Richard O. Hynes

Integrins: bidirectional, allosteric signaling machines.

In their roles as major adhesion receptors, integrins signal across the plasma membrane in both directions. Recent structural and cell biological data suggest models for how integrins transmit signals between their extracellular ligand binding adhesion sites and their cytoplasmic domains, which link to the cytoskeleton and to signal transduction pathways. Long-range conformational changes couple these functions via allosteric equilibria.

Integrins: A family of cell surface receptors

What do cell adhesion, cell migration during embryogenesis, thrombosis, and lymphocyte help and killing have in common? It has recently become clear that these ostensibly unrelated processes all involve related cell surface receptors. This realization comes from sequence data, which show that cell surface receptors for extracellular matrix proteins, a major glycoprotein complex on platelet cell surfaces, and two groups of glycoprotein antigens on lymphoid and myeloid cells all contain homologous subunits. In this brief review, I summarize data from these diverse fields and attempt to clarify the relationships among the members of this family of cell surface receptors.

Genomic analysis of metastasis reveals an essential role for RhoC

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.

Thrombospondin-1 Is a Major Activator of TGF-β1 In Vivo

The activity of TGF-beta1 is regulated primarily extracellularly where the secreted latent form must be modified to expose the active molecule. Here we show that thrombospondin-1 is responsible for a significant proportion of the activation of TGF-beta1 in vivo. Histological abnormalities in young TGF-beta1 null and thrombospondin-1 null mice were strikingly similar in nine organ systems. Lung and pancreas pathologies similar to those observed in TGF-beta1 null animals could be induced in wild-type pups by systemic treatment with a peptide that blocked the activation of TGF-beta1 by thrombospondin-1. Although these organs produced little active TGF-beta1 in thrombospondin null mice, when pups were treated with a peptide derived from thrombospondin-1 that could activate TGF-beta1, active cytokine was detected in situ, and the lung and pancreatic abnormalities reverted toward wild type.

Leukocyte rolling and extravasation are severely compromised in P selectin-deficient mice.

P selectin, expressed on surfaces of activated endothelial cells and platelets, is an adhesion receptor for leukocytes. We report that P selectin-deficient mice, generated by gene targeting in embryonic stem cells, exhibit a number of defects in leukocyte behavior, including elevated numbers of circulating neutrophils, virtually total absence of leukocyte rolling in mesenteric venules, and delayed recruitment of neutrophils to the peritoneal cavity upon experimentally induced inflammation. These results clearly demonstrate a role for P selectin in leukocyte interactions with the vessel wall and in the early steps of leukocyte recruitment at sites of inflammation. These mutant mice should prove useful in deciphering the contributions of P selectin in various inflammatory responses as well as in platelet functions.

Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts

Id proteins may control cell differentiation by interfering with DNA binding of transcription factors. Here we show that targeted disruption of the dominant negative helix–loop–helix proteins Id1 and Id3 in mice results in premature withdrawal of neuroblasts from the cell cycle and expression of neural-specific differentiation markers. The Id1–Id3 double knockout mice also display vascular malformations in the forebrain and an absence of branching and sprouting of blood vessels into the neuroectoderm. As angiogenesis both in the brain and in tumours requires invasion of avascular tissue by endothelial cells, we examined the Id knockout mice for their ability to support the growth of tumour xenografts. Three different tumours failed to grow and/or metastasize in Id1+/-Id3-/- mice, and any tumour growth present showed poor vascularization and extensive necrosis. Thus, the Id genes are required to maintain the timing of neuronal differentiation in the embryo and invasiveness of the vasculature. Because the Id genes are expressed at very low levels in adults, they make attractive new targets for anti-angiogenic drug design.

Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons

What determines whether cells remain in one place, retaining their associations with their neighbors, or dissociate from these associations and move elsewhere? And, if they move, what determines where they go, where and when they stop, and whether or not they associate with like or unlike cells? These questions arise about many events during development, especially but not exclusively the development of the nervous system. The same questions arise when considering normal physiological processes, such as lymphocyte traffic, wound healing, and hemostasis, and pathologies such as invasion and metastasis, thrombosis, and inflammation. All these processes involve alterations in cell associations, many involve cellular (or axonal) migration, and all must be conducted with appropriate specificity to ensure proper development and function of the organism. Yn recent years there have been major advances in deciphering the molecular bases for the cell adhesion events underlying these biological phenomena. Perhaps it is not surprising that there are many different adhesive molecules involved; at times there appears to be a bewildering variety. However, common themes are emerging, and many of the molecules fall into families whose members appear in the different processes performing similar roles. This means that insights obtained in one system can be applied in others, and this cross fertilization among disciplines is proving very fruitful. In this review we will discuss current ideas concerning the roles of cell adhesion molecules and cell interactions, both in development and in several physiological and pathological processes, attempting to highlight the general features and commonalities and presenting working hypotheses.

β3-integrin–deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival

beta3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (alphaIIbbeta3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (alphavbeta3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the alphaIIb or the beta3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of beta3 integrins, we have generated a strain of beta3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of beta3 integrins and we report that postnatal neovascularization of the retina appears to be beta3-integrin-independent, contrary to expectations from inhibition experiments.

CD40L stabilizes arterial thrombi by a β3 integrin-dependent mechanism

CD40L, a member of the tumor necrosis factor family of ligands, plays a major role in immune responses via its receptor, CD40. Recently, CD40L has been detected on the surfaces of activated platelets and shown to activate endothelium. Here we further addressed the function of platelet CD40L. We show that absence of CD40L affects the stability of arterial thrombi and delays arterial occlusion in vivo. Infusion of recombinant soluble (rs)CD40L restored normal thrombosis, whereas rsCD40L lacking the KGD integrin-recognition sequence did not. CD40-deficient mice exhibited normal thrombogenesis. rsCD40L specifically bound to purified integrin αIIbβ3 and to activated platelets in a β3-dependent manner and induced platelet spreading. In addition, rsCD40L promoted the aggregation of either human or mouse platelets under high shear rates. Thus, CD40L appears to be an αIIbβ3 ligand, a platelet agonist, and necessary for stability of arterial thrombi.

Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin

We describe the isolation, characterization, and sequence of cDNA clones encoding one subunit of the complex of membrane glycoproteins that forms part of the transmembrane connection between the extracellular matrix and the cytoskeleton. The cDNA sequence encodes a polypeptide of 89 kd that has features strongly suggesting the presence of a large N-terminal extracellular domain, a single transmembrane segment, and a small C-terminal cytoplasmic domain. The extracellular domain contains a threefold repeat of a novel 40 residue cysteine-rich segment, and the cytoplasmic domain contains a tyrosine residue that is a potential site for phosphorylation by tyrosine kinases. We propose the name integrin for this protein complex to denote its role as an integral membrane complex involved in the transmembrane association between the extracellular matrix and the cytoskeleton.