With blood in the joint - what happens next? Could activation of a pro-inflammatory signalling axis leading to iRhom2/TNFα-convertase-dependent release of TNFα contribute to haemophilic arthropathy?

One of the main complications of haemophilia A is haemophilic arthropathy (HA), a debilitating disease with a significant negative impact on motility and quality of life. Despite major advances in the treatment of haemophilia A, many patients still suffer from HA. We wish to develop new treatments for HA, but must first better understand its causes. Our laboratory studies molecular scissors that release the pro-inflammatory cytokine tumour necrosis factor alpha (TNFα) from cells. TNFα is considered the 'fire alarm' of the body - it helps to fight infections, but can also cause diseases such as inflammatory arthritis. We know that the molecular scissors, called TNFα convertase (TACE), and its newly discovered regulator termed iRhom2 can be rapidly activated by small amounts of cytokines, growth factors, and pro-inflammatory mediators present in the blood. We hypothesize that the rapid activation of TACE could help explain one of the unsolved mysteries regarding the development of HA, which is how even small amounts of blood can provoke a persistent inflammatory response. We propose that once blood enters the joint, iRhom2 and TACE are activated to release TNFα and that this could promote the development of HA in a similar manner to that in which it promotes rheumatoid arthritis (RA). We are currently using immune cells stimulated with blood degradation products, and mouse models of HA, to test this hypothesis. If successful, our study could provide the rationale for testing anti-TNF antibodies, which are already used to treat RA, for the treatment of HA. In addition, they might uncover iRhom2 and TACE as attractive new candidate targets for the treatment of HA.

A transforming Src mutant increases the bioavailability of EGFR ligands via stimulation of the cell-surface metalloproteinase ADAM17.

ADAM17 (a disintegrin and metalloproteinase 17, also referred to as TNFα converting enzyme or TACE) is a cell-surface metalloproteinase that regulates signaling via the epidermal growth factor receptor (EGFR) and has important roles in diseases such as cancer and rheumatoid arthritis. ADAM17 can be activated by stimulation of several tyrosine kinase receptors, raising questions about whether oncogenic tyrosine kinases could also enhance EGFR signaling and activation of extracellular signal-regulated kinase (ERK) via stimulation of ADAM17. The main goal of this study was to evaluate the role of Src in activating ADAM17. We provide evidence that a constitutively active transforming form of Src, the E378G mutant, as well as v-Src enhance ADAM17-mediated shedding of the EGFR ligand TGFα. Moreover, we demonstrate that constitutive shedding of TGFα can be reduced by inhibition of Src in several cell lines, including COS7, MCF7 (the human breast cancer cell line), PAE (a pig aortic endothelial cell line) and HaCaT (the human keratinocyte cell line) cells. Src(E378G)-stimulated shedding of TGFα is abolished in Adam17(-/-) cells, but can be rescued by wild-type (wt) ADAM17 and a mutant ADAM17 lacking its cytoplasmic domain. These findings demonstrate that ADAM17 is the principal TGFα sheddase that is activated by Src in a manner that does not require the cytoplasmic domain of ADAM17. Finally, we show that stimulation of ADAM17 by Src(E378G) leads to enhanced paracrine signaling via release of EGFR ligands into the culture supernatant. These results raise the possibility that activation of ADAM17 by oncogenic forms of Src can aid in promoting tumorigenesis by enhancing signaling via the EGFR and ERK in an autocrine and paracrine manner. Enhanced autocrine signaling could further activate tumor cells expressing oncogenic mutants of Src, whereas paracrine signaling could stimulate EGFR and ERK signaling in surrounding non-transformed cells such as stromal cells, thereby contributing to crosstalk between tumor cells and stromal cells.

The waved with open eyelids (woe) locus is a hypomorphic mouse mutation in Adam17.

