TGF-beta signaling preserves RECK expression in activated pancreatic stellate cells

Activated pancreatic stellate cells (PSCs) play a pivotal role in the pathogenesis of pancreatic fibrosis, but the detailed mechanism for dysregulated accumulation of extracellular matrix (ECM) remains unclear. Cultured rat PSCs become activated by profibrogenic mediators, but these mediators failed to alter the expression levels of matrix metalloproteinases (MMPs) to the endogenous tissue inhibitors of metalloproteinases (TIMPs). Here, we examined the expression of RECK, a novel membrane-anchored MMP inhibitor, in PSCs. Although RECK mRNA levels were largely unchanged, RECK protein expression was barely detected at 2, 5 days after plating PSCs, but appeared following continued in vitro culture and cell passage which result in PSC activation. When PSCs at 5 days after plating (PSCs-5d) were treated with pepstatin A, an aspartic protease inhibitor, or TGF-beta1, a profibrogenic mediator, RECK protein was detected in whole cell lysates. Conversely, Smad7 overexpression or suppression of Smad3 expression in PSCs after passage 2 (PSCs-P2) led to the loss of RECK protein expression. These findings suggest that RECK is post-translationally processed in pre-activated PSCs but protected from proteolytic degradation by TGF-beta signaling. Furthermore, collagenolytic activity of PSCs-5d was greatly reduced by TGF-beta1, whereas that of PSCs-P2 was increased by anti-RECK antibody. Increased RECK levels were also observed in cerulein-induced acute pancreatitis. Therefore, our results suggest for the first time proteolytic processing of RECK as a mechanism regulating RECK activity, and demonstrate that TGF-beta signaling in activated PSCs may promote ECM accumulation via a mechanism that preserves the protease inhibitory activity of RECK.     

Downregulation of the RECK-tumor and metastasis suppressor gene in glioma invasiveness

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK downregulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness.     

Matrix metalloproteinases and gastrointestinal cancers: Impacts of dietary antioxidants

The process of carcinogenesis is tightly regulated by antioxidant enzymes and matrix degrading enzymes, namely, matrix metalloproteinases(MMPs). Degradation of extracellular matrix(ECM) proteins like collagen, proteoglycan, laminin, elastin and fibronectin is considered to be the prerequisite for tumor invasion and metastasis. MMPs can degrade essentially all of the ECM components and, most MMPs also substantially contribute to angiogenesis, differentiation, proliferation and apoptosis. Hence, MMPs are important regulators of tumor growth both at the primary site and in distant metastases; thus the enzymes are considered as important targets for cancer therapy. The implications of MMPs in cancers are no longer mysterious; however, the mechanism of action is yet to be explained. Herein, our major interest is to clarify how MMPs are tied up with gastrointestinal cancers. Gastrointestinal cancer is a variety of cancer types, including the cancers of gastrointestinal tract and organs, i.e., esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum and anus. The activity of MMPs is regulated by its endogenous inhibitor tissue inhibitor of metallopro-teinase(TIMP) which bind MMPs with a 1:1 stoichiometry. In addition, RECK(reversion including cysteinerich protein with kazal motifs) is a membrane bound glycoprotein that inhibits MMP-2,-9 and-14. Moreover, α2-macroglobulin mediates the uptake of several MMPs thereby inhibit their activity. Cancerous conditions increase intrinsic reactive oxygen species(ROS) through mitochondrial dysfunction leading to altered protease/anti-protease balance. ROS, an index of oxidative stress is also involved in tumorigenesis by activation of different MAP kinase pathways including MMP induction. Oxidative stress is involved in cancer by changing the activity and expression of regulatory proteins especially MMPs. Epidemiological studies have shown that high intake of fruits that rich in antioxidants is associated with a lower cancer incidence. Evidence indicates that some antioxidants inhibit the growth of malignant cells by inducing apoptosis and inhibiting the activity of MMPs. This review is discussed in six subchapters, as follows.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

Expression of matrix metalloproteinases-2 and -9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.     

