Membrane-type matrix metalloproteinase-1 (MT1-MMP) is a processing enzyme for human laminin gamma 2 chain

Processing of the laminin-5 (Ln-5) gamma 2 chain by membrane-type-1 matrix metalloproteinases (MT1-MMP) promotes migration and invasion of epithelial and tumor cells. We previously demonstrated that MT1-MMP cleaves the rat gamma 2 chain at two sites, producing two major C-terminal fragments of 100 (gamma 2') and 80 (gamma 2 x) kDa and releasing a 30-kDa fragment containing epidermal growth factor (EGF)-like motifs (domain III (DIII) fragment). The DIII fragment bound the EGF receptor (EGF-R) and stimulated cell scattering and migration. However, it is not yet clear whether human Ln-5 is processed in a similar fashion to rat Ln-5 because one of the two MT1-MMP cleavage sites present in rat gamma 2 is not found in human gamma 2. To identify the exact cleavage site for MT1-MMP in human Ln-5, we purified both the whole molecule as well as a monomeric form of human gamma 2 that is frequently expressed by malignant tumor cells. Like rat Ln-5, both the monomer of gamma 2, as well as the gamma 2 derived from intact Ln-5, were cleaved by MT1-MMP in vitro, generating C-terminal gamma 2' (100 kDa) and gamma 2 x (85 kDa) fragments and releasing DIII fragments (25 and 27k Da). In addition to the conserved first cleavage site used to generate gamma 2', two adjacent cleavage sites (Gly(559)-Asp(560) and Gly(579)-Ser(580)) were found that could generate the gamma 2 x and DIII fragments. Two of the three EGF-like motifs present in the rat DIII fragment are present in the 27-kDa human fragment, and like the rat DIII, this fragment can promote breast carcinoma cell migration by engaging the EGF-R. These results suggest that MT1-MMP processing of Ln-5 in human tumors may stimulate the EGF-R, resulting in increased tumor cell scattering and migration that could possibly increase their metastatic potential.     

The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.     

Furin is the major processing enzyme of the cardiac-specific growth factor bone morphogenetic protein 10

Bone morphogenetic protein 10 (BMP10) is a member of the TGF-β superfamily and plays a critical role in heart development. In the postnatal heart, BMP10 is restricted to the right atrium. The inactive pro-BMP10 (～60 kDa) is processed into active BMP10 (～14 kDa) by an unknown protease. Proteolytic cleavage occurs at the RIRR(316)↓ site (human), suggesting the involvement of proprotein convertase(s) (PCs). In vitro digestion of a 12-mer peptide encompassing the predicted cleavage site with furin, PACE4, PC5/6, and PC7, showed that furin cleaves the best, whereas PC7 is inactive on this peptide. Ex vivo studies in COS-1 cells, a cell line lacking PC5/6, revealed efficient processing of pro-BMP10 by endogenous PCs other than PC5/6. The lack of processing of overexpressed pro-BMP10 in the furin- and PACE4-deficient cell line, CHO-FD11, and in furin-deficient LoVo cells, was restored by stable (CHO-FD11/Fur cells) or transient (LoVo cells) expression of furin. Use of cell-permeable and cell surface inhibitors suggested that endogenous PCs process pro-BMP10 mostly intracellularly, but also at the cell surface. Ex vivo experiments in mouse primary hepatocytes (wild type, PC5/6 knock-out, and furin knock-out) corroborated the above findings that pro-BMP10 is a substrate for endogenous furin. Western blot analyses of heart right atria extracts from wild type and PACE4 knock-out adult mice showed no significant difference in the processing of pro-BMP10, implying no in vivo role of PACE4. Overall, our in vitro, ex vivo, and in vivo data suggest that furin is the major convertase responsible for the generation of BMP10.     

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.     

Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages

It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis.     

Regulation of biological activity of laminin-5 by proteolytic processing of gamma2 chain

Laminin-5 (LN5), which regulates both cell adhesion and cell migration, undergoes specific extracellular proteolytic processing at an amino-terminal region of the gamma2 chain as well as at a carboxyl-terminal region of the alpha3 chain. To clarify the biological effect of the gamma2 chain processing, we prepared a human recombinant LN5 with the 150-kDa, non-processed gamma2 chain (GAA-LN5) and natural LN5 with the 105-kDa, processed gamma2 chain (Nat-LN5). Comparison of their biological activities demonstrated that GAA-LN5 had an about five-times higher cell adhesion activity but an about two-times lower cell migration activity than Nat-LN5. This implies that the proteolytic processing of LN5 gamma2 chain converts the LN5 from the cell adhesion type to the cell migration type. It was also found that human gastric carcinoma cells expressing the LN5 with the non-processed gamma2 chain is more adherent but less migratory than the carcinoma cells expressing a mixture of LN5 forms with the processed gamma2 chain and with the unprocessed one. The functional change of LN5 by the proteolytic processing of the gamma2 chain may contribute to elevated cell migration under some pathological conditions such as wound healing and tumor invasion.     