The waved with open eyes (woe) locus is a spontaneous recessive mouse mutation that exhibits wavy fur, eyelids open at birth, and enlarged heart and esophagus. In this study, we confirmed the previously identified woe phenotypes and additionally identified anterior eye segment defects, absence of the meibomian glands, and defects in the semilunar cardiac valves. Positional cloning identified a C794T substitution in the Adam17 gene that ablates a putative exonic splicing enhancer (ESE) sequence in exon 7 resulting in aberrant Adam17 splicing. The predominant woe transcript, Adam17(Delta)(exon7), lacks exon 7 resulting in an in-frame deletion of 90 bp and a putative Adam17(Delta252-281) protein lacking residues 252-281 from the metalloprotease domain. Western blot analysis in woe identified only the precursor form of Adam17(Delta252-281) protein. Absence of cleavage of the prodomain renders Adam17(Delta252-281) functionally inactive; however, constitutive and stimulated shedding of Adam17 substrates was detected in woe at significantly reduced levels. This residual Adam17 shedding activity in woe most likely originates from full-length Adam17(T265M) encoded by the Adam17(C794T) transcript identified expressed at severely reduced levels. These results show that even small amounts of functional Adam17 allow woe mice to survive into adulthood. In contrast to Adam17(-/-) mice that die at birth, the viability of woe mice provides an excellent opportunity for studying the role of Adam17 throughout postnatal development and homeostasis.

ADAM12 is highly expressed in carcinoma-associated stroma and is required for mouse prostate tumor progression.

The interaction between stromal cells and tumor cells is emerging as a critical aspect of tumor progression. Yet there is a paucity of molecular markers for cells participating in such interactions, and only few genes are known to play a critical role in this process. Here, we describe the identification of ADAM12 (a disintegrin and metalloprotease 12) as a novel marker for a subpopulation of stromal cells that are adjacent to epithelial tumor cells in three mouse carcinoma models (models for prostate, breast and colon cancer). Moreover, we show that ADAM12 is essential for tumor development and progression in the W10 mouse model for prostate cancer. These results suggest that ADAM12 might be a useful marker for stromal cells in mouse tumors that are likely to participate in stromal/tumor cell crosstalk, and that ADAM12 is a potential target for design of drugs that prevent carcinoma growth.

Functional and biochemical characterization of ADAMs and their predicted role in protein ectodomain shedding.

Proteolysis on the cell surface and in the extracellular matrix is essential for normal cellular functions during development and in the adult, but it may also have undesirable consequences by promoting diseases such as cancer, arthritis, and Alzheimer's disease. A particularly interesting function of proteolysis on the cell surface is to release ectodomains of membrane proteins from the plasma membrane. This process, which is referred to as protein ectodomain shedding, affects a variety of proteins with important roles in development and in disease, including cytokines, growth factors, receptors, adhesion proteins and other proteins such as the amyloid precursor protein. In principle, protein ectodomain shedding can dramatically change the properties of a substrate protein. For example, membrane anchored growth factors such as transforming growth factor-alpha (TGF-alpha) are only able to activate their receptor, the epidermal growth factor receptor (EGFR), after they are shed from the plasma membrane. Inhibitor studies have implicated zinc-dependent metalloproteases in protein ectodomain shedding, and in particular a family of metalloproteases termed ADAMs (a disintegrin and metalloprotease). The main focus of my lab is to understand the role of different ADAMs in protein ectodomain shedding, and to learn about the functional consequences of protein ectodomain shedding for individual substrates.

Highly enhanced expression of the disintegrin metalloproteinase MDC15 (metargidin) in rheumatoid synovial tissue.

OBJECTIVE: The aim of the study was to analyze the expression of the disintegrin metalloproteinase MDC15 (metargidin, or ADAM15) at the messenger RNA (mRNA) and protein levels in synovial tissue from osteoarthritis (OA) and rheumatoid arthritis (RA) patients compared with normal specimens. METHODS: Conventional immunohistochemistry and confocal laser scanning microscopy of immunofluorescently stained sections, as well as in situ hybridization experiments and reverse transcription-polymerase chain reaction were performed for analyses of MDC15 expression on normal, OA, and RA synovial tissue specimens. RESULTS: In normal synovium, MDC15 expression was detectable at a very low level. MDC15 expression was considerably increased in OA-derived tissue samples, whereas a maximum of signal intensity for MDC15 mRNA and protein was seen in the RA lining layer. The CD68+ macrophage-like synoviocytes (type A) and the CD68- fibroblast-like synoviocytes (type B) were positive for MDC15. Moreover, a very strong expression of MDC15 was also found in CD138+ plasma cells in all RA tissues as well as in OA specimens that contained areas of mononuclear cell infiltrates. CD20+ B cells and CD4+ and CD8+ T cells, however, did not exhibit expression of MDC15, either in the synovial tissue in situ or in preparations of circulating lymphocytes made from the peripheral blood of RA patients or healthy controls. CONCLUSION: Our results demonstrate high levels of MDC15 expression in macrophage-like and fibroblast-like synoviocytes as well as in plasma cells as a histologic feature most prominent in RA synovial tissue compared with normal or OA synovial tissue. This suggests a potential role of MDC15 in the pathogenesis of cartilage destruction in inflammatory joint disease.

Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1).

Fractalkine (CX3CL1) is an unusual member of the chemokine family that is synthesized with its chemokine domain at the end of a mucin-rich, transmembrane stalk. This membrane-bound localization allows fractalkine to function as an adhesion molecule for cells bearing its receptor, CX3CR1. In addition, fractalkine can be proteolytically released from the cell surface, generating a soluble molecule that functions as a chemoattractant similar to the other members of the chemokine family. In this study, we have examined the mechanisms that regulate the conversion between these two functionally distinct forms of fractalkine. We demonstrate that under normal conditions fractalkine is synthesized as an intracellular precursor that is rapidly transported to the cell surface where it becomes a target for metalloproteinase-dependent cleavage that causes the release of a fragment containing the majority of the fractalkine extracellular domain. We show that the cleavage of fractalkine can be markedly enhanced by stimulating cells with phorbol 12-myristate 13-acetate (PMA), and we identify tumor necrosis factor-alpha converting enzyme (TACE; ADAM17) as the protease responsible for this PMA-induced fractalkine release. In addition, we provide data showing that TACE-mediated fractalkine cleavage occurs at a site distinct from the dibasic juxtamembrane motif that had been suggested previously based on protein sequence homologies. The identification of TACE as a major protease responsible for the conversion of fractalkine from a membrane-bound adhesion molecule to a soluble chemoattractant will provide new information for understanding the physiological function of this chemokine.

Biochemical and pharmacological criteria define two shedding activities for TRANCE/OPGL that are distinct from the tumor necrosis factor alpha convertase.

A number of structurally and functionally diverse membrane proteins are released from the plasma membrane in a process termed protein ectodomain shedding. Ectodomain shedding may activate or inactivate a substrate or change its properties, such as converting a juxtacrine into a paracrine signaling molecule. Here we have characterized the activities involved in protein ectodomain shedding of the tumor necrosis factor family member TRANCE/OPGL in different cell types. The criteria used to evaluate these activities include (a) cleavage site usage, (b) response to activators and inhibitors of intracellular signaling pathways, and (c) sensitivity to tissue inhibitors of metalloproteases (TIMPs). At least two TRANCE shedding activities emerged, both of which are distinct from the tumor necrosis factor alpha convertase. One of the TRANCE sheddases is induced by the tyrosine phosphatase inhibitor pervanadate but not by phorbol esters, whereas the other is refractory to both of these stimuli. Furthermore, the pervanadate-regulated sheddase activity is sensitive to TIMP-2 but not TIMP-1, which is consistent with the properties of a membrane type matrix metalloprotease. This study provides insights into the properties of different activities involved in protein ectodomain shedding and has implications for the functional regulation of TRANCE by potentially more than one protease.

Remarkable roles of proteolysis on and beyond the cell surface.

Proteolysis on the cell surface and in the extracellular matrix is essential for normal cellular functions during development and in the adult, but it may also have undesirable consequences, such as promoting cancer, arthritis, and Alzheimer's disease. Recent advances highlight the roles of zinc-dependent metalloproteinases (metzincins) in proper skeletal development, in activating EGF-receptor ligands, in Notch-dependent signaling, and in initiating and promoting tumorigenesis.

Functional processing of fertilin: evidence for a critical role of proteolysis in sperm maturation and activation.

