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Fibronectin (Fn) plays an important part in the branching morphogenesis of salivary gland, lung, and kidney. Here, we examine the effect of the conditional knockout of Fn in the mammary epithelium [Fn(MEp-/-)] on postnatal mammary gland development, using Cre-loxP-mediated gene knockout technology. Our data show that Fn deletion causes a moderate retardation in outgrowth and branching of the ductal tree in 5-week-old mice. These defects are partially compensated in virgin 16-week-old mice. However, mammary glands consisting of Fn-deficient epithelial cells fail to undergo normal lobuloalveolar differentiation during pregnancy. The severity of lobuloalveolar impairment ranged from lobular hypoplasia to aplasia in some cases and was associated with the amount of Fn protein recovered from these glands. Decreased rates of mammary epithelial cell proliferation accounted for delayed ductal outgrowth in virgin and lack of alveologenesis in pregnant Fn(MEp-/-) mice. Concomitant decreased expression of integrin beta(1) (Itgb1) and lack of autophosphorylation of focal adhesion kinase (Fak) suggest that this pathology might, at least in part, be mediated by disruption of the Fn/Itgb1/Fak signaling pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

We have recently demonstrated that ionizing radiation (IR) of cells increased the expression of beta-galactoside alpha-(2,6)-sialyltransferase (ST6Gal I) and the level of glycoprotein sialylation, especially for the key adhesion molecule integrin beta1. In addition, ST6Gal I-mediated sialylation of integrin beta1 contributed to cell adhesion-mediated radioresistance in colon cancer cells. In this study, we examined IR-induced cell adhesion to the extracellular matrix and evaluated the role of integrin beta1-associated downstream signaling molecules, such as paxillin and AKT. IR exposure and ST6Gal I overexpression increased adhesion of SW480 colon cancer cells to fibronectin and contributed to cell survival through the activation of paxillin and AKT. In contrast, knockdown of ST6Gal I or paxillin reduced the level of radiation-induced cell adhesion and increased the level of cell death. These results suggest that integrin beta1 sialylation may play a critical role in promoting adhesion of cancer cells by integrin beta1-mediated paxillin and AKT activation.

We recently showed that extracellular matrix (ECM) proteins, which are abundant in desmoplastic pancreatic tumor, are as potent as growth factors in inhibiting apoptosis in pancreatic cancer (PaCa) cells. Here we show that fibronectin, a major ECM component, engages insulin-like growth factor-I receptor (IGF-IR) to inhibit PaCa cell death. We found that fibronectin-induced protection from apoptosis is fully mediated by IGF-IR and is independent of IGF-I. Pharmacologic and molecular inhibitions of IGF-IR stimulated apoptosis and prevented the prosurvival effect of fibronectin in PaCa cells. Our data indicate that fibronectin protects from apoptosis through trans-activation of IGF-IR. We showed that fibronectin stimulated complex formation between its receptor beta3 integrin and protein-tyrosine phosphatase SHP-2. This process of complex formation, in turn, prevents SHP-2 from dephosphorylating IGF-IR resulting in sustained phosphorylation of IGF-IR and leading to the downstream activation of Akt kinase, up-regulation of antiapoptotic Bcl(xL), and inhibition of apoptosis. Among ECM proteins tested only fibronectin and laminin but not vitronectin and collagen I stimulated trans-activation of IGF-IR. Interaction of fibronectin with beta3 but not beta1 integrin receptors mediates the survival pathway. In contrast, fibronectin-induced adhesion is mediated through beta1 integrin receptor and is IGF-IR-independent. Thus, our results indicate that the prosurvival effect of fibronectin in PaCa cells is mediated by trans-activation of IGF-IR induced by the beta3 integrin receptor. The data suggest IGF-IR as a key target for prevention of the prosurvival effects of ECM proteins and growth factors in pancreatic cancer.

Hematopoietic cells isolated from patients with Bcr-Abl-positive leukemia exhibit multiple abnormalities of cytoskeletal and integrin function. These abnormalities are thought to play a role in the pathogenesis of leukemia; however, the molecular events leading to these abnormalities are not fully understood. We show here that the Abi1 pathway is required for Bcr-Abl to stimulate actin cytoskeleton remodeling, integrin clustering and cell adhesion. Expression of Bcr-Abl induces tyrosine phosphorylation of Abi1. This is accompanied by a subcellular translocation of Abi1/WAVE2 to a site adjacent to membrane, where an F-actin-enriched structure containing the adhesion molecules such as beta1-integrin, paxillin and vinculin is assembled. Bcr-Abl-induced membrane translocation of Abi1/WAVE2 requires direct interaction between Abi1 and Bcr-Abl, but is independent of the phosphoinositide 3-kinase pathway. Formation of the F-actin-rich complex correlates with an increased cell adhesion to fibronectin. More importantly, disruption of the interaction between Bcr-Abl and Abi1 by mutations either in Bcr-Abl or Abi1 not only abolished tyrosine phosphorylation of Abi1 and membrane translocation of Abi1/WAVE2, but also inhibited Bcr-Abl-stimulated actin cytoskeleton remodeling, integrin clustering and cell adhesion to fibronectin. Together, these data define Abi1/WAVE2 as a downstream pathway that contributes to Bcr-Abl-induced abnormalities of cytoskeletal and integrin function.

Previous studies have indicated that the urokinase-type plasminogen activator receptor (uPAR) can functionally interact with integrins thereby modulating integrin activity. We have previously demonstrated that treatment of fibroblasts with the uPAR ligand, P25, results in an increase in the activation of the beta1 integrin and a 35-fold increase in fibronectin matrix assembly (Monaghan, E., Gueorguiev, V., Wilkins-Port, C., and McKeown-Longo, P. J. (2004) J. Biol. Chem. 279, 1400-1407). Experiments were conducted to address the mechanism of uPAR regulation of matrix assembly. Treatment of fibroblasts with P25 led to an increase in the activation of the epidermal growth factor receptor (EGFR) and a colocalization of activated EGFR with beta1 integrins in cell matrix contacts. The effects of P25 on matrix assembly and beta1 integrin activation were inhibited by pretreatment with EGFR or Src kinase inhibitors, suggesting a role for both Src and EGFR in integrin activation by uPAR. Phosphorylation of EGFR in response to P25 occurred on Tyr-845, an Src-dependent phosphorylation site and was inhibited by PP2, the Src kinase inhibitor, consistent with Src kinase lying upstream of EGFR and integrin activation. Cells null for Src kinases also showed a loss of P25-induced matrix assembly, integrin activation, and EGFR phosphorylation. These P25-induced effects were restored following Src re-expression. The effects of P25 were specific for uPAR as enhanced matrix assembly by P25 was not seen in uPAR-/- cells, but was restored upon uPAR re-expression. These data provide evidence for a novel pathway of fibronectin matrix assembly through the uPAR-dependent sequential activation of Src kinase, EGFR, and beta1 integrin.

