Biomedical subjects

D Gullberg

Publications and source records attributed to D Gullberg.

At least 37 records · Page 2Linked to original sources

PubMed1997

Tenascin-C expression correlates with macrophage invasion in Duchenne muscular dystrophy and in myositis.

Tenascin-C (TN-C) is an extracellular matrix protein expressed during development in several tissues, but restricted to only a few areas in normal adult tissues. By immunizing mice with human fetal myoblasts we generated a monoclonal antibody to TN-C and mapped the epitope to the aminoterminal end containing EGF-like repeats. Using this antibody we detected by immunohistochemistry TN-C in the epimysium and perimysium of human fetal muscles, as well as in nonfibrillar deposits in myoblast cultures. In situ hybridization did not reveal any signal within human fetal muscle groups, suggesting that non-muscle cells synthesize the majority of the tenascin that localizes in and around human fetal muscle. Immunohistochemical analysis of muscle biopsies from Duchenne/Becker muscular dystrophy and myositis patients revealed that TN-C is expressed in skeletal muscle. Although the patterns of TN-C immunoreactivity were quite different in the two disease entities, the endomysial TN-C reactivity in both DMD/BMD and in myositis invariably correlated with the presence of macrophages.

Antibodies, Monoclonal↗

PubMed1997

Effects of divalent cations on M-cadherin expression and distribution during primary rat myogenesis in vitro.

In the process of myogenesis, cadherins are thought to be involved in the initial cell-cell recognition and possible initiation of myoblast fusion to form multinucleated myotubes. Of the cadherins, M-cadherin, but not N-cadherin, is down-regulated upon inhibition of myogenesis, suggesting that M-cadherin may be a key receptor involved in myogenesis. M-cadherin binds in a calcium-dependent manner, and depletion of divalent cations inhibits myoblast fusion. We analyzed the regulation of M-cadherin protein and mRNA levels in primary rat myogenic cultures in the presence and absence of divalent cations. In untreated cultures M-cadherin was localized to various myogenic cell-cell contacts. M-cadherin protein and mRNA levels showed a peak at day 2 after the initiation of growth. When divalent cations were removed from the cell culture medium, myoblast fusion was inhibited and immunocytochemical analysis revealed a failure of M-cadherin to localize to cell-cell contacts. Analysis of M-cadherin protein and mRNA in fusion-inhibited cultures still revealed a peak at day 2. However, by day 3, M-cadherin protein levels in the fusion-inhibited cultures were reduced in both the detergent-soluble and -insoluble fractions in comparison with the untreated cultures. Interestingly, beta-catenin, a protein associated with cadherins, was frequently observed at intercellular contacts in the fusion-inhibited cultures. We could also show that the intracellular levels of beta-catenin protein remained constant regardless of the presence or absence of divalent cations. In summary, the dynamic regulation of M-cadherin in muscle-fusion-related events is an indication of the importance of M-cadherin for myoblast fusion and myogenic differentiation.

Animals↗

PubMed1996

Conserved neuron promoting activity in Drosophila and vertebrate laminin alpha1.

Drosophila S2 cells were transfected with constructs that code for two portions of the Drosophila laminin alpha chain. Construct recalphaL coded for domains III, I/II, and G of laminin alpha. Construct recalphaS coded for only the COOH-most 12% of the I/II domain and the G domain. The corresponding polypeptides were isolated and characterized from the culture media. The recalphaL chain partly formed disulfide-linked heterotrimers with the endogenously produced beta and gamma laminin chains. Like normal Drosophila laminin, a substrate coating of either recalphaL or recalphaS supported neuron differentiation and neurite extension of primary Drosophila embryo cell cultures. However, at the same low concentrations, only Drosophila laminin-1, but neither recalphaL nor recalphaS supported myogenesis in these cultures. Previously, an overlapping set of dodecapeptides that covered a region of the murine laminin alpha1 chain similar to recalphaS had been synthesized and tested for cell culture support properties (Nomizu, M., Kim, W. H., Yamamura, K., Utani, A., Otaka, A., Roller, P. P., Kleinman, H. K., and Yamada, Y. (1995) J. Biol. Chem. 270, 20583-20590). The Drosophila laminin alpha homologues of the six most active vertebrate dodecapeptides were now synthesized and tested as substrates for differentiation of primary Drosophila embryo cells. Peptides that contained either the Drosophila sequence SIKVGV or the murine homologue, SIKVAV, provided support for neurite extension.

