Randall H. Kramer

β1 And β3 integrins have different roles in the adhesion and migration of vascular smooth muscle cells on extracellular matrix

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

Expression of the C‐Met/HGF receptor in human breast carcinoma: Correlation with tumor progression

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell motility and tissue remodeling of various epithelial cells through its receptor, the product of the proto‐oncogene c‐met. High levels of HGF/SF have been correlated with poor prognosis in human breast carcinoma. In this study, we examined the expression of the c‐Met receptor in human breast‐carcinoma cells in vivo and in cultured cell lines. Immunohistochemical analysis of biopsy samples of human breast carcinoma indicated that, in normal mammary gland, c‐Met is localized in the ductal epithelium. The level of expression of c‐Met in primary carcinomas was maintained in autologous metastatic lymph‐node lesions in some cases, and in other cases was elevated. Frequently there was evidence of heterogeneity in cellular expression of c‐Met within individual tumors, suggesting that micro‐environmental factors may regulate receptor expression. In an analysis of a panel of human breast‐carcinoma cell lines, we found that moderately differentiated cell lines did not express detectable levels of c‐Met and were not responsive to HGF. In contrast, poorly differentiated and invasive cell lines did express high levels of the receptor and responded to HGF by increased motility and invasiveness. Sensitivity to HGF/SF also correlated with expression of the c‐Met 9‐kb mRNA. No correlation was found between gene copy number and the expression level of c‐Met protein or mRNA. When the moderately differentiated and c‐Met‐negative T47D cell line was transfected with c‐DNA for c‐met, the transfectants showed delayed cell scattering and migratory response to HGF. Thus, over‐expression of c‐Met in moderately differentiated carcinoma cells may be one of several attributes that contribute to an invasive phenotype during the progression of breast cancer. Int. J. Cancer 74:301–309, 1997.

Basic FGF and TGF-β differentially modulate integrin expression of human microvascular endothelial cells☆

Basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) are known to alter the migratory and proliferative capacity of endothelial cells in vitro and to stimulate angiogenesis in vivo. One mechanism by which these cytokines induce their effects may be through the regulation of integrin adhesion receptor expression and activity. We examined the ability of these growth factors to modulate the expression of specific integrins in human microvascular endothelial cells (MEC). Immunoprecipitation of metabolically labeled MEC showed that bFGF upregulated the biosynthesis of alpha 2, alpha 5, beta 1, and beta 3. bFGF induced an increase in the levels of mRNA for alpha 2 and beta 1. TGF-beta increased synthesis of alpha 2, alpha 5, and beta 1. These results suggest that bFGF and TGF-beta selectively alter integrin profiles and influence interactions of MEC with the extracellular matrix during neovascularization. In particular, the upregulation of the collagen/laminin receptor, alpha 2 beta 1, by bFGF may provide activated endothelial cells with an enhanced capacity to migrate through both their underlying basement membrane and the interstitial matrix.

Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor.

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

α7 Integrin Mediates Cell Adhesion and Migration on Specific Laminin Isoforms

The laminin-binding α7β1 integrin receptor is expressed at high levels by skeletal and cardiac muscles and by certain melanocytic cells. We have assessed the potential role of the α7A/B integrin isoforms in mediating cell adhesion and motility and determined the laminin isoform specificity of this integrin. When MCF-7 breast carcinoma cells, normally nonadherent to laminin 1, were stably transfected with cDNA for mouse α7, they adhered with high efficiency and migrated on laminin 1 substrates. Function-perturbing monoclonal antibodies generated to mouse α7 subunit blocked both adhesion and migration of α7 transfectants on laminin 1 substrates. Additional studies with MCF-7 transfectants revealed that α7β1 binds well to laminin 1 and to a mixture of laminin 2 and 4 but not to laminin 5. Importantly, α7β1 was capable of promoting motility on both laminin 1 and laminin 2/4 substrates. However, MCF-7 cells transfected with cDNA for either α7A or α7B showed no significant differences in cell adhesion or motility on laminin 1 substrates. Although the role for the alternatively spliced cytoplasmic variants of α7 remains unknown, the results establish that α7β1 mediates cell adhesive activities on a restricted number of laminin isoforms.

