Caroline J. Pennington

The ADAM metalloproteinases

Abstract The ADAMs (a disintegrin and metalloproteinase) are a fascinating family of transmembrane and secreted proteins with important roles in regulating cell phenotype via their effects on cell adhesion, migration, proteolysis and signalling. Though all ADAMs contain metalloproteinase domains, in humans only 13 of the 21 genes in the family encode functional proteases, indicating that at least for the other eight members, protein–protein interactions are critical aspects of their biological functions. The functional ADAM metalloproteinases are involved in “ectodomain shedding” of diverse growth factors, cytokines, receptors and adhesion molecules. The archetypal activity is shown by ADAM-17 (tumour necrosis factor-α convertase, TACE), which is the principal protease involved in the activation of pro-TNF-α, but whose sheddase functions cover a broad range of cell surface molecules. In particular, ADAM-17 is required for generation of the active forms of Epidermal Growth Factor Receptor (EGFR) ligands, and its function is essential for the development of epithelial tissues. Several other ADAMs have important sheddase functions in particular tissue contexts. Another major family member, ADAM-10, is a principal player in signalling via the Notch and Eph/ephrin pathways. For a growing number of substrates, foremost among them being Notch, cleavage by ADAM sheddases is essential for their subsequent “regulated intramembrane proteolysis” (RIP), which generates cleaved intracellular domains that translocate to the nucleus and regulate gene transcription. Several ADAMs play roles in spermatogenesis and sperm function, potentially by effecting maturation of sperm and their adhesion and migration in the uterus. Other non-catalytic ADAMs function in the CNS via effects on guidance mechanisms. The ADAM family are thus fundamental to many control processes in development and homeostasis, and unsurprisingly they are also linked to pathological states when their functions are dysregulated, including cancer, cardiovascular disease, asthma, Alzheimer’s disease. This review will provide an overview of current knowledge of the human ADAMs, discussing their structure, function, regulation and disease involvement.

The role of chondrocyte senescence in osteoarthritis

Replicative senescence occurs when normal somatic cells stop dividing. Senescent cells remain viable, but show alterations in phenotype, e.g. altered expression of matrix metalloproteinases (MMPs); these enzymes are known to be involved in cartilage destruction. It is assumed that cells deplete their replicative potential during aging, and age is a major risk factor for osteoarthritis (OA). Therefore, we hypothesized that chondrocytes in aging or diseased cartilage become senescent with associated phenotypic changes contributing to development or progression of OA.

Histone deacetylase inhibitors modulate metalloproteinase gene expression in chondrocytes and block cartilage resorption

Cartilage destruction in the arthritides is thought to be mediated by two main enzyme families: the matrix metalloproteinases (MMPs) are responsible for cartilage collagen breakdown, and enzymes from the ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) family mediate cartilage aggrecan loss. Many genes subject to transcriptional control are regulated, at least in part, by modifications to chromatin, including acetylation of histones. The aim of this study was to examine the impact of histone deacetylase (HDAC) inhibitors on the expression of metalloproteinase genes in chondrocytes and to explore the potential of these inhibitors as chondroprotective agents. The effects of HDAC inhibitors on cartilage degradation were assessed using a bovine nasal cartilage explant assay. The expression and activity of metalloproteinases was measured using real-time RT-PCR, western blot, gelatin zymography, and collagenase activity assays using both SW1353 chondrosarcoma cells and primary human chondrocytes. The HDAC inhibitors trichostatin A and sodium butyrate potently inhibit cartilage degradation in an explant assay. These compounds decrease the level of collagenolytic enzymes in explant-conditioned culture medium and also the activation of these enzymes. In cell culture, these effects are explained by the ability of HDAC inhibitors to block the induction of key MMPs (e.g. MMP-1 and MMP-13) by proinflammatory cytokines at both the mRNA and protein levels. The induction of aggrecan-degrading enzymes (e.g. ADAMTS4, ADAMTS5, and ADAMTS9) is also inhibited at the mRNA level. HDAC inhibitors may therefore be novel chondroprotective therapeutic agents in arthritis by virtue of their ability to inhibit the expression of destructive metalloproteinases by chondrocytes.