Amino Acid Sequence↗

PubMed1996

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 beta 5 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 alpha v integrin subunit. This subunit is known to associate with beta 5, and we therefore suggest that integrin alpha v beta 5 is involved in epithelial-mesenchymal interactions during tooth development. We also demonstrate that the developing tooth epithelium expresses the alpha 6, beta 1 and beta 4 subunits. The laminin receptors alpha 6 beta 1 and alpha 6 beta 4 may thus in part mediate the effect of basement membranes on tooth epithelial development. Interestingly, the enamel knot region expressed very little alpha 6 integrin subunit, whereas some expression was seen transiently in the condensing mesenchyme. During early tooth development, integrins possessing the alpha 6 subunit might also be involved in cell-cell interactions independently of laminins.

Animals↗

PubMed1996

Distinct alpha 7A beta 1 and alpha 7B beta 1 integrin expression patterns during mouse development: alpha 7A is restricted to skeletal muscle but alpha 7B is expressed in striated muscle, vasculature, and nervous system.

The laminin binding alpha 7 beta 1 integrin has been described as a major integrin in skeletal muscle. The RNA coding for the cytoplasmic domain of alpha 7 integrin undergoes alternative splicing to generate two major forms, denoted alpha 7A and alpha 7B. In the current paper, we have examined the developmental expression patterns of the alpha 7A and alpha 7B splice variants in the mouse. The alpha 7 integrin expression is compared to that of the nonintegrin laminin receptor dystroglycan and to that of laminin-alpha 1 and laminin-alpha 2 chains. Alpha 7A integrin was found by in situ hybridization to be specific to skeletal muscle. Antibodies specific for alpha 7B integrin and in situ hybridization revealed the presence of alpha 7 mRNA and alpha 7B protein in the E10 myotome and later in primary and secondary myotubes. In the heart, alpha 7B 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 alpha 7B in the myocardium. In addition to striated muscle, alpha 7B 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 alpha 7 integrin expression pattern with that of different laminin isoforms and dystroglycan revealed a coordinated temporal expression of dystroglycan, alpha 7 integrin, and laminin-alpha 2, but not laminin-alpha 1, in the forming skeletal muscle. We conclude that the alpha 7A and alpha 7B integrin variants are expressed in a developmentally regulated, tissue-specific pattern suggesting different functions for the two splice forms.

Animals↗

PubMed1996

Beta 1 integrin-dependent and -independent polymerization of fibronectin.

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

Animals↗

PubMed1996

Differentiation and integrity of cardiac muscle cells are impaired in the absence of beta 1 integrin.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting peacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of beta 1 integrin. Fourth, beta 1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with beta 1-null cells contain cell debris. The number of beta 1-null cells decrease from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via beta 1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

Animals↗

PubMed1995

Up-regulation of a novel integrin alpha-chain (alpha mt) on human fetal myotubes.

Integrin expression and distribution was studied in cloned human fetal G6 myoblasts and myotubes. Immunoprecipitation of beta 1 integrins from surface iodinated and metabolically labeled G6 cells typically showed a five-fold induction of a beta 1 integrin associated protein upon differentiation. Under non-reducing conditions this beta 1 associated protein migrated as 145 kD. No such beta 1 associated protein was observed in the myogenic L8 rat cell line, before or after differentiation. The beta 1 integrin associated cell surface protein present in G6 myotubes remained associated with the beta 1 subunit in the presence of 1% Triton X-100 and 0.5 M NaCl. Like integrin alpha-chains, the protein dissociated from the beta 1 integrin subunit at low pH. Immunoprecipitation of G6 myotubes further indicated the presence of alpha 1, alpha 3, alpha 5, and alpha v integrins, and small amounts of alpha 4 and alpha 6 integrins. Immunodepletion with integrin alpha-chain antibodies to alpha 1, alpha 3, alpha 4, alpha 5, alpha 6, and alpha v integrin chains could not deplete the beta 1 integrin associated protein, indicating that it did not interact with any of these known integrin heterodimers. Upon treatment with reducing agents, the beta 1 integrin associated protein migrated in SDS-PAGE as a 155 kD protein. The decreased mobility in SDS-PAGE upon reduction is a feature shared with alpha 1, alpha 2, and alpha 9 integrin alpha-chains. Antibodies to alpha 1 immunoprecipitated an integrin heterodimer distinct from the 155 kD protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Differentiation↗

PubMed1995

Analysis of fibronectin and vitronectin receptors on human fetal skeletal muscle cells upon differentiation.

