Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity.

Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.

Tetranectin in slow intra- and extrafusal chicken muscle fibers.

Tetranectin is a C-type lectin that occurs in the mammalian musculoskeletal system. In the present report we describe the first studies on an avian tetranectin. A full-length chicken tetranectin cDNA was isolated. Comparison of the deduced amino acid sequence of chicken tetranectin with mouse and human tetranectin showed an identity of 67 and 68%, respectively. Northern blot analysis demonstrated broad expression of chicken tetranectin mRNA, which was first detected on embryonic day 4. Tetranectin protein was detected in chicken serum and egg yolk. Since muscle is one of few tissues in which tetranectin protein is retained, we examined the distribution of tetranectin in various muscle types in chicken. Myofibers strongly positive for tetranectin were observed in several muscles including m. tibialis ant. and m. sartorius (from embryonic day 10 to adult). Using antibodies to fast and slow myosin heavy chains (MHC) and double immunostaining techniques, we found that tetranectin was restricted to slow (type I) muscle fibers. Similarly only slow intrafusal fibers accumulated tetranectin. The pattern of immunostaining in chickens differs markedly from that seen in mouse muscles, indicating that tetranectin performs a role in muscle that is not associated with a hitherto recognized muscle type or function.

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.

ADAM 12-S cleaves IGFBP-3 and IGFBP-5 and is inhibited by TIMP-3.

ADAMs are a family of multidomain proteins having proteolytic and cell adhesion activities. We have previously shown that ADAM 12-S, the secreted soluble form of human ADAM 12, is a catalytically active protease. We now describe the purification of full-length recombinant ADAM 12-S and demonstrate that it cleaves insulin-like growth factor binding protein-3 (IGFBP-3). This result supports a role for ADAM 12-S in the degradation of IGFBP-3 in the blood of pregnant women. Furthermore, we tested for proteolysis of other members of the IGF binding protein family and found that ADAM 12-S cleaves IGFBP-5 in addition to IGFBP-3, but does not cleave IGFBP-1, -2, -4, or -6. ADAM 12-S may therefore be the IGFBP-5 protease that is secreted by osteoblasts and other cells. Cleavage of both IGFBP-3 and -5 by ADAM 12-S was inhibited by TIMP-3, raising the possibility that TIMP-3 is a physiological inhibitor of ADAM 12-S.

Trafficking of human ADAM 12-L: retention in the trans-Golgi network.

We have investigated the trafficking of the membrane-anchored form of human ADAM 12 (ADAM 12-L) fused to a green fluorescence protein tag. Subcellular localization of the protein in transiently transfected cells was determined by fluorescence microscopy and trypsin sensitivity. Full-length ADAM 12-L was retained in a perinuclear compartment, which was shown to be the trans-Golgi network. In contrast, ADAM 12-L lacking the cytoplasmic domain reached the cell surface. Based on analysis of deletions and mutations of the cytoplasmic tail of ADAM 12-L, the retention signal is comprised of both the cytoplasmic and transmembrane domains, but not the Src homology 3 domain (SH3) binding sites. These results raise the possibility that a trafficking checkpoint in the trans-Golgi network is one of the cellular mechanisms for regulation of ADAM 12-L function, by allowing a rapid release of ADAM 12-L to the cell surface under specific stimuli.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS).

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).

Regulation of human ADAM 12 protease by the prodomain. Evidence for a functional cysteine switch.

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins that are believed to play key roles in cell-cell and cell-matrix interactions. We have shown recently that human ADAM 12-S (meltrin alpha) is an active metalloprotease. It is synthesized as a zymogen, with the prodomain maintaining the protease in a latent form. We now provide evidence that the latency mechanism of ADAM 12 can be explained by the cysteine switch model, in which coordination of Zn2+ in the active site of the catalytic domain by a cysteine residue in the prodomain is critical for inhibition of the protease. Replacing Cys179 with other amino acids results in an ADAM 12 proform that is proteolytically active, but latency can be restored by placing cysteine at other positions in the propeptide. None of the amino acids adjacent to the crucial cysteine residue is essential for blocking activity of the protease domain. In addition to its latency function, the prodomain is required for exit of ADAM 12 protease from the endoplasmic reticulum. Tissue inhibitor of metalloprotease-1, -2, and -3 were not found to block proteolytic activity of ADAM 12, hence a physiological inhibitor of ADAM 12 protease in the extracellular environment remains to be identified.

Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion.

