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Tissue development is orchestrated by the coordinated activities of both chemical and physical regulators. While much attention has been given to the role that chemical regulators play in driving development, researchers have recently begun to elucidate the important role that the mechanical properties of the extracellular environment play. For instance, the stiffness of the extracellular environment has a role in orienting cell division, maintaining tissue boundaries, directing cell migration, and driving differentiation. In addition, extracellular matrix stiffness is important for maintaining normal tissue homeostasis, and when matrix mechanics become imbalanced, disease progression may ensue. In this article, we will review the important role that matrix stiffness plays in dictating cell behavior during development, tissue homeostasis, and disease progression. [ABSTRACT FROM PUBLISHER]

A recent paper demonstrated that decellularized extracellular matrix (DECM) deposited by synovium-derived stem cells (SDSCs), especially from fetal donors, could rejuvenate human adult SDSCs in both proliferation and chondrogenic potential, in which expanded cells and corresponding culture substrate (such as DECM) were found to share a mutual reaction in both elasticity and protein profiles (see ref.1). It seems that young DECM may assist in the development of culture strategies that optimize proliferation and maintain “stemness” of mesenchymal stem cells (MSCs), helping to overcome one of the primary difficulties in MSC-based regenerative therapies. In this paper, the effects of age on the proliferative capacity and differentiation potential of MSCs are reviewed, along with the ability of DECM from young cells to rejuvenate old cells. In an effort to highlight some of the potential molecular mechanisms responsible for this phenomenon, we discuss age-related changes to extracellular matrix (ECM)'s physical properties and chemical composition. [ABSTRACT FROM PUBLISHER]

Abstract: Matrix metalloproteinase-9 (MMP-9) plays an important role in mediating the invasion and angiogenic process of malignant gliomas. This study was undertaken to determine if an isoflavone metabolite, irisolidone, inhibits MMP-9 expression in human astroglioma cells. Irisolidone was found to inhibit the secretion and protein expression of MMP-9 induced by PMA in U87 MG glioma cells, accompanied by the inhibition of MMP-9 mRNA expression and promoter activity. Further mechanistic studies revealed that irisolidone inhibits the binding of NF-κB and AP-1 to the MMP-9 promoter and suppresses the PMA-induced phosphorylation of ERK and JNK, which are upstream signaling molecules in MMP-9 expression. The Matrigel-invasion assay showed that irisolidone suppresses the in vitro invasiveness of glioma cells. Therefore, the strong inhibition of MMP-9 expression by irisolidone might be a potential therapeutic modality for controlling the growth and invasiveness of gliomas. [Copyright &y& Elsevier]

Abstract: We studied the effect of TNF-α stimulation on a cholangiocarcinoma cell line, CCKS1. CCKS1 expressed only one type TNF receptor, TNFR2. Treatment of CCKS1 with TNF-α substantially activated NFκB, MAPK and Akt signalings which in turn activated matrix metalloproteinase-9 (MMP-9) secretion and in vitro invasiveness of CCKS1. Pretreatment of cells with anti-TNFR2 neutralizing antibody inhibited the TNF-α-dependent signaling and MMP-9 secretion and subsequently blocked invasion in vitro. Moreover, an inhibitor for matrix metalloproteinase, Galardin, suppressed the invasion in a dose-dependent manner. Similarly, pharmacological inhibition of signaling clearly suppressed the TNF-α dependent MMP-9 secretion. These results strongly suggest that TNF-α-TNFR2 signaling plays an important role to convert the cholangiocarcinoma cells to be more aggressive one. [Copyright &y& Elsevier]

Our previous studies demonstrated that a synthetic peptide encompassing residues 185–203 of the noncollagenous (NC1) domain of the α3 chain of type IV collagen, named tumstatin, inhibits in vitro melanoma cell proliferation and migration. In the present study, B16F1 melanoma cells were stably transfected to overexpress the complete tumstatin domain (Tum 1–232) or its C-terminal part, encompassing residues 185–203 (Tum 183–232). Tumstatin domain overexpression inhibited B16F1 in vitro cell proliferation, anchorage-independent growth, and invasive properties. For studying the in vivo effect of overexpression, representative clones were subcutaneously injected into the left side of C57BL6 mice. In vivo tumor growth was decreased by −60% and −56%, respectively, with B16F1 cells overexpressing Tum 1–232 or Tum 183–232 compared to control cells. This inhibitory effect was associated with a decrease of in vivo cyclin D1 expression. We also demonstrated that the overexpression of Tum 1–232 or Tum 183–232 induced an in vivo down-regulation of proteolytic cascades involving matrix metalloproteinases (MMPs), especially the production or activation of MMP-2, MMP-9, MMP-13, as well as MMP-14. The plasminogen activation system was also altered in tumors with a decrease of urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA) and a strong increase of plasminogen activator inhibitor-1 (PAI-1). Collectively, our results demonstrate that tumstatin or its C-terminal antitumor fragment, Tum 183–232, inhibits in vivo melanoma progression by triggering an intracellular transduction pathway, which involves a cyclic AMP (cAMP)-dependent mechanism. [Copyright &y& Elsevier]

The mechanism that regulates the plasticity of bone marrow cells (BMCs) into hepatocytes is poorly understood. We developed a green fluorescent protein/carbon tetrachloride model to find that BMC transplantation recovered liver damage. Serum albumin level and liver fibrosis were recovered by BMC transplantation. To understand the mechanism, we used DNA-chip technology to profile the change of transient gene expression before and after BMC transplantation. On the basis of gene expression with self-organizing map using specific equation, genes were classified into 153 clusters. The information is useful to understand the dramatic gene activation during the process of the plasticity of BMC. [Copyright &y& Elsevier]