The role of fibronectin (FN) and vitronectin (VN) receptors for cell adhesion and matrix assembly was analyzed during human fetal myogenesis in vivo and in vitro. In human fetal muscle at 10 weeks gestational age FN and laminin are present in the extracellular matrix. Analysis of the integrin repertoire at this developmental stage reveals that the differentiated muscle cells in vivo express alpha 5 and alpha 6 integrins, but not alpha v, alpha 1, and alpha 3 integrins. However, in vitro cultured myoblasts (G6) isolated from the same gestational age express alpha v, alpha 1, and alpha 3 integrins in addition to alpha 5 and alpha 6 integrins. A more detailed analysis of FN and VN receptors in vitro shows that the localization of different alpha v heterodimers into focal contacts is differently regulated. Alpha v beta 1, and alpha v beta 3, are present at focal contacts throughout in vitro myogenesis whereas alpha v beta 5 appears to depend on an endogenously produced factor to localize to focal contacts. The alpha v beta 1, alpha v beta 5, and alpha 3 beta 1 heterodimers, often reported not to focalize, did form focal contacts in G6 cells, indicating that these myoblasts possess components facilitating the formation of cytoskeletal linkages containing these integrins. Alpha 5 beta 1 colocalized with FN in myoblast cultures, whereas myotubes lacked both FN and alpha 5 beta 1 on the cell surface. In summary, we show that concomitant with in vitro differentiation of G6 cells, FN matrix contacts are abolished, but vitronectin receptors continue to fulfill an anchoring function during the differentiation process in vitro. Further studies are needed to assess the relative importance of the FN and VN binding integrins for the differentiation process in comparison with the laminin-binding integrins alpha 6 and alpha 7, also present on these cells.

Antigens, CD↗

PubMed1995

Extracellular matrix and its receptors during development.

Extracellular matrix (ECM) components are essential for morphogenesis of virtually all tissues. The ECM interacts with the cell surface by binding to specific receptors. The first family of receptors for the ECM that was identified was the integrin family. Integrins are composed of an alpha and a beta-chain, both of which are single pass transmembrane proteins. In muscle cells the dystroglycan complex forms another important receptor system for ECM. It is a complex composed of many proteins. Recent studies have shown that dystroglycan is expressed by embryonic epithelial cells as well. The nature of constituents of the dystroglycan complex is well known for muscle, whereas the detailed composition of the dystroglycan complex in embryonic epithelium is not yet well known. We here review the evidence that binding of ECM to integrins and the dystroglycan complex could be essential for muscle and epithelial cell development and function. It is likely that integrins and the dystroglycan complex have distinct roles during development. It will be an interesting task to study the signal transduction pathways elicited by the interactions between ECM and the two receptor systems during muscle and epithelial morphogenesis.

Animals↗

PubMed1994

Differentiation, extracellular matrix synthesis, and integrin assembly by Drosophila embryo cells cultured on vitronectin and laminin substrates.

Two contrasting substrates, Drosophila laminin and human vitronectin, caused determined primary Drosophila embryo cells to follow alternate intermediate differentiation steps without affecting the final outcome of differentiation. Integrin alpha PS2 beta PS3 was essential for the initial spreading of myocytes on vitronectin: focal contacts rich in beta PS3 integrins formed and were connected by actin- and myosin-containing stress fibers. While alpha PS2 beta PS3 was unnecessary for myotube formation on laminin, it was required for the subsequent change to a sarcomeric cytoarchitecture. The differentiating primary cultures synthesized integrins and assembled them into detergent-insoluble, cytoskeleton-associated complexes. Collagen IV, laminin, glutactin, papilin, and other extracellular matrix proteins were made primarily by hemocytes and were secreted into the medium. Further differentiation within the cultures was influenced by secreted components and by later addition of vitronectin or bovine serum. Comparison of the differentiation of various cell types on the two substrates showed that vitronectin provided a selective advantage for the differentiation of myocytes, with enrichment over epithelia, epidermal cells, and neurites.

Animals↗

PubMed1994

Alpha v integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development.