HPV16 oncoproteins induce MMPs/RECK-TIMP-2 imbalance in primary keratinocytes: possible implications in cervical carcinogenesis

Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome.     

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

Matrix metalloproteinases (MMPs) are secreted endopeptidases that play an essential role in remodeling the extracellular matrix (ECM). MMPs are primarily active during development, when the majority of ECM remodeling events occurs. In adults, elevated MMP activity has been observed in many pathological conditions such as cancer and osteoarthritis. The proteolytic activity of MMPs is controlled by their natural inhibitors - the tissue inhibitor of metalloproteinases (TIMPs). In addition to blocking MMP-mediated proteolysis, TIMPs have a number of MMP-independent functions including binding to cell surface proteins thereby stimulating signaling cascades. TIMP-2, the most studied member of the family, can both inhibit and activate MMPs directly, as well as inhibit MMP activity indirectly by upregulating expression of RECK, a membrane anchored MMP regulator. While TIMP-2 has been shown to play important roles in breast cancer, we describe how the MMP-independent effects of TIMP-2 can modulate the invasiveness of MCF-7, T47D and MDA-MB-231 breast cancer cells. Using an ALA + TIMP-2 mutant which is devoid of MMP inhibition, but still capable of initiating specific cell signaling cascades, we show that TIMP-2 can differentially affect MMP activity and cellular invasiveness in both an MMP dependent and independent manner. More specifically, MMP activity and invasiveness is increased with the addition of exogenous TIMP-2 in poorly invasive cell lines whereas it is decreased in highly invasive cells lines (MDA-MB-231). Conversely, the addition of ALA + TIMP-2 resulted in decreased invasiveness regardless of cell line.     

Reversion inducing cysteine rich protein with Kazal motifs and cardiovascular diseases: The RECKlessness of adverse remodeling

The Reversion Inducing Cysteine Rich Protein With Kazal Motifs (RECK) is a glycosylphosphatidylinositol (GPI) anchored membrane-bound regulator of matrix metalloproteinases (MMPs). It is expressed throughout the body and plays a role in extracellular matrix (ECM) homeostasis and inflammation. In initial studies, RECK expression was found to be downregulated in various invasive cancers and associated with poor prognostic outcome. Restoring RECK, however, has been shown to reverse the metastatic phenotype. Downregulation of RECK expression is also reported in non-malignant diseases, such as periodontal disease, renal fibrosis, and myocardial fibrosis. As such, RECK induction has therapeutic potential in several chronic diseases. Mechanistically, RECK negatively regulates various matrixins involved in cell migration, proliferation, and adverse remodeling by targeting the expression and/or activation of multiple MMPs, A Disintegrin And Metalloproteinase Domain-Containing Proteins (ADAMs), and A Disintegrin And Metalloproteinase With Thrombospondin Motifs (ADAMTS). Outside of its role in remodeling, RECK has also been reported to exert anti-inflammatory effects. In cardiac diseases, for example, it has been shown to counteract several downstream effectors of Angiotensin II (Ang-II) that play a role in adverse cardiac and vascular remodeling, such as Interleukin-6 (IL-6)/IL-6 receptor (IL-6R)/glycoprotein 130 (IL-6 signal transducer) signaling and Epidermal Growth Factor Receptor (EGFR) transactivation. This review article focuses on the current understanding of the multifunctional effects of RECK and how its downregulation may contribute to adverse cardiovascular remodeling.     

Tgat oncoprotein functions as a inhibitor of RECK by association of the unique C-terminal region

We identified RECK, a membrane-anchored glycoprotein negatively regulating the activities of MMPs, as a molecule interacting with Tgat oncoprotein consisting of RhoGEF domain and the unique C-terminal 15 amino acids. The Tgat increased the invasive potential of NIH3T3 cells expressing endogenous mouse RECK and this effect was partially inhibited by the co-expression of human RECK. On the contrary, the expression of exogenous human RECK in HT1080 cell line lacking the endogenous RECK expression reduced its invasive activity, which was recovered by the Tgat co-expression. Moreover, a Tgat mutant lacking the C-terminal region lost the potential to compete the function of RECK in HT1080 cells. These findings indicate that Tgat is the functional inhibitor of RECK, and the activation of MMPs induced by Tgat is likely to enhance invasive activities of cancer cells expressing Tgat.     

