Inhibition of matrix metalloproteinase 14 (MMP-14)-mediated cancer cell migration

Matrix metalloproteinases (MMPs) have been shown to be key players in both extracellular matrix remodeling and cell migration during cancer metastasis. MMP-14, a membrane-anchored MMP, in particular, is closely associated with these processes. The hemopexin (PEX) domain of MMP-14 has been proposed as the modulating region involved in the molecular cross-talk that initiates cell migration through homodimerization of MMP-14 as well as heterodimerization with the cell surface adhesion molecule CD44. In this study, minimal regions required for function within the PEX domain were investigated through a series of substitution mutations. Blades I and IV were found to be involved in cell migration. We found that blade IV is necessary for MMP-14 homodimerization and that blade I is required for CD44 MMP-14 heterodimerization. Cross-talk between MMP-14 and CD44 results in phosphorylation of EGF receptor and downstream activation of the MAPK and PI3K signaling pathways involved in cell migration. Based on these mutagenesis analyses, peptides mimicking the essential outermost strand motifs within the PEX domain of MMP-14 were designed. These synthetic peptides inhibit MMP-14-enhanced cell migration in a dose-dependent manner but have no effect on the function of other MMPs. Furthermore, these peptides interfere with cancer metastasis without affecting primary tumor growth. Thus, targeting the MMP-14 hemopexin domain represents a novel approach to inhibit MMP-14-mediated cancer dissemination.     

Epigenetic regulation by histone methylation and histone variants

Epigenetics is the study of heritable changes in gene expression that are not mediated at the DNA sequence level. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and chromatin/histone modifications. With the identification of key histone-modifying enzymes, the biological functions of many histone posttranslational modifications are now beginning to be elucidated. Histone methylation, in particular, plays critical roles in many epigenetic phenomena. In this review, we provide an overview of recent findings that shape the current paradigms regarding the roles of histone methylation and histone variants in heterochromatin assembly and the maintenance of the boundaries between heterochromatin and euchromatin. We also highlight some of the enzymes that mediate histone methylation and discuss the stability and inheritance of this modification.     

Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1–32], thus revealing a self-amplifying loop for ET-1[1–32] generation. ET-1[1–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work. Published: October 28, 2002     

Redox regulation of matrix metalloproteinase gene family in small cell lung cancer cells

It has been implicated that reactive oxygen species (ROS) play important roles in modulating tumor progression. However, the mechanisms by which redox-regulated tumor progression are largely unknown. We previously demonstrated that reduced intracellular redox conditions could be achieved in stably transfected small cell lung cancer cells with gamma-glutamylcysteine synthetase (gamma-GCSh) cDNA which encodes a rate-limiting enzyme in the biosynthesis of glutathione (GSH), a major physiological redox regulator. In the present study, using DNA microarray analyses, we compared the expression profiles between the gamma-GCSh-transfected cells and their nontransfected counterpart. We observed downregulation of several matrix metalloproteinases (MMPs), i.e., MMPI and MMP3, and MMP10 in the transfected cells. Dot blot and Northern blot hybridizations confirmed that, among the 18 MMP gene family members and four tissue inhibitors of matrix metalloprotein family (TIMP) analyzed, the expression levels of these three MMPs were consistently reduced. Transiently increased gamma-GCSh expression using tetracycline-inducible gamma-GCSh adenoviral expression system also showed down-regulation of MMP3 and MMP10, but not MMP1. Our results demonstrated that redox regulation of MMP1, MMP3 and MMP10 expression depend upon different modes of redox manipulation. These results bear implication that antioxidant modulation of antitumor progression may be contributed at least in part by the downregulation of a subset of metrix metalloproteins.     

Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography

Remodeling of the extracellular matrix (ECM) during development, angiogenesis, wound healing, tumor metastasis, and other morphogenetic processes depends on the exquisitely regulated activities of matrix metalloproteinases (MMPs). Yet very little is known about the activity patterns of these proteases in vivo. We have employed fluorescent MMP-substrates, both in vitro and in vivo, to characterize patterns of MMP activity in the zebrafish embryo. Qualitatively similar patterns of degradation are detected using native Type I or Type IV collagen substrates, suggesting that multiple MMPs are being regulated concomitantly. MMP activity is observed primarily in ECM-rich structures predicted to be undergoing active remodeling, such as the perichordal sheath and somite boundaries. Patterns of Type I and Type IV collagen hydrolysis are similar, but not identical in embryos of any given stage. Conventional gelatin zymography shows MMPs present in embryos as early as 3-somites (11 h) and our in vivo assays detect Type IV collagen degradation at somite boundaries as early as 4-somites (11.5 h). However, we are unable to detect significant in vitro activity using homogenates made from embryos prior to Prim-16 (31 h). Mixed lysate assays demonstrate that this is the result of endogenous inhibitors present in early embryos, suggesting a model of matrix remodeling regulated by spatially heterogeneous MMP inhibition.     

