EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle.

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G(1) phase. Conversely, dominant negative Rac prevents these events on fibronectin. Mitogens promote activation of Rac on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells.

Tyrosine phosphorylation of the beta 4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes.

Ligation of the alpha(6)beta(4) integrin induces tyrosine phosphorylation of the beta(4) cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta(4) that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr(1440), or secondarily Tyr(1422), interacts with the SH2 domain of Shc, whereas Tyr(1526), or secondarily Tyr(1642), interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha(6)beta(4). In addition, mutation of beta(4) Tyr(1526), which binds to the PTB domain of Shc, but not of Tyr(1422) and Tyr(1440), which interact with its SH2 domain, abolishes the activation of ERK by alpha(6)beta(4). Phenylalanine substitution of the beta(4) tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha(6)beta(4) in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta(4). This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta(4) containing phenylalanine substitutions at Tyr(1422) and Tyr(1440), at Tyr(1526) and Tyr(1642), or at all four tyrosine phosphorylation sites. These results suggest that beta(4) Tyr(1526) interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling.

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

Distinct roles of the adaptor protein Shc and focal adhesion kinase in integrin signaling to ERK.

It has been proposed that integrins activate ERK through the adaptor protein Shc independently of focal adhesion kinase (FAK) or through FAK acting on multiple target effectors, including Shc. We show that disruption of the actin cytoskeleton by cytochalasin D causes a complete inhibition of FAK but does not inhibit Shc signaling and activation of ERK. We have then generated primary fibroblasts carrying a targeted deletion of the segment of beta(1) subunit cytoplasmic domain required for activation of FAK. Analysis of these cells indicates that FAK is not necessary for efficient tyrosine phosphorylation of Shc, association of Shc with Grb2, and activation of ERK in response to matrix adhesion. In addition, integrin-mediated activation of FAK does not appear to be required for signaling to ERK following growth factor stimulation. To examine if FAK could contribute to the activation of ERK in a cell type-specific manner through the Rap1/B-Raf pathway, we have used Swiss-3T3 cells, which in contrast to primary fibroblasts express B-Raf. Dominant negative studies indicate that Shc mediates the early phase and peak, whereas FAK, p130(CAS), Crk, and Rap1 contribute to the late phase of integrin-dependent activation of ERK in these cells. In addition, introduction of B-Raf enhances and sustains integrin-mediated activation of ERK in wild-type primary fibroblasts but not in those carrying the targeted deletion of the beta(1) cytoplasmic domain. Thus, the Shc and FAK pathways are activated independently and function in a parallel fashion. Although not necessary for signaling to ERK in primary fibroblasts, FAK may enhance and prolong integrin-mediated activation of ERK through p130(CAS), Crk, and Rap1 in cells expressing B-Raf.

The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4.

This study was undertaken to characterize the antigenic determinants recognized by the autoantibodies of patients with ocular cicatricial pemphigoid (OCP). OCP is a subepithelial, blistering, autoimmune disease that mainly affects the conjunctiva and other mucous membranes. We previously demonstrated that a cDNA clone, isolated from a keratinocyte expression library by using immunoaffinity-purified OCP autoantibody, encoded the cytoplasmic domain of beta 4 integrin subunit. Our subsequent studies showed that sera from all the OCP patients that were tested recognize the human beta 4 integrin subunit. To identify the prevalent epitopes of the anti-beta 4 autoantibodies of OCP, we have used cell lines transfected with vectors encoding a wild-type beta 4 subunit, a tailless beta 4 subunit, or a beta 4 subunit lacking the extracellular domain. Nontransfected cell lines were used as controls. Lysates from these cell lines were analyzed with OCP sera, IgG fractions from OCP sera, and immunoaffinity-purified OCP autoantibodies. Abs to extracellular and cytoplasmic domains of human beta 4 integrin were used as positive controls, whereas normal human sera and normal human IgG fractions were used as negative controls. The reactivity of OCP Abs was determined by using immunoblotting, immunoprecipitation, and FACS analysis. The results of this study indicate that OCP sera, OCP IgG fractions, and immunoaffinity-purified OCP autoantibodies react with the intracellular and not the extracellular domain of human beta 4 integrin subunit. In vitro cell culture experiments demonstrated that OCP autoantibody binds to the cytoplasm of the cells. The relevance of these findings to the pathogenesis of OCP is discussed.

Integrin signaling.

Cells reside in a protein network, the extracellular matrix (ECM), which they secrete and mold into the intercellular space. The ECM exerts profound control over cells. The effects of the matrix are primarily mediated by integrins, a family of cell surface receptors that attach cells to the matrix and mediate mechanical and chemical signals from it. These signals regulate the activities of cytoplasmic kinases, growth factor receptors, and ion channels and control the organization of the intracellular actin cytoskeleton. Many integrin signals converge on cell cycle regulation, directing cells to live or die, to proliferate, or to exit the cell cycle and differentiate.

Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH2-terminal kinase and progression through the G1 phase of the cell cycle.

The extracellular matrix exerts a stringent control on the proliferation of normal cells, suggesting the existence of a mitogenic signaling pathway activated by integrins, but not significantly by growth factor receptors. Herein, we provide evidence that integrins cause a significant and protracted activation of Jun NH2-terminal kinase (JNK), while several growth factors cause more modest or no activation of this enzyme. Integrin-mediated stimulation of JNK required the association of focal adhesion kinase (FAK) with a Src kinase and p130(CAS), the phosphorylation of p130(CAS), and subsequently, the recruitment of Crk. Ras and PI-3K were not required. FAK-JNK signaling was necessary for proper progression through the G1 phase of the cell cycle. These findings establish a role for FAK in both the activation of JNK and the control of the cell cycle, and identify a physiological stimulus for JNK signaling that is consistent with the role of Jun in both proliferation and transformation.

