Selective modulation of the interaction of α 7 β 1 integrin with fibronectin and laminin by L-14 lectin during skeletal muscle differentiation (original) (raw)
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Endogenous muscle lectin inhibits myoblast adhesion to laminin
The Journal of Cell Biology, 1991
L-14, a dimeric lactose-binding lectin with subunits of 14 kD, is expressed in a wide range of vertebrate tissues. Several functions have been postulated for this lectin, but definitive evidence for a specific biological role has been elusive. In muscle, L-14 is secreted during differentiation and accumulates with laminin in basement membrane surrounding each myofiber. Here we present evidence that laminin is a major glycoprotein ligand for L-14 in differentiating mouse C2C12 muscle cells and that binding of secreted L-14 to polylactosamine oligosaccharides of substrate laminin induces loss of cell-substratum adhesion. These results suggest that one function of L-14 is to regulate myoblast detachment from laminin during differentiation and fusion into tubular myofibers.
Journal of Biological Chemistry, 1993
Within the integrin family, there are two groups of receptors that bind laminin. One of these groups comprises the heterodimers a381, a6@1, and a781, all of which bind the E8 fragment of laminin, and whose a subunits show significant homology at the amino acid sequence level. a3 and a6 exist as isoforms with dis-* This work was supported by National Institutes of Health Grants CA47858 and GM46902. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sepuence(s) reported in thispaper has been submitted L16544.
The α7β1 Integrin Mediates Adhesion and Migration of Skeletal Myoblasts on Laminin
Experimental Cell Research, 1997
Prominent among the cell adhesion molecules are sev-Many aspects of myogenesis are believed to be regu-eral members of the integrin receptor family [1] which lated by myoblast interactions with specific compo-recognize specific ECM components such as laminin or nents of the extracellular matrix. For example, laminin fibronectin, resulting in pronounced effects on myoblast has been found to promote adhesion, migration, and behavior. For instance, laminin promotes adhesion, proproliferation of mammalian myoblasts. Based on afliferation, motility, and differentiation [2 -7], whereas finity chromatography, the a7b1 integrin has been prefibronectin promotes adhesion, but only weakly prosumed to be the major receptor mediating myoblast motes motility [2, 3]. The studies reported here were interactions with laminin. We have prepared a monoaimed at further elucidating the role of specific recepclonal antibody, O26, that specifically reacts with both tors in regulating myoblast responses to laminin.
Integrin on developing and adult skeletal muscle
Experimental Cell Research, 1989
Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components.
1996
The role of integrins in muscle differentiation was addressed by ectopic expression of integrin o~ subunits in primary quail skeletal muscle, a culture system particularly amenable to efficient transfection and expression of exogenous genes. Ectopic expression of either the human a5 subunit or the chicken et6 subunit produced contrasting phenotypes. The ot5-transfected myoblasts remain in the proliferative phase and are differentiation inhibited even in confluent cultures. In contrast, myoblasts that overexpress the or6 subunit exhibit inhibited proliferation and substantial differentiation. Antisense suppression of endogenous quail et6 expression inhibits myoblast differentiation resulting in sustained proliferation. These effects of ectopic a subunit expression are mediated, to a large extent, by the cytoplasmic domains. Ectopic expression of chimeric a subunits, ct5ex/6cyto and a6ex/5¢yto, produced phenotypes opposite to those observed with ectopic or5 or or6 expression. Myoblasts that express a5ex/6mo show decreased proliferation while differentiation is partially restored. In contrast, the et6ex/5¢yto transfectants remain in the proliferative phase unless allowed to become confluent for at least 24 h. Furthermore, expression of human ct5 subunit cytoplasmic domain truncations, before and after the conserved GFFKR motif, shows that this sequence is important in a5 regulation of differentiation. Ectopic c~5 and o~6 expression also results in contrasting responses to the mitogenic effects of serum growth factors. Myoblasts expressing the human et5 subunit differentiate only in the absence of serum while differentiation of untransfected and ot6-transfected myoblasts is insensitive to serum concentration. Addition of individual, exogenous growth factors to a5transfected myoblasts results in unique responses that differ from their effects on untransfected ceils. Both bFGF or TGFI3 inhibit the serum-free differentiation of a5-transfected myoblasts, but differ in that bFGF stimulates proliferation whereas TGF-13 inhibits it. Insulin or TGF-ot promote proliferation and differentiation of ot5-transfected myoblasts; however, insulin alters myotube morphology. TGF-o~ or PDGF-BB enhance muscle ct-actinin organization into myofibrils, which is impaired in differentiated or5 cultures. With the exception of TGF-ot, these growth factor effects are not apparent in untransfected myoblasts. Finally, myoblast survival under serum-free conditions is enhanced by ectopic ct5 expression only in the presence of bFGF and insulin while TGF-et and TGF-~ promote survival of untransfected myoblasts. Our observations demonstrate (1) a specificity for integrin a subunits in regulating myoblast proliferation and differentiation; (2) that the ratio of integrin expression can affect the decision to proliferate or differentiate; (3) a role for the et subunit cytoplasmic domain in mediating proliferative and differentiative signals; and (4) the regulation of proliferation, differentiation, cytoskeletal assembly, and cell survival depend critically on the expression levels of different integrins and the growth factor environment in which the cells reside.
