Key metalloproteinases are expressed by specific cell types in experimental autoimmune encephalomyelitis

Metalloproteinases (MPs) include matrix metalloproteinases (MMPs) and metalloproteinase-disintegrins (ADAMs). Their physiological inhibitors are tissue inhibitor of metalloproteinases (TIMPs). MPs are thought to be mediators of cellular infiltration in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). We used real-time RT-PCR to profile the expression of all 22 known mouse MMPs, seven ADAMs, and all four known TIMPs in spinal cord from SJL/J mice and mice with adoptively transferred myelin basic protein (MBP)-specific EAE. A significant and >3-fold alteration in expression was observed for MMP-8, MMP-10, MMP-12, ADAM-12, and TIMP-1, which were up-regulated, and for MMP-15, which was down-regulated. Expression levels correlated with disease course, with all but ADAM-12 returning toward control levels in remission. To examine potential cellular sources of these strongly affected proteins in the inflamed CNS, we isolated macrophages, granulocytes, microglia, and T cells by cell sorting from the CNS of mice with EAE and analyzed their expression by real-time RT-PCR. This identified macrophages as a major source of MMP-12 and TIMP-1. Granulocytes were a major source of MMP-8. ADAM-12 was expressed primarily by T cells. Cellular localization of MMP-10, TIMP-1, and ADAM-12 in perivascular infiltrates was confirmed by immunostaining or in situ hybridization. Microglia from control mice expressed strong signal for MMP-15. Strikingly, the expression of MMP-15 by microglia was significantly down-regulated in EAE, which was confirmed by immunostaining. Our study identifies the cellular sources of key MPs in CNS inflammation.     

Matrix metalloproteinases and their tissue inhibitors in the developing neonatal mouse uterus

Postnatal development of the mouse uterus involves differentiation and development of the endometrial glands as well as the myometrium. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in extracellular matrix breakdown and morphogenesis of many epitheliomesenchymal organs. As a first step to understanding their roles in postnatal mouse uterine development, MMPs and TIMPs found to be expressed in the neonatal mouse uterus by microarray analysis were localized by in situ hybridization. The MMP-2 mRNA was detected only in the uterine stroma, whereas the MMP-10 mRNA was present only in the uterine epithelium from Postnatal Day (PND) 3 to PND 9. All other MMPs (MMP-11, MMP-14, and MMP-23) as well as TIMP-1, TIMP-2, and TIMP-3 were detected in both epithelial and stromal cells of the endometrium, but not in the myometrium. Uterine extracts were then analyzed by gelatin and casein gel zymography to detect active gelatinases and stromelysins, respectively. Five major gelatinase bands of activity were detected and inhibited by the MMP inhibitors, EDTA or 1,10-phenanthroline, but not by PMSF, a serine protease inhibitor. Western blot analysis confirmed the presence of MMP-2 and MMP-9 proteins in the uterus. Immunoreactive MMP-9 protein was detected only in the endometrial stroma, whereas immunoreactive MMP-2 protein was detected in both the stroma and epithelium of the uterus. Casein zymography detected three major bands of activity ( approximately 54, 63, and 80 kDa) that were inhibited by the serine protease inhibitor, PMSF, but not by the MMP inhibitors, EDTA or 1,10-phenanthroline, suggesting that they were serine proteases. These results support the hypothesis that MMPs and TIMPs regulate postnatal development of the mouse uterus.     

基质金属蛋白酶

基质金属蛋白酶是一类分解细胞外基质组分的锌蛋白酶⒚它们在有机体生长发育中的细胞外基质逆转与重塑以及疾病中的病理损害起着极为重要的作用⒚基质金属蛋白酶的表达和活性在不同细胞水平受到严密调控,如细胞因子、生长因子以及激素的调节⒚基质金属蛋白酶以酶原形式分泌,随后被其它蛋白酶如胞浆素或非蛋白酶类化学物质如有机汞所激活⒚所有基质金属蛋白酶都受到天然抑制剂 金属蛋白酶组织抑制剂所抑制⒚两者的不平衡导致许多疾病的发生,如肿瘤侵入及转移⒚合成基质金属蛋白酶组织抑制剂所抑制,如 Ｍ ａｒｉｍ ａｓｔａｔ 能控制肿瘤转移的发生及进一步扩散⒚本文将对基质金属蛋白酶的特征、分子区域结构、底物特性、激活机制、调控方式等方面进行最新概述⒚     

