Reidar Albrechtsen

A NOVEL, SECRETED FORM OF HUMAN ADAM 12 (MELTRIN ALPHA ) PROVOKES MYOGENESIS IN VIVO

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

Cysteine-Rich Domain of Human ADAM 12 (Meltrin α) Supports Tumor Cell Adhesion

The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions. By immunostaining, ADAM 12 (meltrin α) was up-regulated in several human carcinomas and could be detected along the tumor cell membranes. Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion. Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support tumor cell adhesion. We found that the disintegrin-like domain of human ADAM 15 supported adhesion of αvβ3-expressing A375 melanoma cells. In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion of a panel of carcinoma cell lines. On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate proteoglycan to attach. Finally, we demonstrated that authentic full-length human ADAM 12 could bind to heparin Sepharose. Together these results suggest a novel role of the cysteine-rich domain of ADAM 12 — that of supporting tumor cell adhesion.

ADAM12 induces actin cytoskeleton and extracellular matrix reorganization during early adipocyte differentiation by regulating β1 integrin function

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 β1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/β1 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 β1-activating antibodies. A reduced and re-organized fibronectin-rich extracellular matrix accompanied these changes. In addition, β1 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 β1 integrins and, consequently, alters the organization of the actin cytoskeleton and extracellular matrix. These events may be necessary for early adipocyte differentiation.

A Role for ADAM12 in Breast Tumor Progression and Stromal Cell Apoptosis

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.

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 ADAM12s 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 ADAM12s role in both normal physiology and pathology, particularly in cancer, and discusses important areas for future investigation.

Regulation of Human ADAM 12 Protease by the Prodomain EVIDENCE FOR A FUNCTIONAL CYSTEINE SWITCH

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

Molecular Profiling of ADAM12 in Human Bladder Cancer

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.

ADAM 12 protease induces adipogenesis in transgenic mice.

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

Proteomic maps of breast cancer subtypes.

Systems-wide profiling of breast cancer has almost always entailed RNA and DNA analysis by microarray and sequencing techniques. Marked developments in proteomic technologies now enable very deep profiling of clinical samples, with high identification and quantification accuracy. We analysed 40 oestrogen receptor positive (luminal), Her2 positive and triple negative breast tumours and reached a quantitative depth of >10,000 proteins. These proteomic profiles identified functional differences between breast cancer subtypes, related to energy metabolism, cell growth, mRNA translation and cell–cell communication. Furthermore, we derived a signature of 19 proteins, which differ between the breast cancer subtypes, through support vector machine (SVM)-based classification and feature selection. Remarkably, only three proteins of the signature were associated with gene copy number variations and eleven were also reflected on the mRNA level. These breast cancer features revealed by our work provide novel insights that may ultimately translate to development of subtype-specific therapeutics.

ADAM12 Alleviates the Skeletal Muscle Pathology in mdx Dystrophic Mice

Muscular dystrophy is characterized by muscle degeneration and insufficient regeneration and replacement of muscle fibers by connective tissue. New therapeutic strategies directed toward various forms of muscular dystrophy are needed to preserve muscle mass and promote regeneration. In this study we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals, as evidenced by less muscle cell necrosis and inflammation, lower levels of serum creatine kinase, and less uptake of Evans Blue dye into muscle fibers. These studies demonstrate that ADAM12 directly or indirectly contributes to muscle cell regeneration, stability, and survival.