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Sample records for coordinately up-regulates protein

  1. Coordinately up-regulated genes in ovarian cancer.

    PubMed

    Hough, C D; Cho, K R; Zonderman, A B; Schwartz, D R; Morin, P J

    2001-05-15

    A better understanding of the molecular circuitry in normal ovarian tissues and in ovarian cancer will likely provide new targets for diagnosis and therapy. Recently, much has been learned about the genes expressed in ovarian cancer through studies with cDNA arrays and serial analysis of gene expression. However, these methods do not allow highly quantitative analysis of gene expression on a large number of specimens. Here, we have used quantitative real-time RT-PCR in a panel of 39 microdissected ovarian carcinomas of various subtypes to systematically analyze the expression of 13 genes, many of which were previously identified as up-regulated in a subset of ovarian cancers by serial analyses of gene expression. The genes analyzed are glutathione peroxidase 3 (GPX3), apolipoprotein J/clusterin, insulin-like growth factor-binding protein 2, epithelial cell adhesion molecule/GA733-2, Kop protease inhibitor, matrix gla protein, tissue inhibitor of metalloproteinase 3, folate receptor 1, S100A2, signal transducer and activator of transcription 1, secretory leukocyte protease inhibitor, apolipoprotein E, and ceruloplasmin. All of the genes were found overexpressed, some at extremely high levels, in the vast majority of ovarian carcinomas irrespective of the subtype. Interestingly, GPX3 was found at much higher levels in tumors with clear cell histology and may represent a biomarker for this subtype. Some of the genes studied here may thus represent targets for early detection ovarian cancer. The gene expression patterns were not associated with age at diagnosis, stage, or K-ras mutation status in ovarian cancer. We find that several genes are coordinately regulated in ovarian cancer, likely representing the fact that many genes are activated as part of common signaling pathways or that extensive cross-talk exists between several pathways in ovarian cancer. A statistical analysis shows that genes commonly up-regulated in ovarian cancer may result from the aberrant

  2. Nrf2 Protein Up-regulates Antiapoptotic Protein Bcl-2 and Prevents Cellular Apoptosis*

    PubMed Central

    Niture, Suryakant K.; Jaiswal, Anil K.

    2012-01-01

    Nuclear transcription factor Nrf2 regulates the expression and coordinated induction of a battery of genes encoding cytoprotective and drug transporter proteins in response to chemical and radiation stress. This leads to reduced apoptosis, enhanced cell survival, and increased drug resistance. In this study, we investigated the role of Nrf2 in up-regulation of antiapoptotic protein Bcl-2 and its contribution to stress-induced apoptosis and cell survival. Exposure of mouse hepatoma (Hepa-1) and human hepatoblastoma (HepG2) cells to antioxidant tert-butylhydroquinone led to induction of Bcl-2. Mutagenesis and transfection assays identified an antioxidant response element between nucleotides −3148 and −3140 on the reverse strand of the Bcl-2 gene promoter that was essential for activation of Bcl-2 gene expression. Band/supershift and ChIP assays demonstrated binding of Nrf2 to Bcl-2 antioxidant response element. Alterations in Nrf2 led to altered Bcl-2 induction and cellular apoptosis. Moreover, dysfunctional/mutant inhibitor of Nrf2 (INrf2) in human lung cancer cells failed to degrade Nrf2, resulting in an increased Bcl-2 level and decreased etoposide- and UV/γ radiation-mediated DNA fragmentation. In addition, siRNA-mediated down-regulation of Nrf2 also led to decreased apoptosis and increased cell survival. Furthermore, the specific knockdown of Bcl-2 in Nrf2-activated tumor cells led to increased etoposide-induced apoptosis and decreased cell survival and growth/proliferation. These data provide the first evidence of Nrf2 in control of Bcl-2 expression and apoptotic cell death with implications in antioxidant protection, survival of cancer cells, and drug resistance. PMID:22275372

  3. Up-regulation of heat shock proteins is essential for cold survival during insect diapause

    PubMed Central

    Rinehart, Joseph P.; Li, Aiqing; Yocum, George D.; Robich, Rebecca M.; Hayward, Scott A. L.; Denlinger, David L.

    2007-01-01

    Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the fly's heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupa's ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects. PMID:17522254

  4. Histone Hyperacetylation Up-regulates Protein Kinase Cδ in Dopaminergic Neurons to Induce Cell Death

    PubMed Central

    Jin, Huajun; Kanthasamy, Arthi; Harischandra, Dilshan S.; Kondru, Naveen; Ghosh, Anamitra; Panicker, Nikhil; Anantharam, Vellareddy; Rana, Ajay; Kanthasamy, Anumantha G.

    2014-01-01

    The oxidative stress-sensitive protein kinase Cδ (PKCδ) has been implicated in dopaminergic neuronal cell death. However, little is known about the epigenetic mechanisms regulating PKCδ expression in neurons. Here, we report a novel mechanism by which the PKCδ gene can be regulated by histone acetylation. Treatment with histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) induced PKCδ expression in cultured neurons, brain slices, and animal models. Several other HDAC inhibitors also mimicked NaBu. The chromatin immunoprecipitation analysis revealed that hyperacetylation of histone H4 by NaBu is associated with the PKCδ promoter. Deletion analysis of the PKCδ promoter mapped the NaBu-responsive element to an 81-bp minimal promoter region. Detailed mutagenesis studies within this region revealed that four GC boxes conferred hyperacetylation-induced PKCδ promoter activation. Cotransfection experiments and Sp inhibitor studies demonstrated that Sp1, Sp3, and Sp4 regulated NaBu-induced PKCδ up-regulation. However, NaBu did not alter the DNA binding activities of Sp proteins or their expression. Interestingly, a one-hybrid analysis revealed that NaBu enhanced transcriptional activity of Sp1/Sp3. Overexpression of the p300/cAMP-response element-binding protein-binding protein (CBP) potentiated the NaBu-mediated transactivation potential of Sp1/Sp3, but expressing several HDACs attenuated this effect, suggesting that p300/CBP and HDACs act as coactivators or corepressors in histone acetylation-induced PKCδ up-regulation. Finally, using genetic and pharmacological approaches, we showed that NaBu up-regulation of PKCδ sensitizes neurons to cell death in a human dopaminergic cell model and brain slice cultures. Together, these results indicate that histone acetylation regulates PKCδ expression to augment nigrostriatal dopaminergic cell death, which could contribute to the progressive neuropathogenesis of Parkinson disease. PMID:25342743

  5. HMGB1 and HMGB2 proteins up-regulate cellular expression of human topoisomerase IIα

    PubMed Central

    Štros, Michal; Polanská, Eva; Štruncová, Soňa; Pospíšilová, Šárka

    2009-01-01

    Topoisomerase IIα (topo IIα) is a nuclear enzyme involved in several critical processes, including chromosome replication, segregation and recombination. Previously we have shown that chromosomal protein HMGB1 interacts with topo IIα, and stimulates its catalytic activity. Here we show the effect of HMGB1 on the activity of the human topo IIα gene promoter in different cell lines. We demonstrate that HMGB1, but not a mutant of HMGB1 incapable of DNA bending, up-regulates the activity of the topo IIα promoter in human cells that lack functional retinoblastoma protein pRb. Transient over-expression of pRb in pRb-negative Saos-2 cells inhibits the ability of HMGB1 to activate the topo IIα promoter. The involvement of HMGB1 and its close relative, HMGB2, in modulation of activity of the topo IIα gene is further supported by knock-down of HMGB1/2, as evidenced by significantly decreased levels of topo IIα mRNA and protein. Our experiments suggest a mechanism of up-regulation of cellular expression of topo IIα by HMGB1/2 in pRb-negative cells by modulation of binding of transcription factor NF-Y to the topo IIα promoter, and the results are discussed in the framework of previously observed pRb-inactivation, and increased levels of HMGB1/2 and topo IIα in tumors. PMID:19223331

  6. Follistatin-like protein 1 promotes arthritis by up-regulating IFN-gamma.

    PubMed

    Clutter, Suzanne D; Wilson, David C; Marinov, Anthony D; Hirsch, Raphael

    2009-01-01

    Follistatin-like protein-1 (FSTL-1) is a poorly characterized protein that is up-regulated in the early stage of collagen-induced arthritis and that exacerbates arthritis when delivered by gene transfer. The current study was designed to determine the mechanism by which FSTL-1 promotes arthritis. FSTL-1 was injected into mouse paws, resulting in severe paw swelling associated with up-regulation of IFN-gamma transcript and the IFN-gamma-induced chemokine, CXCL10. Mice depleted of T cells were protected. A central role for IFN-gamma was confirmed by the finding that mice deficient in IFN-gamma failed to exhibit paw swelling in response to injection of FSTL-1. Furthermore, IFN-gamma secretion from mouse spleen cells exposed to a weak TCR signal was increased 5-fold in the presence of FSTL-1. FSTL-1 could be induced by innate immune signals, including TLR4 agonists and the arthritogenic cytokine, IL-1beta, via an NFkappaB pathway. Finally, FSTL-1 was found to be overexpressed in human arthritis and its neutralization inhibited murine collagen-induced arthritis and suppressed IFN-gamma and CXCL10 production in arthritic joints. These findings demonstrate that FSTL-1 plays a critical role in arthritis by enhancing IFN-gamma signaling pathways and suggest a mechanism by which FSTL-1 bridges innate and adaptive immune responses. PMID:19109154

  7. Perforin and granzyme B. Cytolytic proteins up-regulated during rejection of rat small intestine allografts.

    PubMed

    McDiarmid, S V; Farmer, D G; Kuniyoshi, J S; Robert, M; Khadavi, A; Shaked, A; Busuttil, R W

    1995-03-15

    Perforin and granzyme B are 2 cytolytic proteins specific to activated killer cells, particularly CTL. We have studied the mRNA expression of these 2 proteins by a reverse transcriptase polymerase chain reaction method in a unidirectional model of rat small intestine transplant rejection. The allograft group consisted of Lewis x Brown Norway F1 donors into Lewis recipients. The isograft controls were Lewis donors into Lewis recipients. Grafts were placed heterotopically and no immunosuppression was given. Five animals in each group were killed at postoperative days (POD) 3, 5, 7, 8, 9, 10, 12, and 14. mRNA was extracted and a semiquantitative reverse transcriptase polymerase chain reaction was performed. For the semiquantitative analysis, we compared scintillation counts from excised bands. Results were expressed as a percent activity compared with beta-actin. From the same tissue samples, a histologic evaluation was made and rejection was graded according to severity. The isograft controls showed no evidence of histologic rejection and a very low expression of mRNA for perforin and granzyme B from POD 3-14. In contrast, the allograft group began to show histologic evidence of mild rejection on POD 5. By day 7, rejection was moderately severe and associated with a significant up-regulation of perforin and granzyme B in the allografts compared with the controls (P < 0.01), which persisted through POD 14. Peak expression for perforin and granzyme B was on POD 10 and 8, respectively. We conclude that the up-regulation of perforin and granzyme B in rat small intestine transplant allografts is a useful marker of clinically important rejection. PMID:7886805

  8. Curcumin Inhibits Prostate Cancer Bone Metastasis by Up-Regulating Bone Morphogenic Protein-7 in Vivo

    PubMed Central

    Dorai, Thambi; Diouri, Janane; O'Shea, Orla; Doty, Stephen B.

    2014-01-01

    A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-β signaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP- 7). This enhancement of BMP-7 in the context of TGF-βin the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regu- lated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-βsignaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic

  9. Curcumin Inhibits Prostate Cancer Bone Metastasis by Up-Regulating Bone Morphogenic Protein-7 in Vivo.

    PubMed

    Dorai, Thambi; Diouri, Janane; O'Shea, Orla; Doty, Stephen B

    2014-04-01

    A number of studies have focused on the beneficial properties of Curcumin (diferuloyl methane, used in South Asian cuisine and traditional medicine) such as the chemoprevention of cancer. Recent studies have also indicated that this material has significant benefits for the treatment of cancer and is currently undergoing several clinical trials. We have been interested in the application of this compound as a therapeutic agent for advanced prostate cancer, particularly the skeletal complications in this malignancy. Our earlier work indicated that this compound could inhibit the osteomimetic properties which occur in castration resistant prostate cancer cells, by interfering with the common denominators between these cancer cells and the bone cells in the metastatic tumor microenvironment, namely the osteoblasts and the osteoclast. We predicted that curcumin could break the vicious cycle of reciprocal stimulation that results in uncontrolled osteolysis in the bony matrix. In this work, we have evaluated the potential of this compound in inhibiting the bone metastasis of hormone refractory prostate cancer cells in an established animal model. Our results strongly suggest that curcumin modulates the TGF-β signaling that occurs due to bone matrix degradation by up-regulating the metastasis inhibitory bone morphogenic protein-7 (BMP- 7). This enhancement of BMP-7 in the context of TGF-βin the tumor microenvironment is shown to enhance the mesenchymal-to-epithelial transition. Most importantly, we show that as a result of BMP-7 up-regulation, a novel brown/beige adipogenic differentiation program is also up-regu- lated which plays a role in the inhibition of bone metastasis. Our results suggest that curcumin may subvert the TGF-βsignaling to an alternative adipogenic differentiation program in addition to the previously established interference with the osteomimetic properties, thus inhibiting the bone metastatic processes in a chemopreventive as well as therapeutic

  10. Hypoxia Induces Autophagy through Translational Up-Regulation of Lysosomal Proteins in Human Colon Cancer Cells

    PubMed Central

    Lai, Ming-Chih; Chang, Chiao-May; Sun, H. Sunny

    2016-01-01

    Hypoxia occurs in a wide variety of physiological and pathological conditions, including tumorigenesis. Tumor cells have to adapt to hypoxia by altering their gene expression and protein synthesis. Here, we showed that hypoxia inhibits translation through activation of PERK and inactivation of mTOR in human colon cancer HCT116 cells. Prolonged hypoxia (1% O2, 16 h) dramatically inhibits general translation in HCT116 cells, yet selected mRNAs remain efficiently translated under such a condition. Using microarray analysis of polysome- associated mRNAs, we identified a large number of hypoxia-regulated genes at the translational level. Efficiently translated mRNAs during hypoxia were validated by polysome profiling and quantitative real-time RT-PCR. Pathway enrichment analysis showed that many of the up-regulated genes are involved in lysosome, glycan and lipid metabolism, antigen presentation, cell adhesion, and remodeling of the extracellular matrix and cytoskeleton. The majority of down-regulated genes are involved in apoptosis, ubiquitin-mediated proteolysis, and oxidative phosphorylation. Further investigation showed that hypoxia induces lysosomal autophagy and mitochondrial dysfunction through translational regulation in HCT116 cells. The abundance of several translation factors and the mTOR kinase activity are involved in hypoxia-induced mitochondrial autophagy in HCT116 cells. Our studies highlight the importance of translational regulation for tumor cell adaptation to hypoxia. PMID:27078027

  11. Uncoupling protein-2 up-regulation and enhanced cyanide toxicity are mediated by PPAR{alpha} activation and oxidative stress

    SciTech Connect

    Zhang, X.; Li, L.; Prabhakaran, K.; Zhang, L.; Leavesley, H.B.; Borowitz, J.L.; Isom, G.E.

    2007-08-15

    Uncoupling protein 2 (UCP-2) is an inner mitochondrial membrane proton carrier that modulates mitochondrial membrane potential ({delta}{psi}{sub m}) and uncouples oxidative phosphorylation. We have shown that up-regulation of UCP-2 by Wy14,643, a selective peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) agonist, enhances cyanide cytotoxicity. The pathway by which Wy14,643 up-regulates UCP-2 was determined in a dopaminergic cell line (N27 cells). Since dopaminergic mesencephalic cells are a primary brain target of cyanide, the N27 immortalized mesencephalic cell was used in this study. Wy14,643 produced a concentration- and time-dependent up-regulation of UCP-2 that was linked to enhanced cyanide-induced cell death. MK886 (PPAR{alpha} antagonist) or PPAR{alpha} knock-down by RNA interference (RNAi) inhibited PPAR{alpha} activity as shown by the peroxisome proliferator response element-luciferase reporter assay, but only partially decreased up-regulation of UCP-2. The role of oxidative stress as an alternative pathway to UCP-2 up-regulation was determined. Wy14,643 induced a rapid surge of ROS generation and loading cells with glutathione ethyl ester (GSH-EE) or pre-treatment with vitamin E attenuated up-regulation of UCP-2. On the other hand, RNAi knockdown of PPAR{alpha} did not alter ROS generation, suggesting a PPAR{alpha}-independent component to the response. Co-treatment with PPAR{alpha}-RNAi and GSH-EE blocked both the up-regulation of UCP-2 by Wy14,643 and the cyanide-induced cell death. It was concluded that a PPAR{alpha}-mediated pathway and an oxidative stress pathway independent of PPAR{alpha} mediate the up-regulation of UCP-2 and subsequent increased vulnerability to cyanide-induced cytotoxicity.

  12. Continuous up-regulation of heat shock proteins in larvae, but not adults, of a polar insect

    PubMed Central

    Rinehart, Joseph P.; Hayward, Scott A. L.; Elnitsky, Michael A.; Sandro, Luke H.; Lee, Richard E.; Denlinger, David L.

    2006-01-01

    Antarctica's terrestrial environment is a challenge to which very few animals have adapted. The largest, free-living animal to inhabit the continent year-round is a flightless midge, Belgica antarctica. Larval midges survive the lengthy austral winter encased in ice, and when the ice melts in summer, the larvae complete their 2-yr life cycle, and the wingless adults form mating aggregations while subjected to surprisingly high substrate temperatures. Here we report a dichotomy in survival strategies exploited by this insect at different stages of its life cycle. Larvae constitutively up-regulate their heat shock proteins (small hsp, hsp70, and hsp90) and maintain a high inherent tolerance to temperature stress. High or low temperature exposure does not further up-regulate these genes nor does it further enhance thermotolerance. Such “preemptive” synthesis of hsps is sufficient to prevent irreversible protein aggregation in response to a variety of common environmental stresses. Conversely, adults exhibit no constitutive up-regulation of their hsps and have a lower intrinsic tolerance to high temperatures, but their hsps can be thermally activated, resulting in enhanced thermotolerance. Thus, the midge larvae, but not the adults, have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat that is more thermally stable than that of the adults but a habitat subjected frequently to freeze-thaw episodes and bouts of pH, anoxic, and osmotic stress. PMID:16968769

  13. Deficits in coordinated motor behavior and in nigrostriatal dopaminergic system ameliorated and VMAT2 expression up-regulated in aged male rats by administration of testosterone propionate.

    PubMed

    Wang, Li; Kang, Yunxiao; Zhang, Guoliang; Zhang, Yingbo; Cui, Rui; Yan, Wensheng; Tan, Huibing; Li, Shuangcheng; Wu, Baiyila; Cui, Huixian; Shi, Geming

    2016-06-01

    The effects of testosterone propionate (TP) supplements on the coordinated motor behavior and nigrostriatal dopaminergic (NSDA) system were analyzed in aged male rats. The present study showed the coordinated motor behavioral deficits, the reduced activity of NSDA system and the decreased expression of vesicular monoamine transporter 2 (VMAT2) in 24month-old male rats. Long term TP treatment improved the motor coordination dysfunction with aging. Increased tyrosine hydroxylase and dopamine transporter, as well as dopamine and its metabolites were found in the NSDA system of TP-treated 24month-old male rats, indicative of the amelioratory effects of TP supplements on NSDA system of aged male rats. The enhancement of dopaminergic (DAergic) activity of NSDA system by TP supplements might underlie the amelioration of the coordinated motor dysfunction in aged male rats. TP supplements up-regulated VMAT2 expression in NSDA system of aged male rats. Up-regulation of VMAT2 expression in aged male rats following chronic TP treatment might be involved in the maintenance of DAergic function of NSDA system in aged male rats. PMID:26956479

  14. Pregnancy-associated plasma protein A up-regulated by progesterone promotes adhesion and proliferation of trophoblastic cells.

    PubMed

    Wang, Jiao; Liu, Shuai; Qin, Hua-Min; Zhao, Yue; Wang, Xiao-Qi; Yan, Qiu

    2014-01-01

    Embryo implantation and development is a complex biological process for the establishment of the successful pregnancy. Progesterone is a critical factor in the regulation of embryo adhesion to uterine endometrium and proliferation. Although it has been reported that pregnancy-associated plasma protein A (PAPPA) is increased in pregnant women, the relationship between progesterone and PAPPA, and the effects of PAPPA on embryo adhesion and proliferation are still not clear. The present results showed that the serum level of progesterone and PAPPA was closely correlated by ELISA assay (p<0.01). PAPPA was detected in the villi of early embryo by RT-PCR, Western blot, immunohistochemistry and immunofluorescent staining. Moreover, PAPPA was significantly up-regulated by progesterone in trophoblastic (JAR) cells by Real-time PCR and ELISA assay (p<0.01); while the expression was decreased by the progesterone receptor inhibitor RU486. The down-regulation of PAPPA by siRNA transfection or up-regulation of PAPPA by progesterone treatment significantly decreased or increased the adhesion rate of trophoblastic cells to human uterine epithelial cell lines (RL95-2 and HEC-1A), respectively (p<0.01), as well as the proliferation of trophoblastic cells. In conclusion, PAPPA is up-regulated by progesterone, which promotes the adhesion and proliferation potential of trophoblastic cells. PMID:24817938

  15. Nitrosative stress leads to protein glutathiolation, increased s-nitrosation, and up-regulation of peroxiredoxins in the heart.

    PubMed

    Reinartz, Michael; Ding, Zhaoping; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2008-06-20

    Nitric oxide (NO) is produced by different isoforms of nitric oxide synthases (NOSs) and operates as a mediator of important cell signaling pathways, such as the cGMP signaling cascade. Another mechanism by which NO exerts biological effects is mediated through S-nitrosation of target proteins. To explore thiol-based protein modifications in a situation of defined nitrosative stress, we used a transgenic mouse model with cardiac specific overexpression of inducible nitric oxide synthase (iNOS) and concomitant myoglobin deficiency (iNOS(+)/myo(-/-)). In comparison with the wild type hearts, protein glutathiolation detected by immunoblotting was significantly enhanced in iNOS(+)/myo(-/-) hearts, whereas protein S-nitrosation as measured by the biotin switch assay and two-dimensional PAGE revealed that nearly all of the detected proteins ( approximately 60) remained unchanged with the exception of three proteins. Tandem mass spectrometry revealed these proteins to be peroxiredoxins (Prxs), which are known to possess peroxidase activity, whereby hydrogen peroxide, peroxynitrite, and a wide range of organic hydroperoxides are reduced and detoxified. Immunoblotting with specific antibodies revealed up-regulation of Prx VI in the iNOS(+)/myo(-/-) hearts, whereas expression of Prx II and Prx III remained unchanged. Furthermore, the analysis of the cardiac S-nitrososubproteome identified several new proteins possibly being involved in NO-signaling pathways. Our data indicate that S-nitrosation and glutathiolation of cardiac proteins may contribute to the phenotype of NO-induced heart failure. The up-regulation of antioxidant proteins like Prx VI appears to be an additional mechanism to antagonize an excess of reactive oxygen/nitrogen species. Furthermore, S-nitrosation of Prxs may serve a new function in the signaling cascade of nitrosative stress. PMID:18426799

  16. Estrogen-dependent up-regulation of TRPA1 and TRPV1 receptor proteins in the rat endometrium.

    PubMed

    Pohóczky, Krisztina; Kun, József; Szalontai, Bálint; Szőke, Éva; Sághy, Éva; Payrits, Maja; Kajtár, Béla; Kovács, Krisztina; Környei, József László; Garai, János; Garami, András; Perkecz, Anikó; Czeglédi, Levente; Helyes, Zsuzsanna

    2016-02-01

    Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors expressed predominantly in sensory nerves are activated by inflammatory stimuli and mediate inflammation and pain. Although they have been shown in the human endometrium, their regulation and function are unknown. Therefore, we investigated their estrogen- and progesterone-dependent alterations in the rat endometrium in comparison with the estrogen-regulated inflammatory cytokine macrophage migration inhibitory factor (MIF). Four-week-old (sexually immature) and four-month-old (sexually mature) female rats were treated with the non-selective estrogen receptor (ER) agonist diethylstilboestrol (DES), progesterone and their combination, or ovariectomized. RT-PCR and immunohistochemistry were performed to determine mRNA and protein expression levels respectively. Channel function was investigated with ratiometric [Ca(2+)]i measurement in cultured primary rat endometrial cells. Both TRP receptors and MIF were detected in the endometrium at mRNA and protein levels, and their localizations were similar. Immunostaining was observed in the immature epithelium, while stromal, glandular and epithelial positivity were observed in adults. Functionally active TRP receptor proteins were shown in endometrial cells by activation-induced calcium influx. In adults, Trpa1 and Trpv1 mRNA levels were significantly up-regulated after DES treatment. TRPA1 increased after every treatment, but TRPV1 remained unchanged following the combined treatment and ovariectomy. In immature rats, DES treatment resulted in increased mRNA expression of both channels and elevated TRPV1 immunopositivity. MIF expression changed in parallel with TRPA1/TRPV1 in most cases. DES up-regulated Trpa1, Trpv1 and Mif mRNA levels in endometrial cell cultures, but 17β-oestradiol having ERα-selective potency increased only the expression of Trpv1. We provide the first evidence for TRPA1/TRPV1 expression and their estrogen-induced up-regulation

  17. Up-regulation of endothelial monocyte chemoattractant protein-1 by coplanar PCB77 is caveolin-1-dependent

    SciTech Connect

    Majkova, Zuzana; Smart, Eric; Toborek, Michal; Hennig, Bernhard

    2009-05-15

    Atherosclerosis, the primary cause of heart disease and stroke is initiated in the vascular endothelium, and risk factors for its development include environmental exposure to persistent organic pollutants. Caveolae are membrane microdomains involved in regulation of many signaling pathways, and in particular in endothelial cells. We tested the hypothesis that intact caveolae are required for coplanar PCB77-induced up-regulation of monocyte chemoattractant protein-1 (MCP-1), an endothelium-derived chemokine that attracts monocytes into sub-endothelial space in early stages of the atherosclerosis development. Atherosclerosis-prone LDL-R{sup -/-} mice (control) or caveolin-1{sup -/-}/LDL-R{sup -/-} mice were treated with PCB77. PCB77 induced aortic mRNA expression and plasma protein levels of MCP-1 in control, but not caveolin-1{sup -/-}/LDL-R{sup -/-} mice. To study the mechanism of this effect, primary endothelial cells were used. PCB77 increased MCP-1 levels in endothelial cells in a time- and concentration-dependent manner. This effect was abolished by caveolin-1 silencing using siRNA. Also, MCP-1 up-regulation by PCB77 was prevented by inhibiting p38 and c-Jun N-terminal kinase (JNK), but not ERK1/2, suggesting regulatory functions via p38 and JNK MAPK pathways. Finally, pre-treatment of endothelial cells with the aryl hydrocarbon receptor (AhR) inhibitor {alpha}-naphthoflavone ({alpha}-NF) partially blocked MCP-1 up-regulation. Thus, our data demonstrate that coplanar PCB77 can induce MCP-1 expression by endothelial cells and that this effect is mediated by AhR, as well as p 38 and JNK MAPK pathways. Intact caveolae are required for these processes both in vivo and in vitro. This further supports a key role for caveolae in vascular inflammation induced by persistent organic pollutants.