Integrin Engagement Differentially Modulates Epithelial Cell Motility by RhoA/ROCK and PAK1

Integrin-ligand binding regulates tumor cell motility and invasion. Cell migration also involves the Rho GTPases that control the interplay between adhesion receptors and the cytoskeleton. We evaluated how specific extracellular matrix ligands modulate Rho GTPases and control motility of human squamous cell carcinoma cells. On laminin-5 substrates, the epithelial cells rapidly spread and migrated, but on type I collagen the cells spread slowly and showed reduced motility. We found that RhoA activity was suppressed in cells attached to laminin-5 through the α3 integrin receptor. In contrast, RhoA was strongly activated in cells bound to type I collagen and this was mediated by the α2 integrin. Inhibiting the RhoA pathway by expression of a dominant-negative RhoA mutant or by directly inhibiting ROCK, reduced focal adhesion formation and enhanced cell migration on type I collagen. Cdc42 and Rac and their downstream target PAK1 were activated following adhesion to laminin-5. PAK1 activation induced by laminin-5 was suppressed by expression of a dominant-negative Cdc42. Moreover, constitutively active PAK1 stimulated migration on collagen I substrates. Our results indicate that in squamous epithelial cells, collagen-α2β1 integrin binding activates RhoA, slowing cell locomotion, whereas laminin-5-α3β1 integrin interaction inhibits RhoA and activates PAK1, stimulating cell migration. The data demonstrate that specific ligand-integrin pairs regulate cell motility differentially by selectively modulating activities of Rho GTPases and their effectors.

Integrin receptors on aortic smooth muscle cells mediate adhesion to fibronectin, laminin, and collagen.

Extracellular matrix receptors on vascular smooth muscle cells help in anchoring the cells during contraction and in promoting cellular migration after vessel injury. We found that rat aortic smooth muscle cells attach to surfaces coated with fibronectin, laminin, and collagen types I and IV. Cell attachment to these substrates appears to be mediated by members of the beta 1 integrin family of extracellular matrix receptors. Antibodies to the beta 1 subunit not only demonstrated the presence of integrin complexes in focal adhesion plaques but also blocked cell adhesion to the different substrates. Ligand-affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis isolated a series of receptor complexes that were recognized by antisera to beta 1 integrin receptors. Each of the receptors appeared to be a heterodimer in which one of several alpha subunits shared a common 120-kDa (nonreduced) beta 1 subunit protein. The rat aortic smooth muscle cells had one alpha subunit (150 kDa nonreduced, 140 kDa reduced) that bound exclusively to fibronectin. There was a second alpha subunit (150 kDa nonreduced, 160 kDa reduced) that bound exclusively to collagen type I. In addition, there was a third alpha subunit (185 kDa nonreduced, 200 kDa reduced) that was promiscuous and bound to collagen types I and IV as well as to laminin; the 185-kDa alpha subunit appeared to bind to collagen more efficiently than it did to laminin. Thus, smooth muscle cells express multiple integrin receptors with different ligand specificities that appear to mediate cell interactions with the extracellular matrix.

Integrin expression in malignant melanoma

Invasion of melanoma cells into the underlying interstitial stromal matrix is the initial step for subsequent local and distant metastasis. The invading tumor cell must interact with the extracellular matrix during the early stages of invasion and later during penetration of lymphatic and blood vessels. This interaction with different types of extracellular matrix predicts that the invasive cell must possess surface adhesion receptors with diverse ligand specificities, including the capacity to bind different types of collagens and adhesive glycoproteins. Metastatic melanoma cells do in fact express multiple adhesion receptors, including several of the receptors from the integrin family of heterodimers. The integrin receptors can be either extremely specific for a single ligand or capable of binding multiple ligands. It is likely that the tumor cells repertoire of adhesion receptors may influence not only its adhesive properties but its metastatic characteristics as well. There is evidence that normal melanocytes have an integrin profile distinct from that of melanoma cells. In particular, melanocytes adhere poorly to laminin while metastatic melanoma cells bind well to this ligand. This difference in adhesion between the two cell types appears to reflect the fact that melanoma cells express a melanoma-specific integrin (% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiGc9yrFr0xXdbba91rFfpec8Eeeu0x% Xdbba9frFj0-OqFfea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs% 0dXdbPYxe9vr0-vr0-vqpWqaaeaabiGaciaacaqabeaadaqaaqGaaO% qaaiaadggadaWgaaWcbaGaamODaaqabaGccqaHYoGydaWgaaWcbaGa% aG4maaqabaaaaa!40CF!\[a_7 \beta _1 \]) that binds laminin and is not detectable in normal melanocytes. The presence of increased laminin receptors and enhanced laminin binding in melanoma cells may contribute to the malignant phenotype.