Spatial analysis of RECK,MT1-MMP,and TIMP-2 proteins during early Xenopus laevis development

Proper cell-cell and cell-ECM interactions are vital for cell migration and patterning of the vertebrate embryo. MMPs and their inhibitors, RECK and TIMPs, are all differentially expressed during embryogenesis to regulate such ECM remodeling and cell interactions. MT1-MMP, RECK, and TIMP-2 are unique amongst other ECM-regulating proteins as they act in the pericellular space. Past studies have shown that RECK and TIMP-2 interact with MT1-MMP on the cell surface, thereby influencing cell behaviour as well as the microenvironment immediately surrounding the cells. We investigated the localization of RECK, TIMP-2, and MT1-MMP proteins throughout early X. laevis development using immunohistochemistry. We found that during neural tube formation, axis elongation, and organogenesis, RECK, TIMP-2, and MT1-MMP proteins show highly similar localization patterns, particularly in the ectoderm and in the dorsal-ventral differentiation of the neural tube. Our data suggests they function together during patterning events in early Xenopus development.     

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.     

The role of the mesangial cell and its matrix in the pathogenesis of diabetic nephropathy.

Mesangial cells are pericyte-like cells which are found the glomeruli of the kidney. It is well known that they have important contractile and synthetic properties regulating the function of the glomerulus. During diabetes the synthesis of various extracellular matrix (ECM) components by mesangial cells are increased. In recent years it has been recognized that degradation of ECM may also be decreased in diabetes, contributing to the process of mesangium accumulation. The major enzymes responsible for ECM degradation are a large group of enzymes collectively known as matrix metalloproteinases (MMPs). The physiology of MMPs is complex and their activity is tightly regulated at many levels. The MMPs are synthesized as proenzymes and require activation via catalytic cleavage to become fully active. In this regard it is of importance that the mesangial cell and its pericellular matrix have a very active plasminogen cascade that can liberate plasmin locally to mediate matrix degradation both directly and indirectly, by activating the MMPs. In addition, the MMPs are regulated by transforming growth factor beta (TGF-beta). There is evidence that each of these pathways regulating the matrix degradation is affected by the diabetic environment and this will be the subject of this contribution.     

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.     

Matrix metalloproteinases in liver injury,repair and fibrosis

The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression.     

Matrix metalloproteinases in squamous cell carcinoma

Controlled degradation of extracellular matrix (ECM) is essential in many physiological situations including developmental tissue remodeling, angiogenesis, tissue repair, and normal turnover of ECM. In addition, degradation of matrix components is an important feature of tumor growth, invasion, metastasis, and tumor-induced angiogenesis. Matrix metallo-proteinases (MMPs) are a family of zinc-dependent neutral endopeptidases, which are collectively capable of degrading essentially all ECM components. MMPs apparently play an important role in all the above mentioned aspects of tumor development. In addition, there is recent evidence that MMP activity is required for tumor cell survival. At present, several MMP inhibitors are in clinical trials of malignant tumors of different histogenetic origin. In this review we discuss the current view on the role of MMPs and their inhibitors in development and invasion of squamous cell carcinomas, as a basis for prognostication and therapeutic intervention in these tumors.     

