The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However,like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites,distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.     

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zine-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events.The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation,and is controlled by growth factors,cytokines,hormones,as well as interactions with the ECM macromolecules.Furthermore,the activity of the MMPs is regulated by their natural endogenous inhibitors,which are members of the tissue inhibitor of metalloproteinases (TIMP) family.In the normal mammary gland,MMPs are expressed during ductal development,lobulo-alveolar development in pregnancy and involution after lactation.Under pathological conditions,such as tumorigenesis,the dysregulated expression of MMPs play a role in tumor initiation,progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.     

Effect of Bcl—2 and caspase—3 on calcium distribution in apoptosis of HL—60 cells

Apoptosis manifests in two major execution programs downstream of the death signal:the caspase pathway and organelle dysfunction.An important antiapoptosis factor,Bcl-2 protein,contributes in caspase pathway of apoptosis.Calcium,an important intracellular signal element in cells,is also observed to have changes during apoptosis,which maybe affected by Bcl-2 protein.We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells,there‘s change of intracellular calcium distribution,oving from cytoplast especially Golgi‘s apparatus to nucleus and accumulating there with the highest concentration.We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells,which can be inhibited by overexpression of Bcl-2 protein.No sign of apoptosis or intracellular calcium movement from Golgi‘s apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO,a specific inhibitor of caspase-3.The results indicate that activated caspase-2 can promote the movement of intracellular calcium from Golgi‘s apparatus to nucleus,and the process is inhibited by Ac-DEVD-CHO(inhibitor of caspase-3),and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3.Calcium relocalization in apoptosis seems to be irreversible,which is different from the intracellular calcium changes caused by growth factor.     

Vital roles of stem cells and biomaterials in skin tissue engineering

Tissue engineering essentially refers to technology for growing new human tissue and is distinct from regenerative medicine. Currently, pieces of skin are already being fabricated for clinical use and many other tissue types may be fabricated in the future.Tissue engineering was first defined in 1987 by the United States National Science Foundation which critically discussed the future targets of bioengineering research and its consequences. The principles of tissue engineering are to initiate cell cultures in vitro, grow them on scaffolds in situ and transplant the composite into a recipient in vivo. From the beginning, scaffolds have been necessary in tissue engineering applications. Regardless, the latest technology has redirected established approaches by omitting scaffolds. Currently, scientists from diverse research institutes are engineering skin without scaffolds. Due to their advantageous properties, stem cells have robustly transformed the tissue engineering field as part of an engineered bilayered skin substitute that will later be discussed in detail. Additionally, utilizing biomaterials or skin replacement products in skin tissue engineering as strategy to successfully direct cell proliferation and differentiation as well as to optimize the safety of handling during grafting is beneficial. This approach has also led to the cells’ application in developing the novel skin substitute that will be briefly explained in this review.     

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.     

Myelopoiesis during Zebrafish Early Development

Myelopoiesis is the process of producing all types of myeloid cells including monocytes/macrophages and granulocytes.Myeloid cells are known to manifest a wide spectrum of activities such as immune surveillance and tissue remodeling.Irregularities in myeloid cell development and their function are known to associate with the onset and the progression of a variety of human disorders such as leukemia.In the past decades,extensive studies have been carried out in various model organisms to elucidate the molecular mechanisms underlying myelopoiesis with the hope that these efforts will yield knowledge translatable into therapies for related diseases.Zebrafish has recently emerged as a prominent animal model for studying myelopoiesis,especially during early embryogenesis,largely owing to its unique properties such as transparent embryonic body and external development.This review introduces the methodologies used in zebrafish research and focuses on the recent research progresses of zebrafish myelopoiesis.     

Structure,expression,and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processesranging from the break-down of extracellular matrix to the processing of signaltransduction-related proteins.These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states.Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution.In this regard,studies from vari-ous laboratories have reported that a number of classes of metalloproteinases are found in hydra,a member of Cnidaria,the second oldest of existing animal phyla.These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1)astacin class,2)matrix met-alloproteinase class,and 3)neprilysin class.Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and cell differentiation as well as specialized functions in adult polyps.This article will review the structure,expression,and function of these metalloproteinases in hydra.     