Microglia are immune cells in the brain; their activation, migration, and proliferation have pivotal roles in brain injuries and diseases. Microglia are known to attach firmly to fibronectin, the upregulation of which is associated with several pathological conditions in the CNS, through beta1 integrin and become activated. Extracellular nucleotides can serve as potent signaling molecules. Recently, ATP and ADP were revealed to possess chemoattractive properties to microglia via Gi-coupled P2Y receptors. In the present study, we report that the ADP-induced chemotaxis of microglia is mediated by P2Y12/13 receptors and is beta1 integrin-dependent in the presence of fibronectin. Signals from P2Y12/13 receptors also cause beta1 integrin translocation to the membrane ruffle regions, but this redistribution was lost when the intracellular cyclic AMP (cAMP) was increased by forskolin or dibutyryl cAMP. This inhibitory effect of cAMP-elevating agents did not appear when microglia were co-incubated with a protein kinase A (PKA) inhibitor, KT-5720, suggesting that PKA is a negative regulator of the beta1 integrin translocation. We also show that the engagement of beta1 integrin enhanced microglial proliferation. Signals from P2Y12/13 receptors attenuated the proliferation, whereas ADP itself had no effect on microglial growth. Furthermore, beta1 integrin-induced proliferation is positively regulated by the cAMP-dependent PKA. Together, these results indicate the involvement of beta1 integrin in microglial proliferation and chemotaxis, both of which have clinical importance. The data also suggest that PKA is inversely involved in these two cellular functions., ((c) 2005 Wiley-Liss, Inc.)

Background and Objectives: Multiple myeloma is characterized by an accumulation of malignant plasma cells in the bone marrow. Inside the bone marrow, adhesion of myeloma cells to extracellular matrix proteins such as fibronectin may promote cell survival and induce drug resistance. In this work we examined the effect of hepatocyte growth factor (HGF) on the adhesion of myeloma cells and the signaling pathways involved., Design and Methods: Cell adhesion experiments were performed with the human myeloma cell line INA-6 and primary myeloma cells. The HGF signaling pathway was studied in INA-6 cells with the use of antibodies against VLA-4 integrin, and with inhibitors of various intracellular signaling molecules., Results: We found that HGF stimulated adhesion of myeloma cells to fibronectin. This event was dependent on the alpha4 and beta1 integrin subunits (VLA-4), but HGF did not increase the expression of integrins on the cell surface. Our findings suggest that HGF promotes myeloma cells to adhere via activation of the phosphatidylinositol 3-kinase (PI3K) pathway independently of AKT, but possibly through the involvement of nuclear factor kappa B (NF-kappaB). INA-6 cells adhered to fibronectin after stimulation by insulin-like growth factor or stromal cell-derived factor 1alpha, but this adhesion was less dependent on PI3K than HGF-mediated adhesion., Interpretation and Conclusions: his work points to HGF as a pro-adhesive factor in cell adherence to the bone marrow matrix protein fibronectin, an event known to promote cancer cell survival and drug resistance. Inhibiting HGF, its receptor c-Met or the VLA-4 integrin may be beneficial to the myeloma patient.

To elucidate mechanisms responsible for mechanotransduction in the heart and define the effects of remodeling of the extracellular matrix, we cultured neonatal rat ventricular myocytes on native type I collagen, fibronectin, or denatured collagen and subjected them to uniaxial, pulsatile stretch. Changes in expression of the cardiac gap junction protein, Cx43, were measured by confocal microscopy and immunoblotting. Cells grown on fibronectin or denatured collagen exhibited significantly greater Cx43 expression than cells grown on native collagen. Stretch induced a approximately 2-fold increase in Cx43 expression in cells grown on native collagen but no increase in cells grown on fibronectin or denatured collagen. Incubation of cells on native collagen with a peptide containing the arginine-glycine-aspartate (RGD) motif upregulated Cx43 expression equivalent to that induced by stretch. Nonselective activation of integrin signaling with MnCl2 also upregulated Cx43 expression in cells grown on native collagen. This effect was blocked completely by pretreatment with anti-beta1 integrin antibody but not by anti-beta3 integrin antibody. Stretch led to a marked increase in beta1 integrin immunofluorescent signal in cells grown on native collagen but not in cells grown on fibronectin or denatured collagen. Stretch-induced upregulation of Cx43 was also blocked by anti-beta1 integrin antibody. Thus, matrix protein-myocyte interactions regulate Cx43 expression via beta1 integrin signaling initiated by mechanical stimulation in cells grown on native type I collagen, or by RGD-integrin signaling independent of mechanical stress in cells grown on fibronectin or denatured collagen. Changes in the composition of the extracellular matrix may affect electrical coupling in cardiac myocytes.

Altered airway smooth muscle (ASM) function and enrichment of the extracellular matrix (ECM) with interstitial collagen and fibronectin are major pathological features of airway remodeling in asthma. We have previously shown that these ECM components confer enhanced ASM proliferation in vitro, but their action on its newly characterized secretory function is unknown. Here, we examined the effects of fibronectin and collagen types I, III, and V on IL-1beta-dependent secretory responses of human ASM cells, and characterized the involvement of specific integrins. Cytokine production (eotaxin, RANTES, and GM-CSF) was evaluated by ELISA, RT-PCR, and flow cytometry. Function-blocking integrin mAbs and RGD (Arg-Gly-Asp)-blocking peptides were used to identify integrin involvement. IL-1beta-dependent release of eotaxin, RANTES, and GM-CSF was enhanced by fibronectin and by fibrillar and monomeric type I collagen, with similar changes in mRNA abundance. Collagen types III and V had no effect on eotaxin or RANTES release but did modulate GM-CSF. Analogous changes in intracellular cytokine accumulation were found, but in <25% of the total ASM cell population. Function-blocking Ab and RGD peptide studies revealed that alpha2beta1, alpha5beta1, alphavbeta1, and alphavbeta3 integrins were required for up-regulation of IL-1beta-dependent ASM secretory responses by fibronectin, while alpha2beta1 was an important transducer for type I collagen. Thus, fibronectin and type I collagen enhance IL-1beta-dependent ASM secretory responses through a beta1 integrin-dependent mechanism. Enhancement of cytokine release from ASM by these ECM components may contribute to airway wall inflammation and remodeling in asthma.

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.

