Cell-surface regulation of beta 1-integrin activity on developing retinal neurons.

Integrins are a family of alpha beta heterodimeric receptors that mediate cell-cell and cell-substratum interactions. Integrin binding to extracellular ligands regulates cell adhesion, shape, motility, intracellular signalling and gene expression. Mechanisms that regulate integrin function are, therefore, central to the participation of integrins in a diverse set of cellular events. Here we report the identification of TASC, a monoclonal antibody to a novel epitope on the integrin beta 1 subunit, which inhibits cell adhesion to vitronectin but promotes adhesion to laminin and collagen types I and IV. We show that developing retinal neurons that have lost responsiveness to laminin regain the ability to bind laminin in the presence of TASC. Thus, beta 1-class integrins are likely to occupy multiple affinity states that can be modulated at the cell surface.     

Laminin receptor on platelets is the integrin VLA-6

Adhesion of platelets to the subendothelial matrix of an injured vessel wall is an essential step in triggering the formation of a haemostatic plug. Fibronectin, collagen and laminin are three major components of the subendothelial matrix which support platelet adhesion. Receptors for fibronectin and collagen have been identified on platelets and are included in the integrin family. Here we report that adhesion of platelets to laminin is inhibited by a rat monoclonal antibody against the integrin family member, VLA-6. This antibody does not affect platelet adhesion to fibrinogen, fibronectin or to type I and III collagen. Binding to laminin does not require platelet activation and is not inhibited by fibronectin and laminin cell-attachment peptides. Platelet adhesion to laminin is supported by Mn2+, Co2+ and Mg2+, but not by Ca2+, Zn2+ and Cu2+. This cation preference is distinct from that characteristic for other platelet-adhesive glycoproteins.     

Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation.

Increasing evidence indicates that the integrin family of cell adhesion receptors can transduce biochemical signals from the extracellular matrix to the cell interior to modulate cell growth and differentiation. We have shown that integrin/ligand interactions can trigger tyrosine phosphorylation of a protein of M(r) 120,000 (pp120), so it is possible that signal transduction by integrins might involve activation of intracellular protein tyrosine kinases as an early event in cell binding to the extracellular matrix. Here we report that pp120 is identical to the focal adhesion-associated protein tyrosine kinase pp125FAK (refs 3, 4). We show that tyrosine phosphorylation of this protein is modulated both by cell adhesion and transformation by pp60v-src, and that these changes in phosphorylation are correlated with increased pp125FAK tyrosine kinase activity. A model is proposed to relate these findings to the molecular basis of anchorage-independent growth of transformed cells.     

Structural basis for vinculin activation at sites of cell adhesion

Vinculin is a highly conserved intracellular protein with a crucial role in the maintenance and regulation of cell adhesion and migration. In the cytosol, vinculin adopts a default autoinhibited conformation. On recruitment to cell-cell and cell-matrix adherens-type junctions, vinculin becomes activated and mediates various protein-protein interactions that regulate the links between F-actin and the cadherin and integrin families of cell-adhesion molecules. Here we describe the crystal structure of the full-length vinculin molecule (1,066 amino acids), which shows a five-domain autoinhibited conformation in which the carboxy-terminal tail domain is held pincer-like by the vinculin head, and ligand binding is regulated both sterically and allosterically. We show that conformational changes in the head, tail and proline-rich domains are linked structurally and thermodynamically, and propose a combinatorial pathway to activation that ensures that vinculin is activated only at sites of cell adhesion when two or more of its binding partners are brought into apposition.     

ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.     

