Preferred SH3 domain partners of ADAM metalloproteases include shared and ADAM-specific SH3 interactions.

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.

Myocardial infarction induces early increased remote ADAM8 expression of rat hearts after cardiac arrest.

BACKGROUND: A disintegrin and metalloproteinase-8 (ADAM8) is a potential surrogate of inflammation which has recently been associated with myocardial infarction. We evaluated in a rat cardiac transplantation model whether ischemia-reperfusion injury alone (IRI) or with early regional myocardial infarction (MI) would suffice to induce inflammatory myocardial remodeling and ADAM8 expression. MATERIAL AND METHODS: Isogenic heterotopic cardiac transplantation after cardiac arrest was performed to 48 Fischer 344 rats to induce ischemia-reperfusion injury (IRI), of which 27 rats also underwent ligation of the left anterior coronary artery (LAD) of the heart to yield MI. Histology was performed at 0.5, 24 and 48 h after transplantation. ADAM8 was evaluated by qRT-PCR after graft harvesting. RESULTS: After 0.5 and 48 h respectively, edematous intramyocardial artery nuclei and periadventitial inflammation were more prominent in MI after transplantation, as compared with IRI alone and Controls (57.0 vs 40.0 and 5.0; 1.9 vs 1.1 and 0.9, point score units, p < 0.05, respectively). The expression of ADAM-8 was increased in MI as compared with Controls (1.9 vs 1.0, 1.9 fold increase) at 48 h. In grafts with MI, ADAM8 was localized using immunohistochemistry to the vicinity of the area corresponding to the developing infarction as well as in intramyocardial arteries remote to the infarction area. CONCLUSIONS: Remote histopathological changes of ischemic cardiac grafts are associated with increased expression of ADAM8 thus emphasizing a global myocardial impact of MI.

A disintegrin and metalloprotease -8 and -15 and susceptibility for ascending aortic dissection.

BACKGROUND. Dilatation of the ascending aorta (AA) is affected by extra-cellular matrix modifications and inflammation. A disintegrin and metalloproteases (ADAMs) may reveal differences between AA and ascending aortic dissection (AD). We characterized the inflammatory histology of AD and AA and examined the role of ADAM8 and -15 in these diseases. MATERIAL AND METHODS. Aortic wall histology and immunohistochemistry for leukocytes, T- and B-lymphocytes, plasma cells, macrophages, endothelial cells, smooth muscle cells, cell proliferation, elastase and Van-Gieson-staining were performed to 40 consecutive patients that underwent surgery for AA or AD. The expressions of ADAM8 and -15 mRNA and proteins were evaluated using QRT-PCR and immunohistochemistry. RESULTS. Thirty-four patients were enrolled, of which 29 had AA and five had AD of the ascending aorta. B-cells throughout the aortic wall and intimal plasma cells were more numerous during AD as compared with AA (p < 0.05). The gene expressions for ADAM8 and -15 were notably lower in AA as compared with AD. The median for down-regulation of ADAM8 and -15 in AA was -2.7 and -1.8, respectively. ADAM8 and -15 were mainly found in the media layer in patients with AD. Two of the patients with AA and increased ADAMs developed AD of the remaining aorta. CONCLUSIONS. The involvement of ADAM8 and -15 together with inflammation consisting of B-cells may indicate active remodelling of the aortic wall leading to AD.

Common variation in the ADAM8 gene affects serum sADAM8 concentrations and the risk of myocardial infarction in two independent cohorts.

OBJECTIVE: The single nucleotide polymorphism (SNP) rs2995300 in the metalloproteinase-disintegrin gene ADAM8 has been shown to affect the areas of complicated coronary plaques and the risk of fatal myocardial infarction (MI) in men. This study was set up to further investigate the role of ADAM8 in MI. AIM: To investigate the possible association of the ADAM8 SNPs rs2995300 and rs2275725 with ADAM8 mRNA levels, serum soluble ADAM8 (sADAM8) concentrations, and MI risk. METHODS: Samples from the Finnish cardiovascular study (FINCAVAS, N=2156) and the angiography and genes study (ANGES, N=1000) were genotyped. Serum sADAM8 concentrations were determined with ELISA (N=443). ADAM8 mRNA levels in atherosclerotic plaques were analysed from the tampere vascular study (TVS, N=53) samples. RESULTS: A significantly increased MI risk for carriers of the rs2995300C allele and the rs2275725 A allele was revealed in the meta-analysis of the ANGES and FINCAVAS patient data (OR=1.42, P<0.001 and OR=1.43, P<0.001). The risk increase was comparable to that caused by smoking in these cohorts. The risk allele carriers also had higher sADAM8 serum concentrations. CONCLUSIONS: The risk alleles of the investigated ADAM8 SNPs were associated with elevated sADAM8 serum levels and MI risk. The present results implicate ADAM8 in the development of CVDs and suggest its prognostic and therapeutic potential.

