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PubMed1993

Evidence for integrins other than beta 2 on polymorphonuclear neutrophils: expression of alpha 6 beta 1 heterodimer.

The expression of non-beta 2 integrins on polymorphonuclear neutrophils (PMNs) was analyzed by immunoprecipitation and flow cytometry using platelet-free PMN preparations and anti-Fc gamma R blocking mAbs. No beta 3 integrin was detected with six anti-beta 3 mAbs. Conversely, integrin beta 1 chain was present on PMNs, although at low level, and could be distinguished from platelet beta 1 by SDS-PAGE. The MW differences disappeared after N-glycanase treatment. PMNs express only 2500 molecules of beta 1 per cell and this expression is not modulated by agonists such as phorbol myristate acetate, formylmethionyl-leucyl-phenylalanine, granulocyte-macrophage colony-stimulating factor, or tumor necrosis factor alpha, which enhance CD11b expression, or by interferon-gamma or transforming growth factor beta. PMNs were found to express alpha 6 associated with beta 1, and no reactivity was observed with various anti-alpha 1, anti-alpha 2, anti-alpha 3, anti-alpha 4, anti-alpha 5 or anti-alpha V mAbs. In conclusion, although other leukocytes express various beta 1 integrins, which mediate cell interactions with ECM proteins, PMNs appear to express only the laminin receptor alpha 6 beta 1. PMN interactions with non-laminin ECM ligands thus seem to be mediated either exclusively by beta 2 integrins or by nonintegrin molecules.

Antibodies, Monoclonal↗

PubMed1997

Transendothelial migration induces rapid expression on neutrophils of granule-release VLA6 used for tissue infiltration.

Little is known of the mechanisms allowing neutrophils to infiltrate tissue after transendothelial migration. We postulated that VLA6 might be involved in neutrophil infiltration because it revealed as the most expressed beta1 integrins among VLA5, VLA4, and VLA3, which also appeared to define subsets within the blood neutrophil population. Transendothelial migration up-regulated by threefold (5,000 to 15,000 receptors) VLA6 expression on neutrophils. VLA6 up-regulation was transient, peaking in 6 min and returning to baseline in 1 h when tested in response to N-formyl-methionyl-leucyl-phenylalanine. Neutrophil degranulation experiments revealed a steady correlation between expression of VLA6 and granule content release, notably beta-glucuronidase, indicating that VLA6 molecules were preformed and stored mostly in azurophilic granules. Migration across fibroblast monolayers of neutrophils preactivated for VLA6 up-regulation was blocked when they were preincubated with anti-VLA6. However, anti-VLA6 had no effect on transfibroblast migration of non-preactivated neutrophils. These results indicate that VLA6 was functional only on activated neutrophils that used their up-regulated VLA6 to cross fibroblasts. Activation of neutrophils by transendothelial migration induces rapid expression of granule release VLA6 that appears to be a key adhesion mechanism used by a subset of neutrophils to infiltrate tissue.

Cell Degranulation↗

PubMed1996

CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the alpha 6/beta 1 integrin and the neural adhesion molecule L1.

We describe here a novel monoclonal antibody (mab H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25-27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N-terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11-day-old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9-immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate-coated laminin, but not on substrate-coated poly-L-lysine. Addition of mab H6 elicited an increase in intracellular Ca2+ concentration in these cells on substrate-coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the alpha 6/beta 1 integrin. Moreover, preparations of CD9 immunoaffinity-purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the alpha 6/beta 1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and alpha 6/beta 1 integrin to influence neural cell interactions in vitro.

Amino Acid Sequence↗

PubMed2001

VLA-6 integrin distribution and calcium signalling in capacitated boar sperm.

