Donald Gullberg

Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF.

The attachment of primary rat hepatocytes and fibroblasts to collagen type I is mediated by non-RGD-dependent beta 1 integrin matrix receptors. In this report we describe a novel 96-well microtiter plate assay for the quantification of fibroblast-mediated contraction of floating collagen type I gels. Fetal calf serum and platelet-derived growth factor (PDGF), but not transforming growth factor-beta 1, stimulated primary rat heart fibroblasts and normal human diploid fibroblasts (AG 1518) to contract collagen gels to less than 10% of the initial gel volume within a 24-h incubation period. Rabbit polyclonal antibodies directed to the rat hepatocyte integrin beta 1-chain inhibited the PDGF-stimulated collagen gel contraction. The inhibitory activity on contraction of the anti-beta 1 integrin IgG could be overcome by adding higher doses of PDGF. The contraction process was not blocked by anti-fibronectin IgG nor by synthetic peptides containing the tripeptide Arg-Gly-Asp (RGD), in concentrations that readily blocked fibroblast attachment to fibronectin-coated planar substrates. Autologous fibronectin or control peptides containing the tripeptide Arg-Gly-Glu were without effect. Immunofluorescence microscopy on fibroblasts grown within collagen gels revealed a punctate distribution of the beta 1 integrin and a lack of detectable levels of endogenously produced fibronectin. Collectively these data suggest a role for integrin collagen receptors with affinity for collagen fibers, distinct from the previously described RGD-dependent fibronectin receptors, in the fibronectin-independent PDGF-stimulated collagen gel contraction process.

Expression of collagen binding integrins during cardiac development and hypertrophy.

The interaction between components of the extracellular matrix and the cell surface of cardiac myocytes appears to be regulated in part by receptors belonging to the integrin superfamily. The expression of the integrins was investigated at different stages of development of the heart as well as during cardiac hypertrophy. The characterization of the membrane proteins showed that a beta 1-integrin and associated alpha-chains were responsible for the interaction with collagen, laminin, and fibronectin. Immunoprecipitation data indicated that the presence of specific alpha-chains varied with development. These data were correlated with the ability of the isolated myocytes to attach to specific components of the extracellular matrix. The expression of the alpha 1-chain was prominently associated with the recognition of interstitial collagens. The presence of the alpha 1-chain was also associated with stages when collagen synthesis was increased, especially during fetal and neonatal growth and cardiac hypertrophy. Immunohistochemical localization with the antiserum against beta 1-integrin demonstrated its specific localization near the Z lines of cardiac myocytes. The localization both in vitro and in vivo indicated that the beta 1-integrin may play a role in myofibrillogenesis during development. The present immunohistochemical, cell adhesion, and biochemical data clearly indicate that integrins play a major role in the regulation of the interaction between cardiac myocytes and the extracellular matrix during development and disease.

Analysis of alpha 1 beta 1, alpha 2 beta 1 and alpha 3 beta 1 integrins in cell--collagen interactions: identification of conformation dependent alpha 1 beta 1 binding sites in collagen type I.

Integrins can mediate the attachment of cells to collagen type I. In the present study we have investigated the possible differences in collagen type I recognition sites for the alpha 1 beta 1 and alpha 2 beta 1 integrins. Different cyanogen bromide (CB) fragments of the alpha 1 (I) collagen chain were used in cell attachment experiments with three rat cell types, defined with regard to expression of collagen binding integrins. Primary rat hepatocytes expressed alpha 1 beta 1, primary rat cardiac fibroblasts alpha 1 beta 1 and alpha 2 beta 1, and Rat‐1 cells only alpha 2 beta 1. All three cell types expressed alpha 3 beta 1 but this integrin did not bind to collagen‐‐Sepharose or to immobilized collagen type I in a radioreceptor assay. Hepatocytes and cardiac fibroblasts attached to substrata coated with alpha 1(I)CB3 and alpha 1(I)CB8; Rat‐1 cells attached to alpha 1(I)CB3 but only poorly to alpha 1(I)CB8‐coated substrata. Cardiac fibroblasts and Rat‐1 cells spread and formed beta 1‐integrin‐containing focal adhesions when grown on substrata coated with native collagen or alpha 1(I)CB3; focal adhesions were also detected in cardiac fibroblasts cultured on alpha 1(I)CB8. The rat alpha 1 specific monoclonal antibody 3A3 completely inhibited hepatocyte attachment to alpha 1(I)CB3 and alpha 1(I)CB8, as well as the attachment of cardiac fibroblasts to alpha 1(I)CB8, but only partially inhibited the attachment of cardiac fibroblasts to alpha 1(I)CB3. 3A3 IgG did not inhibit the attachment of Rat‐1 cells to collagen type I or to alpha 1(I)CB3.(ABSTRACT TRUNCATED AT 250 WORDS)

cDNA cloning and chromosomal localization of human alpha(11) integrin. A collagen-binding, I domain-containing, beta(1)-associated integrin alpha-chain present in muscle tissues.

