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Integrins, a family of heterodimeric adhesion receptors are implicated in cell migration, development and cancer progression. They can adopt conformations that reflect their activation states and thereby impact adhesion strength and migration. Integrins in an intermediate activation state may be optimal for migration and we have shown previously that fully activated integrin α9β1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9β1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9β1-specific ligands led to upregulation of fibronectin matrix assembly and tyrosine phosphorylation of cortactin on tyrosine 470 (Y470). Specifically, cortactin phosphorylated on Y470, but not Y421, redistributed together with α9β1 to focal adhesions where active β1 integrin also localises, upon integrin activation. This was commensurate with reduced migration. The localisation and phosphorylation of cortactin Y470 was regulated by Yes kinase and PTEN phosphatase. Cortactin levels influenced fibronectin matrix assembly and active β1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9β1 integrin that regulates cell-extracellular matrix interactions.

Matrix metalloproteinases (MMPs), in particular MMP-2, MMP-9 and MMP-14, play a key role in various aspects of cancer pathology. Likewise, ADAMs (a disintegrin and metalloproteinases), including ADAM12, are upregulated in malignant tumors and contribute to the pathology of cancers. Here, we show that there is a positive correlation between MMP-14 and ADAM12 expression in human breast cancer. We demonstrated that in 293-VnR and human breast cancer cells expressing ADAM12 at the cell surface, endogenous MMP-14 was recruited to the cell surface, resulting in its activation. Subsequent to this activation, gelatin degradation was stimulated and tumor cell apoptosis was decreased, with reduced expression of the pro-apoptotic proteins BCL2L11 and BIK. The effect on gelatin degradation was abrogated by inhibition of the MMP-14 activity and appeared to be dependent on cell surface αVβ3 integrin localization, but neither the catalytic activity of ADAM12 nor the cytoplasmic tail of ADAM12 were required. The significance of ADAM12-induced activation of MMP-14 was underscored by a reduction in MMP-14-mediated gelatin degradation and abolition of apoptosis-protective effects by specific monoclonal antibodies against ADAM12. Furthermore, orthotopic implantation of ADAM12-expressing MCF7 cells in nude mice produced tumors with increased levels of activated MMP-14 and confirmed that ADAM12 protects tumor cells against apoptosis, leading to increased tumor progression. In conclusion, our data suggest that a ternary protein complex composed of ADAM12, αVβ3 integrin and MMP-14 at the tumor cell surface regulates the function of MMP-14. This interaction might point to a novel concept for the development of MMP-14-targeting drugs in treating cancer.

ADAM (a disintegrin and metalloproteinase) 12 is a metalloprotease implicated in cancer progression. ADAM12 can activate membrane-anchored proteins, such as sonic hedgehog, Delta-like 1 and certain epidermal growth factor receptor ligands, through a process called ectodomain shedding. We screened several membrane-anchored proteins to further dissect the substrate profile of ADAM12-mediated ectodomain shedding, and found shedding of five previously unreported substrates [Kitl1, VE-cadherin (vascular endothelial cadherin), Flk-1 (fetal liver kinase 1), Tie-2, and VCAM-1 (vascular cell adhesion molecule 1)], of which the latter four are specifically expressed by endothelial cells. We also observed that ADAM12 expression was increased in the tumour vasculature of infiltrating ductal carcinoma of the human breast as compared with little to no expression in normal breast tissue vasculature, suggesting a role for ADAM12 in tumour vessels. These results prompted us to further evaluate ADAM12-mediated shedding of two endothelial cell proteins, VE-cadherin and Tie-2. Endogenous ADAM12 expression was very low in cultured endothelial cells, but was significantly increased by cytokine stimulation. In parallel, the shed form of VE-cadherin was elevated in such cytokine-stimulated endothelial cells, and ADAM12 siRNA (small interfering RNA) knockdown reduced cytokine-induced shedding of VE-cadherin. In conclusion, the results of the present study demonstrate a role for ADAM12 in ectodomain shedding of several membrane-anchored endothelial proteins. We speculate that this process may have importance in tumour neovascularization or/and tumour cell extravasation.

Cancer-associated changes in cellular behavior, such as modified cell-cell contact, increased migratory potential, and generation of cellular force, all require alteration of the cytoskeleton. Two homologous mammalian serine/threonine kinases, Rho-associated protein kinases (ROCK I and II), are key regulators of the actin cytoskeleton acting downstream of the small GTPase Rho. ROCK is associated with cancer progression, and ROCK protein expression is elevated in several types of cancer. ROCKs exist in a closed, inactive conformation under quiescent conditions, which is changed to an open, active conformation by the direct binding of guanosine triphosphate (GTP)-loaded Rho. In recent years, a number of ROCK isoform-specific binding partners have been found to modulate the kinase activity through direct interactions with the catalytic domain or via altered cellular localization of the kinases. Thus, these findings demonstrate additional modes to regulate ROCK activity. This review describes the molecular mechanisms of ROCK activity regulation in cancer, with emphasis on ROCK isoform-specific regulation and interaction partners, and discusses the potential of ROCKs as therapeutic targets in cancer.

Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, α9β1 integrin is one of the least studied. α9, together with α4, comprise a more recent evolutionary sub-family of integrins that is only found in vertebrates. Since α9 was thought to have similar functions as α4, due to many shared ligands, it was a rather overlooked integrin until recently, when its importance for survival after birth was highlighted upon investigation of the α9 knockout mouse. α9β1 is expressed on a wide variety of cell types, interacts with many ligands for example fibronectin, tenascin-C and ADAM12, and has been shown to have important functions in processes such as cell adhesion and migration, lung development, lymphatic and venous valve development, and in wound healing. This has sparked an interest to investigate α9β1-mediated signaling and its regulation. This review gives an overview of the recent progress in α9β1-mediated biological and pathological processes, and discusses its potential as a target for cancer diagnosis and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Collapsin response mediator protein 2 (CRMP-2) is known as a regulator of neuronal polarity and differentiation through microtubule assembly and trafficking. Here, we show that CRMP-2 is ubiquitously expressed and a splice variant (CRMP-2L), which is expressed mainly in epithelial cells among nonneuronal cells, regulates myosin II-mediated cellular functions, including cell migration. While the CRMP-2 short form (CRMP-2S) is recognized as a substrate of the Rho-GTP downstream kinase ROCK in neuronal cells, a CRMP-2 complex containing 2L not only bound the catalytic domain of ROCK II through two binding domains but also trapped and inhibited the kinase. CRMP-2L protein levels profoundly affected haptotactic migration and the actin-myosin cytoskeleton of carcinoma cells as well as nontransformed epithelial cell migration in a ROCK activity-dependent manner. Moreover, the ectopic expression of CRMP-2L but not -2S inhibited fibronectin matrix assembly in fibroblasts. Underlying these responses, CRMP-2L regulated the kinase activity of ROCK II but not ROCK I, independent of GTP-RhoA levels. This study provides a new insight into CRMP-2 as a controller of myosin II-mediated cellular functions through the inhibition of ROCK II in nonneuronal cells.