Stromal fibroblasts influence oral squamous‐cell carcinoma cell interactions with tenascin‐C

In this study we identified tenascin‐C (TN‐C) and one of its integrin receptors, αvβ6, in oral squamous‐cell carcinoma (SCC) specimens. Neither TN‐C nor αvβ6 are expressed in normal oral mucosa. We also studied 2 human oral squamous‐cell carcinoma cell lines: the highly invasive HSC‐3 cells, and the poorly invasive SCC‐25 cells. We determined that adhesion of these cells to TN‐C involves both α2 and αv integrins. Migration on TN‐C by oral SCC cells required fibroblast‐conditioned medium and did not occur in its absence. This migration was blocked by anti‐α2 and anti‐αv antibodies and was partially inhibited by antibodies to hepatocyte growth factor, epidermal growth factor and transforming growth factor‐β1. When seeded on TN‐C, the poorly invasive SCC‐25 cells formed αvβ6‐positive focal contacts; the HSC‐3 cells did not. HSC‐3, SCC‐25 and PTF cells secrete TN‐C into the culture medium, as determined by Western blot. However, when HSC‐3 cells were inoculated into the floor of the mouth of nude mice, only murine TN‐C could be identified in the reactive stroma adjacent to the resulting tumor nests, demonstrating that in vivo, HSC‐3 cells do not secrete TN‐C. Our results demonstrate that αvβ6 and tenascin‐C are neo‐expressed in oral squamous‐cell carcinoma, and that the tumor stromal environment is influential in oral SCC behavior. Int. J. Cancer 72:369–376, 1997.

Growth factor regulation of integrin-mediated cell motility

SummaryCell motility, a primary component of tumor cell invasion, is a continuum of sequential events in which the cell extends pseudopodia, forms nascent attachments, assembles and contracts the cytoskeleton, and finally, as it translocates forward, disengages distal adhesions. What triggers cells to move? Substratum contact mediated by integrin adhesion receptors is important, but other signals such as chemokinetic factors appear to be required for continued crawling. It is now apparent that integrins do not simply bind cells to matrix in a Velcrolike fashion, but also are potent signaling molecules. Initial engagement of integrins induces their condensation into focal contacts, forming anchors to the extracellular matrix and discrete signal-transducing complexes on the cytoplasmic surface. A number of growth factors, through either autocrine or paracrine pathways, can activate the cellular machinery that mobilizes the cell. Thus, these two classes of receptors - the integrin receptors that bind specific extracellular adhesion molecules, and growth factor receptors that bind their respective ligands - can regulate cell locomotion. Not surprisingly, there is ‘cross-talk’ between integrin and growth factor receptors that occurs through their common intracellular signaling pathways. In this way, each receptor type can either amplify or attenuate the others signal and downstream response. An example of growth factor-induced motility is the epithelial-mesenchymal transition induced by hepatocyte growth factor/scatter factor (HGF/SF). When bound to its receptor, the c-met proto-oncogene product, HGF/SF induces a phenotypic conversion that appears to be an important aspect of tumor progression in malignant carcinomas. The motogenic response produced by HGF/SF in carcinoma cells occurs in discrete steps in which integrins and focal adhesion kinase (p125FAK) are first recruited to focal contacts. This is rapidly followed by cell spreading, disruption of focal adhesions and cell-cell contacts, and, finally, cell crawling. The precise mechanism by which growth factors such as HGF/SF and its receptor induce this motogenic response and modulate integrin function has not been clearly defined but appears to involve several signaling pathways. Understanding the process by which growth factor and integrin receptors interact and regulate motility may suggest novel targets for therapeutic intervention.