We previously identified a novel integrin α-chain in human fetal muscle cells (Gullberg, D., Velling, T., Sjöberg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57–65). We have now isolated the full-length cDNA for this integrin subunit, α11. The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains seven conserved FG-GAP repeats, an I domain with a metal ion-dependent adhesion site motif, a short transmembrane region, and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. α11, like other I domain integrins, lacks a dibasic cleavage site for generation of a heavy chain and a light chain, and it contains three potential divalent cation binding sites in repeats 5–7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804–826) distinguishes the α11 integrin sequence from other integrin α-chains. Amino acid sequence comparisons reveal the highest identity of 42% with the α10 integrin chain. Immunoprecipitation with antibodies to α11 integrin captures a 145-kDa protein distinctly larger than the 140-kDa α2 integrin chain when analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Fluorescence in situ hybridization maps the integrin α11 gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting, integrin α11 mRNA levels are high in the adult human uterus and in the heart and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, α11 mRNA and protein are up-regulated. Studies of ligand binding properties show that α11β1binds collagen type I-Sepharose, and cultured muscle cells localize α11β1 into focal contacts on collagen type I. Future studies will reveal the importance of α11β1 for muscle development and integrity in adult muscle and other tissues.

Laminins during muscle development and in muscular dystrophies.

Abstract. Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. Laminins constitute a growing family of proteins with complex expression patterns in forming basement membranes during muscle development. In skeletal muscle, laminins constitute major ligands for cell surface receptors involved in the transmission of force from the cell interior, but laminins might also influence signal transmission events during muscle formation and in muscle regeneration. During myogenesis the laminin α1 chain is present around the epithelial somite; but later, in forming muscle, the laminin α1 chain is restricted to the myotendinous junction. The laminin α2,α4 and α5 chains are major laminin chains in the muscle basement membrane during muscle formation, but laminin α4 and α5 chains are absent in adult muscle. The importance of laminins for muscle integrity is manifested in congenital muscular dystrophies with defects in the laminin α2 chain. There is no good evidence for the presence of laminin α1 chain in dystrophic muscle, but some other fetal muscle laminins can be detected in dystrophic muscle. Characterization of laminin expression patterns in muscular dystrophies might be of diagnostic and therapeutic value. In this paper, we review the recent publications on the biological functions of muscle laminins and discuss their roles in skeletal muscle.

Collagen-binding I domain integrins--what do they do?

Collagens are the most abundant proteins in the mammalian body and it is well recognized that collagens fulfill an important structural role in the extracellular matrix in a number of tissues. Inactivation of the collagen alpha 1(I) gene in mice results in embryonic lethality and collagen mutations in humans cause defects leading to disease. Integrins constitute a major group of receptors for extracellular matrix components, including collagens. Currently four collagen-binding I domain-containing integrins are known, namely alpha 1 beta 1, alpha 2 beta 1, alpha 10 beta 1 and alpha 11 beta 1. Unlike the undisputed role of collagens as structural elements, the biological importance of integrin mediated cell-collagen interactions is far from clear. This is in part due to the limited information available on the most recent additions of the integrin family, alpha 10 beta 1 and alpha 11 beta 1. Future studies using gene inactivation of individual and multiple integrin genes will allow testing of the hypothesis that collagen-binding integrins have redundant functions but will also shed light on their importance in pathological conditions. In this review we will describe what is currently known about the collagen-binding integrins and discuss their biological functions.

Integrins During Muscle Development and in Muscular Dystrophies

Cellular interactions with the extracellular matrix (ECM) have been shown to be important for a number of developmental events from the time of fertilization up till the maturation of the organism. In the following review we will discuss what is currently known about these interactions with special emphasis on the role of integrins during the formation of skeletal muscle. The importance of cell-ECM interactions will also be illustrated by a discussion of what happens when these interactions go awry, as happens in muscular dystrophies.

