Increased expression of tenascin-C-binding epithelial integrins in human bullous keratopathy corneas.

We previously found an abnormal deposition of an extracellular matrix glycoprotein, tenascin-C (TN-C), in human corneas with pseudophakic/aphakic bullous keratopathy (PBK/ABK). In this work, we studied cellular TN-C receptors in normal and PBK/ABK corneas. Cryostat sections of normal and PBK/ABK corneas were stained by immuno-fluorescence for TN-C receptors: alpha2, alpha8, alpha9, alphaVbeta3, beta1, and beta6 integrins, and annexin II. Beta6 integrin mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) using beta2-microglobulin gene to normalize the samples. In PBK/ABK compared to normal corneas, relatively minor changes were observed for alpha2 and beta1 integrins, and for annexin II. Alpha8, alpha9, and beta6 subunits of TN-C receptors, alpha8beta1 alpha9beta1, and alphaVbeta6, respectively, were absent from normal central corneas but were found in the central epithelium of PBK/ABK corneas. Beta6 integrin showed the most significant accumulation. It correlated best with the expression of TN-C rather than with the expression of other alphaVbeta6 ligands, fibronectin, and vitronectin. RT-PCR analysis also showed elevated levels of beta6 mRNA in PBK/ABK compared to normal corneas. Therefore, accumulation of TN-C in PBK/ABK corneas was accompanied by an increased expression of its three binding integrins, especially alphaVbeta6 in the corneal epithelium. The interaction of tenascin-C with these integrins may contribute to the fibrotic process that occurs in PBK/ABK corneas.

Role of the alpha(v)beta6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro.

Expression of the alpha(v)beta6 integrin is strikingly upregulated in several types of carcinoma, including human oral squamous cell carcinoma (SCC). Employing a neutralizing monoclonal antibody to alpha(v)beta6, we investigated its role in cell adhesion, proliferation, migration, and in vivo growth of an invasive human SCC line, termed HSC-3. We found that alpha(v)beta6 is strictly required for HSC-3 cell growth in a three-dimensional collagen gel and also prominently contributes to cell migration in two different assay systems. In addition, the anti-alpha(v)beta6 antibody inhibited the invasive growth of HSC-3 cells transorally injected into nude mice. In the presence of the coinjected antibody, the average tumor size at 10 days was reduced by 59%. Histologically, antibody-treated tumors appeared less invasive than control tumors. Furthermore, intravenous application of a neutralizing antibody to the alpha(v) integrin subunit retarded HSC-3 tumor growth. These results point to a possible critical role of the alpha(v)beta6 integrin in controlling growth and invasion of human oral cancer cells.

Distinct roles for astrocyte alphavbeta5 and alphavbeta8 integrins in adhesion and migration.

The alphav integrins are likely to be an important group of molecules for regulating astrocyte behaviour within the central nervous system. Together with their ligand vitronectin, they are expressed by astrocytes in vivo and are further upregulated during neurological disease. Here we have characterised the expression of alphav integrins on primary astrocytes from both rat and mouse, and shown that they express just two members, alphavbeta5 and alphavbeta8. By using RGD peptides and function-blocking antibodies against the beta1 integrins and alphavbeta5, we find that both alphavbeta5 and alphavbeta8 can act as functional receptors for vitronectin. However, while alphavbeta5 is largely responsible for astrocyte adhesion to vitronectin this integrin appears to play no role in migration on vitronectin, with alphavbeta8 playing the dominant role in promoting migration on this substrate. beta1 integrins are not involved in mediating interactions between astrocytes and vitronectin. These results were confirmed in experiments with astrocytes derived from mice in which the beta5 gene had been deleted by homologous recombination. beta5 null astrocytes attached to vitronectin at a reduced rate, but showed no defect in migration on vitronectin relative to wild-type astrocytes. These data provide the first evidence that alphavbeta8 regulates migration and show that astrocyte alphavbeta5 and alphavbeta8 integrins have distinct functions.

Expression of the human integrin beta6 subunit in alveolar type II cells and bronchiolar epithelial cells reverses lung inflammation in beta6 knockout mice.

