Collagen type I modulates the platelet-derived growth factor (PDGF) regulation of the growth and expression of beta1 integrins by rat mesangial cells

Mesangial cell (MC) proliferation and the deposition of collagen type I (collagen I) are the major pathological features in many types of glomerulonephritis (GN). Recent work suggested that beta-integrins play a critical role in the cell proliferation and extracellular matrix (ECM) remodeling observed in tissue repair after injury. To examine the involvement of beta-integrins in MC proliferation in association with the interaction of MCs with pathological collagen I, we investigated the effect of a prominent mitogen, platelet-derived growth factor-BB (PDGF-BB) on the growth and expression of beta-integrins by MCs cultured on plastic or in a three-dimensional collagen I gel. Immunoprecipitation using 35S-metabolic labeling, flow cytometry and a 3H-thymidine-uptake analysis demonstrated that PDGF-BB stimulated the cell mitogenicity and the expression of alpha5beta1 integrin (a fibronectin receptor), but not alpha1beta1 integrin (a collagen and laminin receptor) of MCs on plastic, in a dose-dependent manner. In contrast, MCs in the collagen I gels showed no significant changes in mitogenicity or alpha1beta1 and alpha5beta1 integrin expression, but increased alpha1beta1 integrin-mediated gel contraction was observed after PDGF-BB stimulation. Thus, the parallel up-regulation of MC-mitogenicity and alpha5beta1 integrin expression by PDGF-BB suggested that alpha5beta1 integrin is an important ECM receptor involved in the proliferative phenotype of MC. A spatial interaction between MCs and pathological collagen I in GN may influence the PDGF regulation of the MC phenotype regarding the cell growth and the expression of beta1 integrins.     

Mesodermal defects and cranial neural crest apoptosis in alpha5 integrin-null embryos

Alpha5beta1 integrin is a cell surface receptor that mediates cell-extracellular matrix adhesions by interacting with fibronectin. Alpha5 subunit-deficient mice die early in gestation and display mesodermal defects; most notably, embryos have a truncated posterior and fail to produce posterior somites. In this study, we report on the in vivo effects of the alpha5-null mutation on cell proliferation and survival, and on mesodermal development. We found no significant differences in the numbers of apoptotic cells or in cell proliferation in the mesoderm of alpha5-null embryos compared to wild-type controls. These results suggest that changes in overall cell death or cell proliferation rates are unlikely to be responsible for the mesodermal deficits seen in the alpha5-null embryos. No increases in cell death were seen in alpha5-null embryonic yolk sac, amnion and allantois compared with wild-type, indicating that the mutant phenotype is not due to changes in apoptosis rates in these extraembryonic tissues. Increased numbers of dying cells were, however, seen in migrating cranial neural crest cells of the hyoid arch and in endodermal cells surrounding the omphalomesenteric artery in alpha5-null embryos, indicating that these subpopulations of cells are dependent on alpha5 integrin function for their survival. Mesodermal markers mox-1, Notch-1, Brachyury (T) and Sonic hedgehog (Shh) were expressed in the mutant embryos in a regionally appropriate fashion. Both T and Shh, however, showed discontinuous expression in the notochords of alpha5-null embryos due to (1) degeneration of the notochordal tissue structure, and (2) non-maintenance of gene expression. Consistent with the disorganization of notochordal signals in the alpha5-null embryos, reduced Pax-1 expression and misexpression of Pax-3 were observed. Anteriorly expressed HoxB genes were expressed normally in the alpha5-null embryos. However, expression of the posteriormost HoxB gene, Hoxb-9, was reduced in alpha5-null embryos. These results suggest that alpha5beta1-fibronectin interactions are not essential for the initial commitment of mesodermal cells, but are crucial for maintenance of mesodermal derivatives during postgastrulation stages and also for the survival of some neural crest cells.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Integrin alpha 2 beta 1 is a positive regulator of collagenase (MMP-1) and collagen alpha 1(I) gene expression

