3-D collagen-dependent cell surface expression of MT1-MMP and MMP-2 activation regardless of integrin β1 function and matrix stiffness

Matrix metalloproteinases (MMPs) play roles in spatially dynamic processes, including morphogenesis, wound healing, and tumor invasion. Three-dimensional (3-D) type I collagen stimulates cellular activation of MMP-2, however, the mechanisms underlying this are controversial. The present study investigated mechanisms for 3-D collagen-induced MMP-2 activation in highly invasive human malignant mesothelioma cells. MMP-2 was effectively activated by cells cultured in 3-D collagen but not in 2-D collagen, whereas MMP-2 activation was not regulated by the flexibility of collagen. The 3-D collagen did not largely increase the gene expression of MMP-2 and MT1-MMP. However, MT1-MMP exposed to the cell surface was much increased by 3-D collagen, and loss of MT1-MMP abolished MMP-2 activation in response to 3-D collagen. MT1-MMP and integrin β1 translocated to pericellular regions interacting with collagen-coated microbeads, however their localization was different. Importantly, inhibition of integrin β1 function and expression did not affect 3-D collagen-induced cell surface localization of MT1-MMP and MMP-2 activation. Our results strongly suggest that 3-D collagen scaffolding may provide opportunity for direct and multivalent interaction with MT1-MMP, by which MMP-2 activation occur in abundant cell surface MT1-MMP-dependent manner, rather than a manner regulated by matrix stiffness and integrin β1 function.     

EGFR and HER2 exert distinct roles on colon cancer cell functional properties and expression of matrix macromolecules

Background

ErbB receptors, EGFR and HER2, have been implicated in the development and progression of colon cancer. Several intracellular pathways are mediated upon activation of EGFR and/or HER2 by EGF. However, there are limited data regarding the EGF-mediated signaling affecting functional cell properties and the expression of extracellular matrix macromolecules implicated in cancer progression.

Methods

Functional assays, such as cell proliferation, transwell invasion assay and migration were performed to evaluate the impact of EGFR/HER2 in constitutive and EGF-treated Caco-2 cells. Signaling pathways were evaluated using specific intracellular inhibitors. Western blot was also utilized to examine the phosphorylation levels of ERK1/2. Real time PCR was performed to evaluate gene expression of matrix macromolecules.

Results

EGF increases cell proliferation, invasion and migration and importantly, EGF mediates overexpression of EGFR and downregulation of HER2. The EGF–EGFR axis is the main pathway affecting colon cancer's invasive potential, proliferative and migratory ability. Intracellular pathways (PI3K-Akt, MEK1/2-Erk and JAK-STAT) are all implicated in the migratory profile. Notably, MT1- and MT2-MMP as well as TIMP-2 are downregulated, whereas uPA is upregulated via an EGF–EGFR network. The EGF–EGFR axis is also implicated in the expression of syndecan-4 and TIMP-1. However, glypican-1 upregulation by EGF is mainly mediated via HER2.

Conclusions and general significance

The obtained data highlight the crucial importance of EGF on the expression of both receptors and on the EGF–EGFR/HER2 signaling network, reveal the distinct roles of EGFR and HER2 on expression of matrix macromolecules and open a new area in designing novel agents in targeting colon cancer. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi‐square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.     

Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma

To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance.     

盘状结构域受体2胞外区的可溶性表达、纯化和功能鉴定

盘状结构域受体2（discoidin domain receptor 2,DDR2）是一种与肿瘤细胞转移相关的蛋白酷氨酸激酶,其配体为纤维性胶原,胶原对DDR2的活化上调细胞中基质金属蛋白酶1（MMP－1）的表达。为研究DDR2在类风温性关节炎(rteumatoid arthritis,RA）软骨破坏和肿瘤转移中的作用,尝试了在大肠杆菌中表达一段DDR2胞外区（命名DB）,并进行了可溶性部分的纯化和功能鉴定,以备将来用作DDR2的特异性阻断剂。获得了一株表达GST－DB融合蛋白的大肠杆菌克隆;其表达的蛋白质中可溶性部分约占全部融合蛋白的13％;经GST融合蛋白特异性亲和珠纯化后,获得了纯度约86.1%的可溶性GST－DB融合蛋白;竞争结合抑制实验显示,GST－DB具有阻断Ⅱ型胶原和细胞表面天然DDR2受体结合的功能;细胞实验表明,GST－DB有抑制Ⅱ型胶原刺激下的类风湿性关节炎滑膜细胞和NIH3T3细胞分泌MMP－1的作用。以上结果提示,表达的融保蛋白GST－DB具有抑制天然DDR2功能的作用;DDR2在滑膜细胞和NIH3T3细胞中介导Ⅱ型胶原刺激下的MMP－1的分泌。     