Matrix metalloproteinases (MMPs) are a family of neutral proteases with the ability to degrade all components of extracellular matrix. To date, more than 24 different human MMPs have been identified. MMP activity is important in diseases such as arthritis, atherosclerosis, periodontal diseases and cancer. Recent data suggest that MMPs are involved in tissue injury and healing in the human gut. [Copyright &y& Elsevier]

Receptor tyrosine kinases (RTKs) are transactivated by the stimulation of G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P), a ligand of GPCR, is known as a tumor-promoting lipid, but its signaling pathways are not fully understood. We here demonstrated that S1P induces rapid and transient tyrosine phosphorylation of epidermal growth factor receptor (EGFR) and c-Met in gastric cancer cells, both of which have been proposed as prognostic markers of gastric cancers. The pathway of S1P-induced c-Met transactivation is Gi-independent and matrix metalloproteinase-independent, which differs from that of EGFR transactivation. Our results indicate that S1P acts upstream of various RTKs and thus may act as a potent stimulator of gastric cancer. [Copyright &y& Elsevier]

In an attempt to characterize changes in transcription after a sub-chronic spinal cord injury (SCI), we investigated gene expression profiles using cDNA microarray. Among 7523 genes and expressed sequence tags (ESTs) examined, 444 transcripts, including 218 genes and 226 ESTs, were identified to be either up-regulated (373 of 444) or down-regulated (71 of 444) greater than 2.0-fold in the spinal cord at 14 days after a complete spinal transection at the 11th thoracic level in adult rats. Based on their potential function, these differentially expressed genes were categorized into seven classes which include cell division-related protein, channels and receptors, cytoskeletal elements, extracellular matrix proteins, metalloproteinases and inhibitors, growth-associated molecules, metabolism, intracellular transducers and transcription factors, as well as others. Strong expressional changes were found in all classes revealing the complexity and diversity of gene expression profiles following SCI. We verified array results with RT-PCR for eight genes, Northern blotting for nine genes, and in situ hybridization for one gene and immunohistochemistry for four genes. These analyses confirmed, to a large extent, that the array results have accurately reflected the molecular changes occurring at 14 days post-SCI. Importantly, the current study has identified a number of genes, including annexins, heparin-binding growth-associated protein (HB-GAM), P9ka (S100A4), matrix metalloproteinases, and lysozyme, that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using the large-scale microarray analysis may lead to a better understanding of underlying mechanisms, thus, the development of new repair strategies for SCI. [Copyright &y& Elsevier]

The inhibition of carcinogenesis by tea and tea polyphenols has been demonstrated in different animal models by many investigators. The mechanisms of this inhibitory activity have also been investigated extensively, mostly in cell culture systems, but no clear conclusion can be reached concerning the cancer preventive mechanisms in vivo. In this article, we reviewed the possible mechanisms, which include the inhibition of specific protein kinase activities, blocking receptor-mediated functions, and inhibition of proteases. These events may lead to cell cycle regulation, growth inhibition, enhanced apoptosis, inhibition of angiogenesis, and inhibition of invasion and metastases. The possible complications of translating results obtained in cell culture studies to animals and humans are discussed. It is likely that multiple signal transduction pathways are involved in the inhibition of carcinogenesis by tea constituents. The relative importance of these pathways needs to be determined in vivo. [Copyright &y& Elsevier]

We studied the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in fibrosis formation in the transition from hypertrophy to heart failure (HF) as well as the cellular source of MMPs and TIMPs.Human pressure-overloaded hearts are characterized by a significant increase in cardiac fibrosis. However, the contribution of the proteolytic/antiproteolytic system in aortic stenosis (AS) during hypertrophy progression has not yet been elucidated.Three groups of AS patients (I: EF >50%, n = 12; II: EF 50% to 30%, n = 10; III: EF <30%, n = 12) undergoing aortic valve replacement and seven controls were studied. Tissue samples were investigated by immunoconfocal microscopy, Western blotting, and zymography.Quantitative analysis by immunoconfocal microscopy and Western blotting showed an upregulation of MMP-1, -2, -3, -9, -13, and -14 in group I and further increases in later stages. Tissue inhibitors of metalloproteinase-1 and -2 were enhanced and TIMP-4 was decreased in comparison to control. Gelatinolytic activity of MMP-2 significantly (p < 0.05) increased 1.2-fold (group I), 1.5-fold (group II), and 1.6-fold (group III) over control. The level of collagen I was significantly upregulated in all AS groups. Immunoconfocal microscopy showed that MMPs and TIMPs are produced predominantly by fibroblasts. The number of proliferating fibroblasts was significantly elevated during the transition to HF (0.67 n/mm2-control, 5.03-group III, p < 0.05).In human hearts a continuous turnover of the extracellular matrix occurs during the progression from compensated hypertrophy to HF that is characterized by the upregulation of MMPs and inadequate inhibition by TIMPs. The altered balance between proteolysis/antiproteolysis with accompanying proliferation of fibroblasts results in fibrosis progression. [Copyright &y& Elsevier]

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications. [Copyright &y& Elsevier]

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial. [Copyright &y& Elsevier]

