Matrix Metalloproteinase 2 Is Required for Ovulation and Corpus Luteum Formation in Drosophila

Ovulation is critical for successful reproduction and correlates with ovarian cancer risk, yet genetic studies of ovulation have been limited. It has long been thought that the mechanism controlling ovulation is highly divergent due to speciation and fast evolution. Using genetic tools available in Drosophila, we now report that ovulation in Drosophila strongly resembles mammalian ovulation at both the cellular and molecular levels. Just one of up to 32 mature follicles per ovary pair loses posterior follicle cells (“trimming”) and protrudes into the oviduct, showing that a selection process prefigures ovulation. Follicle cells that remain after egg release form a “corpus luteum (CL)” at the end of the ovariole, develop yellowish pigmentation, and express genes encoding steroid hormone biosynthetic enzymes that are required for full fertility. Finally, matrix metalloproteinase 2 (Mmp2), a type of protease thought to facilitate mammalian ovulation, is expressed in mature follicle and CL cells. Mmp2 activity is genetically required for trimming, ovulation and CL formation. Our studies provide new insights into the regulation of Drosophila ovulation and establish Drosophila as a model for genetically investigating ovulation in diverse organisms, including mammals.     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Modulation of the Membrane Type 1 Matrix Metalloproteinase Cytoplasmic Tail Enhances Tumor Cell Invasion and Proliferation in Three-dimensional Collagen Matrices

Increasing evidence suggests that the cytoplasmic tail of membrane type 1 matrix metalloproteinase (MT1-MMP) is subject to phos pho ryl a tion and that this modification may influence its enzymatic activity at the cell surface. In this study, phos pho ryl a ted MT1-MMP is detected using a phospho-specific antibody recognizing a protein kinase C consensus sequence (phospho-TXR), and a MT1-MMP tail peptide is phos pho ryl a ted by exogenous protein kinase C. To characterize the potential role of cytoplasmic residue Thr⁵⁶⁷ in these processes, mutants that mimic a state of either constitutive (T567E) or defective phos pho ryl a tion (T567A) were expressed and analyzed for their functional effects on MT1-MMP activity and cellular behavior. Phospho-mimetic mutants of Thr⁵⁶⁷ exhibit enhanced matrix invasion as well as more extensive growth within a three-dimensional type I collagen matrix. Together, these findings suggest that MT1-MMP surface action is regulated by phos pho ryl a tion at cytoplasmic tail residue Thr⁵⁶⁷ and that this modification plays a critical role in processes that are linked to tumor progression.Largely composed of a mixture of collagens, laminins, and vitronectin, the extracellular matrix (ECM)² serves as both a physical scaffold and a barrier against cell invasion. It has become increasingly evident that the structural condition of the ECM plays a unique role in regulating cell behavior. Proteolysis of integral components of the basement membrane disturbs the barrier provided by the ECM. Without physical restriction, cells invade the surrounding environment in an unregulated manner. The ability of matrix metalloproteinases (MMPs) to collectively degrade nearly all ECM constituents allows this class of enzymes to function in a diverse range of