Type I gamma phosphatidylinositol phosphate kinase targets and regulates focal adhesions

The ability of cells to form cell contacts, adhere to the extracellular matrix, change morphology, and migrate is essential for development, wound healing, metastasis, cell survival and the immune response. These events depend on the binding of integrin to the extracellular matrix, and assembly of focal adhesions, which are complexes comprising scaffolding and signalling proteins organized by adhesion to the extracellular matrix. Phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) regulates interactions between these proteins, including the interaction of vinculin with actin and talin. The binding of talin to beta-integrin is strengthened by PtdIns(4,5)P(2), suggesting that the basis of focal adhesion assembly is regulated by this lipid mediator. Here we show that the type I phosphatidylinositol phosphate kinase isoform-gamma 661 (PIPKI gamma 661), an enzyme that makes PtdIns(4,5)P(2), is targeted to focal adhesions by an association with talin. PIPKI gamma 661 is tyrosine phosphorylated by focal adhesion associated kinase signalling, increasing both the activity of phosphatidylinositol phosphate kinase and its association with talin. This defines a mechanism for spatial generation of PtdIns(4,5)P(2) at focal adhesions.     

Structural basis for allostery in integrins and binding to fibrinogen-mimetic therapeutics

Integrins are important adhesion receptors in all Metazoa that transmit conformational change bidirectionally across the membrane. Integrin alpha and beta subunits form a head and two long legs in the ectodomain and span the membrane. Here, we define with crystal structures the atomic basis for allosteric regulation of the conformation and affinity for ligand of the integrin ectodomain, and how fibrinogen-mimetic therapeutics bind to platelet integrin alpha(IIb)beta3. Allostery in the beta3 I domain alters three metal binding sites, associated loops and alpha1- and alpha7-helices. Piston-like displacement of the alpha7-helix causes a 62 degrees reorientation between the beta3 I and hybrid domains. Transmission through the rigidly connected plexin/semaphorin/integrin (PSI) domain in the upper beta3 leg causes a 70 A separation between the knees of the alpha and beta legs. Allostery in the head thus disrupts interaction between the legs in a previously described low-affinity bent integrin conformation, and leg extension positions the high-affinity head far above the cell surface.     

Conservation of the sizes for one but not another class of exons in two chick collagen genes

Type III collagen is often found in the same tissues as type I collagen, yet the function and nature of the fibrils formed by the two collagens differ markedly. To understand the evolutionary history of the collagen gene family in more detail, we isolated the gene for type III collagen and compared its structure with that of the gene for alpha 2(I) collagen. This comparison points to a remarkable conservation in the size distribution of the exons coding for the helical part of these two collagen polypeptides: equivalent amino acid segments in the helical domain of each polypeptide are encoded by exons of equal sizes in each gene. This suggests that after the interstitial collagen genes had been duplicated from a common ancestor about 2-5 X 10(8) years ago, no recombinations between these exons were tolerated, although the same recombinational phenomena must have played an important part in shaping the structure of the progenitor for these genes. This fixation of the size distribution of the exons which code for the interstitial collagen helical domains is found despite the persistence in these exons of sequence elements that should have favoured recombinational rearrangements, and contrasts with the variations in the pattern of sizes of some exons coding for the amino and carboxyl propeptides of these collagens.     

Helicobacter exploits integrin for type IV secretion and kinase activation

Integrins are important mammalian receptors involved in normal cellular functions as well as pathogenesis of chronic inflammation and cancer. We propose that integrins are exploited by the gastric pathogen and type-1 carcinogen Helicobacter pylori for injection of the bacterial oncoprotein cytotoxin-associated gene A (CagA) into gastric epithelial cells. Virulent H. pylori express a type-IV secretion pilus that injects CagA into the host cell; CagA then becomes tyrosine-phosphorylated by Src family kinases. However, the identity of the host cell receptor involved in this process has remained unknown. Here we show that the H. pylori CagL protein is a specialized adhesin that is targeted to the pilus surface, where it binds to and activates integrin alpha5beta1 receptor on gastric epithelial cells through an arginine-glycine-aspartate motif. This interaction triggers CagA delivery into target cells as well as activation of focal adhesion kinase and Src. Our findings provide insights into the role of integrins in H.-pylori-induced pathogenesis. CagL may be exploited as a new molecular tool for our further understanding of integrin signalling.     