Expression analysis of the entire MMP and TIMP gene families during mouse tissue development

Matrix metalloproteinases (MMPs) and adamalysins (ADAMs) cleave many extracellular proteins, including matrix, growth factors, and receptors. We profiled the RNA levels of every MMP, several ADAMs, and inhibitors of metalloproteinases (TIMPs and RECK) in numerous mouse tissues during development and in the uterus during pregnancy. Observations include: most secreted MMPs are expressed at low to undetectable levels in tissues, whereas membrane‐bound MMPs, ADAMs and inhibitors are abundant; almost every proteinase and inhibitor is present in the uterus or placenta at some time during gestation; the mouse collagenases mColA and mColB are found exclusively in the uterus and testis; and each tissue has its unique signature of proteinase and inhibitor expression.

Identification of degradome components associated with prostate cancer progression by expression analysis of human prostatic tissues

Extracellular proteases of the matrix metalloproteinase (MMP) and serine protease families participate in many aspects of tumour growth and metastasis. Using quantitative real-time RT–PCR analysis, we have undertaken a comprehensive survey of the expression of these enzymes and of their natural inhibitors in 44 cases of human prostate cancer and 23 benign prostate specimens. We found increased expression of MMP10, 15, 24, 25 and 26, urokinase plasminogen activator-receptor (uPAR) and plasminogen activator inhibitor-1 (PAI1), and the newly characterised serine proteases hepsin and matriptase-1 (MTSP1) in malignant tissue compared to benign prostate tissue. In contrast, there was significantly decreased expression of MMP2 and MMP23, maspin, and the protease inhibitors tissue inhibitor of metalloproteinase 3 (TIMP3), TIMP4 and RECK (reversion-inducing cysteine-rich protein with Kazal motifs) in the cancer specimens. The expression of MMP15 and MMP26 correlated positively with Gleason score, whereas TIMP3, TIMP4 and RECK expression correlated negatively with Gleason score. The cellular localisation of the expression of the deregulated genes was evaluated using primary malignant epithelial and stromal cell cultures derived from radical prostatectomy specimens. MMP10 and 25, hepsin, MTSP1 and maspin showed predominantly epithelial expression, whereas TIMP 3 and 4, RECK, MMP2 and 23, uPAR and PAI1 were produced primarily by stromal cells. These data provide the first comprehensive and quantitative analysis of the expression and localisation of MMPs and their inhibitors in human prostate cancer, leading to the identification of several genes involved in proteolysis as potential prognostic indicators, in particular hepsin, MTSP1, MMP26, PAI1, uPAR, MMP15, TIMP3, TIMP4, maspin and RECK.

Determinants of Human B Cell Migration Across Brain Endothelial Cells

Circulating B cells enter the CNS as part of normal immune surveillance and in pathologic states, including the common and disabling illness multiple sclerosis. However, little is known about the molecular mechanisms that mediate human B cell interaction with the specialized brain endothelial cells comprising the blood-brain barrier (BBB). We studied the molecular mechanisms that regulate the migration of normal human B cells purified ex vivo, across human adult brain-derived endothelial cells (HBECs). We found that B cells migrated across HBECs more efficiently than T cells from the same individuals. B cell migration was significantly inhibited by blocking Abs to the adhesion molecules ICAM-1 and VLA-4, but not VCAM-1, similar to the results previously reported for T cells. Blockade of the chemokines monocyte chemoattractant protein-1 and IL-8, but not RANTES or IFN-γ-inducible protein-10, significantly inhibited B cell migration, and these results were correlated with the chemokine receptor expression of B cells measured by flow cytometry and by RNase protection assay. Tissue inhibitor of metalloproteinase-1, a natural inhibitor of matrix metalloproteinases, significantly decreased B cell migration across the HBECs. A comprehensive RT-PCR comparative analysis of all known matrix metalloproteinases and tissue inhibitors of metalloproteinases in human B and T cells revealed distinct profiles of expression of these molecules in the different cell subsets. Our results provide insights into the molecular mechanisms that underlie human B cell migration across the BBB. Furthermore, they identify potential common, and unique, therapeutic targets for limiting CNS B cell infiltration and predict how therapies currently developed to target T cell migration, such as anti-VLA-4 Abs, may impact on B cell trafficking.