Alternative splicing of ADAM15 regulates its interactions with cellular SH3 proteins.

A Disintegrin And Metalloprotease (ADAM15) is a member of the adamalysin protein family and has been associated with cancer, possibly via its role in ectodomain shedding of cadherins. Alternative mRNA splicing generates several ADAM15 isoforms containing different combinations of putative Src homology-3 (SH3) domain binding sites in their cytosolic tails. Here we present a comprehensive characterization of SH3 binding potential of different ADAM15 isoforms. Alternative use of ADAM15 exons was found to profoundly influence selection of SH3-containing cellular partner proteins, including the avid interactions with nephrocystin and sorting nexin-33 (SNX33 a.k.a. SNX30). Specifically, strong co-precipitation of nephrocystin from cell lysates was specific to ADAM15 isoforms i4, i5, and i6. These isoforms contain one or both of the two almost identical proline-rich regions encoded by exons 20 and 21, wherein the residues RxLPxxP were found to be indispensable for nephrocystin SH3 binding. Similarly, robust cellular association with SNX33 was observed only for ADAM15 isoforms containing the most carboxyterminal proline cluster lacking in isoforms i1 and i3. Thus, alternative mRNA splicing provides a versatile mechanism for regulation of intracellular protein interactions and thereby likely the cellular functions of ADAM15, which could explain the association with cancer of some but not all ADAM15 isoforms.

ADAM8 and its single nucleotide polymorphism 2662 T/G are associated with advanced atherosclerosis and fatal myocardial infarction: Tampere vascular study.

OBJECTIVE: Previously, we scanned all 23,000 human genes for differential expression between normal and atherosclerotic tissues and found the involvement of ADAM8. METHODS: We investigated the expression of ADAM8 mRNA and protein level in human atherosclerotic tissues and non-atherosclerotic internal thoracic arteries as well as the association of ADAM8 2662 T/G single nucleotide polymorphism (SNP) with the extent of coronary atherosclerosis and with the risk of fatal myocardial infarction. RESULTS: ADAM8 mRNA was up-regulated in carotid, aortic, and femoral atherosclerotic plaques (n=24) when compared with non-atherosclerotic arteries. ADAM8 protein expression was increased in advanced atherosclerotic plaques as compared to control vessels wherein it was localized to macrophages and smooth muscle cells The G allele carriers of the ADAM8 2662 T/G SNP had significantly larger areas of fibrotic, calcified, and complicated plaques in coronary arteries (P=0.027, P=0.011, and P=0.011, respectively) and significantly higher occurrence of myocardial infarction (MI) (P=0.004) and fatal pre-hospital MI (P=0.003) than did the TT homozygotes. CONCLUSION: ADAM8 is a promising candidate to be involved in atherosclerosis, and its 2662 T/G allelic variant significantly associates with advanced atherosclerotic lesion areas and MI.

ADAM-9, ADAM-15, and ADAM-17 are upregulated in macrophages in advanced human atherosclerotic plaques in aorta and carotid and femoral arteries--Tampere vascular study.