Experimental Cell Research, 2000
The expression of laminin isoforms and lamininbinding integrin receptors known to occur in muscle was investigated during myogenic regeneration after crush injury. Comparisons were made between dystrophic 129ReJ dy/dy mice, which have reduced laminin ␣2 expression, and their normal littermates. The overall histological pattern of regeneration after crush injury was similar in dy/dy and control muscle, but proceeded faster in dy/dy mice. In vitro studies revealed a greater yield of mononuclear cells extracted from dy/dy muscle and a reduced proportion of desmin-positive cells upon in vitro cultivation, reflecting the presence of inflammatory cells and "preactivated" myoblasts due to ongoing regenerative processes within the endogenous dystrophic lesions. Laminin ␣1 was not detectable in skeletal muscle. Laminin ␣2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin ␣2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin ␣ chains in early myogenic differentiation, such as laminin ␣4 and ␣5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin 1 was expressed on endothelial cells, muscle fibers, and peripheral nerves in uninjured muscle and broadened after crush injury to the interstitium where it occurred on myogenic and nonmyogenic cells. Integrin ␣3 was not expressed in uninjured or regenerating muscle, while integrin ␣6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin ␣6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin ␣6 occurred on some newly formed myotubes. Integrin ␣7 was expressed on muscle fibers at the myo-tendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin ␣7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin ␣7 negative. No marked difference was observed in integrin ␣7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin ␣4 and integrin ␣6 expression patterns were notably different between dy/dy and control muscle. Expression of both molecules was more extensive in dy/dy muscle, especially in the interstitium of regenerating areas and on newly formed myotubes. In view of the faster myogenic regeneration observed in dy/dy mice, the data suggest that laminin ␣4 and integrin ␣6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin ␣2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.
Experimental Cell Research, 1999
␣71 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the ␣7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with ␣7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the ␣71. ␣7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of ␣7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the ␣51 fibronectin receptor. Although cell surface expression of ␣51 was reduced by a factor of 20-25% in ␣7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125 I-fibronectin for its surface receptor was decreased by 50% in ␣7 transfectants, indicating that the ␣51 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn 2؉ , a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn 2؉ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in ␣7 transfectants. These data indicate that ␣7 expression leads to the functional down regulation of ␣51 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between ␣7 and ␣5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.
Acta Histochemica, 2001
MyoD is a member of a skeletal muscle specific family of transcription factors which directs the events of myogenesis during development and regeneration. Muscle cells that lack MyoD show delayed fusion in vivo and in vitro and defects have been observed in vitro in the attachment of MyoD(±/±) myoblasts to complex substrates such as Matrigel. Since interactions with the extracellular matrix (ECM) are important during myoblast fusion (i. e. myotube formation), it was hypothesised that expression of ECM molecules or their receptors may be altered in MyoD(±/±) muscle. The production of basement membrane molecules such as collagen type IV and several laminins, the interstitial molecules fibronectin and tenascin-C, and the cell surface molecules integrin a5 and a6 were quantitated in vitro using ELISA on cultured cells from MyoD(±/±) and wild type mice. Differences were observed in the production of fibronectin, tenascin-C, collagen type IV, laminin-1 and integrin a5 between control and MyoD(±/±) myotubes in vitro. This corresponded with delayed fusion of myoblasts in MyoD(±/±) cultures. On the basis of these findings with respect to matrix expression in vitro, fluorescent immunohisto-chemistry was carried out on adult whole muscle autografts to examine whether the expression of these molecules, as well as integrin a7, was altered in the complex in vivo environment. Some minor differences in expression patterns were observed in MyoD(±/±) as compared to normal BALB/c autografts. The overall expression of matrix components was consistent with the delayed onset of myotube formation. These results suggest that the delay in myotube formation in MyoD(±/±) muscle is not a direct result of altered expression of the matrix molecules collagen type IV, laminins, fibronectin, tenascin-C, and integrins a5, a6 or a7.
Laminins promote the locomotion of skeletal myoblasts via the alpha 7 integrin receptor
Journal of Cell Science, 1996
The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 myoblasts differentiated; myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse myoblasts on laminin 1. Other studies with MM14 myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking ant...