Inactivation of the integrin beta6 subunit gene in mice resulted in an unexpected phenotype-functionally significant inflammation of the skin and lungs. These findings suggested a role for ligation of the alphav beta6 integrin on epithelial cells in downregulating epithelial inflammation. However, the results of gene inactivation could have been due to inactivation of adjacent genes and provided no information about the role of this integrin in specific populations of epithelial cells. In the current study, we used transgenic mice constitutively expressing the human beta6 subunit in alveolar type II cells and bronchiolar epithelial cells to examine directly the significance of alphav beta6 in these cells. Expression of this transgene largely inhibited the increases in airspace lymphocytes and macrophages and the lymphocyte and macrophage activation caused by inactivation of the beta6 subunit gene, and reduced the peribronchial and perivascular accumulations of lymphocytes. In the genetically mixed mice used for this study, we identified airway eosinophilia as an additional effect of beta6 inactivation. This effect was also partially inhibited by limited expression of the human transgene. These results definitively identify a role for distal lung epithelial alphav beta6 in downregulating pulmonary inflammation and suggest that interventions augmenting beta6 expression or function in these cells could influence the course of inflammatory lung diseases.

Characterization of human fibroleukin, a fibrinogen-like protein secreted by T lymphocytes.

We have recently cloned the human homologue of the murine pT49 cDNA (hpT49h), a transcript encoding a protein homologous to the beta- and gamma-chains of fibrinogen. Here, we report the identification of the hpT49h gene product using mAbs generated against a peptide corresponding to the carboxyl-terminal end of the deduced protein and a recombinant protein fragment expressed in Escherichia coli. mAbs 23A6, 7B12, and 3F4 specifically recognized a protein of 70 kDa in reducing SDS-PAGE in the culture supernatant of 293T cells transiently transfected with the full length hpT49h cDNA and freshly isolated PBMC. Under nonreducing conditions, the material migrated with a molecular mass of 250 to 300 kDa, indicating that the 70-kDa protein forms a disulfide bonded complex. Because of its homology with fibrinogen, we have termed this protein fibroleukin. Fibroleukin is spontaneously secreted in vitro by freshly isolated CD4+ and CD8+ T lymphocytes. RT-PCR analysis revealed preferential expression of fibroleukin mRNA in memory T lymphocytes (CD3+/CD45R0+) compared with naive T lymphocytes (CD3+/CD45RA+). Fibroleukin production by PBMC was rapidly lost in culture. Production could be partially maintained in the presence of IFN-gamma, while T lymphocyte activation had no effect. To demonstrate fibroleukin production in vivo, we analyzed colon mucosa by immunohistology. Fibroleukin staining was detected in the extracellular matrix of the T lymphocyte-rich upper portion of the lamina propria mucosa. While the exact function of fibroleukin remains to be defined, these data suggest that fibroleukin may play a role in physiologic lymphocyte functions at mucosal sites.

Neural precursor cell chain migration and division are regulated through different beta1 integrins.

Proliferation and tangential migration of neural precursor cells are essential determinants of CNS development. We have established cell culture models of both these processes using neural precursor cells grown as neurospheres. The pattern of migration that we observe in these cells is homotypic and occurs in the absence of a glial or neuronal scaffold, and is therefore equivalent to that previously described as chain migration. To determine the role of integrins in proliferation and migration, we have analysed the expression pattern of integrins on neurosphere cells and then performed blocking peptide and antibody experiments. Neurosphere cells express five major integrins, alpha5 beta1, alpha 6Abeta1, alphav beta1, alphav beta5 and alpha vbeta8 and, in addition, express low levels of alpha 6Bbeta1. Chain migration is inhibited by blocking the alpha 6beta1 integrin. Proliferation, by contrast, is inhibited by blocking the other beta1 integrins, alphav beta1 and alpha5 beta1. These results show that integrins are important regulators of neural precursor cell behaviour, with distinct beta1 integrins regulating proliferation and migration. They also demonstrate a novel role for the alpha6 beta1 integrin in the cell-cell interactions underlying homotypic chain migration.

Synaptic and glial localization of the integrin alphavbeta8 in mouse and rat brain.

Integrins are a large family of cell adhesion receptors mediating cell-extracellular matrix (ECM) interactions and are widely distributed in tissues. The beta8 integrin subunit mRNA has been shown to be expressed at higher levels in the central nervous system (CNS) than in other organs [M. Moyle, M.A. Napier, J.W. McLean, Cloning and expression of a divergent integrin subunit beta8, J. Biol. Chem. 266 (29) (1991) 19650-19658] but its cellular and subcellular localization in the CNS are unknown. In this report, we demonstrate that beta8 pairs exclusively with the alphav subunit in the CNS to form the alphavbeta8 heterodimer. Immunohistochemical analysis of the distribution of beta8 in adult mouse and rat brains revealed that the protein is expressed in several regions of the hippocampal formation and in the molecular layer and glomeruli of the granular cell layer of the cerebellum. Punctate and diffuse immunolabeling was observed occasionally surrounding neuronal pericarya and extensively throughout dendritic fields suggesting both pre- and post-synaptic localization and/or expression in non-neuronal cells. By immunoelectron microscopy, beta8 immunoreactivity was detected in dendritic spines where it was often localized at post-synaptic densities, occasionally in axon terminals and in glial processes. Association of beta8 with synaptic membranes was further supported by its enrichment in synaptosomal preparations as detected by immunoblotting. These results demonstrate that alphavbeta8 is present in mature synapses and therefore may play a role in synaptic function.