  18. Coordinate up-regulation of low-density lipoprotein receptor and cyclo-oxygenase-2 gene expression in human colorectal cells and in colorectal adenocarcinoma biopsies

    NASA Technical Reports Server (NTRS)

    Lum, D. F.; McQuaid, K. R.; Gilbertson, V. L.; Hughes-Fulford, M.

    1999-01-01

    Many colorectal cancers have high levels of cyclo-oxygenase 2 (COX-2), an enzyme that metabolizes the essential fatty acids into prostaglandins. Since the low-density lipoprotein receptor (LDLr) is involved in the uptake of essential fatty acids, we studied the effect of LDL on growth and gene regulation in colorectal cancer cells. DiFi cells grown in lipoprotein-deficient sera (LPDS) grew more slowly than cells with LDL. LDLr antibody caused significant inhibition of tumor cell growth but did not affect controls. In addition, LDL uptake did not change in the presence of excess LDL, suggesting that ldlr mRNA lacks normal feedback regulation in some colorectal cancers. Analysis of the ldlr mRNA showed that excess LDL in the medium did not cause down-regulation of the message even after 24 hr. The second portion of the study examined the mRNA expression of ldlr and its co-regulation with cox-2 in normal and tumor specimens from patients with colorectal adenocarcinomas. The ratio of tumor:paired normal mucosa of mRNA expression of ldlr and of cox-2 was measured in specimens taken during colonoscopy. ldlr and cox-2 transcripts were apparent in 11 of 11 carcinomas. There was significant coordinate up-regulation both of ldlr and of cox-2 in 6 of 11 (55%) tumors compared with normal colonic mucosa. There was no up-regulation of cox-2 without concomitant up-regulation of ldlr. These data suggest that the LDLr is abnormally regulated in some colorectal tumors and may play a role in the up-regulation of cox-2. Copyright 1999 Wiley-Liss, Inc.

  19. Protein malnutrition up-regulates growth hormone receptor expression in rat splenic B lymphocytes.

    PubMed

    Mejía-Naranjo, Wilson; Sánchez-Gomez, Myriam

    2004-12-01

    The reciprocal interaction between the endocrine and immune systems has been the subject of active research during the last decade, and an important body of evidence has accumulated supporting the role of the GH/IGF axis in immune function. More recently, the GH/IGF axis has been postulated as playing an important role in the modulation of stress conditions, such as catabolic stages, aging-related disorders, immunodeficient aids patients and malnutrition. Whether these effects are exerted through endocrine, autocrine or paracrine mechanisms remains to be determined for different immune cell types and tissues. The aim of the current study was to define which specific subsets of lymphocytes are the primary targets for GH action. In addition, the regulatory role of stress induced by protein restriction was investigated with respect to the relative distribution of GH receptor positive lymphoid cells. Normal growing rats were fed isocaloric diets with variable protein content (0, 4, 8, 12 and 20%) for a period of 14 days. The lymphoid cells were then separated from spleen, lymph nodes and peripheral blood lymphocytes. Flow cytometry analysis measured the binding characteristics of Fluos-rrGH to lymphocytes together with specific PE-labelled mAbs defining CD4+ and CD8+ T cells and B lymphocytes. The pattern of expression of the GH receptor differed among the lymphoid tissues and cell subsets. Spleen was the most responsive organ to protein deprivation with highest GH receptor expression in B lymphocytes, followed by CD4+ T cells. As the protein intake was decreased from 20% to 0%, the percentage of GHR positive cells increased from 12% to 52% in splenic B lymphocytes and from 8% to 17% in CD4+ T cells. In contrast, only 10%-13% of lymphocytes in lymph nodes and 2%-4% in circulation, showed binding sites to GH associated with protein deprivation. In conclusion, the increase in GH receptors on lymphocytes under catabolic stress induced by protein malnutrition gives support

  20. Uncoupling protein-2 is an antioxidant that is up-regulated in the enamel organ of fluoride-treated rats.

    PubMed

    Suzuki, Maiko; Sierant, Megan L; Antone, Jerry V; Everett, Eric T; Whitford, Gary M; Bartlett, John D

    2014-08-01

    Dental fluorosis is characterized by subsurface hypomineralization and retention of enamel matrix proteins. Fluoride (F(-)) exposure generates reactive oxygen species (ROS) that can cause endoplasmic reticulum (ER)-stress. We therefore screened oxidative stress arrays to identify genes regulated by F(-) exposure. Vitamin E is an antioxidant so we asked if a diet high in vitamin E would attenuate dental fluorosis. Maturation stage incisor enamel organs (EO) were harvested from F(-)-treated rats and mice were assessed to determine if vitamin E ameliorates dental fluorosis. Uncoupling protein-2 (Ucp2) was significantly up-regulated by F(-) (∼1.5 & 2.0 fold for the 50 or 100 ppm F(-) treatment groups, respectively). Immunohistochemical results on maturation stage rat incisors demonstrated that UCP2 protein levels increased with F(-) treatment. UCP2 down-regulates mitochondrial production of ROS, which decreases ATP production. Thus, in addition to reduced protein translation caused by ER-stress, a reduction in ATP production by UCP2 may contribute to the inability of ameloblasts to remove protein from the hardening enamel. Fluoride-treated mouse enamel had significantly higher quantitative fluorescence (QF) than the untreated controls. No significant QF difference was observed between control and vitamin E-enriched diets within a given F(-) treatment group. Therefore, a diet rich in vitamin E did not attenuate dental fluorosis. We have identified a novel oxidative stress response gene that is up-regulated in vivo by F(-) and activation of this gene may adversely affect ameloblast function. PMID:25158175

  1. Antithrombin Up-regulates AMP-activated Protein Kinase Signaling during Myocardial Ischemia/Reperfusion Injury

    PubMed Central

    Ma, Yina; Wang, Jinli; Gao, Junjie; Yang, Hui; Wang, Yanqing; Manithody, Chandrashekhara; Li, Ji; Rezaie, Alireza R.

    2014-01-01

    Summary Antithrombin (AT) is a protein of the serpin superfamily involved in regulation of the proteolytic activity of the serine proteases of the coagulation system. AT is known to exhibit anti-inflammatory and cardioprotective properties when it binds to heparan sulfate proteoglycans (HSPGs) on vascular cells. AMP-activated protein kinase (AMPK) plays an important cardioprotective role during myocardial ischemia and reperfusion (I/R). To determine whether the cardioprotective signaling function of AT is mediated through the AMPK pathway, we evaluated the cardioprotective activities of wild-type AT and its two derivatives, one having high affinity and the other no affinity for heparin, in an acute I/R injury model in C57BL/6J mice in which the left anterior descending coronary artery was occluded. The serpin derivatives were given 5 min before reperfusion. The results showed that AT-WT can activate AMPK in both in vivo and ex vivo conditions. Blocking AMPK activity abolished the cardioprotective function of AT against I/R injury. The AT derivative having high affinity for heparin was more effective in activating AMPK and in limiting infraction, but the derivative lacking affinity for heparin was inactive in eliciting AMPK-dependent cardioprotective activity. Activation of AMPK by AT inhibited the inflammatory c-Jun N-terminal protein kinase (JNK) pathway during I/R. Further studies revealed that the AMPK activity induced by AT also modulates cardiac substrate metabolism by increasing glucose oxidation but inhibiting fatty acid oxidation during I/R. These results suggest that AT binds to HSPGs on heart tissues to invoke a cardioprotective function by triggering cardiac AMPK activation, thereby attenuating JNK inflammatory signaling pathways and modulating substrate metabolism during I/R. PMID:25230600

  2. Gelatinases promote calcification of vascular smooth muscle cells by up-regulating bone morphogenetic protein-2.

    PubMed

    Zhao, Yong-Gang; Meng, Fan-Xing; Li, Bing-Wei; Sheng, You-Ming; Liu, Ming-Ming; Wang, Bing; Li, Hong-Wei; Xiu, Rui-Juan

    2016-02-01

    Matrix metalloproteinase-2 (MMP-2), also known as gelatinase A, is involved in vascular calcification. Another member of gelatinases is MMP-9 (gelatinase B). However, the role of gelatinases in the pathogenesis of vascular calcification is not well understood. The current study aims to clarify the relationship between gelatinases and vascular calcification and to elucidate the underlying mechanism. Beta-glycerophosphate (β-GP) was used to induce calcification of vascular smooth muscle cells (VSMCs) with or without 2-[[(4-Phenoxyphenyl)sulfonyl]methyl]-thiirane (SB-3CT), a specific gelatinases inhibitor. Levels of calcification were determined by assessing calcium content and calcification area of VSMCs. Phenotype transition of VSMCs was observed by assessing expressions of alkaline phosphatase (ALP), smooth muscle α-actin (SM-α-actin) and desmin. Gelatin zymography was applied to determine the activities of gelatinases, and western blot was applied to determine expressions of gelatinases, bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2) and msh homeobox homolog 2 (Msx-2). Gelatinases inhibition by SB-3CT alleviated calcification and phenotype transition of VSMCs induced by β-GP. Increased gelatinases expression and active MMP-2 were observed in calcifying VSMCs. Gelatinases inhibition reduced expression of RUNX2, Msx-2 and BMP-2. BMP-2 treatment increased expressions of RUNX2 and Msx-2, while noggin, an antagonist of BMP-2, decreased expressions of RUNX2 and Msx-2. Gelatinases promote vascular calcification by upregulating BMP-2 which induces expression of RUNX2 and Msx-2, two proteins associated with phenotype transition of VSMCs in vascular calcification. Interventions targeting gelatinases inhibition might be a proper candidate for ameliorating vascular calcification. PMID:26797522

  3. Identification of up-regulated proteins potentially involved in the antagonism mechanism of Bacillus amyloliquefaciens G1.

    PubMed

    Cao, Haipeng; Zheng, Weidong; He, Shan; Wang, Hao; Wang, Tu; Lu, Liqun

    2013-06-01

    The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1's antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens. PMID:23483288

  4. Up-Regulation of MUC2 Mucin Expression by Serum Amyloid A3 Protein in Mouse Colonic Epithelial Cells

    PubMed Central

    SHIGEMURA, Hiroaki; ISHIGURO, Naotaka; INOSHIMA, Yasuo

    2014-01-01

    ABSTRACT Serum amyloid A (SAA) proteins are acute-phase proteins and are classified into multiple isoforms; however, the biological functions of each SAA isoform are not fully understood. In this study, to clarify the roles of SAA3 in the intestine, we characterized mRNA expression in mouse colonic epithelial CMT-93 cells treated with rotavirus, Toxoplasma, Staphylococcus aureus, and Escherichia coli, as well as lipopolysaccharide (LPS) and recombinant murine SAAs (rSAAs). E. coli together with LPS, but not the other pathogens, enhanced SAA3 mRNA expression. The mRNA expression of SAA3 by dead E. coli was higher than that by living E. coli, and the mRNA expression by E. coli and LPS increased in a dose-dependent manner. In contrast, mRNA expressions of SAA1 and/or SAA2 were not stimulated by any of the treatments. In comparisons of cell treatments with rSAA1 or rSAA3, rSAA3 significantly up-regulated the mRNA expression of mucin 2 (MUC2), a major component of the mucus layer of the intestines that acts as an epithelial cell barrier against pathogens, while MUC2 mRNA expression was not significantly increased by E. coli and LPS. Furthermore, treatment with rSAAs intensively induced tumor necrosis factor-α mRNA expression. These results suggest that SAA3 plays a role in host innate immunity in the colon by up-regulating MUC2 mucin production, which builds a physiological barrier of colonic epithelia against bacterial invasion. PMID:24694941

  5. S100B protein stimulates microglia migration via RAGE-dependent up-regulation of chemokine expression and release.

    PubMed

    Bianchi, Roberta; Kastrisianaki, Eirini; Giambanco, Ileana; Donato, Rosario

    2011-03-01

    The Ca(2+)-binding protein of the EF-hand type, S100B, is abundantly expressed in and secreted by astrocytes, and release of S100B from damaged astrocytes occurs during the course of acute and chronic brain disorders. Thus, the concept has emerged that S100B might act an unconventional cytokine or a damage-associated molecular pattern protein playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases. S100B proinflammatory effects require relatively high concentrations of the protein, whereas at physiological concentrations S100B exerts trophic effects on neurons. Most if not all of the extracellular (trophic and toxic) effects of S100B in the brain are mediated by the engagement of RAGE (receptor for advanced glycation end products). We show here that high S100B stimulates murine microglia migration in Boyden chambers via RAGE-dependent activation of Src kinase, Ras, PI3K, MEK/ERK1/2, RhoA/ROCK, Rac1/JNK/AP-1, Rac1/NF-κB, and, to a lesser extent, p38 MAPK. Recruitment of the adaptor protein, diaphanous-1, a member of the formin protein family, is also required for S100B/RAGE-induced migration of microglia. The S100B/RAGE-dependent activation of diaphanous-1/Rac1/JNK/AP-1, Ras/Rac1/NF-κB and Src/Ras/PI3K/RhoA/diaphanous-1 results in the up-regulation of expression of the chemokines, CCL3, CCL5, and CXCL12, whose release and activity are required for S100B to stimulate microglia migration. Lastly, RAGE engagement by S100B in microglia results in up-regulation of the chemokine receptors, CCR1 and CCR5. These results suggests that S100B might participate in the pathophysiology of brain inflammatory disorders via RAGE-dependent regulation of several inflammation-related events including activation and migration of microglia. PMID:21209080

  6. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR.

    PubMed

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-03-01

    As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  7. Six1 induces protein synthesis signaling expression in duck myoblasts mainly via up-regulation of mTOR

    PubMed Central

    Wang, Haohan; Li, Xinxin; Liu, Hehe; Sun, Lingli; Zhang, Rongping; Li, Liang; Wangding, Mincheng; Wang, Jiwen

    2016-01-01

    Abstract As a critical transcription factor, Six1 plays an important role in the regulation of myogenesis and muscle development. However, little is known about its regulatory mechanism associated with muscular protein synthesis. The objective of this study was to investigate the effects of overexpression ofSix1 on the expression of key protein metabolism-related genes in duck myoblasts. Through an experimental model where duck myoblasts were transfected with a pEGFP-duSix1 construct, we found that overexpression of duckSix1 could enhance cell proliferation activity and increase mRNA expression levels of key genes involved in the PI3K/Akt/mTOR signaling pathway, while the expression of FOXO1, MuRF1and MAFbx was not significantly altered, indicating thatSix1 could promote protein synthesis in myoblasts through up-regulating the expression of several related genes. Additionally, in duck myoblasts treated with LY294002 and rapamycin, the specific inhibitors ofPI3K and mTOR, respectively, the overexpression of Six1 could significantly ameliorate inhibitive effects of these inhibitors on protein synthesis. Especially, the mRNA expression levels of mTOR and S6K1 were observed to undergo a visible change, and a significant increase in protein expression of S6K1 was seen. These data suggested that Six1plays an important role in protein synthesis, which may be mainly due to activation of the mTOR signaling pathway. PMID:27007909

  8. Elevated Expression of Hepatoma Up-Regulated Protein Inhibits γ-Irradiation-Induced Apoptosis of Prostate Cancer Cells.

    PubMed

    Hassan, Mohamed; El Khattouti, Abdelouahid; Ejaeidi, Ahmed; Ma, Tangeng; Day, William A; Espinoza, Ingrid; Vijayakumar, Srinivasan; Gomez, Christian R

    2016-06-01

    Despite progression in diagnosis and treatment, prostate cancer (PCa) still represents the main cause of cancer-related mortality and morbidity in men. Although radiation therapy offers clinical benefit over other therapeutic modalities, the success of this therapeutic modality is commonly hampered by the resistance of advanced tumors. So far, the mechanisms governing tumor resistance to radiotherapy are not discussed in detail. Here, we demonstrate for the first time that the resistance of PCa to radiation therapy is attributed to elevated expression of Hepatoma Up-Regulated Protein (HURP). In PCa cells, the induction of HURP expression suppresses γ-irradiation-induced apoptosis. γ-irradiation-induced apoptosis of PCa cells is associated with expression of E2F1, p53, p21 proteins together with the phosphorylation of apoptosis signal-regulating kinase1 (ASK1), c-jun-N-terminal kinase (JNK) and Ataxia-telangiectasia mutated (ATM) and histone family member X (H2AX). Whereas, the induction of HURP expression is able to suppress γ-irradiation-induced effects on E2F1, p53, p21, ATM, ASK1, JNK and ATM, and H2AX. Also, inhibition of γ-irradiation-induced- cytochrome c release, cleavage of caspase-9, caspase-3, PARP, and reactive oxygen species (ROS) were noted in PCa cells induced for HURP expression. The observed radio-resistance of PCa is thought to be the consequence of HURP-mediated destabilization of p53 and ATM proteins that are essential for the modulation of γ-irradiation-induced apoptosis. Thus, based on our findings, PCa resistance to radiation therapy results from the deregulation of ASK1/ JNK; ATM/ H2AX; ATM/p53 and checkpoint kinase 2 (Chk2)/ E2F-1 in response to the elevated expression of HURP. J. Cell. Biochem. 117: 1308-1318, 2016. © 2015 Wiley Periodicals, Inc. PMID:26505164

  9. Up-regulation of Interferon-inducible protein 16 contributes to psoriasis by modulating chemokine production in keratinocytes

    PubMed Central

    Cao, Tianyu; Shao, Shuai; Li, Bing; Jin, Liang; Lei, Jie; Qiao, Hongjiang; Wang, Gang

    2016-01-01

    Psoriasis is a common chronic inflammatory skin disease characterized by epidermal hyperplasia and dermal inflammation. Keratinocyte activation is known to play a critical role in psoriasis, but the underlying mechanism remains unclear. Interferon-inducible protein 16 (IFI16), an innate immune system sensor, is reported to affect keratinocyte function. We therefore hypothesized that IFI16 promotes psoriasis by modulating keratinocyte activation. In the present study, we cinfirmed that IFI16 was overexpressed in epidermal keratinocytes of psoriasis patients. In addition, psoriasis-related cytokines, including IFN-γ, TNF-α, IL-17 and IL-22, induced IFI16 up-regulation in keratinocytes via activation of STAT3 signaling. We also observed that IFI16 activated the TBK1-NF-κB signaling, leading to the production of CXCL10 and CCL20. Importantly, knocking down p204, which is reported as the mouse orthologous of human IFI16, inhibited epidermal hyperplasia in mice with imiquimod-induced psoriasiform dermatitis. These findings indicate that IFI16 plays a critical role in the pathogenesis of psoriasis and may be a potential therapeutic target. PMID:27137868

  10. Coagulation factor Xa drives tumor cells into apoptosis through BH3-only protein Bim up-regulation

    SciTech Connect

    Borensztajn, Keren S. . E-mail: K.S.Borensztajn@amc.uva.nl; Bijlsma, Maarten F.; Groot, Angelique P.; Brueggemann, Lois W.; Versteeg, Henri H.; Reitsma, Pieter H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2007-07-15

    Coagulation Factor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulation factor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells with up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.

  11. Farnesoid X receptor up-regulates expression of Lipid transfer inhibitor protein in liver cells and mice

    SciTech Connect

    Li, Liangpeng; Liu, Hong; Peng, Jiahe; Wang, Yongchao; Zhang, Yan; Dong, Jinyu; Liu, Xiaohua; Guo, Dongmei; Jiang, Yu

    2013-11-29

    Highlights: •FXR up-regulates apoF. •It binds to ER1 element. •It activates apoF gene promoter. -- Abstract: Apolipoprotein F is a component protein mainly secreted by liver and resides on several lipoprotein classes. It can inhibit lipids transfer between different lipoproteins. FXR is a member of the nuclear receptor superfamily which is also highly expressed in the liver. It modulates bile acids synthesis and lipids metabolism by transcriptional regulation. We aimed to determine whether apoF can be regulated by FXR. The FXR agonist Chenodeoxycholic acid (CDCA) and GW4064 both can activate the expression of apoF in liver cell lines and in C57/BL6 mouse liver. This is dependent on the binding of FXR to the FXR element ER1 (−2904 to −2892 bp) in the apoF gene promoter. Taken together, we have identified apoF as likely another target gene of FXR.

  12. Pancreatic stone protein/regenerating protein (PSP/reg): a novel secreted protein up-regulated in type 2 diabetes mellitus.