A classical model for studying the effects of extracellular matrix is to culture cells inside a three-dimensional collagen gel. When surrounded by fibrillar collagen, many cell types decrease the production of type I collagen, and the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is simultaneously induced. To study the role of the collagen-binding integrins alpha 1 beta 1 and alpha 2 beta 1 in this process, we used three different osteogenic cell lines with distinct patterns of putative collagen receptors: HOS cells, which express only alpha 1 beta 1 integrin, MG-63 cells, which express only alpha 2 beta 1 integrin, and KHOS-240 cells, which express both. Inside collagen gels, alpha 1 (I) collagen mRNA levels were decreased in HOS and KHOS-240 cells but not in MG-63 cells. In contrast, MMP-1 expression was induced in KHOS-240 and MG-63 cells but not in HOS cells. Transfection of MG-63 cells with alpha 2 integrin cDNA in an antisense orientation reduced the expression level of alpha 2 integrin. These cell clones showed induction and reduction of mRNA levels for MMP-1, respectively. HOS cells normally lacking alpha 2 beta 1 integrin were forced to express it, and this prevented the down-regulation in the levels of alpha 1 (I) collagen mRNA when cells were grown inside collagen gels. The data indicate that the level of MMP-1 expression is regulated by the collagen receptor alpha 2 beta 1 integrin. The down-regulation of collagen alpha 1 (I) is mediated by another receptor. Integrin alpha 2 beta 1 may compete with it and thus be a positive regulator of collagen synthesis.     

Re-expression of the alpha 2 beta 1 integrin abrogates the malignant phenotype of breast carcinoma cells

To assess the role of altered alpha 2 beta 1 integrin expression in breast cancer, we expressed the alpha 2 beta 1 integrin de novo in a poorly differentiated mammary carcinoma that expressed no detectable alpha 2-integrin subunit. Expression of the alpha 2 beta 1 integrin resulted in a dramatic phenotypic alteration from a fibroblastoid, spindle-shaped, non-contact-inhibited, motile, and invasive cell to an epithelioid, polygonal-shaped, contact-inhibited, less motile, and less invasive cell. Although expression of the alpha 2 subunit did not alter adhesion to collagen, it profoundly altered cell spreading. Re-expression of the alpha 2 beta 1 integrin restored the ability to differentiate into gland-like structures in three-dimensional matrices and markedly reduced the in vivo tumorigenicity of the cells. These results indicate that the consequences of diminished alpha 2 beta 1-integrin expression in the development of breast cancer and, presumably, of other epithelial malignancies are increased tumorigenicity and loss of the differentiated epithelial phenotype.     

Chondrocyte survival and differentiation in situ are integrin mediated

Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.     

The role of integrins and extracellular matrix in anchorage-independent growth of a mammary carcinoma cell line

Anchorage-independent growth is a property of malignant cells. Extracellular matrix proteins are present in tumor spheroids but their function is not clearly defined. In this paper we show that a murine mammary carcinoma cell line, SP1, which expresses the fibronectin receptor alpha 5 beta 1 requires fibronectin for anchorage-independent growth in soft agar. Growth factors (hepatocyte growth factor and transforming growth factor-beta) also promote SP1 colony growth. In contrast, collagen types I and IV have an inhibitory effect on SP1 colony growth. A clone isolated from SP1 cells which expresses the collagen/laminin receptor alpha 2 beta 1 as well as the fibronectin receptor alpha 5 beta 1, demonstrates increased colony formation in the presence of fibronectin and collagen. These data suggest a role for both the alpha 5 beta 1 and alpha 2 beta 1 integrin receptors in the regulation of anchorage-independent growth of mammary carcinoma cells.     

Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc, and its complex formation with Grb2 in the human multiple myeloma cell line LP-1

Like many other cytokines and growth factors, interleukin-6 (IL-6) activates p21ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL-6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21ras. In the multiple myeloma cell line LP-1, IL-6 stimulated the tyrosine phosphorylation of Shc in a time- and concentration-dependent manner. This led to the complex formulation of Shc with Grb2, an adaptor protein known to relocate a p21ras-GDP exchange factor. Sos1 (Son-of-sevenless), to the cell membrane. Taken together, these findings suggest that IL-6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL-6 receptor associated non-receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects.     