Tenascin-C matrix assembly in oral squamous cell carcinoma.

We previously showed that the extracellular matrix component tenascin-C (TN-C) is upregulated in oral squamous cell carcinoma (SCC) compared with the normal oral mucosa. In this study we examined oral biopsy specimens of mild to moderate dysplasia or carcinoma in situ to study TN-C expression. We found that carcinoma in situ is the stage at which TN-C becomes widely expressed, suggesting it may be involved in the initial stages of tumor progression. To study TN-C matrix production in vitro, we used an invasive oral SCC cell line (HSC-3) and peri-tumor fibroblasts (PTF). Neither cell type organized a TN-C matrix when cultured alone; however, when co-cultured with HSC-3 cells, PTF were able to assemble a TN-C matrix. PTF retained the ability to organize a TN-C matrix when separated from the HSC-3 cells by a semi-permeable membrane, indicating that cell-cell contact is not necessary for TN-C matrix organization and suggesting that soluble factors may be involved. Moreover, PTF were induced to assemble TN-C matrices when grown in medium conditioned by both the PTF and HSC-3 cells. Antibodies to fibronectin (FN) and to the first FN type III repeat blocked both FN and TN-C matrix assembly, indicating that TN-C matrix organization is dependent on an FN template. Antibodies to alpha5, alphav and beta1 integrins also blocked TN-C matrix formation. When seeded onto FN matrices, the co-cultures were unaffected by the anti-integrin and anti-FN antibodies and were able to organize a TN-C matrix. Our results suggest that progression of malignant oral SCC is accompanied by an alteration of the normal ECM to one rich in TN-C, and that the organization of a TN-C matrix is dependent on soluble cues provided by both the SCC cells and the PTF.

Functional expression of the alpha 7 integrin receptor in differentiated smooth muscle cells.

Expression of the alpha7 integrin is developmentally regulated and is thought to be tissue-specific for both skeletal and cardiac muscles. We now report that alpha7 is also strongly and ubiquitously expressed by various types of smooth muscle, including vascular, gastrointestinal and genitourinary smooth muscles. In addition, alpha7 was surface-expressed by a number of smooth muscle cell lines that maintained their differentiated phenotype following adaptation to culture. Studies with the mouse 9E11G smooth muscle cell line showed that the alpha7 integrin mediated both adhesion and motility of these cells on laminin 1 substrates. Alpha7 expression appears to correlate with the smooth-muscle-differentiated phenotype. The multipotential P19 mouse embryonic stem cell line lacks alpha7 but uses the alpha6 integrin to adhere to laminin 1. Following retinoic acid-induced P19 differentiation predominantly to the smooth muscle cell lineage, high expression of alpha7 was detected along with partial dependence on alpha7 for binding to laminin. The expression of alpha7 paralleled the induction of smooth-muscle-specific alpha-actin, as revealed by dual-labeling flow cytometry. In contrast, alpha7, which initially was highly expressed on the surface of vascular smooth muscle cell explants, was rapidly downregulated in smooth muscle cell outgrowths as they dedifferentiated into their synthetic phenotype. The results indicate that the expression of alpha7 integrin in smooth muscle cells is associated with their differentiated phenotype and mediates their interaction with laminins.

Stromal fibroblasts influence oral squamous-cell carcinoma cell interactions with tenascin-C.