    PubMed

    Yang, Jiayue; Li, Ling; Raptis, Dimitri; Li, Xiaoshan; Li, Fengfei; Chen, Bijun; He, Jiajia; Graf, Rolf; Sun, Zilin

    2015-04-01

    Type 2 diabetes mellitus (T2DM) has insulin resistance (IR) or reduced β-cell mass, partially due to an increased β-cell apoptosis rate. Pancreatic stone protein/regenerating protein (PSP/reg) is a secretory protein produced in the pancreas and up-regulated dramatically during pancreatic disease. Recent studies revealed that β-cells undergoing apoptosis induce PSP/reg expression in surviving neighboring cells. Further experiments demonstrated that PSP/reg was elevated during disease progression in type 1 diabetes mellitus (T1DM). However, the association between PSP/reg and T2DM patients is unknown. The aim of this pilot study was to investigate PSP/reg in different clinical stages of T2DM and evaluate its correlation with chronic complications of diabetes. A total of 1,121 participants (479 males, 642 females; age range 23-80 years) were enrolled in this study. PSP/reg serum values were measured by a newly developed enzyme-linked immunosorbent assay (ELISA). We analyzed its correlation with clinical and biochemical parameters in subjects with T2DM at different clinical phases. Statistical analyses were conducted using SPSS 17.0 software. Correlations of PSP/reg and clinical parameters were performed using Spearman's rank correlation coefficient. Differences between groups were determined by Nemenyi test. PSP/reg was elevated in high-risk and impaired glucose regulation (IGR) patients (p<0.05). PSP/reg was significantly up-regulated in newly diagnosed T2DM patients and long-term diabetes patients with complications (p<0.001). PSP/reg levels correlated with the duration of diabetes (p<0.001). The area under the curve (AUC) for presence of diabetes-onset and its chronic complications was 0.640 and 0.754, respectively. PSP/reg is significantly up-regulated in T2DM patients, and PSP/reg levels are related to the duration of diabetes. Therefore, PSP/reg might be useful as a predictor of T2DM and disease progression. PMID:25234740

  13. The Up-Regulation of Ribosomal Proteins Further Regulates Protein Expression Profile in Female Schistosoma japonicum after Pairing

    PubMed Central

    Sun, Jun; Li, Chen; Wang, Suwen

    2015-01-01

    Background Pairing of Schistosoma males and females leads to and maintains female sexual maturation. However, the mechanism by which pairing facilitates sexual maturation of females is not clear. An increasing body of evidence suggests that ribosomal proteins have regulatory rather than constitutive roles in protein translation. Methodology/Principal Findings To investigate the effect of ribosome regulation on female sex maturation, Solexa and iTRAQ techniques were used to analyze the relationship between ribosomal gene or protein expression and sexual development of Schistosoma females. In the present study, considerably higher number of ribosomal genes or proteins were found to be differentially expressed in paired 23-day-old females. Moreover, mature female-specific proteins associated with egg production, such as ferritin-1 heavy chain and superoxide dismutase, were selectively highly expressed in paired females, rather than higher level of protein synthesis of all transcripts compared with those in unpaired 23-day-old females. Furthermore, other developmental stages were utilized to investigate different expression pattern of ribosomal proteins in females by analysing 18-day-old female schistosomula from single- or double-sex infections to determine the relationship between ribosomal protein expression pattern and development. Results showed that undeveloped 18-day-old females from single- and double-sex infections, as well as 23-day-old unpaired females, possessed similar ribosomal protein expression patterns, which were distinct from those in 23-day-old paired females. Conclusions/Significance Our findings reveal that the pairing of females and males triggers a specialized ribosomal protein expression profile which further regulates the protein profile for sexual maturation in Schistosoma japonicum, based on its gene expression profile. PMID:26070205

  14. Plasma proteome profiling of a mouse model of breast cancer identifies a set of up-regulated proteins in common with human breast cancer cells.

    PubMed

    Pitteri, Sharon J; Faca, Vitor M; Kelly-Spratt, Karen S; Kasarda, A Erik; Wang, Hong; Zhang, Qing; Newcomb, Lisa; Krasnoselsky, Alexei; Paczesny, Sophie; Choi, Gina; Fitzgibbon, Matthew; McIntosh, Martin W; Kemp, Christopher J; Hanash, Samir M

    2008-04-01

    We have applied an in-depth quantitative proteomic approach, combining isotopic labeling extensive intact protein separation and mass spectrometry, for high confidence identification of protein changes in plasmas from a mouse model of breast cancer. We hypothesized that a wide spectrum of proteins may be up-regulated in plasma with tumor development and that comparisons with proteins expressed in human breast cancer cell lines may identify a subset of up-regulated proteins in common with proteins expressed in breast cancer cell lines that may represent candidate biomarkers for breast cancer. Plasma from PyMT transgenic tumor-bearing mice and matched controls were obtained at two time points during tumor growth. A total of 133 proteins were found to be increased by 1.5-fold or greater at one or both time points. A comparison of this set of proteins with published findings from proteomic analysis of human breast cancer cell lines yielded 49 proteins with increased levels in mouse plasma that were identified in breast cancer cell lines. Pathway analysis comparing the subset of up-regulated proteins known to be expressed in breast cancer cell lines with other up-regulated proteins indicated a cancer related function for the former and a host-response function for the latter. We conclude that integration of proteomic findings from mouse models of breast cancer and from human breast cancer cell lines may help identify a subset of proteins released by breast cancer cells into the circulation and that occur at increased levels in breast cancer. PMID:18311905

  15. A novel class of antihyperlipidemic agents with low density lipoprotein receptor up-regulation via the adaptor protein autosomal recessive hypercholesterolemia.

    PubMed

    Asano, Shigehiro; Ban, Hitoshi; Tsuboya, Norie; Uno, Shinsaku; Kino, Kouichi; Ioriya, Katsuhisa; Kitano, Masafumi; Ueno, Yoshihide

    2010-04-22

    We have previously reported compound 2 as a inhibitor of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT) and up-regulator of the low density lipoprotein receptor (LDL-R) expression. In this study we focused on compound 2, a unique LDL-R up-regulator, and describe the discovery of a novel class of up-regulators of LDL-R. Replacement the methylene urea linker in compound 2 with an acylsulfonamide linker kept a potent LDL-R up-regulatory activity, and subsequent optimization work gave compound 39 as a highly potent LDL-R up-regulator (39; EC(25) = 0.047 microM). Compound 39 showed no ACAT inhibitory activity even at 1 microM. The sodium salts of compound 39 reduced plasma total and LDL cholesterol levels in a dose-dependent manner in an experimental animal model of hyperlipidemia. Moreover, we revealed in this study using RNA interference that autosomal recessive hypercholesterolemia (ARH), an adaptor protein of LDL-R, is essential for compound 39 up-regulation of LDL-R expression. PMID:20356098

  16. Chloroquine enhances TRAIL-mediated apoptosis through up-regulation of DR5 by stabilization of mRNA and protein in cancer cells

    PubMed Central

    Park, Eun Jung; Min, Kyoung-jin; Choi, Kyeong Sook; Kubatka, Peter; Kruzliak, Peter; Kim, Dong Eun; Kwon, Taeg Kyu

    2016-01-01

    Chloroquine (CQ), an anti-malarial drug, has immune-modulating activity and lysosomotropic activity. In this study, we investigated CQ sensitizes TRAIL-mediated apoptosis in human renal cancer Caki cells. Combination of CQ and TRAIL significantly induces apoptosis in human renal cancer Caki cells and various human cancer cells, but not in normal mouse kidney cells (TMCK-1) and human mesangial cells (MC). CQ up-regulates DR5 mRNA and protein expression in a dose- and time- dependent manner. Interestingly, CQ regulates DR5 expression through the increased stability in the mRNA and protein of DR5, rather than through the increased transcriptional activity of DR5. Moreover, we found that CQ decreased the expression of Cbl, an E3 ligase of DR5, and knock-down of Cbl markedly enhanced DR5 up-regulation. Other lysosomal inhibitors, including monensin and nigericin, also up-regulated DR5 and sensitized TRAIL-mediated apoptosis. Therefore, this study demonstrates that lysosomal inhibition by CQ may sensitize TRAIL-mediated apoptosis in human renal cancer Caki cells via DR5 up-regulation. PMID:26964637

  17. Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

    SciTech Connect

    Zhang, X.; Li, L.; Zhang, L.; Borowitz, J.L.; Isom, G.E.

    2009-07-01

    Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidative stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.

  18. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  19. Mycoplasma gallisepticum lipid associated membrane proteins up-regulate inflammatory genes in chicken tracheal epithelial cells via TLR-2 ligation through an NF-κB dependent pathway.

    PubMed

    Majumder, Sanjukta; Zappulla, Frank; Silbart, Lawrence K

    2014-01-01

    Mycoplasma gallisepticum-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (R(low)) or a non-virulent (R(high)) strain were incubated with primary TECs or chicken tracheae ex vivo. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. Ex-vivo exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, R(low) exposed epithelial cells both in vitro and ex vivo showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2-3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either R(low) or R(high) exposure. Taken together we conclude that LAMPs isolated from both R(high) and R(low) induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway. PMID

  20. An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death

    NASA Technical Reports Server (NTRS)

    Yang, T.; Poovaiah, B. W.

    2000-01-01

    35S-Labeled calmodulin (CaM) was used to screen a tobacco anther cDNA library. A positive clone (NtER1) with high homology to an early ethylene-up-regulated gene (ER66) in tomato, and an Arabidopsis homolog was isolated and characterized. Based on the helical wheel projection, a 25-mer peptide corresponding to the predicted CaM-binding region of NtER1 (amino acids 796-820) was synthesized. The gel-mobility shift assay showed that the peptide formed a stable complex with CaM only in the presence of Ca(2+). CaM binds to NtER1 with high affinity (K(d) approximately 12 nm) in a calcium-dependent manner. Tobacco flowers at different stages of development were treated with ethylene or with 1-methylcyclopropene for 2 h before treating with ethylene. Northern analysis showed that the NtER1 was rapidly induced after 15 min of exposure to ethylene. However, the 2-h 1-methylcyclopropene treatment totally blocked NtER1 expression in flowers at all stages of development, suggesting that NtER1 is an early ethylene-up-regulated gene. The senescing leaves and petals had significantly increased NtER1 induction as compared with young leaves and petals, implying that NtER1 is developmentally regulated and acts as a trigger for senescence and death. This is the first documented evidence for the involvement of Ca(2+)/CaM-mediated signaling in ethylene action.

  1. Increase in gene-transcript levels as indicators of up-regulation of the unfolded protein response in spontaneous canine tumors

    PubMed Central

    Elliot, Kirsten; MacDonald-Dickinson, Valerie; Linn, Kathleen; Simko, Elemir; Misra, Vikram

    2014-01-01

    The unfolded protein response (UPR), a conserved cellular response to stressors such as hypoxia and nutrient deprivation, is associated with angiogenesis and metastasis in tumor cells. This article discusses a pilot study conducted to determine whether components of the UPR could be identified in spontaneous canine tumors and whether they were up-regulated within tumor tissue compared with adjacent normal tissue. Tissue samples of various spontaneous canine neoplasms were taken from 13 dogs shortly after surgical excision or euthanasia; control samples were taken from adjacent normal tissue. RNA purification and real-time quantitative reverse-transcription polymerase chain reaction were done to measure the expression of 4 genes associated with the UPR (HERP, CHOP, GRP78, and XBP1s). The results indicated that UPR gene expression can be identified in spontaneous canine tumors and that the UPR is up-regulated, as indicated by significantly increased expression of CHOP and GRP78 within the tumor. PMID:24982546

  2. Zyflamend Sensitizes Tumor Cells to TRAIL-Induced Apoptosis Through Up-Regulation of Death Receptors and Down-Regulation of Survival Proteins: Role of ROS-Dependent CCAAT/Enhancer-Binding Protein-Homologous Protein Pathway

    PubMed Central

    Kim, Ji Hye; Park, Byoungduck; Gupta, Subash C.; Kannappan, Ramaswamy; Sung, Bokyung

    2012-01-01

    Abstract Aim: TNF (tumor necrosis factor)-related apoptosis-inducing ligand (TRAIL), is a selective killer of tumor cells, although its potential is limited by the development of resistance. In this article, we investigated whether the polyherbal preparation Zyflamend® can sensitize tumor cells to TRAIL. Results: We found that Zyflamend potentiated TRAIL-induced apoptosis in human cancer cells. Zyflamend manifested its effects through several mechanisms. First, it down-regulated the expression of cell survival proteins known to be linked to resistance to TRAIL. Second, Zyflamend up-regulated the expression of pro-apoptotic protein, Bax. Third, Zyflamend up-regulated the expression of death receptors (DRs) for TRAIL. Up-regulation of DRs was critical as gene-silencing of these receptors significantly reduced the effect of Zyflamend on TRAIL-induced apoptosis. The up-regulation of DRs was dependent on CCAAT/enhancer-binding protein-homologous protein (CHOP), as Zyflamend induced CHOP, its gene-silencing abolished the induction of receptors, and mutation of the CHOP binding site on DR5 promoter abolished Zyflamend-mediated DR5 transactivation. Zyflamend mediated its effects through reactive oxygen species (ROS), as ROS quenching reduced its effect. Further, Zyflamend induced DR5 and CHOP and down-regulated the expression of cell survival proteins in nude mice bearing human pancreatic cancer cells. Innovation: Zyflamend can sensitize tumor cells to TRAIL through modulation of multiple cell signaling mechanisms that are linked to ROS. Conclusion: Zyflamend potentiates TRAIL-induced apoptosis through the ROS-CHOP-mediated up-regulation of DRs, increase in pro-apoptotic protein and down-regulation of cell survival proteins. Antioxid. Redox Signal. 16, 413–427. PMID:22004570

  3. A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots.

    PubMed

    Zamora-Briseño, Jesús Alejandro; de Jiménez, Estela Sánchez

    2016-04-01

    Late embryogenesis abundant (LEA) proteins are hydrophilic proteins that accumulate to high concentrations during the late stages of seeds development, which are integral to desiccation tolerance. LEA proteins also play a protective role under other abiotic stresses. We analyzed in silico a maize protein predicted to be highly hydrophilic and intrinsically disordered. This prediction was experimentally corroborated by solubility assays under denaturing conditions. Based on its amino acid sequence, we propose that this protein belongs to group four of the LEA proteins. The accumulation pattern of this protein was similar to that of dehydrins during the desiccation process that takes place during seed development. This protein was induced by exogenous abscisic acid in immature embryos, but during imbibition was down-regulated by gibberellins. It was also induced in maize roots under osmotic stress. So far, this is the first member of the LEA proteins belonging to group four to be characterized in maize, and it plays a role in the response to osmotic stress. PMID:26922182

  4. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma

    PubMed Central

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-01-01

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p < 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes. PMID:27070597

  5. The freshwater Amazonian stingray, Potamotrygon motoro, up-regulates glutamine synthetase activity and protein abundance, and accumulates glutamine when exposed to brackish (15 per thousand) water.

    PubMed

    Ip, Y K; Loong, A M; Ching, B; Tham, G H Y; Wong, W P; Chew, S F

    2009-12-01

    This study aimed to examine whether the stenohaline freshwater stingray, Potamotrygon motoro, which lacks a functional ornithine-urea cycle, would up-regulate glutamine synthetase (GS) activity and protein abundance, and accumulate glutamine during a progressive transfer from freshwater to brackish (15 per thousand) water with daily feeding. Our results revealed that, similar to other freshwater teleosts, P. motoro performed hyperosmotic regulation, with very low urea concentrations in plasma and tissues, in freshwater. In 15 per thousand water, it was non-ureotelic and non-ureoosmotic, acting mainly as an osmoconformer with its plasma osmolality, [Na+] and [Cl-] comparable to those of the external medium. There were significant increases in the content of several free amino acids (FAAs), including glutamate, glutamine and glycine, in muscle and liver, but not in plasma, indicating that FAAs could contribute in part to cell volume regulation. Furthermore, exposure of P. motoro to 15 per thousand water led to up-regulation of GS activity and protein abundance in both liver and muscle. Thus, our results indicate for the first time that, despite the inability to synthesize urea and the lack of functional carbamoyl phosphate synthetase III (CPS III) which uses glutamine as a substrate, P. motoro retained the capacity to up-regulate the activity and protein expression of GS in response to salinity stress. Potamotrygon motoro was not nitrogen (N) limited when exposed to 15 per thousand water with feeding, and there were no significant changes in the amination and deamination activities of hepatic glutamate dehydrogenase. In contrast, P. motoro became N limited when exposed to 10 per thousand water with fasting and could not survive well in 15 per thousand water without food. PMID:19915125

  6. Roscovitine up-regulates p53 protein and induces apoptosis in human HeLaS(3) cervix carcinoma cells.

    PubMed

    Wesierska-Gadek, Józefa; Wandl, Stefanie; Kramer, Matthias P; Pickem, Christian; Krystof, Vladimir; Hajek, Susanne B

    2008-12-01

    Exposure of human HeLaS(3) cervix carcinoma cells to high doses of conventional cytostatic drugs, e.g. cisplatin (CP) strongly inhibits their proliferation. However, most cytostatic agents are genotoxic and may generate a secondary malignancy. Therefore, therapeutic strategy using alternative, not cytotoxic drugs would be beneficial. Inhibition of cyclin-dependent kinases (CDKs) by pharmacological inhibitors became recently a promising therapeutic option. Roscovitine (ROSC), a selective CDK inhibitor, efficiently targets human malignant cells. ROSC induces cell cycle arrest and apoptosis in human MCF-7 breast cancer cells. ROSC also activates p53 protein. Activation of p53 tumor suppressor protein is essential for induction of apoptosis in MCF-7 cells. Considering the fact that in HeLaS(3) cells wt p53 is inactivated by the action of HPV-encoded E6 oncoprotein, we addressed the question whether ROSC would be able to reactivate p53 protein in them. Their exposure to ROSC for 24 h induced cell cycle arrest at G(2)/M and reduced the number of viable cells. Unlike CP, ROSC in the used doses did not induce DNA damage and was not directly cytotoxic. Despite lack of detectable DNA lesions, ROSC activated wt p53 protein. The increase of p53 levels was attributable to the ROSC-mediated protein stabilization. Further analyses revealed that ROSC induced site-specific phosphorylation of p53 protein at Ser46. After longer exposure, ROSC induced apoptosis in HeLaS(3) cells. These results indicate that therapy of HeLaS(3) cells by ROSC could offer an advantage over that by CP due to its increased selectivity and markedly reduced risk of generation of a secondary cancer. PMID:18846503

  7. C23 protein meditates bone morphogenetic protein-2-mediated EMT via up-regulation of Erk1/2 and Akt in gastric cancer.

    PubMed

    Yang, Yonggang; Yang, Chunyan; Zhang, Jianping

    2015-03-01

    In our previous study, the epithelial-to-mesenchymal transition (EMT) has been identified to be involved in gastric cancer progression. Notably, nuclear protein C23 and bone morphogenetic protein-2 (BMP2) have been linked into EMT. However, the specific mechanisms underlying BMP2 pathway-mediated EMT are not still unraveled. In this study, we adopted immunohistochemistry and immunoblotting to determine the expression of C23 and BMP2 receptor II (BMPR-II) in 90 gastric cancer samples and cell lines. Subsequently, relevant cell lines were selected to be treated with si-C23 or si-BMPRII and the detection of in vitro assay. Our results revealed that both C23 and BMPRII were aberrantly and constitutively expressed in gastric cancer specimens and cell lines, whose expression was positively associated with metastasis, stage and differentiation, and portended poor survival outcome of gastric cancer patients. In vitro assay validated the increased expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2 in BMP2-stimulated MGC803 cells, which was in a dose-dependent manner. By contrast, si-C23 treatment attenuated the BMP2-stimulated expression of p-Erk1/2, p-Akt, vimentin, N-cadherin, and MMP2. Also, the treatment of either si-C23 or si-BMPRII decreased the ability of migration and invasion of MGC803 cells. In conclusion, C23 mediates BMP2-induced EMT progression via the up-regulation of Erk1/2 and Akt signaling pathway in gastric cancer, which indicated both C23 and BMPRII pathway could be recommended as prospective targets or biomarkers to antagonize the progression of gastric cancer. PMID:25698539

  8. Up-regulation of hypertonicity-activated myo-inositol transporter SMIT1 by the cell volume-sensitive protein kinase SGK1.

    PubMed

    Klaus, F; Palmada, M; Lindner, R; Laufer, J; Jeyaraj, S; Lang, F; Boehmer, C

    2008-03-15

    Mechanisms of regulatory cell volume increase following cell shrinkage include accumulation of organic osmolytes such as betaine, taurine, sorbitol, glycerophosphorylcholine (GPC) and myo-inositol. Myo-inositol is taken up by the sodium-myo-inositol-transporter SMIT1 (SLC5A3) expressed in a wide variety of cell types. Hypertonicity induces the transcription of the SMIT1 gene upon binding of the transcription factor tonicity enhancer binding protein (TonEBP) to tonicity responsive enhancers (TonE) in the SMIT1 promoter region. However, little is known about post-translational regulation of the carrier protein. In this study we show that SMIT1 is modulated by the serum- and glucocorticoid-inducible kinase SGK1, a protein genomically up-regulated by hypertonicity. As demonstrated by two-electrode voltage-clamp in the Xenopus oocyte expression system, SMIT1-mediated myo-inositol-induced currents are up-regulated by coexpression of wild type SGK1 and constitutively active (S422D)SGK1 but not by inactive (K127N)SGK1. The increase in SMIT1 activity is due to an elevated cell surface expression of the carrier while its kinetic properties remain unaffected. According to the decay of SMIT1 activity in the presence of brefeldin A, SGK1 stabilizes the SMIT1 protein in the plasma membrane. The SGK isoforms SGK2, SGK3 and the closely related protein kinase B (PKB) are similarly capable of activating SMIT1 activity. SMIT1-mediated currents are decreased by coexpression of the ubiquitin-ligase Nedd4-2, an effect counteracted by additional coexpression of SGK1. In conclusion, the present observations disclose SGK isoforms and protein kinase B as novel regulators of SMIT1 activity. PMID:18202099

  9. Up-regulated 14-3-3β and 14-3-3ζ proteins in prefrontal cortex of subjects with schizophrenia: effect of psychotropic treatment.

    PubMed

    Rivero, Guadalupe; Gabilondo, Ane M; García-Sevilla, Jesús A; La Harpe, Romano; Morentín, Benito; Meana, J Javier

    2015-02-01

    14-3-3 is a family of conserved regulatory proteins that bind to a multitude of functionally diverse signalling proteins. Various genetic studies and gene expression and proteomic analyses have involved 14-3-3 proteins in schizophrenia (SZ). On the other hand, studies about the status of these proteins in major depressive disorder (MD) are still missing. Immunoreactivity values of cytosolic 14-3-3β and 14-3-3ζ proteins were evaluated by Western blot in prefrontal cortex (PFC) of subjects with schizophrenia (SZ; n=22), subjects with major depressive disorder (MD; n=21) and age-, gender- and postmortem delay-matched control subjects (n=52). The modulation of 14-3-3β and 14-3-3ζ proteins by psychotropic medication was also assessed. The analysis of both proteins in SZ subjects with respect to matched control subjects showed increased 14-3-3β (Δ=33±10%, p<0.05) and 14-3-3ζ (Δ=29±6%, p<0.05) immunoreactivity in antipsychotic-free but not in antipsychotic-treated SZ subjects. Immunoreactivity values of 14-3-3β and 14-3-3ζ were not altered in MD subjects. These results show the specific up-regulation of 14-3-3β and 14-3-3ζ proteins in PFC of SZ subjects and suggest a possible down-regulation of both proteins by antipsychotic treatment. PMID:25549848

  10. Stabilization of cellular mRNAs and up-regulation of proteins by oligoribonucleotides homologous to the Bcl2 adenine-uridine rich element motif.

    PubMed

    Bevilacqua, Annamaria; Ghisolfi, Laura; Franzi, Sara; Maresca, Giovanna; Gherzi, Roberto; Capaccioli, Sergio; Nicolin, Angelo; Canti, Gianfranco

    2007-02-01

    Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs. PMID:17077270

  11. Cell-penetrable mouse forkhead box protein 3 alleviates experimental arthritis in mice by up-regulating regulatory T cells.

    PubMed

    Liu, Xia; Ji, Baoju; Sun, Mengyi; Wu, Weijiang; Huang, Lili; Sun, Aihua; Zong, Yangyong; Xia, Sheng; Shi, Liyun; Qian, Hui; Xu, Wenrong; Shao, Qixiang

    2015-07-01

    Regulatory T cells (T(regs)) have potential applications in clinical disease therapy, such as autoimmune diseases and transplant rejection. However, their numbers are limited. Forkhead box protein 3 (FoxP3) is a key transcription factor that controls T(reg) development and function. Here, we generated a cell-permeable fusion protein, protein transduction domain (PTD)-conjugated mouse FoxP3 protein (PTD-mFoxP3), and evaluated whether PTD-mFoxp3 can alleviate rheumatoid arthritis (RA) in the collagen-induced arthritis (CIA) mouse model. As expected, PTD-mFoxP3 was transduced into cells effectively, and inhibited T cell activation and attenuated the cell proliferation. It decreased interleukin (IL) 2 and interferon (IFN)-γ expression, and increased IL-10 expression in activated CD4(+)CD25(-) T cells. PTD-mFoxP3-transduced CD4(+)CD25(-) T cells attenuated proliferation of activated CD4(+)CD25(-) T cells. In addition, PTD-mFoxP3 blocked the Th17 differentiation programme in vitro and down-regulated IL-17 production from T cells by modulating induction and levels of retinoid-related orphan receptor gamma t (RORγt). Intra-articular delivery of PTD-mFoxP3 delayed disease incidence remarkably and alleviated autoimmune symptoms of CIA mice. Moreover, protective effects of PTD-mFoxP3 were associated with regulating the balance of T helper type 17 (Th17) and T(regs). These results suggest that PTD-mFoxP3 may be a candidate for RA therapy. PMID:25809415

  12. Protein kinase C activation is involved in ultraviolet B irradiation-induced endothelial cell ICAM-1 up-regulation and lymphocyte-endothelium interaction in vitro.