Wnt signaling control of bone cell apoptosis

Wnts are a large family of growth factors that mediate essential biological processes like embryogenesis, morpho- genesis and organogenesis. These proteins also play a role in oncogenesis, and they regulate apoptosis in many tissues. Wnts bind to a membrane receptor complex comprised of a frizzled （FZD） G-protein-coupled receptor and a low-density lipoprotein （LDL） receptor-related protein （LRP）. The formation of this ligand-receptor complex initiates a number of signaling cascades that include the canonical/beta-catenin pathway as well as several noncanonical pathways. In recent years, canonical Wnt signaling has been reported to play a significant role in the control of bone formation. Clinical studies have found that mutations in LRP-5 are associated with reduced bone mineral density （BMD） and fractures. Investigations of knockout and transgenic mouse models of Wnt pathway components have shown that canonical Wnt signaling modulates most aspects ofosteoblast physiology including proliferation, differentiation, function and apoptosis. Transgenic mice expressing a gain of function mutant of LRP-5 in bone, or mice lacking the Wnt antagonist secreted frizzled-related protein-l, exhibit elevated BMD and suppressed osteoblast apoptosis. In addition, preclinical studies with pharmacologic compounds such as those that inhibit glycogen synthase kinase-3β support the importance of the canonical Wnt pathway in modulation of bone formation and osteoblast apoptosis.     

Structure, expression, and developmental function of early divergent forms of metalloproteinases in Hydra

Metalloproteinases have a critical role in a broad spectrum of cellular processes ranging from the breakdown of extracellular matrix to the processing of signal transduction-related proteins. These hydrolytic functions underlie a variety of mechanisms related to developmental processes as well as disease states. Structural analysis of metalloproteinases from both invertebrate and vertebrate species indicates that these enzymes are highly conserved and arose early during metazoan evolution. In this regard, studies from various laboratories have reported that a number of classes of metalloproteinases are found in hydra, a member of Cnidaria, the second oldest of existing animal phyla. These studies demonstrate that the hydra genome contains at least three classes of metalloproteinases to include members of the 1) astacin class, 2) matrix met-alloproteinase class, and 3) neprilysin class. Functional studies indicate that these metalloproteinases play diverse and important roles in hydra morphogenesis and ce     

Differential regulation of three thyroid hormone-responsive matrix metalloproteinase genes implicates distinct functions during frog embryogenesis.

Matrix metalloproteinases (MMPs) are a family of Zn(2+)-dependent extracellular proteases capable of degrading various proteinaceous components of the extracellular matrix (ECM). They are expressed in developmental and pathological processes such as postlactation mammary gland involution and tumor metastasis. Relatively few studies have been carried out to investigate the function of MMPs during embryogenesis and postembryonic organ development. Using Xenopus development as a model system, we and others have previously isolated three MMP genes as thyroid hormone response genes. They have distinct temporal and organ-specific regulations during thyroid hormone-dependent metamorphosis. We demonstrate here that three MMPs-stromelysin-3 (ST3), collagenases-3 (Col3), and collagenases-4 (Col4)-also have distinct spatial and temporal expression profiles during embryogenesis. Consistent with earlier suggestions that ST3 is a direct thyroid hormone response gene whereas Col3 and Col4 are not, we show that precocious overexpression of thyroid hormone receptors in the presence of thyroid hormone lead to increased expression of ST3, but not Col3. Furthermore, our whole-mount in situ hybridizations reveal a tight but distinct association of individual MMPs with tissue remodeling in different regions of the animal during embryogenesis. These results suggest that ST3 is likely to play a role in ECM remodeling that facilitate apoptotic tissue remodeling or resorption, whereas Col3 and Col4 appear to participate in connective tissue degradation during development.     