Matrix metalloproteinases (MMPs) are endopeptidases that contribute to growth, development and wound healing as well as to pathologies such as arthritis and cancer. Until recently, it has been thought that MMPs participate in these processes simply by degrading extracellular matrix (ECM) molecules. However, it is now clear that MMP activity is much more directed and causes the release of cryptic information from the ECM. By precisely cleaving large insoluble ECM components and ECM-associated molecules, MMPs liberate bioactive fragments and growth factors and change ECM architecture, all of which influence cellular behavior. Thus, MMPs have become a focal point for understanding matrix biology. [Copyright &y& Elsevier]

Delivery of growth factors for tissue (e.g. bone, cartilage) or cell repair (e.g. nerves) is about to gain important potential as a future therapeutic tool. Depending on the targeted cell type and its state of differentiation, growth factors can activate or regulate a variety of cellular functions. Therefore, strictly localized delivery regimens at well-defined kinetics appear to be logical prerequisites to assure safe and efficacious therapeutic use of such factors and avoid unwanted side effects and toxicity, a major hurdle in the clinical development of growth factor therapies so far. This review summarizes various approaches for localized growth factor delivery as focused on bone repair. Similar considerations may apply to other growth factors and therapeutic indications. Considering the vast number of preclinical studies reported in the area of growth factor-assisted bone repair, it surprises though that only two medical products for bone repair have so far been commercialized, both consisting of a collagen matrix impregnated with a bone morphogenetic protein. The marked diversity of the reported growth factors, delivery concepts and not yet standardized animal models adds to the complexity to learn from past preclinical studies presented in the literature. Nonetheless, it is now firmly established from the available information that the type, dose and delivery kinetics of growth factors all play a decisive role for the therapeutic success of any such approach. Very likely, all of these parameters have to be adapted and optimized for each animal model or clinical case. In the future, systems for localized growth factor delivery thus need to be designed in such a way that their modular components are readily adaptable to the individual pathology. To make such customized systems feasible, close cooperative networks of biomedical and biomaterials engineers, pharmaceutical scientists, chemists, biologists and clinicians need to be established. [Copyright &y& Elsevier]

Background/Aims: Several studies have indicated increased expression of the Ras protooncogenes in liver cirrhosis. In a previous study in rats, we have shown that a synthetic Ras antagonist, S-farnesylthiosalicylic acid (FTS), could inhibit the development of liver cirrhosis. The aim of the current study was to examine whether FTS will accelerate the resolution of liver cirrhosis induced in rats by thioacetamide.Methods: Cirrhosis was induced in male Wistar rats by intraperitoneal (i.p.) administration of thioacetamide (200 mg/kg twice weekly for 12 weeks). In the treated group, the Ras antagonist FTS (5 mg/kg, i.p./3 times/week) was administered for 8 weeks after liver cirrhosis has already been established. Control cirrhotic rats received PBS injections for 8 weeks.Results: Rats treated with FTS for 8 weeks had lower histopathologic score of fibrosis (P=0.01), lower hepatic hydroxyproline levels (P=0.0002) and lower spleen weight (P=0.02) than the cirrhotic rats treated with PBS. Following FTS treatment, the MMP-2 and MMP-9-induced collagenolytic activity and TIMP-2 expression, were increased in FTS-compared to PBS-treated rats. TUNEL assay of liver sections performed 8 weeks after thioacetamide withdrawal showed increased apoptotic figures in both groups (P=NS).Conclusions: These results indicate that the Ras antagonist FTS accelerates the regression of experimentally-induced hepatic cirrhosis. The mechanism may involve increased collagenolytic activity. [Copyright &y& Elsevier]

The reversion-inducing–cysteine-rich protein with Kazal motifs (RECK) was initially isolated as a transformation-suppressor gene by expression cloning and found to encode a membrane-anchored regulator of the matrix metalloproteinases (MMPs). Experimental studies have shown that RECK can suppress tumour – invasion, metastasis and angiogenesis. However, the clinical impact of RECK remains unclear. To assess the clinical significance of RECK-expression in non-small cell lung cancer (NSCLC), a total of 171 patients with completely resected pathological stage (p-stage) I-IIIA NSCLC were retrospectively examined. Expression of RECK and vascular endothelial growth factor (VEGF) in tumour tissues was assessed by immunohistochemical staining (IHS). Intratumoural microvessel density (IMVD), a measurement of angiogenesis, was also determined by IHS using an anti-CD34 antibody. A significant inverse correlation between RECK-expression and tumour angiogenesis was documented; the mean IMVD in tumours with strong RECK-expression (157.1) was significantly lower than that observed in tumours with weak RECK-expression (194.5; P=0.008). Interestingly, this inverse correlation was seen only when VEGF was strongly expressed, which suggests that RECK could suppress the angiogenesis induced by VEGF. The 5-year survival rate for patients with tumours with strong RECK-expression (75.8%) was significantly higher than that for patients with weakly expressing tumours (54.3%; P=0.016). Subset analyses showed that the prognostic impact of RECK-status was evident in patients with either adenocarcinoma, poorly differentiated tumours, or p-stage IIIA disease. A multivariate analysis confirmed that reduced RECK-expression was an independent and significant factor in predicting a poor prognosis (P=0.009; Hazard ratio (HR), 0.474 with a 95% Confidence interval (CI) of 0.271–0.830). In conclusion, RECK-status is a significant prognostic factor correlated with tumour angiogenesis in NSCLC patients. [Copyright &y& Elsevier]

In this study, we focused on the influence of progesterone and its receptor in invasion and MMPs on endometrial carcinoma cells. The growth of Ishikawa cells, to which an progesterone receptor form B (PR-B) expressing vector was transfected, was inhibited by progesterone as was the inhibition of the expression of cyclin D1. By invasion assay, in conditions with progesterone, the invasiveness of Ishikawa cells was inhibited as well as the expression of (metalloproteinase) MMP-1, -2, -7 and -9 and Ets-1 decreased. These results suggest that activation of PR-B by progesterone results in tumor suppression by inhibiting cell growth and invasiveness via suppression of the expression of MMPs. [Copyright &y& Elsevier]

Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the ∼20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by ∼70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70–80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, ∼65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen α-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin. [Copyright &y& Elsevier]

Unstimulated human fibrosarcoma cells (HT1080) constitutively secrete matrix metalloproteinase 2 (MMP 2) as a proenzyme requiring proteolytic cleavage by membrane type-1 MMP (MT1 MMP) for activation. Physiological and pharmacological stimuli induce clustering of MT1 MMP/tissue inhibitor of MMP 2 “receptors”, promoting binding and activation of MMP 2. We now report that cholesterol depleted HT1080 cells accumulated MT1 MMP on the cell surface and activated MMP 2. A specific inhibitor of mitogen activated protein kinase kinase 1/2 inhibited both MMP 2 activation and extracellular signal-related kinase phosphorylation induced by cholesterol depletion. Our data indicate that the cholesterol content of unstimulated cells is critical for secretion of MMP 2 as an inactive zymogen and control of pericellular proteolysis. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Carbamylation refers to chemical modification of protein side chains by cyanate derived e.g. from urea. It alters their structural and functional properties. We have studied the influence of the carbamylation of type I collagen in vitro on its interactions with elutriated human monocytes, and its potential role in atherosclerosis. Adhesion of monocytes onto carbamylated collagen was significantly enhanced compared to native collagen. There was no change in superoxide anion production. On the other hand, there was an increase in the production and the activation of matrix metalloproteinase-9. No effect was found on tissue inhibitor of metalloproteinase-1 production. Thus, the presence of carbamylated collagen may stimulate the remodelling of extracellular matrix mediated by activated monocytes. Such alterations may contribute to enhanced atherosclerosis in renal insufficiency, a pathological condition associated with elevated levels of carbamylation. [Copyright &y& Elsevier]

Ischemic damage to the retina is a multifaceted process that results in irreversible loss of ganglion cells and blinding disease. Although the mechanisms underlying ischemia-induced ganglion cell death in the retina are not clearly understood, we have recently reported that retinal damage induced by ligation of the optic nerve results in increased matrix metalloproteinase-9 (MMP-9) synthesis and promotes ganglion cell loss. In this study, we have investigated the roles of IL-1beta and mitogen activated protein kinases in MMP-9 induction in the retina. Optic nerve ligation led to a transient increase in IL-1beta and MMP-9 levels and phosphorylation of p42/p44 mitogen activated protein kinases (extracellular signal-regulated kinases, ERK1 and ERK2) in the retina. We found no significant increase in phosphorylation of p38 MAP kinase or c-jun N-terminal kinases indicating that ERK1/2 plays a major role in MMP-9 induction. Intravitreal injection of IL-1 receptor antagonist (IL-1Ra) or MAP kinase inhibitor U0126 significantly decreased both ERK1/2 phosphorylation and MMP-9 induction suggesting that interruption of this cascade might attenuate retinal damage. In support of this, intravitreal injection of IL-1Ra and U0126 offered significant protection against optic nerve-induced retinal damage. These results suggest that optic nerve ligation-induced IL-1beta promotes retinal damage by increasing MMP-9 synthesis in the retina. [Copyright &y& Elsevier]

Membrane-type matrix metalloproteinases (MT-MMPs) have attracted strong attention, because four of them can activate a key player in the tumor scenario, proMMP-2/progelatinase A. In addition to this indirect effect on the cellular environment, these MT-MMPs degrade extracellular matrix proteins, and their overproduction is associated with tumor growth. We have solved the structure of the catalytic domain (cd) of MT3-MMP/MMP-16 in complex with the hydroxamic acid inhibitor batimastat. CdMT3–MMP exhibits a classical MMP-fold with similarity to MT1-MMP. Nevertheless, it also shows unique properties such as a modified MT-specific loop and a closed S1′ specificity pocket, which might help to design specific inhibitors. Some MT-MMP-specific features, derived from the crystal structures of MT-1-MMP determined previously and MT3-MMP, and revealed in recent mutagenesis experiments, explain the impaired interaction of the MT-MMPs with TIMP-1. Docking experiments with proMMP-2 show some exposed loops including the MT-loop of cdMT3–MMP involved in the interaction with the proMMP-2 prodomain in the activation encounter complex. This model might help to understand the experimentally proven importance of the MT-loop for the activation of proMMP-2. [Copyright &y& Elsevier]

Crk-associated substrate (Cas) is highly phosphorylated by v-Src and plays a critical role in v-Src-induced cell transformation. In this study, we found that the Src homology (SH) 3 domain of Cas blocked v-Src-stimulated anchorage-independent cell growth, Matrigel invasion, and tumor growth in nude mice. Biochemical analysis revealed that the Cas SH3 domain selectively inhibited v-Src-stimulated activations of AKT and JNK, but not ERK and STAT3. Attenuation of the AKT pathway by the Cas SH3 domain rendered v-Src-transformed cells susceptible to apoptosis. Inhibition of the JNK pathway by the Cas SH3 domain led to suppression of v-Src-stimulated invasion. Taken together, our results indicate that the Cas SH3 domain has an anti-tumor function, which severely impairs the transforming potential of v-Src. [Copyright &y& Elsevier]