physiological processes (1, 2). Of the anchored MMPs, membrane type 1 matrix metalloproteinase (MT1-MMP) was the first to be discovered and has been most thoroughly characterized. Unlike soluble MMPs, MT1-MMP has a stretch of hydrophobic amino acids that traverse the cell membrane, followed by a short cytoplasmic tail composed of 20 amino acids (3). The advantage of cell surface localization is 2-fold. Surface restriction allows MT1-MMP to modify the immediate pericellular environment, overcoming physical constraints imposed by the ECM (2). Localization at the cell surface also places tethered MMPs in an optimal position to function at invadapodia, highly specialized areas of the cell membrane that form during focalized cell invasion (4). Although information regarding the role of the cytoplasmic tail is relatively limited (5, 6), this domain may function as a bridge to the intracellular machinery.MT1-MMP has an essential role in matrix remodeling during physiological processes (7, 8). Conversely, its enzymatic activity is key to acquiring a metastatic phenotype in a variety of tumor cells, including lung, colon, breast, and cervical carcinomas (2, 9–11). The ability to alter the physical structure of the pericellular environment, while triggering the activation and modification of several cell surface proteins, identifies a central role for MT1-MMP in influencing cellular behavior (12). In return, stringent cellular regulation of MT1-MMP enzymatic activity is necessary to prevent aberrant proteolysis.Increasing evidence suggests that the cytoplasmic tail of MT1-MMP may regulate its activity at the cell surface. It has been demonstrated that MT1-MMP is internalized from the cell surface and that this process requires the presence of the cytoplasmic domain (5, 6). Tail truncation restricts MT1-MMP to the cell surface, suggesting that this domain contains sequence(s) that either mediate internalization or are required for physical interaction with another protein that facilitates its internalization (5, 6). The mechanism regulating this process has yet to be determined. Interestingly, both invasion and migration are down-regulated in cells where MT1-MMP is restricted to the cell surface (5, 6). These data suggest a correlation between internalization and matrix turnover, where MT1-MMP activity is either abrogated or enhanced under appropriate stimuli.Reversible phosphorylation is widely recognized as a key post-translational modification that regulates protein function. The cytoplasmic domain of MT1-MMP has three potential phosphorylation sites: Thr⁵⁶⁷, Tyr⁵⁷³, and Ser⁵⁷⁷. Recent work by Nyalendo et al. (13) indicates that MT1-MMP is phosphorylated at tyrosine residue Tyr⁵⁷³, and that this modification influences cell migration. Several surface proteins are regulated by phosphorylation at multiple residues. In the MT1-MMP cytoplasmic tail, Thr⁵⁶⁷ has homology with the consensus sequence for both protein kinase C (TXR) and ERK1/2 (XTP) (14), suggesting the possibility that active MT1-MMP might also be regulated through phosphorylation of this cytoplasmic tail residue. In the present study, we report that MT1-MMP bears a threonine phosphorylation site in its cytoplasmic tail and that this modification plays an important role in regulating several aspects of carcinoma cell behavior, including invasion and three-dimensional growth.     

