Impact of Flavonoids on Matrix Metalloproteinase Secretion and Invadopodia Formation in Highly Invasive A431-III Cancer Cells

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.     

银丹心脑通抑制泡沫细胞形成及巨噬细胞分泌基质金属蛋白酶

目的:银丹心脑临床治疗冠心病心绞痛疗效显著,但其机理尚不明确.本实验通过观察银丹心脑通对巨噬细胞分泌基质金属蛋白酶的作用,探讨银丹心脑通的作用机理.方法:生长状态良好的单核细胞分化成巨噬细胞,加入不同浓度的药物.24h后收集细胞上清,ELISA检测细胞上清中MMP-2、MMP-9的含量.结果:给药组不同剂量均能抑制MMP-2、MMP-9的表达(P＜0.01-0.05),其中高剂量组效果最为显著.结论:银丹心脑通能够抑制巨噬细胞基质金属蛋白酶的分泌,具有明显的抗炎作用,此实验为其在临床上的进一步应用提供了依据.     

The Role of Collagen Charge Clusters in the Modulation of Matrix Metalloproteinase Activity

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-l-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, k_cat/K_m values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P₂₃–P₂₃′ subsites of collagenous substrates.     

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.     

The Role of Progestogens in Regulating Matrix Metalloproteinase Activity in Macrophages and Microglial Cells

Although the systemic effects of progestogens have been extensively studied, little is known in regards to the cellular effects of these compounds. Using a cellular model for vascular (macrophages) and brain (microglial) cells, we studied the effects of various progestogens, either alone or in combination with 17β-estradiol (E₂) on the activity of matrix metalloproteinase-9 (MMP-9), a proteolytic enzyme involved in vascular remodeling and plaque destabilization in cardiovascular events, blood–brain barrier breakdown in stroke and brain regeneration and neurovascular remodeling during repair phases of brain injury. In the absence of E₂, medroxyprogesterone acetate (MPA), a synthetic progestogen and progesterone (PG) metabolites tended to increase MMP-9 enzyme activity in macrophages and microglial cells, whereas PG decreased such activity in macrophages; exceptions being that MPA and the PG metabolite, pregnanediol (Pdiol) had no effect on macrophage MMP-9 enzyme activity and PG had no effect on microglial cell MMP-9 enzyme activity. In the presence of E₂, an opposite affect was observed whereby MPA and the PG metabolites tended to decrease MMP-9 enzyme activity from macrophages and microglial cells, whereas PG had no effect; exceptions being that MPA and Pdiol had no effect on macrophage MMP-9 enzyme activity. In conclusion, these results demonstrate that the effects of PG, PG metabolites and MPA on MMP-9 enzyme activity differ across vascular and brain cells when administered alone or in combination with E₂ which could have important mechanistic implications for hormone therapy.     

The Role of Structural Dynamics of Actin in Class-Specific Myosin Motility

The structural dynamics of actin, including the tilting motion between the small and large domains, are essential for proper interactions with actin-binding proteins. Gly146 is situated at the hinge between the two domains, and we previously showed that a G146V mutation leads to severe motility defects in skeletal myosin but has no effect on motility of myosin V. The present study tested the hypothesis that G146V mutation impaired rotation between the two domains, leading to such functional defects. First, our study showed that depolymerization of G146V filaments was slower than that of wild-type filaments. This result is consistent with the distinction of structural states of G146V filaments from those of the wild type, considering the recent report that stabilization of actin filaments involves rotation of the two domains. Next, we measured intramolecular FRET efficiencies between two fluorophores in the two domains with or without skeletal muscle heavy meromyosin or the heavy meromyosin equivalent of myosin V in the presence of ATP. Single-molecule FRET measurements showed that the conformations of actin subunits of control and G146V actin filaments were different in the presence of skeletal muscle heavy meromyosin. This altered conformation of G146V subunits may lead to motility defects in myosin II. In contrast, distributions of FRET efficiencies of control and G146V subunits were similar in the presence of myosin V, consistent with the lack of motility defects in G146V actin with myosin V. The distribution of FRET efficiencies in the presence of myosin V was different from that in the presence of skeletal muscle heavy meromyosin, implying that the roles of actin conformation in myosin motility depend on the type of myosin.     