Characterization and expression of the laminin gamma3 chain: a novel, non-basement membrane-associated, laminin chain

Laminins are heterotrimeric molecules composed of an alpha, a beta, and a gamma chain; they have broad functional roles in development and in stabilizing epithelial structures. Here, we identified a novel laminin, composed of known alpha and beta chains but containing a novel gamma chain, gamma3. We have cloned gene encoding this chain, LAMC3, which maps to chromosome 9 at q31-34. Protein and cDNA analyses demonstrate that gamma3 contains all the expected domains of a gamma chain, including two consensus glycosylation sites and a putative nidogen-binding site. This suggests that gamma3-containing laminins are likely to exist in a stable matrix. Studies of the tissue distribution of gamma3 chain show that it is broadly expressed in: skin, heart, lung, and the reproductive tracts. In skin, gamma3 protein is seen within the basement membrane of the dermal-epidermal junction at points of nerve penetration. The gamma3 chain is also a prominent element of the apical surface of ciliated epithelial cells of: lung, oviduct, epididymis, ductus deferens, and seminiferous tubules. The distribution of gamma3-containing laminins on the apical surfaces of a variety of epithelial tissues is novel and suggests that they are not found within ultrastructurally defined basement membranes. It seems likely that these apical laminins are important in the morphogenesis and structural stability of the ciliated processes of these cells.     

Monoclonal anti-mouse laminin antibodies: AL-1 reacts with laminin alpha1 chain, AL-2 with laminin beta1 chain, and AL-4 with the coiled-coil domain of laminin beta1 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.     

Bone morphogenetic protein 1 prodomain specifically binds and regulates signaling by bone morphogenetic proteins 2 and 4

Highly purified fractions of bone extracts capable of inducing ectopic bone formation have been reported to contain peptides corresponding to the mature active regions of the TGF-beta-like bone morphogenetic proteins (BMPs) 2-7, and to the prodomain region of the metalloproteinase BMP1. Co-purification of BMPs 2-7 with BMP1 prodomain sequences through the multiple biochemical steps used in these previous reports has suggested the possibility of interactions between the BMP1 prodomain and BMPs 2-7. Here we demonstrate that the BMP1 prodomain binds BMPs 2 and 4 with high specificity and with a KD of approximately 11 nM, in the physiological range. It is further demonstrated that the BMP1 prodomain is capable of modulating signaling by BMPs 2 and 4 in vitro and in vivo, and that endogenous BMP1 prodomain-BMP4 complexes exist in cell culture media and in tissues.     

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.     

The cyclooxygenase inhibitor indomethacin modulates gene expression and represses the extracellular matrix protein laminin gamma1 in human glioblastoma cells

The induction of cyclooxygenase-2 (COX-2) expression is associated with more aggressive gliomas and poor survival. Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX activity and have antitumorigenic properties. In this report, our initial aim was to determine if indomethacin would alter gene expression as measured by suppression subtractive hybridization (SSH). Three up-regulated and four down-regulated genes by indomethacin treatment were identified. Laminin gamma1, an extracellular matrix molecule, was the most significantly repressed gene. The repression of laminin gamma1 by indomethacin was confirmed by Northern and Western blot analyses and occurred in a concentration- and time-dependent manner at the protein level. Among several NSAIDs tested, only sulindac sulfide and indomethacin suppressed laminin gamma1 protein expression, and this repression was observed in both COX-expressing and -deficient cell lines, suggesting that laminin gamma1 repression by COX inhibitors was independent of COX. Indomethacin, at a concentration that represses laminin gamma1, inhibited glioblastoma cell invasion that was partially restored with additional human laminin protein containing gamma1 chain. The repression of laminin gamma1 by NSAIDs may be related to attenuation of invasion of brain tumors. These findings are important in understanding the chemopreventive activity of some NSAIDs and could be relevant for designing therapeutic strategies against glioblastoma.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Characterization of an enzyme that is capable of processing pro-gonadotropin-releasing hormone protein

A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.     

The synaptic vesicle protein SV2 is complexed with an alpha5-containing laminin on the nerve terminal surface

Interactions between growing axons and synaptic basal lamina components direct the formation of neuromuscular junctions during nerve regeneration. Isoforms of laminin containing alpha5 or beta2 chains are potential basal lamina ligands for these interactions. The nerve terminal receptors are unknown. Here we show that SV2, a synaptic vesicle transmembrane proteoglycan, is complexed with a 900-kDa laminin on synaptosomes from the electric organ synapse that is similar to the neuromuscular junctions. Although two laminins are present on synaptosomes, only the 900-kDa laminin is associated with SV2. Other nerve terminal components are absent from this complex. The 900-kDa laminin contains an alpha5, a beta1, and a novel gamma chain. To test whether SV2 directly binds the 900-kDa laminin, we looked for interaction between purified SV2 and laminin-1, a laminin isoform with a similar structure. We find SV2 binds with high affinity to purified laminin-1. Our results suggest that a synaptic vesicle component may act as a laminin receptor on the presynaptic plasma membrane; they also suggest a mechanism for activity-dependent adhesion at the synapse.     

