Expression of alpha 1 integrin, a laminin-collagen receptor, during myogenesis and neurogenesis in the avian embryo.

In this study, we have examined the spatiotemporal distribution of the alpha 1 integrin subunit, a putative laminin and collagen receptor, in avian embryos, using immunofluorescence microscopy and immunoblotting techniques. We used an antibody raised against a gizzard 175 x 10(3) M(r) membrane protein which was described previously and which we found to be immunologically identical to the chicken alpha 1 integrin subunit. In adult avian tissues, alpha 1 integrin exhibited a very restricted pattern of expression; it was detected only in smooth muscle and in capillary endothelial cells. In the developing embryo, alpha 1 integrin subunit expression was discovered in addition to smooth muscle and capillary endothelial cells, transiently, in both central and peripheral nervous systems and in striated muscles, in association with laminin and collagen IV. alpha 1 integrin was practically absent from most epithelial tissues, including the liver, pancreas and kidney tubules, and was weakly expressed by tissues that were not associated with laminin and collagen IV. In the nervous system, alpha 1 integrin subunit expression occurred predominantly at the time of early neuronal differentiation. During skeletal muscle development, alpha 1 integrin was expressed on myogenic precursors, during myoblast migration, and in differentiating myotubes. alpha 1 integrin disappeared from skeletal muscle cells as they became contractile. In visceral and vascular smooth muscles, alpha 1 integrin appeared specifically during early smooth muscle cell differentiation and, later, was permanently expressed after cell maturation. These results indicate that (i) the expression pattern of alpha 1 integrin is consistent with a function as a laminin/collagen IV receptor; (ii) during avian development, expression of the alpha 1 integrin subunit is spatially and temporally regulated; (iii) during myogenesis and neurogenesis, expression of alpha 1 integrin is transient and correlates with cell migration and differentiation.     

Human smooth muscle VLA-1 integrin: purification, substrate specificity, localization in aorta, and expression during development

A membrane glycoprotein complex was isolated and purified from human smooth muscle by detergent solubilization and affinity chromatography on collagen-Sepharose. The complex was identified as VLA-1 integrin and consisted of two subunits of 195 and 130 kD in SDS-PAGE. Liposomes containing the VLA-1 integrin adhered to surfaces coated with type I, II, III, and IV collagens, Clq subcomponent of the first component of the complement, and laminin. The liposomes specifically adhered to these proteins in a Ca2+, Mg2(+)-dependent manner, but did not bind to gelatin, fibronectin, and thrombospondin substrates. The expression of VLA-1 integrin in different human tissues and cell types, and during aorta smooth muscle development was studied by SDS-PAGE, and subsequent quantitative immunoblotting was performed with antibodies recognizing alpha 1 and beta 1 subunits of the VLA-1 integrin. A high level of VLA- 1 integrin expression was an exceptional feature of smooth muscles. Fibroblasts, endothelial cells, keratinocytes, striated muscles, and platelets contained trace amounts of VLA-1 integrin. In the 10-wk-old human fetal aorta, VLA-1 integrin was found only in smooth muscle cells whereas mesenchymal cells, surrounding aortic smooth muscle cells, were VLA-1 integrin negative. By the 24th wk of gestation, the amount of VLA- 1 integrin was significantly reduced in the aortic media (4.3-fold for alpha 1 subunit and 2.5-fold for beta 1 subunit) compared with that in the 10-wk-old aortic smooth muscle cells. After birth, the expression of VLA-1 integrin increased and in the 1.5-yr-old child aorta the VLA-1 integrin level was almost the same as in adult aortic media. Smooth muscle cells from intimal thickening of adult aorta express five times less alpha 1 subunit of VLA integrin that smooth muscle cells from adult aortic media. In primary culture of aortic smooth muscle cells, the content of the VLA-1 integrin was dramatically reduced and subcultured cells did not contain VLA-1 integrin at all.     

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.     

