A functional role for specific spliced variants of the alpha7beta1 integrin in acetylcholine receptor clustering

The clustering of acetylcholine receptors (AChR) on skeletal muscle fibers is an early event in the formation of neuromuscular junctions. Recent studies show that laminin as well as agrin can induce AChR clustering. Since the alpha7beta1 integrin is a major laminin receptor in skeletal muscle, we determined if this integrin participates in laminin and/or agrin-induced AChR clustering. The alternative cytoplasmic domain variants, alpha7A and alpha7B, and the extracellular spliced forms, alpha7X1 and alpha7X2, were studied for their ability to engage in AChR clustering. Immunofluorescence microscopy of C2C12 myofibers shows that the alpha7beta1 integrin colocalizes with laminin-induced AChR clusters and to a much lesser extent with agrin-induced AChR clusters. However, together laminin and agrin promote a synergistic response and all AChR colocalize with the integrin. Laminin also induces the physical association of the integrin and AChR. High concentrations of anti-alpha7 antibodies inhibit colocalization of the integrin with AChR clusters as well as the enhanced response promoted by both laminin and agrin. Engaging the integrin with low concentrations of anti-alpha7 antibody initiates cluster formation in the absence of agrin or laminin. Whereas both the alpha7A and alpha7B cytoplasmic domain variants cluster with AChR, only those isoforms containing the alpha7X2 extracellular domain were active. These results demonstrate that the alpha7beta1 integrin has a physiologic role in laminin-induced AChR clustering, that alternative splicing is integral to this function of the alpha7 chain, and that laminin, agrin, and the alpha7beta1 integrin interact in a common or convergent pathway in the formation of neuromuscular junctions.     

The alpha7beta1 integrin mediates adhesion and migration of skeletal myoblasts on laminin

Many aspects of myogenesis are believed to be regulated by myoblast interactions with specific components of the extracellular matrix. For example, laminin has been found to promote adhesion, migration, and proliferation of mammalian myoblasts. Based on affinity chromatography, the alpha7beta1 integrin has been presumed to be the major receptor mediating myoblast interactions with laminin. We have prepared a monoclonal antibody, O26, that specifically reacts with both the X1 and the X2 extracellular splice variants of the alpha7 integrin chain. This antibody completely and selectively blocks adhesion and migration of rat L8E63 myoblasts on laminin-1, but not on fibronectin. In contrast, a polyclonal antibody to the fibronectin receptor, alpha5beta1 integrin, blocks myoblast adhesion on fibronectin, but not on laminin-1. The alpha7beta1 integrin also binds to a mixture of laminin-2 and laminin-4, the major laminin isoforms in developing and adult skeletal muscle, but O26 is a much less potent inhibitor of myoblast adhesion on the laminin-2/4 mixture than on laminin-1. Based on affinity chromatography, we suggest that this may be due to higher affinity binding of alpha7X1 to laminin-2/4 than to laminin-1.     

Expression of laminin alpha1, alpha2, alpha4, and alpha5 chains, fibronectin, and tenascin-C in skeletal muscle of dystrophic 129ReJ dy/dy mice