    PubMed

    Funk, J O; Holler, E; Kohlhuber, F; Ueffing, M; Bornkamm, G W; Kind, P; Eissner, G

    1996-10-01

    Lymphocyte-endothelium interactions are pivotal steps in mediating inflammatory responses. The authors have analysed the influence of ultraviolet B (UVB) irradiation on intercellular adhesion molecule (ICAM)-1 expression on cells of the human microvascular endothelial cell line (HMEC)-1 and the intracellular signalling pathways involved. Flow cytometry revealed dose-dependent ICAM-1 up-regulation with maximum induced expression 24h after sublethal UVB irradiation of 10 mJ/cm2. While anti-tumour necrosis factor (TNF)-alpha antibodies or recombinant human interleukin (IL)-10 did not influence this response, anti-interferon (IFN)-gamma antibodies blocked the UVB-induced ICAM-1 up-regulation. Significant induction of intracellular/membrane-bound IFN-gamma was measured as early as 6 h post-UVB. Since previous work has shown a differential role of protein kinase C (PKC) in cytokine induced ICAM-1 expression, the effect of a selective bisindolylmaleimide-derived PKC-inhibitor (GF109203X) was studied. Ultraviolet B-induced ICAM-1 up-regulation was effectively blocked by the PKC-inhibitor, whereas a PKA-inhibitor was ineffective. Moreover, immunofluorescence analysis showed a radiation-induced membrane translocation of PKC-alpha, indicative of enzyme activation, in HMEC-1 cells already 30 min post-UVB. The functional relevance of the UVB-induced ICAM-1 expression and involvement of PKC in this process was demonstrated in an adhesion assay with peripheral blood mononuclear cells. In conclusion, UVB-induced ICAM-1 expression on human endothelial cells involves PKC-dependent pathways and can be prevented by a PKC-inhibitor. The use of PKC-inhibitors as additive modulators in immune reactions may bear clinical potential. The mechanisms of IFN-gamma induction in endothelial cells by UVB deserve further investigation. PMID:8845028

  13. Up-regulation of Alzheimer's disease-associated proteins may cause enflurane anesthesia induced cognitive decline in aged rats.

    PubMed

    Liu, Haijian; Weng, Hao

    2014-02-01

    Isoflurane anesthesia can cause post-operative cognitive dysfunction in elderly patients. As an isomer of isoflurane, enflurane may also account for cognitive dysfunction. However, the mechanism of enflurane-induced cognitive dysfunction remains obscure. In this study, we investigated the effects of enflurane anesthesia on cognitive function and the possible roles of β-amyloid protein and phosphorylated tau protein in response to enflurane anesthesia in aged rats. After intraperitoneal injection of enflurane, the Morris water maze and the step-down passive avoidance tests were conducted to test the cognitive ability and memory. The enflurane group showed prolonged escape latency, extended space exploration time and increased number of errors at early stage after enflurane anesthesia, suggesting that enflurane should be responsible for the impairment of cognition in aged rats. In addition, we analyzed the expression level of β-amyloid and phosphorylation level of tau in the hippocampus by immunoblotting. Interestingly, the levels of β-amyloid and phosphorylated tau in the hippocampus increased significantly at early stage and then restored to pre-anesthetic levels. Taken together, our results suggest that increasing of β-amyloid and phosphorylation of tau are important to cause cognitive decline in aged rats during initial stage after enflurane anesthesia. PMID:23934553

  14. Yes-Associated Protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy.

    PubMed

    Goodman, Craig A; Dietz, Jason M; Jacobs, Brittany L; McNally, Rachel M; You, Jae-Sung; Hornberger, Troy A

    2015-06-01

    Mechanically-induced skeletal muscle growth is regulated by mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Yes-Associated Protein (YAP) is a mechanically-sensitive, and growth-related, transcriptional co-activator that can regulate mTORC1. Here we show that, in skeletal muscle, mechanical overload promotes an increase in YAP expression; however, the time course of YAP expression is markedly different from that of mTORC1 activation. We also show that the overexpression of YAP induces hypertrophy via an mTORC1-independent mechanism. Finally, we provide preliminary evidence of possible mediators of YAP-induced hypertrophy (e.g. increased MyoD and c-Myc expression, and decreased Smad2/3 activity and muscle ring finger 1 (MuRF1) expression). PMID:25959868

  15. Involvement of YODA and mitogen activated protein kinase 6 in Arabidopsis post-embryogenic root development through auxin up-regulation and cell division plane orientation.

    PubMed

    Smékalová, Veronika; Luptovčiak, Ivan; Komis, George; Šamajová, Olga; Ovečka, Miroslav; Doskočilová, Anna; Takáč, Tomáš; Vadovič, Pavol; Novák, Ondřej; Pechan, Tibor; Ziemann, Anja; Košútová, Petra; Šamaj, Jozef

    2014-09-01

    The role of YODA MITOGEN ACTIVATED PROTEIN KINASE KINASE KINASE 4 (MAPKKK4) upstream of MITOGEN ACTIVATED PROTEIN KINASE 6 (MPK6) was studied during post-embryonic root development of Arabidopsis thaliana. Loss- and gain-of-function mutants of YODA (yda1 and ΔNyda1) were characterized in terms of root patterning, endogenous auxin content and global proteomes. We surveyed morphological and cellular phenotypes of yda1 and ΔNyda1 mutants suggesting possible involvement of auxin. Endogenous indole-3-acetic acid (IAA) levels were up-regulated in both mutants. Proteomic analysis revealed up-regulation of auxin biosynthetic enzymes tryptophan synthase and nitrilases in these mutants. The expression, abundance and phosphorylation of MPK3, MPK6 and MICROTUBULE ASSOCIATED PROTEIN 65-1 (MAP65-1) were characterized by quantitative polymerase chain reaction (PCR) and western blot analyses and interactions between MAP65-1, microtubules and MPK6 were resolved by quantitative co-localization studies and co-immunoprecipitations. yda1 and ΔNyda1 mutants showed disoriented cell divisions in primary and lateral roots, abortive cytokinesis, and differential subcellular localization of MPK6 and MAP65-1. They also showed deregulated expression of TANGLED1 (TAN1), PHRAGMOPLAST ORIENTING KINESIN 1 (POK1), and GAMMA TUBULIN COMPLEX PROTEIN 4 (GCP4). The findings that MPK6 localized to preprophase bands (PPBs) and phragmoplasts while the mpk6-4 mutant transformed with MPK6AEF (alanine (A)-glutamic acid (E)-phenylanine (F)) showed a root phenotype similar to that of yda1 demonstrated that MPK6 is an important player downstream of YODA. These data indicate that YODA and MPK6 are involved in post-embryonic root development through an auxin-dependent mechanism regulating cell division and mitotic microtubule (PPB and phragmoplast) organization. PMID:24923680

  16. Constitutive shedding of the amyloid precursor protein ectodomain is up-regulated by tumour necrosis factor-alpha converting enzyme.

    PubMed Central

    Slack, B E; Ma, L K; Seah, C C

    2001-01-01

    The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells. PMID:11463349

  17. CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) express early apoptotic markers but avoid programmed cell death by up-regulation of antiapoptotic proteins

    PubMed Central

    Pfannes, Loretta; Chen, Gubin; Shah, Simant; Solomou, Elena E.; Barrett, John; Young, Neal S.

    2007-01-01

    CD34 cells from patients with trisomy 8 myelodysplastic syndrome (MDS) are distinguished from other MDS cells and from normal hematopoietic cells by their pronounced expression of apoptotic markers. Paradoxically, trisomy 8 clones can persist in patients with bone marrow failure and expand following immunosuppression. We previously demonstrated up-regulation of c-myc and CD1 by microarray analysis. Here, we confirmed these findings by real-time polymerase chain reaction (PCR), demonstrated up-regulation of survivin, c-myc, and CD1 protein expression, and documented comparable colony formation by annexin+ trisomy 8− CD34+ and annexin− CD34 cells. There were low levels of DNA degradation in annexin+ trisomy 8 CD34 cells, which were comparable with annexin− CD34 cells. Trisomy 8 cells were resistant to apoptosis induced by gamma irradiation. Knock-down of survivin by siRNA resulted in preferential loss of trisomy 8 cells. These results suggest that trisomy 8 cells undergo incomplete apoptosis and are nonetheless capable of colony formation and growth. PMID:17090657

  18. Proteins involved on TGF-β pathway are up-regulated during the acute phase of experimental Chagas disease.

    PubMed

    Ferreira, Roberto Rodrigues; de Souza, Elen Mello; de Oliveira, Fabiane Loiola; Ferrão, Patrícia Mello; Gomes, Leonardo Henrique Ferreira; Mendonça-Lima, Leila; Meuser-Batista, Marcelo; Bailly, Sabine; Feige, Jean Jacques; de Araujo-Jorge, Tania Cremonini; Waghabi, Mariana Caldas

    2016-05-01

    Studies developed by our group in the last years have shown the involvement of TGF-β in acute and chronic Chagas heart disease, with elevated plasma levels and activated TGF-β cell signaling pathway as remarkable features of patients in the advanced stages of this disease, when high levels of cardiac fibrosis is present. Imbalance in synthesis and degradation of extracellular matrix components is the basis of pathological fibrosis and TGF-β is considered as one of the key regulators of this process. In the present study, we investigated the activity of the TGF-β signaling pathway, including receptors and signaling proteins activation in the heart of animals experimentally infected with Trypanosoma cruzi during the period that mimics the acute phase of Chagas disease. We observed that T. cruzi-infected animals presented increased expression of TGF-β receptors. Overexpression of receptors was followed by an increased phosphorylation of Smad2/3, p38 and ERK. Furthermore, we correlated these activities with cellular factors involved in the fibrotic process induced by TGF-β. We observed that the expression of collagen I, fibronectin and CTGF were increased in the heart of infected animals on day 15 post-infection. Correlated with the increased TGF-β activity in the heart, we found that serum levels of total TGF-β were significantly higher during acute infection. Taken together, our data suggest that the commitment of the heart associates with increased activity of TGF-β pathway and expression of its main components. Our results, confirm the importance of this cytokine in the development and maintenance of cardiac damage caused by T. cruzi infection. PMID:26852285

  19. Inhibitory effect of spinal mGlu(5) receptor antisense oligonucleotide on the up-regulated expression of spinal G protein associated with chronic morphine treatment.

    PubMed

    Chen, Moxi; Zhang, Xiaoli; Xu, Hao; Ma, Xiaqing; Jiang, Wei; Xu, Tao

    2014-01-15

    Knockdown of spinal metabotropic glutamate 5 (mGlu5) receptor was shown to inhibit the development of intrathecal morphine antinociceptive tolerance. The present work was designed to evaluate the expression of spinal G-protein during morphine tolerance and knockdown of spinal mGlu5 receptor with antisense oligonucleotide (ODN). Rats were treated with saline, morphine, mGlu5 receptor antisense or mismatch ODN intrathecally. Behavioral tests were employed to test the thermal and mechanical pain thresholds. Five days later, rats were scarified and spinal expression of spinal Gαi, Gαo, Gαq and Gβ were detected. Consistent with the previous results, knockdown of spinal mGlu5 receptor could inhibit spinal morphine antinociceptive tolerance in behavioral tests (P<0.05). The mGlu5 receptor antisense ODN produced a significant reduction in mGlu5 receptor protein of about 56.6% compared with the control group (P<0.05). Expression of spinal Gαi, Gαo, Gαq and Gβ were up-regulated while morphine tolerance developed (P<0.05). Antisense ODN of spinal mGlu5 receptor, but not mismatched ODN, reduced the spinal dorsal horn levels of Gαi, Gαo, Gαs, Gαq and Gβ (P<0.05). We conclude that expression of spinal G (αi, αo, αs, αq and β) protein may be up-regulated after chronic morphine treatment which could be attenuated by knockdown of spinal mGlu5 receptor with antisense ODN. PMID:24296320

  20. Accumulated SET protein up-regulates and interacts with hnRNPK, increasing its binding to nucleic acids, the Bcl-xS repression, and cellular proliferation

    SciTech Connect

    Almeida, Luciana O.; Garcia, Cristiana B.; Matos-Silva, Flavia A.; Curti, Carlos; Leopoldino, Andréia M.

    2014-02-28

    Highlights: • hnRNPK is a new target of SET. • SET regulates hnRNPK. • SET and hnRNPK accumulation promotes tumorigenesis. • SET accumulation is a potential model to study genes regulated by SET-hnRNPK. - Abstract: SET and hnRNPK are proteins involved in gene expression and regulation of cellular signaling. We previously demonstrated that SET accumulates in head and neck squamous cell carcinoma (HNSCC); hnRNPK is a prognostic marker in cancer. Here, we postulate that SET and hnRNPK proteins interact to promote tumorigenesis. We performed studies in HEK293 and HNSCC (HN6, HN12, and HN13) cell lines with SET/hnRNPK overexpression and knockdown, respectively. We found that SET and/or hnRNPK protein accumulation increased cellular proliferation. SET accumulation up-regulated hnRNPK mRNA and total/phosphorylated protein, promoted hnRNPK nuclear location, and reduced Bcl-x mRNA levels. SET protein directly interacted with hnRNPK, increasing both its binding to nucleic acids and Bcl-xS repression. We propose that hnRNPK should be a new target of SET and that SET–hnRNPK interaction, in turn, has potential implications in cell survival and malignant transformation.

  1. Knockout of the abundant Trichomonas vaginalis hydrogenosomal membrane protein TvHMP23 increases hydrogenosome size but induces no compensatory up-regulation of paralogous copies.

    PubMed

    Brás, Xavier Pereira; Zimorski, Verena; Bolte, Kathrin; Maier, Uwe-G; Martin, William F; Gould, Sven B

    2013-05-01

    The Trichomonas vaginalis genome encodes up to 60000 genes, many of which stem from genome duplication events. Paralogous copies thus accompany most T. vaginalis genes, a phenomenon that limits genetic manipulation. We characterized one of the parasite's most abundant hydrogenosomal membrane proteins, TvHMP23, which is phylogenetically distinct from canonical metabolite carriers, and which localizes to the inner hydrogenosomal membrane as shown through sub-organellar fractionation and protease protection assays. Knockout of Tvhmp23 through insertion of the selectable neomycin marker led to a size increase of hydrogenosomes, the first knockout-induced phenotypes reported for Trichomonas, but no growth impairment. The transcriptional response of its four paralogous copies then analyzed revealed that they are not up-regulated, and hence do not compensate for the Tvhmp23 knockout. PMID:23499435

  2. Integrated Proteomics Identified Up-Regulated Focal Adhesion-Mediated Proteins in Human Squamous Cell Carcinoma in an Orthotopic Murine Model

    PubMed Central

    Granato, Daniela C.; Zanetti, Mariana R.; Kawahara, Rebeca; Yokoo, Sami; Domingues, Romênia R.; Aragão, Annelize Z.; Agostini, Michelle; Carazzolle, Marcelo F.; Vidal, Ramon O.; Flores, Isadora L.; Korvala, Johanna; Cervigne, Nilva K.; Silva, Alan R. S.; Coletta, Ricardo D.; Graner, Edgard; Sherman, Nicholas E.; Leme, Adriana F. Paes

    2014-01-01

    Understanding the molecular mechanisms of oral carcinogenesis will yield important advances in diagnostics, prognostics, effective treatment, and outcome of oral cancer. Hence, in this study we have investigated the proteomic and peptidomic profiles by combining an orthotopic murine model of oral squamous cell carcinoma (OSCC), mass spectrometry-based proteomics and biological network analysis. Our results indicated the up-regulation of proteins involved in actin cytoskeleton organization and cell-cell junction assembly events and their expression was validated in human OSCC tissues. In addition, the functional relevance of talin-1 in OSCC adhesion, migration and invasion was demonstrated. Taken together, this study identified specific processes deregulated in oral cancer and provided novel refined OSCC-targeting molecules. PMID:24858105

  3. Fibroblast growth factor 2 inhibits up-regulation of bone morphogenic proteins and their receptors during osteoblastic differentiation of human mesenchymal stem cells

    SciTech Connect

    Biver, Emmanuel; Soubrier, Anne-Sophie; Thouverey, Cyril; Cortet, Bernard; Broux, Odile; Caverzasio, Joseph; Hardouin, Pierre

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer FGF modulates BMPs pathway in HMSCs by down-regulating BMP/BMPR expression. Black-Right-Pointing-Pointer This effect is mediated by ERK and JNK MAPKs pathways. Black-Right-Pointing-Pointer Crosstalk between FGF and BMPs must be taken into account in skeletal bioengineering. Black-Right-Pointing-Pointer It must also be considered in the use of recombinant BMPs in orthopedic and spine surgeries. -- Abstract: Understanding the interactions between growth factors and bone morphogenic proteins (BMPs) signaling remains a crucial issue to optimize the use of human mesenchymal stem cells (HMSCs) and BMPs in therapeutic perspectives and bone tissue engineering. BMPs are potent inducers of osteoblastic differentiation. They exert their actions via BMP receptors (BMPR), including BMPR1A, BMPR1B and BMPR2. Fibroblast growth factor 2 (FGF2) is expressed by cells of the osteoblastic lineage, increases their proliferation and is secreted during the healing process of fractures or in surgery bone sites. We hypothesized that FGF2 might influence HMSC osteoblastic differentiation by modulating expressions of BMPs and their receptors. BMP2, BMP4, BMPR1A and mainly BMPR1B expressions were up-regulated during this differentiation. FGF2 inhibited HMSCs osteoblastic differentiation and the up-regulation of BMPs and BMPR. This effect was prevented by inhibiting the ERK or JNK mitogen-activated protein kinases which are known to be activated by FGF2. These data provide a mechanism explaining the inhibitory effect of FGF2 on osteoblastic differentiation of HMSCs. These crosstalks between growth and osteogenic factors should be considered in the use of recombinant BMPs in therapeutic purpose of fracture repair or skeletal bioengineering.

  4. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells

    PubMed Central

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  5. Global quantitative proteomics reveal up-regulation of endoplasmic reticulum stress response proteins upon depletion of eIF5A in HeLa cells.

    PubMed

    Mandal, Ajeet; Mandal, Swati; Park, Myung Hee

    2016-01-01

    The eukaryotic translation factor, eIF5A, is a translation factor essential for protein synthesis, cell growth and animal development. By use of a adenoviral eIF5A shRNA, we have achieved an effective depletion of eIF5A in HeLa cells and undertook in vivo comprehensive proteomic analyses to examine the effects of eIF5A depletion on the total proteome and to identify cellular pathways influenced by eIF5A. The proteome of HeLa cells transduced with eIF5A shRNA was compared with that of scramble shRNA-transduced counterpart by the iTRAQ method. We identified 972 proteins consistently detected in three iTRAQ experiments and 104 proteins with significantly altered levels (protein ratio ≥1.5 or ≤0.66, p-value ≤0.05) at 72 h and/or 96 h of Ad-eIF5A-shRNA transduction. The altered expression levels of key pathway proteins were validated by western blotting. Integration of functional ontology with expression data of the 104 proteins revealed specific biological processes that are prominently up- or down-regulated. Heatmap analysis and Cytoscape visualization of biological networks identified protein folding as the major cellular process affected by depletion of eIF5A. Our unbiased, quantitative, proteomic data demonstrate that the depletion of eIF5A leads to endoplasmic reticulum stress, an unfolded protein response and up-regulation of chaperone expression in HeLa cells. PMID:27180817

  6. Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition

    PubMed Central

    2010-01-01

    Background Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. Methods Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. Results The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. Conclusion In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor. PMID

  7. The Aspergillus fumigatus StuA Protein Governs the Up-Regulation of a Discrete Transcriptional Program during the Acquisition of Developmental CompetenceD⃞

    PubMed Central

    Sheppard, Donald C.; Doedt, Thomas; Chiang, Lisa Y.; Kim, H. Stanley; Chen, Dan; Nierman, William C.; Filler, Scott G.

    2005-01-01

    Members of the Asm1p, Phd1p, Sok2p, Efg1p, and StuAp (APSES) family of fungal proteins regulate morphogenesis and virulence in ascomycetes. We cloned the Aspergillus fumigatus APSES gene encoding StuAp and demonstrated that stuA transcription is markedly up-regulated after the acquisition of developmental competence. A. fumigatus ΔstuA mutants were impaired in their ability to undergo asexual reproduction. Conidiophore morphology was markedly abnormal, and only small numbers of dysmorphic conidia were produced, which exhibited precocious germination. Whole genome transcriptional analysis during the onset of developmental competence was performed and identified a subset of developmentally regulated genes that were stuA dependent, including a cluster of putative secondary metabolite biosynthesis genes, genes encoding proteins implicated in the regulation of morphogenesis, and genes encoding allergens and other antigenic proteins. Additionally, hyphae of the ΔstuA mutant displayed reduced expression of the catalase gene CAT1 and were hypersusceptible to hydrogen peroxide. PMID:16207816

  8. Deferoxamine-mediated up-regulation of HIF-1α prevents dopaminergic neuronal death via the activation of MAPK family proteins in MPTP-treated mice.

    PubMed

    Guo, Chuang; Hao, Li-Juan; Yang, Zhao-Hui; Chai, Rui; Zhang, Shuai; Gu, Yu; Gao, Hui-Ling; Zhong, Man-Li; Wang, Tao; Li, Jia-Yi; Wang, Zhan-You

    2016-06-01

    Accumulating evidence suggests that an abnormal accumulation of iron in the substantia nigra (SN) is one of the defining characteristics of Parkinson's disease (PD). Accordingly, the potential neuroprotection of Fe chelators is widely acknowledged for the treatment of PD. Although desferrioxamine (DFO), an iron chelator widely used in clinical settings, has been reported to improve motor deficits and dopaminergic neuronal survival in animal models of PD, DFO has poor penetration to cross the blood-brain barrier and elicits side effects. We evaluated whether an intranasal administration of DFO improves the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of dopaminergic neurons in the nigrostriatal axis and investigated the molecular mechanisms of intranasal DFO treatment in preventing MPTP-induced neurodegeneration. Treatment with DFO efficiently alleviated behavioral deficits, increased the survival of tyrosine hydroxylase (TH)-positive neurons, and decreased the action of astrocytes in the SN and striatum in an MPTP-induced PD mouse model. Interestingly, we found that DFO up-regulated the expression of HIF-1α protein, TH, vascular endothelial growth factor (VEGF), and growth associated protein 43 (GAP43) and down-regulated the expression of α-synuclein, divalent metal transporter with iron-responsive element (DMT1+IRE), and transferrin receptor (TFR). This was accompanied by a decrease in iron-positive cells in the SN and striatum of the DFO-treated group. We further revealed that DFO treatment significantly inhibited the MPTP-induced phosphorylation of the c-Jun N-terminal kinase (JNK) and differentially enhanced the phosphorylation of extracellular regulated protein kinases (ERK) and mitogen-activated protein kinase (MAPK)/P38 kinase. Additionally, the effects of DFO on increasing the Bcl-2/Bax ratio were further validated in vitro and in vivo. In SH-SY5Y cells, the DFO-mediated up-regulation of HIF-1α occurred via the activation of

  9. How precise are reported protein coordinate data?

    PubMed

    Konagurthu, Arun S; Allison, Lloyd; Abramson, David; Stuckey, Peter J; Lesk, Arthur M

    2014-03-01

    Atomic coordinates in the Worldwide Protein Data Bank (wwPDB) are generally reported to greater precision than the experimental structure determinations have actually achieved. By using information theory and data compression to study the compressibility of protein atomic coordinates, it is possible to quantify the amount of randomness in the coordinate data and thereby to determine the realistic precision of the reported coordinates. On average, the value of each C(α) coordinate in a set of selected protein structures solved at a variety of resolutions is good to about 0.1 Å. PMID:24598758

  10. Amphetamine Up-Regulates AGS1 mRNA and Protein Levels in Rat Frontal Cortex: The Role of Dopamine and Glucocorticoid Receptors

    PubMed Central

    Schwendt, Marek; McGinty, Jacqueline F.