A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer.

Expression of ADAM12 is low in most normal tissues but is markedly increased in numerous human cancers, including breast carcinomas. We have previously shown that overexpression of ADAM12 accelerates tumor progression in a mouse model of breast cancer (PyMT). In this study, we found that ADAM12 deficiency reduces breast tumor progression in the PyMT model. However, the catalytic activity of ADAM12 seems to be dispensable for its tumor-promoting effect. Interestingly, we show that ADAM12 endogenously expressed in tumor-associated stroma in the PyMT model does not influence tumor progression, but that ADAM12 expression by tumor cells is necessary for tumor progression in these mice. This finding is consistent with our observation that in human breast carcinoma, ADAM12 is almost exclusively located in tumor cells and, only rarely, seen in the tumor-associated stroma. We hypothesized, however, that the tumor-associated stroma may stimulate ADAM12 expression in tumor cells, on the basis of the fact that TGF-β1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-β1 stimulation of ADAM12-negative Lewis lung tumor cells induced ADAM12 synthesis, and growth of these cells in vivo induced more than 200-fold increase in ADAM12 expression. Our observation that ADAM12 expression is significantly higher in the terminal duct lobular units (TDLU) adjacent to human breast carcinoma compared with TDLUs found in normal breast tissue supports our hypothesis that tumor-associated stroma triggers ADAM12 expression., (Mol Cancer Res; 9(11); 1449-61. ©2011 AACR.)

Invadopodia are dynamic actin structures at the cell surface that degrade extracellular matrix and act as sites of signal transduction. The biogenesis of invadopodia, including the mechanisms regulating their formation, composition, and turnover is not entirely understood. Here, we demonstrate that antibody ligation of ADAM12, a transmembrane disintegrin and metalloprotease, resulted in the rapid accumulation of invadopodia with extracellular matrix-degrading capacity in epithelial cells expressing the αvβ3 integrin and active c-Src kinase. The induction of invadopodia clusters required an intact c-Src interaction site in the ADAM12 cytoplasmic domain, but was independent of the catalytic activity of ADAM12. Caveolin-1 and transmembrane protease MMP14/MT1-MMP were both present in the ADAM12-induced clusters of invadopodia, and cholesterol depletion prevented their formation, suggesting that lipid-raft microdomains are involved in the process. Importantly, our data demonstrate that ADAM12-mediated ectodomain shedding of epidermal growth factor receptor ligands can occur within these invadopodia. Such localized growth factor signalling offers an interesting novel biological concept highly relevant to the properties of carcinoma cells, which often show upregulated ADAM12 and β3 integrin expression, together with high levels of c-Src kinase activity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.

ADAM12 is an active metalloprotease playing an important role in tumour progression. Human ADAM12 exists in two splice variants: a long transmembrane form, ADAM12-L, and a secreted form, ADAM12-S. The subcellular localization of ADAM12-L is tightly regulated and involves intracellular interaction partners and signalling proteins. We demonstrate here a c-Src-dependent redistribution of ADAM12-L from perinuclear areas to actin-rich Src-positive structures at the cell periphery, and identified two separate c-Src binding sites in the cytoplasmic tail of ADAM12-L that interact with the SH3 domain of c-Src with different binding affinities. The association between ADAM12-L and c-Src is transient, but greatly stabilized when the c-Src kinase activity is disrupted. In agreement with this observation, kinase-active forms of c-Src induce ADAM12-L tyrosine phosphorylation. Interestingly, ADAM12-L was also found to enhance Src kinase activity in response to external signals, such as integrin engagement. Thus, we suggest that activated c-Src binds, phosphorylates, and redistributes ADAM12-L to specific sites at the cell periphery, which may in turn promote signalling mechanisms regulating cellular processes with importance in cancer.

Cell surface integrins are the primary receptors for cell migration on extracellular matrix, and exist in several activation states regulated in part by ectodomain conformation. The alpha9 integrin subunit, which pairs only with beta1, has specific roles in the immune system and may regulate cell migration. Melanoma cells express abundant alpha9beta1 integrin, and its role in cell migration was assessed. Ligands derived from Tenascin-C and ADAM12 supported alpha9beta1 integrin-mediated cell attachment and GTP-Rac dependent migration, but not focal adhesion formation. Manganese ions induced alpha9beta1 integrin- and Rho kinase-dependent focal adhesion and stress fibre formation, suggesting that the activation status of alpha9beta1 integrin was altered. The effect of manganese ions in promoting focal adhesion formation was reproduced by beta1 integrin activating antibody. The alpha9beta1 integrin translocated to focal adhesions, where active beta1 integrin was also detected by conformation-specific antibodies. Focal adhesion assembly was commensurate with reduced cell migration. Endogenous alpha9beta1 integrin-mediated adhesion was sensitive to the PP1 chemical inhibitor and an inhibitor of endosomal vesicle recycling, but not inhibitors of protein kinase C or the small GTPase Rho. Our results demonstrated that although alpha9beta1 integrin can induce and localise to focal adhesions in a high activation state, its intermediate activity state normally supports cell adhesion consistent with migration.

ADAM12/meltrin alpha is a type I transmembrane multidomain protein involved in tumor progression and other severe diseases, including osteoarthritis, and as such could be considered as a potential drug target. In addition to protease activity, ADAM12 possesses cell binding and cell signaling properties. This functional trinity is reflected in the structure of ADAM12, which can be divided into head, body, and tail. The head of the protein (consisting of the pro and catalytic domains) mediates processing of growth factors and cytokines and has been implicated in epidermal growth factor (EGF) and insulin-like growth factor receptor signaling. The body of the protein (consisting of the disintegrin, cysteine-rich, and EGF-like domains) is involved in contacts with the extracellular matrix and other cells through interactions with integrins and syndecans. Finally, the tail of the protein (consisting of the cytoplasmic domain) is engaged in interactions with intracellular signaling molecules. In many studies, ADAM12 overexpression has been correlated with disease, and ADAM12 has been shown to promote tumor growth and progression in cancer. On the other hand, protective effects of ADAM12 in disease have also been reported. Future investigations should address the precise mechanisms of ADAM12 in disease and biology in order to counterbalance the benefits from targeting ADAM12 therapeutically with possible side effects. This review describes the biology of ADAM12, its association with disease, and evaluates the possible approaches to targeting ADAM12 in human disease.