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.     

Primary structure of the mouse laminin B2 chain and comparison with laminin B1

One of the major components of basement membranes is the glycoprotein laminin, made up of three disulfide-bonded subunits, the A, B1, and B2 chains. We have isolated and sequenced overlapping mouse laminin B2 chain cDNA clones covering 7562 base pairs. The deduced amino acid sequence predicts that the mature B2 chain consists of 1572 residues, has an unglycosylated molecular weight of 173,541, and possesses 14 potential N-linked glycosylation sites. Analysis of the predicted secondary structure shows the presence of six domains, two rich in alpha-helical structure, two composed of homologous cysteine-rich repeat units, and two globular regions. The organization of the molecule is very similar to that of the mouse laminin B1 chain, and significant sequence homology between the B1 and B2 chains was found in their two cysteine-rich domains and in their amino-terminal globular domains.     

Cloning of laminin gamma2 cDNA and chromosome mapping of the genes for the dog adhesion ligand laminin 5

Overexpression of the gamma2 chain of laminin-5 has been linked to tumor invasion and an unfavorable prognostic value, but the role of this adhesion molecule in cancer progression remains unclear. Because dog models of human cancers provide the opportunity of clarifying the relation between laminin-5 and tumor malignancy we have isolated and characterized the cDNA of dog gamma2 chain. Comparative analysis of the nucleotide sequence revealed high identity between the dog and the human gamma2, including the intermolecular molecule binding sites and the regulatory promoter sequences. Moreover, expression of a recombinant human gamma2 chain in dog keratinocytes results in assembly and secretion of hybrid laminin-5 molecules, which underscore the functional relevance of the gamma2 conserved domains. We have also determined the syntenic location of the dog laminin-5 loci on CFA7. Our study provides a basis for therapeutical approaches of epithelial cancers of gamma2 using dogs as large animal models.     

Bone morphogenetic protein-1 processes insulin-like growth factor-binding protein 3

The bone morphogenetic protein-1 (BMP1)-like metalloproteinases play key roles in extracellular matrix formation, by converting precursors into mature functional proteins involved in forming the extracellular matrix. The BMP1-like proteinases also play roles in activating growth factors, such as BMP2/4, myostatin, growth differentiation factor 11, and transforming growth factor β1, by cleaving extracellular antagonists. The extracellular insulin-like growth factor-binding proteins (IGFBPs) are involved in regulating the effects of insulin-like growth factors (IGFs) on growth, development, and metabolism. Of the six IGFBPs, IGFBP3 has the greatest interaction with the large pool of circulating IGFs. It is also produced locally in tissues and is itself regulated by proteolytic processing. Here, we show that BMP1 cleaves human and mouse IGFBP3 at a single conserved site, resulting in markedly reduced ability of cleaved IGFBP3 to bind IGF-I or to block IGF-I-induced cell signaling. In contrast, such cleavage is shown to result in enhanced IGF-I-independent ability of cleaved IGFBP3 to block FGF-induced proliferation and to induce Smad phosphorylation. Consistent with in vivo roles for such cleavage, it is shown that, whereas wild type mouse embryo fibroblasts (MEFs) produce cleaved IGFBP3, MEFs doubly null for the Bmp1 gene and for the Tll1 gene, which encodes the related metalloproteinase mammalian Tolloid-like 1 (mTLL1), produce only unprocessed IGFBP3, thus demonstrating endogenous BMP1-related proteinases to be responsible for IGFBP3-processing activity in MEFs. Similarly, in zebrafish embryos, overexpression of Bmp1a is shown to reverse an Igfbp3-induced phenotype, consistent with the ability of BMP1-like proteinases to cleave IGFBP3 in vivo.     

Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia japonica

In order to clarify the function of the Djbmp (Dugesia japonica bone morphogenetic protein) gene in planarian body patterning, we carried out knockdown of this gene by RNA interference. When the planarians were treated with double-stranded RNA of Djbmp, a bulge formed on the dorsal side, with a dent in the middle of the bulge, and the body surface inside the dent was smoothened and less pigmented. In situ hybridization of the DjIFb gene, which is expressed in the body margin, revealed that the additional body margin was formed ectopically at the region surrounding the dent. The Djbmp-knockdown planarians often had a pair of incomplete nerve cords in the dorsal side, in addition to the original pair of ventral nerve cords. Taken together, we concluded that the Djbmp-knockdown induced formation of an ectopic ventral side, suggesting that Djbmp is required for the dorso-ventral body patterning in the planarian.