Beta 1 and beta 3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix.

Extracellular matrix receptors on ductus arteriosus smooth muscle cells (SMC) must enable the cells to migrate through both interstitial and basement membrane matrices to form intimal mounds during postnatal ductus closure. We examined the role of beta 1 and beta 3 integrin receptors on SMC adhesion and migration. Using a new assay to measure cell migration, we found that lamb ductus arteriosus SMC attach to and migrate over surfaces coated with fibronectin (FN), laminin (LN), vitronectin (VN), and collagens I (I) and IV (IV). Blocking antibodies, specific to different integrin complexes, showed that SMC adhesion to FN, LN, I, and IV depended exclusively on functioning beta 1 integrins with little, if any, contribution by the alpha V beta 3 integrin; on the other hand, cell migration over these substrates depended to a large extent on the alpha V beta 3 receptor. Immunofluorescent staining demonstrated that during the early phase of SMC migration, the beta 1 integrins organized rapidly into focal plaques that, with time, gradually covered the cell's basal surface; on the other hand, the beta 3 receptor remained concentrated at all times at the cell's margins. Ligand affinity chromatography and immunoprecipitation techniques identified a unique series of beta 1 integrins binding to each matrix component: FN (alpha 5 beta 1, alpha 3 beta 1, alpha V beta 1), LN (alpha 1 beta 1, alpha 7 beta 1), VN (alpha V beta 1), I (alpha 1 beta 1, alpha 2 beta 1), and IV (alpha 1 beta 1). In contrast, the beta 3 integrin, alpha V beta 3, bound to all the substrates tested: FN, LN, VN, I, and IV. The results indicate that beta 1 and beta 3 integrins may play different roles in attachment and migration as SMC move through the vascular extracellular matrix to produce obliteration of the ductus arteriosus lumen.     

Integrin αvβ3, metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect

Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin α_vβ₃, evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the α_vβ₃ blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin α_vβ₃ interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin α_vβ₃, uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin α_vβ₃ and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.     

An abundant chick gizzard integrin is the avian α1β1 integrin heterodimer and functions as a divalent cation-dependent collagen IV receptor

The α-subunit of an abundant chick gizzard integrin was isolated ([12.], J. Biol. Chem. 262, 17,189–17,199) and fragmented by proteolytic digestion. The N-terminal sequences of the intact polypeptide and of several internal peptides were determined and were found to be highly homologous to the mammalian integrin α₁-subunit. Monoclonal antibodies to the chick integrin β₁-chain react on immunoblots with the gizzard integrin β-subunit ([28.], J. Biol. Chem. 265, 14,561–14,565). The chain composition of the abundant chick gizzard integrin is therefore α₁β₁. Polyclonal antibodies to the avian integrin α₁-subunit block attachment of embryonic gizzard cells to human and chick collagen IV completely and inhibit attachment to mouse Engelbreth-Holm-Swarm (EHS) tumor laminin partially. In ELISA-style receptor assays, the isolated α₁β₁ integrin bound to human and chick collagen IV and to mouse EHS tumor and chick heart laminin. While the binding to collagen IV was abolished by removal of divalent cations, the binding to laminin was not sensitive to EDTA under the conditions used. Collagen I bound the isolated avian α₁β₁ integrin only weakly. As collagen IV was the only extracellular matrix protein for which a consistent, divalent cation-dependent, binding to the avian α₁β₁ integrin could be demonstrated in both cellular and molecular assays we suggest that it is a preferred ligand for this integrin.     

αvβ3 Integrin Mediates the Cell-adhesive Capacity and Biological Activity of Basic Fibroblast Growth Factor (FGF-2) in Cultured Endothelial Cells

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38–61 and 82–101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-α_vβ₃ antibodies prevent cell adhesion to FGF-2. Also, purified human α_vβ₃ binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Finally, anti-α_vβ₃ monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with α_vβ₃ integrin and that this interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells.     