Matrix metalloproteinases (MMPs) are a family of endopeptidases that degrade extracellular matrix components. Membrane-type 5 MMP (MT5-MMP/MMP-24) was identified as neuron-specific, and is believed to contribute to neuronal circuit formation and plasticity. To elucidate its function in vivo, we have generated mice lacking MT5-MMP by gene targeting. MT5-MMP-deficient mice were born without obvious morphological abnormalities. No apparent histological defects were observed in the nervous system either. However, MT5-MMP-deficient mice did not develop neuropathic pain with mechanical allodynia after sciatic nerve injury, though responses to acute noxious stimuli were normal. Neuropathic pain induced by peripheral nerve lesions is known to accompany structural reorganization of the nervous system. Intraneural injection of cholera toxin B subunit, a transganglionic tracer, into the injured sciatic nerve of wild-type mice revealed that the myelinated Aβ-fiber primary afferents sprouted from laminae III–VI of the dorsal horn of the spinal cord and invaded lamina II. However, no such sprouting and invasion of Aβ-fibers were observed in MT5-MMP-deficient mice. These findings suggest that MT5-MMP is essential for the development of mechanical allodynia and plays an important role in neuronal plasticity in this mouse model. [Copyright &y& Elsevier]

The inhibitor of angiogenesis, thrombospondin 2 (TSP2), belongs to a group of matricellular proteins that are induced in response to injury and modulate the healing of dermal wounds. Thus, TSP-2-null mice display abnormal connective tissue architecture and increased angiogenesis in the dermis, and heal wounds at an accelerated rate. In this study, we report that the content of TSP2 is increased in the uninjured skin of aged mice. Furthermore, in primary dermal fibroblasts, TSP2 expression is increased both as a function of the age of the donor and days in culture. To determine the significance of the increased TSP2 in aged mice (two years or older), we performed full-thickness excisional wounds and compared their healing in aged and young (3–4 months) wild-type and TSP2-null mice. Gross morphological examination of wounds indicated that aged TSP2-null mice healed faster than their aged wild-type counterparts, but healing in aged mice was always sub-optimal in comparison to that in young animals. Surprisingly, despite the increase in TSP2, a potent inhibitor of angiogenesis, in wounds in aged mice, the vascular density of these wounds was not reduced in comparison to that in young animals. However, immunohistochemical analysis of healing wounds revealed a shift in the peak content of TSP2, from day 10 in young mice to day 14 or later in aged mice, and there was a corresponding delay in the expected increase in matrix metalloproteinase (MMP) 2 levels in aged TSP2-null mice. We suggest that the delay in expression of TSP2 and MMP2 in the wounds of aged mice could contribute to their impaired rate of wound healing. [Copyright &y& Elsevier]

Aggrecanases are considered to play a key role in the destruction of articular cartilage during the progression of arthritis. Here we report that the N-terminal inhibitory domain of tissue inhibitor of metalloproteinases 3 (N-TIMP-3), but not TIMP-1 or TIMP-2, inhibits glycosaminoglycan release from bovine nasal and porcine articular cartilage explants stimulated with interleukin-1α or retinoic acid in a dose-dependent manner. This inhibition is due to the blocking of aggrecanase activity induced by the catabolic factors. Little apoptosis of primary porcine chondrocytes is observed at an effective concentration of N-TIMP-3. These results suggest that TIMP-3 may be a candidate agent for use against cartilage degradation. [Copyright &y& Elsevier]

Background/Aims: Hybrid bioartificial liver devices supporting a large mass of metabolically active hepatocytes are thought to be necessary for the successful treatment of patients with severe acute liver failure. However, it is very difficult to obtain cells with both growth activity and differentiated functions. Rat small hepatocytes (SHs), which are hepatic progenitor cells, can differentiate into mature hepatocytes and reconstruct a hepatic organoid by interacting with hepatic nonparenchymal cells (NPCs).Methods: Colonies of SHs were collected and replated on a collagen sponge. Hepatic functions were examined by ELISA, immunoblotting, and Northern blotting. Cells in the sponge were characterized by immunocytochemistry and transmission electron microscopy. Urea synthesis was measured and metabolization of fluorescein diacetate was examined.Results: SHs could proliferate and expand to form a hepatic organoid in the sponge. Albumin secretion and other hepatic protein production of the cells in the sponge increased with time in culture and the amounts were much larger than for those obtained from cells grown on dishes. Morphologically and functionally differentiated hepatocytes were observed and some CK19-positive cells formed duct-like structures within the sponge. Excretion of fluorescein was observed in bile canaliculi.Conclusions: Hepatic organoids can be rapidly reconstructed in a collagen sponge by rat SHs and NPCs. [Copyright &y& Elsevier]

A tissue inhibitor of metalloproteinases-2 (TIMP-2)-independent mechanism for generating the first activational cleavage of pro-matrix metalloproteinase-2 (MMP-2) was identified in membrane type-1 MMP (MT1-MMP)-transfected MCF-7 cells and confirmed in TIMP-2-deficient fibroblasts. In contrast, the second MMP-2-activational step was found to be TIMP-2 dependent in both systems. MMP-2 hemopexin C-terminal domain was found to be critical for the first step processing, confirming a need for membrane tethering. We propose that the intermediate species of MMP-2 forms the well-established trimolecular complex (MT1-MMP/TIMP-2/MMP-2) for further TIMP-2-dependent autocatalytic cleavage to the fully active species. This alternate mechanism may supplement the traditional TIMP-2-mediated first step mechanism. [Copyright &y& Elsevier]

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease–disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFα converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites. [Copyright &y& Elsevier]