Le^Y寡糖单克隆抗体对小鼠胚泡基质金属蛋白酶表达和分泌的影响

以LeY 寡糖特异性单克隆抗体AH6为工具中和胚泡表面LeY 寡糖后 ,通过RT PCR、明胶酶谱法、免疫印迹法等方法 ,在体外研究了着床前小鼠胚泡表面LeY 寡糖抗原与其基质金属蛋白酶 (MMP)、金属蛋白酶组织抑制因子 (TIMP1)的表达和分泌之间的关系。结果显示 :胚泡表面LeY 寡糖抗原被中和后仅 1.5h ,胚泡MMP2和MMP9基因转录表达明显下降、而TIMP1基因转录表达则略有升高 ;随后抗体中和引起胚泡MMP2、MMP9的分泌减少 ,而TIMP1的分泌则未见明显变化。结果表明胚泡表面的LeY 寡糖抗原对着床前胚泡的MMP的合成和分泌具有调节作用 ,而且这种作用可能主要是通过调节相应的MMP2和MMP9基因的表达而引起的     

Heme Oxygenase-1 Regulates Matrix Metalloproteinase MMP-1 Secretion and Chondrocyte Cell Death via Nox4 NADPH Oxidase Activity in Chondrocytes

Interleukin-1β (IL-1β) activates the production of reactive oxygen species (ROS) and secretion of MMPs as well as chondrocyte apoptosis. Those events lead to matrix breakdown and are key features of osteoarthritis (OA). We confirmed that in human C-20/A4 chondrocytes the NADPH oxidase Nox4 is the main source of ROS upon IL-1β stimulation. Since heme molecules are essential for the NADPH oxidase maturation and activity, we therefore investigated the consequences of the modulation of Heme oxygenase-1 (HO-1), the limiting enzyme in heme catabolism, on the IL-1β signaling pathway and more specifically on Nox4 activity. Induction of HO-1 expression decreased dramatically Nox4 activity in C-20/A4 and HEK293 T-REx™ Nox4 cell lines. Unexpectedly, this decrease was not accompanied by any change in the expression, the subcellular localization or the maturation of Nox4. In fact, the inhibition of the heme synthesis by succinylacetone rather than heme catabolism by HO-1, led to a confinement of the Nox4/p22^phox heterodimer in the endoplasmic reticulum with an absence of redox differential spectrum highlighting an incomplete maturation. Therefore, the downregulation of Nox4 activity by HO-1 induction appeared to be mediated by carbon monoxide (CO) generated from the heme degradation process. Interestingly, either HO-1 or CO caused a significant decrease in the expression of MMP-1 and DNA fragmentation of chondrocytes stimulated by IL-1β. These results all together suggest that a modulation of Nox4 activity via heme oxygenase-1 may represent a promising therapeutic tool in osteoarthritis.     

基质金属蛋白酶与血管壁细胞外基质重建

细胞外基质（ECM）不仅维持血管壁的完整性,而且还为血管细胞传递增殖,迁移,分化和凋亡的调控信号,基质金属蛋白酶（MMP）及其内源性抑制剂（TIMP）通过调节ECM合成与降解之间的动态平衡,使ECM维持正常的结构与功能。在高血压,动脉粥样硬化与血管再狭窄的发生与发展过程中,MMP和TIMP的合成与分泌出现异常,由此所引起的ECM合成与降解失调使ECM迅速发生重建。     

Role of ATP-Hydrolysis in the Dynamics of a Single Actin Filament

We study the stochastic dynamics of growth and shrinkage of single actin filaments taking into account insertion, removal, and ATP hydrolysis of subunits either according to the vectorial mechanism or to the random mechanism. In a previous work, we developed a model for a single actin or microtubule filament where hydrolysis occurred according to the vectorial mechanism: the filament could grow only from one end, and was in contact with a reservoir of monomers. Here we extend this approach in two ways—by including the dynamics of both ends and by comparing two possible mechanisms of ATP hydrolysis. Our emphasis is mainly on two possible limiting models for the mechanism of hydrolysis within a single filament, namely the vectorial or the random model. We propose a set of experiments to test the nature of the precise mechanism of hydrolysis within actin filaments.     