    2010-01-01

    Acute and chronic exposure to psychostimulants results in altered function of G-protein-coupled receptors in the forebrain. It is believed that neuroadaptations in G-protein signaling contribute to behavioral sensitivity to psychostimulants that persists over a prolonged drug-free period. Proteins termed activators of G-protein signaling (AGS) have been characterized as potent modulators of both receptor-dependent and receptor-independent G-protein signaling. Nevertheless, the regulation of AGS gene and protein expression by psychostimulants remains poorly understood. In the present study, we investigated amphetamine (AMPH)-induced changes in expression patterns of several forebrain-enriched AGS proteins. A single exposure to AMPH (2.5 mg/kg i.p.) selectively induced gene expression of AGS1, but not Rhes or AGS3 proteins, in the rat prefrontal cortex (PFC) as measured 3h later. Induction of AGS1 mRNA in the PFC by acute AMPH was transient and dose-dependent. Even repeated treatment with AMPH for 5 days did not produce lasting changes in AGS1 mRNA and protein levels in the PFC as measured three weeks post treatment. However, at this time point, a low dose AMPH challenge (1 mg/kg, i.p.) induced a robust behavioral response and up-regulated AGS1 expression in the PFC selectively in animals with an AMPH history. The effects of AMPH on AGS1 expression in the PFC were blocked by a D2, but not D1, dopamine receptor antagonist and partially by a glucocorticoid receptor antagonist. Collectively, the present study suggests that (1) AGS1 represents a regulator of G-protein signaling that is rapidly inducible by AMPH in the frontal cortex, (2) AGS1 regulation in the PFC parallels behavioral activation by acute AMPH in drug-naïve animals and hypersensitivity to AMPH challenge in sensitized animals, and (3) D2 dopamine and glucocorticoid receptors regulate AMPH effects on AGS1 in the PFC. Changes in AGS1 levels in the PFC may result in abnormal receptor-to-G-protein coupling

  11. Three-Dimensional Collagen Type I Matrix Up-Regulates Nuclear Isoforms of the Microtubule Associated Protein Tau Implicated in Resistance to Paclitaxel Therapy in Ovarian Carcinoma

    PubMed Central

    Gurler, Hilal; Yu, Yi; Choi, Jacqueline; Kajdacsy-Balla, Andre A.; Barbolina, Maria V.

    2015-01-01

    Epithelial ovarian carcinoma is the deadliest gynecologic malignancy. One reason underlying treatment failure is resistance to paclitaxel. Expression of the microtubule associated protein tau has recently been proposed as a predictor of response to paclitaxel in ovarian carcinoma patients. Expression of tau was probed using immunohistochemistry in 312 specimens of primary, and 40 specimens of metastatic, ovarian carcinoma. Serous epithelial ovarian carcinoma cell line models were used to determine the expression of tau by Western blot and immunofluorescence staining. Subcellular fractionation and Western blot were employed to examine nuclear and cytoplasmic localization of tau. Gene silencing and clonogenic assays were used to evaluate paclitaxel response. Tau was expressed in 44% of all tested cases. Among the primary serous epithelial ovarian carcinoma cases, 46% were tau-positive. Among the metastatic serous epithelial ovarian carcinomas, 63% were tau-positive. Cell culture experiments demonstrated that tau was expressed in multiple isoforms. Three-dimensional collagen I matrix culture conditions resulted in up-regulation of tau protein. Silencing of tau with specific siRNAs in a combination with three-dimensional culture conditions led to a significant decrease of the clonogenic ability of cells treated with paclitaxel. The data suggest that reduction of tau expression may sensitize ovarian carcinoma to the paclitaxel treatment. PMID:25658796

  12. Survivin enhances telomerase activity via up-regulation of specificity protein 1- and c-Myc-mediated human telomerase reverse transcriptase gene transcription

    SciTech Connect

    Endoh, Teruo; Tsuji, Naoki; Asanuma, Koichi; Yagihashi, Atsuhito; Watanabe, Naoki . E-mail: watanabn@sapmed.ac.jp

    2005-05-01

    Suppression of apoptosis is thought to contribute to carcinogenesis. Survivin, a member of the inhibitor-of-apoptosis family, blocks apoptotic signaling activated by various cellular stresses. Since elevated expression of survivin observed in human cancers of varied origin was associated with poor patient survival, survivin has attracted growing attention as a potential target for cancer treatment. Immortalization of cells also is required for carcinogenesis; telomere length maintenance by telomerase is required for cancer cells to proliferate indefinitely. Yet how cancer cells activate telomerase remains unclear. We therefore examined possible interrelationships between survivin expression and telomerase activity. Correlation between survivin and human telomerase reverse transcriptase (hTERT) expression was observed in colon cancer tissues, and overexpression of survivin enhanced telomerase activity by up-regulation of hTERT expression in LS180 human colon cancer cells. DNA-binding activities of specificity protein 1 (Sp1) and c-Myc to the hTERT core promoter were increased in survivin gene transfectant cells. Phosphorylation of Sp1 and c-Myc at serine and threonine residues was enhanced by survivin, while total amounts of these proteins were unchanged. Further, 'knockdown' of survivin by a small inhibitory RNA decreased Sp1 and c-Myc phosphorylation. Thus survivin participates not only in inhibition of apoptosis, but also in prolonging cellular lifespan.

  13. Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

    SciTech Connect

    Liu, Fabao; You, Xiaona; Chi, Xiumei; Wang, Tao; Ye, Lihong; Niu, Junqi; Zhang, Xiaodong

    2014-02-07

    Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

  14. Immunologically induced, complement-dependent up-regulation of the prion protein in the mouse spleen: follicular dendritic cells versus capsule and trabeculae.

    PubMed

    Lötscher, Marius; Recher, Mike; Hunziker, Lukas; Klein, Michael A

    2003-06-15

    The expression of the prion protein (PrP) in the follicular dendritic cell network of germinal centers in the spleen is critical for the splenic propagation of the causative agent of prion diseases. However, a physiological role of the prion protein in the periphery remains elusive. To investigate the role and function of PrP expression in the lymphoid system we treated naive mice i.v. with preformed immune complexes or vesicular stomatitis virus. Immunohistochemistry and Western blot analysis of the spleen revealed that 8 days after immunization, immune complexes and vesicular stomatitis virus had both induced a strong increase of PrP expression in the follicular dendritic cell network. Remarkably, this up-regulation did not occur in mice that lack an early factor of the complement cascade, C1q, a component which has been shown previously to facilitate early prion pathogenesis. In addition to the variable PrP level in the germinal centers, we detected steady and abundant PrP expression in the splenic capsule and trabeculae, which are structural elements that have not been associated before with PrP localization. The abundant trabeculo-capsular PrP expression was also evident in spleens of Rag-1-deficient mice, which have been shown before to be incapable of prion expansion. We conclude that trabeculocapsular PrP is not sufficient for splenic prion propagation. Furthermore, our observations may provide important clues for a physiological function of the prion protein and allow a new view on the role of complement and PrP in peripheral prion pathogenesis. PMID:12794132

  15. The neuroprotective action of the mood stabilizing drugs lithium chloride and sodium valproate is mediated through the up-regulation of the homeodomain protein Six1

    SciTech Connect

    Plant, Kathryn E.; Anderson, Elizabeth; Simecek, Nicole; Brown, Richard; Forster, Sam; Spinks, Jenny; Toms, Nick; Gibson, G. Gordon; Lyon, Jon; Plant, Nick

    2009-02-15

    The mood stabilizing agents lithium chloride (LiCl) and sodium valproate (VPA) have recently gained interest as potential neuroprotective therapeutics. However, exploitation of these therapeutic applications is hindered by both a lack of molecular understanding of the mode of action, and a number of sub-optimal properties, including a relatively small therapeutic window and variable patient response. Human neuroblastoma cells (SH-SY5Y) were exposed to 1 mM lithium chloride or 1 mM sodium valproate for 6 h or 72 h, and transcriptomes measured by Affymetrix U133A/B microarray. Statistically significant gene expression changes were identified using SAM software, with selected changes confirmed at transcript (TaqMan) and protein (Western blotting) levels. Finally, anti-apoptotic action was measured by an in vitro fluorescent assay. Exposure of SH-SY5Y cells to therapeutically relevant concentrations of either lithium chloride or sodium valproate elicited 936 statistically significant changes in gene expression. Amongst these changes we observed a large (maximal 31.3-fold) increase in the expression of the homeodomain protein Six1, and have characterized the time- and dose-dependent up-regulation of this gene in response to both drugs. In addition, we demonstrate that, like LiCl or VPA treatment, Six1 over-expression protects SH-SY5Y cells from staurosporine-induced apoptosis via the blockade of caspsase-3 activation, whereas removal of Six1 protein via siRNA antagonises the ability of LiCl and VPA to protect SH-SY5Y cells from STS-induced apoptosis. These results provide a novel mechanistic rationale underlying the neuroprotective mechanism of LiCl and VPA, suggesting exciting possibilities for the development of novel therapeutic agents against neurodegenerative diseases such as Alzheimer's or Parkinsonism.

  16. Hyperbaric Oxygen Preconditioning Induces Tolerance against Oxidative Injury and Oxygen-Glucose Deprivation by Up-Regulating Heat Shock Protein 32 in Rat Spinal Neurons

    PubMed Central

    Xu, Li; Wang, Shifeng; Li, Runping; Liu, Kan; Zheng, Juan; Cai, Zhiyu; Zhang, Kun; Luo, Yuandeng; Xu, Weigang

    2014-01-01

    Objective Hyperbaric oxygen (HBO) preconditioning (HBO-PC) has been testified to have protective effects on spinal cord injury (SCI). However, the mechanisms remain enigmatic. The present study aimed to explore the effects of HBO-PC on primary rat spinal neurons against oxidative injury and oxygen-glucose deprivation (OGD) and the relationship with heat shock proteins (HSPs). Methods Primary rat spinal neurons after 7 days of culture were used in this study. HSPs were detected in rat spinal neurons following a single exposure to HBO at different time points by Western blot. Using lactate dehydrogenase release assay and cell counting kit-8 assay, the injuries induced by hydrogen peroxide (H2O2) insult or OGD were determined and compared among neurons treated with HBO-PC with or without HSP inhibitors. Results The results of Western blot showed that HSP27, HSP70 and HSP90 have a slight but not significant increase in primary neurons following HBO exposure. However, HSP32 expression significantly increased and reached highest at 12 h following HBO exposure. HBO-PC significantly increased the cell viability and decreased the medium lactate dehydrogenase content in cultures treated with H2O2 or OGD. Pretreatment with zinc protoporphyrin IX, a specific inhibitor of HSP32, significantly blocked the protective effects of HBO-PC. Conclusions These results suggest that HBO-PC could protect rat spinal neurons in vitro against oxidative injury and OGD mostly by up-regulating of HSP32 expression. PMID:24465817

  17. Fluoxetine up-regulates expression of cellular FLICE-inhibitory protein and inhibits LPS-induced apoptosis in hippocampus-derived neural stem cell

    SciTech Connect

    Chiou, S.-H. . E-mail: shchiou@vghtpe.gov.tw; Chen, S.-J. . E-mail: sjchen@vghtpe.gov.tw; Peng, C-H.; Chang, Y.-L.; Ku, H.-H.; Hsu, W.-M.; Ho, Larry L.-T.; Lee, C.-H.

    2006-05-05

    Fluoxetine is a widely used antidepressant compound which inhibits the reuptake of serotonin in the central nervous system. Recent studies have shown that fluoxetine can promote neurogenesis and improve the survival rate of neurons. However, whether fluoxetine modulates the proliferation or neuroprotection effects of neural stem cells (NSCs) needs to be elucidated. In this study, we demonstrated that 20 {mu}M fluoxetine can increase the cell proliferation of NSCs derived from the hippocampus of adult rats by MTT test. The up-regulated expression of Bcl-2, Bcl-xL and the cellular FLICE-inhibitory protein (c-FLIP) in fluoxetine-treated NSCs was detected by real-time RT-PCR. Our results further showed that fluoxetine protects the lipopolysaccharide-induced apoptosis in NSCs, in part, by activating the expression of c-FLIP. Moreover, c-FLIP induction by fluoxetine requires the activation of the c-FLIP promoter region spanning nucleotides -414 to -133, including CREB and SP1 sites. This effect appeared to involve the phosphatidylinositol-3-kinase-dependent pathway. Furthermore, fluoxetine treatment significantly inhibited the induction of proinflammatory factor IL-1{beta}, IL-6, and TNF-{alpha} in the culture medium of LPS-treated NSCs (p < 0.01). The results of high performance liquid chromatography coupled to electrochemical detection further confirmed that fluoxentine increased the functional production of serotonin in NSCs. Together, these data demonstrate the specific activation of c-FLIP by fluoxetine and indicate the novel role of fluoxetine for neuroprotection in the treatment of depression.

  18. The Mutant KRAS Gene Up-regulates BCL-XL Protein via STAT3 to Confer Apoptosis Resistance That Is Reversed by BIM Protein Induction and BCL-XL Antagonism.

    PubMed

    Zaanan, Aziz; Okamoto, Koichi; Kawakami, Hisato; Khazaie, Khashayarsha; Huang, Shengbing; Sinicrope, Frank A

    2015-09-25

    In colorectal cancers with oncogenic GTPase Kras (KRAS) mutations, inhibition of downstream MEK/ERK signaling has shown limited efficacy, in part because of failure to induce a robust apoptotic response. We studied the mechanism of apoptosis resistance in mutant KRAS cells and sought to enhance the efficacy of a KRAS-specific MEK/ERK inhibitor, GDC-0623. GDC-0623 was shown to potently up-regulate BIM expression to a greater extent versus other MEK inhibitors in isogenic KRAS HCT116 and mutant KRAS SW620 colon cancer cells. ERK silencing enhanced BIM up-regulation by GDC-0623 that was due to its loss of phosphorylation at Ser(69), confirmed by a BIM-EL phosphorylation-defective mutant (S69G) that increased protein stability and blocked BIM induction. Despite BIM and BIK induction, the isogenic KRAS mutant versus wild-type cells remained resistant to GDC-0623-induced apoptosis, in part because of up-regulation of BCL-XL. KRAS knockdown by a doxycycline-inducible shRNA attenuated BCL-XL expression. BCL-XL knockdown sensitized KRAS mutant cells to GDC-0623-mediated apoptosis, as did the BH3 mimetic ABT-263. GDC-0623 plus ABT-263 induced a synergistic apoptosis by a mechanism that includes release of BIM from its sequestration by BCL-XL. Furthermore, mutant KRAS activated p-STAT3 (Tyr(705)) in the absence of IL-6 secretion, and STAT3 knockdown reduced BCL-XL mRNA and protein expression. These data suggest that BCL-XL up-regulation by STAT3 contributes to mutant KRAS-mediated apoptosis resistance. Such resistance can be overcome by potent BIM induction and concurrent BCL-XL antagonism to enable a synergistic apoptotic response. PMID:26245900

  19. Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires De Novo Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

    PubMed Central

    Rivera-Pagán, Aixa F.; Rivera-Aponte, David E.; Melnik-Martínez, Katya V.; Zayas-Santiago, Astrid; Kucheryavykh, Lilia Y.; Martins, Antonio H.; Cubano, Luis A.; Skatchkov, Serguei N.; Eaton, Misty J.

    2015-01-01

    Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role. PMID:25886567

  20. Human cutaneous fatty acid-binding protein induces metastasis by up-regulating the expression of vascular endothelial growth factor gene in rat Rama 37 model cells.

    PubMed

    Jing, C; Beesley, C; Foster, C S; Chen, H; Rudland, P S; West, D C; Fujii, H; Smith, P H; Ke, Y

    2001-06-01

    PSV-VEGF-2 cells when compared with that of the primary tumors developed from the control pSV-R37 cells. Therefore, we suggest that overexpression of the C-FABP gene in the original transfectants induces metastasis through up-regulation of expression of the VEGF gene in this rat Rama 37 model system, and thus VEGF may play a crucial role in this particular metastatic cascade. PMID:11389060

  1. Protein kinase signalling pathways involved in the up-regulation of the rat alpha1(I) collagen gene by transforming growth factor beta1 and bone morphogenetic protein 2 in osteoblastic cells.

    PubMed Central

    Palcy, S; Goltzman, D

    1999-01-01

    Transforming growth factor beta (TGFbeta) family members are known for their important role in bone physiology. TGFbeta(1) and, to a smaller extent, bone morphogenetic protein 2 (BMP-2) have been reported to regulate the gene expression of different osteoblast markers in vitro. However, little is known about the molecular mechanisms involved in these actions. Here we report that BMP-2, like TGFbeta(1), up-regulated alpha1(I) collagen mRNA expression in ROS 17/2.8 osteoblastic cells. This was mediated through an increase in the transcriptional rate of the gene rather than through the stabilization of alpha1(I) collagen mRNA, and required new protein synthesis. In addition, TGFbeta(1)- and BMP-2-induced increases in alpha1(I) collagen mRNA levels were both dependent on protein kinase C and protein tyrosine kinase activities. Furthermore, the mitogen-activated protein kinase (MAPK) [MAPK/extracellular signal-regulated protein kinase kinase 1/extracellular signal-regulated protein kinase (MEK-1/ERK)] pathway participated in the up-regulation of alpha1(I) collagen gene expression by TGFbeta(1) and BMP-2. In response to either TGFbeta(1) or BMP-2, the stimulation of alpha1(I) collagen mRNA levels was paralleled by an early increase in extracellular signal-regulated kinase protein activity. Moreover, the effects of both TGFbeta(1) and BMP-2 on alpha1(I) collagen gene expression were markedly decreased in transfected ROS 17/2.8 cells expressing a dominant-negative MEK-1. Our findings therefore show that TGFbeta(1) and BMP-2, which signal through discrete cell-surface receptors, are able to trigger analogous, if not identical, protein-phosphorylation-transducing cascades leading to comparable actions on the transcription of the alpha1(I) collagen gene in osteoblastic cells. PMID:10493907

  2. Up-regulation of lipolysis genes and increased production of AMP-activated protein kinase protein in the skeletal muscle of rats after resistance training

    PubMed Central

    An, Jae-Heung; Yoon, Jin-Hwan; Suk, Min-Hwa; Shin, Yun-A

    2016-01-01

    The purpose of this study was to investigate the expression of lipogenesis- and lipolysis-related genes and proteins in skeletal muscles after 12 weeks of resistance training. Sprague-Dawley rats (n=12) were randomly divided into control (resting) and resistance training groups. A tower-climbing exercise, in which rats climbed to the top of their cage with a weight applied to their tails, used for resistance training. After 12 weeks, rats from the resistance training group had lower body weights (411.66±14.71 g vs. 478.33±24.63 g in the control), there was no significant difference between the two groups in the concentrations of total cholesterol, and high or low density lipoprotein cholesterol. However, the concentration of triglyceride was lower in resistance-trained rats (59.83±14.05 μg/mL vs 93.33±33.89 μg/mL in the control). The mRNA expression levels of the lipogenesis-related genes sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase were not significantly different between the resistance-trained and control rats; however, mRNA expression of the lipolysis-related carnitine palmitoyl transferase 1 and malonyl-CoA decarboxylase increased significantly with resistance training. AMP-activated protein kinase protein levels also significantly increased in resistance training group compared with in the control group. These results suggested that resistance exercise training contributing to reduced weight gain may be in part be due to increase the lipolysis metabolism and energy expenditure in response to resistance training. PMID:27419110

  3. Up-regulation of lipolysis genes and increased production of AMP-activated protein kinase protein in the skeletal muscle of rats after resistance training.

    PubMed

    An, Jae-Heung; Yoon, Jin-Hwan; Suk, Min-Hwa; Shin, Yun-A

    2016-06-01

    The purpose of this study was to investigate the expression of lipogenesis- and lipolysis-related genes and proteins in skeletal muscles after 12 weeks of resistance training. Sprague-Dawley rats (n=12) were randomly divided into control (resting) and resistance training groups. A tower-climbing exercise, in which rats climbed to the top of their cage with a weight applied to their tails, used for resistance training. After 12 weeks, rats from the resistance training group had lower body weights (411.66±14.71 g vs. 478.33±24.63 g in the control), there was no significant difference between the two groups in the concentrations of total cholesterol, and high or low density lipoprotein cholesterol. However, the concentration of triglyceride was lower in resistance-trained rats (59.83±14.05 μg/mL vs 93.33±33.89 μg/mL in the control). The mRNA expression levels of the lipogenesis-related genes sterol regulatory element binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase were not significantly different between the resistance-trained and control rats; however, mRNA expression of the lipolysis-related carnitine palmitoyl transferase 1 and malonyl-CoA decarboxylase increased significantly with resistance training. AMP-activated protein kinase protein levels also significantly increased in resistance training group compared with in the control group. These results suggested that resistance exercise training contributing to reduced weight gain may be in part be due to increase the lipolysis metabolism and energy expenditure in response to resistance training. PMID:27419110

  4. Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism*

    PubMed Central

    Kakoi, Hironori; Maeda, Shingo; Shinohara, Naohiro; Matsuyama, Kanehiro; Imamura, Katsuyuki; Kawamura, Ichiro; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-01-01

    Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism. PMID:24505141

  5. Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

    PubMed Central

    Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

    2013-01-01

    Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

  6. Herpes simplex virus induces the marked up-regulation of the zinc finger transcriptional factor INSM1, which modulates the expression and localization of the immediate early protein ICP0

    PubMed Central

    2011-01-01

    Background Herpes simplex viruses (HSVs) rapidly shut off macromolecular synthesis in host cells. In contrast, global microarray analyses have shown that HSV infection markedly up-regulates a number of host cell genes that may play important roles in HSV-host cell interactions. To understand the regulatory mechanisms involved, we initiated studies focusing on the zinc finger transcription factor insulinoma-associated 1 (INSM1), a host cell protein markedly up-regulated by HSV infection. Results INSM1 gene expression in HSV-1-infected normal human epidermal keratinocytes increased at least 400-fold 9 h after infection; INSM1 promoter activity was also markedly stimulated. Expression and subcellular localization of the immediate early HSV protein ICP0 was affected by INSM1 expression, and chromatin immunoprecipitation (ChIP) assays revealed binding of INSM1 to the ICP0 promoter. Moreover, the role of INSM1 in HSV-1 infection was further clarified by inhibition of HSV-1 replication by INSM1-specific siRNA. Conclusions The results suggest that INSM1 up-regulation plays a positive role in HSV-1 replication, probably by binding to the ICP0 promoter. PMID:21609490

  7. ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response

    PubMed Central

    Oskolkova, Olga V.; Afonyushkin, Taras; Leitner, Alexander; von Schlieffen, Elena; Gargalovic, Peter S.; Lusis, Aldons J.; Binder, Bernd R.

    2008-01-01

    We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques. PMID:18451308

  8. Inhibitory heterotrimeric GTP-binding proteins inhibit hydrogen peroxide-induced apoptosis by up-regulation of Bcl-2 via NF-{kappa}B in H1299 human lung cancer cells

    SciTech Connect

    Seo, Mi Ran; Nam, Hyo-Jung; Kim, So-Young; Juhnn, Yong-Sung

    2009-04-03

    Inhibitory heterotrimeric GTP-binding proteins (Gi proteins) mediate a variety of signaling pathways by coupling receptors and effectors to regulate cellular proliferation, differentiation, and apoptosis. However, the role of Gi proteins in the modulation of hydrogen peroxide-induced apoptosis is not clearly understood. Thus, we investigated the effect of Gi proteins on hydrogen peroxide-induced apoptosis and the underlying mechanisms in H1299 human lung cancer cells. The stable expression of constitutively active alpha subunits of Gi1 (G{alpha}i1QL), Gi2, or Gi3 inhibited hydrogen peroxide-induced apoptosis. The expression of G{alpha}i1QL up-regulated Bcl-2 expression, and the knockdown of Bcl-2 with siRNA abolished the anti-apoptotic effect of G{alpha}i1QL. G{alpha}i1 induced the transcription of Bcl-2 by activation of NF-{kappa}B, which resulted from an increase in NF-{kappa}B p50 protein. We conclude that G{alpha}i1 inhibits hydrogen peroxide-induced apoptosis of H1299 lung cancer cells by up-regulating the transcription of Bcl-2 through a p50-mediated NF-{kappa}B activation.

  9. Role of protein kinase D2 phosphorylation on Tyr in modulation by ghrelin of Helicobacter pylori-induced up-regulation in gastric mucosal matrix metalloproteinase-9 (MMP-9) secretion.

    PubMed

    Slomiany, B L; Slomiany, A

    2016-06-01

    Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase associated with host reaction to microbial endotoxins and also characterizes gastric mucosal inflammatory response to H. pylori infection. Here, we report on the factors involved in gastric mucosal MMP-9 secretion in response to H. pylori LPS, and the effect of hormone, ghrelin. We show that both the LPS-elicited induction in MMP-9 secretion and also the modulatory influence of ghrelin occur at the level of MMP-9 processing between the endoplasmic reticulum (ER) and Golgi. Further, we demonstrate that the LPS effect is associated with up-regulation in the activation of Arf1, a small GTPase of the ADP-ribosylation factor family, and the recruitment and phosphorylation of protein kinase D2 (PKD2), involved in the secretory cargo processing in the Golgi. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. Thus, our findings demonstrate the role of Arf1/PKD2 in mediation of H. pylori LPS-induced up-regulation in gastric mucosal MMP-9 secretion and suggest the modulatory mechanism of ghrelin action. PMID:27209313

  10. Aβ(1-42) oligomer-induced leakage in an in vitro blood-brain barrier model is associated with up-regulation of RAGE and metalloproteinases, and down-regulation of tight junction scaffold proteins.