The mechanisms of action of Ewing’s sarcoma (EWS) associated EWS-ETS oncoproteins have largely remained unresolved. Here, we analyzed how two EWS-ETS proteins, EWS-ER81 and EWS-Fli-1, in vitro activate the matrix metalloproteinase (MMP)-1 promoter that is upregulated in a subset of EWSs. EWS-ER81 and EWS-Fli-1 interact with and thereby activate the MMP-1 promoter, which is potentiated by the cofactor p300 and the proto-oncoprotein c-Jun. Further, EWS-ER81 binds to c-Jun in vitro and in vivo. The interaction between c-Jun, p300 and EWS-ER81 or EWS-Fli-1 may also be relevant to the regulation of other yet-to-be-identified genes that are responsible for EWS formation. [Copyright &y& Elsevier]

Objective: The pathogenesis of thoracic aortic aneurysms (TAA) is still unclear. A recent investigation indicated that angiotensin II, a potent activator of NADH/NADPH oxidase, plays an important role in aneurysmal formation. We investigated the potential role of p22phox-based NADH/NADPH oxidase in the pathogenesis of TAA. Methods: Human thoracic aneurysmal (n=40) and non-aneurysmal (control, n=39) aortic sections were examined, and the localization of p22phox, an essential component of the oxidase, and its expressional differences were investigated by immunohistochemistry and Western blot. In situ reactive oxygen species (ROS) generation was examined by the dihydroethidium method, and the impact of medical treatment on p22phox expression was investigated by multiple regression analysis. Results: In situ production of ROS and the expression of p22phox increased markedly in TAA throughout the wall, and Western blot confirmed the enhanced expression of p22phox. The expression was more intense in the regions where monocytes/macrophages accumulated. In these inflammatory regions, numerous chymase-positive mast cells and angiotensin converting enzyme-positive macrophages were present. Their localization closely overlapped the in situ activity of matrix metalloproteinase and the expression of p22phox. Multiple regression analysis revealed that medical treatment with statin and angiotensin II type 1 receptor blocker (ARB) suppressed p22phox expression in TAA. Conclusion: Our findings indicate the role of p22phox-based NADH/NADPH oxidase and the local renin–angiotensin system in the pathogenesis of TAA. Statin and ARB might have inhibitory effects on the formation of aneurysms via hrough the suppression of NADH/NADPH oxidase. [Copyright &y& Elsevier]

Matrix metalloproteinases (MMPs) have been implicated in the disruption of atherosclerotic plaques that leads to acute coronary events. The present study investigates the effect of thiazolidinediones (TZDs), new antidiabetic drugs, on MMP-1 expression by human vascular endothelial cells. Results show that troglitazone, but not pioglitazone and rosiglitazone, stimulated MMP-1 secretion and mRNA expression in both human umbilical vein and aortic endothelial cells, but had no effect on TIMP-1 and TIMP-2 secretion. Interestingly, troglitazone at high concentrations (≥30 μmol/l) inhibited MMP-1 protein synthesis despite a marked stimulation on MMP-1 mRNA. Further studies revealed that troglitazone at higher concentrations inhibits de novo protein synthesis as determined by 35S-methionine/cysteine incorporation, suggesting that the inhibition of MMP-1 synthesis by troglitazone is due to the suppression of total protein synthesis. Finally, our studies showed that high concentrations of troglitazone inhibited the translation initiation factor 4E (eIF4E), but not eIF4G. In summary, the present study demonstrates that insulin sensitizers have different effects on MMP-1 expression, and troglitazone stimulates MMP-1 mRNA expression and protein synthesis at the pharmacological concentrations, but inhibits MMP-1 synthesis at higher doses. This study also suggests that supra-pharmacological concentrations of troglitazone that could be attained in body tissues may inhibit protein synthesis and cause cytotoxicity. [Copyright &y& Elsevier]

Angiogenesis, which is essential for tumor growth, is regulated by various angiogenic factors. Osteopontin (OPN) is expressed in various human tumors and is postulated to be involved in tumor progression. We have recently reported that culture medium with murine neuroblastoma C1300 cells transfected with OPN gene significantly stimulates human umbilical vein endothelial cell migration and induces neovascularization in mice by dorsal air sac assay. However, the effect of OPN on tumorigenesis as an angiogenic factor remains to be clarified. In this study, we injected the OPN-transfected C1300 cells and control cells into the nude mice subcutaneously. OPN-overexpressing C1300 cells significantly formed rapidly growing tumor as compared to the control cells in mice, although in vitro and in vivo cell growth rates were similar. In vivo tumorigenecity of these cells correlated with the amount of secreted OPN protein. In addition, neovascularization of OPN-transfected tumor was significantly increased in comparison with those of control cells by immunohistochemistry for CD31. In vitro chemoinvasiveness and gene expression of proteases including uPA, MMP2, 9, MT1-MMP, and cathepsin B, D, L, were not different between OPN-transfected and control cells determined with matrigel invasion assay and cDNA expression macroarray, respectively. Conclusively, these results strongly imply that OPN plays an important role in tumor growth through the enhancement of angiogenesis in vivo. [Copyright &y& Elsevier]

The mitogen-activated protein kinase p38 pathway was originally identified as a signalling cascade activated by pro-inflammatory stimuli and cellular stresses, and playing a critical role in the translational regulation of pro-inflammatory cytokine synthesis. In almost a decade since this discovery, a great deal has been learned about the role of the p38 pathway in the post-transcriptional regulation of pro-inflammatory gene expression. However, important questions remain to be answered concerning the specificity and mechanism or mechanisms of action of p38. This review describes recent progress and remaining puzzles in the field of post-transcriptional regulation by p38. [Copyright &y& Elsevier]