The cell surface: the stage for matrix metalloproteinase regulation of migration

Matrix metalloproteinases are important for the turnover of extracellular matrix in tissue. Recent studies have expanded their roles well beyond extracellular matrix degradation - they also cleave many growth factors, cytokines and cell adhesion molecules in the extracellular milieu, modulating their functions irreversibly. In particular, some matrix metalloproteinases that associate with the cell surface have arisen as intriguing regulators of cellular functions, including migration.     

Progress in matrix metalloproteinase research

Matrix metalloproteinases (MMPs) are now acknowledged as key players in the regulation of both cell-cell and cell-extracellular matrix interactions. They are involved in modifying matrix structure, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury and in the progression of diseases such as arthritis, cancer and cardiovascular disease. Because of their wide spectrum of activities and expression sites, the elucidation of their potential as drug targets in disease or as important features of the repair process will be dependent upon careful analysis of their role in different cellular locations and at different disease stages. Novel approaches to the specific regulation of individual MMPs in different contexts are also being developed.     

A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

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Synthesis and characterization of dextran-peptide-methotrexate conjugates for tumor targeting via mediation by matrix metalloproteinase II and matrix metalloproteinase IX

We designed and synthesized new dextran-peptide-methotrexate conjugates for tumor-targeted delivery of chemotherapeutics via the mediation of matrix metalloproteinase II (MMP-2) and matrix metalloproteinase IX (MMP-9), both being widely known tumor-associated enzymes. A robust and flexible synthesis procedure and process monitoring chromatography assays were developed. The linker chemistry and the backbone charge were optimized to allow high sensitivity of the conjugates toward the targeted enzymes. The optimal conjugate carries Pro-Val-Gly-Leu-Ile-Gly as the peptide linker, and the charge on the dextran backbone is fully neutralized. In the presence of the targeted enzymes, the peptide was cleaved and peptidyl methotrexate was released, with a kcat/Km value of 1.21 x 10(5) M(-1) s(-1) for MMP-2 and 3.60 x 10(3) M(-1) s(-1) for MMP-9, respectively. Satisfactory stability of the new conjugates was demonstrated in serum containing conditions, suggesting the conjugates can remain intact in systemic circulation. These findings supported the tumor targeting capability of the new conjugates and warranted further investigation with in vivo study.     

Autogenous regulation of histone mRNA decay by histone proteins in a cell-free system.

We tested the hypothesis that histone mRNA turnover is accelerated in the presence of free histone proteins. In an in vitro mRNA decay system, histone mRNA was degraded four- to sixfold faster in reaction mixtures containing core histones and a cytoplasmic S130 fraction than in reaction mixtures lacking these components. The decay rate did not change significantly when histones or S130 was added separately, suggesting either that the histones were modified and thereby activated by S130 or that additional factors besides histones were required. RecA, SSB (single-stranded binding), and histone proteins all formed complexes with histone mRNA, but only histones induced accelerated histone mRNA turnover. Therefore, the effect was not the result of random RNA-protein interactions. Moreover, histone proteins did not induce increased degradation of gamma globin mRNA, c-myc mRNA, or total poly(A)- or poly(A)+ polysomal mRNAs. This autoregulatory mechanism is consistent with the observed accumulation of cytoplasmic histone proteins in cells after DNA synthesis stops, and it can account, in part, for the rapid disappearance of histone mRNA at the end of S phase.     

肺结核并发纤维化患者BALF和血清MMP及TIMP-1检测的临床意义

目的:探讨肺结核并发纤维化患者支气管肺泡灌洗液(BALF)及血清中基质金属蛋白9(MMP-9)及基质金属蛋白酶组织抑制剂l(TIMP-1)含量与肺纤维化及肺功能的关系.方法:酶联免疫(ELISA)法检测我院2009年3月至2009年9月68例肺结核并发纤维化患者(纤维化组)及30例无肺部疾病患者(对照组)的支气管肺泡灌洗液及血清中MMP-9及TIMP-1的含量,同时进行肺功能检查.结果:纤维化组患者的BALF及血清MMP-9的含量与对照组怠者相比无统计学意义,但纤维化组患者的BALF及血清TIMP-1的含量显著高于对照组(P＜0.05),且纤雏化组患者的BALF及血清MMP-9/TIMP-1比值低于对照组(P＜0.05).患者的动脉血氧分压、肺活量等肺功能指标与TIMP-1呈正相关,而与MMP-9/TIMP-1比值呈负相关(P＜0.05).结论:肺结核患者肺纤维化的发生可能与TIMP-1水平升高及MMP-9/TIMP-1比值降低相关,降低TIMP-9的水平可能是防止肺结核惠者并发肺纤维化的一种新途径.