    PubMed

    Wan, Wenbin; Cao, Lan; Liu, Lumei; Zhang, Chunyan; Kalionis, Bill; Tai, Xiantao; Li, Yaming; Xia, Shijin

    2015-07-01

    Accumulating evidence indicates that abnormal deposition of amyloid-β (Aβ) peptide in the brain is responsible for endothelial cell damage and consequently leads to blood-brain barrier (BBB) leakage. However, the mechanisms underlying BBB disruption are not well described. We employed an monolayer BBB model comprising bEnd.3 cell and found that BBB leakage was induced by treatment with Aβ(1-42), and the levels of tight junction (TJ) scaffold proteins (ZO-1, Claudin-5, and Occludin) were decreased. Through comparisons of the effects of the different components of Aβ(1-42), including monomer (Aβ(1-42)-Mono), oligomer (Aβ(1-42)-Oligo), and fibril (Aβ(1-42)-Fibril), our data confirmed that Aβ(1-42)-Oligo is likely to be the most important damage factor that results in TJ damage and BBB leakage in Alzheimer's disease. We found that the incubation of bEnd.3 cells with Aβ(1-42) significantly up-regulated the level of receptor for advanced glycation end-products (RAGE). Co-incubation of a polyclonal antibody to RAGE and Aβ(1-42)-Oligo in bEnd.3 cells blocked RAGE suppression of Aβ(1-42)-Oligo-induced alterations in TJ scaffold proteins and reversed Aβ(1-42)-Oligo-induced up-regulation of RAGE, matrix metalloproteinase (MMP)-2, and MMP-9. Furthermore, we found that these effects induced by Aβ(1-42)-Oligo treatment were effectively suppressed by knockdown of RAGE using small interfering RNA (siRNA) transfection. We also found that GM 6001, a broad-spectrum MMP inhibitor, partially reversed the Aβ(1-42)-Oligo-induced inhibitor effects in bEnd.3 cells. Thus, these results suggested that RAGE played an important role in Aβ-induced BBB leakage and alterations of TJ scaffold proteins, through a mechanism that involved up-regulation of MMP-2 and MMP-9. PMID:25866188

  11. Differential up-regulation of Vesl-1/Homer 1 protein isoforms associated with decline in visual performance in a preclinical glaucoma model

    PubMed Central

    Kaja, Simon; Naumchuk, Yuliya; Grillo, Stephanie L.; Borden, Priscilla K.; Koulen, Peter

    2014-01-01

    Glaucoma is a multifactorial progressive ocular pathology, clinically presenting with damage to the retina and optic nerve, ultimately leading to blindness. Retinal ganglion cell loss in glaucoma ultimately results in vision loss. Vesl/Homer proteins are scaffolding proteins that are critical for maintaining synaptic integrity by clustering, organizing and functionally regulating synaptic proteins. Current anti-glaucoma therapies target IOP as the sole modifiable clinical parameters. Long-term pharmacotherapy and surgical treatment do not prevent gradual visual field loss as the disease progresses, highlighting the need for new complementary, alternative and comprehensive treatment approaches. Vesl/Homer expression was measured in the retinae of DBA/2J mice, a preclinical genetic glaucoma model with spontaneous mutations resulting in a phenotype reminiscent of chronic human pigmentary glaucoma. Vesl/Homer proteins were differentially expressed in the aged, glaucomatous DBA/2J retina, both at the transcriptional and translational level. Immunoreactivity for the long Vesl-1L/Homer 1c isoform, but not of the immediate early gene product Vesl-1S/Homer 1a was increased in the synaptic layers of the retina. This increased protein level of Vesl-1L/Homer 1c was correlated with phenotypes of increased disease severity and a decrease in visual performance. The increased expression of Vesl-1L/Homer 1c in the glaucomatous retina likely results in increased intracellular Ca2+ release through enhancement of synaptic coupling. The ensuing Ca2+ toxicity may thus activate neurodegenerative pathways and lead to the progressive loss of synaptic function in glaucoma. Our data suggest that higher levels of Vesl-1L/Homer 1c generate a more severe disease phenotype and may represent a viable target for therapy development. PMID:24219919

  12. High Doses of Ursodeoxycholic Acid Up-Regulate the Expression of Placental Breast Cancer Resistance Protein in Patients Affected by Intrahepatic Cholestasis of Pregnancy

    PubMed Central

    Azzaroli, Francesco; Raspanti, Maria Elena; Simoni, Patrizia; Montagnani, Marco; Lisotti, Andrea; Cecinato, Paolo; Arena, Rosario; Simonazzi, Giuliana; Farina, Antonio; Rizzo, Nicola; Mazzella, Giuseppe

    2013-01-01

    Background Ursodeoxycholic acid (UDCA) administration in intrahepatic cholestasis of pregnancy (ICP) induces bile acids (BA) efflux from the foetal compartment, but the molecular basis of this transplacental transport is only partially defined. Aim To determine if placental breast cancer resistance protein (BCRP), able to transport BA, is regulated by UDCA in ICP. Methods 32 pregnant women with ICP (14 untreated, 34.9±5.17 years; 18 treated with UDCA - 25 mg/Kg/day, 32.7±4.62 years,) and 12 healthy controls (33.4±3.32 years) agreed to participate in the study. Placentas were obtained at delivery and processed for membrane extraction. BCRP protein expression was evaluated by immunoblotting techniques and chemiluminescence quantified with a luminograph measuring emitted photons; mRNA expression with real time PCR. Statistical differences between groups were evaluated by ANOVA with Dunn’s Multiple Comparison test. Results BCRP was expressed only on the apical membrane of the syncytiotrophoblast. A significant difference was observed among the three groups both for mRNA (ANOVA, p = 0.0074) and protein (ANOVA, p<0.0001) expression. BCRP expression was similar in controls and in the untreated ICP group. UDCA induced a significant increase in placental BCRP mRNA and protein expression compared to controls (350.7±106.3 vs 100±18.68% of controls, p<0.05 and 397.8±56.02 vs 100±11.44% of controls, p<0.001, respectively) and untreated ICP (90.29±17.59% of controls, p<0.05 and 155.0±13.87%, p<0.01). Conclusion Our results confirm that BCRP is expressed only on the apical membrane of the syncytiotrophoblast and show that ICP treatment with high dose UDCA significantly upregulates placental BCRP expression favouring BA efflux from the foetal compartment. PMID:23717540

  13. Mucosal acidification increases hydrogen sulfide release through up-regulating gene and protein expressions of cystathionine gamma-lyase in the rat gastric mucosa

    PubMed Central

    Mard, Seyyed Ali; Veisi, Ali; Ahangarpour, Akram; Gharib-Naseri, Mohammad Kazem

    2016-01-01

    Objective(s): This study was performed to investigate the effects of mucosal acidification on mRNA expression and protein synthesis of cystathionine gamma lyase (CSE), cystathionine beta synthase (CBS), and mucosal release of H2S in gastric mucosa in rats. Materials and Methods: Thirty-two rats were randomly assigned into 4 groups (8 in each), including: the control group, HCl (10 mM) treated group, HCl (100 mM) treated group, and one group to study the effect of Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME). Anesthetized rats underwent tracheostomy and midline laparotomy. Ninety min after the instillation of neutral or acidic solutions, animals were sacrificed and the gastric mucosa was collected to measure the H2S concentration by ELISA method and to quantify mRNA expression of CSE and CBS by quantitative real-time PCR (qRT-PCR). Protein synthesis was also detected by Western blot method. Results: Mucosal acidification with 10 and 100 HCl, significantly increased mucosal levels of H2S (P<0.01 and P<0.001) and mRNA (P<0.01 and P<0.001) and protein expressions of CSE (P<0.01 and P<0.001). L-NAME treatment reversed H2S release to control level. Conclusion: Our findings indicated that mucosal acidification with HCl increased mucosal release of H2S through upregulation of CSE gene and its protein expression. This effect is mainly mediated through the involvement of nitric oxide. PMID:27081462

  14. 6-Hydroxydopamine lesions of rat substantia nigra up-regulate dopamine-induced phosphorylation of the cAMP-response element-binding protein in striatal neurons.

    PubMed Central

    Cole, D G; Kobierski, L A; Konradi, C; Hyman, S E

    1994-01-01

    Destruction of the substantia nigra produces striatal D1 dopamine receptor supersensitivity without increasing receptor number or affinity, thus implicating postreceptor mechanisms. The nature of these mechanisms is unknown. Increased striatal c-fos expression ipsilateral to a unilateral lesion of the substantia nigra in rats treated with appropriate dopamine agonists provides a cellular marker of D1 receptor supersensitivity. D1 receptors are positively linked to adenylate cyclase and therefore to cAMP-dependent protein kinase. Because expression of the c-fos gene in response to cAMP- and Ca2+/calmodulin-regulated protein kinases depends on phosphorylation of cAMP-response element-binding protein (CREB) at Ser-133, we examined CREB phosphorylation after dopaminergic stimulation in cultured striatal neurons and in the striatum of rats after unilateral 6-hydroxydopamine ablation of the substantia nigra. Using an antiserum specific for CREB phosphorylated at Ser-133, we found that dopamine increases CREB phosphorylation in cultured striatal neurons. This effect was blocked by a D1 antagonist. L-Dopa produced marked CREB phosphorylation in striatal neurons in rats ipsilateral, but not contralateral, to a 6-hydroxydopamine lesion. This response was blocked by a D1 antagonist, but not a D2 antagonist, and was reproduced by a D1 agonist, but not a D2 agonist. These findings are consistent with the hypothesis that D1 receptor supersensitivity is associated with upregulated activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinases, or both, following dopamine denervation of striatal neurons. Images PMID:7937819

  15. Up-regulation of telomere-binding proteins, TRF1, TRF2, and TIN2 is related to telomere shortening during human multistep hepatocarcinogenesis.

    PubMed

    Oh, Bong-Kyeong; Kim, Young-Joo; Park, Chanil; Park, Young Nyun

    2005-01-01

    The telomeric repeat-binding factor 1 (TRF1), TRF2, and the TRF1-interacting nuclear protein 2 (TIN2) are involved in telomere maintenance. We describe the regulation of expression of these genes along with their relationship to telomere length in hepatocarcinogenesis. The transcriptional expression of these genes, TRF1 protein, and telomere length was examined in 9 normal livers, 14 chronic hepatitis, 24 liver cirrhosis, 5 large regenerative nodules, 14 low-grade dysplastic nodules (DNs), 7 high-grade DNs, 10 DNs with hepatocellular carcinoma (HCC) foci, and 31 HCCs. The expression of TRF1, TRF2, TIN2 mRNA, and TRF1 protein was gradually increased according to the progression of hepatocarcinogenesis with a marked increase in high-grade DNs and DNs with HCC foci and a further increase in HCCs. There was a gradual shortening of telomere during hepatocarcinogenesis with a significant reduction in length in DNs. Most nodular lesions (52 of 67) had shorter telomeres than their adjacent chronic hepatitis or liver cirrhosis, and the telomere lengths were inversely correlated with the mRNA level of these genes (P

  16. Osteopontin, a chemotactic protein with cytokine-like properties, is up-regulated in muscle injury caused by Bothrops lanceolatus (fer-de-lance) snake venom.

    PubMed

    Barbosa-Souza, Valéria; Contin, Daniel Kiss; Filho, Waldemar Bonventi; de Araújo, Albetiza Lôbo; Irazusta, Silvia Pierre; da Cruz-Höfling, Maria Alice

    2011-10-01

    Osteopontin (OPN) is a chemotactic, adhesive protein whose receptors include some integrins and matrix proteins known to have role in inflammatory and repair processes. We examined the time course of OPN expression at acute and chronic stages after intramuscular injection of Bothrops lanceolatus venom in rats. Additionally, we examined the expression of CD68 (a marker for phagocytic macrophages) and the myogenic factors, myoD and myogenin. There was a biphasic upregulation of OPN (6-48 h and 3-14 days post-venom), i.e., during acute inflammation and myogenic cell proliferation and differentiation phases. OPN was detected in CD68 + macrophages, fibroblasts, normal and damaged myofibers, myoblasts and myotubes. Myogenin was expressed in the cytoplasm (atypical pattern) and nucleus of myoblasts and myotubes from 18 h to 7 days, after which it was expressed only in nuclei. Macrophage numbers, OPN and myogenin expression were still elevated at 7, 14 and 7 days. At 3 days, when OPN achieved the peak, some clusters of myoblasts were within regions of intense collagen deposition. Fibrosis may represent limitation for repairing processes and may explain the small diameter of regenerated fibers at 21 days post-venom. The expression of OPN in the course of venom-induced damage and regeneration suggests stages-specific mediation role along the whole process. PMID:21839764

  17. Nanoparticles up-regulate tumor necrosis factor-{alpha} and CXCL8 via reactive oxygen species and mitogen-activated protein kinase activation

    SciTech Connect

    Lee, Hye-Mi; Shin, D.-M.; Song, Hwan-Moon; Yuk, Jae-Min; Lee, Zee-Won; Lee, Sang-Hee; Hwang, Song Mei; Kim, Jin-Man; Lee, Chang-Soo Jo, Eun-Kyeong

    2009-07-15

    Evaluating the toxicity of nanoparticles is an integral aspect of basic and applied sciences, because imaging applications using traditional organic fluorophores are limited by properties such as photobleaching, spectral overlaps, and operational difficulties. This study investigated the toxicity of nanoparticles and their biological mechanisms. We found that nanoparticles, quantum dots (QDs), considerably activated the production of tumor necrosis factor (TNF)-{alpha} and CXC-chemokine ligand (CXCL) 8 through reactive oxygen species (ROS)- and mitogen-activated protein kinases (MAPKs)-dependent mechanisms in human primary monocytes. Nanoparticles elicited a robust activation of intracellular ROS, phosphorylation of p47phox, and nicotinamide adenine dinucleotide phosphate oxidase activities. Blockade of ROS generation with antioxidants significantly abrogated the QD-mediated TNF-{alpha} and CXCL8 expression in monocytes. The induced ROS generation subsequently led to the activation of MAPKs, which were crucial for mRNA and protein expression of TNF-{alpha} and CXCL8. Furthermore, confocal and electron microscopy analyses showed that internalized QDs were trapped in cytoplasmic vesicles and compartmentalized inside lysosomes. Finally, several repeated intravenous injections of QDs caused an increase in neutrophil infiltration in the lung tissues in vivo. These results provide novel insights into the QD-mediated chemokine induction and inflammatory toxic responses in vitro and in vivo.

  18. The Wheat Abscisic Acid-Responsive Protein Kinase mRNA, PKABA1, Is Up-Regulated by Dehydration, Cold Temperature, and Osmotic Stress.

    PubMed Central

    Holappa, L. D.; Walker-Simmons, M. K.

    1995-01-01

    The effects of dehydration, cold-temperature treatment, and osmotic and salt stress on the expression of an abscisic acid-responsive protein kinase mRNA (PKABA1) were determined in wheat (Triticum aestivum L.) seedlings. The PKABA1 transcript was detectable at basal levels in tissues of nonstressed plants and accumulated to higher levels in shoot, scutellar, and root tissues of stressed plants. PKABA1 transcript accumulated rapidly within 2 h following dehydration and within 24 h following other treatments (cold, osmotic stress, and high salt). The accumulation of PKABA1 mRNA could not be separated temporally from that of a wheat group 3 late embryogenesis abundant mRNA during dehydration and cold treatment. High PKABA1 mRNA levels were observed in field-grown plants growing under cold winter conditions but not under warmer summer conditions. A recent GenBank data base search indicated that other plant protein kinases with similar acidic amino acid stretches as in PKABA1 have been identified, and some of these kinases are responsive to environmental signals. These results suggest that PKABA1 may be part of general environmental stress responses in wheat. PMID:12228537

  19. Cysteine-Rich Secretory Protein-3 (CRISP3) Is Strongly Up-Regulated in Prostate Carcinomas with the TMPRSS2-ERG Fusion Gene

    PubMed Central

    Costa, Vera L.; Barros-Silva, João D.; Ramalho-Carvalho, João; Jerónimo, Carmen; Henrique, Rui; Lind, Guro E.; Skotheim, Rolf I.; Lothe, Ragnhild A.; Teixeira, Manuel R.

    2011-01-01

    A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (n = 16) and without (n = 8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (rs = 0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene. PMID:21814574

  20. Restoration of adipose function in obese glucose-tolerant men following pioglitazone treatment is associated with CCAAT enhancer-binding protein β up-regulation.

    PubMed

    Powell, Lesley A; Crowe, Paul; Kankara, Chenchi; McPeake, Jennifer; McCance, David R; Young, Ian S; Trimble, Elisabeth R; McGinty, Ann

    2012-08-01

    Obese AT (adipose tissue) exhibits increased macrophage number. Pro-inflammatory CD16+ peripheral monocyte numbers are also reported to increase with obesity. The present study was undertaken to simultaneously investigate obesity-associated changes in CD16+ monocytes and ATMs (AT macrophages). In addition, a pilot randomized placebo controlled trial using the PPAR (peroxisome-proliferator-activated receptor) agonists, pioglitazone and fenofibrate was performed to determine their effects on CD14+/CD16+ monocytes, ATM and cardiometabolic and adipose dysfunction indices. Obese glucose-tolerant men (n=28) were randomized to placebo, pioglitazone (30 mg/day) and fenofibrate (160 mg/day) for 12 weeks. A blood sample was taken to assess levels of serum inflammatory markers and circulating CD14+/CD16+ monocyte levels via flow cytometry. A subcutaneous AT biopsy was performed to determine adipocyte cell surface and ATM number, the latter was determined via assessment of CD68 expression by IHC (immunohistochemistry) and real-time PCR. Subcutaneous AT mRNA expression of CEBPβ (CCAAT enhancer-binding protein β), SREBP1c (sterol-regulatory-element-binding protein 1c), PPARγ2, IRS-1 (insulin receptor substrate-1), GLUT4 (glucose transporter type 4) and TNFα (tumour necrosis factor α) were also assessed. Comparisons were made between obese and lean controls (n=16) at baseline, and pre- and post-PPAR agonist treatment. Obese individuals had significantly increased adipocyte cell surface, percentage CD14+/CD16+ monocyte numbers and ATM number (all P=0.0001). Additionally, serum TNF-α levels were significantly elevated (P=0.017) and adiponectin levels reduced (total: P=0.0001; high: P=0.022) with obesity. ATM number and percentage of CD14+/CD16+ monocytes correlated significantly (P=0.05). Pioglitazone improved adiponectin levels significantly (P=0.0001), and resulted in the further significant enlargement of adipocytes (P=0.05), without effect on the percentage CD14+/CD16

  1. Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol.

    PubMed

    Jeng, Y J; Hinko, A; Soloff, M S

    1995-11-01

    Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs. PMID:8527507

  2. Magnesium modification up-regulates the bioactivity of bone morphogenetic protein-2 upon calcium phosphate cement via enhanced BMP receptor recognition and Smad signaling pathway.

    PubMed

    Ding, Sai; Zhang, Jing; Tian, Yu; Huang, Baolin; Yuan, Yuan; Liu, Changsheng

    2016-09-01

    Efficient presentation of growth factors is one of the great challenges in tissue engineering. In living systems, bioactive factors exist in soluble as well as in matrix-bound forms, both of which play an integral role in regulating cell behaviors. Herein, effect of magnesium on osteogenic bioactivity of recombinant human bone morphogenetic protein-2 (rhBMP-2) was investigated systematically with a series of Mg modified calcium phosphate cements (xMCPCs, x means the content of magnesium phosphate cement wt%) as matrix model. The results indicated that the MCPC, especially 5MCPC, could promote the rhBMP-2-induced in vitro osteogenic differentiation via Smad signaling of C2C12 cells. Further studies demonstrated that all MCPC substrates exhibited similar rhBMP-2 release rate and preserved comparable conformation and biological activity of the released rhBMP-2. Also, the ionic extracts of MCPC made little difference to the bioactivity of rhBMP-2, either in soluble or in matrix-bound forms. However, with the quartz crystal microbalance (QCM), we observed a noticeable enhancement of rhBMP-2 mass-uptake on 5MCPC as well as a better recognition of the bound rhBMP-2 to BMPR IA and BMPR II. In vivo results demonstrated a better bone regeneration capacity of 5MCPC/rhBMP-2. From the above, our results demonstrated that it was the Mg anchored on the underlying substrates that tailored the way of rhBMP-2 bound on MCPC, and thus facilitated the recognition of BMPRs to stimulate osteogenic differentiation. The study will guide the development of Mg-doped bioactive bone implants for tissue regeneration. PMID:27156155

  3. Human gonadotropin-releasing hormone receptor gene transcription: up-regulation by 3',5'-cyclic adenosine monophosphate/protein kinase A pathway.

    PubMed

    Cheng, K W; Leung, P C

    2001-07-01

    Transient transfection of mouse gonadotrope-derived (alphaT3-1) cells with a 2297 bp human GnRHR promoter-luciferase construct (p2300-LucF) showed a dose- and time-dependent increase in the human gonodotropin-releasing hormone receptor (GnRHR) promoter activity after forskolin treatment. An average of 4.8-fold increase in promoter activity was observed after 12 h of 10 microM forskolin treatment. This effect was mimicked by administration of cholera toxin, cAMP analog or pituitary adenylate cyclase activating polypeptide 38 (PACAP). A specific adenylate cyclase (AC) inhibitor (ACI) or protein kinase A (PKA) inhibitor (PKAI) pretreatment reversed the forskolin- and PACAP-induced increase in the human GnRHR promoter activity. These results not only confirm the stimulatory effect of Cyclic adenosine monophosphate (cAMP) in human GnRHR promoter activation, but also suggest that hormones or neurotransmitters that activate adenylate cyclase in pituitary gonadotropes may increase the expression of human GnRHR gene in transcriptional level. Progressive 5' deletion assays identified a 412 bp fragment (-577 to 167) in the human GnRHR 5'-flanking region that is essential in maintaining the basal responsiveness to cAMP. Mutagenesis coupled with functional studies have identified two putative AP-1/CREB binding sites, namely hGR-AP/CRE-1 and hGR-AP/CRE-2 that participated in mediating the cAMP-stimulatory effect. Mutation of the putative hGR-AP/CRE-1 and hGR-CRE-2 resulted in a 38 and 32% decrease in the forskolin-induced stimulation. However, mutation of both binding sites did not completely abolish the cAMP-stimulatory effect, suggesting that multiple transcription factor binding sites were involved in full response in cAMP stimulation. The binding of CREB to these motifs was confirmed by gel mobility shift assay and antibody supershift assay. PMID:11476937

  4. Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients.

    PubMed

    Buga, Ana-Maria; Ciobanu, Ovidiu; Bădescu, George Mihai; Bogdan, Catalin; Weston, Ria; Slevin, Mark; Di Napoli, Mario; Popa-Wagner, Aurel

    2016-04-01

    Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke. PMID:27013593

  5. Up-regulation of endoplasmic reticulum stress induced genes of the unfolded protein response in the liver of periparturient dairy cows

    PubMed Central

    2014-01-01

    Background In dairy cows, the periparturient phase is a stressful period, which is commonly associated with strong metabolic adaptations and the development of pathophysiologic conditions and disorders. Some of the symptoms occurring in the liver, such as the development of fatty liver, are similar to those observed under the condition of endoplasmic reticulum (ER) stress. Therefore, we hypothesized, that in the liver of dairy cows ER stress is induced during the periparturient phase, which in turn leads to an induction of the unfolded protein response (UPR). In order to investigate this hypothesis, we determined relative mRNA concentrations of 14 genes of the ER stress-induced UPR in liver biopsy samples of 13 dairy cows at 3 wk antepartum and 1, 5 and 14 wk postpartum. Results We found, that the mRNA concentrations of 13 out of the 14 genes involved in the UPR in the liver were significantly increased (1.9 to 4.0 fold) at 1 wk postpartum compared to 3 wk antepartum. From 1 wk postpartum to later lactation, mRNA concentrations of all the genes considered were declining. Moreover, at 1 wk postpartum, mRNA concentration of the spliced variant of XBP1 was increased in comparison to 3 wk antepartum, indicating that splicing of XBP1 – a hallmark of ER stress - was induced following the onset of lactation. Conclusion The present study reveals, that ER stress might be induced during the periparturient phase in the liver of dairy cows. We assume that the ER stress-induced UPR might contribute to the pathophysiologic conditions commonly observed in the liver of periparturient cows, such as the development of fatty liver, ketosis or inflammation. PMID:24555446

  6. Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra.

