Laminin Beta1 Chain Research Articles

Insulin-Induced Laminin Expression Promotes a Hypercontractile Airway Smooth Muscle Phenotype

Airway smooth muscle (ASM) plays a key role in the development of airway hyperresponsiveness and remodeling in asthma, which may involve maturation of ASM cells to a hypercontractile phenotype. In vitro studies have indicated that long-term exposure of bovine tracheal smooth muscle (BTSM) to insulin induces a functional hypercontractile, hypoproliferative phenotype. Similarly, the extracellular matrix protein laminin has been found to be involved in both the induction and maintenance of a contractile ASM phenotype. Using BTSM, we now investigated the role of laminins in the insulin-induced hypercontractile, hypoproliferative ASM phenotype. The results demonstrate that insulin-induced hypercontractility after 8 days of tissue culture was fully prevented by combined treatment of BTSM-strips with the laminin competing peptides Tyr-Ile-Gly-Ser-Arg (YIGSR) and Arg-Gly-Asp-Ser (RGDS). YIGSR also prevented insulin-induced increases in sm-myosin expression and abrogated the suppressive effects of prolonged insulin treatment on platelet-derived growth factor-induced DNA synthesis in cultured cells. In addition, insulin time-dependently increased laminin alpha2, beta1, and gamma1 chain protein, but not mRNA abundance in BTSM strips. Moreover, as previously found for contractile protein accumulation, signaling through PI3-kinase- and Rho kinase-dependent pathways was required for the insulin-induced increase in laminin abundance and contractility. Collectively, our results indicate a critical role for beta1-containing laminins, likely laminin-211, in the induction of a hypercontractile, hypoproliferative ASM phenotype by prolonged insulin exposure. Increased laminin production by ASM could be involved in the increased ASM contractility and contractile protein expression in asthma. Moreover, the results may be of interest for the use of inhaled insulin administrations by diabetics.

Molecular genetic analysis of podocyte genes in focal segmental glomerulosclerosis—a review

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin β2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

Laminin peptide YIGSR and its receptor regulate sensory axonal response to the chemoattractive guidance cue in the chick embryo

During early development, centrally projecting dorsal root ganglion (DRG) neurons extend their axons toward the dorsal spinal cord. We previously reported the involvement of dorsal spinal cord-derived chemoattraction in this projection (Masuda et al. [ 2007] Neuroreport 18:1645-1649). However, the molecular nature of this attraction is not clear. Here we show that laminin-1 (alpha1beta1gamma1) is expressed strongly along the pathway of DRG axons and that its 67-kDa receptor (67LR) is present on DRG cells. This evidence suggests that laminin-1-67LR signaling may be involved in DRG axonal guidance. By employing culture assays, we show that laminin-1 or the YIGSR peptide, a soluble peptide of the laminin beta1 chain, promotes the DRG axonal response to dorsal spinal cord-derived chemoattraction. By using a function-blocking antibody against 67LR, we show that the anti-67LR antibody blocks the modulation of DRG axonal response by the YIGSR peptide in vitro. Furthermore, the in ovo injection of the anti-67LR antibody inhibits the DRG axonal growth toward the dorsal spinal cord. These results provide evidence that the YIGSR peptide promotes dorsal spinal cord-derived chemoattraction via 67LR to contribute to the formation of the initial trajectories of developing DRG axons.

Variable phenotype of Pierson syndrome

Pierson syndrome is caused by mutations in the LAMB2 gene, which encodes the laminin beta2 chain, and is clinically characterized by congenital nephrotic syndrome (CNS) and bilateral microcoria. Here, we describe two cases of Pierson syndrome involving atypical phenotypes. Patient 1 presented with congenital microcoria and infantile nephrotic syndrome. Despite persistent nephrotic syndrome, her renal function was maintained normally until she was 6 years old. Genetic analysis revealed two frame-shifting deletions (truncating mutations) in the LAMB2 gene. Patient 2 presented with isolated CNS without ocular involvement. Her renal function deteriorated progressively over several months, and retinal detachment in the right eye developed when she was aged 10 months. LAMB2 analysis revealed a missense mutation in one allele and a frame-shifting deletion in the other allele. Electron microscopy of a renal biopsy revealed irregular lamellation of the glomerular basement membrane (GBM) in both patients. The phenotypes of Pierson syndrome vary widely, and the severity of the renal phenotype is not always parallel to that of the ocular phenotype. The phenotypic variability likely reflects genotype-phenotype correlations, but unknown genetic or environmental modifiers may play an additional role. Ultrastructural changes of the GBM are a useful diagnostic indicator.