Osteopontin-Stimulated Vascular Smooth Muscle Cell Migration Is Mediated by β3 Integrin

Osteopontin (OPN), a 41-kDa phosphorylated glycoprotein, has been detected in rat aorta and carotid arteries, and expression of its mRNA in blood vessels is strongly increased in response to vascular injury. To investigate the potential role of OPN in vascular pathophysiology, we studied the effect of rat OPN on aortic smooth muscle cell migration and proliferation in vitro. OPN enhanced the migration of rat smooth muscle cells in a time- and concentration-dependent manner with an EC₅₀ value of 46 ± 11 nmol/liter (n = 5). The maximal increase in cell migration by OPN was 29-fold over basal levels. OPN-induced smooth muscle cell migration was inhibited in a concentration-dependent manner by the monoclonal antibody F11, which recognizes the rat integrin subunit β₃. In contrast, polyclonal antiserum recognizing the rat integrin β₁ subunit did not inhibit smooth muscle cell migration in response to OPN, but did block fibronectin-promoted migration. Moreover, OPN-induced smooth muscle cell migration was dependent on the presence of extracellular divalent cations and was significantly inhibited by anti-OPN antibodies. OPN did not stimulate [³H]thymidine incorporation into cultured smooth muscle cells, indicating that it selectively enhanced migration. In view of the pathological significance of arterial smooth muscle cell migration in the formation of intimal thickening, our results suggest that smooth muscle cell recognition of OPN, probably through the vitronectin receptor, α_vβ₃, could play a role in the cells' response to vascular injury and especially neointima formation.     

Regulation of Tenascin-C, a Vascular Smooth Muscle Cell Survival Factor that Interacts with the αvβ3 Integrin to Promote Epidermal Growth Factor Receptor Phosphorylation and Growth

Tenascin-C (TN-C) is induced in pulmonary vascular disease, where it colocalizes with proliferating smooth muscle cells (SMCs) and epidermal growth factor (EGF). Furthermore, cultured SMCs require TN-C for EGF-dependent growth on type I collagen. In this study, we explore the regulation and function of TN-C in SMCs. We show that a matix metalloproteinase (MMP) inhibitor (GM6001) suppresses SMC TN-C expression on native collagen, whereas denatured collagen promotes TN-C expression in a β3 integrin– dependent manner, independent of MMPs. Floating type I collagen gel also suppresses SMC MMP activity and TN-C protein synthesis and induces apoptosis, in the presence of EGF. Addition of exogenous TN-C to SMCs on floating collagen, or to SMCs treated with GM6001, restores the EGF growth response and “rescues” cells from apoptosis. The mechanism by which TN-C facilitates EGF-dependent survival and growth was then investigated. We show that TN-C interactions with α_vβ₃ integrins modify SMC shape, and EGF- dependent growth. These features are associated with redistribution of filamentous actin to focal adhesion complexes, which colocalize with clusters of EGF-Rs, tyrosine-phosphorylated proteins, and increased activation of EGF-Rs after addition of EGF. Cross-linking SMC β₃ integrins replicates the effect of TN-C on EGF-R clustering and tyrosine phosphorylation. Together, these studies represent a functional paradigm for ECM-dependent cell survival whereby MMPs upregulate TN-C by generating β₃ integrin ligands in type I collagen. In turn, α_vβ₃ interactions with TN-C alter SMC shape and increase EGF-R clustering and EGF-dependent growth. Conversely, suppression of MMPs downregulates TN-C and induces apoptosis.     

β1 Integrin-mediated collagen gel contraction is stimulated by PDGF

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent β₁ integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-β1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin β₁-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-β₁ integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the β₁ integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.     

The β4Integrin Subunit Is Expressed in Mouse Fibroblasts and Modulated by Transforming Growth Factor-β1

Integrin β₄subunit is present in association with α₆chain on both normal and transformed epithelial cells. Recently α₆β₄heterodimer was found on the endothelium of medium-sized blood vessels and on immature thymocytes. In this report we show, by Northern blotting, indirect immunofluorescence, immunoprecipitation, and Western blotting, that β₄subunit is expressed also on cells of mesenchymal origin such as fibroblasts, myoblasts, and myotubes. Increased expression of α₆β₄has been related to the aggressive metastatic phenotype of human and murine carcinomas. The transforming growth factor β₁(TGF-β₁) has been found to modulate the expression of several integrins and intracellular matrix proteins, as well as to stimulate cell invasion and metastatic potential. To evaluate whether α₆β₄expression is modulated by TGF-β₁, we transfected 3T3 fibroblasts with an expression vector carrying the human TGF-β₁cDNA driven by the SV40 early promoter. We observed by indirect immunofluorescence a modification in the subcellular distribution of β₄subunit, which acquires a perinuclear localization. This finding suggests this integrin subunit correlates with the cytoskeletal reorganization induced by TGF-β₁.     

Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Disruption of integrin α5β1 signaling does not impair PDGF-BB-mediated stimulation of the extracellular signal-regulated kinase pathway in smooth muscle cells

Smooth muscle cell (SMC) proliferation is dependent on both anchorage to the extracellular matrix by integrins and the presence of growth factors. Integrins and growth factor receptors transduce signals that seem to converge on the extracellular signal-regulated (ERK) pathway, but the molecular basis for this interaction is not known. SMC proliferation has previously been shown to be supported by culture on fibronectin (FN), whereas cells cultured on laminin (LN) are growth inhibited. In the present study, we examined the mitogenic response to platelet-derived growth factor BB (PDGF-BB) in baboon SMCs cultured on FN vs. LN. Induction of DNA synthesis and the activity of ERK and the ERK activating kinase MKK-1 were reduced only slightly after stimulation with PDGF-BB in cells cultured on LN vs. those cultured on FN. We tested the possibility that endogenous FN secretion contributes to the ability of the cells to respond to PDGF stimulation during culture on LN. Inhibition of interactions between FN and integrin α₅β₁ by the competitive GRGDSP-peptide or anti-α₅ integrin antibody restricted cell spreading, reduced cell-surface staining for α₅β₁ and FN fibrils, and inhibited PDGF-BB-induced DNA synthesis. These results showed that SMC growth on LN required a provisional FN matrix. Although disruption of interactions between α₅β₁ and FN by the GRGDSP-peptide prevented PDGF-BB-induced DNA synthesis, neither ERK activity nor translocation of ERKs into the nucleus was inhibited. These results show that integrins regulate SMC growth through pathways that function in parallel with, but distinct from, growth factor-mediated ERK signaling. J. Cell. Physiol. 172:109–116, 1997. © 1997 Wiley-Liss, Inc.     

PGE2 is a more potent vasodilator of the lamb ductus arteriosus than is either PGI2 or 6 keto PGFα

It has been shown in vitro that the lamb ductus arteriosus forms prostaglandins PGE₂, PGF_2α, 6 keto PGF_1α (and its unstable precursor PGI₂). In this study the relative potencies of these endogenous prostaglandins were investigated on isolated lamb ductus arteriosus preparations contracted by exposure to elevated PO₂ and indomethacin. All the prostaglandins (except PGF_2α) relaxed the vessel. This is consistent with the hypothesis that endogenous prostaglandins inhibit the tendency of the vessel to contract in response to oxygen. Only PGE₂, however, relaxed the vessel at concentrations below 10⁻⁸M. PGI₂ and 6 keto PGF_1α had approximately 0.001 and 0.0001 times the activity of PGE₂. Although PGE₂ has been observed to be a minor product of prostaglandin production in the lamb ductus arteriosus, the tissue's marked sensitivity to PGE₂ might make it the most significant prostaglandin in regulating the patency of the vessel.     

Activation of Integrin αVβ3 Regulates Cell Adhesion and Migration to Bone Sialoprotein

