Transforming growth factor-beta1 modulates matrix metalloproteinase-9 production through the Ras/MAPK signaling pathway in transformed keratinocytes

Mouse transformed keratinocytes cultured in the presence of transforming growth factor-beta1 (TGF-beta1) acquire a set of morphological and functional properties giving rise to a more motile phenotype that expresses mesenchymal markers. In this work, we present evidence showing that TGF-beta1 stimulates cellular production of MMP-9 (Gelatinase B), a metalloproteinase that plays an important role in tumoral invasion. Our results demonstrate that TGF-beta1stimulates MMP-9 production and MMP-9 promoter activity in a process that depends of the activation of the Ras-ERK1,2 MAP kinase pathway. The latter was demonstrated by cellular transfection of TGF-beta1-sensitive cells with a RasN17 mutant gene, using PD 098059, a MEK 1,2 inhibitor, and treating cells with anti-sense oligodeoxinucleotides. The enhanced MMP-9 production proved to be an important factor in the acquisition of migratory and invasive properties as shown by the use of a specific inhibitor of MMP-9 (GM6001) that inhibits the TGF-beta1-stimulated invasive and migratory properties of these transformed keratinocytes.     

FGF-2 在细胞凋亡中的作用机制

成纤维细胞生长因子2(fibroblast growth factor 2,FGF-2)具有多种细胞生物学功能。FGF-2在肿瘤组织中呈高水平表达状态,且可抑制多种化疗药物的促凋亡作用,从而曾为肿瘤细胞存活的重要刺激因素。但也有研究表明FGF-2可诱导部分细胞的分化和凋亡。鉴于FGF-2在肿瘤的发生发展中发挥的重要作用,FGF-2与细胞凋亡的关系及其相应的调节机制成为有待于深入研究和迫切需要解决的问题。本文主要阐述在细胞凋亡通路中,FGF-2关键分子的作用机制及其最新研究进展。     

A permeable FGF-1 nuclear localization sequence peptide induces DNA synthesis independently of Ras activation

A 26-amino-acid peptide (designated PFNP) composed of the nuclear localization signal of fibroblast growth factor (FGF)-1 and a membrane-permeable peptide is known to mimic FGF-1's ability to stimulate DNA synthesis in various cell types at low cell densities. The underlying molecular mechanism is unknown, however. Here we show that PFNP activity is inhibited in murine fibroblasts by a tyrosine kinase inhibitor, that PFNP does not bind to the FGF receptor, and that PFNP does not induce phosphorylation of the FGF receptor substrate. In addition, expression of a dominant-negative form of Ras, which abolished the activities of epidermal growth factor (EGF) and heparin-binding EGF, had no affect on PFNP-induced DNA synthesis. Despite this apparent Ras independence, PFNP activity correlated with phosphorylation of ERK1/2 MAP kinases and was concentration dependently inhibited by inhibitors of ERK1/2 MAP kinase phosphorylation. These results indicate that whereas Ras activation is dispensable for PFNP-induced DNA synthesis, activation of tyrosine kinases and ERK1/2 kinases, albeit independently of the FGF receptor system, is crucial. Interestingly, FGF-1 signaling was predominantly Ras-independent when the cell density was optimum for PFNP, suggesting that PFNP and FGF-1 share the same signaling mechanism.     

Curcumin suppresses phorbol ester-induced matrix metalloproteinase-9 expression by inhibiting the PKC to MAPK signaling pathways in human astroglioma cells

The aberrant expression of matrix metalloproteinase-9 (MMP-9) is implicated in the invasion and angiogenesis process of brain tumor. This study has investigated the effects of curcumin on MMP-9 expression in human astroglioma cell lines. Curcumin significantly inhibited the MMP-9 enzymatic activity and protein expression that was induced by PMA. The inhibitory effect of curcumin on MMP-9 expression correlates with the decreased MMP-9 mRNA level and the suppression of MMP-9 promoter activity. The curcumin-mediated inhibition of MMP-9 gene expression appears to occur via NF-kappaB and AP-1 because their DNA binding activities were suppressed by curcumin. Furthermore, curcumin strongly repressed the PMA-induced phosphorylation of ERK, JNK, and p38 MAP kinase, which were dependent on the PKC pathway. Therefore, the inhibition of MMP-9 expression by curcumin might have therapeutic potential for controlling the growth and invasiveness of brain tumor.     