Extracellular metalloproteinases in neural crest development and craniofacial morphogenesis

Abstract

The neural crest (NC) is a population of migratory stem/progenitor cells that is found in early vertebrate embryos. NC cells are induced during gastrulation, and later migrate to multiple destinations and contribute to many types of cells and tissues, such as craniofacial structures, cardiac tissues, pigment cells and the peripheral nervous system. Recently, accumulating evidence suggests that many extracellular metalloproteinases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), and ADAMs with thrombospondin motifs (ADAMTSs), play important roles in various stages of NC development. Interference with metalloproteinase functions often causes defects in craniofacial structures, as well as in other cells and tissues that are contributed by NC cells, in humans and other vertebrates. In this review, we summarize the current state of the field concerning the roles of these three families of metalloproteinases in NC development and related tissue morphogenesis, with a special emphasis on craniofacial morphogenesis.     

Tissue inhibitor of matrix metalloproteinase-3 expression in the mouse uterus during implantation and artificially induced decidualization

During implantation in mice, tissue inhibitor of matrix metalloproteinases-3 is believed to play a key role in inhibiting matrix metalloproteinase activity associated with embryo invasion and tissue remodeling. The first objective of this study was to quantitatively compare the steady-state mRNA levels of tissue inhibitors of matrix metalloproteinases between segments of the mouse uterus undergoing decidualization compared to those that are not during early pregnancy plus oil-induced decidualization. Steady-state tissue inhibitor of metalloproteinase-3 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 6 and 7 of pregnancy and in stimulated compared to nonstimulated uterine horns at 48 and 72 hr after artificial induction of decidualization. Steady-state tissue inhibitor of metalloproteinase-1 mRNA levels were significantly greater in implantation compared to interimplantation areas on days 5-8 of pregnancy and in stimulated compared to nonstimulated uterine horns at 24, 48, and 72 hr after oil stimulation. Therefore, the steady-state mRNA levels of tissue inhibitors of metalloproteinase-1 and -3 increased in the uterus during decidualization. The second objective of this study was to determine if transforming growth factor-beta1 influences tissue inhibitors of metalloproteinase mRNA concentrations in mouse endometrial stromal cells. As determined by Northern blot analyses, transforming growth factor beta1 significantly increased tissue inhibitors of matrix metalloproteinases-1 and -3 mRNA levels in cultured mouse endometrial stromal cells isolated from uteri sensitized for decidualization. On the other hand, interleukin-1, epidermal growth factor, and leukemia inhibitory factor had no effect. The results of this study further characterize the tissue inhibitor of metalloproteinase expression in the uterus during implantation and artificially induced decidualization and the potential control of their expression in the stroma by transforming growth factor.     

Crystal structures of MMPs in complex with physiological and pharmacological inhibitors

Matrix Metalloproteinases (MMPs) are a family of multidomain zinc endopeptidases that function in the extracellular space or attached to the cell membrane. Their proteolytic activity is controlled by the presence of endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), alpha-macroglobulin and others. Disruption of the proteinase-inhibitor balance is observed in serious diseases such as arthritis, tumor growth and metastasis, rendering the MMPs attractive targets for drug intervention by pharmacological inhibitors. The determination of MMP structures is of critical importance in order to understand their substrate preferences, dimerization events, and their association with matrix components and inhibitors. Thus, MMP structures may contribute significantly to the development of specific MMP inhibitors, which should allow precise control of individual members of the MMP family without affecting all members or the closely related metalloproteinases such as ADAMs and ADAMTSs.     

