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PubMed2002

ADAM 12 protease induces adipogenesis in transgenic mice.

ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane-anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did not exhibit increased serum insulin, cholesterol, or triglycerides. Male transgenics were slightly overweight and also developed herniation but did not become obese. Transgenic mice expressing a truncated form of ADAM 12-S lacking the prodomain and the metalloprotease domain did not develop this adipogenic phenotype, suggesting a requirement for ADAM 12 protease activity. This is the first in vivo demonstration that an ADAM protease is involved in adipogenesis.

ADAM Proteins↗
PubMed1999

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.

ADAM Proteins↗
PubMed2001

Activation of ADAM 12 protease by copper.

Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism for ADAM 12 involving both furin cleavage and copper binding.

ADAM Proteins↗
PubMed2000

Analysis for transcript expression of meltrin alpha in normal, regenerating, and denervated rat muscle.

Meltrin alpha (a disintegrin and metalloprotease (ADAM) 12) is a recently discovered molecule of the metalloprotease-disintegrin family which has been shown to participate in myotube formation in vitro and in myogenesis in vivo. In this study we investigated meltrin alpha in regenerating rat muscle, which is a condition where satellite cells (SC) contribute to myofiber growth by fusing with one another and with myotubes or muscle fibers. We studied meltrin alpha mRNA expression by RT-PCR and in situ-hybridization in normal adult muscle, in soleus muscle regenerating for 2, 5, or 10 days, and in muscle which had been denervated 1 week, 4 weeks, or 6 months previously. SC do not fuse after denervation. They detach from the principal muscle fiber. Immunohistochemistry using an antibody against M-cadherin was performed in parallel in order to identify SC. Messenger RNA as revealed by RT-PCR was absent in normal adult muscle, but present in regenerating and also in denervated muscle. Meltrin alpha transcript detected by in situ-hybridization was present in regenerating muscle only, not in normal or denervated muscle. It was localized to SC. Taken together, meltrin alpha is absent in normal muscle, and localized to SC in fusing conditions. After denervation, the transcript is upregulated. However, it is so lowly abundant that it fails to be detected by in situ-hybridization. This expression profile suggests a role for meltrin alpha in the fusion of SC with myotubes or muscle fibers, but not in SC adhesion to the adjacent myofiber in normal adult muscle.

ADAM Proteins↗
PubMed1995

A metalloprotease-disintegrin participating in myoblast fusion.

Skeletal muscle development involves the formation of multi-nucleated myotubes. This is thought to proceed by the induction of differentiation (acquisition of fusion competence) of myoblast cells, their aggregation, and union of their plasma membranes. Various membrane proteins including N- and M-cadherins, N- and V-CAMs and integrins participate in myotube formation, but the molecular mechanisms of muscle cell fusion are poorly understood. Here we report the identification of three new, myoblast-expressed gene products, meltrin-alpha, beta and gamma, with homology to both viper haemorrhagic factors and fertilin (PH-30), a membrane protein involved in egg-sperm fusion. Meltrin-alpha, a member of the metalloproteinase/disintegrin protein family, appears to be required for myotube formation. Involvement of a fertilin-related protein in myogenesis suggests that there are common mechanisms in gamete and myoblast fusion.

ADAM Proteins↗
PubMed2006

Expression of a disintegrin and metalloprotease (ADAM and ADAMTS) enzymes in human non-small-cell lung carcinomas (NSCLC).

A Disintegrin and Metalloprotease (ADAM) are transmembrane proteases displaying multiple functions. ADAM with ThromboSpondin-like motifs (ADAMTS) are secreted proteases characterised by thrombospondin (TS) motifs in their C-terminal domain. The aim of this work was to evaluate the expression pattern of ADAMs and ADAMTS in non small cell lung carcinomas (NSCLC) and to investigate the possible correlation between their expression and cancer progression. Reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemical analyses were performed on NSCLC samples and corresponding nondiseased tissue fragments. Among the ADAMs evaluated (ADAM-8, -9, -10, -12, -15, -17, ADAMTS-1, TS-2 and TS-12), a modulation of ADAM-12 and ADAMTS-1 mRNA expression was observed. Amounts of ADAM-12 mRNA transcripts were increased in tumour tissues as compared to the corresponding controls. In sharp contrast, ADAMTS-1 mRNA levels were significantly lower in tumour tissues when compared to corresponding nondiseased lung. These results were corroborated at the protein level by Western blot and immunohistochemistry. A positive correlation was observed between the mRNA levels of ADAM-12 and those of two vascular endothelial growth factor (VEGF)-A isoforms (VEGF-A(165) and VEGF-A(121)). Taken together, these results providing evidence for an overexpression of ADAM-12 and a lower expression of ADAMTS-1 in non-small-cell lung cancer suggest that these proteases play different functions in cancer progression.