The disintegrin and metalloproteases (ADAMs) are emerging as therapeutic targets in human disease, but specific drug design is hampered by potential redundancy. Unlike other metzincins, ADAM prodomains remain bound to the mature enzyme to regulate activity. Here ADAM12, a protease that promotes tumor progression and chondrocyte proliferation in osteoarthritic cartilage, is shown to possess a prodomain/catalytic domain cationic molecular switch, regulated by exogenous heparan sulfate and heparin but also endogenous cell surface proteoglycans and the polyanion, calcium pentosan polysulfate. Sheddase functions of ADAM12 are regulated by the switch, as are proteolytic functions in placental tissue and sera of pregnant women. Moreover, human heparanase, an enzyme also linked to tumorigenesis, can promote ADAM12 sheddase activity at the cell surface through cleavage of the inhibitory heparan sulfate. These data present a novel concept that might allow targeting of ADAM12 and suggest that other ADAMs may have specific regulatory activity embedded in their prodomain and catalytic domain structures.

The ADAMs (a disintegrin and metalloproteases) are an important class of enzymes in the regulation of human disease. The pro domains of ADAMs are responsible for the latency and secretion of mature enzymes. Unlike other metzincins, ADAM pro domains remain bound to the mature enzyme after secretion. To understand the functions of human ADAM pro domains and to determine three-dimensional structures, we have screened promising targets for expression and purification properties when using Escherichia coli as the host. The pro domain of ADAM22 (ADAM22-P) expressed in E. coli was folded, as determined by CD and NMR spectroscopy. An ADAM22-P fragment encoding residues 26-199 could be expressed in high amounts, remained soluble above 1 mM, and was suitable for structural studies by NMR spectroscopy. CD spectroscopy and predictions suggest that the secondary structure in ADAM22-P consists of beta-strands. Furthermore, our data indicate that the pro domains of ADAMs are expressed as two subdomains. The most N-terminal subdomain (ADAM22-P(N)) was found to be susceptible to proteolysis and was required for folding stability of the second subdomain (ADAM22-P(C)).

Human ADAM12 (a disintegrin and metalloproteinase) is a multidomain zinc metalloproteinase expressed at high levels during development and in human tumors. ADAM12 exists as two splice variants: a classical type 1 membrane-anchored form (ADAM12-L) and a secreted splice variant (ADAM12-S) consisting of pro, catalytic, disintegrin, cysteine-rich, and EGF domains. Here we present a novel activity of recombinant ADAM12-S and its domain deletion mutants on S-carboxymethylated transferrin (Cm-Tf). Cleavage of Cm-Tf occurred at multiple sites, and N-terminal sequencing showed that the enzyme exhibits restricted specificity but a consensus sequence could not be defined as its subsite requirements are promiscuous. Kinetic analysis revealed that the noncatalytic C-terminal domains are important regulators of Cm-Tf activity and that ADAM12-PC consisting of the pro domain and catalytic domain is the most active on this substrate. It was also observed that NaCl inhibits ADAM12. Among the tissue inhibitors of metalloproteinases (TIMP) examined, the N-terminal domain of TIMP-3 (N-TIMP-3) inhibits ADAM12-S and ADAM12-PC with low nanomolar Ki(app) values while TIMP-2 inhibits them with a slightly lower affinity (9-44 nM). However, TIMP-1 is a much weaker inhibitor. N-TIMP-3 variants that lack MMP inhibitory activity but retained the ability to inhibit ADAM17/TACE failed to inhibit ADAM12. These results indicate unique enzymatic properties of ADAM12 among the members of the ADAM family of metalloproteinases.

ADAM12 belongs to the large family of ADAMs (a disintegrin and metalloproteases) and possesses extracellular metalloprotease and cell-binding functions, as well as intracellular signaling capacities. Interest in ADAM12 has increased recently because its expression is related to tumor progression and it is a potential biomarker for breast cancer. It is therefore important to understand ADAM12's functions. Many cellular roles for ADAM12 have been suggested. It is an active metalloprotease, and has been implicated in insulin-like growth factor (IGF) receptor signaling, through cleavage of IGF-binding proteins, and in epidermal growth factor receptor (EGFR) pathways, via ectodomain shedding of membrane-tethered EGFR ligands. These proteolytic events may regulate diverse cellular responses, such as altered cell differentiation, proliferation, migration, and invasion. ADAM12 may also regulate cell-cell and cell-extracellular matrix contacts through interactions with cell surface receptors - integrins and syndecans - potentially influencing the actin cytoskeleton. Moreover, ADAM12 interacts with several cytoplasmic signaling and adaptor molecules through its intracellular domain, thereby directly transmitting signals to or from the cell interior. These ADAM12-mediated cellular effects appear to be critical events in both biological and pathological processes. This review presents current knowledge on ADAM12 functions gained from in vitro and in vivo observations, describes ADAM12's role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.

Muscular dystrophies are characterized by insufficient restoration and gradual replacement of the skeletal muscle by fat and connective tissue. ADAM12 has previously been shown to alleviate the pathology of young dystrophin-deficient mdx mice, a model for Duchenne muscular dystrophy. The observed effect of ADAM12 was suggested to be mediated via a membrane-stabilizing up-regulation of utrophin, alpha7B integrin, and dystroglycans. Ectopic ADAM12 expression in normal mouse skeletal muscle also improved regeneration after freeze injury, presumably by the same mechanism. Hence, it was suggested that ADAM12 could be a candidate for nonreplacement gene therapy of Duchenne muscular dystrophy. We therefore evaluated the long-term effect of ADAM12 overexpression in muscle. Surprisingly, we observed loss of skeletal muscle and accelerated fibrosis and adipogenesis in 1-year-old mdx mice transgenically overexpressing ADAM12 (ADAM12(+)/mdx mice), even though their utrophin levels were mildly elevated compared with age-matched controls. Thus, membrane stabilization was not sufficient to provide protection during prolonged disease. Consequently, we reinvestigated skeletal muscle regeneration in ADAM12 transgenic mice (ADAM12(+)) after a knife cut lesion and observed that the regeneration process was significantly impaired. ADAM12 seemed to inhibit the satellite cell response and delay myoblast differentiation. These results discourage long-term therapeutic use of ADAM12. They also point to impaired regeneration as a possible factor in development of muscular dystrophy.