Background: We have shown previously that fibronectin (FN) together with megakaryocyte (Mk) growth and development factor (MGDF) enhanced generation of Mk progenitors determined by colony-forming unit (CFU)-Mk assay. MGDF can activate beta(1)-integrins on progenitor cells and increase binding to FN. We studied the role of beta(1)-integrin-tetraspanin complexes by which FN may enhance megakaryopoiesis., Methods: Cord blood CD34(+) cells were cultured for up to 8 days in serum-free medium containing IL-3, IL-6 and SCF with or without MGDF in the presence or absence of FN. Immunofluorescence flow cytometry was used to monitor changes in beta(1)-integrin-tetraspanin complexes. CFU-Mk assay was used to assess Mk commitment., Results: The cocktail of cytokines irrespective of the presence of MGDF altered the percentage expression of beta(1)-integrins CD49d and CD49e on CD34(+) cells. CD49d expression fell initially (98% to 15%) and then rose to 75%, whereas CD49e progressively increased over the 8 days of culture, from 5.4% to 22%. However, with the addition of FN, similar changes in the expression of beta(1)-integrins were observed but the expression was maintained at a higher level. MGDF and FN increased expression of tetraspanin molecules, CD63 and CD151, as well as their co-expression with the beta(1)-integrins on both the CD34(+) and CD34(-) cells (e.g. and increase from 0% to 80% co-expression of CD49d and CD151 on CD34(+)). Blocking of beta(1)-integrins or the tetraspanin CD151 with the respective MAb reduced Mk progenitor generation in a stromal cell model., Discussion: FN enhanced Mk progenitor generation through modulation of beta(1)-integrin-tetraspanin complexes, such as CD151/CD49d.

Eph receptor tyrosine kinases and their ligands, the ephrins, have been primarily described in the nervous system for their roles in axon guidance, development, and cell intermingling. Here we address whether Eph receptors may also regulate dendritic cell (DC) trafficking. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that DCs derived from CD34+ progenitors, but not from monocytes, expressed several receptors, in particular EphA2, EphA4, EphA7, EphB1, and EphB3 mRNA. EphB3 was specifically expressed by Langerhans cells, and EphA2 and EphA7 were expressed by both Langerhans- and interstitial-type DCs. EphA and EphB protein expression on DCs generated in vitro was confirmed by staining with ephrin-A3-Fc and ephrin-B3-Fc fusion proteins that bind to different Eph members, in particular EphA2 and EphB3. Immunostaining with anti-EphA2 antibodies demonstrated the expression of EphA2 by immature DCs and by skin Langerhans cells isolated ex vivo. Interestingly, ephrin expression was detected in epidermal keratinocytes and also in DCs. Adhesion of CD34+-derived DCs to fibronectin, but not to poly-l-lysine, was increased in the presence of ephrin-A3-Fc, a ligand of EphA2, through a beta1 integrin activation pathway. As such, EphA2/ephrin-A3 interactions may play a role in the localization and network of Langerhans cells in the epithelium and in the regulation of their trafficking.

Recently, it has been shown that Fas ligand (FasL) interacts with the extracellular matrix (ECM) protein fibronectin (FN), and that the bound FasL retains its cytotoxic efficacy. Herein, we examined the ramifications of FasL-ECM protein interactions throughout a specific time period, in the absence or presence of additional activating molecules, assuming that these complexed interactions occur during inflammation. We found that exposure of purified human T cells to FN-associated recombinant FasL for as brief as 5-10 min at 0.1-100 ng/ml induced their adhesion in beta(1) integrin- and FasR-dependent manners while activating the intracellular protein kinase, Pyk-2. The FN-associated FasL stops the CXCL12 (stromal cell-derived factor 1alpha)-induced chemotaxis of T cells by inhibiting the chemokine-induced extracellular signal-regulated kinase signaling and cytoskeletal rearrangement. This short term exposure of T cells to the FN-bound FasL (1 ng/ml), which was followed by T cell activation via the CD3 complex, resulted in 1) increased secretion of IFN-gamma (measured after 24 h), and 2) enhanced T cell apoptosis (measured after 72 h). Thus, in the context of inflamed ECM and depending on the time after FasL activation, its concentration, and the nature of other contextual mediators, FasL initially retains effector T cells at sites of inflammation and, later, induces T cell apoptosis and return to homeostasis.

Human epidermal keratinocytes are an important target for gene therapy because they can be easily expanded in culture and used to generate skin substitutes for the treatment of wounds, genetic diseases of the skin, and for delivery of proteins to the systemic circulation. Although retroviral transduction results in permanent genetic modification, differentiation and loss of transduced cells from the epidermis results in temporary transgene expression. To ensure permanent genetic modification, epidermal stem cells must be transduced with high efficiency. We evaluated gene transfer on two different substrates and found that the efficiency of gene transfer is substantially higher on a substrate of recombinant fibronectin (FN), when compared to tissue culture plastic (TCP). The rate of retroviral transduction on FN is four times faster than transduction on tissue culture plates and is independent of polybrene (PB). The transduction efficiency correlates with the levels of expression of integrin subunits alpha5, alpha2, and beta1, which have been shown to correlate with stem cell phenotype. Notably, cells that adhere rapidly to FN are transduced more efficiently than slowly adherent cells. In addition, integrin-blocking antibodies decrease the efficiency of gene transfer in a dose-dependent manner. Our results suggest that FN may enhance retroviral gene transfer to the least differentiated cells, thereby increasing the potential of genetically modified keratinocytes to treat short- and long-term disease states.

The region of fibronectin (FN) surrounding the two type II modules of FN binds type I collagen. However, little is known about interactions of this collagen binding domain with other collagen types or extracellular matrix molecules. Among several expressed recombinant (r) human FN fragments from the collagen binding region of FN, only rI6-I7, which included the two type II modules and both flanking type I modules, bound any of several tested collagens. The rI6-I7 interacted specifically with both native and denatured forms of types I and III collagen as well as denatured types II, IV, V and X collagen with apparent K(d) values of 0.2-3.7 x 10(-7) M. Reduction with DTT disrupted the binding to gelatin verifying the functional requirement for intact disulfide bonds. The FN fragments showed a weak, but not physiologically important, binding to heparin, and did not bind elastin or laminin. The broad, but selective range of ligand interactions by rI6-I7 mirrored our prior observations for the collagen binding domain (rCBD) from matrix metalloproteinase-2 (MMP-2) [J. Biol. Chem. 270 (1995) 11555]. Subsequent experiments showed competition between rI6-I7 and rCBD for binding to gelatin indicating that their binding sites on this extracellular matrix molecule are identical or closely positioned. Two collagen binding domain fragments supported cell attachment by a beta1-integrin-dependent mechanism although neither protein contains an Arg-Gly-Asp recognition sequence. Furthermore, activation of MMP-2 and MMP-9 was greatly reduced for HT1080 fibrosarcoma cells cultured on either of the fibronectin fragments compared to full-length FN. These observations imply that the biological activities of FN in the extracellular matrix may involve interactions with a broad range of collagen types, and that exposure to pathologically-generated FN fragments may substantially alter cell behavior and regulation., (Copyright 2002 Elsevier Science B.V. and International Society of Matrix Biology)