INTEGRIN SUBUNIT EXPRESSION ASSOCIATED WITH EPITHELIAL-MESENCHYMAL INTERACTIONS DURING MURINE TOOTH DEVELOPMENT

The initial information for patterning of early tooth development resides in the epithelium. Later, this is shifted to the mesenchyme. The process is governed by multiple epithelial‐mesenchymal interactions. Integrins are cell surface receptors for extracellular matrix components. Expression of the β5 integrin subunit alternates between epithelium and mesenchyme during early tooth development (Yamada et al. [1994] Int. J. Dev. Biol. 38: 553–556). By immunofluorescence and in situ hybridization we show here a remarkably similar oscillating expression pattern of the αv integrin subunit. This subunit is known to associate with β5, and we therefore suggest that integrin αvβ5 is involved in epithelial‐mesenchymal interactions during tooth development. We also demonstrate that the developing tooth epithelium expresses the α6, β1 and β4 subunits. The laminin receptors α6β1 and α6β4 may thus in part mediate the effect of basement membranes on tooth epithelial development. Interestingly, the enamel knot region expressed very little α6 integrin subunit, whereas some expression was seen transiently in the condensing mesenchyme. During early tooth development, integrins possessing the α6 subunit might also be involved in cell‐cell interactions independently of laminins.

Distinct α7Aβ1 and α7Bβ1 integrin expression patterns during mouse development: α7A is restricted to skeletal muscle but α7B is expressed in striated muscle, vasculature, and nervous system

The laminin binding α7β1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of α7 integrin undergoes alternative splicing to generate two major forms, denoted α7A and α7B. In the current paper, we have examined the developmental expression patterns of the α7A and α7B splice variants in the mouse. The α7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin‐α1 and laminin‐α2 chains. α7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for α7B integrin and in situ hybridization revealed the presence of α7 mRNA and α7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, α7B integrin was not detectable in the endocardium or myocardium during embryonic and fetal heart development. Northern blot analysis and immunohistochemistry revealed a postnatal induction of α7B in the myocardium. In addition to striated muscle, α7B integrin was localized to previously unreported nonmuscle locations such as a subset of vascular endothelia and restricted sites in the nervous system. Comparison of the α7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, α7 integrin, and laminin‐α2, but not laminin‐α1, in the forming skeletal muscle. We conclude that the α7A and α7B integrin variants are expressed in a developmentally regulated, tissue‐specific pattern suggesting different functions for the two splice forms.

Different β1-integrin collagen receptors on rat hepatocytes and cardiac fibroblasts

Detergent extracts of primary rat hepatocytes and neonatal cardiac fibroblasts were applied to collagen type I-Sepharose in the presence of 1 mM MnCl2. Elution of bound proteins by 10 mM EDTA yielded one beta 1-integrin heterodimer from hepatocytes with an Mr of 180,000/115,000 under nonreducing conditions. Two beta 1-integrins with Mrs (nonreduced) of 180,000/115,000 and 145,000/115,000 could be isolated from surface-iodinated fibroblasts. A monoclonal antibody, 3A3, directed against the rat homolog of the human integrin VLA-1, precipitated the affinity-purified Mr 180,000/115,000 heterodimer, establishing the relatedness of the Mr 180,000 subunit to the alpha 1-chain of the beta 1-integrin subfamily. Both the alpha 1 beta 1-integrin and the 145,000/beta 1-integrin heterodimers bound specifically to Sepharose beads derivatized with the collagen fragment alpha 1(I) CB3, which lacks RGD sequences. Immunofluorescence staining using the 3A3 monoclonal antibody revealed that the rat alpha 1 beta 1-integrin was present at focal adhesion sites of fibroblasts grown on native collagen type I- but not on fibronectin-coated substrates, although both types of substrates supported the formation of beta 1-integrin containing focal adhesions. Similarly, hepatocytes cultured on substrata coated with collagen type I (but not fibronectin) were stained in a patchy pattern localized to the cell periphery by 3A3 IgG. Furthermore, 3A3 IgG completely inhibited the attachment of hepatocytes to collagen type I, whereas under identical conditions the attachment of fibroblasts to these substrates was inhibited only by approximately 40%. The attachment of both hepatocytes and cardiac fibroblasts to fibronectin was unaffected by the presence of the 3A3 antibody. Collectively these data show that a rat homolog of the human VLA-1 heterodimer both biochemically and functionally fulfills the criteria of a single collagen receptor on rat hepatocytes. In contrast, rat cardiac fibroblasts utilize two different collagen-binding integrins to adhere to collagen, one of which is the rat homolog of the human VLA-1 heterodimer. Furthermore alpha 1(I) CB3 contains cell binding sites for beta 1-integrins.