ADAM Proteins↗
PubMed2000

Meltrin alpha cytoplasmic domain interacts with SH3 domains of Src and Grb2 and is phosphorylated by v-Src.

Meltrin alpha/ADAM12 is a member of the ADAM/MDC family proteins characterized by the presence of metalloprotease and disintegrin domains. This protein also contains a single transmembrane domain and a relatively long cytoplasmic domain containing several proline-rich sequences. These sequences are compatible with the consensus sequences for binding the Src homology 3 (SH3) domains. To determine whether the proline-rich sequences interact with SH3 domains in several proteins, binding of recombinant SH3 domains to the meltrin alpha cytoplasmic domain was analysed by pull-down assays. The SH3 domains of Src and Yes bound strongly, but that of Abl or phosphatidylinositol 3-kinase p85 subunit did not. Full-length Grb2/Ash bound strongly, whereas its N-terminal SH3 domain alone did less strongly. Src and Grb2 in bovine brain extracts also bound to meltrin alpha cytoplasmic domain on affinity resin. Furthermore, immunoprecipitation with a monoclonal antibody to meltrin alpha resulted in coprecipitation of Src and Grb2 with meltrin alpha in cell extracts, suggesting that Src and Grb2 are associated in vivo with meltrin alpha cytoplasmic domain. This notion was also supported by the findings that exogenously expressed meltrin cytoplasmic domain coexisted with Src and Grb2 on the membrane ruffles. The C-terminal Tyr901 of meltrin alpha was phosphorylated both in vitro and in cultured cells by v-Src. These results may imply that meltrin alpha cytoplasmic domain is involved in a signal transduction for some biological function through the interaction with SH3-containing proteins.

ADAM Proteins↗
PubMed2001

Metalloprotease-disintegrin ADAM 12 interacts with alpha-actinin-1.

ADAM 12, a member of the ADAM family of proteins (containing A Disintegrin And Metalloprotease domain), has been implicated in differentiation and fusion of myoblasts. While the extracellular domain of ADAM 12 contains an active metalloprotease and a region involved in cell adhesion, the function of the cytoplasmic tail of ADAM 12 has been less clear. Here we show that the cytoplasmic domain of ADAM 12 interacts in vitro and in vivo with alpha-actinin-1, an actin-binding and cross-linking protein. Green fluorescent protein fused to ADAM 12 cytoplasmic domain co-localizes with alpha-actinin-1-containing actin stress fibres in C2C12 cells. The interaction between ADAM 12 and alpha-actinin-1 is direct and involves the 58-amino acid C-terminal fragment of ADAM 12 and the 27 kDa N-terminal domain of alpha-actinin-1. Consistently, expression of the 27 kDa fragment of alpha-actinin-1 in C2C12 cells using a mitochondrial targeting system results in recruitment of the co-expressed ADAM 12 cytoplasmic domain to the mitochondrial surface. Moreover, alpha-actinin-1 co-purifies with a transmembrane, His6-tagged form of ADAM 12 expressed in C2C12 myoblasts, indicating that the transmembrane ADAM 12 forms a complex with alpha-actinin-1 in vivo. These results indicate that the actin cytoskeleton may play a critical role in ADAM 12-mediated cell-cell adhesion or cell signalling during myoblast differentiation and fusion.

ADAM Proteins↗
PubMed2005

Abundance of ADAM-8, -9, -10, -12, -15 and -17 and ADAMTS-1 in mouse uterus during the oestrous cycle.

The aim of the present study was to determine whether a disintegrin and metalloproteinase (ADAM)-8, -9, -10, -12, -15 and -17 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1 are involved in the remodelling process of the mouse uterus during the oestrous cycle. The mRNA expression of ADAM was observed in all uterine tissues throughout the entire cycle. The levels of ADAM-8 mRNA were maximal at pro-oestrus, whereas the expression of ADAM-9 and ADAMTS-1 mRNA was maximal at oestrus. The minimum mRNA level of all ADAM genes always occurred at dioestrus. The mRNA levels of ADAM-10, -12, -15 and -17 did not vary significantly, regardless of the stage of the oestrous cycle. Immunoblot analyses demonstrated the presence of all ADAM proteins throughout the cycle. In terms of protein intensities, ADAM-8, -12 and -17 were maximal at pro-oestrus, whereas ADAM-10 and ADAMTS-1 were maximal at metoestrus and ADAM-9 was maximal at oestrus. Regardless of the ADAM species, minimal protein expression always occurred at dioestrus. Immunohistochemical studies showed ADAM protein expression in luminal and glandular epithelial layers, but not in the stromal layer. Moreover, ADAM proteins were found to be heterogeneously localised and their individual localisations depended on the stage of the oestrous cycle. From these observations, we suggest that the ADAM genes play an important role in mouse uterine tissue remodelling during the oestrous cycle.