    PubMed

    St Martin, Jessie L; Klucken, Jochen; Outeiro, Tiago F; Nguyen, Paul; Keller-McGandy, Christine; Cantuti-Castelvetri, Ippolita; Grammatopoulos, Tom N; Standaert, David G; Hyman, Bradley T; McLean, Pamela J

    2007-03-01

    Several transgenic mouse lines with altered alpha-synuclein expression have been developed that show a variety of Parkinson's disease-like symptoms without specific loss of dopaminergic neurons. Targeted over-expression of human alpha-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates leads to dopaminergic cell loss and the formation of alpha-synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno-associated virus (AAV) to over-express wild type human alpha-synuclein in the substantia nigra of mice. We hypothesized that this over-expression would recapitulate pathological hallmarks of Parkinson's disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing alpha-synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress-related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over-expression of alpha-synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson's disease pathogenesis can be further elucidated. PMID:17241127

  7. Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients

    PubMed Central

    Bădescu, George Mihai; Bogdan, Catalin; Weston, Ria; Slevin, Mark; Di Napoli, Mario; Popa-Wagner, Aurel

    2016-01-01

    Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke. PMID:27013593

  8. Platycodon grandiflorum root attenuates vascular endothelial cell injury by oxidized low-density lipoprotein and prevents high-fat diet-induced dyslipidemia in mice by up-regulating antioxidant proteins.

    PubMed

    Chung, Mi Ja; Kim, Soo-Hyun; Park, Jeong-Won; Lee, Young Jin; Ham, Seung-Shi

    2012-05-01

    We hypothesized that a Platycodon grandiflorum root (PG) ethyl acetate extract (PGEA) would help reduce the vascular cell injury caused by oxidized low-density lipoprotein (oxLDL) and prevent high-fat (HF) diet-induced dyslipidemia and oxidative stress by up-regulating antioxidant proteins. We investigated the protective effects of PGEA against vascular endothelial cell injury induced by oxLDL and dyslipidemia induced by an HF diet, and the mechanisms underlying these effects were studied. The protective effects of PGEA were investigated with respect to calf pulmonary arterial endothelial (CPAE) cell viability and the lactate dehydrogenase release during oxLDL treatment. The in vivo effects of PGEA were examined using C57BL/6 mice, which were fed an HF diet for 9 weeks. The HF diet was supplemented with 0, 25, or 75 mg/kg PGEA during the last 4 weeks of the experimental period. Histologic analyses of hepatic lipid accumulation were performed. The changes in antioxidant protein levels induced by PGEA, which protects against HF diet-induced oxidative stress, were measured using a proteomics approach. We found that PGEA exhibited antioxidant activity. In CPAE cells, PGEA inhibited both oxLDL-induced cell death and lactate dehydrogenase release. In the HF diet-induced obese mice that received PGEA, we observed significantly reduced plasma and hepatic lipid levels, demonstrating that PGEA has beneficial effects on hyperlipidemia. In addition, we found that PGEA caused the up-regulation of antioxidant proteins. These findings suggest that the antioxidant effects of PGEA may protect against oxidative stress-related diseases. PMID:22652376

  9. Proteomic analysis of common bean seed with storage protein deficiency reveals up-regulation of sulfur-rich proteins and starch and raffinose metabolic enzymes, and down-regulation of the secretory pathway.

    PubMed

    Marsolais, Frédéric; Pajak, Agnieszka; Yin, Fuqiang; Taylor, Meghan; Gabriel, Michelle; Merino, Diana M; Ma, Vanessa; Kameka, Alexander; Vijayan, Perumal; Pham, Hai; Huang, Shangzhi; Rivoal, Jean; Bett, Kirstin; Hernández-Sebastià, Cinta; Liu, Qiang; Bertrand, Annick; Chapman, Ralph

    2010-06-16

    A deficiency in major seed storage proteins is associated with a nearly two-fold increase in sulfur amino acid content in genetically related lines of common bean (Phaseolus vulgaris). Their mature seed proteome was compared by an approach combining label-free quantification by spectral counting, 2-DE, and analysis of selective extracts. Lack of phaseolin, phytohemagglutinin and arcelin was mainly compensated by increases in legumin, alpha-amylase inhibitors and mannose lectin FRIL. Along with legumin, albumin-2, defensin and albumin-1 were major contributors to the elevated sulfur amino acid content. Coordinate induction of granule-bound starch synthase I, starch synthase II-2 and starch branching enzyme were associated with minor alteration of starch composition, whereas increased levels of UDP-glucose 4-epimerase were correlated with a 30% increase in raffinose content. Induction of cell division cycle protein 48 and ubiquitin suggested enhanced ER-associated degradation. This was not associated with a classical unfolded protein response as the levels of ER HSC70-cognate binding protein were actually reduced in the mutant. Repression of rab1 GTPase was consistent with decreased traffic through the secretory pathway. Collectively, these results have implications for the nutritional quality of common bean, and provide information on the pleiotropic phenotype associated with storage protein deficiency in a dicotyledonous seed. PMID:20353836

  10. A novel antitumor activity of deguelin targeting the insulin-like growth factor (IGF) receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer

    PubMed Central

    Suh, Young-Ah; Kim, Jai-Hyun; Sung, Myung A; Boo, Hye-Jin; Yun, Hye Jeong; Lee, Sun-Hye; Lee, Hyo-Jong; Suh, Young-Ger; Kim, Kyu-Won; Lee, Ho-Young

    2013-01-01

    In this study, we investigated the antitumor effects of deguelin in several human breast cancer cells in vitro and in vivo. Deguelin inhibited cell viability and the anchorage-dependent and anchorage-independent colony formation of triple-negative (MDA-MB-231 and MDA-MB-468) and triple-positive (MCF-7) breast cancer cells, and it significantly reduced the growth of MCF-7 cell xenograft tumors. The induction of apoptosis, inhibition of insulin-like growth factor-1 receptor (IGF-1R) signaling activation, and up-regulation of IGF-binding protein-3 (IGFBP-3) expression may be associated with deguelin-mediated antitumor effects. Our findings suggest a potential therapeutic use for deguelin in patients with triple-negative breast cancer and for those with breast cancers who are sensitive to endocrine- and HER2-targeted therapies. PMID:23348700

  11. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro; Hayashi, Hidetoshi; Itoh, Saotomo; Takii, Takemasa; Onozaki, Kikuo

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  12. The receptor protein tyrosine phosphatase HmLAR1 is up-regulated in the CNS of the adult medicinal leech following injury and is required for neuronal sprouting and regeneration.

    PubMed

    Sethi, Jasmine; Zhao, Bailey; Cuvillier-Hot, Virginie; Boidin-Wichlacz, Céline; Salzet, Michel; Macagno, Eduardo R; Baker, Michael W

    2010-12-01

    LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (P(D)) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level. PMID:20708686

  13. Peroxisome proliferator-activated receptor gamma up-regulates the Bcl-2 anti-apoptotic protein in neurons and induces mitochondrial stabilization and protection against oxidative stress and apoptosis.

    PubMed

    Fuenzalida, Karen; Quintanilla, Rodrigo; Ramos, Patricio; Piderit, Daniela; Fuentealba, Rodrigo A; Martinez, Gabriela; Inestrosa, Nibaldo C; Bronfman, Miguel

    2007-12-21

    Peroxisome proliferator-activated receptor gamma (PPARgamma) has been proposed as a therapeutic target for neurodegenerative diseases because of its anti-inflammatory action in glial cells. However, PPARgamma agonists preventbeta-amyloid (Abeta)-induced neurodegeneration in hippocampal neurons, and PPARgamma is activated by the nerve growth factor (NGF) survival pathway, suggesting a neuroprotective anti-inflammatory independent action. Here we show that the PPARgamma agonist rosiglitazone (RGZ) protects hippocampal and dorsal root ganglion neurons against Abeta-induced mitochondrial damage and NGF deprivation-induced apoptosis, respectively, and promotes PC12 cell survival. In neurons and in PC12 cells RGZ protective effects are associated with increased expression of the Bcl-2 anti-apoptotic protein. NGF-differentiated PC12 neuronal cells constitutively overexpressing PPARgamma are resistant to Abeta-induced apoptosis and morphological changes and show functionally intact mitochondria and no increase in reactive oxygen species when challenged with up to 50 microM H2O2. Conversely, cells expressing a dominant negative mutant of PPARgamma show increased Abeta-induced apoptosis and disruption of neuronal-like morphology and are highly sensitive to oxidative stress-induced impairment of mitochondrial function. Cells overexpressing PPARgamma present a 4- to 5-fold increase in Bcl-2 protein content, whereas in dominant negative PPARgamma-expressing cells, Bcl-2 is barely detected. Bcl-2 knockdown by small interfering RNA in cells overexpressing PPARgamma results in increased sensitivity to Abeta and oxidative stress, further suggesting that Bcl-2 up-regulation mediates PPARgamma protective effects. PPARgamma prosurvival action is independent of the signal-regulated MAPK or the Akt prosurvival pathways. Altogether, these data suggest that PPARgamma supports survival in neurons in part through a mechanism involving increased expression of Bcl-2. PMID:17965419

  14. cAMP Response Element-binding Protein (CREB) and Nuclear Factor κB Mediate the Tamoxifen-induced Up-regulation of Glutamate Transporter 1 (GLT-1) in Rat Astrocytes*

    PubMed Central

    Karki, Pratap; Webb, Anton; Smith, Keisha; Lee, Kyuwon; Son, Deok-Soo; Aschner, Michael; Lee, Eunsook

    2013-01-01

    Tamoxifen (TX), a selective estrogen receptor modulator, exerts antagonistic effects on breast tissue and is used to treat breast cancer. Recent evidence also suggests that it may act as an agonist in brain tissue. We reported previously that TX enhanced the expression and function of glutamate transporter 1 (GLT-1) in rat astrocytes, an effect that was mediated by TGF-α. To gain further insight into the mechanisms that mediate TX-induced up-regulation of GLT-1 (EAAT2 in humans), we investigated its effect on GLT-1 at the transcriptional level. TX phosphorylated the cAMP response element-binding protein (CREB) and recruited CREB to the GLT-1 promoter consensus site. The effect of TX on astrocytic GLT-1 was attenuated by the inhibition of PKA, the upstream activator of the CREB pathway. In addition, the effect of TX on GLT-1 promoter activity was abolished by the inhibition of the NF-κB pathway. Furthermore, TX recruited the NF-κB subunits p65 and p50 to the NF-κB binding domain of the GLT-1 promoter. Mutation of NF-κB (triple, −583/-282/-251) or CRE (-308) sites on the GLT-1 promoter led to significant repression of the promoter activity, but neither mutant completely abolished the TX-induced GLT-1 promoter activity. Mutation of both the NF-κB (-583/-282/-251) and CRE (-308) sites led to a complete abrogation of the effect of TX on GLT-1 promoter activity. Taken together, our findings establish that TX regulates GLT-1 via the CREB and NF-κB pathways. PMID:23955341

  15. Cell type-dependent modulation of the gene encoding heat shock protein HSPA2 by hypoxia-inducible factor HIF-1: Down-regulation in keratinocytes and up-regulation in HeLa cells.

    PubMed

    Habryka, Anna; Gogler-Pigłowska, Agnieszka; Sojka, Damian; Kryj, Mariusz; Krawczyk, Zdzisław; Scieglinska, Dorota

    2015-09-01

    HSPA2 belongs to the multigene HSPA family, whose members encode chaperone proteins. Although expression and function of HSPA2 is mainly associated with spermatogenesis, recent studies demonstrated that in humans, the gene is active in various cancers, as well as in normal tissues, albeit in a cell type-specific manner. In the epidermis, HSPA2 is expressed in keratinocytes in the basal layer. Currently, the mechanisms underlying the regulation of HSPA2 expression remain unknown. This study was aimed at determining whether HIF-1 and its binding site, the hypoxia-response element (HRE) located in the HSPA2 promoter, are involved in HSPA2 regulation. As a model system, we used an immortal human keratinocyte line (HaCaT) and cervical cancer cells (HeLa) grown under control or hypoxic conditions. Using an in vitro gene reporter assay, we demonstrated that in keratinocytes HSPA2 promoter activity is reduced under conditions that facilitate stabilization of HIF-1α, whereas HIF-1 inhibitors abrogated the suppressive effect of hypoxia on promoter activity. Chromatin immunoprecipitation revealed that HIF-1α binds to the HSPA2 promoter. In keratinocytes, hypoxia or overexpression of a stable form of HIF-1α attenuated the expression of endogenous HSPA2, whereas targeted repression of HIF-1α by RNAi increased transcription of HSPA2 under hypoxia. Conversely, in HeLa cells, HSPA2 expression increased under conditions that stimulated HIF-1α activity, whereas inhibition of HIF-1α abrogated hypoxia-induced up-regulation of HSPA2 expression. Taken together, our results demonstrate that HIF-1 can exert differential, cell context-dependent regulatory control of the HSPA2 gene. Additionally, we also showed that HSPA2 expression can be stimulated during hypoxia/reoxygenation stress. PMID:26164067

  16. Up-regulation of Survivin during Immortalization of Human Myofibroblasts Is Linked to Repression of Tumor Suppressor p16INK4a Protein and Confers Resistance to Oxidative Stress*

    PubMed Central

    Kan, Chin-Yi; Petti, Carlotta; Bracken, Lauryn; Maritz, Michelle; Xu, Ning; O'Brien, Rosemary; Yang, Chen; Liu, Tao; Yuan, Jun; Lock, Richard B.; MacKenzie, Karen L.

    2013-01-01

    Survivin is an essential component of the chromosomal passenger complex and a member of the inhibitor of apoptosis family. It is expressed at high levels in a large variety of malignancies, where it has been implicated in drug resistance. It was also shown previously that survivin is up-regulated during telomerase-mediated immortalization, which occurs at a relatively early stage during carcinogenesis. This study shows that up-regulation of survivin during immortalization of human myofibroblasts is an indirect consequence of the repression of p16INK4a. Survivin and p16INK4a were functionally linked by assays that showed that either the up-regulation of survivin or repression of p16INK4a rendered telomerase-transduced MRC-5 myofibroblasts resistant to oxidative stress. Conversely, siRNA-mediated down-regulation of survivin activated caspases and enhanced the sensitivity of immortal MRC-5 cells to oxidative stress. The E2F1 transcription factor, which is negatively regulated by the pRB/p16INK4a tumor suppressor pathway, was implicated in the up-regulation of survivin. Using the ChIP assay, it was shown that E2F1 directly interacted with the survivin gene (BIRC5) promoter in cells that spontaneously silenced p16INK4a during telomerase-mediated immortalization. E2F1 binding to the BIRC5 was also enhanced in telomerase-transduced cells subjected to shRNA-mediated repression of p16INK4a. Together, these data show that repression of p16INK4a contributes to the up-regulation of survivin and thereby provides a survival advantage to cells exposed to oxidative stress during immortalization. The up-regulation of survivin during immortalization likely contributes to the vulnerability of immortal cells to transformation by oncogenes that alter intracellular redox state. PMID:23449974

  17. Endogenous mouse interleukin-10 is up-regulated by exogenously administered recombinant human interleukin-10, but does not contribute to the efficacy of the human protein in mouse models of endotoxemia.

    PubMed

    Smith, S R; Terminelli, C; Denhardt, G; Manfra, D; Davies, L; Narula, S

    1999-02-01

    In murine models of experimental endotoxemia, inflammatory cytokines as well as antiinflammatory interleukin-10 (IL-10) appear in the circulation after the injection of lipopolysaccharide (LPS). There is considerable experimental evidence to suggest that the major function of endogenously produced IL-10 is to down-regulate inflammatory cytokine production. Indeed, the protective effects of exogenously administered IL-10 against murine endotoxin lethality have been shown to correlate with its ability to inhibit the LPS-induced production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). While mouse IL-10 (mIL-10) has been used in the majority of studies in murine endotoxemia, we have found the human homolog to be equally effective in suppressing inflammatory cytokine production and in protecting mice from endotoxin lethality. However, we have recently observed that the LPS-induced endogenous IL-10 response is enhanced when mice are treated with recombinant human IL-10 (rhuIL-10). The upregulation of endogenous IL-10 by exogenously administered rhuIL-10 is particularly evident in mice that are primed with Corynebacterium partum (Proprionibacterium acnes). In the present study, we have examined the potential contributions of the increased circulating levels of mouse IL-10 to the inhibitory effects seen with rhuIL-10 on inflammatory cytokine production and endotoxin lethality. We show that pretreatment with a neutralizing anti-mouse IL-10 monoclonal antibody (mAb) has no effect on the ability of rhuIL-10 to suppress an LPS-induced inflammatory cytokine response in these mice. In contrast, the suppressive effects of the human protein on inflammatory cytokine responses are blocked completely by pretreating the animals with an anti-huIL-10 mAb. These data show that despite the up-regulated endogenous IL-10 response, it is the exogenously administered rhuIL-10 that is directly responsible for the suppressed inflammatory cytokine responses that

  18. 5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt's lymphoma line rael.

    PubMed Central

    Masucci, M G; Contreras-Salazar, B; Ragnar, E; Falk, K; Minarovits, J; Ernberg, I; Klein, G

    1989-01-01

    Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells. Images PMID:2470924

  19. Protein flexibility: coordinate uncertainties and interpretation of structural differences

    SciTech Connect

    Rashin, Alexander A.; Rashin, Abraham H. L.; Jernigan, Robert L.

    2009-11-01

    Criteria for the interpretability of coordinate differences and a new method for identifying rigid-body motions and nonrigid deformations in protein conformational changes are developed and applied to functionally induced and crystallization-induced conformational changes. Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent ‘noise’, showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function.

  20. Protein flexibility: coordinate uncertainties and interpretation of structural differences.

    PubMed

    Rashin, Alexander A; Rashin, Abraham H L; Jernigan, Robert L

    2009-11-01

    Valid interpretations of conformational movements in protein structures determined by X-ray crystallography require that the movement magnitudes exceed their uncertainty threshold. Here, it is shown that such thresholds can be obtained from the distance difference matrices (DDMs) of 1014 pairs of independently determined structures of bovine ribonuclease A and sperm whale myoglobin, with no explanations provided for reportedly minor coordinate differences. The smallest magnitudes of reportedly functional motions are just above these thresholds. Uncertainty thresholds can provide objective criteria that distinguish between true conformational changes and apparent 'noise', showing that some previous interpretations of protein coordinate changes attributed to external conditions or mutations may be doubtful or erroneous. The use of uncertainty thresholds, DDMs, the newly introduced CDDMs (contact distance difference matrices) and a novel simple rotation algorithm allows a more meaningful classification and description of protein motions, distinguishing between various rigid-fragment motions and nonrigid conformational deformations. It is also shown that half of 75 pairs of identical molecules, each from the same asymmetric crystallographic cell, exhibit coordinate differences that range from just outside the coordinate uncertainty threshold to the full magnitude of large functional movements. Thus, crystallization might often induce protein conformational changes that are comparable to those related to or induced by the protein function. PMID:19923711

  1. AMP-activated Protein Kinase Up-regulates Mitogen-activated Protein (MAP) Kinase-interacting Serine/Threonine Kinase 1a-dependent Phosphorylation of Eukaryotic Translation Initiation Factor 4E.

    PubMed

    Zhu, Xiaoqing; Dahlmans, Vivian; Thali, Ramon; Preisinger, Christian; Viollet, Benoit; Voncken, J Willem; Neumann, Dietbert

    2016-08-12

    AMP-activated protein kinase (AMPK) is a molecular energy sensor that acts to sustain cellular energy balance. Although AMPK is implicated in the regulation of a multitude of ATP-dependent cellular processes, exactly how these processes are controlled by AMPK as well as the identity of AMPK targets and pathways continues to evolve. Here we identify MAP kinase-interacting serine/threonine protein kinase 1a (MNK1a) as a novel AMPK target. Specifically, we show AMPK-dependent Ser(353) phosphorylation of the human MNK1a isoform in cell-free and cellular systems. We show that AMPK and MNK1a physically interact and that in vivo MNK1a-Ser(353) phosphorylation requires T-loop phosphorylation, in good agreement with a recently proposed structural regulatory model of MNK1a. Our data suggest a physiological role for MNK1a-Ser(353) phosphorylation in regulation of the MNK1a kinase, which correlates with increased eIF4E phosphorylation in vitro and in vivo. PMID:27413184

  2. Using diastereopeptides to control metal ion coordination in proteins.

    PubMed

    Peacock, Anna F A; Hemmingsen, Lars; Pecoraro, Vincent L

    2008-10-28

    Here, we report a previously undescribed approach for controlling metal ion coordination geometry in biomolecules by reorientating amino acid side chains through substitution of L- to D-amino acids. These diastereopeptides allow us to manipulate the spatial orientation of amino acid side chains to alter the sterics of metal binding pockets. We have used this approach to design the de novo metallopeptide, Cd(TRIL12L(D)L16C)(3)(-), which is an example of Cd(II) bound to 3 L-Cys as exclusively trigonal CdS(3), as characterized by a combination of (113)Cd NMR and (111m)Cd PAC spectroscopy. We subsequently show that the physical properties of such a site, such as the high pK(a2) for Cd(II) binding of 15.1, is due to the nature of the coordination number and not the ligating group. Further more this approach allowed for the design of a construct, GRANDL12L(D)L16CL26AL30C, capable of independently binding 2 equivalents of Cd(II) to 2 very similar Cys sites as exclusively 3- and 4-, CdS(3) and CdS(3)O, respectively. Demonstrating that we are capable of controlling the Cd(II) coordination number in these 2 sites solely by varying the nature of a noncoordinating second coordination sphere amino acid, with D-leucine and L-alanine resulting in exclusively 3- and 4-coordinate structures, respectively. Cd(II) was found to selectively bind to the 4-coordinate CdS(3)O site, demonstrating that a protein can be designed that displays metal-binding selectivity based solely on coordination number control and not on the chemical identity of coordinating ligands. PMID:18940928

  3. Infiltrating cellular pattern in kidney graft biopsies translates into forkhead box protein 3 up-regulation and p16INK4α senescence protein down-regulation in patients treated with belatacept compared to cyclosporin A

    PubMed Central

    Furuzawa-Carballeda, J; Lima, G; Alberú, J; Palafox, D; Uribe-Uribe, N; Morales-Buenrostro, L E; Reyes Acevedo, R; Mondragón, G; Chevaile, A; Llorente, L

    2012-01-01

    Renal allograft survival is related directly to cell senescence. In the transplantation scenario many cellular events – participating as immunological and non-immunological factors – could contribute to accelerate this biological process, responsible for the ultimate fate of the graft. Mechanisms concerned in tolerance versus rejection are paramount in this outcome. For this reason, immunosuppressive treatment constitutes an extremely important decision to prevent organ dysfunction and, finally, graft loss. This study was conducted to document the proportion of CD4+/interleukin (IL)-17A+-, CD16+/indoleamine 2, 3-dioxygenase (IDO+)-, forkhead box protein P3 (FoxP3+)-expressing cells, senescent cells (p16INK4α) and the percentage of interstitial fibrosis (IF) in graft biopsies of kidney transplant recipients participating in the BENEFIT (Bristol-Myers Squibb IM103008) study. CD4+/IL-17A+, CD16+/IDO+, FoxP3+ and p16INK4α+ cells were evaluated by immunohistochemistry, and the percentage of IF by morphometry on graft biopsies obtained at time 0 (pre-implantation) and at 12 months post-transplant. Senescent cells and CD4+/IL-17A+ cells were increased among graft biopsies in subjects receiving cyclosporin A (CsA) compared to those under belatacept treatment. Meanwhile, CD16+/IDO+ and FoxP3+-expressing cells were lower in biopsies from CsA treatment compared to patients treated with Belatacept. Histological morphometric analyses disclosed more IF in 12-month CsA-treated patients in comparison to pre-implantation biopsy findings. Summing up, renal biopsies from patients receiving belatacept showed greater amounts of FoxP3+ cells and lower amounts of CD4+/IL-17A+ and senescent cells compared to patients under CsA treatment. Along with these findings, an increase in IF in annual CsA-treated-patients biopsies compared to pre-implantation and belatacept-treated patients were observed. PMID:22236010