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and α-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, α-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5–7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing α-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3–7 days progressively reduced their expression of mRNA for type I procollagen and α-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix. [Copyright &y& Elsevier]

The high-affinity inhibition of stromelysin 1 (MMP-3) by tissue inhibitor of metalloproteinases 1 (TIMP-1) helps control tissue remodeling and tumor development. The interaction of N-TIMP-1 with the catalytic domain of MMP-3 has been investigated by titration calorimetry and 15N NMR. Their unfavorable enthalpy of binding of +6.5 kcal mol−1 is unusual among protein–protein associations, deviates from structure-based prediction, and is compensated by a net entropy increase providing at least 18 kcal mol−1 of favorable free energy of binding at a 1 M reference state. The small heat capacity of binding agrees well with the heat capacity predicted from 65% of the surface buried on binding being polar, and suggests that the hydrophobic effect can account for only part of the entropy of binding. Using NMR, binding-induced changes in the backbone of N-TIMP-1 were checked as one possible source of conformational entropy changes. MMP binding slightly increases rigidity in some contact sites in TIMP-1 but increases mobility remotely in the otherwise rigid β-barrel core of N-TIMP-1, increasing 15N relaxation evidence of pico- to nanosecond and micro- to millisecond fluctuations of β-strands A–F. Residual dipolar couplings suggest dynamic deviations from X-ray coordinates of the complex. These suggest that the β-barrel has small backbone conformational fluctuations, while segments of strands βB, βE and βF might experience fluctuations only in their backbone environment. This is a distinctive example of affinity between two well-structured proteins being enhanced by increased conformational entropy in the reservoir of a folding core. [Copyright &y& Elsevier]

Side-chain flexibility of ligand-binding sites needs to be considered in the rational design of novel inhibitors. We have developed a method to generate conformational ensembles that efficiently sample local side-chain flexibility from a single crystal structure. The rotamer-based approach is tested here for the S1′ pocket of human collagenase-1 (MMP-1), which is known to undergo conformational changes in multiple side-chains upon binding of certain inhibitors. First, a raw ensemble consisting of a large number of conformers of the S1′ pocket was generated using an exhaustive search of rotamer combinations on a template crystal structure. A combination of principal component analysis and fuzzy clustering was then employed to successfully identify a core ensemble consisting of a low number of representatives from the raw ensemble. The core ensemble contained geometrically diverse conformers of stable nature, as indicated in several cases by a relative energy lower than that of the minimised template crystal structure. Through comparisons with X-ray crystallography and NMR structural data we show that the core ensemble occupied a conformational space similar to that observed under experimental conditions. The synthetic inhibitor RS-104966 is known to induce a conformational change in the side-chains of the S1′ pocket of MMP-1 and could not be docked in the template crystal structure. However, the experimental binding mode was reproduced successfully using members of the core ensemble as the docking target, establishing the usefulness of the method in drug design. [Copyright &y& Elsevier]

The human transferrin receptor (TfR) is proteolytically cleaved at R100 within the juxtamembrane stalk and to a lesser extent at an alternative site. We examined the effect of stalk mutations on human TfR shedding in transfected CHO cells. Point mutations at R100 led to an increase in alternative shedding while the R100 cleavage product was undetectable. Replacing the TfR-stalk by the corresponding sequences from tumor necrosis factor-α or interleukin-6 receptor also led to TfR ectodomain shedding. These results show that cleavage at alternative sites can compensate for suppressed cleavage at the major site and inhibitor studies reveal that at least three metalloproteases are involved in the shedding process. [Copyright &y& Elsevier]

The Ets family of transcription factors characterized by an evolutionarily-conserved DNA-binding domain regulates expression of a variety of viral and cellular genes by binding to a purine-rich GGAA/T core sequence in cooperation with other transcriptional factors and co-factors. Most Ets family proteins are nuclear targets for activation of Ras-MAP kinase signaling pathway and some of them affect proliferation of cells by regulating the immediate early response genes and other growth-related genes. Some of them also regulate apoptosis-related genes. Several Ets family proteins are preferentially expressed in specific cell lineages and are involved in their development and differentiation by increasing the enhancer or promoter activities of the genes encoding growth factor receptors and integrin families specific for the cell lineages. Many Ets family proteins also modulate gene expression through protein-protein interactions with other cellular partners. Deregulated expression or formation of chimeric fusion proteins of Ets family due to proviral insertion or chromosome translocation is associated with leukemias and specific types of solid tumors. Several Ets family proteins also participate in malignancy of tumor cells including invasion and metastasis by activating the transcription of several protease genes and angiogenesis-related genes. [Copyright &y& Elsevier]

The complex structure of articular cartilage, the connective tissue lining diarthrodial joints, enables this tissue to dissipate compressive loads but also appears to hinder its repair ability. At best, both natural and surgical repair attempts replace the highly ordered extracellular matrix of native articular cartilage with fibrous repair tissue of inferior mechanical properties. Numerous bioactive molecules closely regulate the cellular processes in healthy and degenerative articular cartilage. Accordingly, this review outlines the roles of important signaling molecules in cartilage tissue. In addition, drug delivery strategies, aimed at utilizing these bioactive agents to prevent inflammation, to regulate extracellular matrix metabolism, and to control cellular activities, are discussed. As scientists gain further insight into the complex signaling cascades of articular cartilage, continued refinement of drug delivery systems is necessary to develop effective clinical therapies for articular cartilage repair. [Copyright &y& Elsevier]