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-beta type I receptor and the TGF-beta type II receptor (TbetaRII). Upon ligand binding, TGF-beta type I receptor activated by TbetaRII propagates signals to Smad proteins, which mediate the activation of TGF-beta target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-beta signaling pathway that acts through association with TbetaRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-beta signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TbetaRII in early endosomal vesicles and stabilizes the TbetaRII protein presumably by suppressing the association of TbetaRII with Smad7. These results define ADAM12 as a new partner of TbetaRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.

Background: ADAM 12 is a placenta-derived glycoprotein that is involved in growth and differentiation. The maternal serum concentration of ADAM 12 is a potential first-trimester maternal serum marker of Down syndrome (DS). Here we examine the potential of ADAM 12 as a second-trimester maternal serum marker of DS., Materials and Methods: The concentration of ADAM 12 was determined in gestational week 14-19 in 88 DS pregnancies and 341 matched control pregnancies. Medians of normal pregnancies were established by polynomial regression and the distribution of log(10) MoM ADAM 12 values in DS pregnancies and controls determined. Correlations with alpha-fetoprotein (AFP) and free beta-human chorionic gonadotrophin (free beta-hCG) were established and used to model the performance of maternal serum screening with ADAM 12 in combination with other second-trimester serum markers., Results: The ADAM 12 maternal serum concentration was significantly increased with a median MoM of 1.85 and a mean log(10) MoM (SD) of 0.268 (0.2678) compared to a mean log(10) MoM (SD) of 0.013 (0.4318) in controls. ADAM 12 correlated with maternal weight and ethnicity (with the serum concentration increased in Afro-Caribbeans), but neither with maternal age nor gestational age, and only marginally with AFP (r(DS) = 0.078, r(controls) = 0.093) and free beta-hCG (r(DS) = 0.073, r(controls) = 0.144. The increase in detection rate-for a false positive rate of 5%--by adding ADAM 12 to the double test (AFP + free beta-hCG) was 4%, similar to that of adding uE3 to the double test., Conclusion: ADAM 12 is an efficient second-trimester marker for DS. Further studies should be conducted to determine whether it may be a useful additional or alternative marker to those currently used in the second-trimester.

Purpose: We have previously found ADAM12, a disintegrin and metalloprotease, to be an interesting biomarker for breast cancer. The purpose of this study was to determine the gene and protein expression profiles of ADAM12 in different grades and stages of bladder cancer., Experimental Design: ADAM12 gene expression was evaluated in tumors from 96 patients with bladder cancer using a customized Affymetrix GeneChip. Gene expression in bladder cancer was validated using reverse transcription-PCR, quantitative PCR, and in situ hybridization. Protein expression was evaluated by immunohistochemical staining on tissue arrays of bladder cancers. The presence and relative amount of ADAM12 in the urine of cancer patients were determined by Western blotting and densitometric measurements, respectively., Results: ADAM12 mRNA expression was significantly up-regulated in bladder cancer, as determined by microarray analysis, and the level of ADAM12 mRNA correlated with disease stage. Reverse transcription-PCR, quantitative PCR, and in situ hybridization validated the gene expression results. Using immunohistochemistry, we found ADAM12 protein expression correlated with tumor stage and grade. Finally, ADAM12 could be detected in the urine by Western blotting; ADAM12 was present in higher levels in the urine from patients with bladder cancer compared with urine from healthy individuals. Significantly, following removal of tumor by surgery, in most bladder cancer cases examined, the level of ADAM12 in the urine decreased and, upon recurrence of tumor, increased., Conclusions: ADAM12 is a promising biomarker of bladder cancer.

Background: A Disintegrin And Metalloprotease 12 (ADAM 12) is a glycoprotein synthesised by placenta and it has been shown to be a potential first-trimester maternal serum marker for Down syndrome (DS) in two small series. Here we analyse further, the potential of ADAM 12 as a marker for DS in a large collection of first-trimester serum samples., Materials and Methods: The concentration of ADAM 12 was determined in 10-14-week pregnancy sera from 218 DS pregnancies and 389 gestational age-matched control pregnancies, which had been collected as part of routine prospective first-trimester screening programs (DS = 105) or as part of previous research studies (DS = 113). ADAM 12 was measured using a semi-automated time resolved immunofluorometric assay and median values for normal pregnancies were established by polynomial regression. These medians were then used to determine population distribution parameters for DS and normal pregnancy groups. Correlation with previously established PAPP-A and free beta-hCG multiple of the medians (MoMs) and delta nuchal translucency (NT) were determined and used to model the performance of first-trimester screening with ADAM 12 in combination with other first-trimester markers at various time periods across the first trimester. The benefits of a contingent testing model incorporating early measurement of PAPP-A and ADAM 12 were also explored., Results: The maternal serum concentration of ADAM 12 was significantly reduced (p = 0.0049) with an overall median MoM of 0.79 in the DS cases and a log(10) MoM SD of 0.3734 in the DS cases and 0.3353 in the controls. There was a significant correlation of ADAM 12 MoM in DS cases with gestational age (r = 0.375) and the median MoM increased from 0.50 at 10-11 weeks to 1.38 at 13 weeks. ADAM 12 was correlated with maternal weight (r(controls) = 0.283), PAPP-A (r(controls) = 0.324, r(DS) = 0.251) but less so with free beta-hCG (r(controls) = 0.062, r(DS) = 0.049) and delta NT (r(controls) = 0.110, r(DS) = 0.151). ADAM 12 was significantly (p = 0.026) lower in smokers (0.87 vs 1.00) and elevated in Afro-Caribbean women compared to Caucasian women (1.34 vs 1.00). Population modelling using parameters from this and an earlier study showed that a combination of ADAM 12 and PAPP-A measured at 8-9 weeks and combined with NT and free beta-hCG measured at 12 weeks could achieve a detection rate of 97% at a 5% false-positive rate or 89% at a 1% false-positive rate. PAPP-A and ADAM 12 alone at 8-9 weeks could identify 91% of cases at a 5% false-positive rate. Using this as part of a contingent-screening model to select an intermediate risk group of women for NT and free beta-hCG at 11-12 weeks would enable the detection of 92% of cases with a 1% false-positive rate at a cost of providing NT and free beta-hCG for 6% of women with 94% of women having completed screening by the 10th week of pregnancy., Conclusion: ADAM 12 in early first trimester is a very efficient marker of DS. In combination with existing markers, it offers enhanced screening efficiency in a two-stage sequential first-trimester screening program or in a contingent-screening model, which may have benefits in health economies where universal access to high quality ultrasound is difficult. More data on early first-trimester cases with DS are required to establish more secure population parameters by which to assess further the validity of these models.