NDRG4 Is Required for Cell Cycle Progression and Survival in Glioblastoma Cells

NDRG4 is a largely unstudied member of the predominantly tumor suppressive N-Myc downstream-regulated gene (NDRG) family. Unlike its family members NDRG1–3, which are ubiquitously expressed, NDRG4 is expressed almost exclusively in the heart and brain. Given this tissue-specific expression pattern and the established tumor suppressive roles of the NDRG family in regulating cellular proliferation, we investigated the cellular and biochemical functions of NDRG4 in the context of astrocytes and glioblastoma multiforme (GBM) cells. We show that, in contrast to NDRG2, NDRG4 expression is elevated in GBM and NDRG4 is required for the viability of primary astrocytes, established GBM cell lines, and both CD133⁺ (cancer stem cell (CSC)-enriched) and CD133⁻ primary GBM xenograft cells. While NDRG4 overexpression has no effect on cell viability, NDRG4 knockdown causes G₁ cell cycle arrest followed by apoptosis. The initial G₁ arrest is associated with a decrease in cyclin D1 expression and an increase in p27^Kip1 expression, and the subsequent apoptosis is associated with a decrease in the expression of XIAP and survivin. As a result of these effects on cell cycle progression and survival, NDRG4 knockdown decreases the tumorigenic capacity of established GBM cell lines and GBM CSC-enriched cells that have been implanted intracranially into immunocompromised mice. Collectively, these data indicate that NDRG4 is required for cell cycle progression and survival, thereby diverging in function from its tumor suppressive family member NDRG2 in astrocytes and GBM cells.The N-Myc downstream-regulated gene (NDRG)⁵ family consists of four genes (NDRG1–4) that can be divided into two subfamilies based on sequence homology: NDRG1 and NDRG3 are in the first subfamily, and NDRG2 and NDRG4 make up the second subfamily. Although the four NDRG family members show distinct spatiotemporal expression patterns during embryonic development and in adult tissues (1–10), all four are highly expressed in the brain (4). To date, however, NDRG2 is the only NDRG family member that has been studied in the context of GBM cells and astrocytes. NDRG2 mRNA and protein levels are lower in GBM than in normal brain tissue, normal glial cells, and low grade astrocytomas (11–14), suggesting a tumor suppressive function. Data from experimental and clinical studies support this hypothesis: NDRG2 overexpression inhibits GBM cell proliferation (15), and decreased NDRG2 expression correlates with decreased GBM patient survival (13).In contrast to its subfamily member NDRG2, NDRG4 has not been studied in GBM cells or astrocytes. Nevertheless, available evidence supports the hypothesis that NDRG4 has an important role in this context that is similar to the role of NDRG2. First, unlike the relatively ubiquitous expression patterns of NDRG1–3, NDRG4 expression is restricted to a small number of tissues including the brain, where it is expressed at particularly high levels (7, 10). This restricted expression pattern suggests that NDRG4 plays an important role within the central nervous system. Second, NDRG4 is more than 60% identical in amino acid sequence to NDRG2. This sequence similarity is likely behind the overlapping functions of these two proteins in certain cell types within the brain. For example, in PC12 neuronal cells, both NDRG4 and NDRG2 promote neurite extension (16–18). In combination with the brain-specific expression pattern of NDRG4, these functional and sequence similarities suggest that NDRG4 may recapitulate the tumor suppressive function of NDRG2 in primary brain neoplasms.To determine if the similarities between NDRG2 and NDRG4 extend to the context of GBM, we investigated the role of NDRG4 in GBM cell lines and primary human astrocytes. In contrast to the established roles of NDRG2 and other NDRG family members, we found that the role of NDRG4 in GBM is not tumor suppressive. On the contrary, both astrocytes and GBM cells require the presence of NDRG4 for cell cycle progression and survival.     

Expression of Metalloproteinase 2 in the Cell Response to Porous Demineralized Bovine Bone Matrix

Summary The purpose of the study was to analyze the involvement of metalloproteinase 2 (MMP-2) and macrophages in the tissue and cell response to the organic graft material produced from bovine cancellous bone. Thirty adult male white Wistar rats (Rattus norvegicus) received implants of blocks of demineralized bovine bone matrix between the fasciae of the quadriceps muscle. The specimens collected at 3, 7, 14, 21 and 28 days after implantation (n = 6/period). Sections of 6 μm thick were stained with hematoxylin and eosin and immunolabeled with anti-MMP-2 and anti-CD68 using standard avidin–biotin–peroxidase method. The tissue response to the material was initially mediated by polymorphonuclear neutrophils, evolving to a mononuclear inflammatory infiltrate with macrophages and few lymphocytes and plasma cells and presence of inflammatory multinucleated giant cells (GC) in contact with the material that exhibited signs of resorption. The number of cells immunolabeled to MMP-2 was highest at day 7 (103.2 ± 39.1), but significantly decreased (F = 3.67; p = 0.044) until day 28 (45.9 ± 13.1). CD68 immunostaining also significantly decreased (F = 6.75; p = 0.007) from day 7 (49.5 ± 10.4) to day 28 (19.5 ± 8.9). A positive and statistically significant correlation was observed between the evolutions of these two variables. The material had been almost completely resorbed at day 28. Among cells present at the granuloma, anti-MMP-2 immunostaining was predominant and more intense in macrophages, yet lightly immunolabeled multinucleated giant cells were found in close contact with the material. Thus, considering the experimental limitations of this study, we concluded that MMP-2 produced by macrophages participates in the resorption of demineralized bovine bone.     