Subtle structural alterations in the chains of type I procollagen produce osteogenesis imperfecta type II

Although the perinatal lethal form of osteogenesis imperfecta (OI type II) occasionally results from large rearrangements within the genes encoding type I collagen, most mutations are far more subtle. The complexity of the human collagen genes precludes cloning and sequencing each gene from every patient, and we have therefore developed an approach to localizing mutations at the protein level. We report here that cells cultured from 15 infants with OI type II synthesized both normal type I procollagen and a form that was unstable, poorly secreted and excessively modified. Abnormal procollagen from different strains was overmodified to different extents. The patterns of overmodification we observed are best explained by mutations that disrupt the Gly-X-Y sequence of pro alpha chains, and thus alter the rate of propagation of triple helix from COOH-terminus to NH2-terminus. As a consequence, a given mutation allows overmodification of all three chains in a molecule NH2-terminal to its position in the triple helix.     

Vinculin activation by talin through helical bundle conversion

Vinculin is a conserved component and an essential regulator of both cell-cell (cadherin-mediated) and cell-matrix (integrin-talin-mediated focal adhesions) junctions, and it anchors these adhesion complexes to the actin cytoskeleton by binding to talin in integrin complexes or to alpha-actinin in cadherin junctions. In its resting state, vinculin is held in a closed conformation through interactions between its head (Vh) and tail (Vt) domains. The binding of vinculin to focal adhesions requires its association with talin. Here we report the crystal structures of human vinculin in its inactive and talin-activated states. Talin binding induces marked conformational changes in Vh, creating a novel helical bundle structure, and this alteration actively displaces Vt from Vh. These results, as well as the ability of alpha-actinin to also bind to Vh and displace Vt from pre-existing Vh-Vt complexes, support a model whereby Vh functions as a domain that undergoes marked structural changes that allow vinculin to direct cytoskeletal assembly in focal adhesions and adherens junctions. Notably, talin's effects on Vh structure establish helical bundle conversion as a signalling mechanism by which proteins direct cellular responses.     

High cell density alters the ratio of type III to I collagen synthesis by fibroblasts

HUMAN fibroblasts in culture synthesise both type I and type III collagen(1), with type I accounting for 70-90% of the total(2). In culture, the rates at which these proteins are synthesised is constant and apparently rather rigidly controlled(3). However, the proportions of these collagens differs in cells cultured with increased amounts of serum (increased type III/I)(4) as well as in cells obtained from patients with certain diseases. Cells from patients with the Ehlers-Danlos type IV syndrome make little or no type III collagen(5,6), whereas cells from patients with osteogenesis imperfecta have an increased type III/I (refs 7, 8). We have found that cells from some patients with systemic sclerosis (scleroderma), have a reduced type III/I ratio. However, as previously reported, these cells grew to a lower density than control cells(9). We report here that normal fibroblasts from human and guinea pig skin produce proportionally more type III collagen at high cell density, probably because of a reduction in the synthesis of type I collagen.     

Perinatal lethal osteogenesis imperfecta in transgenic mice bearing an engineered mutant pro-alpha 1(I) collagen gene

Substitutions of single glycine residues of alpha 1(I) collagen have previously been associated with the inherited disease osteogenesis imperfecta type II. Transgenic mice bearing a mutant alpha 1(I) collagen gene into which specific glycine substitutions have been engineered show a dominant lethal phenotype characteristic of the human disease, and demonstrate that as little as 10% mutant gene expression can disrupt normal collagen function.     