In this study we identified tenascin-C (TN-C) and one of its integrin receptors, alpha(v)beta6, in oral squamous-cell carcinoma (SCC) specimens. Neither TN-C nor alpha(v)beta6 are expressed in normal oral mucosa. We also studied 2 human oral squamous-cell carcinoma cell lines: the highly invasive HSC-3 cells, and the poorly invasive SCC-25 cells. We determined that adhesion of these cells to TN-C involves both alpha2 and alpha(v) integrins. Migration on TN-C by oral SCC cells required fibroblast-conditioned medium and did not occur in its absence. This migration was blocked by anti-alpha2 and anti-alpha(v) antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor-beta1. When seeded on TN-C, the poorly invasive SCC-25 cells formed alpha(v)beta6-positive focal contacts; the HSC-3 cells did not. HSC-3, SCC-25 and PTF cells secrete TN-C into the culture medium, as determined by Western blot. However, when HSC-3 cells were inoculated into the floor of the mouth of nude mice, only murine TN-C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC-3 cells do not secrete TN-C. Our results demonstrate that alpha(v)beta6 and tenascin-C are neo-expressed in oral squamous-cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior.

Protein tyrosine phosphatases mediate cell readhesion in alveolar epithelial cells mechanically separated from in vitro matrix.

Alveolar epithelial type II cells are the progenitor cells for restoring the alveolar epithelial barrier after acute lung injury. During repair of lung injury, the alveolar epithelial type II cells reepithelialize denuded air spaces, a process that involves breaking and reforming cell adhesions. A novel technique of mechanical separation of cultured alveolar epithelial cells from in vitro matrix was used to examine the intracellular signals that result when alveolar epithelial cell adhesions are broken. The results show that the tyrosine phosphorylation levels of focal adhesion kinase, paxillin, and pp60(src) decreased immediately after mechanical separation of the cells. Levels returned to nearly normal by 24 h after mechanical separation. Paxillin and pp60(scr) coprecipitated with focal adhesion kinase regardless of their phosphorylation state. Interestingly, the tyrosine phosphorylation level of the mitogen-activated protein kinase, p42(erk2), increased 15 min after mechanical separation. Preincubation of cell monolayers with phenylarsine oxide, a protein tyrosine phosphatase inhibitor, blocked the decrease in tyrosine phosphorylation levels of focal adhesion kinase, paxillin and pp60(src). Phenylarsine oxide incubation also prevented readhesion of mechanically separated cells at 24 h, but genistein, a tyrosine kinase inhibitor, had no effect. We conclude that protein tyrosine phosphatases are activated immediately after cultured alveolar epithelial cells are mechanically separated from in vitro matrix, and their activation is required for alveolar epithelial cell readhesion.

Division of labor of Schwann cell integrins during migration on peripheral nerve extracellular matrix ligands.

Myelination of the peripheral nervous system (PNS) requires the migration of Schwann cells during both development and regeneration. We have characterized the expression pattern of Schwann cell integrins and analyzed their role in migration on different ECM substrates known to be present within the PNS. We found that Schwann cells in cell culture express four beta1 integrins, alpha1 beta1, alpha2 beta1, alpha6 beta1, and another unidentified beta1 integrin, as well as two alpha v integrins, alpha v beta3 and alpha v beta8. Using the Varani migration assay, we found that laminin-1, laminin-2 (merosin), and fibronectin increased Schwann cell migration, while vitronectin and collagen did not increase migration compared to an uncoated plastic substrate. Schwann cell migration on laminin-1 and laminin-2 (merosin) was blocked by antibodies against beta1 integrins, but not affected by RGD peptides or antibodies against beta3 integrins. In contrast, migration on fibronectin was unaffected by antibodies against beta1 and beta3 integrins but was blocked by RGD peptides. This in vitro study shows that there is a division of labor of Schwann cell integrins in the regulation of migration on peripheral nerve ECM components; beta1 integrins mediate migration on laminin-1 and laminin-2 (merosin), while alpha v integrins mediate migration on fibronectin. Taken together, these results suggest that multiple interactions between Schwann cell integrins and ECM within the PNS will contribute to Schwann cell migration during myelination of the PNS.

Expression of alpha vbeta3 and alpha vbeta8 integrins during oligodendrocyte precursor differentiation in the presence and absence of axons.