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

BackgroundCysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT1 or CysLT2, mediate this effect.MethodsCultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D4 (LTD4) and E4 (LTE4) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.ResultsLTD4 and LTE4 significantly augmented the expression of Tn, whereas LTD4, distinctly from LTE4, was able to increase also the Ln β2 chain. Although the expression of CysLT2 prevailed over that of CysLT1, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT1-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.ConclusionThese findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT1 receptor. The results provide a novel aspect to support the use of CysLT1 receptor antagonists in the anti-remodeling treatment of asthma.

Determination of disulfide bond patterns in laminin β1 chain N‐terminal domains by nano‐high‐performance liquid chromatography/matrix‐assisted laser desorption/ionization time‐of‐flight/time‐of‐flight mass spectrometry

The disulfide bonding patterns in the N-terminal (LN) domains of the basement membrane protein laminin beta1 have not been investigated so far. We report an in-depth mass spectrometric analysis using offline nano-high-performance liquid chromatography/matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (nano-HPLC/MALDI-TOF/TOF-MS) for determining the disulfide bond patterns in the LN-domain of recombinant mouse laminin beta1 chain for the first time. Mass spectra were recorded and the putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the disulfide bond. Screening the fragment ion mass spectra of disulfide-linked peptides for characteristic 66-amu patterns (34 u +32 u), arising from symmetric and asymmetric cleavage of disulfide bonds, facilitated their identification. Using various enzymes for proteolytic digestion of a recombinant laminin beta1 chain N-terminal protein fragment, a linear bonding pattern of the eight cysteine residues in the LN-domain of the laminin beta1 chain was observed with a (1-2, 3-4, 5-6, 7-8) connectivity of cysteines. The identical disulfide-bonding pattern was found in E4, the N-terminal laminin beta1 chain fragment derived by elastase digestion of mouse tumor laminin-111, confirming that this pattern also occurs in native laminin.

Compositional Differences between Infant and Adult Human Corneal Basement Membranes

Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development. Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components. Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from alpha1-alpha2 to alpha3-alpha4 chains after 3 years of life; in the adult, alpha1-alpha2 chains were retained only in the limbal BM. Laminin alpha2 and beta2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, beta2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin gamma3 chain. In the infant DM, type IV collagen alpha1-alpha6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years. The distribution of laminin gamma3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.

Impaired Glomerular Maturation and Lack of VEGF165b in Denys-Drash Syndrome

Individuals with Denys-Drash syndrome (DDS) develop diffuse mesangial sclerosis, ultimately leading to renal failure. The disease is caused by mutations that affect the zinc finger structure of the Wilms' tumor protein (WT1), but the mechanisms whereby these mutations result in glomerulosclerosis remain largely obscure. How WT1 regulates genes is likely to be complex, because it has multiple splice forms, binds both DNA and RNA, and associates with spliceosomes. Herein is described that in DDS podocytes, the ratio of both WT1 +KTS isoforms C to D differs considerably from that of normal child and adult control podocytes and more closely resembles fetal profiles. Aside from the delay in podocyte maturation, DDS glomeruli show swollen endothelial cells, reminiscent of endotheliosis, together with incompletely fused capillary basement membranes; a dramatic decrease in collagen alpha4(IV) and laminin beta2 chains; and the presence of immature or activated mesangial cells that express alpha-smooth muscle actin. Because appropriate vascular endothelial growth factor A (VEGF-A) expression is known to be essential for the development and maintenance of glomerular architecture and function, this article addresses the question of whether VEGF-A expression is deregulated in DDS. The data presented here show that DDS podocytes express high levels of the proangiogenic isoform VEGF165, but completely lack the inhibitory isoform VEGF165b. The VEGF165/VEGF165b ratio in DDS resembles that of fetal S-shaped bodies, rather than that of normal child or adult control subjects. The alteration in VEGF-A expression presented here may provide a mechanistic insight into the pathogenesis of DDS.

Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin alpha5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin alpha4, beta1, beta2 and gamma1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin alpha5 chain and Lutheran glycoprotein, a receptor for the laminin alpha5 chain. In normal lymphatic capillaries, the presence of primarily alpha4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin alpha5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.

Malignancy-related 67 kDa laminin receptor in adenoid cystic carcinoma. Effect on migration and β-catenin expression

Adenoid cystic carcinoma is a malignant salivary gland neoplasm with recurrence and metastasis. We studied the expression of a malignancy-related non-integrin laminin receptor, the 67LR, in this neoplasm. Immunohistochemistry showed 67LR in adenoid cystic carcinoma. This receptor binds a sequence of laminin beta1 chain, the YIGSR peptide. We studied the effect of 67LR and YIGSR in cells (CAC2) from adenoid cystic carcinoma. Three-dimensional cultures of cells embedded into either laminin-111 gel (controls) or YIGSR-enriched laminin-111 (treated) were prepared and studied by light microscopy. CAC2 cells treated with YIGSR appeared fibroblast-like, while control cells were epithelioid. Blockage of 67LR by antibody abolished YIGSR effect in three-dimensional cultures. We analysed the relevance of 67LR and YIGSR on beta-catenin expression in CAC2 cells. Immunofluorescence and immunoblot showed that YIGSR decreased beta-catenin, while blockage of 67LR restored the presence of this molecule. The 67LR and YIGSR induced fibroblast-like morphology in CAC2 cells, with disruption of cell-cell contacts and decrease of beta-catenin. These features resemble epithelial-mesenchymal transition (EMT). EMT also increases cell migration. In monolayer assays YIGSR increased migration of CAC2 cells. We conclude that 67LR and YIGSR are involved in epithelial-mesenchymal transition, modulation of beta-catenin expression, and migratory activity of CAC2 cells.

Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin α1 chain, AL-2 with laminin β1 chain, and AL-4 with the coiled-coil domain of laminin β1 chain

We analyzed the reactivity of three different commercially available rat monoclonal antibodies raised against mouse laminin-alpha1beta1gamma1 (laminin-111), AL-1, AL-2, and AL-4. Using ELISA assays, Western blot analysis and immunostainings we present refined epitope maps for these three laminin monoclonals. AL-1 reacted, as predicted with laminin alpha1 chain. AL-4 has also been marketed as an alpha1 chain specific probe, but we show here that AL-4 detects mouse laminin beta1 chain, in the distal part of the coiled-coil region. AL-2 was predicted to react with all three chains near the cross-region, but seems to primarily react with laminin beta1 chain.

Laminin α2 Chain-Positive Vessels and Epidermal Growth Factor in Lung Neuroendocrine Carcinoma: A Model of a Novel Cooperative Role of Laminin-2 and Epidermal Growth Factor in Vessel Neoplastic Invasion and Metastasis

Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors.

Segment‐specific but pathologic laminin isoform profiles in human labial salivary glands of patients with Sjögren's syndrome

To assess the laminins in basement membrane of the labial salivary glands of patients with Sjogren's syndrome (SS) and healthy controls. Labeling of laminin alpha1-alpha5, beta1, beta2, gamma1, and gamma2 chains was performed using immunohistochemistry with chain-specific monoclonal antibodies and pattern recognition analysis of the labeled specimens. Laminin alpha1, alpha2, and alpha4 chains were detected exclusively in the acinar basement membranes, whereas laminin alpha3, alpha5, beta1, gamma1, and gamma2 chains were also detected in ductal basement membranes. Laminin beta2 chain was not found. In patients with SS, laminin alpha1 and alpha2 chains were weakly labeled, but laminin alpha4 labeling was also intense in areas not infiltrated by lymphocytes. Pattern recognition analysis suggested that laminin alpha1, alpha2, and alpha4 chains were associated with acinar cells, myoepithelial cells, and tissue damage/repair, respectively. All salivary gland basement membranes signal through laminin 5, laminin 6, and laminin 10 trimers, but acinar basement membranes also signal through laminin 1, laminin 2, and laminin 8 trimers. Laminin alpha1 chain/laminin 1 may play a central role in the maintenance of acinar cells in healthy glands. This possibility was supported by the finding of variable expression of laminin alpha1 chain/laminin 1 in acinar basement membrane and its weak expression in SS with acinar cell atrophy. The impairment of myoepithelial laminin alpha2 chain/laminin 2 in patients with SS indicates a double defect in the acinar compartment and pathologic extracellular matrix-to-cell signaling, which may contribute to structural deterioration and functional abnormality of the exocrine glands.