Changes in expression and organization of smooth-muscle-specific α-actin during fibronectin-mediated modulation of arterial smooth muscle cell phenotype

The spreading of freshly isolated arterial smooth muscle cells on a substrate of fibronectin is mediated by an integrin receptor on the cell surface. It is associated with organization of actin filaments in stress fibers and marked changes in cell morphology and function, collectively referred to as a transition from a contractile to a synthetic phenotype. To study further how extracellular matrix components affect smooth muscle phenotype, we have analyzed the expression and organization of smooth-muscle-specific alpha-actin in freshly isolated rat aortic smooth muscle cells cultured on a substrate of fibronectin under serum-free conditions. Northern-blot analysis showed that the expression of mRNA for smooth muscle alpha-actin, but not for nonmuscle actin, was strongly repressed during primary culture. On the other hand, the cellular content of alpha-actin was only moderately changed during the same period. Indirect immunofluorescence staining revealed that nonmuscle actin was rapidly organized in stress fibers, which did not stain with a monoclonal antibody against smooth muscle alpha-actin. Filament bundles containing alpha-actin were most prominent in the central parts of the cytoplasm and gradually disappeared as the spreading of the cells progressed. In contrast to the situation with nonmuscle actin, there was no apparent overlap in the staining for alpha-actin and the fibronectin receptor (alpha 5 beta 1), indicating that this receptor interacted with nonmuscle actin during the initial spreading process. Taken together, the results show that the expression and organization of smooth muscle alpha-actin are changed during interaction of the cells with fibronectin early in primary culture. They support the notion that integrin-mediated interactions between extracellular matrix components and arterial smooth muscle cells take part in the control of smooth muscle phenotype.     

Laminin alpha5 chain is required for intestinal smooth muscle development

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice. In the subepithelial basement membrane, loss of alpha5 expression was paralleled by ectopic or accelerated deposition of laminin alpha2 and alpha4 chains; this may explain why no obvious defects were observed in the villous form and enterocytic differentiation. This compensation process is attributable to mesenchyme-derived molecules as assessed by chick/mouse alpha5(-/-) grafted associations. Lack of the laminin alpha5 chain was accompanied by a decrease in epithelial alpha3beta1 integrin receptor expression adjacent to the epithelial basement membrane and of Lutheran blood group glycoprotein in the smooth muscle cells, indicating that these receptors are likely mediating interactions with laminin alpha5-containing molecules. Taken together, the data indicate that the laminin alpha5 chain is essential for normal development of the intestinal smooth muscle and point to possible mesenchyme-derived compensation to promote normal intestinal morphogenesis when laminin alpha5 is absent.     

Tetraspanin CD82 attenuates cellular morphogenesis through down-regulating integrin alpha6-mediated cell adhesion

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.     

Alpha 6.beta 1 integrin (laminin receptor) is down-regulated by tumor necrosis factor alpha and interleukin-1 beta in human endothelial cells.

Endothelial cells from human umbilical vein (HEC) express several distinct integrin complexes that mediate the interaction with the basal membrane components. In this paper we show that treatment with tumor necrosis factor alpha (TNF alpha) down-regulates the expression of the laminin receptor alpha 6.beta 1 integrin in cultured HEC. After 48 h of treatment with TNF alpha, the level of expression of the alpha 6.beta 1 complex reached 20% of the control value. The down-regulation of the alpha 6.beta 1 integrin is caused by a decreased expression of the alpha 6 subunit, whereas the synthesis of the beta 1 subunit remains constant. Northern blot analysis shows that the decreased level of alpha 6 subunit synthesis is caused by down-regulation of alpha 6 mRNA in TNF alpha-treated HEC. TNF alpha treatment does not alter the expression of alpha 2, alpha 3, and alpha 5 integrins, also present on endothelial cell surface, thus showing that this cytokine has a selective action on distinct integrin complexes. Down-regulation of alpha 6.beta 1 correlates with pronounced reduction in adhesion of TNF alpha-treated HEC to laminin, but not to fibronectin-coated culture dishes. In addition to TNF alpha, interleukin-1 beta also decreases the expression of the alpha 6.beta 1 integrin and reduces adhesion to laminin, thus suggesting that this regulation plays an important role in inflammation.     