基质金属蛋白酶2抑制剂的筛选及鉴定

以原核表达的具有明胶水解活性的人基质金属蛋白酶 2的催化区 (MCD)为靶标 ,筛选噬菌体随机环七肽库和十二肽库 .找到 6种与MCD特异结合的小肽 ,将 6种小肽基因分别与GST表达质粒重组 ,进行GST融合表达 ,制备融合蛋白 .采用Glutathione Sepharose 4B亲和层析法纯化融合蛋白 ,通过酶抑制实验、体外侵袭实验检测融合蛋白的活性 .结果表明 ,GST C71能够抑制MCD水解 β酪蛋白的活性 ,并且对人纤维肉瘤细胞HT10 80的体外侵袭有明显的抑制作用     

Hsp70-2 is Required for Tumor Cell Growth and Survival

Heat shock protein 70 (Hsp70) family consists of at least eight chaperone proteins that differ from each other by their pattern of expression and intracellular localization. Whereas ample experimental and clinico-pathological data has implicated the major stress-inducible Hsp70-1 as a protein required for cancer cell survival, the study of the other family members has been limited by the lack of experimental tools to differentiate between the highly homologous family members. This limitation has been recently overcome by the RNA interference technology that for the first time allows targeted knockdown of the individual Hsp70 family members. Data based on this technology has revealed that also Hsp70-2, a protein essential for spermatogenesis, is required for cancer cell growth and survival. Remarkably, the highly homologous Hsp70 proteins enhance cancer cell growth and survival by distinct molecular mechanisms.     

Rab24 is Required for Normal Cell Division

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co‐localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i. e. chromatin bridges). Furthermore, in CHO cells stably transfected with GFP‐Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.     

Calpain 2 is required for sister chromatid cohesion

Calpains form a family of Ca²⁺-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2 depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion.     

Calpain2对肝癌细胞浸润和转移能力的影响

目的：肝细胞癌（HCC）是一类常见的恶性肿瘤,主要表现为进展迅速、易复发及预后不良。侵袭转移作为肝癌的最主要的恶性表型,是造成较高致死率的主要原因。Calpain是钙激活中性蛋白酶,广泛参与了细胞多种生命过程。其中Calpainl和Calpain2是Calpain家族主要成员,对于维持肿瘤细胞恶性表型有重要作用。本研究通过RNA干涉技术下调人肝癌Huh7细胞中Calpain2基因的表达,检测下调Calpain2对人肝癌Huh7细胞黏附,侵袭和迁移能力的影响,明确Calpain2在人肝癌细胞浸润和转移过程中的作用。方法：合成Calpain2的RNAi片段,瞬时转染人肝癌细胞Huh7,降低Hull7细胞中Calpain2的表达,运用细胞黏附实验,细胞侵袭实验及划痕愈合实验检测干涉Calpain2对肝癌细胞的黏附,侵袭和迁移能力的影响。结果：合成Calpain2的RNAi片段。瞬时转染人肝癌细胞Huh73,36小时后,细胞中Calpain2的蛋白水平明显下降,干涉Calpain2后人肝癌细胞Huh7的黏附率,侵袭率及划痕修复率的显著下降。结论：以上实验结果表明Calpain2能够促进肝癌细胞黏附,侵袭及划痕修复能力,Calpain2能够促进肝癌细胞的浸润和转移的作用,是肝癌发生发展过程中的肿瘤促进因子。因此,Calpain2可以作为抑制肝癌侵袭和转移的潜在靶点,靶向Call＇ain2的药物可能成为治疗肝癌侵袭转移的新方法。     

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 .     