Retinoic acid (RA) affects many cell types by either promoting their survival or inducing their differentiation. Dehydroepiandrosterone (DHEA), a precursor for both androgenic and estrogenic steroids and abundantly produced by brain, is known as an inhibitor of cell proliferation. Differentiation of a human neuroblastoma cell line (SK-N-BE) was evaluated measuring growth rate, motility, neurite extension and GAP-43 expression. We report that DHEA enhances the differentiating effect of RA on neuroblastoma cells via a signalling that is not RA receptor-mediated. Instead, we show a differential expression of matrix metalloproteinases: RA enhances the activity of MMP-2, whereas MMP-9 expression is up-regulated by DHEA. The concerted modulation of these proteinases may support the neurite outgrowth observed after co-treatment with the two drugs. [Copyright &y& Elsevier]

Transforming growth factor (TGF)-β1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen-activated protein kinase (MAPK) and/or Smad pathways are involved in TGF-β1-stimulated collagenase-3 expression in MDA-MB231 cells. Biochemical blockade of extracellular regulated kinase-1/2 and p38 MAPK pathways partially abolished TGF-β1-stimulated collagenase-3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF-β1-response. These data indicate that TGF-β1-induced MAPK and Smad pathways are involved in TGF-β1-stimulated collagenase-3 expression in MDA-MB231 cells. [Copyright &y& Elsevier]

The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr∼33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of ∼28.5–29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin''s deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin''s C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site. [Copyright &y& Elsevier]

Ovotransferrin (OTF) is an acute phase protein in chickens, serum levels of which increase in inflammation and infections. To understand the significance of OTF in inflammation, we studied its in vitro effects on HD11 cells, a macrophage cell line, and heterophils isolated from blood using a panel of variables indicative of cellular activation. These included the production of interleukin-6 (IL-6), nitrite, matrix metalloproteinase (MMP), oxidation of dichlorofluorescein diacetate for respiratory burst and the degranulation of heterophils by the loss of fluorescein isothiocyanate positive cytoplasmic granules. The results show that ovotransferrin stimulates the production of IL-6, nitrite and MMP by HD11 cells and augments phorbol ester-induced respiratory burst. Ovotransferrin stimulated heterophils to produce IL-6, and MMP, but failed to produce nitrite, enhanced respiratory burst activity and degranulation. These results suggest that ovotransferrin can modulate macrophage and heterophil functions in chickens. [Copyright &y& Elsevier]

Astacin (EC 3.4.24.21) is a prototype for the astacin family and for the metzincin superfamily of zinc peptidases, which comprise membrane-bound and secreted enzymes involved in extracellular proteolysis during tissue development and remodelling. Generally, metzincins are translated as pro-enzymes (zymogens), which are activated by removal of an N-terminal pro-peptide. In astacin, however, the mode of zymogen activation has been obscured, since the pro-form does not accumulate in vivo. Here we report the detection of pro-astacin in midgut glands of brefeldin A-treated crayfish (Astacus astacus) by immunoprecipitation and mass spectrometry. We demonstrate that the pro-peptide is able to shield the active site of mature astacin as a transient inhibitor, which is degraded slowly. In vitro studies with recombinant pro-astacin in the absence of another protease reveal a potential of auto-proteolytic activation. The initial cleavage in this autoactivation appears to be an intramolecular event. This is supported by the fact that the mutant E93A-pro-astacin is incapable of autoactivation, and completely resistant to cleavage by mature astacin. However, this mutant is cleaved by Astacus trypsin within the pro-peptide. This probably reflects the in vivo situation, where Astacus trypsin and astacin work together during pro-astacin activation. In a first step, trypsin produces amino-terminally truncated pro-astacin derivatives. These are trimmed subsequently by each other and by astacin to yield the mature amino terminus, which forms a salt-bridge with Glu103 in the active site. The disruption of this salt-bridge in the mutants E103A and E103Q results in extremely heat labile proteins, whose catalytic activities are not altered drastically, however. This supports a concept according to which the linkage of Glu103 to the precisely trimmed amino terminus is a crucial structural prerequisite throughout the astacin family. [Copyright &y& Elsevier]

We have characterized a novel human matrix metalloproteinase (MMP-21) from human placenta DNA complementary to RNA (cDNA). The 569 amino acid translation of the cDNA includes all the typical features of an MMP family member, namely a signal sequence, a prodomain with a PRCGVPD motif, a zinc-binding catalytic domain with an HEIGHVLGL sequence, and a hemopexin-like domain flanked by two cysteine residues. Furthermore, MMP-21 has a furin activation sequence, but no transmembrane sequence nor a cytoplasmic domain. As in Xenopus laevis and Cynops pyrrhogaster there is an additional insertion of approximately 30 amino acids between the prodomain and the catalytic domain, which is poorly conserved between the species and is in human MMP-21 especially proline rich. The MMP-21 gene has seven exons and is located in chromosome 10. This new MMP is the human orthologue for XMMP and CyMMP expressed during gastrulation of X. laevis and C. pyrrhogaster, respectively. A 2.5 kb messenger RNA was observed in fetal liver by Northern analysis. By reverse transcription-polymerase chain reaction, MMP-21 is expressed in various human fetal and adult tissues as well as in cancer cell lines. MMP-21 protein can also be detected in malignancies such as ovarian and colon carcinomas by immunohistochemical staining. Our findings suggest that MMP-21 functions in embryogenesis and tumor progression. [Copyright &y& Elsevier]