Alpha v integrin subunit can dimerize with different beta subunits to form receptors for several matrix proteins. The function of these receptors in vivo is still largely unknown. We examined the localization of alpha v integrin during mouse development and showed that its distribution is dynamically regulated in the glia of the central nervous system and in skeletal muscle. Immunoreactivity in the neural tube was firstly localized at embryonic day 10.5 (E10.5) around cell bodies lining the lumen and along tiny fibres extending towards the outer margin. At E12.5 alpha v distribution follows the highly defined pattern of the radial glia: fascicles of immunoreactive fibres form parallel palisades, in particular along the hindbrain and the spinal cord. At E15.5, although with weaker intensity, alpha v was still detectable in radial glia fibres, and it codistributed with glial fibrillary acidic protein positive fascicles. After birth (P8) alpha v immunoreactivity in the brain and spinal cord decreased dramatically, but remained high in the radial glia of the cerebellum. In adult mice alpha v reactivity in the central nervous system disappeared. During myogenesis alpha v appears at E10.5 in myotomal cells and from E12.5 alpha v was evident in myoblasts and in myotubes. In the developing skeletal muscle of E15.5 embryos, immunoreactivity became more concentrated in the apical portion of the myotubes. In adult striated muscle the amount of alpha v subunit dramatically declined and immunostaining was no longer detectable. During development, alpha v was weakly evident in other sites including heart and endothelia of blood vessels, mesonephric tubula, smooth muscle of the digestive tract, and bronchia. Comparative analysis of the localization of alpha v, alpha 3, and alpha 5 integrin subunits indicated that alpha v has a unique and highly regulated distribution pattern. The distribution in the nervous system is consistent with a role of alpha v in neuron-glia interaction during the organization of the neuronal layers in the brain cortex and in the cerebellum. Moreover, alpha v is likely to be involved in the myotendinous junction during embryonic life, suggesting a dual functional role of this integrin in muscle and nervous tissue.

Amino Acid Sequence↗

PubMed1994

Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alpha PS2 beta PS integrins.

Genetic and other studies of Drosophila integrins have implicated these extracellular matrix receptors in various morphogenetic events, but identification of their endogenous ligands has been elusive. We report the biochemical purification and cloning of tiggrin, a novel extracellular matrix protein from Drosophila. This 255 x 10(3) M(r) polypeptide contains the potential integrin recognition sequence Arg-Gly-Asp (RGD) and 16 repeats of a novel 73-77 amino acid motif. The tiggrin gene is at chromosome locus 26D1-2 and is expressed by embryonic hemocytes and fat body cells. Tiggrin protein is detected in matrices, especially at muscle attachment sites that also strongly express integrins. Tiggrin-coated surfaces support primary embryo cell culture and provide excellent substrates for alpha PS2 beta PS integrin-mediated cell spreading. Soluble RGD-peptides inhibit this cell spreading.

Amino Acid Sequence↗

PubMed1992

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)

Amino Acid Sequence↗

PubMed1992

Neuritogenesis on collagen substrates. Involvement of integrin-like matrix receptors in retinal fibre outgrowth on collagen.

Extracellular matrix molecules such as laminin, fibronectin and collagen promote neurite outgrowth in vitro. We have investigated the capacity of hydrated gels of collagen types I-III and monomeric collagen types I-VI on plastic surfaces to support neuritogenesis. The attachment and survival of explants from the day 6 chick embryo were studied and neurite outgrowth measured as mean elongation rate and maximal neurite length. Collagen types I and III, both as three-dimensional gels or as native monomers supported neuritogenesis equal to or better than laminin. Collagen type V also supported neurite out-growth although less effectively. Collagen types II, IV and VI, as well as denatured collagens of all types tested, did not support outgrowth. The monoclonal anti-beta 1 integrin antibody (CSAT), as well as rabbit polyclonal antibodies directed to the integrin beta 1-chain, effectively inhibited neurite outgrowth on permissive collagenous substrata, indicating that collagen-binding integrins were involved in the neuritogenesis. These beta 1-integrins were independent of Arg-Gly-Asp (RGD) since neurite formation proceeded in the presence of synthetic RGD-containing peptides. Fluorescence immunohistochemistry revealed the presence of the integrin beta 1-chain on the outgrowing neurites. The results suggest a possible function of collagen and collagen-binding integrins in the development of the visual system.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

Different beta 1-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 Mr's (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.

Animals↗

PubMed1990

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.

Actins↗