The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor cell membranes. Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion. Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion of a panel of carcinoma cell lines. On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate proteoglycan to attach. Finally, we demonstrated that authentic full-length human ADAM 12 could bind to heparin Sepharose. Together these results suggest a novel role of the cysteine-rich domain of ADAM 12 -- that of supporting tumor cell adhesion.

Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation.

The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus. These results exclude Lama5 as a candidate gene for the Ra mutation.

Tetranectin is a novel marker for myogenesis during embryonic development, muscle regeneration, and muscle cell differentiation in vitro.

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.

Human ADAM 12 (meltrin alpha) is an active metalloprotease.

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.

A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo.

The ADAM (A Disintegrin And Metalloprotease) family of cell-surface proteins may have an important role in cellular interactions and in modulating cellular responses. In this report we describe a novel, secreted form of human ADAM 12 (meltrin alpha), designated ADAM 12-S (S for short), and a larger, membrane-bound form designated ADAM 12-L (L for long form). These two forms arise by alternative splicing of a single gene located on chromosome 10q26. Northern blotting demonstrated that mRNAs of both forms are abundant in human term placenta and are also present in some tumor cell lines. The ADAM 12-L transcript can also be detected in normal human adult skeletal, cardiac, and smooth muscle. Human A204 embryonal rhabdomyosarcoma cells that do not differentiate into muscle cells and do not express any form of ADAM 12 were stably transfected with an ADAM 12-S minigene encoding the disintegrin domain, the cysteine-rich domain, and the unique 34 amino acid carboxyl terminus. Nude mouse tumors derived from these transfected cells contained ectopic muscle cells of apparent mouse origin as shown by species-specific markers. These results may have potential applications in the development of muscle-directed gene and cell therapies.

The integrin alpha 6 beta 1 promotes the survival of metastatic human breast carcinoma cells in mice.

The role of the integrin alpha 6 beta 1 in breast carcinoma progression was studied by targeted elimination of this integrin in MDA-MB-435 cells, a human breast carcinoma cell line that is highly metastatic in athymic mice. The strategy used is based on the finding that expression of a cytoplasmic domain deletion mutant of the beta 4-integrin subunit (beta 4-delta CYT) in MDA-MB-435 cells eliminates formation of the alpha 6 beta 1 heterodimer. MDA-MB-435 cells that lacked alpha 6 beta 1 expression (beta 4-delta CYT transfectants) formed tumors in athymic mice that were suppressed in their growth and that exhibited a significant increase in apoptosis in comparison to the control tumors. Unlike the control MDA-MB-435 cells, the beta 4-delta CYT transfectants were unable to establish metastatic foci in the lungs. Also, the control transfectants grew substantially better than the beta 4-delta CYT transfectants in the liver after intrahepatic injection because of extensive apoptosis in the beta 4-delta CYT transfectants. These data suggest that a major function of the alpha 6 beta 1 integrin in breast carcinoma is to facilitate tumorigenesis and promote tumor cell survival in distant organs.

Extrasynaptic location of laminin beta 2 chain in developing and adult human skeletal muscle.

We have investigated the distribution of the laminin beta 2 chain (previously s-laminin) in human fetal and adult skeletal muscle and compared it to the distribution of laminin beta 1. Immunoblotting and transfection assays were used to characterize a panel of monoclonal and polyclonal antibodies to the laminin beta 2 chain. We found that laminin beta 1 chain was detected at all times during development from 10 weeks of gestation. Laminin beta 2 chain was first detected in 15 to 22-week-old fetal skeletal muscle as distinct focal immunoreactivity in the sarcolemmal basement membrane area of some myofibers. In the adult skeletal muscle, laminin beta 2 chain immunoreactivity was found along the entire perimeter of each of the individual myofibers in a large series of different muscles studied. Laminin beta 2 chain was similarly found in the skeletal muscle basement membranes in patients with Duchenne and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present results demonstrate a prominent extrasynaptic localization of laminin beta 2 in the human muscle, suggesting that it may have an important function in the sarcolemmal basement membrane.

Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation.

To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.

Assignment of the gene for human tetranectin (TNA) to chromosome 3p22-->p21.3 by somatic cell hybrid mapping.

Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human connective tissue disorders that map to the short arm of chromosome 3, MFS2 and LRS1.

Structural organization of the human and mouse laminin beta2 chain genes, and alternative splicing at the 5' end of the human transcript.

We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span

Laminin beta 2 chain and adhalin deficiency in the skeletal muscle of Walker-Warburg syndrome (cerebro-ocular dysplasia-muscular dystrophy).

Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin beta 2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.