Chemokine and integrin receptors must work in concert when circulating leukocytes mobilize toward a site of tissue inflammation or infection. In a previous study, we reported that ligation of the alpha5beta1 integrin with a 120-kDa cell-binding fibronectin fragment (120-kDa FN) in suspensions of human polymorphonuclear leukocytes (PMNLs) inhibited chemotaxis toward the chemokine called interleukin-8 (IL-8). Binding of chemokines to their receptors on leukocytes leads to the activation of heterotrimeric G proteins that initiate multiple signaling cascades, including p38 and p42/p44 mitogen-activated protein kinase (MAPK) pathways. In the present study, we examine the potential interaction of beta1 integrin ligation on chemokine-mediated MAPK signaling in human PMNLs. We demonstrate that blockade of the p42/p44 MAPK signaling pathway by the inhibitor PD98059 suppresses IL-8-mediated PMNL chemotaxis. Furthermore, when PMNLs are pretreated with 120-kDa FN or an activating antibody to beta1 integrins (TS2/16), IL-8-mediated phosphorylation of p42/p44 MAPK is also inhibited. In contrast, pretreating PMNL with a specific ligand (laminin-1) for the alpha6beta1 integrin does not suppress IL-8-mediated phosphorylation of p42/p44 MAPK. These observations demonstrate a desensitization of IL-8-mediated p42/p44 MAPK signaling in response to ligation of the alpha5beta1 integrin in PMNL. Also, they suggest an interplay between integrin and chemokine signaling during PMNL migration through the extracellular matrix.

During corneal development, neural crest derivatives from the periocular mesenchyme migrate into the cornea and differentiate into corneal fibroblasts. During this time, these cells interact with a variety of extracellular matrices for proper orientation and development. In the present studies, we have examined the interaction of beta(1) integrins on periocular mesenchyme cells (POM) and corneal fibroblasts (CF) with fibronectin and tenascin by perturbing the function of this integrin. POM and CF attached and spread to a much greater extent on fibronectin than on tenascin. An antibody against beta(1) integrin, CSAT, decreased spreading and attachment, and resulted in a lack of immuno-detectable beta(1) integrin in focal adhesions on fibronectin; few beta(1) positive focal adhesions were observed in cells grown on tenascin. An anti-sense retroviral construct decreased endogenous levels of beta(1) integrin protein, and caused decreased attachment and spreading as well as sparse, disorganized focal adhesions. These data indicate that in vitro, both POM and CF have beta(1) integrins that interact with fibronectin and allow them to attach and spread, while tenascin is anti-adhesive. Further studies using both of these experimental paradigms will clarify whether these interactions also occur in vivo., (Copyright 2001 Elsevier Science Ltd.)

Adhesion of human salivary gland (HSG) epithelial cells to fibronectin- or collagen I gel-coated substrates, mediated by beta1 integrins, has been shown to upregulate the expression of more than 30 genes within 3-6 h. Adhesion of HSG cells to fibronectin or collagen I for 6 h also enhanced total protein kinase C (PKC) activity by 1.8-2.3-fold. HSG cells expressed PKC-alpha, gamma, delta, epsilon, mu, and zeta. Adhesion of HSG cells to fibronectin or collagen I specifically activated PKC-gamma and PKC-delta. Cytoplasmic PKC-gamma and PKC-delta became membrane-associated, and immunoprecipitated PKC-gamma and PKC-delta kinase activities were enhanced 2.5-4.0-fold in HSG cells adherent to fibronectin or collagen I. In addition, adhesion of fibronectin-coated beads to HSG monolayers co-aggregated beta1 integrin and PKC-gamma and PKC-delta but not other PKC isoforms. Thus, integrin-dependent adhesion of HSG cells to fibronectin or collagen I activated PKC-gamma and PKC-delta. The role of this PKC upregulation on adhesion-responsive gene expression was then tested. HSG cells were treated with the specific PKC inhibitor bisindolylmaleimide I, cultured on non-precoated, fibronectin- or collagen I-coated substrates, and analyzed for changes in adhesion-responsive gene expression. Bisindolylmaleimide I strongly inhibited the expression of seven adhesion-responsive genes including calnexin, decorin, S-adenosylmethionine decarboxylase, steroid sulfatase, and 3 mitochondrial genes. However, the expression of two adhesion-responsive genes was not affected by bisindolylmaleimide I. Treatment with bisindolylmaleimide I did not affect cell spreading and did not significantly affect the actin cytoskeleton. These data suggest that adhesion of HSG cells to fibronectin or collagen I induces PKC activity and that this induction contributes to the upregulation of a variety of adhesion-responsive genes., (Copyright 2001 Wiley-Liss, Inc.)

Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.

The extracellular matrix molecule fibronectin (FN) is a glycoprotein whose major functional property is to support cell adhesion. FN contains at least two distinct cell-binding domains: the central cell-binding domain and the HepII/IIICS region. The HepII region comprises type III repeats 12-14 and contains proteoglycan-binding sites, while the alternatively spliced IIICS segment possesses the major alpha4beta1 integrin-binding sites. Both cell surface proteoglycans and integrins are important for mediating the adhesion of cells to this region of FN. By comparing heparin binding to different recombinant splice variants of the HepII/IIICS region, evidence was obtained for the existence of a novel heparin-binding site in the centre of the IIICS. Site-directed mutagenesis of basic amino acid sequences in this region reduced heparin binding to recombinant HepII/IIICS proteins and, in conjunction with mutations in the HepII region, caused a synergistic loss of activity. Using the H/120 variant of FN, which contains type III repeats 12-15 and the full-length IIICS region, and the H/95 variant of FN, which contains type III repeats 12-15 but lacks the high affinity integrin-binding LDV sequence, the relative roles played by cell-surface proteoglycans and integrins in mediating cell adhesion have been investigated. This was achieved by studying the effects of anti-integrin antibodies and exogenous heparin on A375 melanoma cell attachment to the wild-type and three different mutants of H/120 and H/95 in which the potential proteoglycan-binding sites were partially or completely removed. A375 cell adhesion to H/120 and its mutants was found to involve the co-operative action of both integrin and cell-surface proteoglycan binding, although integrin made a dominant contribution. Anti-integrin antibodies and exogenous heparin were capable of inhibiting melanoma cell adhesion to H/95 and in this case adhesion was due primarily to cell-surface proteoglycan and not integrin binding.