Previous investigations showed that VLA-6 integrin present on boar sperm membrane can induce acrosome reaction upon exposure to laminin accumulated in expanded cumuli (Mattioli et al., 1998. To further investigate this novel sperm egg-recognition system, the authors studied the distribution of VLA-6 integrin on the membrane of boar sperm throughout capacitation and following acrosome reaction, and analyzed intracellular Ca(2+) changes occurring in spermatozoa exposed to laminin. Immunofluorescent localisation of VLA-6 revealed a low proportion (nearly 22%) of positive cells in freshly ejaculated sperm, with integrin mainly concentrated in clustered spots. After 3 hr incubation most of the spermatozoa showed integrin molecules on the membrane, with three different labeling patterns: fluorescence localised on the edge of the acrosome (58.2 +/- 14.2% of the cells); fluorescence uniformly spread over the whole sperm head (5.0 +/- 1.9%) and finally fluorescence concentrated in clustered spots (7.6 +/- 5.6%), as recorded in freshly ejaculated sperm. Twenty-nine percent of cells did not show any distinct fluorescence. Following acrosome reaction sperm with fluorescence on the acrosomal region virtually disappeared and the proportion of unstained cells rose from 29.2 +/- 9.2 to 69.0 +/- 10.1%. Electron microscopy demonstrated that VLA-6 integrin was exclusively located on the sperm membrane of intact spermatozoa. Confocal analysis showed that laminin triggers distinct Ca(2+) raises, and that sperm exposed and kept in the presence of laminin fully retained their ability to rise intracellular Ca(2+) in response to zona pellucida proteins. These data indicate that boar sperm accumulate VLA-6 integrin on the membrane and concentrate it on the acrosomal region as capacitation progresses. Probably due to this compartmentalisation, sperm exposed to laminin experience a Ca(2+) raise that originates in the anterior sperm head where it is more adequate for the induction of acrosome reaction. Mol. Reprod. Dev. 59:322-329, 2001.

Animals↗

PubMed1994

Anti-beta 1 integrin antibody inhibits Schwann cell myelination.

Schwann cells (SCs) co-cultured with sensory neurons require ascorbate supplementation for basal lamina assembly and differentiation into myelinating cells. The ascorbate requirement can be bypassed by adding a purified basal lamina component, laminin, to SC/neuron co-cultures. We have examined the role of laminin receptors, namely, the beta 1 subfamily of integrins, in the process of myelination. We demonstrate by immunostaining or immunoprecipitation that undifferentiated SCs in contact with axons express large amounts of the beta 1 subunit in association with the alpha 1 or alpha 6 subunit. In co-cultures of myelinating SCs, alpha 1 beta 1 is no longer present, alpha 6 beta 1 is still present but at reduced levels, and alpha 6 beta 4 is expressed at much higher levels than in co-cultures of undifferentiated SCs. Immunogold labelling at the electron microscope level suggested that beta 1 integrins are randomly distributed on undifferentiated SCs, become localized to the SC surface contacting basal lamina in differentiating SCs before the onset of myelination, and are not detected on myelinating SCs. Fab fragments of beta 1 function-blocking antibody block both attachment of isolated SCs to laminin and formation of myelin sheaths by SCs co-cultured with neurons in ascorbate-supplemented medium. SCs unable to myelinate in the presence of the anti-beta 1 antibody assemble patchy basal lamina that is only loosely attached to the cell surface and in some cases appears to be detaching from the membrane. In contrast, an alpha 1 beta 1 function-blocking antibody only partially blocks attachment of isolated SCs to laminin but has no inhibitory effect on SC myelination. These results are consistent with the hypothesis that a member of the beta 1 subfamily of integrins other than alpha 1 beta 1 binds laminin present in basal lamina to the SC surface and transduces signals that are critical for initiation of SC differentiation into a myelinating cell.

Animals↗

PubMed1996

Extracellular matrix components of the mouse thymus microenvironment. V. Interferon-gamma modulates thymic epithelial cell/thymocyte interactions via extracellular matrix ligands and receptors.

Extracellular matrix (ECM) proteins influence cell migration and differentiation in a variety of cell systems. Within the thymus, the ECM distribution pattern is conserved among various mammalian species, but its physiological role is not completely understood. Interferon-gamma (IFN-gamma), a cytokine produced by thymocytes, is able to in vitro modulate ECM production by thymic epithelial cells (TEC) in a dose-dependent biphasic pattern. In the same model, we determined herein that the expression of VLA-5 and VLA-6 (fibronectin and laminin receptors, respectively) were upregulated by low doses of IFN-gamma, whereas high doses of the cytokine induced an opposite effect. In a second in vitro system, we evidenced that thymocyte adhesion to a TEC line was also modulated by IFN-gamma. Importantly, such effects were due to the biphasic modulation of ECM ligands and receptors since they could be specifically prevented by preincubating the TEC cultures with anti-ECM or anti-ECM receptor antibodies. Additionally, spontaneous thymocyte release from thymic nurse cells was similarly biphasically modulated by IFN-gamma. Our data provide support for the notion that thymocyte-derived products can play a role in the dialogue that exists in the thymus, involving differentiating thymocytes and microenvironmental cells. Moreover, we bring arguments indicating that one of the implicated mechanisms involved is the modulation of ECM ligands and receptors by the thymic epithelium.