Transforming growth factor beta 1 suppresses transformation in hepatocytes by regulating alpha 1 beta 1 integrin expression

We previously reported that (a) treatment of the ras-transformed hepatocyte cell line NR4 with transforming growth factor (TGF) beta 1 suppresses many characteristics associated with the transformed phenotype including altered morphology, actin cytoskeleton reorganization, and anchorage-independent growth such that the cells more closely resemble the immortalized CWSV1 parent cell line; (b) transformed NR4 cells expressed significantly less alpha 1 integrin RNA than the immortalized CWSV1 cells; and (c) TGF-beta 1 treatment of NR4 cells stimulated the expression of alpha 1 and beta 1 integrin RNAs. In this report, the role of the alpha 1 beta 1 integrin in TGF-beta 1-mediated suppression of the ras-transformed phenotype was investigated. We determined that (a) the cell surface integrin that increased in response to TGF-beta 1 treatment of NR4 cells was alpha 1 integrin; (b) TGF-beta 1 altered the ability of NR4 cells to attach to collagen and laminin, the extracellular matrix components that interact with the alpha 1 beta 1 integrin receptor; (c) TGF-beta 1 treatment resulted in relocalization of the alpha 1 integrin on the NR4 cell surface; and (d) TGF-beta 1-mediated inhibition of anchorage-independent growth was blocked by the presence of alpha 1 integrin antibody. A cell line that overexpresses alpha 1 integrin was derived from NR4 cells; characterization of these cells indicated that they continued to express H-ras RNA but were less transformed than the parent NR4 cells. Specifically, they had an altered morphology, an organized actin cytoskeleton, and reduced ability to demonstrate anchorage-independent growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

A peptidomimetic antagonist of the integrin alpha(v)beta3 inhibits Leydig cell tumor growth and the development of hypercalcemia of malignancy

The integrin alpha(v)beta3 interacts with the arginine-glycine-aspartic acid (RGD) tripeptide recognition sequence of a variety of extracellular matrix proteins. Recent studies show that alpha(v)beta3 plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha(v)beta3 inhibit angiogenic processes that include endothelial cell adhesion and migration. Consequently, we reasoned that an RGD-based peptidomimetic antagonist of alpha(v)beta3 might inhibit tumor angiogenesis and tumor growth in vivo. An RGD-peptidomimetic library was screened to identify antagonists of vitronectin binding to alpha(v)beta3, and the compounds chosen were modified to produce selective and potent inhibitors of alpha(v)beta3. One of these compounds, beta-[[2-2-[[[3-[(aminoiminomethyl)amino]-phenyl]carbonyl]amino]ac etyl]amino]-3,5-dichlorobenzenepropanoic acid (SC-68448), inhibited vitronectin binding to both alpha(v)beta3 and the closely related platelet receptor, alpha(IIb)beta3, in a dose-responsive manner. SC-68448 inhibited vitronectin binding to alpha(v)beta3 (IC50, 1 nM) and fibrinogen binding to the platelet receptor alpha(IIb)beta3 (IC50, >100 nM), demonstrating that SC-68448 was 100-fold more potent as an inhibitor of alpha(v)beta3 versus alpha(IIb)beta3. In cell-based studies, SC-68448 inhibited alpha(v)beta3-mediated endothelial cell proliferation in a dose-dependent manner but did not inhibit tumor cell proliferation, suggesting that effects on endothelial cell proliferation were not due to SC-68448-induced cytotoxicity. In accord with these results, SC-68448 inhibited angiogenesis in vivo in a basic fibroblast growth factor-induced rat corneal neovascularization model. A xenogeneic severe combined immune deficiency mouse/rat Leydig cell tumor model was developed for testing SC-68448 as an inhibitor of tumor growth in vivo. Rat Leydig cell tumors grew rapidly in severe combined immune deficiency mice and produced humoral hypercalcemia of malignancy. SC-68448 inhibited the growth of the tumors in mice by up to 80% and completely blocked the development of hypercalcemia. Together, these results demonstrate the feasibility of antitumor therapies based upon the development of nontoxic small molecule pharmacological antagonists of integrin alpha(v)beta3.     