Expression of matrix metalloproteinase-26 (MMP-26) mRNA in mouse uterus during the estrous cycle and early pregnancy

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the remodeling of extracellular matrix (ECM). MMP-26, also called endometase or matrilysin-2, is a novel member of the MMP family. The present study was to investigate the temporal and spatial expression of MMP-26 mRNA in mouse uterus during the estrous cycle and early pregnancy by using in situ hybridization and semi-quantitative RT-PCR. In this study, MMP-26 mRNA was found to be localized to the luminal and glandular epithelium at proestrus and estrus, and the expression level was decreased significantly from metestrus to dioestrus. During pre-implantation period, MMP-26 mRNA was predominantly expressed in luminal and glandular epithelium at much higher level; whereas it switched to stroma during peri-implantation period, and also appeared in the blastocysts and the implantation sites. The results suggested that MMP-26 might play a role in the cycling changes of mouse uterus during the estrous cycle and embryo implantation.     

Matrix Metalloproteinases of Normal Human Tissues

This review considers biochemical properties of the family of matrix metalloproteinases (MMPs) of normal human tissues and the involvement of these enzymes in morphogenesis. Four main MMP subfamilies are characterized, and a group of other MMPs is described. Data on mechanisms of activation and inhibition of MMPs in certain tissues during various physiological processes (embryogenesis, angiogenesis, tissue growth and involution) are considered. Information about tissue inhibitors of MMP is presented, and the ability of these inhibitors to regulate the activity of MMPs is analyzed.     

Potential clinical implications of recent matrix metalloproteinase inhibitor design strategies

Analysis of matrix metalloproteinases (MMPs) expression profiles in various pathologies correlated with their presence in promoting disease progression. Drugs were designed to inhibit MMPs in an extreme manner by chelating the active site zinc ion. This approach did not distinguish between the 24 members of the MMP family and had devastating consequences during clinical trials. Subsequent knockout mouse studies showed that some MMPs are beneficial in regulating tumor growth, metastasis and indirectly stimulating the immune system. The broad-spectrum inhibitor approach was rethought and modified in order to increase specificity by taking into account the non-conserved secondary binding sites or differences in structures within MMPs and also generating antibodies. These showed interesting results in vitro and in vivo. The recent technological advances that allow us to better understand the function and structure of MMPs are aiding in the development of selective inhibitors.     

Membrane type-1 matrix metalloproteinases and tissue inhibitor of metalloproteinases-2 RNA levels mimic each other during Xenopus laevis metamorphosis

Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, using RT-PCR analysis, we examine the metamorphic RNA levels of two membrane-type MMPs (MT1-MMP, MT3-MMP), two TIMPs (TIMP-2, TIMP-3) and a potent gelatinase (Gel-A) that can be activated by the combinatory activity of a MT-MMP and a TIMP. In the metamorphic tail and intestine the RNA levels of TIMP-2 and MT1-MMP mirror each other, and closely resemble that of Gel-A as all three are elevated during periods of cell death and proliferation. Conversely, MT3-MMP and TIMP-3 do not have similar RNA level patterns nor do they mimic the RNA levels of the other genes examined. Intriguingly, TIMP-3, which has been shown to have anti-apoptotic activity, is found at low levels in tissues during periods of apoptosis.     

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

A disintegrin and metalloproteinases (ADAMs) are a recently discovered family of proteins that share the metalloproteinase domain with matrix metalloproteinases (MMPs). Among this family, structural features distinguish the membrane-anchored ADAMs and the secreted ADAMs with thrombospondin motifs referred to as ADAMTSs. By acting on a large panel of membrane-associated and extracellular substrates, they control several cell functions such as adhesion, fusion, migration and proliferation. The current review addresses the contribution of these proteinases in the positive and negative regulation of cancer progression as mainly mediated by the regulation of growth factor activities and integrin functions.     

Cyclic changes in the matrix metalloproteinase system in the ovary and uterus

With each estrous or menstrual cycle, extensive alterations occur in the extracellular matrix and connective tissue of the ovary and uterus. In the ovary, these changes occur during follicular development, breakdown of the follicular wall and extrusion of the oocyte, as well as during the formation and regression of the corpus luteum. In the uterus, the endometrium undergoes dramatic connective tissue turnover associated with tissue breakdown and subsequent regrowth during each menstrual cycle. These changes in the ovarian and uterine extracellular architecture are regulated, in part, by the matrix metalloproteinase (MMP) system. This system is comprised of both a proteolytic component, the MMPs, and associated inhibitors, and it is involved in connective tissue remodeling processes throughout the body. The current review highlights the key features of the MMP system and focuses on the changes in the MMPs and the tissue inhibitors of metalloproteinases during the dynamic remodeling that takes place in the ovary and uterus during the estrous and menstrual cycles.     