α_Vβ₃, a broadly distributed member of the integrin family of adhesion receptors, has been implicated in a variety of physiological and pathophysiological events, including control of bone density, angiogenesis, apoptosis, tumor growth, and metastasis. Recently, it has been shown that activation of α_Vβ₃, its transition from a low- to a high-affinity/avidity state, influences its recognition of certain ligands. Bone sialoprotein (BSP) is recognized as an important ligand for α_Vβ₃ in processes ranging from bone formation to the homing of metastatic tumor cells. Here, the influence of α_Vβ₃ activation on the adhesion and migration of relevant cells to BSP has been examined. Stimulation of lymphoblastoid, osteoblastoid, and human umbilical vein endothelial cells (HUVEC) with PMA or Mn²⁺ markedly enhanced α_Vβ₃-dependent adhesion to BSP. α_Vβ₃-mediated migration of HUVEC or osteoblastic cells to BSP was substantially enhanced by stimulation, demonstrating that α_Vβ₃ activation enhances both adhesive and migratory responses. However, adhesion and/or migration of certain tumor cell lines, including M21 melanoma and MDA MB435 and SKBR3 breast carcinoma cell lines, to BSP was constitutively high and was not augmented by α_Vβ₃-activating stimuli. Inhibitors of the intracellular signaling molecules, phosphatidylinositol 3-kinase with wortmannin, hsp90-dependent kinases with geldanamycin, and calpain with calpeptin, but not MAPKK with PD98059, reduced the high spontaneous adhesion and migration of the M21 cells to BSP, consistent with the constitutive activation of the receptor on these tumor cells. These results indicate that the activation state of α_Vβ₃ can regulate cell migration and adhesion to BSP and, by extension, to other ligands of this receptor. The constitutive activation of α_Vβ₃ on neoplastic cells may contribute to tumor growth and metastatic potential.     

Levels of the bcl-2 protein, fibronectin and α5β1 fibronectin receptor in HIV-1-infected patients with Kaposi’s sarcoma

Kaposi's sarcoma (KS) is an angioproliferative disease characterized by proliferation of neoplastic cells (spindle cells) mixed with endothelial and inflammatory cells. In this study we evaluated the role of the adhesive glycoprotein, fibronectin (FN) and its receptor α₅β₁ (FNR), and the proto-oncogene bcl-2, an anti-apoptotic protein. Significantly decreased serum levels of FN were noted in HIV-1-infected patients with KS, whereas serum levels of FNR were significantly increased in the same patients. Furthermore, increased FNR expression was observed on CD4 cells from KS patients. Serum levels of bcl-2 protein were significantly decreased in asymptomatic seropositive patients, whereas HIV-1-infected patients with KS showed increased serum levels of bcl-2. These results provide further information about interaction between integrins and the extracellular matrix and bcl-2 protein that can support cell survival either of neoplastic cells or endothelial and inflammatory cells.     

A simple method for preparation of valency hybrid hemoglobins, (αβ)2 and (αβ)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

Human G Protein γ11 and γ14 Subtypes Define a New Functional Subclass

The mammalian γ subunit family consists of a minimum of 12 members. Analysis of the amino acid sequence conservation suggests that the γ subunit family can be divided into three distinct subclasses. The division of the γ subunit family into these classes is based not only on amino acid homology, but also to some extent on functional similarities. In the present study, two new members of the γ subunit family, the γ₁₁ and γ₁₄ subunits, are identified and characterized in terms of their expression and function. The γ₁₁ and γ₁₄ subunits are most closely related to the γ₁ subunit and share similar biochemical properties, suggesting their inclusion in class I. However, despite their close phylogenetic relationship and similar biochemical properties, the γ₁, γ₁₁, and γ₁₄ subunits exhibit very distinct expression patterns, suggesting that class I should be further subdivided and that the signaling functions of each subgroup are distinct. In this regard, the γ₁₁ and γ₁₄ subunits represent a new subgroup of farnesylated γ subunits that are expressed outside the retina and have functions other than phototransduction.     