The dy/dy mouse is an animal model for human merosin-negative congenital muscular dystrophy (CMD), which has been reported to have reduced or no expression of the basement membrane protein laminin alpha2. We here investigate various myogenic and nonmyogenic tissues of mature dy/dy and control 129ReJ mice histologically and for laminin alpha2 expression. In addition, expression patterns of laminin alpha1, alpha2, alpha4, and alpha5 chains, the interstitial proteins fibronectin and tenascin-C, and the adhesion molecules VCAM-1, ICAM-1, and alpha4 integrin were characterized in skeletal muscle of 1- and 7-day and mature (>6 weeks old) dy/dy and control 129ReJ mice. The laminin alpha2 chain remained detectable in myogenic tissues of dy/dy mice by immunofluorescence using two different monoclonal antibodies and by Northern blot analysis. However, laminin alpha2 expression was significantly reduced or not detectable in nonmyogenic tissues of dy/dy mice, including skin, lung, kidney, brain, thymus, and eye. Focal lesions were observed in mature skeletal muscle only, characterized by necrotic tissue, isolated VCAM-1- and ICAM-1-positive cells indicative of inflammatory processes, and regenerating muscle fibers surrounded by intense tenascin-C and fibronectin expression. In contrast to studies on human CMD muscle, laminin alpha1 was not detectable in either dy/dy or control skeletal muscle using immunofluorescence or Northern blot analysis. Immunofluorescence localized laminin alpha4 to basement membranes of blood vessels, the endoneurium of the intramuscular nerves, and the neuromuscular junction in skeletal muscle of 1- and 7-day-old dy/dy and control mice. In mature muscle, laminin alpha4 expression shifted to the perineurium of intramuscular nerves in both dy/dy and control mice. Furthermore, strong upregulation of laminin alpha4 in the basement membranes of blood vessels, the perineurium of intramuscular nerves, and of isolated regenerating muscle fibers in the dy/dy mice was apparent. Investigation of 1-day-old animals revealed expression of laminin alpha5 in skeletal muscle fiber basement membranes of dy/dy but not control animals. This difference between dy/dy and control animals was no longer apparent at 7 days after birth, indicating a temporary shift in expression pattern of laminin alpha5 in dy/dy animals. Analysis of the extracellular matrix components of 1- and 7-day-old dy/dy and control skeletal muscle revealed an early onset of the dystrophy, even before histopathological features of the disease were evident. Our data confirm the absence of laminin alpha1 chain in myogenic tissues of both dy/dy and control mice and suggest compensation for reduced laminin alpha2 in dy/dy skeletal muscle by laminin alpha4 and, in early development, also laminin alpha5. These results have significant ramifications in the diagnosis of human merosin-negative CMD.     

The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function

alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 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 alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 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 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.     

Primary structure, developmental expression, and immunolocalization of the murine laminin alpha4 chain

The complete primary structure of the mouse laminin alpha4 chain was derived from cDNA clones. The translation product contains a 24-residue signal peptide preceding the mature alpha4 chain of 1,792 residues. Northern analysis on whole mouse embryos revealed that the expression was weak at day 7, but it later increased and peaked at day 15. In adult tissues the strongest expression was observed in lung and cardiac and skeletal muscles. Weak expression was also seen in other adult tissues such as brain, spleen, liver, kidney, and testis. By in situ hybridization of fetal and newborn tissues, expression of the laminin alpha4 chain was mainly localized to mesenchymal cells. Strong expression was seen in the villi and submucosa of the developing intestine, the mesenchymal stroma surrounding the branching lung epithelia, and the external root sheath of vibrissae follicles, as well as in cardiac and skeletal muscle fibers. In the developing kidney, intense but transient expression was associated with the differentiation of epithelial kidney tubules from the nephrogenic mesenchyme. Immunohistologic staining with affinity-purified IgG localized the laminin alpha4 chain primarily to lung septa, heart, and skeletal muscle, capillaries, and perineurium.     

Presence of laminin alpha5 chain and lack of laminin alpha1 chain during human muscle development and in muscular dystrophies

There is currently a great interest in identifying laminin isoforms expressed in developing and regenerating skeletal muscle. Laminin alpha1 has been reported to localize to human fetal muscle and to be induced in muscular dystrophies based on immunohistochemistry with the monoclonal antibody 4C7, suggested to recognize the human laminin alpha1 chain. Nevertheless, there seems to be no expression of laminin alpha1 protein or mRNA in developing or dystrophic mouse skeletal muscle fibers. To address the discrepancy between the results obtained in developing and dystrophic human and mouse muscle we expressed the E3 domain of human laminin alpha1 chain as a recombinant protein and made antibodies specific for human laminin alpha1 chain (anti-hLN-alpha1G4/G5). We also made antibodies to the human laminin alpha5 chain purified from placenta. In the present report we show that hLN-alpha1G4/G5 antibodies react with a 400-kDa laminin alpha1 chain and that 4C7 reacts with a 380-kDa laminin alpha5 chain. Immunohistochemistry with the hLN-alpha1G4/G5 antibody and 4C7 revealed that the two antibodies stained human kidney, developing and dystrophic muscle in distinct patterns. Our data indicate that the previously reported expression patterns in developing, adult, and dystrophic human muscle tissues with 4C7 should be re-interpreted as an expression of laminin alpha5 chain. Our data are also consistent with earlier work in mouse, indicating that laminin alpha1 is largely an epithelial laminin chain not present in developing or dystrophic muscle fibers.     

Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells

An arginine-specific mono-ADP-ribosyltransferase is expressed on the surface of differentiated mouse skeletal muscle cells and is anchored in the membrane via a glycosylphosphatidylinositol tail. Following incubation of intact cells with [adenylate-32P]NAD and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), a 97-kDa [32P]ADP-ribosylated protein was observed under reducing conditions and a 140-kDa complex under nonreducing conditions. The ADP-ribosylated protein was purified on a laminin affinity column. Based on its N-terminal sequence (FNLDVM-GAIRKEGEPGSLFGF) and a partial internal sequence (GLMRSEELSFVAGAP), the modified protein was identified as integrin alpha 7. Following partial trypsin digestion, a 39-kDa/79-kDa radiolabeled fragment was produced (reduced/nonreduced SDS-PAGE), narrowing the ADP-ribosylation site to a 39-kDa segment in the extracellular domain of integrin alpha 7. Labeling under optimal conditions was at least 0.4 mol of ADP-ribose/mol of integrin alpha 7. Selective expression of both ADP-ribosyltransferase and integrin alpha 7 in cardiac and skeletal muscle, a similar developmental appearance, and the apparently specific ADP-ribosylation, are consistent with a regulatory association between these proteins. ADP-ribosylation may modulate integrin receptor signaling and could play a significant role in the regulation of muscle cell function by the extracellular matrix.     

Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development

Two splice variants of the alpha6 integrin subunit, alpha6A and alpha6B, with different cytoplasmic domains, have previously been described. While alpha6B is expressed throughout the development of the mouse, the expression of alpha6A begins at 8.5 days post coitum and is initially restricted to the myocardium. Later in ontogeny, alpha6A is found in various epithelia and in certain cells of the immune system. In this study, we have investigated the function of alpha6A in vivo by generating knockout mice deficient for this splice variant. The Cre- loxP system of the bacteriophage P1 was used to specifically remove the exon encoding the cytoplasmic domain of alpha6A in embryonic stem cells, and the deletion resulted in the expression of alpha6B in all tissues that normally express alpha6A. We show that alpha6A-/- mice develop normally and are fertile. The substitution of alpha6A by alpha6B does not impair the development and function of the heart, hemidesmosome formation in the epidermis, or keratinocyte migration. Furthermore, T cells differentiated normally in alpha6A-/- mice. However, the substitution of alpha6A by alpha6B leads to a decrease in the migration of lymphocytes through laminin-coated Transwell filters and to a reduction of the number of T cells isolated from the peripheral and mesenteric lymph nodes. Lymphocyte homing to the lymph nodes, which involves various types of integrin-ligand interactions, was not affected in the alpha6A knockout mice, indicating that the reduced number of lymph node cells could not be directly attributed to defects in lymphocyte trafficking. Nevertheless, the expression of alpha6A might be necessary for optimal lymphocyte migration on laminin in certain pathological conditions.     

Biochemical characterization of human osteoclast integrins. Osteoclasts express alpha v beta 3, alpha 2 beta 1, and alpha v beta 1 integrins

The study of osteoclast integrins has been previously hampered by the lack of a source of large numbers of purified osteoclasts. Osteoclastoma, a human giant cell tumor of bone, supplied a rich source of osteoclasts within a tissue containing many diverse cell types. Osteoclastoma integrin immunostaining confirmed the presence of the integrin alpha v beta 3 complex and the alpha 2 and beta 1 integrin subunits on osteoclasts. However, weak integrin expression, for example with alpha v beta 5, was difficult to interpret. Purification with magnetic beads coated with vitronectin receptor monoclonal antibody (13C2) enabled osteoclast membranes to be isolated with high purity and yield (57%) from osteoclastoma tissue. Positively (osteoclast-enriched) selected membranes were biochemically assessed for integrin expression by immunoprecipitation and visualization by non-radioactive enhanced chemiluminescence. alpha 1, alpha 4, alpha 6, alpha 8, alpha M, alpha X, gpIIb, beta 4, beta 6, and beta 8 integrin chains were undetectable at a sensitivity of 1 ng. alpha 3, alpha 5, alpha L, beta 2, and alpha v beta 5 were found in the negatively selected osteoclastoma tissue but not in the positively purified osteoclast membranes. The presence of alpha v beta 1 and alpha 2 beta 1 dimers was demonstrated biochemically on the immunoisolated osteoclast membranes. Osteoclast alpha v beta 3 isolation by Arg-Gly-Asp (RGD) affinity chromatography for NH2-terminal amino acid sequencing confirmed that the osteoclast vitronectin receptor was identical to that previously characterized on other cell types. In situ hybridization using human alpha v riboprobes in osteoclasts from human and rodent bone further demonstrated the high level and specificity of expression of alpha v vitronectin receptor in osteoclasts.     