The role of TIMPs in regulation of extracellular matrix proteolysis

Tissue inhibitors of metalloproteinases (TIMPs), which inhibit matrix metalloproteinases (MMPs) as well as the closely related, a disintegrin and metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs), were traditionally thought to control extracellular matrix (ECM) proteolysis through direct inhibition of MMP-dependent ECM proteolysis. This classical role for TIMPs suggests that increased TIMP levels results in ECM accumulation (or fibrosis), whereas loss of TIMPs leads to enhanced matrix proteolysis. Mice lacking TIMP family members have provided support for such a role; however, studies with these TIMP deficient mice have also demonstrated that loss of TIMPs can often be associated with an accumulation of ECM. Collectively, these studies suggest that the divergent roles of TIMPs in matrix accumulation and proteolysis, which together can be referred to as ECM turnover, are dependent on the TIMP, specific tissue, and local tissue environment (i.e. health vs. injury/disease). Ultimately, these combined factors dictate the specific metalloproteinases being regulated by a given TIMP, and it is likely the diversity of metalloproteinases and their physiological substrates that determines whether TIMPs inhibit matrix proteolysis or accumulation. In this review, we discuss the evidence for the dichotomous roles of TIMPs in ECM turnover highlighting some of the common findings between different TIMP family members. Importantly, while we now have a better understanding of the role of TIMPs in regulating ECM turnover, much remains to be determined. Data on the specific metalloproteinases inhibited by different TIMPs in vivo remains limited and must be the focus of future studies.     

The isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition

ADAM (a disintegrin and metalloproteinase) 10 is a key member of the ADAM family of disintegrin and metalloproteinases which process membrane-associated proteins to soluble forms in a process known as 'shedding'. Among the major targets of ADAM10 are Notch, EphrinA2 and CD44. In many cell-based studies of shedding, the activity of ADAM10 appears to overlap with that of ADAM17, which has a similar active-site topology relative to the other proteolytically active ADAMs. The tissue inhibitors of metalloproteinases, TIMPs, have proved useful in the study of ADAM function, since TIMP-1 inhibits ADAM10, but not ADAM17; however, both enzymes are inhibited by TIMP-3. In the present study, we show that, in comparison with ADAM17 and the MMPs (matrix metalloproteinases), the N-terminal domains of TIMPs alone are insufficient for the inhibition of ADAM10. This knowledge could form the basis for the design of directed inhibitors against different metalloproteinases.     

Up-regulation of multiple serine proteinases during earthworm tail regeneration

Summary

Previous studies have shown that spatiotemporal regulation of extracellular matrix (ECM) by proteinases is implicated in the initial step of regeneration. In amphibian regeneration, the up-regulation of proteinases such as metalloproteinases (MMPs) and cathepsin D, and proteinase-related proteins such as proteinase tissue inhibitors and activators has been demonstrated. Since the earthworm could provide a unique and valuable model to investigate the mechanism of regeneration, we studied the developmental change in proteinase expression during earthworm tail regeneration. Zymographic analysis revealed that proteinase activities began to increase within 1 h after amputation and reached a maximum at 7 days post-amputation. This peak in activity was approximately 22-fold greater than the unamputated controls. Thereafter, the proteinase activities tended to decrease followed by another peak at 30 days before returning to control levels. At least four types of proteinase were distinguishable at 7 and 30 days post-amputation, with molecular weights of 25, 28, 38, and 44 kDa, respectively. All proteinase activities were strongly inhibited by addition of phenylmethylsulfonyl fluoride (PMSF) and aprotinin, specific inhibitors for serine proteinase. Pepstatin A, E-64, iodoacetamide and a metal ion-free medium were not effective inhibitors, indicating that proteinases expressed during earthworm tail regeneration would be serine proteinases. In addition, we were able to detect two types of plasminogen activator (PA) with molecular weights of 40 and 47 kDa, respectively. PA activities were predominantly expressed at 1, 5, and 25 days post-amputation, which preceded two peaks of serine proteinase activities appearing at approximately 7 and 30 days after amputation, respectively. This fact supports the view that serine proteinases expressed in respond to tail amputation may be plasmin-like proteinases activated by PA.     