Fertilin is a sperm surface protein with an essential role in fertilization. It is required for the migration of spermatozoa through the oviduct, for binding to the zona pellucida, and for efficient binding to the egg plasma membrane. Fertilin consists of two subunits, fertilin alpha and beta, both of which belong to the metalloprotease-disintegrin protein family (ADAMs). Fertilin alpha and beta are made as larger precursors that are processed proteolytically at different stages of sperm maturation in the testis and epididymis. Fertilin alpha is processed first, most likely by a pro-protein convertase in the secretory pathway of testicular cells. Fertilin beta is processed later, while spermatozoa are in transit through the epididymis. The processing of fertilin beta in the epididymis correlates with the acquisition of fertilization competence in spermatozoa, exposes an epitope that has a role in sperm-egg interactions, and triggers the relocalization of fertilin from the whole sperm head to the posterior head. These findings indicate that the proteolytic processing of fertilin and perhaps also other sperm proteins plays an important role in sperm maturation and activation in the epididymis. Further evaluation of the functional significance of proteolysis for sperm maturation should lead to new and exciting insights into the mechanism of sperm maturation, and may also uncover the cause of certain types of male infertility. The identification of the responsible proteases could provide novel targets for contraceptive drugs.

Cloning and characterization of ADAM28: evidence for autocatalytic pro-domain removal and for cell surface localization of mature ADAM28.

The metalloprotease disintegrins are a family of membrane-anchored glycoproteins with diverse functions in fertilization, myoblast fusion, neurogenesis and protein ectodomain shedding. Here we report a cDNA sequence, encoding a metalloprotease disintegrin, termed ADAM28 ('a disintegrin and metalloprotease 28'), which was cloned from mouse lung. From protein sequence comparisons, ADAM28 is more closely related to snake venom metalloproteases (SVMPs) than to other ADAMs, and hence may cleave similar substrates to SVMPs, perhaps including components of the extracellular matrix. Northern blot analysis of selected mouse tissues revealed that ADAM28 is expressed highly and in alternatively spliced forms in the epididymis, suggesting a possible role in sperm maturation, and at lower levels in lung. The intracellular maturation of ADAM28 expressed in COS-7 cells resembles that of other ADAMs, in that ADAM28 is made as a precursor and processed to a mature form in a late Golgi compartment of the secretory pathway. Most or all of the mature, and thus presumably catalytically active, form of ADAM28 in COS-7 cells is accessible to cell surface trypsinization, suggesting that ADAM28 functions mainly on the cell surface. A mutation converting the catalytic-site glutamate residue into alanine abolishes pro-domain removal, even though this mutant form of ADAM28 can be transported to the cell surface in a manner similar to the wild-type protein. This suggests that pro-domain removal and maturation of ADAM28 may be, at least in part, autocatalytic. This is in contrast with several other ADAMs, for which furin-like proprotein convertases are involved in pro-domain removal, and in which a glutamate-to-alanine mutation in the catalytic site does not alter pro-domain removal.

Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE).

Tumour necrosis factor alpha convertase (TACE) is a metalloprotease/disintegrin involved in the ectodomain shedding of several proteins, a process thought to be important in inflammation, rheumatoid arthritis and murine development. The characterization of the intracellular maturation and subcellular localization of endogenous TACE is decribed in the present study. Similarly to other proteolytically active metalloprotease/disintegrins, two forms of TACE are found in cells; a full-length precursor and a mature form lacking the prodomain. Prodomain removal occurs in a late Golgi compartment, consistent with the proposed role of a furin type proprotein convertase in this process. An additional form of TACE, lacking the pro and cytoplasmic domains, is detected when cell lysates are prepared in the presence of EDTA instead of a hydroxamate-based metalloprotease inhibitor or 1,10-phenanthroline. This form appears to be generated by mature TACE cleaving its own cytoplasmic tail and may explain why little mature TACE has been detected in previous studies. In cell-surface labelling experiments, mature TACE was detected on the cell surface but immunofluorescence data indicate that TACE is predominantly localized to a perinuclear compartment similar to that described for tumour necrosis factor (TNF)alpha. This raises the possibility that TACE-mediated ectodomain shedding may occur in an intracellular compartment in addition to the cell surface.

Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding.

Metalloprotease-disintegrins (ADAMs) have captured our attention as key players in fertilization and in the processing of the ectodomains of proteins such as tumor necrosis factor (&agr;) (TNF(&agr;)), and because of their roles in Notch-mediated signaling, neurogenesis and muscle fusion. ADAMs are integral membrane glycoproteins that contain a disintegrin domain, which is related to snake-venom integrin ligands, and a metalloprotease domain (which can contain or lack a catalytic site). Here, we review and critically discuss current topics in the ADAMs field, including the central role of fertilin in fertilization, the role of the TNF(&agr;) convertase in protein ectodomain processing, the role of Kuzbanian in Notch signaling, and links between ADAMs and processing of the amyloid-precursor protein.