Background: The first type III repeat of fibronectin is known to be involved in fibronectin matrix assembly, and recombinant proteins from this type III repeat can inhibit cell proliferation, tumor metastasis and angiogenesis. We have analyzed the way rat aortic smooth muscle cells (RASMCs) interact with a recombinant protein encompassing a C-terminal portion of the first type III repeat of fibronectin (protein III1-C)., Results: Cells are able to adhere to and spread on III1-C coated on a dish. Both beta1 integrins and cell surface heparan sulfate proteoglycans serve as receptors for III1-C. For example, cell attachment to III1-C is partially inhibited by agents that block beta1 integrins or by heparin. Complete inhibition of cell attachment is seen only when integrin blocking agents are combined with heparin. Affinity chromatography revealed the binding of proteins that likely represent the integrin beta1 and alpha5 submits to a III1-C column. Cell adhesion to III1-C results in robust ERK1/2 activation that is blocked by integrin-blocking agents. In addition, cell adhesion to III1-C and ERK1/2 activation by III1-C are both inhibited by heparan sulfate but not by chondroitin sulfate. Moreover, heparitinase treatment, but not chondroitinase treatment of RASMCs results in reduced cell adhesion and ERK1/2 activation. Affinity chromatography experiments demonstrated that 35SO4-labeled cell surface heparan sulfate proteoglycans bound specifically to III1-C., Conclusions: The results suggest that the 1st type III repeat of fibronectin contains a previously unrecognized cell adhesion domain that stimulates robust ERK1/2 activation in RASMCs. Cells interact with this domain through cell surface heparan sulfate proteoglycans and integrins, and both classes of receptors are required for optimal cell adhesion and ERK1/2 activation.

We show that tyrosine phosphorylation of FAK was increased as precartilage condensation occurred, followed by a subsequent decrease in proliferation of in vitro micromass culture of wing bud mesenchymal cells. FAK was associated with fibronectin and paxillin, which were maximal at day 3 of culture. FAK was also associated with signaling molecules such as PLC-gamma and PI3-kinase through c-Src. The beta1 integrin antibody and several inhibitors of signaling molecules such as herbimycin A, U73122, LY294002, as well as cytochalasin D, an actin depolymerizing agent, remarkably decreased tyrosine phosphorylation of FAK and its association with fibronectin and paxillin during condensation. resulting in a marked inhibition of condensation and chondrogenesis. Taken together, our findings suggest that beta1 integrin-mediated interaction of mesenchymal cells and fibronectin signals to accelerate the precartilage condensation through tyrosine phosphorylation of FAK and its association with paxillin. This signaling pathway is required for precartilage condensation and subsequent cartilage nodule formation in chondrogenesis., (Copyright 2000 Academic Press.)

The relationship between activation-dependent changes in beta1 integrin conformation, T cell adhesion to immobilized fibronectin, and T cell migration in vitro was analyzed in this study. Stimulation of Jurkat T cells and peripheral T cells with Mn(2+), the activating beta1 integrin-specific monoclonal antibody (mAb) TS2 /16, CD2, or CD28 stimulation led to increased adhesion, soluble fibronectin (FN) binding and expression of the activation epitope defined by the beta1 integrin mAb HUTS-21. Phorbol 12-myristate 13-acetate treatment increased adhesion, but not soluble FN binding or HUTS-21 epitope expression. In peripheral T cells, CD3 or CD7 stimulation also led to increased adhesion, soluble FN binding and HUTS-21 epitope expression. Soluble FN blocked peripheral T cell adhesion induced by Mn(2+) or TS2/16, but had no effect on adhesion induced by the other integrin-activating signals. In contrast, migration induced by TS2/16, CD2, CD3, CD7 or CD28 stimulation was blocked by excess soluble FN. Phosphoinositide 3-OH kinase (PI 3-K) inhibitors blocked receptor-mediated increases in cell adhesion, but not soluble FN binding or HUTS-21 expression. Migration was similarly unaffected by PI 3-K inhibitors, with the exception of CD7- and CD28-induced migration, which was specifically blocked by LY294,002. These results suggest that activation-dependent changes in beta1 integrin conformation are PI 3-K-independent and are involved in T cell migration but not adhesion.

Tumor invasion and metastasis development is a multistep process involving adhesion molecules as well as tumor proteases. It has been reported that tumor cells lacking fibronectin (FN) expression and engineered to re-express FN showed a marked reduction in metastatic ability. Besides its effects on cell adhesion and migration, FN could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we analyzed the production of urokinase-type plasminogen activator (uPA), and its receptor (uPAR), 2 molecules involved in the invasive phenotype, in cells over-expressing RGD wild-type FN (FNwt clones) or RGD-mutated FN (FN RGD-minus clones). Secreted uPA activity and antigen were significantly up-regulated in FN-expressing clones, although RGD-minus cells secreted approximately 50% less uPA than the FNwt ones. Interestingly, while control and FN RGD-minus clones were able to readily bind uPA to their surface, FNwt clones exhibited impaired uPA binding. Furthermore, treatment of the parental cell line as well as the control and FN-expressing clones with exogenous purified FN or RGD peptides induced up-regulation of uPA production and the reduction of uPA membrane binding, which was associated with lower expression of uPAR. This modulation by FN was found to be dependent on RGD sequence and beta1 integrin. These results strongly suggest a novel activity for the multifunctional glycoprotein FN regarding the regulation of uPA production as well as the capacity of tumor cells to bind uPA., (Copyright 1999 Wiley-Liss, Inc.)

FRT thyroid epithelial cells synthesize fibronectin and organize a network of fibronectin fibrils at the basal surface of the cells. Fibronectin fibril formation is enhanced by the overexpression of the ubiquitous beta1A integrin and is inhibited by the expression of the dominant-negative beta1B subunit. We tested the hypotheses that RhoA activity might mediate the integrin-dependent fibronectin fibrillogenesis and might counteract beta1B integrin inhibitory effect. FRT-beta1A cells were transfected with a vector carrying a dominant negative form of RhoA (RhoAN19) or treated with the C3 transferase exoenzyme. Both treatments inhibited fibronectin assembly and caused loss of actin microfilaments and adhesion plaques. On the other hand, FRT-beta1B cells were transfected with the constitutively activated form of RhoA (RhoAV14) or treated with the E. coli cytotoxic necrotizing factor 1, which directly activates RhoA. Either treatment restored microfilament and adhesion plaque assembly and promoted fibronectin fibril organization. A great increase in fibronectin fibril assembly was also obtained by treatment of FRT-beta1B cells with TGF-beta. Our data indicate that RhoA is required to promote fibronectin matrix assembly in FRT cells and that the activation of the signal transduction pathway downstream of RhoA can overcome the inhibitory effect of beta1B integrin.

