Extracellular matrix proteins and their cellular receptors, integrins, play a fundamental role in keratinocyte adhesion and migration. During wound healing, keratinocytes detach, migrate until the two epithelial sheets confront, and then regenerate the basement membrane. We examined the expression of different integrins and their putative ligands in keratinocytes during human mucosal wound healing. Migrating keratinocytes continuously expressed kalinin but not the other typical components of the basement membrane zone: type IV collagen, laminin, and type VII collagen. When the epithelial sheets confronted each other, these missing basement membrane components started to appear gradually through the entire wound area. The expression of integrin 81 subunit was increased in keratinocytes during migration. The #,B-associated a2 and a3 subunits were expressed constantly by wound keratinocytes whereas the a5 subunit was present only in keratinocytes during reepithelialization. Furthermore, migrating cells started to express a,-integrins which were not present in the nonaffected epithelium. All keratinocytes also expressed the a684 integrin during migration. In the migrating cells, the distribution of integrins was altered. In normal mucosa, ,61-integrins were located mainly on the lateral plasma membrane and aQ84 at the basal surface of basal keratinocytes in the nonaffected tissue. In wounds, integrins were found in filopodia of migrating keratinocytes, and also surrounding cells in several cell layers of the migrating sheet. The results indicate that migrating keratinocytes in deep human wounds enlarge their integrin repertoire. The changes in integrin expression take place concomitantly with changes in the basement membrane composition, suggesting a close interplay of these two groups of molecules during wound healing. (J. Clin. Invest. 1993. 92:1425-1435

The basal lamina of muscle fibers plays a crucial role in the development and function of skeletal muscle. An important laminin receptor in muscle is integrin alpha7beta1D. Integrin beta1 is expressed throughout the body, while integrin alpha7 is more muscle-specific. To address the role of integrin alpha7 in human muscle disease, we determined alpha7 protein expression in muscle biopsies from 117 patients with unclassified congenital myopathy and congenital muscular dystrophy by immunocytochemistry. We found three unrelated patients with integrin alpha7 deficiency and normal laminin alpha2 chain expression. To determine if any of these three patients had mutations of the integrin alpha7 gene, ITGA7, we cloned and sequenced the full-length human ITGA7 cDNA, and screened the patients for mutations. One patient had splice mutations on both alleles; one causing a 21-bp insertion in the conserved cysteine-rich region, and the other causing a 98-bp deletion. A second patient was a compound heterozygote for the same 98-bp deletion, and had a 1-bp frame-shift deletion on the other allele. A third showed marked deficiency of ITGA7 mRNA. Clinically, these patients showed congenital myopathy with delayed motor milestones. Our results demonstrate that mutations in ITGA7 are involved in a form of congenital myopathy.

Integrin-basement membrane interactions provide essential signals that promote survival and growth of epithelial cells, whereas loss of such adhesions triggers programmed cell death. We found that HSC-3 human squamous carcinoma cells survived and grew readily as monolayers, but when they were suspended as single cells, they ceased proliferating and entered into the apoptotic death pathway, characterized by DNA fragmentation. In contrast, if the suspended carcinoma cells were permitted to form E-cadherin-mediated multicellular aggregates, they not only survived but proliferated. However, aggregated normal keratinocytes were unable to survive in suspension culture and rapidly became apoptotic. Anchorage independence and resistance to apoptosis of HSC-3 cell aggregates required high levels of extracellular Ca 2؉ and was inhibited with function-perturbing anti-E-cadherin antibody. Resistance to suspension-induced apoptosis in cell aggregates paralleled the up-regulation of Bcl-2 but occurred in the absence of focal adhesion kinase activation. Analysis of suspension-induced death in a set of cloned squamous epithelial cell lines with different levels of E-cadherin expression revealed that receptor-positive cell clones evaded apoptosis and proliferated in three-dimensional aggregate culture, whereas cadherin-negative clones failed to survive. Collectively, these observations indicate that cadherin-mediated intercellular adhesions generate a compensatory mechanism that promotes anchorage-independent growth and suppresses apoptosis.