Mapping of the α4 subunit gene (GABRA4) to human chromosome 4 defines an α2—α4—β1—γ1 gene cluster: further evidence that modern GABAA receptor gene clusters are derived from an ancestral cluster

We demonstrated previously that an α₁—β₂—γ₂ gene cluster of the γ-aminobutyric acid (GABA_A) receptor is located on human chromosome 5q34–q35 and that an ancestral α—β—γ gene cluster probably spawned clusters on chromosomes 4, 5, and 15. Here, we report that the α₄ gene (GABRA4) maps to human chromosome 4p14–q12, defining a cluster comprising the α₂, α₄, β₁, and γ₁ genes. The existence of an α₂—α₄—β₁—γ₁ cluster on chromosome 4 and an α₁—α₆—β₂—γ₂ cluster on chromosome 5 provides further evidence that the number of ancestral GABA_A receptor subunit genes has been expanded by duplication within an ancestral gene cluster. Moreover, if duplication of the α gene occurred before duplication of the ancestral gene cluster, then a heretofore undiscovered subtype of α subunit should be located on human chromosome 15q11–q13 within an α₅—α_x—β₃—γ₃ gene cluster at the locus for Angelman and Prader—Willi syndromes.     

Integrin α5 during early development of Xenopus laevis

The full length sequence of the Xenopus integrin α₅ subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of α₅ mRNA occurs in the embryo. Furthermore, a variant form of the α₅ mRNA is expressed which encodes an integrin α₅ subunit with a truncated cytoplasmic domain. Integrin α₅ mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of α₅ mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the α₅ protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin α₅ subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of α subunit expression in early vertebrate embryos.     

Differential Modulation of Integrin Receptors and Extracellular Matrix Laminin by Transforming Growth Factor-β1 in Rat Alveolar Epithelial Cells

The transforming growth factors-β (TGFs-β) family of genes plays important roles in cell growth and differentiation in many cell types. TGFβ modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGFβ is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGFβ may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGFβ on modulation of laminin and its integrin receptors α₆β₁ and α₃β₁ in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGFβ₁ (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin β2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGFβ₁ induced dose-dependent increases in α₆ and β₁ mRNA levels. TGFβ₁ also increased the expression of laminin β2 chain mRNA at 12-16 h poststimulation. In contrast, TGFβ decreased α₃ mRNA expression. Immunoprecipitation studies of TGFβ₁-treated cells showed increased surface expression of both α₆ and β₁ protein while surface expression of the α₃ integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGFβ₁ may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.     

Foot-and-Mouth Disease Virus Virulent for Cattle Utilizes the Integrin αvβ3 as Its Receptor

Adsorption and plaque formation of foot-and-mouth disease virus (FMDV) serotype A₁₂ are inhibited by antibodies to the integrin α_vβ₃ (A. Berinstein et al., J. Virol. 69:2664–2666, 1995). A human cell line, K562, which does not normally express α_vβ₃ cannot replicate this serotype unless cells are transfected with cDNAs encoding this integrin (K562-α_vβ₃ cells). In contrast, we found that a tissue culture-propagated FMDV, type O₁BFS, was able to replicate in nontransfected K562 cells, and replication was not inhibited by antibodies to the endogenously expressed integrin α₅β₁. A recent report indicating that cell surface heparan sulfate (HS) was required for efficient infection of type O₁ (T. Jackson et al., J. Virol. 70:5282–5287, 1996) led us to examine the role of HS and α_vβ₃ in FMDV infection. We transfected normal CHO cells, which express HS but not α_vβ₃, and two HS-deficient CHO cell lines with cDNAs encoding human α_vβ₃, producing a panel of cells that expressed one or both receptors. In these cells, type A₁₂ replication was dependent on expression of α_vβ₃, whereas type O₁BFS replicated to high titer in normal CHO cells but could not replicate in HS-deficient cells even when they expressed α_vβ₃. We have also analyzed two genetically engineered variants of type O₁Campos, vCRM4, which has greatly reduced virulence in cattle and can bind to heparin-Sepharose columns, and vCRM8, which is highly virulent in cattle and cannot bind to heparin-Sepharose. vCRM4 replicated in wild-type K562 cells and normal, nontransfected CHO (HS⁺ α_vβ_3⁻) cells, whereas vCRM8 replicated only in K562 and CHO cells transfected with α_vβ₃ cDNAs. A similar result was also obtained in assays using a vCRM4 virus with an engineered RGD→KGE mutation. These results indicate that virulent FMDV utilizes the α_vβ₃ integrin as a primary receptor for infection and that adaptation of type O₁ virus to cell culture results in the ability of the virus to utilize HS as a receptor and a concomitant loss of virulence.