Temporospatial expression of tissue inhibitors of matrix metalloproteinases-1, -2 and -3 during development, growth and aging of the mouse skeleton

Proteolytic degradation of collagen-rich extracellular matrices is a key feature in the development, growth and aging of skeleton. Matrix metalloproteinases (MMPs) are a family of enzymes capable of performing this function, whereas tissue inhibitors of MMPs (TIMPs) are believed to play an important role in regulating their activity. To better understand the roles of TIMP-1, -2 and -3, we have studied their mRNA levels in several different mouse tissues with special emphasis on the skeleton and the developing eye. A systematic analysis of TIMP-1, -2 and -3 mRNA levels in mouse knee joints during growth and aging demonstrated markedly different expression patterns for each TIMP. Immunohistochemical analysis revealed several time-dependent changes in the distribution of TIMP-1 and -2 in articular and growth cartilages, synovial tissue and bone. The data suggest that upon aging synovial tissue becomes the major source of synovial fluid TIMPs. In articular cartilage these inhibitors were mainly found in the deep layer and in subchondral bone. Compared with epiphyseal growth plate, the amounts of TIMP-1 and -2 in articular cartilage were quite low. These findings suggest that the capacity of articular cartilage chondrocytes to inhibit MMP activities by local production of TIMPs is limited, which may be of consequence during osteoarthritic cartilage degeneration.     

Specific targeting of metzincin family members with small-molecule inhibitors: progress toward a multifarious challenge

Zn-metalloproteinases are an important class of hydrolytic enzymes that are characterized by the presence of a catalytic zinc(II) atom in their active center which is fundamental for proteolytic activity. Metzincins, a superfamily of Zn-metalloproteinases with many structural and functional commonalities among its members, are responsible for the fine tuning of key physiological functions in mammals and the deregulation of their activity is directly connected to numerous inflammatory and degenerative diseases such as arthritis or cancer. Development of small-molecule exogenous inhibitors of metzincins able to re-establish normal proteolytic activity in pathological conditions has been a field of intense research effort for many years but applications in the clinic were not always successful. One of the main reasons for this failure is the uncontrolled action of these inhibitors on target as well as anti-target metzincin family members. Current medicinal efforts have been shifted to the discovery of target-specific inhibitors that will help to improve our understanding of metzincins biological function and provide the basis for the development of safer pharmaceutical agents. This review focuses on the cases of certain medicinally important metzincins [matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), ADAMs with thrombospondin motifs (ADAMTSs), and procollagen C-proteinase (PCP)] and summarizes the latest advances on the discovery of inhibitors of these enzymes that display improved selectivity profiles.     

Tissue distribution, inhibition and activation of gelatinolytic activities in Atlantic cod (Gadus morhua)

Gelatinolytic activities in fish tissues with properties like matrix metalloproteinases (MMPs) have been paid little attention. However, they have been proposed to participate in post mortem degradation during storage and the disintegration of pericellular connective tissue during spawning. In this paper the distribution of gelatinolytic activities in liver, heart, muscle, gill, and male and female gonad of Atlantic cod (Gadus morhua) was studied by using gelatin SDS-PAGE, proteinase inhibitors, gelatin and lentil lectin Sepharose affinity chromatography. The amount of gelatin degrading enzymes varied from tissue to tissue. Most of the gelatin binding enzymes were found to be matrix metalloproteinases by adding galardin, a broad range MMP inhibitor, to the incubation buffer. A 72 kDa form of cod gelatin degrading enzyme had properties similar to human proMMP-2, as it could be activated by p-aminophenylmercuric acetate and trypsin. Like the human MMP-2 it did not bind to lentil lectin. An 83 kDa cod gelatin degrading enzyme had properties similar to the 92 kDa progelatinase B (proMMP-9). These properties were also similar to that of the 72 kDa form, except that the 83 kDa cod gelatinase was bound to lentil lectin, showing that it is a glycoprotein like MMP-9.