Alteration in gene expression profile and biological behavior in human lung cancer cell line NL9980 by nm23-H1 gene silencing

Lung cancer is the leading cause of cancer death in both men and women. Tumor metastasis is an essential aspect of lung cancer progression. nm23-H1 is a metastasis suppressor gene. The molecular mechanism by which nm23-H1 suppresses the metastasis is still unclear. Here, we compared the gene expression profile of human large cell lung cancer cell line NL9980 by nm23-H1 gene silencing with that of negative control cells to comprehensively investigate nm23-H1-mediated changes in gene expression of NL9980 cells. Microarray assay revealed that expression of 733-known genes (1.9%, 733/38,500) were altered in response to nm23-H1 gene silencing, including 466 upregulated genes and 267 downregulated. real-time PCR assay of the expression changes indicated that 81.82% (45/55) of verified genes were consistent with that observed in microarray assay. The upregulated genes included MMP-1, -2, SNAI2, CXCL1, 2, 3, PAI-2, while the downregulated genes included cystatin B, TIMP-2, E-cadherin, centrin-2, all of which have been associated with tumor metastasis. Furthermore, we confirmed by Western blot that the expression of MMP-1 and -2 were significantly increased while that of cystatin B was dramatically decreased in NL9980-nm23-H1 silencing cells. The NL9980-nm23-H1 silencing cells exhibited significantly more S phase growth and invasive ability. Thus, silencing of nm23-H1 gene caused metastasis-related gene expression changes in lung cancer cells. The knockdown of nm23-H1 expression may change the lung cancer cells to a more invasive phenotype through alteration in the expression of a set of genes.     

水通道蛋白1-C末端肽段/GST融合蛋白的原核表达(简报)

水通道蛋白(Aquaporin,AQP)是一类选择性高效转运水分子的细胞膜通道蛋白,广泛存在于原核和真核生物细胞的细胞膜上,主要介导自由水分子的被动跨膜转运,对保持细胞内外液环境的稳态平衡起着重要的作用.     

Cellular overexpression of Aquaporins slows down the natural HIF-2α degradation during prolonged hypoxia

Overexpression of cell membrane aquaporins (AQPs) has recently been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the hypoxia inducible factor (HIF) family has been extensively implicated in tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic hypoxia, characteristic of a growing tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different proteins (AQP1, AQP3, AQP5, G6PD, and GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16 h at 1% O₂, in wt-PC12 and in PC12 overexpressing non-AQP proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to cobalt chloride or dimethyloxallyl glycine. Normal proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3, VEGF, and PGK1) were 2–4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more tumor-like phenotype.     

A novel monoclonal antibody DC63 reveals that inhibitor 1 of protein phosphatase 2A is preferentially nuclearly localised in human brain

Abnormal phosphorylation of tau protein represents one of the major candidate pathological mechanisms leading to Alzheimer's disease (AD) and related tauopathies. Altered phosphorylation status of neuronal tau protein may result from upregulation of tau-specific kinases or from inhibition of tau-specific phosphatases. Increased expression of the protein inhibitor 1 of protein phosphatase 2A (I1PP2A) could therefore indirectly regulate the phosphorylation status of tau. As an important step towards elucidation of the role of I1PP2A in the physiology and pathology of tau phosphorylation, we developed a novel monoclonal antibody, DC63, which recognizes I1PP2A. Specificity of the antibody was examined by mass spectrometry and Western blot. This analysis supports the conclusion that the antibody does not recognize any of the other proteins of the 9-member leucine-rich acidic nuclear phosphoprotein family to which I1PP2A belongs. Immunoblot detection revealed that the inhibitor I1PP2A is expressed throughout the brain, including the hippocampus, temporal cortex, parietal cortex, subcortical nuclei and brain stem. The cerebellum displayed significantly higher levels of expression of I1PP2A than was seen elsewhere in the brain. Imunohistochemical analysis of normal human brain showed that I1PP2A is expressed in both neurons and glial cells and that the protein is preferentially localized to the nucleus. We conclude that the novel monoclonal antibody DC63 could be successfully employed as a mass spectrometry-validated molecular probe that may be used for in vitro and in vivo qualitative and quantitative studies of physiological and pathological pathways involving I1PP2A.     