A role for Src in signal relay by the platelet-derived growth factor alpha receptor

Previous studies have shown that Src is required for platelet-derived growth factor (PDGF)-dependent cell cycle progression in fibroblasts. Since fibroblasts usually express both PDGF receptors (PDGFRs), these findings suggested that Src was mandatory for signal relay by both the alpha and betaPDGFRs. In this study, we have focused on the role of Src in signal relay by the alphaPDGFR. In response to stimulation with PDGF-AA, which selectively engages the alphaPDGFR, Src family members (Src) associated with the alphaPDGFR and Src kinase were activated. A mutant receptor, in which tyrosines 572 and 574 were replaced with phenylalanine (F72/74), failed to efficiently associate with Src or activate Src. The wild type (WT) and F72/74 receptors induced the expression of c-myc and c-fos to comparable levels. Furthermore, an equivalent extent of PDGF-dependent soft agar growth was observed in cells expressing the WT or the F72/74 alphaPDGFR. Comparing the ability of these two receptors to initiate tyrosine phosphorylation of signaling molecules indicated that both receptors mediated phosphorylation of the receptor itself, phospholipase Cgamma 1, and SHP-2 to similar levels. In contrast, the F72/74 receptor triggered phosphorylation of Shc to 1 and 20% of the WT levels for the 55- and 46-kDa Shc isoforms, respectively. These findings indicate that after exposure of cells to PDGF-AA, Src stably associates with the alphaPDGFR, and Src activity is increased. Furthermore, Src is required for the PDGF-dependent phosphorylation of signaling molecules such as Shc. Finally, activation of Src during the G0/G1 transition does not appear to be required for latter cell cycle events such as induction of c-myc or cell proliferation.     

Enhancement of cell interactions with collagen/glycosaminoglycan matrices by RGD derivatization

The interaction of three cell types important to the wound repair process with collagen/glycosaminoglycan (GAG) dermal regeneration matrices covalently modified with an Arg-Gly-Asp (RGD)-containing peptide was characterized. Function-blocking monoclonal antibodies directed against various integrin subunits were used to demonstrate that human fibroblasts attached to the unmodified matrix through the integrin, alpha2beta1. Human endothelial cells and human keratinocytes, however, attached minimally to the unmodified matrix. After modification of the collagen/GAG matrix with RGD-containing peptide, endothelial cells and keratinocytes attached and spread well on the matrix. This attachment was RGD dependent as evidenced by essentially complete inhibition with competing soluble peptide. In terms of overall cell number, fibroblast cell attachment remained unchanged on the RGD peptide-modified matrix compared to the unmodified material. Antibody and peptide inhibition studies demonstrate, however, that attachment to the modified matrix was mediated by both alpha2beta1 and RGD-binding integrins. We have successfully introduced a specific RGD receptor-mediated attachment site on collagen/GAG dermal regeneration matrices, resulting in enhanced cell interaction of important wound healing cell types. This modification could have important implications for the performance of these matrices in promoting dermal regeneration.     

CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize beta1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion

Cell migration may depend on integrin-mediated adhesion to and deadhesion from extracellular matrix ligands. This concept, however, has not yet been confirmed for T lymphocytes migrating in three-dimensional extracellular matrices. We investigated receptor involvement in T cell migration combining a three-dimensional collagen matrix model with time-lapse videomicroscopy, computer-assisted cell tracking and confocal microscopy. In collagen lattices, the migration of CD4+ T cells (1) involved interactions with collagen fibers at the leading edge and uropod likewise, (2) occurred independently of the co-clustering of beta1, beta2, or beta3 integrins with F-actin, focal adhesion kinase, and phosphotyrosine at interactions with collagen fibers, (3) was counteracted by high-affinity beta1 integrin binding induced by antibody TS2/16; however, (4) the migration could not be blocked by a combination of adhesion-perturbing anti-beta1, -beta2, -beta3, and alpha v integrin antibodies. Integrin blocking neither affected cell polarization, interaction with fibers, beta1 integrin distribution, migration velocity, path structure, nor the number of locomoting cells in spontaneously migrating or concanavalin A-activated cells. Hence, T lymphocytes migrating in three-dimensional collagen matrices may utilize highly transient interactions with collagen fibers of low adhesivity, thereby differing from focal adhesion-dependent migration strategies employed by other cells.     