Osmotic Regulation Is Required for Cancer Cell Survival under Solid Stress

For a solid tumor to grow, it must be able to support the compressive stress that is generated as it presses against the surrounding tissue. Although the literature suggests a role for the cytoskeleton in counteracting these stresses, there has been no systematic evaluation of which filaments are responsible or to what degree. Here, using a three-dimensional spheroid model, we show that cytoskeletal filaments do not actively support compressive loads in breast, ovarian, and prostate cancer. However, modulation of tonicity can induce alterations in spheroid size. We find that under compression, tumor cells actively efflux sodium to decrease their intracellular tonicity, and that this is reversible by blockade of sodium channel NHE1. Moreover, although polymerized actin does not actively support the compressive load, it is required for sodium efflux. Compression-induced cell death is increased by both sodium blockade and actin depolymerization, whereas increased actin polymerization offers protective effects and increases sodium efflux. Taken together, these results demonstrate that cancer cells modulate their tonicity to survive under compressive solid stress.     

ANT2 Isoform Required for Cancer Cell Glycolysis

The three adenine nucleotide translocator ({ANT1} to {ANT3}) isoforms, differentially expressed in human cells, play a crucial role in cell bioenergetics by catalyzing ADP and ATP exchange across the mitochondrial inner membrane. In contrast to differentiated tissue cells, transformed cells, and their ρ⁰ derivatives, i.e. cells deprived of mitochondrial DNA, sustain a high rate of glycolysis. We compared the expression pattern of {ANT} isoforms in several transformed human cell lines at different stages of the cell cycle. The level of {ANT2} expression and glycolytic ATP production in these cell lines were in keeping with their metabolic background and their state of differentiation. The sensitivity of the mitochondrial inner membrane potential (Δψ) to several inhibitors of glycolysis and oxidative phosphorylation confirmed this relationship. We propose a new model for ATP uptake in cancer cells implicating the {ANT2} isoform, in conjunction with hexokinase II and the β subunit of mitochondrial ATP synthase, in the Δψ maintenance and in the aggressiveness of cancer cells.     

Diurnal Variation of Tight Junction Integrity Associates Inversely with Matrix Metalloproteinase Expression in Xenopus laevis Corneal Epithelium: Implications for Circadian Regulation of Homeostatic Surface Cell Desquamation

Background and Objectives

The corneal epithelium provides a protective barrier against pathogen entrance and abrasive forces, largely due to the intercellular junctional complexes between neighboring cells. After a prescribed duration at the corneal surface, tight junctions between squamous surface cells must be disrupted to enable them to desquamate as a component of the tissue homeostatic renewal. We hypothesize that matrix metalloproteinase (MMPs) are secreted by corneal epithelial cells and cleave intercellular junctional proteins extracellularly at the epithelial surface. The purpose of this study was to examine the expression of specific MMPs and tight junction proteins during both the light and dark phases of the circadian cycle, and to assess their temporal and spatial relationships in the Xenopus laevis corneal epithelium.

Methodology/Principal Findings

Expression of MMP-2, tissue inhibitor of MMP-2 (TIMP-2), membrane type 1-MMP (MT1-MMP) and the tight junction proteins occludin and claudin-4 were examined by confocal double-label immunohistochemistry on corneas obtained from Xenopus frogs at different circadian times. Occludin and claudin-4 expression was generally uniformly intact on the surface corneal epithelial cell lateral membranes during the daytime, but was frequently disrupted in small clusters of cells at night. Concomitantly, MMP-2 expression was often elevated in a mosaic pattern at nighttime and associated with clusters of desquamating surface cells. The MMP-2 binding partners, TIMP-2 and MT1-MMP were also localized to surface corneal epithelial cells during both the light and dark phases, with TIMP-2 tending to be elevated during the daytime.

Conclusions/Significance

MMP-2 protein expression is elevated in a mosaic pattern in surface corneal epithelial cells during the nighttime in Xenopus laevis, and may play a role in homeostatic surface cell desquamation by disrupting intercellular junctional proteins. The sequence of MMP secretion and activation, tight junction protein cleavage, and subsequent surface cell desquamation and renewal may be orchestrated by nocturnal circadian signals.