Laminin chains in rat and human peripheral nerve: distribution and regulation during development and after axonal injury.

During nerve growth, axons are dependent upon contact with matrix components, such as laminins, for elongation, guidance, and trophic support. Semiquantitative in situ hybridization histochemistry and immunohistochemistry (IHC) were used to identify laminin chains in normal peripheral nerves, during postnatal development, after sciatic nerve transection (SNT), and after sciatic nerve crush (SNC). Laminin alpha2, alpha4, beta1, beta2, and gamma1 chain mRNAs were all expressed at high levels in newborn rat sciatic nerves with declining levels during later developmental stages. At the adult stage, no laminin chain mRNA was detectable. Of interest, the mRNA levels for alpha4 chain declined faster than those for alpha2. After SNT, laminin alpha2, alpha4, beta1, and gamma1 mRNA levels were up-regulated at the site of the injury, with the most profound reaction in the proximal nerve stump. Laminin alpha2 and alpha4 chains differed in that the mRNA levels of alpha4 were up-regulated earlier and declined quicker, whereas alpha2 had a later onset, with high levels remaining even after 6 weeks. After SNC, there was an initial up-regulation of the same laminin chain mRNAs as after SNT in the nerve, however, less intense, and at 6 weeks after SNC, all laminin mRNA levels studied had returned to normal. IHC of adult human normal and transected peripheral nerves stained positive for laminin alpha2, alpha4, beta1, and gamma1 chains in close relation to neurofilament labeled axons. Laminin alpha3, alpha4, alpha5, beta1, beta2, and gamma1 chains were found in blood vessel-like structures and alpha3, alpha4, alpha5, beta2, and gamma1 in the perineurium. These results and a previously published description of integrin regulation in spinal motoneurons suggest that both laminin-2 (alpha2beta1gamma1) and laminin-8 (alpha4beta1gamma1) are important for the postnatal nerve development and axonal regeneration after injury and that laminin-8 may have important functions especially early postnatally and early after adult nerve lesion.

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases.

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.

Domain IV of mouse laminin beta1 and beta2 chains.

Domain IV, consisting of about 230 residues, represents a particular protein module so far found only in laminin beta1 and beta2 chains. Both domains were obtained by recombinant production in mammalian cells. They showed a globular structure, as expected from electron microscopic examination of laminins. Fragment beta1IV was obtained as a monomer and a disulfide-bonded dimer, and both were modified to approximately 50% by a single chondroitin sulfate chain attached to Ser721 of an SGD consensus sequence. Dimerization is caused by an odd number of cysteines, with three of them having a partial thiol character. Whether both modifications also occur in tissue forms of laminin remains to be established. Fragment beta2IV was only obtained as a monomer, as it lacked one crucial cysteine and the SGD sequence. It required, however, the presence of two adjacent LE modules for proper folding. Polyclonal antibodies raised against both fragments showed no cross-reaction with each other and allowed establishment of beta chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 5-25-fold lower content of beta2 compared with beta1 chains in various tissue extracts of adult mice. Tissues derived from beta2-deficient mice failed to react with the beta2-specific antibodies but showed a twofold higher content of beta1 than heterozygotes. The antibodies to beta2 showed broader tissue staining than reported previously, including in particular a distinct reaction with the extrasynaptic endomysium of skeletal muscle. Immunogold staining localized both beta chains primarily to basement membranes of kidney, muscle and various other tissues.

Regulation of laminin beta2 chain gene expression in human cancer cell lines.

The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.

Properly formed but improperly localized synaptic specializations in the absence of laminin alpha4.

