Geldanamycin derivative inhibition of HGF/SF-mediated Met tyrosine kinase receptor-dependent urokinase-plasminogen activation

Ansamycins, including geldanamycin and the derivative 17-allylamino-17-demethoxygeldanamycin, and radicicol are known for their ability to tightly bind to the ATP-binding site of the amino-terminal domain region of heat shock protein 90. We have found that geldanamycin and some of its derivatives can inhibit hepatocyte growth factor/scatter factor-mediated Met tyrosine kinase receptor-dependent urokinase-plasminogen activation at femtomolar levels. Assessment is made of structural requirements for such an activity and evidence is given that distinguishes the target of such an activity from that of heat shock protein 90.     

Gene expression profiling of mouse Sertoli cell lines

The proliferation and differentiation of Sertoli cells is regulated by follicle-stimulating hormone (FSH). The molecular events following FSH stimulation are only partially known. To investigate FSH action in Sertoli cells, we established two novel FSH-responsive mouse Sertoli-cell-derived lines expressing human wild-type (WT) FSH receptor (FSHR) or overexpressing mutated (Asp567Gly) constitutively active FSHR (MUT). Gene expression profiling with commercially available cDNA arrays, including 588 mouse genes, revealed 146 genes expressed in both cell lines. Compared with the expression pattern of WT cells, 20 genes were identified as being either up- or down-regulated (>two-fold) in the MUT cells. We observed a strong differential expression of factors involved in cellular proliferation, e.g. cyclin D2 (repressed to nearly undetectable levels), proliferating cell nuclear antigen (2.5-fold repression) and Eps-8 (six-fold repression), and in genes involved in cellular differentiation, e.g. cytokeratin-18 (13-fold induction). The cDNA array results for six representative genes were confirmed by Northern blotting, which also included the parental SK-11 cell line devoid of FSHR expression. We found no further acute FSH- or forskolin-induced change in expression levels after 3-h stimulations, suggesting that the observed differences between the two cell lines is a consequence of mild, chronically increased, cAMP production in MUT cells. These results provide a platform for the further investigation of selected candidate genes in primary cultures and/or in vivo.Electronic Supplementary Material Supplementary material is available in the online version of this article at This work was supported by grants from the Deutsche Forschungsgemeinschaft (Confocal Research Group The Male Gamete: Production, Maturation, Function, grant FOR 197-3) and from the German Academic Exchange Service (DAAD) to P. Mathur     

Gene expression profiling of mouse embryonic stem cell subpopulations

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.     

A mouse model for spatial and temporal expression of HGF in the heart

In order to study the effects of Hepatocyte Growth Factor (HGF) in the heart, two transgenic mice were developed, one carrying a bidirectional HGF-TetO-GFP responder construct and the other carrying a α-MHC-tTA transactivator construct. Crosses were carried out between heterozygotes, so that litters contained bitransgenic α-MHC-tTA/HGF-TetO-GFP+, thus expressing HGF and GFP exclusively in the heart and only in the absence of Doxycycline. Our data show that the expression of HGF was indeed restricted to the heart and that the expression was limited to the timeframe of the absence of Doxycycline. Surprisingly the expression was variable even between bitransgenic littermates. In the setting of a model of ischemia-reperfusion, the expression of HGF ameliorates cardiac functionality, enhances proliferation and diminishes the scarred area, proving that this is a good model to study the beneficial influences and functional roles of HGF in the heart.     