Requirement for matrix metalloproteinase stromelysin-3 in cell migration and apoptosis during tissue remodeling in Xenopus laevis

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) was originally discovered as a gene whose expression was associated with human breast cancer carcinomas and with apoptosis during organogenesis and tissue remodeling. It has been shown previously, in our studies as well as those by others, that ST3 mRNA is highly upregulated during apoptotic tissue remodeling during Xenopus laevis metamorphosis. Using a function-blocking antibody against the catalytic domain of Xenopus ST3, we demonstrate here that ST3 protein is specifically expressed in the cells adjacent to the remodeling extracellular matrix (ECM) that lies beneath the apoptotic larval intestinal epithelium in X. laevis in vivo, and during thyroid hormone-induced intestinal remodeling in organ cultures. More importantly, addition of this antibody, but not the preimmune antiserum or unrelated antibodies, to the medium of intestinal organ cultures leads to an inhibition of thyroid hormone-induced ECM remodeling, apoptosis of the larval epithelium, and the invasion of the adult intestinal primodia into the connective tissue, a process critical for adult epithelial morphogenesis. On the other hand, the antibody has little effect on adult epithelial cell proliferation. Furthermore, a known MMP inhibitor can also inhibit epithelial transformation in vitro. These results indicate that ST3 is required for cell fate determination and cell migration during morphogenesis, most likely through ECM remodeling.     

A causative role of stromelysin-3 in extracellular matrix remodeling and epithelial apoptosis during intestinal metamorphosis in Xenopus laevis

The matrix metalloproteinases are a family of proteases capable of degrading various components of the extracellular matrix. Expression studies have implicated the involvement of the matrix metalloproteinase stromelysin-3 (ST3) in tissue remodeling and pathogenesis. However, the in vivo role of ST3 has been difficult to study because of a lack of good animal models. Here we used intestinal remodeling during thyroid hormone-dependent metamorphosis of Xenopus laevis as a model to investigate in vivo the role of ST3 during postembryonic organ development in vertebrates. We generated transgenic tadpoles expressing ST3 under control of a heat shock-inducible promoter. We showed for the first time in vivo that wild type ST3 but not a catalytically inactive mutant was sufficient to induce larval epithelial cell death and fibroblast activation, events that normally occur only in the presence of thyroid hormone. We further demonstrated that these changes in cell fate are associated with altered gene expression in the intestine and remodeling of the intestinal basal lamina. These results thus suggest that ST3 regulates cell fate and tissue morphogenesis through direct or indirect ECM remodeling.     

Identification and characterization of a novel collagenase in Xenopus laevis: possible roles during frog development.

Matrix metalloproteinases (MMPs) participate in extracellular matrix remodeling and degradation and have been implicated in playing important roles during organ development and pathological processes. Although it has been hypothesized for > 30 years that collagenase activities are responsible for collagen degradation during tadpole tail resorption, none of the previously cloned amphibian MMPs have been biochemically demonstrated to be collagenases. Here, we report a novel matrix metalloproteinase gene from metamorphosing Xenopus laevis tadpoles. In vitro biochemical studies demonstrate that this Xenopus enzyme is an interstitial collagenase and has an essentially identical enzymatic activity toward a collagen substrate as the human interstitial collagenase. Sequence comparison of this enzyme to other known MMPs suggests that the Xenopus collagenase is not a homologue of any known collagenases but instead represents a novel collagenase, Xenopus collagenase-4 (xCol4, MMP-18). Interestingly, during development, xCol4 is highly expressed only transiently in whole animals, at approximately the time when tadpole feeding begins, suggesting a role during the maturation of the digestive tract. More importantly, during metamorphosis, xCol4 is regulated in a tissue-dependent manner. High levels of its mRNA are present as the tadpole tail resorbs. Similarly, its expression is elevated during hindlimb morphogenesis and intestinal remodeling. In addition, when premetamorphic tadpoles are treated with thyroid hormone, the causative agent of metamorphosis, xCol4 expression is induced in the tail. These results suggest that xCol4 may facilitate larval tissue degeneration and adult organogenesis during amphibian metamorphosis.     

Thyroid-hormone-dependent and fibroblast-specific expression of BMP-4 correlates with adult epithelial development during amphibian intestinal remodeling