We investigated the effects of signaling molecule inhibitors on the expression and function of beta1 integrins in Jurkat cells. Jurkat cells expressed alpha4beta1 and alpha5beta1, with significant levels of constitutively activated beta1 integrins as assessed by labeling with mAb 15/7 that distinguishes between activation states. Adhesion to fibronectin (Fn) was mediated equally through alpha4 and alpha5 subunits, and was potentiated by the beta1 integrin activating mAb 8A2. Fn adhesion was decreased by okadaic acid through effects on both alpha4beta1, and alpha5beta1. Tyrphostin A23 also decreased adhesion but was less potent. Neither inhibitor had any effect on the surface expression of total or activated beta1 integrins. The effect of tyrphostin was completely reversed by 8A2; the effect of okadaic acid was only partially reversed. Using Calyculin A, we determined that Jurkat adhesion to Fn was regulated via protein phosphatase 1, independent of the levels of integrins or integrin activation epitopes. Activation of Jurkat cells with a CD3-stimulating mAb enhanced adhesion to Fn and was partially blocked by okadaic acid. These data demonstrate different regulatory pathways for constitutive versus activation-dependent adhesion via beta1 integrins, and implicate both tyrosine kinases and serine-threonine phosphatases in integrin function.

Human CD34+ bone marrow cells were committed to the megakaryocytic lineage in serum-free liquid cultures by the following cytokines: thrombopoietin, erythropoietin, and IL-6. Megakaryocyte maturation has been described as being regulated by the extracellular matrix. These cells express receptors for laminin, collagen, and vitronectin, but they selectively adhere to and spread on fibronectin, a major component of the bone marrow environment. Function-perturbing antibodies against beta 1 integrins totally abolished the adhesion of megakaryocytes on fibronectin, whereas antibodies to beta 3 did not, suggesting that beta 1 integrins were responsible for the adhesive phenotype of these polyploid cells. beta 1-positive clusters were visualized in close contact with the extremities of stress fibers at the cell surface. In the course of cell spreading, we observed morphological modifications such as the disorganization of the compact nuclei structure and the appearance of holes in the cytoplasm leading to the release of alpha IIb beta 3-positive cellular fragments. This process appeared to be a specific feature of megakaryocytes and is correlated neither to apoptosis nor to integrin signaling.

Objective: This study is to identify the expression of integrin beta 1 in human dental pulp cells and the role of integrin beta 1 in pulp cell adhesion on extracellular matrix protein laminin and fibronectin., Study Design: Immunoblot detection of integrin beta 1 in human pulp cells was with the use of monoclonal anti-beta 1 antibody. Dental pulp cell adhesion assay on extracellular matrix protein laminin and fibronectin and blocking cell adhesion was performed with monoclonal anti-beta 1 antibody., Result: Integrin beta 1 was identified in human dental pulp cells. Pulp cells adhered and spread on both laminin and fibronectin. Monoclonal anti-beta 1 antibody inhibited human dental pulp cells adhesion on laminin but not on fibronectin., Conclusions: Integrin beta 1 was expressed on human dental pulp cells and mediated cell adhesion on laminin. Human dental pulp cells also adhered on fibronectin but the adhesion was not regulated by beta 1 integrin.

Many cell-surface and extracellular matrix proteins contain multiple modular domains known as fibronectin type III (FNIII) repeats. Cells adhere to the extracellular matrix proteins fibronectin and tenascin in part by the interaction of certain integrins with the Arg-Gly-Asp (RGD) sequence, displayed on specific FNIII repeats. We have found that, after experimental activation of beta1 integrins, a number of cell types adhere and spread on FNIII repeats lacking RGD, derived from extracellular matrix proteins and cytokine receptors. Interaction between individual FNIII domains and beta1 integrins mediates focal adhesion kinase phosphorylation and subsequent stress fiber and focal contact formation. These data suggest that many FNIII-containing proteins may bind and signal through activated beta1 integrins, dramatically expanding the potential for integrin-dependent intercellular and cell-matrix communication.

Lymphocyte recirculation is dependent on families of adhesion molecules expressed on lymphocytes and their sequential interaction with ligands expressed on high endothelial venules in secondary lymphoid organs such as peripheral lymph nodes. By binding its carbohydrate-based ligands, L-selectin initiates this cascade of molecular interactions, supporting the rolling of lymphocytes along high endothelial venules. Subsequent activation of lymphocyte integrins leads to cell arrest followed by lymphocyte extravasation. Here, we demonstrate stimulated adhesion of PBL and Jurkat T cells to immobilized fibronectin following treatment with (1) GlyCAM-1, a physiologic ligand for L-selectin, and (2) cross-linked anti-L-selectin mAbs. We also utilize a solution binding assay to detect early changes in integrin activity, including affinity modulation and/or integrin clustering, and distinguish these from later postreceptor binding events such as changes in cell shape and spreading. With the Jurkat cell line, GlyCAM-1 and fucoidin (an L-selectin ligand mimetic) induce the binding of soluble fibronectin. In contrast, stimulation through the Jurkat TCR fails to promote binding to soluble ligand even though TCR cross-linking markedly enhances adhesion to immobilized fibronectin. These data suggest that L-selectin and the TCR promote adhesion through distinct mechanisms. Finally, we demonstrate that beta1 integrins are preferentially activated on naive T cells through the L-selectin pathway. Together with our previous studies showing similar activation of beta2 integrins on the naive T cell subset, these data suggest that signals delivered though L-selectin participate in the preferential recruitment of these cells to peripheral lymph nodes.