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As beta1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin alpha subunits known to form dimers with beta1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin alpha1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while alpha7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus alpha1 and alpha7 integrins are present in synaptic membranes. Expression of the alpha7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the alpha7 gene (A, B, and C), all of which were expressed in muscle. The alpha7A and alpha7B isoforms were confined to synaptic sites in adult muscle, while alpha7C was present both synaptically and extrasynaptically. In developing muscle, alpha7A appeared postnatally and specifically at the synapse; alpha7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and alpha7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the alpha7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the alpha7 integrins. In normal mice, laminin beta2 is concentrated in synaptic basal lamina. In beta2-null mutant mice, alpha7A was still present at synaptic sites, but alpha7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

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The interactions of tumorigenic and nontumorigenic human and rodent cells with vascular endothelial cells and their underlying extracellular matrix were studied in culture. Gospodarowicz (9)(10)(11). Endothelial cells were used at passage 4-8. BAE cells were routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf bovine serum (Irvine Scientific), and human umbilical cord endothelial cells were cultured in medium 199 plus 20% fetal bovine serum. Fibroblast growth factor was purified as described (11) and added every other day at a concentration of 100-500 ng/ml. At confluency the serum concentration was reduced to 5% and fibroblast growth factor at 5 ng/ml was added every other day.Tumorigenic and nontumorigenic cell lines were obtained and grown as referenced in Table 1. Human foreskin and mouse embryo fibroblasts were obtained from D. Cunningham and cultured in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum.Assays and Electron Microscopy. The adhesion and invasion assays were performed as follows: Completely confluent monolayers of BAE or human umbilical cord endothelial cells were seeded with suspensions of tumorigenic or nontumorigenic cells at 370C in Dulbecco's modified Eagle's medium plus 10% calf serum (or, in the case of human endothelial cells, in medium 199 plus 20% fetal calf serum) at 2-5 X 105 cells per 16-mm culture dish. Single cell suspensions of adherent cell lines were prepared after a 10-to 15-min incubation with 2 mM EDTA in Ca2+,Mg2+-free Dulbecco's phosphate-buffered saline (28). After incubation for various times at 370C, the culture dishes were examined by time-lapse or phase-contrast microscopy (see legend to Table 1). Some of the endothelial monolayers were carefully washed with phosphate buffered saline (28) at 370C by aspiration and fixed in phosphate-buffered saline/1.5% glutaraldehyde for 10 min at 370C and then for 1-3 hr at 220C. The glutaraldehyde-fixed monolayers were prepared for scanning or transmission electron microscopy after postfixation in 1% osmium tetroxide/1 mM CaCl2/0.1 M sodium phosphate, buffer, pH 7.2, for 0.5 hr at room temperature. For scanning electron microscopy, monolayer samples were dehydrated through a graded series of ethanol, transferred to Freon 113, and critical-point dried (29). After they were coated with 50-100 A of gold/palladium (Hummer II, Technics)

Cell adhesion receptors of the integrin family play a major role during re-epithelialization of human wounds. We have previously documented that the expression of alpha v family integrins is induced in keratinocytes of mucosal wounds [1]. In the present investigation, we extended these studies to determine whether alpha v beta 6 integrin is expressed during wound healing in humans. Mucosal and epidermal wound sections from 1- to 7-day-old wounds were used for immunolocalization of integrins and their putative ligands. In addition, freshly isolated epidermal keratinocytes were used to study integrin expression in vitro. Expression of alpha v beta 6 integrin appeared relatively late during mucosal and dermal wound healing. Maximal expression was seen in 7-day-old wounds in which epithelial sheets had fused and granulation tissue was present. Fibronectin and tenascin, both possible ligands for alpha v beta 6 integrin, were found concentrated underneath the basal epithelial cells expressing this receptor, and the maximal expression of tenascin coincided with that of alpha v beta 6 integrin. Freshly isolated epidermal keratinocytes did not stain for alpha v beta 6 integrin but began to express this integrin after subculturing. Our results suggest that the expression of alpha v beta 6 integrin, a putative binding integrin for fibronectin and tenascin, is induced in keratinocytes when epithelial sheets fuse during wound healing.

The survival and growth of squamous epithelial cells require signals generated by integrin-matrix interactions. After conversion to squamous cell carcinoma, the cells remain sensitive to detachment-induced anoikis, yet in tumor cell aggregates, which are matrix-deficient, these cells are capable of suprabasal survival and proliferation. Their survival is enhanced through a process we call synoikis, whereby junctional adhesions between neighboring cells generate specific downstream survival signals. Here we show that in squamous cell carcinoma cells, E-cadherin-mediated cell-cell contacts specifically induce activation of epidermal growth factor receptor (EGFR). EGFR activation in turn triggers the ERK/MAPK signaling module, leading to elevation of anti-apoptotic Bcl-2. After intercellular adhesion, formation of adherens junctions triggers the formation of E-cadherin-EGFR complexes, correlating with EGFR transactivation. Analysis of the process with a dominant-negative EGFR mutant indicated that activation of EGFR is ligand-independent. Our data implicate cell-cell adhesion-induced activation of EGFR as a cooperative mechanism that generates compensatory survival signaling, protecting malignant cells from detachment-induced death.