Resting smooth muscle cells as a model for studying vascular cell activation

Vascular smooth muscle (VSM) cells constitute the main structural components of tunica media. Under physiological conditions, these cells display a contractile phenotype and a low proliferative activity. However, they may also acquire a synthetic phenotype and become predominantly proliferative if stimulated under certain stress conditions. This capacity plays a major role in the inception and progression of such cardiovascular diseases as atherosclerosis, hypertension and restenosis. Porcine coronary smooth muscle (PCSM) cells exhibit a synthetic phenotype (ON cells) under standard culturing conditions, but they can be reverted to a contractile phenotype (OFF cells) in a serum-free medium. However, OFF cells can also re-acquire a synthetic phenotype (OFF/ON cells) upon serum administration. In the present study, proliferative and contractile behaviors were characterized by expression of specific differentiation markers. Taken together, these results demonstrate that porcine vascular smooth muscle cells can retain their phenotypic plasticity in culture, and thus mimic in vitro their in vivo differentiation states. OFF cells may thus provide a suitable model system in studying the mechanism(s) by which either known or unknown serum factors may trigger vascular smooth muscle activation. In the present study, this possibility was actually tested by exposing OFF cells to fetal bovine serum (FBS), PDGF-BB and IGF-I. Data show that only FBS could induce a synthetic phenotype in OFF cells, while both PDGF-BB and IGF-I failed to induce any VSM activation.     

Laminin receptors in the retina: sequence analysis of the chick integrin alpha 6 subunit. Evidence for transcriptional and posttranslational regulation.

The integrin alpha 6 beta 1 is a prominent laminin receptor used by many cell types. In the present work, we isolate clones and determine the primary sequence of the chick integrin alpha 6 subunit. We show that alpha 6 beta 1 is a prominent integrin expressed by cells in the developing chick retina. Between embryonic days 6 and 12, both retinal ganglion cells and other retinal neurons lose selected integrin functions, including the ability to attach and extend neurites on laminin. In retinal ganglion cells, we show that this is correlated with a dramatic decrease in alpha 6 mRNA and protein, suggesting that changes in gene expression account for the developmental regulation of the interactions of these neurons with laminin. In other retinal neurons the expression of alpha 6 mRNA and protein remains high while function is lost, suggesting that the function of the alpha 6 beta 1 heterodimer in these cells is regulated by posttranslational mechanisms.     

Cib2 binds integrin alpha7Bbeta1D and is reduced in laminin alpha2 chain-deficient muscular dystrophy

Mutations in the gene encoding laminin alpha2 chain cause congenital muscular dystrophy type 1A. In skeletal muscle, laminin alpha2 chain binds at least two receptor complexes: the dystrophin-glycoprotein complex and integrin alpha7beta1. To gain insight into the molecular mechanisms underlying this disorder, we performed gene expression profiling of laminin alpha2 chain-deficient mouse limb muscle. One of the down-regulated genes encodes a protein called Cib2 (calcium- and integrin-binding protein 2) whose expression and function is unknown. However, the closely related Cib1 has been reported to bind integrin alphaIIb and may be involved in outside-in-signaling in platelets. Since Cib2 might be a novel integrin alpha7beta1-binding protein in muscle, we have studied Cib2 expression in the developing and adult mouse. Cib2 mRNA is mainly expressed in the developing central nervous system and in developing and adult skeletal muscle. In skeletal muscle, Cib2 colocalizes with the integrin alpha7B subunit at the sarcolemma and at the neuromuscular and myotendinous junctions. Finally, we demonstrate that Cib2 is a calcium-binding protein that interacts with integrin alpha7Bbeta1D. Thus, our data suggest a role for Cib2 as a cytoplasmic effector of integrin alpha7Bbeta1D signaling in skeletal muscle.