Precise apposition of pre- to postsynaptic specializations is required for optimal function of chemical synapses, but little is known about how it is achieved. At the skeletal neuromuscular junction, active zones (transmitter release sites) in the nerve terminal lie directly opposite junctional folds in the postsynaptic membrane. Few active zones or junctional folds form in mice lacking the laminin beta2 chain, which is normally concentrated in the synaptic cleft. beta2 and the broadly expressed gamma1 chain form heterotrimers with alpha chains, three of which, alpha2, alpha4 and alpha5, are present in the synaptic cleft. Thus, alpha2beta2gamma1, alpha4beta2gamma1 and alpha5beta2gamma1 heterotrimers are all lost in beta2 mutants. In mice lacking laminin alpha4, active zones and junctional folds form in normal numbers, but are not precisely apposed to each other. Thus, formation and localization of synaptic specializations are regulated separately, and alpha4beta2gamma1 (called laminin-9) is critical in the latter process.

Purification and Characterization of Human Laminin-8: LAMININ-8 STIMULATES CELL ADHESION AND MIGRATION THROUGH α3β1 AND α6β1INTEGRINS

Recently identified laminin isoforms containing the alpha4 chain have been shown to be expressed in the basement membrane of restricted organs such as heart, skeletal muscle, and blood vessels, especially those in embryos. We screened 38 human cell lines for the expression of the laminin alpha4 chain by reverse transcriptase-polymerase chain reaction and found that T98G glioblastoma cells express only alpha4, but not other alpha chains. Laminin-8, an isoform containing the alpha4 and beta1 chains, was purified from conditioned medium of T98G cells by gel filtration and immunoaffinity chromatography using a monoclonal antibody against laminin beta1 chain. The purified laminin isoform was composed of disulfide-linked 230-, 220-, and 200-kDa subunits, which immunoblot analysis identified as the beta1, gamma1, and alpha4 chains. Purified laminin-8 had cell adhesive activity comparable to laminin-1 but significantly weaker than laminin-5 and laminin-10/11. T98G cells adhering to laminin-8 became more elongated than those adhering to other laminin isoforms and extended multiple pseudopods. Cell adhesion to laminin-8 was abolished by an antibody against the integrin beta1 subunit or a combination of antibodies against the integrin alpha3 and alpha6 subunits, but not by either anti-alpha3 or anti-alpha6 antibody alone, suggesting that both alpha3beta1 and alpha6beta1 integrins serve as adhesion receptors for laminin-8. Consistent with these observations, K562 erythroleukemic cells transfected with either integrin alpha3 or alpha6 cDNA were capable of adhering to laminin-8 when beta1 integrins were stimulated by the beta1-activating antibody 8A2. Despite its moderate cell adhesive activity, laminin-8 was significantly potent in promoting cell migration when compared with other laminin isoforms and fibronectin. Cell migration on laminin-8 was completely inhibited by a combination of antibodies against alpha3 and alpha6 integrins, and substantially inhibited by anti-alpha3 antibody alone, suggesting that laminin-8-mediated cell migration is predominantly mediated by alpha3beta1 integrin. Given its potency to stimulate cell migration and preferential localization to the basement membrane of capillaries and embryonic tissues, laminin-8 may play a role in processes requiring enhanced cell migration during development, wound healing, and angiogenesis.

Laminin chains in developing and adult human myotendinous junctions.

In addition to being the specialized site for transmission of force from the muscle to the tendon, the myotendinous junction (MTJ) also plays an important role in muscle splitting during morphogenesis. An early event in the formation of the MTJ is a regional deposition of basement membranes. We used immunocytochemistry to investigate the distribution of laminin chains during the development of MTJs in human limb muscle at 8-22 weeks of gestation (wg) and in adult MTJs. We used polyclonal antibodies and a new monoclonal antibody (MAb) against the human laminin alpha1 G4/G5 domains. At 8-10 wg, laminin alpha1 and laminin alpha5 chains were specifically localized to the MTJ. Laminin alpha1 chain remained restricted to the MTJ at 22 wg as the laminin beta2 chain had appeared, whereas the laminin alpha5 chain became deposited along the entire length of the myotubes from 12 wg. In the adult MTJ, only vestigial amounts of laminin alpha1 and laminin alpha5 chains could be detected. On the basis of co-distribution data, we speculate that laminin alpha1 chain in the forming MTJ undergoes an isoform switch from laminin 1 to laminin 3. Our data indicate a potentially important role for laminin alpha1 chain in skeletal muscle formation. (J Histochem Cytochem 48:201-209, 2000)