Beta1 integrins are widely expressed in human tissues but their presence and function on malignant mesothelioma cells have not been examined. In this study, we have investigated the expression and function of beta1 integrins in 7 human malignant mesothelioma cell lines. Immunofluorescence staining and FACS analysis showed similar expression of beta1 integrins with strongest expression of alpha3beta1 in all investigated mesothelioma cell lines. Using the Boyden chamber assay, we found that mesothelioma cell lines migrated to soluble (chemotaxis) and substrate-bound (haptotaxis) fibronectin, laminin and collagen type IV. In order to investigate the biological function of integrins in mesothelioma cells, we pre-incubated the cells with blocking anti-integrin monoclonal antibodies (MAbs) prior to the adhesion and migration assays. Anti-beta1 antibodies inhibited cell adhesion, chemotaxis and haptotaxis in all cell lines. Generally, anti-alpha2 integrin antibodies inhibited cell adhesion, chemotactic and haptotactic migration to collagen type IV, whereas antibodies to the alpha5 and alpha6 subunits inhibited cell adhesion and migration to fibronectin and laminin, respectively. Preincubation of mesothelioma cells with anti-alpha3 antibodies inhibited the migration to either collagen type IV, laminin or fibronectin in all cell lines. Interestingly, in 3 cell lines anti-alpha3 antibodies inhibited cell migration to laminin and collagen type IV without affecting the ability of the cells to adhere to these proteins. Furthermore, in 2 cell lines, antibodies to the alpha3 chain inhibited chemotaxis but not haptotaxis to collagen type IV, indicating the presence of distinct signalling pathways.

The Yersinia pseudotuberculosis invasin protein was found to be a potent inducer of pseudopodia formation and chemotactic and haptotactic migration in human T lymphocytes. Checkerboard analysis confirmed that migration was directional. The Yersinia invasin triggered migration of otherwise poorly migratory normal T cells on fibronectin and in particular on collagen type IV, and augmented the migration of leukemic T cell lines on these components. Invasin-induced lymphocyte migration was inhibited by staurosporin that selectively prevented pseudopodia formation but, noteworthy, augmented adhesion. The motogenic and attractant properties of invasin (Inv) were mediated via beta1-integrins, as shown by lack of effect of Inv on the motility of a beta1-integrin-negative lymphoid cell line and inhibition of invasin-induced lymphocyte motility by anti-beta1 Abs. Inv was markedly more effective than the extracellular matrix components fibronectin, collagen type IV, and laminin, which also interact with lymphocyte beta1-integrins, with respect to induction of pseudopodia, chemotaxis, and haptotaxis. Thus, Yersinia invasin is a model ligand for induction of lymphocyte motility via beta1-integrins. The extraordinary capacity of Inv to trigger and guide T lymphocyte motility and potentiate lymphocyte migration to extracellular matrix components may be of pathogenetic significance for the movement of lymphocytes to extraintestinal sites secondary to Yersinia infection.

Acute lung injury leads to type I alveolar epithelial cell (AEC) death, denudation of the alveolar basement membrane, and formation of an alveolar provisional matrix from fibronectin, fibrinogen, and type I collagen. The provisional matrix provides a scaffold for alveolar repair. To restore normal lung architecture, surviving type II AECs must reepithelialize denuded alveoli. We examined whether AECs migrate on provisional matrix proteins and whether integrins mediate this migration using a Boyden chemotaxis chamber. Cultured AECs migrated on fibronectin-coated filters by haptotaxis (defined as movement on a solid-phase substrate) more than one type I collagen-coated filters, and they did not migrate on fibrinogen-coated filters. Soluble fibronectin augmented migration on type I collagen-coated filters, but not on fibronectin-coated filters. Anti-alpha v beta 3-integrin monoclonal antibody (MAb) inhibited migration on substrate-bound fibronectin by 62-77%, whereas anti-beta 1-integrin MAb inhibited migration by 48%. Anti-alpha 2-integrin MAb almost completely inhibited migration on substrate-bound type I collagen, but not on fibronectin. The novel findings in this study are as follows: 1) AECs migrate by haptotaxis more effectively on substrate-bound fibronectin than on type I collagen; 2) alpha v beta 3- and beta 1-integrins partially mediate AEC haptotaxis on fibronectin; and 3) the alpha 2 beta 1-integrin mediates AEC migration on type I collagen. These results support the importance of type II cell migration on provisional matrix proteins during repair of lung injury.

After injury, the wound space is filled with a fibrin/fibronectin clot containing growth factors released by platelets and monocytes. In response to these factors, fibroblasts migrate into the fibrin clot and contribute to the formation of granulation tissue. The functional mechanisms allowing fibroblasts to leave the collagenous matrix of normal connective tissue and invade the provisional matrix of the fibrin clot have not been fully defined. To investigate these mechanisms we established a new in vitro model which simulates specific aspects of early wound healing, that is, the migration of fibroblasts from a three-dimensional collagen matrix into a fibrin clot. This transmigration could be induced by physiological concentrations of platelet releasate or platelet-derived growth factor BB (PDGF-BB) in a concentration-dependent manner. At 24 hours irradiated fibroblasts invaded the fibrin gel almost as well as non-irradiated cells, indicating that transmigration was independent of proliferation. Plasminogen and its activators appear to be necessary for invasion of the fibrin clot since protease inhibitors decreased the amount of migration. These serine proteases, however, were not necessary for exit from the collagen gel as fibroblasts migrated out of the collagen gel onto a surface coated with fibrin fibrils even in the presence of inhibitors. Removal of fibronectin (FN) from either the collagen gel or the fibrin gel markedly decreased the number of migrating cells, suggesting that FN provides a conduit for transmigration. Cell movement in the in vitro model was inhibited by RGD peptide, and by monoclonal antibodies against the subunits of the alpha5 beta1 and alpha v beta3 integrin receptor. Thus, the functional requirements for fibroblast transmigration from collagen-rich to fibrin-rich matrices, such as occurs in early wound healing, have been partially defined using an in vitro paradigm of this important biologic process.

The state of integrin activation can be assessed by monoclonal antibodies (mAb) that selectively recognize integrins in their active form. We demonstrate herein that the expression of the epitope recognized by mAb HUTS-21 is induced on T lymphoblasts upon binding of soluble vascular cell adhesion molecule (VCAM)-1 and an 80-kDa tryptic fragment of fibronectin (FN80) to the beta1 integrins very late activation antigen (VLA)-4 and VLA-5, and that this effect is dependent on ligand concentration and is specific for beta1 integrins. On T lymphoblasts adhering to immobilized fibronectin, the HUTS-21 epitope localized exclusively to sites of integrin binding to fibronectin. These results indicate that mAb HUTS-21 recognizes a ligand-induced binding site (LIBS) on the common beta1 subunit of VLA proteins. Engagement of beta1 integrins through this LIBS epitope inhibited T lymphoblast movement on fibronectin, as determined by quantitative time-lapse video microscopy studies. Furthermore, the HUTS-21 mAb also prevented T lymphoblast-directed migration through gradients of substratum-immobilized beta1 integrin ligands such as fibronectin or VCAM-1, whereas it did not affect migration on intercellular adhesion molecule (ICAM)-1. This anti-LIBS mAb stimulated cell adhesion through postreceptor events, without affecting receptor affinity for ligand, and appears to interfere with cell migration by a mechanism distinct from that of other anti-beta1 activating antibodies.