Interactions between growth factors and integrins: latent forms of transforming growth factor-beta are ligands for the integrin alphavbeta1.

The multipotential cytokine transforming growth factor-beta (TGF-beta) is secreted in a latent form. Latency results from the noncovalent association of TGF-beta with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-beta-binding protein (LTBP) produces the most common form of latent TGF-beta, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-beta. LTBP and the LAP propeptides of TGF-beta (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-beta function in the ECM, we determined whether latent TGF-beta1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits alphav and beta1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. alphavbeta1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of alphavbeta5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was alphavbeta1 dependent. These results establish alphavbeta1 as a LAP-beta1 receptor. Interactions between latent TGF-beta and alphavbeta1 may localize latent TGF-beta to the surface of specific cells and may allow the TGF-beta1 gene product to initiate signals by both TGF-beta receptor and integrin pathways.

A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth.

Caveolin-1 functions as a membrane adaptor to link the integrin alpha subunit to the tyrosine kinase Fyn. Upon integrin ligation, Fyn is activated and binds, via its SH3 domain, to Shc. Shc is subsequently phosphorylated at tyrosine 317 and recruits Grb2. This sequence of events is necessary to couple integrins to the Ras-ERK pathway and promote cell cycle progression. These findings reveal an unexpected function of caveolin-1 and Fyn in integrin signaling and anchorage-dependent cell growth.

Cell cycle and adhesion defects in mice carrying a targeted deletion of the integrin beta4 cytoplasmic domain.

The cytoplasmic domain of the integrin beta4 subunit mediates both association with the hemidesmosomal cytoskeleton and recruitment of the signaling adaptor protein Shc. To examine the significance of these interactions during development, we have generated mice carrying a targeted deletion of the beta4 cytoplasmic domain. Analysis of homozygous mutant mice indicates that the tail-less alpha6beta4 binds efficiently to laminin 5, but is unable to integrate with the cytoskeleton. Accordingly, these mice display extensive epidermal detachment at birth and die immmediately thereafter from a syndrome resembling the human disease junctional epidermolysis bullosa with pyloric atresia (PA-JEB). In addition, we find a significant proliferative defect. Specifically, the number of precursor cells in the intestinal epithelium, which remains adherent to the basement membrane, and in intact areas of the skin is reduced, and post-mitotic enterocytes display increased levels of the cyclin-dependent kinase inhibitor p27(Kip). These findings indicate that the interactions mediated by the beta4 tail are crucial for stable adhesion of stratified epithelia to the basement membrane and for proper cell-cycle control in the proliferative compartments of both stratified and simple epithelia.

Integrin alpha1beta1 mediates a unique collagen-dependent proliferation pathway in vivo.

Activation of integrins upon binding to extracellular matrix proteins is believed to be a crucial step for the regulation of cell survival and proliferation. We have used integrin alpha1-null mice to investigate the role of this collagen receptor in the regulation of cell growth and survival in vivo. alpha1-deficient animals, which are viable and fertile, have a hypocellular dermis and a deficiency in dermal fibroblast proliferation as embryos. In vitro analysis of alpha1-null embryonic fibroblasts has revealed that their proliferation rate is markedly reduced when plated on collagenous substrata, despite normal attachment and spreading. Moreover, on the same collagenous matrices, alpha1-null fibroblasts fail to recruit and activate the adaptor protein Shc. The failure to activate Shc is accompanied by a downstream deficiency in recruitment of Grb2 and subsequent mitogen-activated protein kinase activation. Taken together with the growth deficiency observed on collagens, this finding indicates that the alpha1beta1 is the sole collagen receptor which can activate the Shc mediated growth pathway. Thus, integrin alpha1 has a unique role among the collagen receptors in regulating both in vivo and in vitro cell proliferation in collagenous matrices.

Integrin signaling: specificity and control of cell survival and cell cycle progression.

Integrin-mediated adhesion to the extracellular matrix plays an important role in regulating cell survival and proliferation. There is now increasing evidence that integrins activate shared as well as subgroup-specific signaling pathways. The signals from these adhesion receptors are integrated with those originating from growth factor and cytokine receptors in order to organize the cytoskeleton, stimulate mitogen-activated protein kinase cascades, and regulate immediate early gene expression. The repertoire of integrins and composition of the extracellular matrix appear to dictate whether a cell will survive, proliferate or exit the cell cycle and differentiate in response to soluble factors.

The coupling of alpha6beta4 integrin to Ras-MAP kinase pathways mediated by Shc controls keratinocyte proliferation.

The signaling pathways linking integrins to nuclear events are incompletely understood. We have examined intracellular signaling by the alpha6beta4 integrin, a laminin receptor expressed in basal keratinocytes and other cells. Ligation of alpha6beta4 in primary human keratinocytes caused tyrosine phosphorylation of Shc, recruitment of Grb2, activation of Ras and stimulation of the MAP kinases Erk and Jnk. In contrast, ligation of the laminin- and collagen-binding integrins alpha3beta1 and alpha2beta1 did not cause these events. While the stimulation of Erk by alpha6beta4 was suppressed by dominant-negative Shc, Ras and RhoA, the activation of Jnk was inhibited by dominant-negative Ras and Rac1 and by the phosphoinositide 3-kinase inhibitor Wortmannin. Adhesion mediated by alpha6beta4 induced transcription from the Fos serum response element and promoted cell cycle progression in response to mitogens. In contrast, alpha3beta1- and alpha2beta1-dependent adhesion did not induce these events. These findings suggest that the coupling of alpha6beta4 integrin to the control of cell cycle progression mediated by Shc regulates the proliferation of basal keratinocytes and possibly other cells which are in contact with the basement membrane in vivo.