Gene expression profiling of rewarding effect in methamphetamine treated Bax-deficient mouse

Methamphetamine is an illicit drug that is often abused and can cause neuropsychiatric and neurotoxic damage. Repeated administration of psychostimulants such as methamphetamine induces a behavioral sensitization. According to a previous study, Bax was involved in neurotoxicity by methamphetamine, but the function of Bax in rewarding effect has not yet been elucidated. Therefore, we have studied the function of Bax in a rewarding effect model. In the present study, we treated chronic methamphetamine exposure in a Bax-deficient mouse model and examined behavioral change using a conditioned place preference (CPP) test. The CPP score in Bax knockout mice was decreased compared to that of wild-type mice. Therefore, we screened for Bax-related genes that are involved in rewarding effect using microarray technology. In order to confirm microarray data, we applied the RT-PCR method to observe relative changes of Bcl2, a pro-apoptotic family gene. As a result, using our experiment microarray, we selected genes that were associated with Bax in microarray data, and eventually selected the Tgfbr2 gene. Expression of the Tgfbr2 gene was decreased by methamphetamine in Bax knockout mice, and the gene was overexpressed in Bax wild-type mice. Additionally, we confirmed that Creb, FosB, and c-Fos were related to rewarding effect and Bax using immunohistochemistry.     

Gene expression profiling of the developing mouse kidney and embryo

The metanephros is formed from the reciprocal inductive interaction of two precursor tissues, the metanephric mesenchyme (MM) and the ureteric bud (UB). The UB induces MM to condense and differentiate forming the glomerulus and renal tubules, whilst the MM induces the UB to differentiate into the collecting tubules of the mature nephron. Uninduced MM is considered the progenitor cell population of the developing metanephros because of its potential to differentiate into more renal cell types than the UB. Previous studies have identified the phenotype of renal precursor cells; however, expression of candidate marker genes have not been analysed in other tissues of the murine embryo. We have assayed up to 19 candidate genes in eight embryonic tissues at five gestation stages of the mouse embryo to identify markers definitively expressed by renal cells during metanephric induction and markers developmentally regulated during kidney maturation. We then analysed their expression in other developing tissues. Results show Dcn, Hoxc9, Mest, Wt1 and Ywhaq were expressed at moderate to high levels during the window of metanephric specification and early differentiation (E10.5-E12.5 dpc), and Hoxc9, Ren1 and Wt1 expression was characteristic of mature renal cells. We demonstrated Cd24a, Cdh11, Mest, Scd2 and Sim2 were regulated during brain development, and Scd2, Cd24a and Sip1 expression was enriched in developing liver. These markers may be useful negative markers of kidney development. Use of a combination of highly expressed and negative markers may aid in the identification and removal of non-renal cells from heterogeneous populations of differentiating stem cells.     

Gene expression profiling revealed specific spermatogonial stem cell genes in mouse

Mammalian spermatogenesis originates from spermatogonial stem cells (SSCs), which undergo mitosis, meiosis and spermiogenesis in order to generate mature spermatozoa. SSCs are adult stem cells that can both self‐renew and differentiate. To maintain pluripotency, SSCs are regulated by both extrinsic factors secreted from surrounding somatic cells and intrinsic factors including specific gene expression programs. Using fluorescent labeled germ line stem cells, mouse gonocytes and SSCs were purified up to 97% by improved FACS method. Through microarray analyses, global gene expression profiles of gonocytes, SSCs, and differentiated cells were compared. A large number of distinctive genes were found to be enriched in respective cell populations, indicating different functional requirements of each cell type. Functional clustering analyses revealed that while gonocytes and SSCs preferentially express genes implicated in gene expression regulation and epigenetic modifications, differentiated cells including somatic cells are enriched with genes encoding proteins involved in various cellular activities. Further in situ hybridization and RT‐PCR experiments confirmed SSC specific expression of several genes of which functions have not been characterized in SSCs. The comparative gene expression profiling provides a useful resource for gene discovery in relation to SSC regulation and opens new avenues for the study of molecular mechanisms underlying SSC self‐renewal and differentiation. genesis 51:83–96, 2013. © 2012 Wiley Periodicals, Inc.     