We have shown previously that switching of the alpha v-associated beta1 and beta5 integrin subunits during differentiation of myelin-forming oligodendrocytes may regulate important aspects of cell behaviour such as migration (Milner et al., 1996: J Neurosci 16:7240-7252). In this study we have examined the developmental regulation of other alpha v-associated beta subunits in oligodendroglial cell cultures and also the control of their expression by neurons, using xenocultures to distinguish glial and neuronal integrins. We have found that oligodendroglia express alpha vbeta8 in addition to the previously-described alpha vbeta1, alpha vbeta3, and alpha vbeta5. Beta8 and beta3 together comprise the 80kD band seen in alpha v immunoprecipitations that represents the most abundant alpha v-associated beta subunit and show reciprocal patterns of expression during development. Alpha vbeta8 is expressed at high levels on oligodendrocyte precursors and differentiated oligodendrocytes but diminishes during the intermediate stages of differentiation. Alpha vbeta3, in contrast, shows an opposite pattern of expression, with the highest levels seen at the intermediate stages of differentiation and little expression on either oligodendrocyte precursors and differentiated oligodendrocytes. The expression of alpha vbeta3 is not altered by coculture with neurons, unlike that of alpha vbeta8, in which the decrease seen at the intermediate stages of differentiation is less marked in the presence of neurones. Our results confirm that switching of alpha v-associated beta subunits is an important feature of oligodendrocyte differentiation and suggest that alpha vbeta8 and alpha vbeta3 have distinct functions during myelination.

Osteopontin N-terminal domain contains a cryptic adhesive sequence recognized by alpha9beta1 integrin.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

Changes in endothelial cell and smooth muscle cell integrin expression during closure of the ductus arteriosus: an immunohistochemical comparison of the fetal, preterm newborn, and full-term newborn rhesus monkey ductus.

Anatomical closure of the ductus arteriosus requires normally quiescent luminal endothelial cells and medial smooth muscle cells to migrate into the subendothelial space forming intimal mounds that eventually coalesce and occlude the vessel's lumen. The migration of endothelial cells and smooth muscle cells requires the presence of integrin receptors that interact with the surrounding matrix. We used immunohistochemical staining to examine the repertoires of integrins expressed by endothelial cells and smooth muscle cells during postnatal closure of the ductus arteriosus in full-term and preterm rhesus monkeys. In the fetal ductus, luminal endothelial cells have a limited repertoire of integrins. During postnatal ductus closure, luminal endothelial cells, of both term and preterm monkeys, change their phenotype and express the full repertoire of integrins found on growing capillary endothelial cells (alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha v beta 1, alpha 6 beta 4, and alpha v beta 5). Similarly, during ductus closure, smooth muscle cells of both term and preterm monkeys expand their integrin repertoire to include the alpha 5 beta 1 and alpha v beta 3 integrins; these two integrins have been shown to be essential for smooth muscle cell migration in vitro. These changes in integrin profile occur at the same time the endothelial and smooth muscle cells invade their neighboring compartments. In contrast, preterm monkeys with a persistently patent ductus lumen fail to develop these changes in integrin expression and fail to develop neointimal mounds. No evidence of intimal thickening occurs in the absence of changes in integrin expression. Therefore, endothelial cells and smooth muscle cells change phenotypes to produce the intimal thickening required for ductus closure.

The human integrin alpha 8 beta 1 functions as a receptor for tenascin, fibronectin, and vitronectin.

The integrin family of adhesion receptors consists of at least 21 heterodimeric transmembrane proteins that differ in their tissue distribution and ligand specificity. The recently identified alpha 8 integrin subunit associates with beta 1 and is predominantly expressed in smooth muscle and other contractile cells in adult tissues, and in mesenchymal and neural cells during development. We now show that alpha 8 beta 1 specifically localizes to focal contacts in cells plated on the extracellular matrix proteins fibronectin or vitronectin. In addition we show that human embryonic kidney cells (293), transfected with alpha 8 cDNA, express alpha 8 beta 1 on their surface and use this receptor for adhesion to fibronectin and vitronectin. Furthermore, alpha 8 beta 1 binds to both fibronectin- and vitronectin-Sepharose and can be specifically eluted from either matrix protein by the arginine-glycine-aspartic acid (RGD)-containing peptide, GRGDSP. Because fibronectin and vitronectin adhesion appeared to be mediated by RGD, we examined additional RGD-containing proteins, including tenascin, fibrinogen, thrombospondin, osteopontin, and denatured collagen type I. We found that only tenascin was able to mediate adhesion of alpha 8-transfected 293 cells. By using recombinant fragments of tenascin in adhesion assays, we were able to localize the alpha 8 beta 1 binding domain of tenascin to the RGD-containing third fibronectin type III repeat. These data strongly suggest that tenascin, fibronectin, and vitronectin are ligands for alpha 8 beta 1 and that this integrin binds to the RGD site in each of these ligands through mechanisms that are distinct and separate from alpha 5- and alpha v-containing integrins.

Sequence of a human transcript expressed in T-lymphocytes and encoding a fibrinogen-like protein.