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a^null blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a^null embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.     

Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR^−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR^−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR^+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of ¹²⁵I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR^−/− and u-PAR^+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR^+/+ cells, whereas it was present in the pericellular space around u-PAR^−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.     

肿瘤细胞外泌体对肿瘤血管新生的调控作用

外泌体可将其内容物蛋白质、脂类、RNA、循环DNA等生物活性分子由供体细胞转运至受体细胞,对细胞与细胞间的通讯发挥重要调控作用。肿瘤细胞可以主动释放包括外泌体在内的胞外囊泡进入周围微环境。血管为肿瘤的生长提供氧气和营养物质,因此血管新生是肿瘤生长所必需的。研究发现,蛋白或非编码RNA在不同肿瘤细胞衍生的外泌体中存在特异性表达的现象。肿瘤外泌体将其内含的非编码RNA以及蛋白转运至内皮细胞,上调促血管新生因子的表达,进而提高内皮细胞的活性,促进其增殖、迁移和管腔形成。     

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.     

Addressing the Role of Cell Adhesion in Tumor Cell Dormancy

The dissemination of tumor cells prior to the surgical resection of early stage tumors poses a serious risk to the disease free survival of cancer patients. This risk arises from the latent capacity of these cells to form solid metastatic lesions after a prolonged period of dormancy, exacerbated by the fact that these cells are often refractory to adjuvant chemotherapeutic protocols. Ensuring the long term survival of cancer patients therefore necessitates an understanding of the mechanisms of tumor cell dormancy and the accompanying drug resistance. Experiments designed to compare the biological behavior of metastatic versus non-metastatic variants of tumor cells provide evidence that there exists a phenomenon of single-cell dormancy which may depend on a reciprocal dialogue between the tumor cell and the tissue microenvironment. Through a combination of 3-dimensional cell culture technique and in vivo models investigators are now beginning to elucidate the molecular mechanisms underlying this phenomenon. Here we review the results of a series of experiments describing the role of cell adhesion events in dictating tumor cell behavior, including the balance between proliferation and dormancy, and the acquisition of drug resistance.     

The Role of Microtubules and Their Dynamics in Cell Migration

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.     

基质力学对肿瘤发生发展及肿瘤细胞生物学行为影响的研究进展

实体瘤的发生发展常伴随着细胞外基质的异常沉积、交联和基质刚度增加.基质刚度增加和肿瘤细胞软化引起肿瘤微环境的力学异质性.基质力学通过影响肿瘤细胞的增殖、迁移、转移、上皮间质转换、肿瘤干细胞特性和耐药性等调控肿瘤的发生、恶性转变和转移.研究基质力学对肿瘤发生发展的影响不仅可深化对肿瘤发展的认识,也可为研究新的诊治方法提供理论基础.本文论述了细胞外基质力学特性对肿瘤发生发展及肿瘤细胞生物学行为影响的研究进展,并展望了其发展前景.     

Role of Actin Filaments in Correlating Nuclear Shape and Cell Spreading

It is well known that substrate properties like stiffness and adhesivity influence stem cell morphology and differentiation. Recent experiments show that cell morphology influences nuclear geometry and hence gene expression profile. The mechanism by which surface properties regulate cell and nuclear properties is only beginning to be understood. Direct transmission of forces as well as chemical signalling are involved in this process. Here, we investigate the formal aspect by studying the correlation between cell spreading and nuclear deformation using Mesenchymal stem cells under a wide variety of conditions. It is observed that a robust quantitative relation holds between the cell and nuclear projected areas, irrespective of how the cell area is modified or when various cytoskeletal or nuclear components are perturbed. By studying the role of actin stress fibers in compressing the nucleus we propose that nuclear compression by stress fibers can lead to enhanced cell spreading due to an interplay between elastic and adhesion factors. The significance of myosin-II in regulating this process is also explored. We demonstrate this effect using a simple technique to apply external compressive loads on the nucleus.