水飞蓟宾抑制TNF-α诱导的MMP-9在胃癌细胞中的表达

目的确定在胃癌细胞株中水飞蓟宾对TNF-α诱导的MMP-9表达的影响。方法应用细胞增殖分析、化学抑制剂处理、免疫印迹、流式细胞分析、腺病毒转移等技术完成实验。结果在SNU216和SNU668胃癌细胞中,MMP-9mRNA和蛋白的表达都被TNF-α剂量依赖性地提高。另-方面,TNF—α诱导的MMP-9表达被水飞蓟宾剂量依赖性地抑制。结论在胃癌细胞中水飞蓟宾可减少TNF-α诱导的MMP-9表达。     

The kinetics of FGF-2 binding to heparan sulfate proteoglycans and MAP kinase signaling

Binding of growth factors to specific cell surface receptors is the first step in initiating cell signaling cascades that ultimately result in diverse activities such as proliferation, differentiation, and apoptosis. Dimerization and phosphorylation of tyrosine kinase transmembrane receptors is the typical paradigm for this activation but, for many growth factors, cell surface interactions are not limited to a single receptor type. In particular, heparin-binding growth factors, such as fibroblast growth factor-2 (FGF-2), bind to heparan sulfate proteoglycans (HSPG) on the cell surface and within the extracellular matrix (ECM), and these molecules have been viewed as accessory co-receptors serving to facilitate tyrosine kinase receptor binding. Recent studies, however, have indicated that HSPG can directly participate in signal transduction in response to FGF-2 binding. Thus, in the present study, we used mathematical modeling to examine whether the kinetics of formation of the various FGF-2 bound complexes on the cell surface correlate with the activation of the downstream mediators of FGF-2 response, Erk1/2. We find that FGF-2 binding to its receptor correlates well with Erk1/2 activation and that HSPG can modulate this response through its ability to stabilize these ligand receptor complexes. Moreover, we also observed that FGF-2 binding to HSPG correlates strongly with Erk1/2 activation under conditions where there is a loss of receptor activity, and we demonstrate that the relative amounts of signaling and non-signaling HSPG on the cell surface, as well as the presence of competing HSPG in the ECM, can impact the signal potential via this pathway. Thus, the selective regulation of specific HSPG might provide a mechanism for fine tuned modulation of heparin-binding growth factor signaling in cells where signal intensity and duration could direct cellular response toward growth, migration or differentiation.     

Synthesis of disaccharides derived from heparin and evaluation of effects on endothelial cell growth and on binding of heparin to FGF-2

The disaccharide beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and other small nonsulfated oligosaccharides related to heparin/heparan sulfate have been shown to bind to FGF and activated the fibroblast growth factor (FGF) signalling pathway in (F32) cells expressing the FGF receptor. Synthetic routes to beta-D-GlcA-(1-->4)-alpha-D-GlcNAc-1-->OMe and a glucose analogue beta-D-Glc-(1-->4)-alpha-D-GlcNAc-1-->OMe are described. The effects of these disaccharides on endothelial cell growth, which is relevant to angiogenesis, were evaluated and it was found they did not mimic the inhibitory effects that were observed for heparin albumin (HA) and that have also been observed by monosaccharide conjugates. They did not alter bovine aortic endothelial cell (BAEC) proliferation, in the presence of FGF-2 in serum free medium or in absence of FGF-2 in serum free and complete medium. Disaccharides (10 microg/mL) reduced by 25-31% the inhibition caused by HA (10 microg/mL) on BAEC growth in serum-free medium but had no effect in complete medium. There was no evidence obtained for the binding of these oligosaccharides to FGF-2 in competition with HA by ELISA.     

同型半胱氨酸对A7r5细胞增殖及MMP-9表达的影响

观察同型半胱氨酸（Hcy）对大鼠胸主动脉平滑肌细胞（A7r5细胞）增殖及对基质金属蛋白酶9（MMP-9）表达的影响。0.25、0.50和1.00 mmol/LHcy分别作用A7r5细胞48 h,倒置显微镜下观察细胞的形态学改变;RT-PCR检测MMP-9 mRNA的表达,Western blot检测MMP-9蛋白的表达。随着Hcy浓度的升高,细胞数目逐渐增多,体积增大,生长旺盛,呈增殖表现。不同浓度Hcy处理组MMP-9 mRNA及蛋白的表达均逐渐增加,各实验组与对照组差异均有统计学意义（P〈0.05）。同时显示正常对照组中MMP-9表达相对较少,为（3.60±1.42）%、（1.60±0.82）%。Hcy能促进A7r5细胞增殖,正常A7r5细胞中MMP-9表达较少;Hcy可诱导A7r5细胞MMP-9的表达,这一效应可能是Hcy致心血管疾病的分子机制之一。提示MMP-9可能成为干预心血管疾病的靶点。     

Role of matrix metalloproteinase-2 and -9 in the development of diabetic retinopathy

Diabetic retinopathy represents the most common causes of vision loss in patients affected by diabetes mellitus. The cause of vision loss in diabetic retinopathy is complex and remains incompletely understood. One of the earliest changes in the development of retinopathy is the accelerated apoptosis of retinal microvascular cells and the formation of acellular capillaries by unknown mechanism. Results of a recent research suggest an important role of matrix metalloproteinases (MMPs) in the development of diabetic retinopathy. MMPs are a large family of proteinases that remodel extracellular matrix components, and under pathological condition, its induction is considered as a negative regulator of cell survival; and in diabetes, latent MMPs are activated in the retina and its capillary cells, and activation of MMP-2 and -9 induces apoptosis of retinal capillary cells. This review will focus on the MMP-2 and MMP-9 in the diabetic retina with special reference to oxidative stress, mitochondria dysfunction, inflammation and angiogenesis, as well as summarizing the current information linking these proteins to pathogenesis of diabetic retinopathy.     

