interaction between alpha5beta1 and OPN. No interaction was seen in the absence of TPA. alpha5beta1 selectively interacted with a GST fusion protein of the N-terminal fragment of OPN (aa17-168), which is generated in vivo by thrombin cleavage of OPN. Expression of the alpha4 integrin in K562 cells (K562-alpha4beta1) stimulated alpha5beta1-dependent binding to aa17-168 in the absence of TPA, suggesting that alpha4beta1 activates alpha5beta1 in K562 cells. Adhesion via alpha5beta1 is mediated by the Arg-Gly-Asp (RGD) motif of OPN, as mutating this sequence to Arg-Ala-Asp (RAD) blocked binding of both cell types. These data demonstrate that thrombin cleavage regulates the adhesive properties of OPN and that alpha5beta1 integrin can interact with thrombin-cleaved osteopontin when in a high activation state.     

The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.     

Activation of p53 function in carcinoma cells by the alpha6beta4 integrin.

The interaction of integrins with extracellular matrix is known to promote cell survival by inhibiting apoptotic signaling. In contrast, we demonstrate here that the alpha6beta4 integrin induces apoptosis in carcinoma cells by stimulating p53 function. Specifically, we show that expression of alpha6beta4 in carcinoma cells that lack this integrin stimulates an increase in the transactivating function of p53 as demonstrated by the ability of this integrin to up-regulate the expression of a p53-sensitive reporter gene as well as the endogenous p53 response gene, bax. In addition, we report that alpha6beta4 triggers apoptosis in carcinoma cells that express wild-type but not mutant p53 and that these alpha6beta4 functions are inhibited by a dominant negative p53 construct. Importantly, we provide a link between integrin signaling and p53 activation by demonstrating that the clustering of alpha6beta4 with a beta4 integrin-specific antibody promotes p53-dependent apoptosis in cells that express both alpha6beta4 and wild-type p53. These studies are the first to demonstrate that a specific integrin can promote apoptosis by activating p53. Moreover, given the ability of alpha6beta4 to stimulate invasion (Shaw, L. M., Rabinovitz, I., Wang, H. F., Toker, A., and Mercurio, A. M. (1997) Cell 91, 949-960), these studies suggest that the ability of alpha6beta4 to promote carcinoma progression will be enhanced in tumor cells that express mutant, inactive forms of p53.     

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated. The integrin alpha 6 beta 4 is expressed along the basal surface of various epithelial cells. We show here that this integrin localizes to hemidesmosomes as determined by immunoelectron microscopy using antibodies directed against both the extra- and intracytoplasmic domains of alpha 6 beta 4. This result, which agrees with a recent study, suggests a functional role for the alpha 6 beta 4 integrin in the hemidesmosomes. We therefore investigated such a potential role for this integrin using the cultured rat bladder carcinoma cell line 804G, which has the uncommon ability to form hemidesmosomes in vitro when maintained on uncoated glass substrates. By immunoprecipitation and immunofluorescence, we show that 804G cells express alpha 6 beta 4 along their basal surface in a punctate pattern that overlaps with the distribution of hemidesmosomal plaque antigens. However, this pattern is altered when cells are plated in the presence of an antiserum directed against alpha 6 beta 4. Furthermore, no hemidesmosomes are detectable at the ultrastructural level in the alpha 6 beta 4 antibody-treated cells compared with control cells. These results indicate that integrins may play a critical role in assembly and adhesive functions of the hemidesmosome.     

Stimulation-dependent recycling of integrin beta1 regulated by ARF6 and Rab11

In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin β1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of β1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.     

The Met receptor and alpha 6 beta 4 integrin can function independently to promote carcinoma invasion

It has been proposed that a constitutive, physical association of the Met receptor and the alpha(6)beta(4) integrin exists on the surface of invasive carcinoma cells and that hepatocyte growth factor (HGF)-mediated invasion is dependent on alpha(6)beta(4) (Trusolino, L., Bertotti, A., and Comoglio, P. M. (2001) Cell 107, 643-654). The potential significance of these results prompted us to re-examine this hypothesis. Using three different carcinoma cell lines that express both Met and alpha(6)beta(4), we were unable to detect the constitutive association of these receptors by co-immunoprecipitation. Moreover, carcinoma cells that lacked expression of alpha(6)beta(4) exhibited Met-dependent invasion toward HGF, and increasing Met expression by viral infection of these cells enhanced invasion without inducing alpha(6)beta(4) expression. Although expression of alpha(6)beta(4) in such cells enhanced their invasion to HGF, it also enhanced their ability to invade toward other chemoattractants such as lysophosphatidic acid, and this latter invasion was not inhibited by a function-blocking Met antibody. Finally, depletion of beta(4) by RNA interference in invasive carcinoma cells that express both receptors reduced the ability of these cells to invade toward HGF by approximately 25%, but it did not abrogate their invasion. These data argue that the invasive function of Met can be independent of alpha(6)beta(4) and that alpha(6)beta(4) has a generic influence on the invasion of carcinoma cells that is not specific to Met.