BACKGROUND AND AIMS: The expression of disintegrin and metalloprotease ADAM-9, ADAM-15, and ADAM-17 has been associated with cell-cell, cell-platelet, and cell-matrix interactions and inflammation. They are possibly implicated in the pathophysiology of atherosclerosis. METHODS AND RESULTS: Whole-genome expression array and quantitative real-time polymerase chain reaction (PCR) analysis confirmed that ADAM-9, ADAM-15, and ADAM-17 are upregulated in advanced human atherosclerotic lesions in samples from carotid, aortic, and femoral territories compared to samples from internal thoracic artery (ITA) free of atherosclerotic plaques. Western analysis indicated that the majority of these ADAMs were in the catalytically active form. ADAM-9, ADAM-15, and ADAM-17-expressing cells were shown to co-localize with CD68-positive cells of monocytic origin in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Co-localization was demonstrated in all vascular territories. In the carotid territory, cells expressing the ADAMs co-distributed also with smooth muscle cells and, in femoral territory, with CD31-positive endothelial cells, indicating that the ADAM expression pattern depends on vascular bed territory. CONCLUSIONS: Present findings provide strong evidence for the involvement of catalytically active ADAM-9, ADAM-15, and ADAM-17 in advanced atherosclerosis, most notably associated with cells of monocytic origin.

ADAM15 gene structure and differential alternative exon use in human tissues.

BACKGROUND: ADAM15 is a metalloprotease-disintegrin implicated in ectodomain shedding and cell adhesion. Aberrant ADAM15 expression has been associated with human cancer and other disorders. We have previously shown that the alternative splicing of ADAM15 transcripts is mis-regulated in cancer cells. To gain a better understanding of ADAM15 regulation, its genomic organization and regulatory elements as well as the alternative exon use in human tissues were characterized. RESULTS: Human ADAM15, flanked by the FLJ32785/DCST1 and ephrin-A4 genes, spans 11.4 kb from the translation initiation codon to the polyadenylation signal, being the shortest multiple-exon ADAM gene. The gene contains 23 exons varying from 63 to 316 bp and 22 introns from 79 to 1283 bp. The gene appeared to have several transcription start sites and their location suggested the promoter location within a CpG island proximal to the translation start. Reporter expression experiments confirmed the location of functional GC-rich, TATAless and CAATless promoter, with the most critical transcription-supporting elements located -266 to -23 bp relative to the translation start. Normal human tissues showed different complex patterns of at least 13 different ADAM15 splice variants arising from the alternative use of the cytosolic-encoding exons 19, 20a/b, and 21a/b. The deduced ADAM15 protein isoforms have different combinations of cytosolic regulatory protein interaction motifs. CONCLUSION: Characterization of human ADAM15 gene and identification of elements involved in the regulation of transcription and alternative splicing provide important clues for elucidation of physiological and pathological roles of ADAM15. The present results also show that the alternative exon use is a physiological post-transcriptional mechanism regulating ADAM15 expression in human tissues.

Shedding light on ADAM metalloproteinases.

ADAM metalloproteinase disintegrins have emerged as the major proteinase family that mediates ectodomain shedding, the proteolytic release of extracellular domains from their membrane-bound precursors. Recent gene-manipulation studies have established the role of ADAM-mediated shedding in mammalian physiology and, in addition, raised the issue of functional redundancy among ADAM sheddases. ADAM sheddases activate, for example, growth factors and cytokines, thus regulating signalling pathways that are important in development and pathological processes such as cancer. The recent studies have also begun to elucidate the substrate specificity and the mechanisms that control ADAM-mediated shedding events that regulate, for example, growth-factor and Notch signalling, and the processing of the amyloid precursor protein.

Aberrant alternative exon use and increased copy number of human metalloprotease-disintegrin ADAM15 gene in breast cancer cells.

ADAM genes have been associated with cancer, with ADAM expression, genomic rearrangements, and, by implication of ADAM proteins in the altered behavior found in tumor cells. In the present study, increased copy number of the ADAM15 gene in human breast cancer cell lines was demonstrated by fluorescence in situ hybridization. This was not reflected in mRNA levels, however. Instead, the use of alternative ADAM15 exons appeared erratic, leading to aberrant combinations of ADAM15 mRNA isoforms in the cancer cells. Clustering analysis indicated that these isoform patterns were nonrandom, suggesting a failure in the regulation mechanism or mechanisms of the alternative exon usage. Altered regulation of alternative exon usage may provide a useful target for cancer diagnostics development. ADAM15 would be particularly appropriate for breast cancer diagnostics because the various combinations of its three alternatively used exons can be readily examined with a simple, straightforward PCR protocol.