Leucine aminopeptidase 3 promotes migration and invasion of breast cancer cells through upregulation of fascin and matrix metalloproteinases-2/9 expression

Overexpression of leucine aminopeptidase 3 (LAP3) is involved in proliferation, migration, and invasion of several tumor cells and plays a crucial role in tumor metastasis. However, the related mechanism remains unknown. In this study, we used MDA-MB-231 and MCF7 breast cancer cell lines to explore the role of LAP3 in the regulation of cancer cell migration and invasion by employing the natural LAP3 inhibitor bestatin and a lentivirus vector that overexpresses or knocks down LAP3. Bestatin inhibited tumor cell migration and invasion in a dose-dependent manner. Western blot assay showed that bestatin and knockdown of LAP3 upregulated phosphorylation of Hsp27 and downregulated expression of fascin. Phosphorylation of Akt and expression of matrix metalloproteinase-2/9 can also be downregulated. LAP3 overexpression showed the opposite results. Immunohistochemistry analysis was conducted to detect expression levels of LAP3 in breast cancer tissues. High LAP3 expression was correlated with the grade of malignancy. Findings of this study uncovered the molecular mechanism of LAP3 on breast cancer metastasis and indicated that LAP3 may act as a potential antimetastasis therapeutic target.     

Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.     

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.     

PKC alpha regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKC alpha, beta II, delta, and epsilon (only wild-type zeta) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKC alpha, beta II, delta, and epsilon revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKC alpha, but not betaI I, delta, epsilon, or zeta induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [(3)H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKC alpha, beta II, delta, and epsilon revealed a necessary role for PKC alpha as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKC epsilon reduced cellular viability. A mechanism whereby PKC alpha might regulate hypertrophy was suggested by the observations that wild-type PKC alpha induced extracellular signal-regulated kinase1/2 (ERK1/2), that dominant negative PKC alpha inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKC alpha-induced hypertrophic growth. These results implicate PKC alpha as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway.     

Estrogen induces phospholipase A2 activation through ERK1/2 to mobilize intracellular calcium in MCF-7 cells

The principal secreted estrogen, 17beta-estradiol rapidly activates signaling cascades that regulate important physiological processes including ion transport across membranes, cytosolic pH and cell proliferation. These effects have been extensively studied in the MCF-7 estrogen-responsive human breast carcinoma cell line. Here, we demonstrate that a physiological concentration of 17beta-estradiol caused a rapid, synchronous and transient increase in intracellular calcium concentration in a confluent monolayer of MCF-7 cells 2-3 min after treatment. This response was abolished when cells were pre-incubated with the phospholipase A(2) (PLA(2)) inhibitor quinacrine or with the cyclooxygenase inhibitor indomethacin. The translocation of GFP-cPLA(2)alpha to perinuclear membranes occurred 1-2 min after 17beta-estradiol treatment; this translocation was concurrent with the transient phosphorylation of cPLA(2)alpha at serine residue 505. The phosphorylation and translocation of cPLA(2) were sensitive to inhibition of the extracellular signal regulated kinase (ERK) signaling cascade and occurred simultaneously with a transient activation of ERK. The phosphorylation of cPLA(2) could be stimulated by membrane impermeable 17beta-estradiol conjugated to bovine serum albumen and was blocked by an antagonist of the classical estrogen receptor. Here we show, for the first time, that PLA(2) and the eicosanoid biosynthetic pathway are involved in the 17beta-estradiol induced rapid calcium responses of breast cancer cells.     

Transient and sustained oxidative stress differentially activate the JNK1/2 pathway and apoptotic phenotype in H9c2 cells

The aim of this study was to investigate the activation of JNK1/2 signalling pathway and the respective cellular phenotype of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation of JNK1/2 pathway by exogenous H₂O₂, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation of c-Jun was observed, coincidently with the activation of nuclear JNK1/2; under sustained stress, these phosphorylation levels remained elevated above basal for up to 6 h, whereas under transient stress they declined to basal ones within 4 h of withdrawal. Furthermore, the JNK1/2 selective inhibitor SP600125 abolished the c-jun phosphorylation induced by oxidative stress. Our results using cell viability assays and light microscopy revealed that sustained H₂O₂ stimulation significantly and time-dependently decreased H9c2 viability, in contrast to transient stimulation; SP600125 (10 μM) abolished cell death induced by sustained as well as cell survival induced by transient oxidative stress. Hoechst staining showed an increase in DNA condensation during sustained, but not during transient stimulation. Moreover, from the antioxidants tested, catalase and superoxide dismutase prevented oxidative stress-induced cell death. Flow cytometry studies reconfirmed that sustained oxidative stress induced apoptosis, whereas transient resulted in the recovery of cardiac myoblasts within 24 h. We conclude that in H9c2 myoblasts, sustained activation of JNK1/2 signalling pathway during oxidative stimulation is followed by an apoptotic phenotype, while transient JNK1/2 activation correlates well with cell survival, suggesting a dual role of this signalling pathway in cell fate determination.