Animals↗

PubMed1996

VLA-5 in the plasma cell line, FR4ds, acts as a common regulator of VLA-4 and VLA-6 in spreading induced by fibronectin and laminin.

VLA-5 recognizes the GRGDSP sequence of fibronectin (FN) in the extracellular matrix (ECM). We examined the role of beta1 integrin in the spreading of the human plasma cell line, FR4ds, induced by FN and laminin (LN). We first examined the role of VLA-5 in the spreading induced by FN. Anti-alpha4 antibody induced 46.4% inhibition, whereas anti-alpha5 had no effect. A combination of anti-alpha4 and anti-alpha5 enhanced the inhibition of spreading significantly. Complementary inhibition was also demonstrated using the GRGDSP peptide plus anti-alpha4 and the GRGDNP peptide of LN plus anti-alpha4. The results suggested that VLA-5 is a regulator of VLA-4 and that it is involved in the recognition of GRGDNP. We then examined the role of VLA-5 in the spreading induced by LN. Anti-alpha6 induced 53.1% inhibition. Anti-alpha5 alone had no effect. A combination of alpha5 and anti-alpha6, however, significantly enhanced the inhibition of spreading. The combination of GRGDSP plus anti-alpha6 and GRGDNP plus anti-alpha6 resulted in complete inhibition. These results suggested that VLA-5 participates in the recognition of LN cooperatively with VLA-6 and that VLA-5 is a common regulator of VLA-4 and the LN receptor, VLA-6.

Cell Movement↗

PubMed2000

Alpha 6 beta 1 integrin (VLA-6) mediates leukocyte tether and arrest on laminin under physiological shear flow.

Polymorphonuclear leukocytes (PMN) were perfused over extracellular matrix protein substrates under laminar shear flow. Under shear below 1.5 dyn/cm(2), many PMN tethered to immobilized laminin but not to fibronectin or vitronectin. Almost all the tethered PMN immediately arrested on laminin. The number of tethered PMN was mostly abrogated by mAbs to integrin alpha 6 or beta 1 chains at concentrations of more than 5 microg/ml. Addition of the two mAbs together produced no further inhibition compared with each mAb alone. In contrast, none of the mAbs to alpha 2, alpha 3, and beta 4 chains showed significant inhibition, indicating that PMN tethering to laminin is mostly dependent on alpha 6 beta 1 integrin. The addition of 10-100 ng/ml IL-8 in the assay medium before perfusion partially reduced PMN tethering to laminin. Stimulation with IL-8 also induced detachment of some tethered PMN within 30 s. Thus, IL-8 partially weakens the adhesiveness of alpha 6 beta 1 integrin on PMN in flow conditions.

Antibodies, Monoclonal↗

PubMed1997

Characterization of adhesion molecule expression in the pathogenesis of homologous collagen-induced arthritis in the DA rat.

We have investigated the inflammatory cell infiltrates and adhesion molecule expression in the synovial fluid (SF) and synovial membranes (SM) of rats with homologous collagen-induced arthritis. Immunohistochemical staining revealed that the majority of the cells in the SF were granulocytes, expressing CD11b and CD11c. In SM, the majority of the cells were monocytes/macrophages. CD49d and CD49f were expressed mainly in the erosion zone in SM, and ICAM-1 was expressed in the lining layer, in the capillaries, and in the erosion zone. In SF 7% of the cells were ICAM-1 positive. CD2 was more abundant in SM than in SF. These findings might explain the difference in granulocyte counts between SF and SM. CD49d and CD49f expression might retain lymphocytes and monocytes in SM, while granulocytes not expressing CD49d and CD49f are not retained.