Interleukin-1beta induces the expression of aquaporin-4 through a nuclear factor-kappaB pathway in rat astrocytes

Interleukin (IL)-1beta is known to play a role in the formation of brain edema after various types of injury. Aquaporin (AQP)4 is also reported to be involved in the progression of brain edema. We tested the hypothesis that AQP4 is induced in response to IL-1beta. We found that expression of AQP4 mRNA and protein was significantly up-regulated by IL-1beta in cultured rat astrocytes, and that intracerebroventricular administration of IL-1beta increased the expression of AQP4 protein in rat brain. The effects of IL-1beta on induction of AQP4 were concentration and time dependent. The effects of IL-1beta on AQP4 were mediated through IL-1beta receptors because they were abolished by co-incubation with IL-1 receptor antagonist. It appeared that IL-1beta increased the level of AQP4 mRNA without involvement of de novo protein synthesis because cycloheximide, a protein synthesis inhibitor, did not inhibit the effects of IL-1beta. Inhibition of the nuclear factor-kappaB (NF-kappaB) pathway blocked the induction of AQP4 by IL-1beta in a concentration-dependent manner. These findings show that IL-1beta induces expression of AQP4 through a NF-kappaB pathway without involvement of de novo protein synthesis in rat astrocytes.     

N-3 PUFAs modulate global gene expression profile in cultured rat cardiomyocytes. Implications in cardiac hypertrophy and heart failure

In cardiac cells the effects of n-3 PUFAs on the whole genome are still unknown despite their recognized cardioprotective effects and ability to modulate gene expression. We have evaluated the effects of n-3 PUFAs supplementation on the global gene expression profile in cultured neonatal rat cardiomyocytes, detecting many genes related to lipid transport and metabolism among the upregulated ones. Many of the downregulated genes appeared related to inflammation, cell growth, extracellular and cardiac matrix remodelling, calcium movements and ROS generation. Our data allow to speculate that the cardioprotective effect of n-3 PUFAs is related to a direct modulation of genes in cardiac cells.     

An extract of Artemisia dracunculus L. enhances insulin receptor signaling and modulates gene expression in skeletal muscle in KK-A mice

An ethanolic extract of Artemisia dracunculus L. (PMI 5011) has been observed to decrease glucose and insulin levels in animal models, but the cellular mechanisms by which insulin action is enhanced in vivo are not precisely known. In this study, we evaluated the effects of PMI 5011 to modulate gene expression and cellular signaling through the insulin receptor in skeletal muscle of KK-A^y mice. Eighteen male KK-A^y mice were randomized to a diet (w/w) mixed with PMI 5011 (1%) or diet alone for 8 weeks. Food intake, adiposity, glucose and insulin were assessed over the study, and at study completion, vastus lateralis muscle was obtained to assess insulin signaling parameters and gene expression. Animals randomized to PMI 5011 were shown to have enhanced insulin sensitivity and increased insulin receptor signaling, i.e., IRS-associated PI-3 kinase activity, Akt-1 activity and Akt phosphorylation, in skeletal muscle when compared to control animals (P<.01, P<.01 and P<.001, respectively). Gene expression for insulin signaling proteins, i.e., IRS-1, PI-3 kinase and Glut-4, was not increased, although a relative increase in protein abundance was noted with PMI 5011 treatment. Gene expression for specific ubiquitin proteins and specific 20S proteasome activity, in addition to skeletal muscle phosphatase activity, i.e., PTP1B activity, was significantly decreased in mice randomized to PMI 5011 relative to control. Thus, the data demonstrate that PMI 5011 increases insulin sensitivity and enhances insulin receptor signaling in an animal model of insulin resistance. PMI 5011 may modulate skeletal muscle protein degradation and phosphatase activity as a possible mode of action.     

Cul4A is an oncogene in malignant pleural mesothelioma

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up‐regulation of p21 and p27 tumour suppressor proteins in a p53‐independent manner in H290, H28 and MS‐1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS‐1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down‐regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down‐regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for MPM.     

Different coexpressions of arthritis-relevant genes between different body organs and different brain regions in the normal mouse population

Structural changes in different parts of the brain in rheumatoid arthritis (RA) patients have been reported. RA is not regarded as a brain disease. Body organs such as spleen and lung produce RA-relevant genes. We hypothesized that the structural changes in the brain are caused by changes of gene expression in body organs. Changes in different parts of the brain may be affected by altered gene expressions in different body organs. This study explored whether an association between gene expressions of an organ or a body part varies in different brain structures. By examining the association of the 10 most altered genes from a mouse model of spontaneous arthritis in a normal mouse population, we found two groups of gene expression patterns between five brain structures and spleen. The correlation patterns between the prefrontal cortex, nucleus accumbens, and spleen were similar, while the associations between the other three parts of the brain and spleen showed a different pattern. Among overall patterns of the associations between body organs and brain structures, spleen and lung had a similar pattern, and patterns for kidney and liver were similar. Analysis of the five additional known arthritis-relevant genes produced similar results. Analysis of 10 nonrelevant-arthritis genes did not result in a strong association of gene expression or clearly segregated patterns. Our data suggest that abnormal gene expressions in different diseased body organs may influence structural changes in different brain parts.