CD44-independent activation of the Met signaling pathway by HGF and InlB

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium responsible for listeriosis. It is able to invade, survive and replicate in phagocytic and non-phagocytic cells. The L. monocytogenes surface protein InlB interacts with c-Met, the hepatocyte growth factor (HGF) receptor, inducing bacterial internalization in numerous non-phagocytic cells. As InlB and HGF are known to trigger similar signaling pathways upon c-Met activation, we investigated the role of CD44, and more specifically its isoform CD44v6, in bacterial internalization in non-phagocytic cells. Indeed, CD44, the hyaluronic acid transmembrane receptor, and more specifically its isoform CD44v6 have been reported as necessary for the activation of c-Met upon the interaction with either the endogenous ligand HGF or the L. monocytogenes surface protein InlB. Our results demonstrate that, in the cell lines that we used, CD44 receptors play no role in the activation of c-Met, neither during L. monocytogenes entry, nor upon HGF activation. Furthermore, none of the CD44 isoforms was recruited at the L. monocytogenes entry site, and depletion by siRNA of total CD44 or of CD44v6 isoform did not reduce bacterial infections. Conversely, the overexpression of CD44 or CD44v6 had no significant effect on L. monocytogenes internalization. Together our results reveal that the activation of c-Met can be largely CD44-independent.     

Gene expression profiling of pituitary melanotrope cells during their physiological activation

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.     

Gene and protein expression profiling of the fat-1 mouse brain

Polyunsaturated fatty acids (PUFAs) are essential structural components of all cell membranes and, more so, of the central nervous system. Several studies revealed that n-3 PUFAs possess anti-inflammatory actions and are useful in the treatment of dyslipidemia. These actions explain the beneficial actions of n-3 PUFAs in the management of cardiovascular diseases, inflammatory conditions, neuronal dysfunction, and cancer. But, the exact molecular targets of these beneficial actions of n-3 PUFAs are not known. Mice engineered to carry a fat-1 gene from Caenorhabditis elegans add a double bond into an unsaturated fatty acid hydrocarbon chain and convert n-6 to n-3 fatty acids. This results in an abundance of n-3 eicosapentaenoic acid and docosapentaenoic acid specifically in the brain and a reduction in n-6 fatty acids of these mice that can be used to evaluate the actions of n-3 PUFAs. Gene expression profile, RT-PCR and protein microarray studies in the hippocampus and whole brain of wild-type and fat-1 transgenic mice revealed that genes and proteins concerned with inflammation, apoptosis, neurotransmission, and neuronal growth and synapse formation are specifically modulated in fat-1 mice. These results may explain as to why n-3 PUFAs are of benefit in the prevention and treatment of diseases such as Alzheimer's disease, schizophrenia and other diseases associated with neuronal dysfunction, low-grade systemic inflammatory conditions, and bronchial asthma. Based on these data, it is evident that n-3 PUFAs act to modulate specific genes and formation of their protein products and thus, bring about their various beneficial actions.     

Gene expression profiling of mouse host response to Listeria monocytogenes infection

The purpose of this study was to evaluate gene expression profiles in the liver and blood for prediction of infection severity from Listeria monocytogenes (LM). Mice were injected with medium broth (control) or a nonlethal or lethal dose of LM and sacrificed 6 h later. Gene expression changes were determined using Affymetrix MGU74Av2 GeneChips and confirmed by real-time polymerase chain reaction analysis. We identified discernable genes whose gene expression profiles can be used in pattern recognition to predict and classify samples in differently treated groups, with >or=90% accuracy in liver samples and 80% accuracy in blood at prediction; however, different genes were predictive in each tissue. Our results suggest that gene expression profiling in response to LM in mice may be able to distinguish samples in groups with varying severity of infection and provide information in finding molecular mechanisms and early biomarkers for subsequent conventional clinical endpoints.     