A murine cDNA sequence (termed pT49), encoding a protein related to the fibrinogen (Fib) beta and gamma chains, has been previously reported [Koyama et al., Proc. Natl. Acad. Sci. USA 84 (1987) 1609-1613]. We used a murine pT49 probe to screen a human small intestine cDNA library, and obtained a set of overlapping cDNA clones encoding a Fib-like protein that is 80% identical with the product of the murine pT49 gene. The deduced amino acid (aa) sequence contains a predicted signal peptide and five consensus motifs for N-linked glycosylation. The presence of conserved Cys residues involved in the assembly of the mature Fib complex and of two alpha-helical regions permissive for coiled-coil formation, suggests that this Fib-like protein may be secreted as a multichain complex. Two mRNA species of approx. 4.5 and approx. 1.5 kb were detected by Northern blot hybridization of a human pT49-homolog cDNA probe with RNA obtained from resting peripheral blood T-lymphocytes. By RT-PCR analysis of purified peripheral blood T-lymphocyte subsets, we found expression of the pT49-homolog transcript in both CD3+/CD4+ and CD3+/CD8+ T-lymphocytes. The coding region of the human pT49-homolog cDNA was fused at its 3' end with a tag-coding sequence and was expressed in CHO cells. The corresponding gene product was immunoprecipitated with an anti-tag antibody from the cell lysate and from the culture supernatant of metabolically labeled transfectants and was identified as approx. 62 and approx. 64-70-kDa proteins, respectively.

Transforming growth factor beta 1 inhibits fetal lamb ductus arteriosus smooth muscle cell migration.

Anatomical closure of the ductus arteriosus (DA) requires normally quiescent smooth muscle cells (SMC) to migrate out of the muscle media into the subendothelial space, forming intimal mounds that eventually coalesce to occlude the vessel's lumen. Transforming growth factor-beta 1 (TGF beta 1), a potent modulator of vascular SMC migration, is found in the wall of the closing DA. We examined the effect of TGF beta 1 on the migration of fetal lamb DA-SMC. Although TGF beta 1 has been shown to be a chemoattractant for other mesenchymal cells, it had no chemotactic effect on DA-SMC; furthermore, TGF beta 1 did not enhance the migration of DA-SMC (as has been reported for aortic SMC). Rather, incubating DA-SMC with TGF beta 1 for 22 h decreased the rate of migration of SMC on extracellular matrix substrata composed of fibronectin, vitronectin, laminin, and collagen I and IV. Exposure of DA-SMC to TGF beta 1 was associated with an increase in the formation of focal adhesion plaques (tight associations between the cells' surface and extracellular matrix). DA-SMC use integrin receptors to attach to and migrate on extracellular matrix components. The decrease in DA-SMC migration was not associated with a significant change in the profile of integrin receptors expressed by the cell. TGF beta 1 had little effect on overall DA-SMC integrin expression, except for a modest increase in the fibronectin receptor (alpha 5 beta 1 integrin). Rather, the decrease in migration and changes in cell morphology were associated with an increased ability of integrin receptors to associate with the cytoskeleton. TGF beta 1 appears to anchor the cell's cytoskeleton to the extracellular matrix, making the cells more adherent and less capable of migrating.

Sequence and tissue distribution of the human integrin alpha 8 subunit: a beta 1-associated alpha subunit expressed in smooth muscle cells.

Integrins are a major family of cell adhesion molecules involved in cell-cell and cell-extracellular matrix interactions. Each integrin is a heterodimeric glycoprotein composed of an alpha and a beta subunit. We now report the cDNA sequence and distribution of a new human integrin alpha subunit. This sequence is 78% identical to the previously reported chicken alpha 8 integrin sequence. Thus, we have designated this subunit as human alpha 8. By northern blot analysis, an alpha 8 probe detects two mRNA species of approximately 6.5 and 4.0 kb in neuroglioma H4 cells. An anti-alpha 8 polyclonal antibody precipitates a protein complex containing the beta 1 subunit associated with the putative alpha 8 subunit, which has an apparent molecular mass of 180 kDa (non-reduced) or 155 kDa and 25 kDa (reduced). Immunohistochemistry with anti-alpha 8 polyclonal antibody in adult rat tissues shows prominent staining in vascular and visceral smooth muscle. In addition, the antibody strongly stained kidney mesangial cells and a cell type in the alveolar wall of the lungs, most likely corresponding to alveolar myofibroblasts. These results suggest that in adult mammalian tissues, alpha 8 is predominantly expressed in smooth muscle and smooth muscle-like contractile cells.

Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model.

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.