Up-regulation of MDC15 (metargidin) messenger RNA in human osteoarthritic cartilage.

OBJECTIVE: The aim of the study was to investigate the messenger RNA (mRNA) expression of the disintegrin metalloproteinase MDC15 (metargidin, or ADAM-15) in normal and osteoarthritic (OA) articular cartilage. METHODS: In situ hybridization experiments and reverse transcription-polymerase chain reaction (RT-PCR) were performed on tissue samples of adult normal and OA articular cartilage. RESULTS: MDC15 mRNA could be detected in normal articular cartilage by RT-PCR using tissue-extracted total RNA as a template. However, the mRNA level remained below the sensitivity of in situ hybridization. In contrast, in situ hybridizations of OA cartilage revealed an intense staining with the MDC15-specific riboprobes. The extension of the analysis to chondrosarcomas showed a strong up-regulation of MDC15 mRNA in these malignant transformed cells. CONCLUSION: Our results demonstrate a markedly strong up-regulation of MDC15 in adult OA and neoplastic cartilage compared with adult normal articular cartilage, indicating a potential role of the disintegrin metalloproteinase in cartilage remodeling.

Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1.

Metalloprotease disintegrins (a disintegrin and metalloprotease (ADAM) and metalloprotease, disintegrin, cysteine-rich proteins (MDC)) are a family of membrane-anchored glycoproteins that function in diverse biological processes, including fertilization, neurogenesis, myogenesis, and ectodomain processing of cytokines and other proteins. The cytoplasmic domains of ADAMs often include putative signaling motifs, such as proline-rich SH3 ligand domains, suggesting that interactions with cytoplasmic proteins may affect metalloprotease disintegrin function. Here we report that two SH3 domain-containing proteins, endophilin I (SH3GL2, SH3p4) and a novel SH3 domain- and phox homology (PX) domain-containing protein, termed SH3PX1, can interact with the cytoplasmic domains of the metalloprotease disintegrins MDC9 and MDC15. These interactions were initially identified in a yeast two-hybrid screen and then confirmed using bacterial fusion proteins and co-immunoprecipitations from eukaryotic cells expressing both binding partners. SH3PX1 and endophilin I both preferentially bind the precursor but not the processed form of MDC9 and MDC15 in COS-7 cells. Since rat endophilin I is thought to play a role in synaptic vesicle endocytosis and SH3PX1 has sequence similarity to sorting nexins in yeast, we propose that endophilin I and SH3PX1 may have a role in regulating the function of MDC9 and MDC15 by influencing their intracellular processing, transport, or final subcellular localization.

Evidence for an interaction of the metalloprotease-disintegrin tumour necrosis factor alpha convertase (TACE) with mitotic arrest deficient 2 (MAD2), and of the metalloprotease-disintegrin MDC9 with a novel MAD2-related protein, MAD2beta.

Metalloprotease-disintegrins are a family of transmembrane glycoproteins that have a role in fertilization, sperm migration, myoblast fusion, neural development and ectodomain shedding. In the present study we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic domain of two metalloprotease-disintegrins, tumour necrosis factor alpha convertase (TACE; ADAM17) and MDC9 (ADAM9; meltrin gamma). We have identified mitotic arrest deficient 2 (MAD2) as a binding partner of the TACE cytoplasmic domain, and a novel MAD2-related protein, MAD2beta, as a binding partner of the MDC9 cytoplasmic domain. MAD2beta has 23% sequence identity with MAD2, which is a component of the spindle assembly (or mitotic) checkpoint mechanism. Northern blot analysis of human tissues indicates that MAD2beta mRNA is expressed ubiquitously. The interaction of the TACE and MDC9 cytoplasmic domains with their binding partners has been confirmed biochemically. The independent identification of MAD2 and MAD2beta as potential interacting partners of distinct metalloprotease-disintegrins raises the possibility of a link between metalloprotease-disintegrins and the cell cycle, or of functions for MAD2 and MAD2beta that are not related to cell cycle control.

Evidence for a role of a tumor necrosis factor-alpha (TNF-alpha)-converting enzyme-like protease in shedding of TRANCE, a TNF family member involved in osteoclastogenesis and dendritic cell survival.

Tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), a member of the TNF family, is a dendritic cell survival factor and is essential for osteoclastogenesis and osteoclast activation. In this report we demonstrate (i) that TRANCE, like TNF-alpha, is made as a membrane-anchored precursor, which is released from the plasma membrane by a metalloprotease; (ii) that soluble TRANCE has potent dendritic cell survival and osteoclastogenic activity; (iii) that the metalloprotease-disintegrin TNF-alpha convertase (TACE) can cleave immunoprecipitated TRANCE in vitro in a fashion that mimics the cleavage observed in tissue culture cells; and (iv) that in vitro cleavage of a TRANCE ectodomain/CD8 fusion protein and of a peptide corresponding to the TRANCE cleavage site by TACE occurs at the same site that is used when TRANCE is shed from cells into the supernatant. We propose that the TRANCE ectodomain is released from cells by TACE or a related metalloprotease-disintegrin, and that this release is an important component of the function of TRANCE in bone and immune homeostasis.

Metalloprotease-disintegrin MDC9: intracellular maturation and catalytic activity.

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that are known to function in fertilization, myoblast fusion, neurogenesis, and ectodomain shedding of tumor necrosis factor (TNF)-alpha. Here we report the analysis of the intracellular maturation and catalytic activity of the widely expressed metalloprotease disintegrin MDC9. Our results suggest that the pro-domain of MDC9 is removed by a furin-type pro-protein convertase in the secretory pathway before the protein emerges on the cell surface. The soluble metalloprotease domain of MDC9 cleaves the insulin B-chain, a generic protease substrate, providing the first evidence that MDC9 is catalytically active. Soluble MDC9 appears to have distinct specificities for cleaving candidate substrate peptides compared with the TNF-alpha convertase (TACE/ADAM17). The catalytic activity of MDC9 can be inhibited by hydroxamic acid-type metalloprotease inhibitors in the low nanomolar range, in one case with up to 50-fold selectivity for MDC9 versus TACE. Peptides mimicking the predicted cysteine-switch region of MDC9 or TACE inhibit both enzymes in the low micromolar range, providing experimental evidence for regulation of metalloprotease disintegrins via a cysteine-switch mechanism. Finally, MDC9 is shown to become phosphorylated when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate, a known inducer of protein ectodomain shedding. This work implies that removal of the inhibitory pro-domain of MDC9 by a furin-type pro-protein convertase in the secretory pathway is a prerequisite for protease activity. After pro-domain removal, additional steps, such as protein kinase C-dependent phosphorylation, may be involved in regulating the catalytic activity of MDC9, which is likely to target different substrates than the related TNF-alpha-convertase.

Intracellular maturation of the mouse metalloprotease disintegrin MDC15.

Metalloprotease disintegrins are a family of membrane-anchored glycoproteins that play a role in fertilization, myoblast fusion, neuronal development, and cleavage of the membrane-anchored cytokine tumor necrosis factor-alpha. Here, we report the cloning and cDNA sequencing of the mouse metalloprotease disintegrin MDC15 and an analysis of its processing in the secretory pathway. A notable difference between mMDC15 and its putative human orthologue (hMDC15, metargidin) is the presence of the peptide sequence TDDC instead of the RGDC found in the disintegrin domain of hMDC15. In a Western blot analysis the majority of mMDC15 was found to lack the pro-domain in all mouse tissues examined. Pulse-chase experiments in transiently transfected COS-7 cells suggest that mMDC15 is processed by a pro-protein convertase in a late Golgi compartment, since (i) addition of brefeldin A or monensin blocks pro-domain removal, (ii) all detectable processed mMDC15 is endoglycosidase H -resistant, and (iii) a recombinant soluble form of the trans-Golgi network pro-protein convertase furin can mimic mMDC15 processing in vitro. Cell-surface trypsinization revealed that more than half of mature mMDC15 is intracellular. Immunolocalization provided evidence for a strong perinuclear accumulation in a region resembling the trans-Golgi network and/or endosomal compartments. This study provides the first characterization of the intracellular processing of a metalloprotease disintegrin, and highlights the potential role of pro-protein convertases in removal of the inhibitory pro-domain. These results further suggest possible intracellular functions for mMDC15, such as in protein maturation, in addition to a potential role in cell-surface proteolysis or cell adhesion.

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

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