Background: Maternal serum ADAM12 is reduced, on average, in early first-trimester Down and Edwards' syndrome pregnancies but the extent of reduction declines with gestation. Here we study levels at 9-12 weeks when the marker might be used concurrently with other established markers., Methods: Samples from 16 Down and 2 Edwards' syndrome cases were retrieved from storage and tested together with 313 unaffected singleton pregnancies using a semi-automated time-resolved immuno-fluorometric assay. Results were expressed in multiples of the gestation-specific median (MoM) based on regression., Results: The median in Down syndrome was 0.94 MoM with a 10th-90th centile range of 0.22-1.63 MoM compared with 1.00 and 0.33-2.24 MoM in unaffected controls (P = 0.21, one-side Wilcoxon Rank Sum Test). The two Edwards' syndrome cases had values 0.31 and 2.17 MoM., Conclusions: ADAM12 cannot be used concurrently with other markers in the late first trimester. However, it does have the potential to be used earlier in pregnancy either concurrently with other early markers or in a sequential or contingent protocol. More data will be required to reliably predict the performance of either approach.

Unlabelled: ADAM12-S transgenic mice exhibit a pronounced increase in the length of bones, such as femur, tibia, and vertebrae. The effect of ADAM12-S on longitudinal bone growth involves the modulation of chondrocyte proliferation and maturation, likely through proteolytic activities and altered cell-extracellular matrix interactions in the growth plate., Introduction: The disintegrin and metalloprotease ADAM12 is expressed in both osteoblasts and osteoclasts, suggesting a regulatory role of ADAM12 in bone. However, thus far, no in vivo function of ADAM12 in the skeleton has been reported., Materials and Methods: Transgenic mice expressing the secreted form of human ADAM12, ADAM12-S, or a truncated metalloprotease-deficient form of ADAM12-S in the circulation were used to study the effects of ADAM12 on the skeleton. In addition, murine chondrocyte cultures were used to study the effect of ADAM12-S on cell-extracellular matrix interactions., Results: ADAM12-S transgenic mice exhibit increased longitudinal bone growth. The increased bone length is progressive and age dependent, with a maximum increase of 17% seen in the femur from 6-month-old transgenic mice. The effect is gene dose dependent, being more pronounced in mice expressing higher levels of the transgene than in a lower-expressing line. Histological analysis revealed no alterations in the growth plate organization, but mean growth plate width was increased. Both the cellular incorporation of bromodeoxyuridine and the width of the collagen type X-positive hypertrophic zone were increased in the growth plate of ADAM12-S transgenic mice. Importantly, mice expressing a truncated form of ADAM12-S that lacked the pro- and metalloprotease domains showed no alterations in bone length, suggesting that protease activity is required for the ADAM12-S effect. In vitro studies showed that ADAM12-S inhibits chondrocyte adhesion to fibronectin and collagen type II., Conclusions: ADAM12-S stimulates bone growth in mice by modulating chondrocyte proliferation and maturation through mechanisms probably involving both metalloprotease and adhesion activities.

The ADAMs (a disintegrin and metalloprotease) comprise a family of multidomain proteins with metalloprotease, cell adhesion, and signaling activities. Human ADAM12, which is implicated in diseases such as cancer, is expressed in two splice forms, the transmembrane ADAM12-L and the shorter and soluble ADAM12-S. ADAM12 is synthesized as a zymogen with the prodomain keeping the metalloprotease inactive through a cysteine-switch mechanism. Maturation and activation of the protease involves the cleavage of the prodomain in the trans-Golgi or possibly at the cell surface by a furin-peptidase. The aim of the present study was to determine the fate of the prodomain following furin cleavage. Here we demonstrate that, following cleavage of the human ADAM12-S prodomain in the trans-Golgi by a furin-peptidase, the prodomain remains non-covalently associated with the mature molecule. Accordingly, both the 68-kDa mature form of ADAM12-S and the 25-kDa prodomain could be detected using domain-specific antisera in immunoprecipitation and Western blot analyses of human serum ADAM12 and purified recombinant human ADAM12. Using electron microscopy after negative staining we have furthermore obtained the first visualization of a full-length ADAM molecule, human ADAM12-S, and report that it appears to be a compact clover composed of four globular domains, one of which is the prodomain. Finally, our data demonstrate that the presence of the metalloprotease domain appears to be sufficient for the prodomain to remain associated with the mature ADAM12-S. Thus, we conclude that the prodomain of human ADAM12-S is an integral domain of the mature molecule and as such might have specific biological functions in the extracellular space.

Oncofetal RNA-binding IMPs have been implicated in mRNA localization, nuclear export, turnover and translational control. To depict the cellular actions of IMPs, we performed a loss-of-function analysis, which showed that IMPs are necessary for proper cell adhesion, cytoplasmic spreading and invadopodia formation. Loss of IMPs was associated with a coordinate downregulation of mRNAs encoding extracellular matrix and adhesion proteins. The transcripts were present in IMP RNP granules, implying that IMPs were directly involved in the post-transcriptional control of the transcripts. In particular, we show that a 5.0 kb CD44 mRNA contained multiple IMP-binding sites in its 3'UTR, and following IMP depletion this species became unstable. Direct knockdown of the CD44 transcript mimicked the effect of IMPs on invadopodia, and we infer that CD44 mRNA stabilization may be involved in IMP-mediated invadopodia formation. Taken together, our results indicate that RNA-binding proteins exert profound effects on cellular adhesion and invasion during development and cancer formation.

Background/aims: In chronic liver injury, quiescent hepatic stellate cells change into proliferative myofibroblast-like cells, which are a main source of fibrosis. We have recently reported that these cells synthesize ADAM12, a disintegrin and metalloprotease whose expression is up-regulated by TGF-beta1 in liver cancers. Here, we studied the role of the serine/threonine p70S6 kinase (p70S6K) in regulating TGF-beta1-induced ADAM12 expression., Results: The phophatidylinositol 3-kinase (PI3K) inhibitor LY294002 and the mitogen-activated protein kinase inhibitor, UO126, decreased the TGF-beta1-dependent ADAM12 expression and prevented the phosphorylation of p70S6K. In addition, TGF-beta1-induced ADAM12 up-regulation was blocked by the Frap/mTOR inhibitor rapamycin, which abrogated the phosphorylation of p70S6K. In untreated cells, LY294002 but not rapamycin diminished the basal ADAM12 expression related to inhibition of Akt and the glycogen synthase kinase-3 phosphorylation., Conclusions: The data suggest that TGF-beta1 induces ADAM12 gene expression through both the PI3K/Frap-mTOR/p70S6K and MEK/ERK pathways. In addition, activation of the PI3 pathway might be involved in the basal ADAM12 expression in cultured hepatic stellate cells. The involvement of PI3K in ADAM12 expression, similar to that previously observed for collagen I and fibronectin, suggests common pathways for gene up-regulation in hepatic stellate cells that occur during liver fibrogenesis and contribute to tumor progression.