Animals↗

PubMed1994

The alpha 6 beta 4 integrin is a receptor for both laminin and kalinin.

Previously, we have establish K562 transfectants that express either alpha 6A beta 1 or alpha 6B beta 1 (K alpha 6A or K alpha 6B) on their surface. Both cell lines bind to laminin and kalinin after treatment with the beta 1-stimulatory antibody TS2/16. Here we introduce the full-length beta 4 cDNA into the alpha 6A- and alpha 6B-expressing K562 cells and selected stably transfected cells. The beta 4 subunit was expressed on the surface of both transfectants and it formed dimers with the alpha 6A or alpha 6B subunits. Immunoprecipitation and preclearing analyses revealed that both transfectants expressed alpha 6 beta 1, in addition to alpha 6 beta 4. While K alpha 6A and K alpha 6B cells required TS2/16 stimulation for binding to laminin or kalinin, adhesion of the unstimulated beta 4-transfected K alpha 6A and K alpha 6B cells to these matrix components was already substantial. This adhesion was mediated by both alpha 6 beta 1 and alpha 6 beta 4 since it was completely blocked by an alpha 6-specific antibody or by a combination of anti-beta 1 and anti-beta 4 antibodies, but only partially by either of these latter two antibodies alone. Adhesion to laminin was completely blocked by an antiserum to laminin fragment E8 as was the adhesion to kalinin by an antibody to kalinin, demonstrating the specificity of adhesion. Both transfectants always adhered more strongly to kalinin than to laminin. Furthermore, binding to kalinin was less well blocked by antibodies to beta 4 than binding to laminin, indicating that the affinity of alpha 6 beta 4 for kalinin is higher than that for laminin. The fact that alpha 6 beta 1 mediated adhesion without TS2/16 stimulation on the beta 4-transfected K alpha 6A and K alpha 6B cells suggests that some activation of alpha 6 beta 1 had occurred in these cells, even though binding was increased when they were actively stimulated by the antibody TS2/16. Finally, we show that Mn2+ induced binding of solubilized alpha 6 beta 4 to matrix containing kalinin, deposited by the murine cell line RAC-11P/SD. This binding was inhibited by the anti-alpha 6 mAb GoH3. Together, these results indicate that both alpha 6 beta 1 and alpha 6 beta 4 are receptors for laminin and kalinin and that there are no differences in ligand specificity between the A and B variants of the alpha 6 subunit when associated with either beta 1 or beta 4.

Animals↗

PubMed1995

Regulation of endothelial cell morphogenesis by integrins, mechanical forces, and matrix guidance pathways.

Basement membrane matrix is known to induce human endothelial cells to form cord-like structures that mimic those observed during early angiogenesis in vivo. Using this model, blocking antibody studies revealed a major role for the alpha 6 beta 1 integrin in cord formation. During this process, two alterations in the Matrigel structure were observed which suggested a mechanism for the precision of cord formation. First, Matrigel contracted and lifted off an agarose support and second, linear distortions became visible in the Matrigel that correspond to the migration pathways of endothelial cell processes. These pathways, which we have termed "matrix guidance pathways," appear to result from the generation of mechanical tension between endothelial cells. The above data support the concept that endothelial cell guidance during morphogenetic events could be controlled by the ability of these cells to exert mechanical forces on the surrounding extracellular matrix to create pathways for migration.

Cell Communication↗

PubMed1995

The cytoplasmic domain of alpha 6A integrin subunit is an in vitro substrate for protein kinase C.

We have investigated whether the cytoplasmic domain of alpha 6A integrin subunit can be phosphorylated by Ser/Thr kinases using synthetic peptides as in vitro substrates. This domain was phosphorylated by protein kinase C (PKC) and cyclic AMP-dependent kinase (protein kinase A, PKA) but not by mitogen-activated protein kinase. While Ser1041 has been shown to be phosphorylated in PMA-stimulated cells in vitro, Ser1048 was phosphorylated by PKA. Furthermore pharmacological agents which induce a rise in cyclic AMP concentration failed to stimulate the phosphorylation of the alpha 6A cytoplasmic domain in intact cells. These results suggest that PKC, but not PKA, is involved in the physiological phosphorylation of the alpha 6A integrin subunit.