基质金属蛋白酶家族介绍(英文)

 当细胞外基质 (ECM)组分被破坏时 ,基质金属蛋白酶 (MMPs)影响发育过程并和许多疾病如关节炎及肿瘤相关联 . ECM的正常转换是发育所需要的 . ECM的调节异常却能引起过多的损伤 ,并导致疾病如关节炎 .因此 ,更好地了解 MMP介导的 ECM的水解作用 ,有可能从机理方面为疾病诊断学与治疗学的介入提供依据 .本文介绍了 MMP生物学以及它的 ECM的相关的转换方面的最新进展 .随着新的 MMPs的发现 ,MMP家族正在迅速地扩大 .并且开始向已经确立的基因结构、潜伏期、底物专一性和功能调节方面的范例提出挑战 .即将完成的基因组测序将无容置疑地确定人类 MMPs的有限的数字 .揭示每个 MMP的功能所进行的努力可能标志我们在寻求最终了解细胞与它们的环境之间的相互作用的开始 ,这个过程对于哺乳类物种例如人类的进化是至关重要的 .     

Metalloproteinases: A parade of functions in matrix biology and an outlook for the future

This issue of Matrix Biology is devoted to exploring how metalloproteinases – here inclusive of related families of extracellular proteinases – act on extracellular matrix (ECM) proteins to influence an astonishing diversity of biological systems and diseases. Since their discovery in the 1960's, matrix metalloproteinases (MMPs) have oft and widely been considered as the principal mediators of ECM destruction. However, as becomes clear from several articles in this issue, MMPs affect processes that both promote and limit ECM assembly, structure, and quantity. Furthermore, it has become increasingly apparent that ECM proteolysis is neither the exclusive function of MMPs nor their only sphere of influence. Thus, other enzymes may be important participants in ECM proteolysis, and indeed they are. The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type 1 repeat) proteinases, BMP/tolloid proteases, and meprins have all emerged as major mechanisms of ECM proteolysis. An aggregate view of proteolysis as an exquisitely specific and crucial post-translational modification of secreted proteins emerges from these reviews. The cumulative evidence strongly suggests that although some MMPs can and do cleave ECM components, notably fibrillar collagens, the majority of these proteinases are not key physiological participants in morphogenesis nor in control of matrix metabolism in homeostasis or disease. In contrast, deficiency of ADAMTS proteases leads to a remarkable array of morphogenetic defects and connective tissue disorders consistent with a specialized role in turnover of the embryonic provisional ECM and in ECM assembly. Astacin-related proteases emerge into crucial positions in ECM assembly and turnover, although they also have numerous roles related to morphogen and growth factor regulation. To further turn the traditional view on its head, it is clear that many MMPs are key participants in many, diverse immune and inflammation processes rather than ECM proteolysis. The overlap in the activities within and between these families leads to the view that ECM proteolysis, which is indispensable for life, was over-engineered to an extraordinary extent during vertebrate evolution. That these proteinases, which likely evolved within networks regulating morphogenesis, immunity and regeneration, also participate in diseases is a side effect of human longevity. Attempts to inhibit metalloproteinases in human diseases thus require continuing appraisal of their biological roles and cautious evaluation of potential new therapeutic opportunities.     

Fibronectin–integrin mediated signaling in human cervical cancer cells (SiHa)

Interaction between cell surface integrin receptors and extracellular matrix (ECM) components plays an important role in cell survival, proliferation, and migration, including tumor development and invasion of tumor cells. Matrix metalloproteinases (MMPs) are a family of metalloproteinases capable of digesting ECM components and are important molecules for cell migration. Binding of ECM to integrins initiates cascades of cell signaling events modulating expression and activity of different MMPs. The aim of this study is to investigate fibronectin–integrin-mediated signaling and modulation of MMPs. Our findings indicated that culture of human cervical cancer cell (SiHa) on fibronectin-coated surface perhaps sends signals via fibronectin–integrin-mediated signaling pathways recruiting focal adhesion kinase (FAK) extracellular signal regulated kinase (ERK), phosphatidyl inositol 3 kinase (PI-3K), integrin-linked kinase (ILK), nuclear factor-kappa B (NF-κB), and modulates expression and activation of mainly pro-MMP-9, and moderately pro-MMP-2 in serum-free culture medium.