Lysyl oxidase is essential for hypoxia-induced metastasis

Metastasis is a multistep process responsible for most cancer deaths, and it can be influenced by both the immediate microenvironment (cell-cell or cell-matrix interactions) and the extended tumour microenvironment (for example vascularization). Hypoxia (low oxygen) is clinically associated with metastasis and poor patient outcome, although the underlying processes remain unclear. Microarray studies have shown the expression of lysyl oxidase (LOX) to be elevated in hypoxic human tumour cells. Paradoxically, LOX expression is associated with both tumour suppression and tumour progression, and its role in tumorigenesis seems dependent on cellular location, cell type and transformation status. Here we show that LOX expression is regulated by hypoxia-inducible factor (HIF) and is associated with hypoxia in human breast and head and neck tumours. Patients with high LOX-expressing tumours have poor distant metastasis-free and overall survivals. Inhibition of LOX eliminates metastasis in mice with orthotopically grown breast cancer tumours. Mechanistically, secreted LOX is responsible for the invasive properties of hypoxic human cancer cells through focal adhesion kinase activity and cell to matrix adhesion. Furthermore, LOX may be required to create a niche permissive for metastatic growth. Our findings indicate that LOX is essential for hypoxia-induced metastasis and is a good therapeutic target for preventing and treating metastases.     

青少年特发性脊柱侧凸椎间盘胶原的异常代谢模式

探寻青少年特发性脊柱侧凸 (简称AIS)患者侧凸区椎间盘胶原代谢模式的异常 .实验组为顶椎为腰椎的特发性脊柱侧凸患者 2 0例 ,其中男性 4例、女性 1 6例 ,年龄 1 2～ 1 9岁 (平均 1 4 .5岁 ) .术前Cobb角 38°～ 90°,平均 60°.1 5例同年龄段先天性脊柱侧凸为对照组 .标本取自脊柱侧凸前路手术 ,取材节段为L1～L2和L2～L3 .该取材节段在实验组为侧凸顶椎区 ,而在对照组则为侧凸的下终椎区 ,胃蛋白酶 -乙酸 (0 .5mol/L)体系提取胶原组织 ,SDS -聚丙烯酰胺凝胶电泳法及抗原抗体Western印记法进行胶原的分类和定量测定 .在GelWork图像分析系统中进行泳道的对比定量分析 .分别计算病变组和正常组纤维环和髓核中Ⅰ型、Ⅱ型胶原含量的均值和标准差 (X±s) ,并对正常组和病变组之间差异进行显著性t检验 .结果显示AIS椎间盘纤维环和髓核含水量与对照组相比显著下降 .Ⅰ型、Ⅱ型胶原在纤维环中的含量在AIS的凹侧明显低于凸侧且有显著性差异 (P <0 .0 5) ,和对照组相比较 ,Ⅰ型、Ⅱ型胶原的含量也有显著下降 (P <0 .0 1 ) .Ⅰ型胶原在AIS椎间盘的髓核中有升高 ,而Ⅱ型胶原在髓核的凹侧凸侧没有明显的差异 ,但是和对照组相比较含量显著降低 (P <0 .0 5) .本实验所采用的胶原提取和定量分析方法具有直观性和精确性     

Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions

The self-renewal and differentiation of haematopoietic stem cells occurs in vivo and in vitro in direct contact with cells making up the haematopoietic microenvironment. In this study we used adhesive ligands and blocking antibodies to identify stromal cell-derived extracellular matrix proteins involved in promoting attachment of murine haematopoietic stem cells. Here we report that day-12 colony-forming-unit spleen (CFU-S12)5 cells and reconstituting haematopoietic stem cells attach to the C-terminal, heparin-binding fragment of fibronectin by recognizing the CS-1 peptide of the alternatively spliced non-type III connecting segment (IIICS) of human plasma fibronectin. Furthermore, CFU-S12 stem cells express the alpha 4 subunit of the VLA-4 integrin receptor, which is known to be a receptor for the CS-1 sequence, and monoclonal antibodies against the integrin alpha 4 subunit of VLA-4 block adhesion of CFU-S12 stem cells to plates coated with the C-terminal fibronectin fragment. Finally, polyclonal antibodies against the integrin beta 1 subunit of VLA-4 inhibit the formation of CFU-S12-derived spleen colonies and medullary haematopoiesis in vivo following intravenous infusion of antibody-treated bone marrow cells.