We have recently shown that overexpression of ADAM12 results in increased muscle regeneration and significantly reduced pathology in mdx, dystrophin deficient mice. In the present study, we tested the effect of overexpressing ADAM12 in dy(W) laminin-deficient mice. dy mice have a very severe clinical phenotype and would be expected to benefit greatly from enhanced regeneration. We found that dy(W) mice overexpressing ADAM12 indeed have increased muscle regeneration, as evidenced by increased numbers of muscle fibers expressing fetal myosin. However, overexpression of ADAM12 had no significant effect on overall health, as evidenced by body weight, and did not improve muscle pathology.

ADAM12, adisintegrin and metalloprotease, has been demonstrated to be upregulated in human malignant tumors and to accelerate the malignant phenotype in a mouse model for breast cancer. ADAM12 is a substrate for beta1 integrins and may affect tumor and stromal cell behavior through its binding to beta1 integrins. Here, we report that cells deficient in beta1 integrin or overexpressing beta3 integrin can bind to recombinant full-length human ADAM12 via beta3 integrin. Furthermore, cell binding to ADAM12 via beta3 integrin results in the formation of focal adhesions, which are not formed upon beta1 integrin-mediated cell attachment. We also show that RhoA is involved in beta3 integrin-mediated focal adhesion formation.

ADAMs (a disintegrin and metalloprotease) comprise a family of cell surface proteins with protease and cell-binding activities. Using different forms and fragments of ADAM12 as substrates in cell adhesion and spreading assays, we demonstrated that alpha9beta1 integrin is the main receptor for ADAM12. However, when alpha9beta1 integrin is not expressed--as in many carcinoma cells--other members of the beta1 integrin family can replace its ligand binding activity. In attachment assays, the recombinant disintegrin domain of ADAM12 only supported alpha9 integrin-dependent tumor cell attachment, whereas full-length ADAM12 supported attachment via alpha9 integrin and other integrin receptors. Cells that attached to full-length ADAM12 in an alpha9 integrin-dependent manner also attached to ADAM12 in which the putative alpha9beta1 integrin-binding motif in the disintegrin domain had been mutated. This attachment was mediated through use of an alternate beta1 integrin. We also found that cell spreading in response to ADAM12 is dependent on the apparent level of integrin activation. Binding of cells to ADAM12 via the alpha9beta1 integrin was Mn(2+)-independent and resulted in attachment of cells with a rounded morphology; attachment of cells with a spread morphology required further activation of the alpha9beta1 integrin. We demonstrated that phosphoinositide-3-kinase appears to be central in regulating alpha9beta1 integrin cell spreading activity in response to ADAM12.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Insulin-like growth factor-II mRNA-binding proteins 1, 2 and 3 (IMP1, IMP2 and IMP3) belong to a family of RNA-binding proteins implicated in mRNA localization, turnover and translational control. We examined their expression pattern during development of murine and human testis and ovaries. In the mouse, IMPs were expressed in male and female gonadal cells at embryonic day 12.5 (E12.5). From E16.5, IMP1 and IMP3 became restricted to the developing germ cells, whereas IMP2 expression persisted in the interstitial cells. In mature mouse and human ovaries, IMP1, IMP2 and IMP3 were detected in resting and growing oocytes and in the granulosa cells. In testis, IMP1 and IMP3 were found mainly in the spermatogonia, whereas IMP2 was expressed in the immature Leydig cells. Moreover, all three IMPs were detected in human semen. The developmental expression pattern of IMP1 and IMP3 in the human testis prompted us to examine their possible involvement in testicular neoplasia. IMPs were detected primarily in germ-cell neoplasms, including preinvasive testicular carcinoma in situ, classical and spermatocytic seminoma, and nonseminomas, with particularly high expression in undifferentiated embryonal carcinoma. The relative expression of IMP1, IMP2 and IMP3 varied among tumor types and only IMP1 was detected in all carcinoma in situ cells. Thus IMPs, and in particular IMP1, may be useful auxiliary markers of testicular neoplasia.

Objectives: The secreted form of ADAM12 is a metalloprotease that may be involved in placental and fetal growth. We examined whether the concentration of ADAM12 in first-trimester maternal serum could be used as a marker for preeclampsia., Methods: We developed a semiautomated, time-resolved, immunofluorometric assay for the quantification of ADAM12 in serum. The assay detected ADAM12 in a range of 78-1248 microg/L. Serum samples derived from women in the first trimester of a normal pregnancy (n = 324) and from women who later developed preeclampsia during pregnancy (n = 160) were obtained from the First Trimester Copenhagen Study. ADAM12 levels were assayed in these serum samples. Serum levels of ADAM12 were converted to multiples of the median (MoM) after log-linear regression of concentration versus gestational age., Results: Serum ADAM12 levels in women who developed preeclampsia during pregnancy had a mean log MoM of -0.066, which was significantly lower than the mean log MoM of -0.001 for ADAM12 levels observed in serum samples from women with normal pregnancy (P = .008). The mean log MoM was even lower in serum derived from preeclamptic women whose infant's weight at birth was less than 2,500 g (n = 27, mean log MoM of -0.120, P = .053)., Conclusion: The maternal serum levels of ADAM12 are significantly lower during the first trimester in women who later develop preeclampsia during pregnancy when compared with levels in women with normal pregnancies. Because the secreted form of ADAM12 cleaves insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5, the IGF axis may play a role in preeclampsia. ADAM12 may be a useful early marker for preeclampsia., Level of Evidence: II-2.

