The Phosphorylation and Distribution of Cortactin Downstream of Integrin α9ß1 Affects Cancer Cell Behaviour.

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 α9ß1 corresponds with less migratory behaviour in melanoma cells. Here, we aimed to identify components associated with the activation status of α9ß1. Using cancer cell lines with naturally occuring high levels of this integrin, activation by α9ß1-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 α9ß1 to focal adhesions where active ß1 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 ß1 integrin on the cell surface, being inversely correlated with migratory behaviour. This study underlines the complex interplay between cortactin and α9ß1 integrin that regulates cell-extracellular matrix interactions.

ADAM12 redistributes and activates MMP-14, resulting in gelatin degradation, reduced apoptosis and increased tumor growth.

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 αVß3 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, αVß3 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.

ADAM12 is expressed in the tumour vasculature and mediates ectodomain shedding of several membrane-anchored endothelial proteins.

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.

Regulation of ROCK activity in cancer.

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.

The newcomer in the integrin family: integrin α9 in biology and cancer.

Integrins are heterodimeric transmembrane receptors regulating cell-cell and cell-extracellular matrix interactions. Of the 24 integrin heterodimers identified in humans, α9ß1 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. α9ß1 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 α9ß1-mediated signaling and its regulation. This review gives an overview of the recent progress in α9ß1-mediated biological and pathological processes, and discusses its potential as a target for cancer diagnosis and therapy.

Functional analysis of a breast cancer-associated mutation in the intracellular domain of the metalloprotease ADAM12.

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.

A collapsin response mediator protein 2 isoform controls myosin II-mediated cell migration and matrix assembly by trapping ROCK II.

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.

ADAM12 produced by tumor cells rather than stromal cells accelerates breast tumor progression.

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-ß1 stimulates ADAM12 expression and is a well-known growth factor released from tumor-associated stroma. TGF-ß1 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.

Aging, longevity and health.

The IARU Congress on Aging, Longevity and Health, held on 5-7 October 2010 in Copenhagen, Denmark, was hosted by Rector Ralf Hemmingsen, University of Copenhagen and Dean Ulla Wewer, Faculty of Health Sciences, University of Copenhagen and was organized by Center for Healthy Aging (CEHA) under the leadership of CEHA Managing Director Lene Juel Rasmussen and Prof. Vilhelm Bohr, National Institute on Aging, NIH, Baltimore, USA (associated to CEHA). The Congress was attended by approximately 125 researchers interested in and/or conducting research on aging and aging-related topics. The opening Congress Session included speeches by Ralf Hemmingsen, Ulla Wewer, and Lene Juel Rasmussen and Keynote Addresses by four world renowned aging researchers: Povl Riis (The Age Forum), Bernard Jeune (University of Southern Denmark), George Martin (University of Washington, USA) and Jan Vijg (Albert Einstein School of Medicine, USA) as well as a lecture discussing the art-science interface by Thomas Söderqvist (Director, Medical Museion, University of Copenhagen). The topics of the first six Sessions of the Congress were: Neuroscience and DNA damage, Aging and Stress, Life Course, Environmental Factors and Neuroscience, Muscle and Life Span and Life Span and Mechanisms. Two additional Sessions highlighted ongoing research in the recently established Center for Healthy Aging at the University of Copenhagen. This report highlights outcomes of recent research on aging-related topics, as described at the IARU Congress on Aging, Longevity and Health.

Extracellular engagement of ADAM12 induces clusters of invadopodia with localized ectodomain shedding activity.

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 αvß3 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 ß3 integrin expression, together with high levels of c-Src kinase activity.

Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2).

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 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity.

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.

Alpha9beta1 integrin in melanoma cells can signal different adhesion states for migration and anchorage.

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.

Impaired cutaneous wound healing in mice lacking tetranectin.

Tetranectin was originally purified from human serum on the basis of plasminogen kringle 4-binding properties. Tetranectin enhances plasminogen activation by a tissue-type plasminogen activator so that it has been suggested to play a role in tissue remodeling. We have generated mice with a targeted disruption of the tetranectin gene to elucidate the biological function of tetranectin. In this study, we showed that wound healing was markedly delayed in tetranectin-null mice compared with wild-type mice. A single full-thickness incision was made in the dorsal skin. By 14 days after the incision, the wounds fully healed in all wild-type mice based on the macroscopic closure; in contrast, the progress of wound healing in the tetranectin null mice appeared to be impaired. In histological analysis, wounds of wild-type mice showed complete reepithelialization and healed by 14 days after the incision. However, those of tetranectin-null mice never showed complete reepithelialization at 14 days. At 21 days after the injury, the wound healed and was covered with an epidermis. These results supported the fact that tetranectin may play a role in the wound healing process.

Heparan sulfate regulates ADAM12 through a molecular switch mechanism.

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.

Expression, purification and insights into structure and folding of the ADAM22 pro domain.

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)).

Cellular roles of ADAM12 in health and disease.

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.

Catalytic properties of ADAM12 and its domain deletion mutants.

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.

Transgenic overexpression of ADAM12 suppresses muscle regeneration and aggravates dystrophy in aged mdx mice.

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.

The disintegrin and metalloproteinase ADAM12 contributes to TGF-beta signaling through interaction with the type II receptor.

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.