Expression of laminin alpha 1, alpha 5 and beta 2 chains during embryogenesis of the kidney and vasculature

Laminins, found predominantly in basement membranes, are large glycoproteins consisting of different subsets of alpha, beta and gamma chain subunits. To resolve conflicting data in the literature concerning coexpression of alpha 1 and beta 2 chains, expression of alpha 1 chain was studied with two different antisera against the E3 fragment of laminin alpha 1 chain. Expression of the alpha 1 chain was seen in several types of epithelial basement membranes throughout development, but its expression in rat glomerular basement membranes and some other types of epithelial basement membranes occurred only during early stages of development. By contrast, beta 2 chains were detected by immunofluorescence only during advanced stages of glomerulogenesis and vascular development. By Northern and Western blots, beta 2 chains were detected somewhat earlier, but in situ hybridization revealed that beta 2 chain was also confined to vasculature during the earlier stages. It thus seems that, in the tissues studied here, the expression of alpha 1 and beta 2 chains was mutually exclusive. To explore whether the newly described alpha 5 chain is expressed in locations lacking alpha 1 chain, expression of alpha 5 chain was studied by Northern blots and in situ hybridization. The alpha 5 chain was not uniformly expressed in all embryonic epithelial cell types but was present mainly in epithelial sheets which produce very little alpha 1 chain. There also appeared to be a developmental trend, with alpha 1 chain appearing early and alpha 5 later, in maturing epithelial sheets. The alpha 5 chain could be a major alpha chain of the adult glomerular basement membrane.     

Sequence-specific DNA alkylation of novel tallimustine derivatives

beta 1D is a recently identified isoform of the beta 1 integrin subunit selectively expressed in skeletal and cardiac muscles. In the present study we determined the temporal expression of beta 1D and its association with alpha subunits during mouse development. By immunohistochemistry and western blot analysis we demonstrated that beta 1D begins to be expressed in skeletal muscles of 17 days embryo (stage E17). Its level progressively increases reaching maximal values few days after birth and remaining high in adult mice. At earlier stages of development (E11-E17) the beta 1A isoform is expressed in skeletal muscle cells. After E17 beta 1A is downregulated and disappears from muscle fibers few days after birth. In cardiac muscle the regulation of the beta 1D expression is different: beta 1D and beta 1A are coexpressed in the heart of E11 embryo. Subsequently expression of beta 1A declines, while beta 1D increases until it becomes the unique beta 1 isoform in cardiomyocytes few days after birth. Previous studies (Belkin et al J. Cell Biol. 132: 211-226, 1996) demonstrated that beta 1D in adult mouse cardiomyocytes is exclusively associated with alpha 7B. Western blot analysis shows that alpha 7B starts to be expressed in the heart only at stage E17, while beta 1D is expressed already at E11 embryo, indicating that alpha subunits other than alpha 7 should associate with beta 1D in early developmental stages. To investigate this aspect, beta 1 associated alpha subunits were identified by western blotting from cardiomyocytes integrin complexes immunoprecipitated with alpha subunit specific antibodies. We found that, during cardiomyocyte development, beta 1D associates with several alpha subunits namely with alpha 5, alpha 6A and alpha 7B. In conclusion these data show that the expression of the beta 1D muscle specific integrin during development occurs much earlier in heart than in skeletal muscle and it can dimerize with different alpha subunits.     