PINCH1 is composed of 5 LIM domains, binds integrin-linked kinase (ILK) and locates to integrin-mediated adhesion sites. In order to investigate PINCH1 function we generated mice and embryonic stem (ES) cell-derived embryoid bodies (EBs) lacking the PINCH1 gene. Similar to mice lacking beta1 integrin or Ilk, loss of PINCH1 arrested development at the peri-implantation stage. In contrast to beta1 integrin or Ilk mutants, however, disruption of the PINCH1 gene produced implantation chambers with visible cell clumps even at embryonic day 9.5. In order to define the phenotype leading to the peri-implantation lethality we made PINCH1-null EBs and found similar but also additional defects not observed in beta1 integrin or Ilk mutant EBs. The similarities included abnormal epiblast polarity, impaired cavitation and detachment of endoderm and epiblast from basement membranes. Additional defects, which were not observed in beta1 integrin- or ILK-deficient mice or EBs, included abnormal cell-cell adhesion of endoderm and epiblast as well as the presence of apoptotic cells in the endodermal cell layer. Although ILK and PINCH1 were shown to be involved in the phosphorylation of serine-473 of PKB/Akt, immunostaining with specific antibodies revealed no apparent alteration of PKB/Akt phosphorylation in PINCH1-deficient EBs. Altogether these data demonstrate an important role of PINCH1 for integrin function, actin organization, cell-cell adhesion and endodermal cell survival during the implanting of mouse embryos.

As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression.

Knowledge on molecular systems involved in myogenic precursor cell (mpc) fusion into myotubes is fragmentary. Previous studies have implicated the a disintegrin and metalloproteinase (ADAM) family in most mammalian cell fusion processes. ADAM12 is likely involved in fusion of murine mpc and human rhabdomyosarcoma cells, but it requires yet unknown molecular partners to launch myogenic cell fusion. ADAM12 was shown able to mediate cell-to-cell attachment through binding alpha9beta1 integrin. We report that normal human mpc express both ADAM12 and alpha9beta1 integrin during their differentiation. Expression of alpha9 parallels that of ADAM12 and culminates at time of fusion. alpha9 and ADAM12 coimmunoprecipitate and participate to mpc adhesion. Inhibition of ADAM12/alpha9beta1 integrin interplay, by either ADAM12 antisense oligonucleotides or blocking antibody to alpha9beta1, inhibited overall mpc fusion by 47-48%, with combination of both strategies increasing inhibition up to 62%. By contrast with blockade of vascular cell adhesion molecule-1/alpha4beta1, which also reduced fusion, exposure to ADAM12 antisense oligonucleotides or anti-alpha9beta1 antibody did not induce detachment of mpc from extracellular matrix, suggesting specific involvement of ADAM12-alpha9beta1 interaction in the fusion process. Evaluation of the fusion rate with regard to the size of myotubes showed that both ADAM12 antisense oligonucleotides and alpha9beta1 blockade inhibited more importantly formation of large (> or =5 nuclei) myotubes than that of small (2-4 nuclei) myotubes. We conclude that both ADAM12 and alpha9beta1 integrin are expressed during postnatal human myogenic differentiation and that their interaction is mainly operative in nascent myotube growth.

Background: ADAM12-S is a pregnancy-associated insulin-like growth factor binding protein-3 (IGFBP-3) and IGFBP-5 protease present in human gestational serum. Recently, maternal serum levels of ADAM12-S were found to be markedly reduced during the first trimester of pregnancies with a Down syndrome (DS) fetus. On the basis of this finding, it was suggested that ADAM12-S might be a useful maternal serum marker of fetal chromosomal disease., Objective: Retrospective examination of the use of ADAM12-S as a marker for fetal trisomy 18., Method: Serum samples were obtained from ten women during the first semester of their pregnancies with fetuses that had trisomy 18. An ELISA was used to determine the levels of ADAM12 in maternal serum. Results were compared to ADAM12-S levels, previously measured in the serum of 170 women carrying normal pregnancies during the first trimester., Results: In all cases, the ADAM12-S concentration in maternal serum samples was lower in trisomy 18 pregnancies than in normal pregnancies, with a median multiple of the median (MoM) of 0.28 (p < 0.001), Conclusion: A reduced concentration of ADAM12-S in maternal serum is a promising marker for foetal trisomy 18, as well as for DS., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

The ADAM (a disintegrin and metalloprotease) family consists of multidomain cell-surface proteins that have a major impact on cell behavior. These transmembrane-anchored proteins are synthesized as proforms that have (from the N terminus): a prodomain; a metalloprotease-, disintegrin-like-, cysteine-rich, epidermal growth factor-like, and transmembrane domain; and a cytoplasmic tail. The 90-kDa mature form of human ADAM12 is generated in the trans-Golgi through cleavage of the prodomain by a furin-peptidase and is stored intracellularly until translocation to the cell surface as a constitutively active protein. However, little is known about the regulation of ADAM12 cell-surface translocation. Here, we used human RD rhabdomyosarcoma cells, which express ADAM12 at the cell surface, in a temporal pattern. We report that protein kinase C (PKC) epsilon induces ADAM12 translocation to the cell surface and that catalytic activity of PKCepsilon is required for this translocation. The following results support this conclusion: 1) treatment of cells with 0.1 microM phorbol 12-myristate 13-acetate (PMA) enhanced ADAM12 cell-surface immunostaining, 2) ADAM12 and PKCepsilon could be co-immunoprecipitated from membrane-enriched fractions of PMA-treated cells, 3) RD cells transfected with EGFP-tagged, myristoylated PKCepsilon expressed more ADAM12 at the cell surface than did non-transfected cells, and 4) RD cells transfected with a kinase-inactive PKCepsilon mutant did not exhibit ADAM12 cell-surface translocation upon PMA treatment. Finally, we demonstrate that the C1 and C2 domains of PKCepsilon both contain a binding site for ADAM12. These studies show that PKCepsilon plays a critical role in the regulation of ADAM12 cell-surface expression.

Insulin-like growth factor II mRNA-binding protein 1 (IMP1) belongs to a family of RNA-binding proteins implicated in mRNA localization, turnover, and translational control. Mouse IMP1 is expressed during early development, and an increase in expression occurs around embryonic day 12.5 (E12.5). To characterize the physiological role of IMP1, we generated IMP1-deficient mice carrying a gene trap insertion in the Imp1 gene. Imp1(-/-) mice were on average 40% smaller than wild-type and heterozygous sex-matched littermates. Growth retardation was apparent from E17.5 and remained permanent into adult life. Moreover, Imp1(-/-) mice exhibited high perinatal mortality, and only 50% were alive 3 days after birth. In contrast to most other organs, intestinal epithelial cells continue to express IMP1 postnatally, and Imp1(-/-) mice exhibited impaired development of the intestine, with small and misshapen villi and twisted colon crypts. Analysis of target mRNAs and global expression profiling at E12.5 indicated that Igf2 translation was downregulated, whereas the postnatal intestine showed reduced expression of transcripts encoding extracellular matrix components, such as galectin- 1, lumican, tenascin-C, procollagen transcripts, and the Hsp47 procollagen chaperone. Taken together, the results demonstrate that IMP1 is essential for normal growth and development. Moreover, IMP1 may facilitate intestinal morphogenesis via regulation of extracellular matrix formation.