ADAM Proteins↗
PubMed1998

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.

ADAM Proteins↗
PubMed1998

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.

ADAM Proteins↗
PubMed1998

Cloning and initial characterization of mouse meltrin beta and analysis of the expression of four metalloprotease-disintegrins in bone cells.

Here we report the cloning and initial biochemical characterization of the mouse metalloprotease/disintegrin/cysteine-rich (MDC) protein meltrin beta and the analysis of the mRNA expression of four MDC genes (meltrin alpha, meltrin beta, mdc9, and mdc15) in bone cells, including osteoclasts and osteoblasts. Like most other MDC proteins, the predicted meltrin beta protein consists of a signal sequence, prodomain, metalloprotease domain with a predicted catalytic site, disintegrin domain, cysteine-rich region, epidermal growth factor repeat, transmembrane domain, and cytoplasmic domain with putative signaling motifs, such as potential SH3 ligand domains. Northern blot analysis indicates that meltrin beta is widely expressed, with the highest expression in bone, heart, and lung. RNase protection studies revealed expression of all four MDC genes analyzed here in osteoblasts, whereas only mdc9 and mdc15 mRNAs were detectable in osteoclast-like cells generated in vitro. Treatment of primary osteoblasts with 10 nM calcitriol increased meltrin beta expression more than 3-fold, and both meltrin alpha and meltrin beta expression is apparently regulated in a differentiation-associated manner in a mouse osteoblastic cell line, MC3T3E1. Collectively, these results suggest that meltrin alpha and meltrin beta may play a role in osteoblast differentiation and/or function but are not likely to be involved in osteoclast fusion.

ADAM Proteins↗
PubMed1999

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.

ADAM Proteins↗
PubMed2000

RGD-independent binding of integrin alpha9beta1 to the ADAM-12 and -15 disintegrin domains mediates cell-cell interaction.

ADAMs (a disintegrin and metalloproteases) mediate several important processes (e.g. tumor necrosis factor-alpha release, fertilization, and myoblast fusion). The ADAM disintegrin domains generally lack RGD motifs, and their receptors are virtually unknown. Here we show that integrin alpha(9)beta(1) specifically interacts with the recombinant ADAMs-12 and -15 disintegrin domains in an RGD-independent manner. We also show that interaction between ADAM-12 or -15 and alpha(9)beta(1) supports cell-cell interaction. Interestingly, the cation requirement and integrin activation status required for alpha(9)beta(1)/ADAM-mediated cell adhesion and cell-cell interaction is similar to those required for known integrin-extracellular matrix interaction. These results are quite different from recent reports that ADAM-2/alpha(6)beta(1) interaction during sperm/egg fusion requires an integrin activation status distinct from that for extracellular matrix interaction. These results suggest that alpha(9)beta(1) may be a major receptor for ADAMs that lack RGD motifs, and that, considering a wide distribution of ADAMs and alpha(9)beta(1), this interaction may be of potential biological and pathological significance.

ADAM Proteins↗
PubMed2000

ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growth factor-binding protein-3.

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may regulate insulin-like growth factor bioavailability. IGFBP-3 is extensively degraded in serum during pregnancy; however, as yet the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloprotease-12 (ADAM 12), a member of a family of metalloprotease disintegrins that is highly expressed in placental tissue, was identified as interacting with IGFBP-3. This interaction involved the cysteine-rich domain of ADAM 12. Unlike other members of this family of disintegrin metalloproteases that are membrane proteins, ADAM 12 exists as an alternatively spliced soluble secreted protein. To verify the interaction between ADAM 12 and IGFBP-3, an expression construct containing an ADAM 12-S cDNA was transfected into COS-1 cells. Co-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 followed by Western blotting with anti-IGFBP-3 or anti-ADAM 12. Although minimal proteolysis of IGFBP-3 was observed in conditioned medium from control cells, this was increased approximately 4-fold in conditioned medium from ADAM 12-S-transfected cells. Recombinant ADAM 12-S partially purified from conditioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The degradation pattern was similar to that seen with pregnancy serum, and the presence of ADAM 12-S in serum during pregnancy was confirmed. The data suggest that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to the IGFBP-3 protease activity present in pregnancy serum.

ADAM Proteins↗