Adhesion to laminin is down-regulated upon retinoic acid-induced F9 cell differentiation: a role for alpha 6/beta 1 integrin

F9 mouse teratocarcinoma cells have a high capacity to adhere to laminin and we identified alpha 6/beta 1 integrin as the principal laminin-binding protein present in these cells. F9 cells differentiated into parietal endoderm when monolayer cultures were treated with retinoic acid and dibutyryl cyclic AMP. In this process a decreased adherence to laminin was observed due to a lower expression of alpha 6/beta 1 integrin on the cell surface.     

Abnormal expression of laminin beta 1 chain in skeletal muscle of adult-onset limb-girdle muscular dystrophy

BACKGROUND: Laminin 2 is a major component of the basal lamina of skeletal muscle cells. It is a heterotrimer composed of 3 chains: merosin (laminin alpha 2 chain), beta 1, and gamma 1. Deficiency of merosin, with or without laminin beta 1 chain reduction, is associated with some forms of congenital muscular dystrophy. Deficient expression of laminin beta 1 chain is also associated with some cases of merosin-positive congenital muscular dystrophy. The expression of laminin 2 subunits has not been well studied in the skeletal muscle of limb-girdle muscular dystrophy (LGMD), nor has much attention been given to the significance of reduction of individual laminin 2 subunits, such as beta 1. OBJECTIVES: To examine the expression of laminin 2 subunits in skeletal muscle in patients with LGMD and to define the clinical features of patients with LGMD who have abnormal expression of laminin 2 subunits. METHODS: We studied muscle biopsy specimens from 18 patients with LGMD using immunofluorescence with antibodies against dystrophin C-terminus, beta-dystroglycan, alpha-sarcoglycan, gamma-sarcoglycan, and the laminin subunits merosin, beta 1, and gamma 1. Of the 18 biopsy specimens, 9 were available for electron microscopic examination of the muscle basement membrane. The clinical features associated with abnormal laminin beta 1 chain immunoreactivity were further described. RESULTS: Laminin beta 1 chain was either barely detectable or severely reduced in 3 cases of patients with LGMD in which the biopsy specimens showed normal staining with the other antibodies. Patients in all 3 cases had common clinical features consistent with a slowly progressive, adult-onset LGMD. Specimens from 2 of the 3 cases that were available for ultrastructural examination showed significant abnormalities of the muscle fiber basement membrane. CONCLUSIONS: Abnormal expression of laminin beta 1 chain without concomitant deficiency of alpha-sarcoglycan in skeletal muscle has not been previously described in LGMD. Reduced laminin beta 1 chain immunoreactivity may potentially serve as a marker for defining subsets of individuals with LGMD, in particular those with slowly progressive, adult-onset pelvifemoral presentation. The abnormality of muscle fiber basement membranes in specimens from cases that were available for ultrastructural study suggests that defects in the extracellular matrix may play a role in the pathogenesis of this subset of LGMD.     

Differential heparin sensitivity of alpha-dystroglycan binding to laminins expressed in normal and dy/dy mouse skeletal muscle

The alpha-dystroglycan binding properties of laminins extracted from fully differentiated skeletal muscle were characterized. We observed that the laminins expressed predominantly in normal adult rat or mouse skeletal muscle bound alpha-dystroglycan in a Ca2+-dependent, ionic strength-sensitive, but heparin-insensitive manner as we had observed previously with purified placental merosin (Pall, E. A., Bolton, K. M., and Ervasti, J. M. 1996 J. Biol. Chem. 271, 3817-3821). Rat skeletal muscle laminins partially purified by heparin-agarose affinity chromatography also bound alpha-dystroglycan without sensitivity to heparin. We also confirm previous studies of dystrophic dy/dy mouse skeletal muscle showing that the alpha2 chain of merosin is reduced markedly and that the laminin alpha1 chain is not up-regulated detectably. However, we further observed a quantitative decrease in the expression of laminin beta/gamma chain immunoreactivity in alpha2 chain-deficient dy/dy skeletal muscle and reduced alpha-dystroglycan binding activity in laminin extracts from dy/dy muscle. Most interestingly, the alpha-dystroglycan binding activity of residual laminins expressed in merosin-deficient dy/dy skeletal muscle was inhibited dramatically (69 +/- 19%) by heparin. These results identify a potentially important biochemical difference between the laminins expressed in normal and dy/dy skeletal muscle which may provide a molecular basis for the inability of other laminin variants to compensate fully for the deficiency of merosin in some forms of muscular dystrophy.