Objectives: The concentration of bioavailable insulin-like growth factor (IGF) I and II is important to foetal growth. It is regulated by insulin-like growth factor binding proteins (IGFBP) 1 through 6. Proteolytic cleavage of IGFBP-3 takes place in human pregnancy serum; accordingly, IGFBP-3 serum levels decrease markedly during pregnancy. ADAM12 (A disintegrin and metalloprotease) is an IGFBP-3 and IGFBP-5 protease and is present in human pregnancy serum. The goal of this study was to determine whether ADAM12 concentration in maternal serum is a useful indicator of foetal health., Methods: We developed an enzyme-linked immunosorbent assay (ELISA) for the quantification of ADAM12 in serum. The assay range was 42 to 667 micro g/L. Recombinant ADAM12 was used as the standard for calibration., Results: We found that ADAM12 was highly stable in serum. Serum concentration increased from 180 micro g/L at week 8 of pregnancy to 670 micro g/L at 16 weeks, and reached 12 000 micro g/L at term. In 18 first-trimester Down syndrome pregnancies, the concentration of ADAM12 was decreased, thus the median multiple of mean (MoM) value was 0.14 (0.01-0.76). A detection rate for foetal Down syndrome of 82% for a screen-positive rate of 3.2% and a 1:400 risk cut-off was found by Monte Carlo estimation using ADAM12 and maternal age as screening markers., Conclusion: ADAM12 is a promising marker for Down syndrome., (Copyright 2003 John Wiley & Sons, Ltd.)

Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of beta1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/beta1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal adhesions, and adhered less efficiently to fibronectin in attachment assays. Moreover, ADAM12-expressing cells were more prone to apoptosis, which could be prevented by treating the cells with beta1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, beta1 integrin was more readily extracted with Triton X-100 from cells overexpressing ADAM12 than from control cells. Collectively, these results show that surface expression of ADAM12 impairs the function of beta1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation.

Mouse models for genetic diseases are among the most powerful tools available for developing and testing new treatment strategies. ADAM12 is a disintegrin and metalloprotease, previously demonstrated to significantly alleviate the pathology of mdx mice, a model for Duchenne muscular dystrophy in humans. More specifically ADAM12 appeared to prevent muscle cell necrosis in the mdx mice as evidenced by morphological analysis and by the reduced levels of serum creatine kinase. In the present study we demonstrated that ADAM12 may compensate for the dystrophin deficiency in mdx mice by increasing the expression and redistribution of several components of the muscle cell-adhesion complexes. First, we analyzed transgenic mice that overexpress ADAM12 and found mild myopathic changes and accelerated regeneration following acute injury. We then analyzed changes in gene-expression profiles in mdx/ADAM12 transgenic mice compared with their littermate controls and found only a few genes with an expression change greater than 2-fold between mdx/ADAM12 and mdx. The small changes in gene expression were unexpected, considering the marked improvement of the mdx pathology when ADAM12 is overexpressed, and suggested that significant changes in mdx/ADAM12 muscle might occur post-transcriptionally. Indeed, by immunostaining and immunoblotting we found an approximately 2-fold increase in expression, and distinct extrasynaptic localization, of alpha 7B integrin and utrophin, the functional homolog of dystrophin. The expression of the dystrophin-associated glycoproteins was also increased. In conclusion, these results demonstrate a novel way to alleviate dystrophin deficiency in mice, and may stimulate the development of new approaches to compensate for dystrophin deficiency in animals and humans.

More than 30 different forms of muscular dystrophy (MD) have been molecularly characterized and can be diagnosed, but progress toward treatment has been slow. Gene replacement therapy has met with great difficulty because of the large size of the defective genes and because of difficulties in delivering a gene to all muscle groups. Cell replacement therapy has also been difficult to realize. Will it even be possible to design specific therapy protocols for all MDs? Or is a more realistic goal to treat some of the secondary manifestations that are common to several forms of MD, such as membrane instability, necrosis, and inflammation, and to promote regeneration? As reviewed here, enhanced expression of a range of proteins provides a boost for degenerating dystrophic muscle in mouse models. Expression of a mini-agrin promotes basement membrane formation instead of laminin alpha2; integrin alpha7, GalNac transferase, and ADAM12 promote cell adhesion and muscle stability in the absence of dystrophin; calpastatin prevents muscle necrosis; and nitric oxide synthase prevents inflammation. ADAM12, IGF-I, and myostatin blockade promote regeneration and reduce fibrosis. One can envision numerous other candidate booster genes which encode proteins that promote survival and/or regeneration of the compromised muscle or proteins that affect post-translational modifications of critical proteins. Finally, fibrosis, which is the curse of many human diseases, may also be attacked. Once the mechanisms of the boosters are better understood, drugs may be developed to provide the boost to muscle. Some of the experiences in models of muscular dystrophy may inspire new approaches in other genetic degenerative diseases as well.

"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT-PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up-regulated by transforming growth factor beta (TGF-beta) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-beta, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression.

Rigid spine muscular dystrophy and the classical form of multiminicore disease are caused by mutations in SEPN1 gene, leading to a new clinical entity referred to as SEPN1-related myopathy. SEPN1 codes for selenoprotein N, a new member of the selenoprotein family, the function of which is still unknown. In a previous study, two isoforms were deduced from SEPN1 transcript analyses. Using polyclonal antibodies directed against SEPN1 and cDNA constructs encoding for the two isoforms, we show that the main SEPN1 gene product corresponds to a 70 kDa protein, containing a single selenocysteine residue. Subcellular fractionation experiments and endoglycosidase H sensitivity indicate that SEPN1 is a glycoprotein-localized within the endoplasmic reticulum. Immunofluorescence analyses confirm this subcellular localization and green fluorescent protein fusion experiments demonstrate the presence of an endoplasmic reticulum-addressing and -retention signal within the N-terminus. SEPN1 is present at a high level in several human fetal tissues and at a lower level in adult ones, including skeletal muscle. Its high expression in cultured myoblasts is also down-regulated in differentiating myotubes, suggesting a role for SEPN1 in early development and in cell proliferation or regeneration.