During implantation, the embryonic trophoblast aggressively invades the uterine stroma. The resulting uterine reaction, decidualization, involves differentiation of new cell morphologies and remodeling of the extracellular matrix. This creates an environment that first permits invasion, then controls this invasion to allow the establishment of the placenta. The production, organization, and cellular interactions with the matrix are thought to underlie decidual functions. We have begun a reductional analysis of the components of the decidual matrix, focusing on extracellular fibronectin (FN). Using decidual cell cultures prepared from day 7 implantation sites, the synthesis, extracellular organization, and details of decidual cell:FN interaction were studied. Employing immunofluorescence, immunoprecipitation, and dot blot analysis, decidualizing cultures showed a constitutive level of FN synthesis and deposition. The differentiating cells organized extracellular FN in patterns similar to that seen in vivo. The predominant, flattened dendritic decidual cells organized FN in long, thin fibrils. Large, rounded decidual cells, limited to the primary decidual zone in vivo, showed FN limited to punctate membrane patches and short, thick fibrils. Using double labeling techniques, FN expression was co-localized with actin microfilament (MF) bundles during the cytoskeletal changes associated with the differentiation of both decidual cell types. The function of MFs in maintaining morphology was demonstrated by cytochalasin B perturbation. Attachment of decidual cells to FN was calcium dependent and gly-arg-gly-asp-ser-pro (GRGDSP) sensitive, with dendritic decidual cells expressing the alpha 5 and beta 1 integrin subunits. This suggests that an integrin system functions to attach decidual MF bundles to extracellular FN. This work shows that during decidual matrix remodeling, constitutive levels of FN are maintained to provide an extracellular framework to stabilize the decidual cytoskeleton and support morphological differentiation of decidual cells.

Fibroblasts that migrate into a wound during the early stages of repair use cell surface integrins to interact with extracellular molecules as they move away from the interstitial matrix of normal tissue and into the provisional matrix of the wound. Therefore, to understand a critical phase of wound healing, it is necessary to understand the details of integrin involvement. Normal adult human dermal fibroblasts in culture express many receptors for the provisional matrix proteins fibronectin, vitronectin, and fibrinogen, including the integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 1, alpha v beta 3, and alpha v beta 5. We used quantitative flow cytometry to estimate the relative numbers of these receptors and immunoprecipitation to confirm the expression of alpha v beta 1. Adult human dermal fibroblasts primarily use beta 1 integrins, alpha 4 beta 1, alpha 5 beta 1, and possibly alpha v beta 1, for attachment to fibronectin. alpha v beta 3 and perhaps other integrins containing the alpha v subunit serve fibroblasts as secondary or auxiliary receptors for fibronectin. In contrast, these cells use alpha v integrins but probably not beta 1 integrins for attachment to vitronectin. alpha v beta 3 and alpha v beta 5 apparently act in concert to mediate attachment to vitronectin, and these two integrins may perform different functions during wound repair. Fibroblast adhesion to certain preparations of fibrinogen occurs, at least partially, through the small amount of fibronectin present in the preparations. Fibroblast attachment to fibrinogen purified free of fibronectin also occurs, and that was demonstrated with a sensitive new assay called electrical cell-substrate impedance sensing. Fibroblast attachment to pure fibrinogen can be inhibited by RGD peptide, suggesting that integrins are involved.

We established an immortalized, nontumorigenic human bronchial epithelial cell line by transfection with the origin of replication-defective SV40 large T plasmid. This line spontaneously became tumorigenic at passage 184 (NL20T), although subsequent passages (passages 189, 200, and 205) failed to form tumors. The tumorigenic cell line NL20T was reinoculated back into athymic nude mice, and the two subsequently derived cell lines (NL20T-A and NL20T-B) have been passaged 85 times in vitro and remain tumorigenic. However, late-passage NL20T cells consistently lose their tumorigenicity when passaged in vitro on tissue culture plastic dishes (NL20T-n cells). Thus, two of the cell lines, NL20 and NL20T, reverted to the nontumorigenic phenotype reproducibly and spontaneously following serial passage on plastic tissue culture plates, whereas cells passaged in mice (NL20T-A and -B) did not. We used these nontumorigenic (NL20 and NL20T-n) and tumorigenic (early passage NL20T, NL20T-A, and NL20T-B) cells to study the role of the alpha 5/beta 1-integrin and attachment to fibronectin in tumorigenicity. The two nontumorigenic cell lines (NL20 and NL20T-n) attached slower to fibronectin-coated plates than the two tumorigenic cell lines in a cellular-extracellular matrix adhesion assay. Attachment was abrogated by exposure to a blocking antibody to the alpha 5/beta 1-integrin, the fibronectin receptor, in the two tumorigenic cell lines. Cell surface expression of the alpha 5/beta 1 cell surface protein by flow cytometry was highest in the tumorigenic NL20T and NL20T-A cells. NL20T-A cells were cultured with an antibody to alpha 5/beta 1 and inoculated s.c. into athymic nude mice; tumorigenicity of the NL20T-A cells was inhibited in a dose-dependent manner. Tumorigenicity was also inhibited partially with monoclonal antibodies to either alpha 5 or beta 1. A mixture of 10% tumorigenic NL20T-A cells and 90% nontumorigenic NL20 cells was cultured on plastic, type IV collagen, laminin, and fibronectin for 9 weeks. Only cells cultured on fibronectin formed tumors when inoculated s.c. into athymic nude mice. We conclude that these data are consistent with the hypothesis that neoplastic transformation in our original cell line arose from in vivo selection of a small mutant clone, which had arisen in culture and was selected subsequently in vivo but was lost with in vitro culture in NL20 cells, and that alpha 5/beta 1-integrin interaction with the extracellular matrix may play a role in tumorigenicity in our system.

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor alpha 4 beta 1, and MDAY-D2 cells in addition also alpha 5 beta 1. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn2+. In ESb cells, this adhesion was mediated by alpha 4 beta 1. In MDAY-D2 cells, however, only alpha 5 beta 1 was involved, despite alpha 4 beta 1 levels similar to ESb cells. The alpha 4 beta 1 integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An alpha 5 beta 1-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by alpha 4 beta 1. These results indicate that alpha 4 beta 1 function in these cells is suppressed in the presence of alpha 5 beta 1. Adhesion of ESb cells to hepatocytes was inhibited by anti-alpha 4 antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that alpha 4 beta 1 and alpha 5 beta 1 function is not activated during this interaction. The 9EG7 antibody against mouse beta 1 integrin was described to inhibit beta 1 integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that 9EG7 stimulated beta 1-integrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.