Metalloproteinase inhibitor TIMP‐1 affects hepatocyte cell cycle via HGF activation in murine liver regeneration

Liver regeneration depends on timely restoration of cellular mass while orchestrating structural matrix remodeling. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the extracellular matrix (ECM) turnover and, more recently, the processing of growth factors and cytokines. We have previously demonstrated that TIMP‐1 inhibits preneoplastic hepatocyte proliferation by attenuating growth factor bioavailability. In the present study, we examined the role of TIMP‐1 in de novo hepatocyte cell division during liver regeneration. Comprehensive real‐time reverse‐transcriptase polymerase chain reaction analyses of regenerating livers revealed significant inductions in the messenger RNA of TIMP‐1, TIMP‐3, TIMP‐4, MMP‐2, MMP‐9, MMP‐13, MMP‐14, and MMP‐24, while MMP‐15 expression was significantly reduced. Induction of TIMP‐1 occurred during the peak of hepatocyte DNA synthesis. Studies using genetically altered mice revealed that TIMP‐1 loss of function accelerated hepatocyte cell cycle progression. This finding was demonstrated by earlier expression of cyclin D1, proliferating cell nuclear antigen, and phosphorylated histone H3, which mark the G1‐S, S, and M phase, respectively. Conversely, TIMP‐1 gain of function delayed cell cycle progression. MMP activity was increased in the absence of Timp‐1. Examination of hepatocyte growth factor (HGF), and its receptor Met, both of which provide a mitogenic signal for hepatocyte division, showed increased HGF activity in Timp‐1−/−–regenerating livers. HGF is released from the ECM and is proteolytically processed to its active form. Active HGF was elevated in Timp‐1−/− mice, leading to increased immunostaining of phosphorylated Met as well as activation of a downstream effector, p38. In conclusion, TIMP‐1 is a novel negative regulator of HGF activity during liver regeneration. (HEPATOLOGY 2005.)

Matrix Metalloproteinase-8 Functions as a Metastasis Suppressor through Modulation of Tumor Cell Adhesion and Invasion

Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human.

Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling.

Balanced expression of proteases and their inhibitors is one prerequisite of tissue homeostasis. Metastatic spread of tumor cells through the organism depends on proteolytic activity and is the death determinant for cancer patients. Paradoxically, increased expression of tissue inhibitor of metalloproteinases-1 (TIMP-1), a natural inhibitor of several endometalloproteinases, including matrix metalloproteinases and a disintegrin and metalloproteinase-10 (ADAM-10), in cancer patients is negatively correlated with their survival, although TIMP-1 itself inhibits invasion of some tumor cells. Here, we show that elevated stromal expression of TIMP-1 promotes liver metastasis in two independent tumor models by inducing the hepatocyte growth factor (HGF) signaling pathway and expression of several metastasis-associated genes, including HGF and HGF-activating proteases, in the liver. We also found in an in vitro assay that suppression of ADAM-10 is in principle able to prevent shedding of cMet, which may be one explanation for the increase of cell-associated HGF receptor cMet in livers with elevated TIMP-1. Similar TIMP-1-associated changes in gene expression were detected in livers of patients with metastatic colorectal cancer. The newly identified role of TIMP-1 to create a prometastatic niche may also explain the TIMP-1 paradoxon.

Cutting Edge: The Metalloproteinase ADAM17/TNF-α-Converting Enzyme Regulates Proteolytic Shedding of the MHC Class I-Related Chain B Protein

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.