We have identified one of the genes that are up-regulated by thyroid hormone (TH) in Xenopus laevis small intestine as the Xenopus homolog of bone morphogenetic protein-4 (BMP-4). To clarify possible roles of BMP-4 in intestinal remodeling during metamorphosis, we have examined its expression in X. laevis intestine by using in situ hybridization and organ culture techniques. At the beginning of metamorphic climax, BMP-4 mRNA first becomes detectable in the connective tissue, concurrently with the appearance of adult epithelial primordia. Subsequently, when the adult epithelial primordia are actively proliferating, BMP-4 mRNA becomes more abundant only in the connective tissue with a gradient toward the epithelium. Thereafter, as the adult primordia differentiate, the level of BMP-4 mRNA gradually decreases. Thus, BMP-4 expression correlates well with cell proliferation and/or initial differentiation of the adult epithelium, but not with apoptosis of the larval epithelium. Furthermore, the present culture study indicates that (1) TH-induced expression of BMP-4 mRNA is higher in the anterior part of the intestine than in the posterior part, which agrees with the better development of the adult epithelium in the more anterior part, and that (2) the expression of BMP-4 mRNA is up-regulated by TH in the presence of epithelium, but not in its absence. Therefore, BMP-4, which is indirectly induced by TH through some epithelial factor(s), probably plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Isolation of connective-tissue-specific genes involved in <Emphasis Type="Italic">Xenopus</Emphasis> intestinal remodeling: thyroid hormone up-regulates Tolloid/BMP-1 expression

To clarify connective-tissue-specific genes involved in adult epithelial development during amphibian intestinal remodeling, we have isolated 16 cDNA clones derived from the anterior part of Xenopus laevis intestine cultured in vitro by using subtractive suppression hybridization. Among four genes identified, the expression of Xtld, a Xenopus homolog of Drosophila Tolloid closely related to bone morphogenic protein-1 (BMP-1), was most remarkably up-regulated during metamorphosis. To further explore the roles of Xtld in intestinal remodeling, we examined its developmental expression in the X. laevis intestine by in situ hybridization and northern blot analysis. Xtld mRNA first became detectable in the connective tissue just before the appearance of adult epithelial primordia. Subsequently, the level of Xtld mRNA reached a high in the connective tissue, concomitantly with adult epithelial development along the anteroposterior axis of the intestine. Thereafter, towards the completion of metamorphosis, the expression of Xtld mRNA was down-regulated. Thus, the expression profile of Xtld mRNA spatiotemporally correlates well with adult epithelial development in vivo. Furthermore, the present culture study has shown that thyroid hormone (TH) up-regulates the expression of Xtld mRNA organ-autonomously in the anterior part of the intestine, but not in its posterior part, and that TH up-regulation of Xtld expression is not mediated by the epithelium. These results suggest that TH directly up-regulates Xtld expression in the connective tissue along the anteroposterior axis, which in turn plays important roles in adult epithelial development during amphibian intestinal remodeling.     

Activity and expression of Xenopus laevis matrix metalloproteinases: identification of a novel role for the hormone prolactin in regulating collagenolysis in both amphibians and mammals

Prolactin (PRL) has long been implicated in Xenopus metamorphosis as an anti-metamorphic and/or juvenilizing hormone. Numerous studies showed that PRL could prevent effects of either endogenous or exogenous thyroid hormone (TH; T(3)). It has been shown that expression of matrix metalloproteinases (MMPs) is induced by TH during Xenopus metamorphosis. Direct in vivo evidence, however, for such anti-TH effects by PRL with respect to MMPs has not been available for the early phase of Xenopus development or metamorphosis. To understand the functional role of PRL, we investigated effects of PRL on Xenopus collagenase-3 (XCL3) and collagenase-4 (XCL4) expression in a cultured Xenopus laevis cell line, XL-177. Northern blot analysis demonstrated that XCL3 and XCL4 expression were not detected in control or T(3)-treated cells, but were differentially induced by PRL in a dose- and time-dependent fashion. Moreover, treatment with IL-1alpha as well as phorbol myristate acetate (PMA), a protein kinase C (PKC) activator, or H8, a protein kinase A (PKA) inhibitor, augmented PRL-induced collagenase expression, suggesting that multiple protein kinase pathways and cytokines may participate in PRL-induced collagenase expression. Interestingly, XCL3 expression could be induced in XL-177 cells by T(3), but only when co-cultured with prometamorphic Xenopus tadpole tails (stage 54/55), suggesting that the tails secrete a required intermediate signaling molecule(s) for T(3)-induced XCL3 expression. Taken together, these data demonstrate that XCL3 and XCL4 can be differentially induced by PRL and T(3) and further suggest that PRL is a candidate regulator of TH-independent collagenase expression during the organ/tissue remodeling which occurs in Xenopus development.     

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.