Amino Acid Sequence↗

PubMed1995

Function and differential regulation of the alpha 6 integrin isoforms during parietal endoderm differentiation.

F9 embryonal carcinoma cells treated with retinoic acid differentiate in monolayer into parietal endoderm (PE) or in suspension into embryoid bodies with an outer layer of visceral endoderm (VE) surrounding a core of largely undifferentiated cells. Previous reports have shown that cell-extracellular matrix interactions mediated by the beta 1 integrins play a critical role in the differentiation and migration of PE. In the present study we investigated the pattern of expression and function of the integrin alpha 6 beta 1 during the differentiation of F9 cells into VE and PE. F9 cells express integrin subunits alpha 3, alpha 5, alpha 6, and beta 1. Cell adhesion and migration assays demonstrate that alpha 6 beta 1 is the major laminin receptor in undifferentiated F9 cells as well as F9-derived PE cells. However, the amount of alpha 6 protein decreases significantly upon F9 cell differentiation into either VE or PE, as revealed by immunofluorescent staining and immunoprecipitation analysis. In contrast, the amount of steady-state alpha 6 message stays constant before and after F9 cell differentiation, suggesting that the down-regulation of alpha 6 beta 1 occurs post-transcriptionally. In view of previous reports of two alpha 6 isoforms generated by alternative RNA processing, we carried out reverse transcription-PCR analysis and show that, while alpha 6B is the major mRNA isoform before and after F9 cell differentiation, alpha 6A mRNA is weakly expressed in undifferentiated F9 cells and is substantially increased following F9 differentiation into PE. Immunoprecipitations using the isoform-specific antibodies show an increase in alpha 6A and a dramatic decrease in alpha 6B protein following PE differentiation. Pulse-chase experiments indicate that, whereas the stability of alpha 6B protein is unaltered, synthesis of alpha 6B protein is decreased at least threefold following PE differentiation. Further experiments demonstrate that alpha 6A localizes to focal contacts in PE cells. The switch from alpha 6B to alpha 6A and the localization of alpha 6A at focal contacts correlate with the acquisition of PE cell motility, which suggests distinct functions for the two alpha 6 isoforms.

Animals↗

PubMed1995

Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells.

The transforming growth factors-beta (TGFs-beta) family of genes plays important roles in cell growth and differentiation in many cell types. TGF beta modulates the synthesis and accumulation of extracellular matrix (ECM) components and the expression of cell surface receptors for ECM components. TGF beta is increased in alveolar lining fluid during inflammatory reactions of the lung and has been identified in alveolar epithelial cells of developing lungs and hyperplastic type II cells during repair. However, little is known about how TGF beta may regulate expression of extracellular matrix proteins and ECM receptors in lung alveolar epithelial cells. Laminin, a major glycoprotein component of epithelial basement membrane, is synthesized and secreted by alveolar epithelial cells. To study the effects of TGF beta on modulation of laminin and its integrin receptors alpha 6 beta 1 and alpha 3 beta 1 in lung alveolar epithelial cells, a rat alveolar type II cell-derived cell line, LM5, was incubated with TGF beta 1 (0-100 pg/ml) in serum-free medium for 0-16 h. We examined the expression of integrin subunits and laminin beta 2 chain (s-laminin) mRNAs and protein expression. By Northern blot analysis, TGF beta 1 induced dose-dependent increases in alpha 6 and beta 1 mRNA levels. TGF beta 1 also increased the expression of laminin beta 2 chain mRNA at 12-16 h poststimulation. In contrast, TGF beta 1 decreased alpha 3 mRNA expression. Immunoprecipitation studies of TGF beta 1-treated cells showed increased surface expression of both alpha 6 and beta 1 protein while surface expression of the alpha 3 integrin subunit was decreased. The same treatment resulted in increased laminin protein expression. These data suggest that TGF beta 1 may regulate alveolar epithelial cell differentiation in part through its modulation of integrins and laminin chains.

Animals↗

PubMed1997

Modulation of alpha 6/beta 1 integrin expression during differentiation of F9 murine embryonal carcinoma cells to parietal endoderm.