Gene expression profiling reveals the mechanism and pathophysiology of mouse liver regeneration

Comprehensive analysis of the changes in gene expression during liver regeneration was carried out by using an in-house microarray composed of 2,304 distinct mouse liver cDNA clones. Mice were subjected to partial two-thirds hepatectomy, and changes in mRNA levels were monitored up to 48 h. Of the 2,304 genes analyzed, 496 genes showed expression levels measurable at all time points after the partial hepatectomy. 317 genes were up- or down-regulated 2-fold or more at least at one time point during liver regeneration and were classified into eight clusters based on their expression patterns. With a more stringent cut-off value of +/-2 S.D., 68 genes were listed and were classified into five clusters. In these two analyses with different clustering criteria, functionally categorized genes showed similar cluster distributions. Genes involved in protein synthesis and posttranslational processing were significantly enriched in the cluster characterized by rapid gene activation and subsequent persistence. This suggests the importance of modulating the efficiency of protein supply and/or altering the composition of protein population from the early phase of hepatocyte proliferation. Genes for two major liver functions, i.e. plasma protein secretion and intermediate metabolism were enriched in distinct clusters exhibiting the features of gradual gene activation and sustained repression, respectively. Therefore, these genes are differentially regulated during the regeneration, possibly leading to changes in the flow of amino acids and energy from enzyme proteins to plasma proteins in their synthesis. Thus, clustering analysis of expression patterns of functionally classified genes gave insights into mechanism and pathophysiology of liver regeneration.     

Gene expression profiling related to the enhanced erythropoiesis in mouse bone marrow cells

Peroxiredoxin II knockout (Prdx II(-/-)) mice had a spontaneous phenotype of hemolytic anemia. In this study, we found that Ter-119(+)CD71(+) cells increased in Prdx II(-/-) mice bone marrow (BM) at 8 weeks of age. We examined the differential expression profiles to bone marrow cells (BMCs) between Prdx II(+/+) and Prdx II(-/-) mice using a cDNA microarray. We identified the 136 candidates were differentially expressed a greater twofold increase or decrease than EPO receptor. In this study, we focused on the up-regulated NBPs during erythropoietic differentiation. According to cDNA microarray results, six NBPs except zfp-127 were up-regulated during erythropoiesis in Prdx II(-/-) mice. Among the six candidates, eIF3-p44, hnRNPH1, G3bp, and Zfpm-1 were dramatically increased at day 7 of the in vitro erythropoietic differentiation of human CD34(+) cells. However, DJ-1 and Rbm3 were slightly increased only at day 12. Our results suggest that up-regulated NBPs might be involved during erythropoietic differentiation.     

Proteomic profiling of aging in the mouse heart: Altered expression of mitochondrial proteins

Using two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry, we have used a systems biology approach to study the molecular basis of aging of the mouse heart. We have identified 8 protein spots whose expression is up-regulated due to aging and 36 protein spots whose expression is down-regulated due to aging (p0.05 as judged by Wilcoxon Rank Sum test). Among the up-regulated proteins, we have characterized 5 protein spots and 2 of them, containing 3 different enzymes, are mitochondrial proteins. Among the down-regulated proteins, we have characterized 27 protein spots and 16 of them are mitochondrial proteins. Mitochondrial damage is believed to be a key factor in the aging process. Our current study provides molecular evidence at the level of the proteome for the alteration of structural and functional parameters of the mitochondria that contribute to impaired activity of the mouse heart due to aging.     

Gene expression profiling of lipoarabinomannan-treated mouse macrophage cultures infected with Mycobacterium bovis BCG

The mannosylated lipoarabinomanan (ManLAM) from mycobacterial species possesses strong anti-apoptotic action. Here we examined the ability of ManLAM isolated from Mycobacterium tuberculosis H37Rv to alter expression profiles of apoptosis-related genes in mouse macrophages infected with Mycobacterium bovis BCG Danish strain. ManLAM suppressed BCG-induced apoptosis and activities of caspase-1, -3, -8 and 9. Mouse Apoptosis Gene Array showed that ManLAM significantly down-regulated pro-apoptotic and proinflammatory genes: caspase-1, -3, -7, -8 and -9, TNF-alpha/TNFSF2, Fas/TNFRSF6, Bax-alpha, as well as IL-12 p35 and iNOS simultaneously up-regulating anti-apoptotic genes such as Bcl-2 and Mcl-1. The effect of ManLAM was contrary to BCG-induced up-regulation of proapoptotic and pro-inflammatory genes and consistent with the functional data.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.