Multiple Grb2-mediated integrin-stimulated signaling pathways to ERK2/mitogen-activated protein kinase: summation of both c-Src- and focal adhesion kinase-initiated tyrosine phosphorylation events

Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.     

Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.     

A collagen peptide motif activates tyrosine kinase-dependent calcium signalling pathways in human osteoblast-like cells

A collagen peptide motif (DGEA) which is a putative alpha 2 beta 1 integrin binding site was examined for its ability to activate Ca2+ signalling pathways in the human osteoblast-like cell line SaOS-2. We show that these cells express both alpha 2 beta 1 integrin subunits (by immunocytochemistry) and that an anti-beta 1 monoclonal antibody (DF5) mobilizes Ca2+ in these cells. DGEA elevated intracellular Ca2+ in fura-2-loaded cells, in a concentration- and sequence-dependent fashion, with an EC50 of 250 microM. The tyrosine kinase inhibitor herbimycin A reduced the number of cells responding to DGEA and to transforming growth factor alpha. Thrombin also stimulated a rise in intracellular Ca2+, but the number of cells responding was not reduced by herbimycin A. The DGEA response was dependent on extracellular Ca2+, but was not due to Ca2+ influx, since it was blocked by thapsigargin and not by lanthanum. Using three different anti-alpha 2 monoclonal antibodies, we were unable to show that the DGEA-induced Ca2+ signal was mediated by the alpha 2 beta 1 integrin. In summary, the DGEA collagen motif does appear to activate receptor-mediated Ca2+ signalling events in SaOS-2 cells, in a divalent cation-dependent manner, but we were unable to demonstrate a role for alpha 2 beta 1 integrin in this response.     

The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function

alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Disruption of fibronectin binding to the alpha 5 beta 1 integrin stimulates the expression of cyclin-dependent kinases and DNA synthesis through activation of extracellular signal-regulated kinase

The alpha 5 alpha 1 integrin, a fibronectin receptor, has been implicated in the control of cell growth and the regulation of gene expression. We report that disruption of ligation between alpha 5 alpha 1 and fibronectin by integrin alpha 5 subunit or fibronectin monoclonal antibodies stimulated DNA synthesis in growth-arrested FET human colon carcinoma cells. This stimulation only occurred when monoclonal antibody was added in the early G1 phase of the cell cycle after release from quiescence by fresh medium. Stimulation of DNA synthesis by alpha 5 or fibronectin antibody was concentration- and time-dependent. FET cells expressed alpha 4 beta 1 integrin (another fibronectin receptor); however, addition of anti-human integrin alpha 4 monoclonal antibody had no effect on DNA synthesis. Treatment with alpha 5 monoclonal antibody led to a marked increase in the expression of CDK4 in G1 phase of the cell cycle and consequently increased the phosphorylation of retinoblastoma protein. alpha 5 monoclonal antibody treatment increased both cyclin A- and cyclin E-associated kinase activity which was accompanied by increased protein levels of CDK2 and cyclin A. Western blotting of immunoprecipitates demonstrated increased CDK2-cyclin E and CDK2-cyclin A complexes in cells treated with alpha 5 monoclonal antibody. Furthermore, disruption of alpha 5 alpha 1/fibronectin ligation activated mitogen-activated protein kinase p44 and p42 (extracellular signal-regulated kinase 1 and 2). Pretreatment of the cells with a specific inhibitor of MEK-1, PD98059, blocked the alpha 5 monoclonal antibody-induced mitogen-activated protein kinase activity. In addition PD98059 prevented alpha 5 monoclonal antibody-induced DNA synthesis. Since alpha 5 alpha 1 ligation to fibronectin is associated with decreased growth parameters, our results indicate that ligation of alpha 5 alpha 1 integrin to fibronectin results in suppressed mitogen-activated protein kinase activity which in turn inhibits cyclin-dependent kinase activity in growth-arrested cells.