In vitro differentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in beta 1 integrin expression, its maturation, and organization on the cell surface in association with alpha 6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the beta 1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of beta 1 expression during F9 cells differentiation occurs first at post-translational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of alpha 6B and its association to a high molecular weight protein, while alpha 6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the beta 1 and alpha 6B integrin subunits from cell-cell contacts to focal contacts where they colocalize with vinculin. On the contrary alpha 6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two alpha 6 isoforms.

Animals↗

PubMed1997

Alpha 6 beta 4 and alpha 6 beta 1 integrins associate with ErbB-2 in human carcinoma cell lines.

Growth factors modulate integrin-mediated cell adhesion and motility, and their receptors are thought to share proteins that mediate intracellular signaling with integrin receptors. The crosstalk between these receptors is thought to play a relevant role in transformation and tumor progression. To highlight possible interactions between growth factors and cell adhesion receptors we investigated whether integrins associate with tyrosine kinase receptors in tumor cells. By affinity chromatography and Western blot analyses of purified immune complexes, we studied the association of laminin receptors (alpha 6 beta 1 and alpha 6 beta 4) with ErbB-2 tyrosine kinase in human carcinoma cell lines. We demonstrated that the alpha 6 beta 4 and alpha 6 beta 1 integrins coprecipitated with ErbB-2 in lysates from carcinoma or NIH3T3 cells overexpressing ErbB-2. Integrin-mediated activation of ErbB-2 receptors suggested that this association is functionally meaningful. Indeed, carcinoma cells treated with a monoclonal antibody to the alpha 6 integrin subunit showed a ligand-dependent increase of ErbB-2-phosphorylated molecules coprecipitated with integrins and an increased DNA synthesis. The interaction between growth factor receptors and integrins was also studied in NIH3T3 cells overexpressing alpha 6 beta 4 receptors and ErbB-2 protein. We report that cells overexpressing both receptors, but not those overexpressing a crippled ErbB-2, showed enhanced proliferation rates and invasiveness, further suggesting that alpha 6 beta 4 integrin and ErbB-2 receptor interaction might contribute to generate a more malignant phenotype in carcinoma cells.

3T3 Cells↗

PubMed2000

Laminin alpha4 and integrin alpha6 are upregulated in regenerating dy/dy skeletal muscle: comparative expression of laminin and integrin isoforms in muscles regenerating after crush injury.

The expression of laminin isoforms and laminin-binding 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 alpha2 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 alpha1 was not detectable in skeletal muscle. Laminin alpha2 was present in basement membranes of mature myofibers and newly formed myotubes in control and dy/dy muscles, albeit weaker in dy/dy. Laminin alpha2-negative myogenic cells were detected in dy/dy and control muscle, suggesting the involvement of other laminin alpha chains in early myogenic differentiation, such as laminin alpha4 and alpha5 which were both transiently expressed in basement membranes of newly formed myotubes of dy/dy and control mice. Integrin beta1 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 alpha3 was not expressed in uninjured or regenerating muscle, while integrin alpha6 was expressed mainly on endothelial cells and peripheral nerves in uninjured muscle. Upon crush injury integrin alpha6 increased in the interstitium mainly on nonmyogenic cells, including infiltrating leukocytes, endothelial cells, and fibroblasts. In dy/dy muscle, integrin alpha6 occurred on some newly formed myotubes. Integrin alpha7 was expressed on muscle fibers at the myotendinous junction and showed weak and irregular expression on muscle fibers. After crush injury, integrin alpha7 expression extended to the newly formed myotubes and some myoblasts. However, many myoblasts and newly formed myotubes were integrin alpha7 negative. No marked difference was observed in integrin alpha7 expression between dy/dy and control muscle, either uninjured or after crush injury. Only laminin alpha4 and integrin alpha6 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 alpha4 and integrin alpha6 support myogenic regeneration. However, whether these accelerated myogenic effects are a direct consequence of the reduced laminin alpha2 expression in dy/dy mice, or an accentuation of the ongoing regenerative events in focal lesions in the muscle, requires further investigation.

Animals↗

PubMed2000

Phosphorylation of the beta1 integrin cytoplasmic domain: toward an understanding of function and mechanism.

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the beta1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of beta1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in beta1 phosphorylation and selective loss of beta1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with beta1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with beta1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with beta1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with beta1. A possible role for regulated beta1 phosphorylation in cell migration is discussed.

Animals↗