  4. Protein kinase C coordinates histone H3 phosphorylation and acetylation

    PubMed Central

    Darieva, Zoulfia; Webber, Aaron; Warwood, Stacey; Sharrocks, Andrew D

    2015-01-01

    The re-assembly of chromatin following DNA replication is a critical event in the maintenance of genome integrity. Histone H3 acetylation at K56 and phosphorylation at T45 are two important chromatin modifications that accompany chromatin assembly. Here we have identified the protein kinase Pkc1 as a key regulator that coordinates the deposition of these modifications in S. cerevisiae under conditions of replicative stress. Pkc1 phosphorylates the histone acetyl transferase Rtt109 and promotes its ability to acetylate H3K56. Our data also reveal novel cross-talk between two different histone modifications as Pkc1 also enhances H3T45 phosphorylation and this modification is required for H3K56 acetylation. Our data therefore uncover an important role for Pkc1 in coordinating the deposition of two different histone modifications that are important for chromatin assembly. DOI: http://dx.doi.org/10.7554/eLife.09886.001 PMID:26468616

  5. ClC-3 Chloride Channel Proteins Regulate the Cell Cycle by Up-regulating cyclin D1-CDK4/6 through Suppressing p21/p27 Expression in Nasopharyngeal Carcinoma Cells

    PubMed Central

    Ye, Dong; Luo, Hai; Lai, Zhouyi; Zou, Lili; Zhu, Linyan; Mao, Jianwen; Jacob, Tim; Ye, Wencai; Wang, Liwei; Chen, Lixin

    2016-01-01

    It was shown in this study that knockdown of ClC-3 expression by ClC-3 siRNA prevented the activation of hypotonicity-induced chloride currents, and arrested cells at the G0/G1 phase in nasopharyngeal carcinoma CNE-2Z cells. Reconstitution of ClC-3 expression with ClC-3 expression plasmids could rescue the cells from the cell cycle arrest caused by ClC-3 siRNA treatments. Transfection of cells with ClC-3 siRNA decreased the expression of cyclin D1, cyclin dependent kinase 4 and 6, and increased the expression of cyclin dependent kinase inhibitors (CDKIs), p21 and p27. Pretreatments of cells with p21 and p27 siRNAs depleted the inhibitory effects of ClC-3 siRNA on the expression of CDK4 and CDK6, but not on that of cyclin D1, indicating the requirement of p21 and p27 for the inhibitory effects of ClC-3 siRNA on CDK4 and CDK6 expression. ClC-3 siRNA inhibited cells to progress from the G1 phase to the S phase, but pretreatments of cells with p21 and p27 siRNAs abolished the inhibitory effects of ClC-3 siRNA on the cell cycle progress. Our data suggest that ClC-3 may regulate cell cycle transition between G0/G1 and S phases by up-regulation of the expression of CDK4 and CDK6 through suppression of p21 and p27 expression. PMID:27451945

  6. FRZB up-regulated in hepatocellular carcinoma bone metastasis

    PubMed Central

    Huang, Jia; Hu, Wenhao; Lin, Xiangjin; Wang, Xuanwei; Jin, Ketao

    2015-01-01

    The clinical relevance of frizzled-related protein (FRZB) in hepatocellular carcinoma (HCC) bone metastasis remains uncertain. The aim of this study was to assess the clinical relationship of FRZB in patients with HCC bone metastasis after surgical resection. FRZB expression was evaluated by immunohistochemistry in formalin-fixed paraffin embedded (FFPE) HCC and paired bone metastasis tissues from 13 patients that underwent surgical resection. The clinical characteristics of 13 HCC patients with synchronous or metachronous bone metastasis received surgery were retrospectively reviewed. We found that FRZB was positive in 9 HCC tissues (69.2%) and in 11 paired bone metastatic tissues (84.6%) among these 13 paired samples. The expression of FRZB in the bone metastases was noticeably higher than that in the paired HCC tissues. The expression of FRZB was up-regulated in 10 (76.9%) paired bone metastases tissues. FRZB expression was up-regulated in HCC bone metastasis tissue, which suggested that FRZB might play a key role in the HCC bone metastasis. PMID:26722540

  7. Proteomic profiles of white sucker (Catostomus commersonii) sampled from within the Thunder Bay Area of Concern reveal up-regulation of proteins associated with tumor formation and exposure to environmental estrogens.

    PubMed

    Simmons, Denina B D; Bols, Niels C; Duncker, Bernard P; McMaster, Mark; Miller, Jason; Sherry, James P

    2012-02-01

    White sucker (Catostomus commersonii) sampled from the Thunder Bay Area of Concern were assessed for health using a shotgun approach to compile proteomic profiles. Plasma proteins were sampled from male and female fish from a reference location, an area in recovery within Thunder Bay Harbour, and a site at the mouth of the Kaministiquia River where water and sediment quality has been degraded by industrial activities. The proteins were characterized using reverse-phase liquid chromatography tandem to a quadrupole-time-of-flight (LC-Q-TOF) mass spectrometer and were identified by searching in peptide databases. In total, 1086 unique proteins were identified. The identified proteins were then examined by means of a bioinformatics pathway analysis to gain insight into the biological functions and disease pathways that were represented and to assess whether there were any significant changes in protein expression due to sampling location. Female white sucker exhibited significant (p = 0.00183) site-specific changes in the number of plasma proteins that were related to tumor formation, reproductive system disease, and neurological disease. Male fish plasma had a significantly different (p < 0.0001) number of proteins related to neurological disease and tumor formation. Plasma concentrations of vitellogenin were significantly elevated in females from the Kaministiquia River compared to the Thunder Bay Harbour and reference sites. The protein expression profiles indicate that white sucker health has benefited from the remediation of the Thunder Bay Harbour site, whereas white sucker from the Kaministiquia River site are impacted by ongoing contaminant discharges. PMID:22260729

  8. Up-regulation of the Sirtuin 1 (Sirt1) and Peroxisome Proliferator-activated Receptor γ Coactivator-1α (PGC-1α) Genes in White Adipose Tissue of Id1 Protein-deficient Mice

    PubMed Central

    Zhao, Ying; Ling, Flora; Griffin, Timothy M.; He, Ting; Towner, Rheal; Ruan, Hong; Sun, Xiao-Hong

    2014-01-01

    Id1, a helix-loop-helix (HLH) protein that inhibits the function of basic HLH E protein transcription factors in lymphoid cells, has been implicated in diet- and age-induced obesity by unknown mechanisms. Here we show that Id1-deficient mice are resistant to a high fat diet- and age-induced obesity, as revealed by reduced weight gain and body fat, increased lipid oxidation, attenuated hepatosteatosis, lower levels of lipid droplets in brown adipose tissue, and smaller white adipocytes after a high fat diet feeding or in aged animals. Id1 deficiency improves glucose tolerance, lowers serum insulin levels, and reduces TNFα gene expression in white adipose tissue. Id1 deficiency also increased expression of Sirtuin 1 and peroxisome proliferator-activated receptor γ coactivator 1α, regulators of mitochondrial biogenesis and energy expenditure, in the white adipose tissue. This effect was accompanied by the elevation of several genes encoding proteins involved in oxidative phosphorylation and fatty acid oxidation, such as cytochrome c, medium-chain acyl-CoA dehydrogenase, and adipocyte protein 2. Moreover, the phenotype for Id1 deficiency was similar to that of mice expressing an E protein dominant-positive construct, ET2, suggesting that the balance between Id and E proteins plays a role in regulating lipid metabolism and insulin sensitivity. PMID:25190816

  9. MEDELLER: homology-based coordinate generation for membrane proteins

    PubMed Central

    Kelm, Sebastian; Shi, Jiye; Deane, Charlotte M.

    2010-01-01

    Motivation: Membrane proteins (MPs) are important drug targets but knowledge of their exact structure is limited to relatively few examples. Existing homology-based structure prediction methods are designed for globular, water-soluble proteins. However, we are now beginning to have enough MP structures to justify the development of a homology-based approach specifically for them. Results: We present a MP-specific homology-based coordinate generation method, MEDELLER, which is optimized to build highly reliable core models. The method outperforms the popular structure prediction programme Modeller on MPs. The comparison of the two methods was performed on 616 target–template pairs of MPs, which were classified into four test sets by their sequence identity. Across all targets, MEDELLER gave an average backbone root mean square deviation (RMSD) of 2.62 Å versus 3.16 Å for Modeller. On our ‘easy’ test set, MEDELLER achieves an average accuracy of 0.93 Å backbone RMSD versus 1.56 Å for Modeller. Availability and Implementation: http://medeller.info; Implemented in Python, Bash and Perl CGI for use on Linux systems; Supplementary data are available at http://www.stats.ox.ac.uk/proteins/resources. Contact: kelm@stats.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online. PMID:20926421

  10. Increased Mitochondrial Pro-oxidant Activity Mediates Up-regulation of Complex I S-glutathionylation via Protein Thiyl Radical in the Murine Heart of eNOS−/−

    PubMed Central

    Kang, Patrick T.; Chen, Chwen-Lih; Chen, Yeong-Renn

    2014-01-01

    In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS•) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 kDa and 75 kDa subunits was detected under the conditions of •O2− production, and mass spectrometry confirmed that formation of Complex I PrS• mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS• (Kang et al Free Radical Biol. Med. 2012; 52: 962–73). In this investigation, we tested our hypothesis in the murine heart of eNOS−/−. The eNOS−/− mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS−/− myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in •O2− generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS−/− murine heart. When Complex I from the mitochondria of the eNOS−/− murine heart was analyzed by immuno-spin trapping and probed with anti-GSH antibody, both PrS• and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS•, supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS• is a unique pathway for the redox regulation of mitochondrial function in vivo. PMID:25445401

  11. Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer's disease.

    PubMed

    Pei, Jin-Jing; Braak, Heiko; An, Wen-Lin; Winblad, Bengt; Cowburn, Richard F; Iqbal, Khalid; Grundke-Iqbal, Inge

    2002-12-30

    The abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braak's protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of beta-amyloid in AD. PMID:12531514

  12. Investigation of reference gene expression during human herpesvirus 6B infection indicates peptidylprolyl isomerase A as a stable reference gene and TATA box binding protein as a gene up-regulated by this virus.

    PubMed

    Engdahl, Elin; Dunn, Nicky; Fogdell-Hahn, Anna

    2016-01-01

    When using relative gene expression for quantification of RNA it is crucial that the reference genes used for normalization do not change with the experimental condition. We aimed at investigating the expressional stability of commonly used reference genes during Human herpesvirus 6B (HHV-6B) infection. Expression of eight commonly used reference genes were investigated with quantitative PCR in a T-cell line infected with HHV-6B. The stability of genes was investigated using the 2(-ΔΔCT) method and the algorithms BestKeeper, GeNorm and NormFinder. Our results indicate that peptidylprolyl isomerase A (PPIA) is the most stably expressed gene while TATA box binding protein (TBP) is the least stably expressed gene during HHV-6B infection. In a confirmatory experiment, TBP was demonstrated to be dose and time dependently upregulated by HHV-6B. The stability of PPIA is in line with other studies investigating different herpesvirus infections whereas the finding that HHV-6B significantly upregulates TBP is novel and most likely specific to HHV-6B. PMID:26542463

  13. Ezrin Inhibition Up-regulates Stress Response Gene Expression.

    PubMed

    Çelik, Haydar; Bulut, Gülay; Han, Jenny; Graham, Garrett T; Minas, Tsion Z; Conn, Erin J; Hong, Sung-Hyeok; Pauly, Gary T; Hayran, Mutlu; Li, Xin; Özdemirli, Metin; Ayhan, Ayşe; Rudek, Michelle A; Toretsky, Jeffrey A; Üren, Aykut

    2016-06-17

    Ezrin is a member of the ERM (ezrin/radixin/moesin) family of proteins that links cortical cytoskeleton to the plasma membrane. High expression of ezrin correlates with poor prognosis and metastasis in osteosarcoma. In this study, to uncover specific cellular responses evoked by ezrin inhibition that can be used as a specific pharmacodynamic marker(s), we profiled global gene expression in osteosarcoma cells after treatment with small molecule ezrin inhibitors, NSC305787 and NSC668394. We identified and validated several up-regulated integrated stress response genes including PTGS2, ATF3, DDIT3, DDIT4, TRIB3, and ATF4 as novel ezrin-regulated transcripts. Analysis of transcriptional response in skin and peripheral blood mononuclear cells from NSC305787-treated mice compared with a control group revealed that, among those genes, the stress gene DDIT4/REDD1 may be used as a surrogate pharmacodynamic marker of ezrin inhibitor compound activity. In addition, we validated the anti-metastatic effects of NSC305787 in reducing the incidence of lung metastasis in a genetically engineered mouse model of osteosarcoma and evaluated the pharmacokinetics of NSC305787 and NSC668394 in mice. In conclusion, our findings suggest that cytoplasmic ezrin, previously considered a dormant and inactive protein, has important functions in regulating gene expression that may result in down-regulation of stress response genes. PMID:27137931

  14. Cotton Benzoquinone Reductase: Up-regulation During Early Cotton Fiber Developement

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme that catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2-D PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but ...

  15. α1-adrenergic receptor signaling in osteoblasts regulates clock genes and bone morphogenetic protein 4 expression through up-regulation of the transcriptional factor nuclear factor IL-3 (Nfil3)/E4 promoter-binding protein 4 (E4BP4).

    PubMed

    Hirai, Takao; Tanaka, Kenjiro; Togari, Akifumi

    2014-06-13

    Several studies have demonstrated that the α1-adrenergic receptor (AR) plays an important role in regulating cell growth and function in osteoblasts. However, the physiological role of α1-AR signaling in bone metabolism is largely unknown. In this study, the stimulation of phenylephrine (PHE), a nonspecific α1-AR agonist, increased the transcriptional factor Nfil3/E4BP4 and led to the rhythmic expression of bone morphogenetic protein 4 (Bmp4) in MC3T3-E1 osteoblastic cells. We also showed that Bmp4 mRNA expression peaked in bone near zeitgeber time 8 in a 24-h rhythm. Furthermore, the expression of Nfil3 and Bmp4 displayed a circadian pattern with opposing phases, which suggested that Nfil3 repressed the expression of the Bmp4 gene during a circadian cycle. On a molecular level, both loss-of-function and gain-of-function experiments demonstrated that Nfil3/E4BP4 negatively regulated Bmp4 expression in osteoblasts. Furthermore, the systemic administration of PHE increased the expression of Nfil3 mRNA in bone, whereas it decreased that of Bmp4 mRNA. The expression of Bmp4 mRNA was decreased significantly by exposure to PHE, and this was concomitant with the increase in Nfil3 binding to the D-box-containing Bmp4 promoter region in MC3T3-E1 cells, which indicates that the expression of Nfil3 by α1-AR signaling can bind directly to the Bmp4 promoter and inhibit Bmp4 expression in osteoblasts. Our results suggest that α1-AR signaling regulates clock genes and Bmp4 expression in osteoblasts. Moreover, α1-AR signaling negatively regulated Bmp4 expression by up-regulating the transcriptional factor Nfil3/E4BP4 in osteoblasts. PMID:24794868

  16. A Plant Gene Up-Regulated at Rust Infection Sites

    PubMed Central

    Ayliffe, Michael A.; Roberts, James K.; Mitchell, Heidi J.; Zhang, Ren; Lawrence, Gregory J.; Ellis, Jeffrey G.; Pryor, Tony J.

    2002-01-01

    Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a β-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%–82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a Δ1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection. PMID:12011348

  17. Wounding coordinately induces cell wall protein, cell cycle and pectin methyl esterase genes involved in tuber closing layer and wound periderm development.

    PubMed

    Neubauer, Jonathan D; Lulai, Edward C; Thompson, Asunta L; Suttle, Jeffrey C; Bolton, Melvin D

    2012-04-15

    Little is known about the coordinate induction of genes that may be involved in agriculturally important wound-healing events. In this study, wound-healing events were determined together with wound-induced expression profiles of selected cell cycle, cell wall protein, and pectin methyl esterase genes using two diverse potato genotypes and two harvests (NDTX4271-5R and Russet Burbank tubers; 2008 and 2009 harvests). By 5 d after wounding, the closing layer and a nascent phellogen had formed. Phellogen cell divisions generated phellem layers until cessation of cell division at 28 d after wounding for both genotypes and harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB) and cyclin-dependent kinase regulatory subunit (StCKS1At) were induced by 1 d after wounding; these expressions coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) were dramatically up-regulated by 1-5 d after wounding, suggesting involvement with closing layer and later phellem cell layer formation. Wounding up-regulated pectin methyl esterase genes (StPME and StPrePME); StPME expression increased during closing layer and phellem cell formation, whereas maximum expression of StPrePME occurred at 5-14 d after wounding, implicating involvement in later modifications for closing layer and phellem cell formation. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine-and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and maturation. Collectively, the genes monitored were wound-inducible and their expression profiles markedly coordinated with closing layer formation and the index for phellogen layer meristematic activity during wound periderm development; results were more

  18. Nutraceutical up-regulation of serotonin paradoxically induces compulsive behavior

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The role of diet in either the etiology or treatment of complex mental disorder is highly controversial in psychiatry. However, physiological mechanisms by which diet can influence brain chemistry – particularly that of serotonin – are well established. Here we show that dietary up-regulation of br...

  19. Microarray and KOG analysis of Acanthamoeba healyi genes up-regulated by mouse-brain passage.

    PubMed

    Moon, Eun-Kyung; Xuan, Ying-Hua; Kong, Hyun-Hee

    2014-08-01

    Long-term cultivation in a laboratory could reduce the virulence of Acanthamoeba. To identify virulence factors of Acanthamoeba, the authors compared the transcription profiles of long-term cultivated Acanthamoeba healyi (OLD) and three times mouse-brain passaged A. healyi (MBP) using microarray analysis and eukaryotic orthologous group (KOG) assignments. Microarray analysis revealed that 601 genes were up-regulated by mouse-brain passage. The results of real-time PCR of 8 randomly selected genes up-regulated in the MBP strain confirmed microarray analysis findings. KOG assignments showed relatively higher percentages of the MBP strain up-regulated genes in T article (signal transduction mechanism), O article (posttranslational modification, protein turnover, chaperones), C article (energy production and conversion), and J article (translation, ribosomal structure and biogenesis). In particular, the MBP strain showed higher expressions of cysteine protease and metalloprotease. A comparison of KOG assignments by microarray analysis and previous EST (expressed sequence tags) analysis showed similar populations of up-regulated genes. These results provide important information regarding the identification of virulence factors of pathogenic Acanthamoeba. PMID:24859526

  20. Inhibition of Epithelial to Mesenchymal Transition by E-cadherin Up-regulation via Repression of Slug Transcription and Inhibition of E-cadherin Degradation

    PubMed Central

    Adhikary, Arghya; Chakraborty, Samik; Mazumdar, Minakshi; Ghosh, Swatilekha; Mukherjee, Shravanti; Manna, Argha; Mohanty, Suchismita; Nakka, Kiran Kumar; Joshi, Shruti; De, Abhijit; Chattopadhyay, Samit; Sa, Gaurisankar; Das, Tanya

    2014-01-01

    The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor. PMID:25086032

  1. SAMe Prevents the Up Regulation of Toll-Like Receptor Signaling in Mallory-Denk Body Forming Hepatocytes

    PubMed Central

    Bardag-Gorce, Fawzia; Oliva, Joan; Lin, Andrew; Li, Jun; French, Barbara A.; French, Samuel W.

    2010-01-01

    Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. In the present study, the role of the toll-like receptor (TLR) signaling pathway was investigated in the mechanism of MDB formation in the DDC-fed mouse model. Microarray analysis data mining, performed on the livers of drug primed mice refed DDC, showed that TLR2/4 gene expression was significantly up regulated by DDC refeeding. SAMe supplementation prevented this up regulation and prevented the formation of MDBs. qRT-PCR analysis confirmed these results. TLR2/4 activates the adapter protein MyD88. The levels of MyD88 were increased by DDC refeeding. The increase of MyD88 was also prevented by SAMe supplementation. Results showed that MyD88-independent TLR3/4-TRIF-IRF3 pathway was not up regulated in the liver of DDC refed mice. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is the down stream protein recruited by the MyD88/IRAK protein complex, and is involved in the regulation of innate immune responses. Results showed a significant increase in the levels of TRAF-6. TRAF-6 activation leads to activation of NFkB and the mitogen-activated protein kinase (MAPK) cascade. The TRAF-6 increase was ameliorated by SAMe supplementation. These results suggest that DDC induces MDB formation through the TLR2/4 and MyD88-dependent signaling pathway. In conclusion, SAMe blocked the over-expression of TLR2/4, and their downstream signaling components MyD88 and TRAF-6. SAMe prevented the DDC-induced up regulation of the TLR signaling pathways, probably by preventing the up regulation of INF-γ receptors by DDC feeding. INFγ stimulates the up regulation of TLR2. The ability of SAMe feeding to prevent TLR signaling up regulation has not been previously described. PMID:20206621

  2. Coordinated and Distinct Functions of Velvet Proteins in Fusarium verticillioides

    PubMed Central

    Lan, Nan; Zhang, Hanxing; Hu, Chengcheng; Wang, Wenzhao; Calvo, Ana M.; Harris, Steven D.; Chen, She

    2014-01-01

    Velvet-domain-containing proteins are broadly distributed within the fungal kingdom. In the corn pathogen Fusarium verticillioides, previous studies showed that the velvet protein F. verticillioides VE1 (FvVE1) is critical for morphological development, colony hydrophobicity, toxin production, and pathogenicity. In this study, tandem affinity purification of FvVE1 revealed that FvVE1 can form a complex with the velvet proteins F. verticillioides VelB (FvVelB) and FvVelC. Phenotypic characterization of gene knockout mutants showed that, as in the case of FvVE1, FvVelB regulated conidial size, hyphal hydrophobicity, fumonisin production, and oxidant resistance, while FvVelC was dispensable for these biological processes. Comparative transcriptional analysis of eight genes involved in the ROS (reactive oxygen species) removal system revealed that both FvVE1 and FvVelB positively regulated the transcription of a catalase-encoding gene, F. verticillioides CAT2 (FvCAT2). Deletion of FvCAT2 resulted in reduced oxidant resistance, providing further explanation of the regulation of oxidant resistance by velvet proteins in the fungal kingdom. PMID:24792348

  3. Cotton Benzoquinon Reductase: Up-Regulation During Early Fiber Development and Heterologous Expresson and Characterization in Pichia Pastoris

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Benzoquinone reductase (BR) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2-D PAGE, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage. These proteins were excis...

  4. Characterization of up-regulated proteases in an industrial recombinant Escherichia coli fermentation.

    PubMed

    Jordan, G L; Harcum, S W

    2002-02-01

    Proteolytic degradation of recombinant proteins is an industry-wide challenge in host organisms such as Escherichia coli. These proteases have been linked to stresses, such as the stringent and heat-shock responses. This study reports the dramatic up-regulation of protease activity in an industrial recombinant E. coli fermentation upon induction. The objective of this project was to detect and characterize up-regulated proteases due to recombinant AXOKINE overexpression upon IPTG induction. AXOKINE is a 22-kDa protein currently in clinical trials as a therapeutic for obesity associated with diabetes. AXOKINE was expressed in both the soluble and inclusion body fractions in E. coli. Sodium dodecyl sulfate gelatin-polyacrylamide gel electrophoresis (SDS-GPAGE) was used to analyze the up-regulated protease activity. Western blot analysis showed degraded AXOKINE in both the soluble and insoluble fractions. Protease inhibitors were used to characterize the proteases. The proteases were ethylenediaminetetraacetic acid (EDTA) sensitive. The protease activity increased in the presence of phenyl-methyl sulfonyl-fluoride (PMSF), a serine protease inhibitor. The incubation buffer composition was varied with respect to Mg2+ and ATP, and the protease activity was ATP independent and Mg2+ dependent. A two-dimensional electrophoresis technique was used to estimate the pI of the proteases to be between 2.9 and 4.0. PMID:12074055

  5. MicroRNA-34a induces a senescence-like change via the down-regulation of SIRT1 and up-regulation of p53 protein in human esophageal squamous cancer cells with a wild-type p53 gene background.

    PubMed

    Ye, Zhimin; Fang, Jun; Dai, Shujun; Wang, Yuezhen; Fu, Zhenfu; Feng, Wei; Wei, Qichun; Huang, Pintong

    2016-01-28

    MiR-34a has been reported as a non-coding RNA universally expressed in normal old cells and a probable suppressor of diverse cancer cells; however, this miRNA's expression and anti-tumor mechanism in esophageal squamous cancer cells (ESCC) remains unclear. We explored these questions in three human ESCC lines, KYSE-450, KYSE-410, and ECa-109, with wild-type p53 and mutant p53 backgrounds. Through a specific stem-loop RT primer for miR-34a, we examined the relevant expression level of miR-34a in these three cell lines using real-time reverse transcription PCR (qRT-PCR). We found that the expression level of miR-34a induced by the DNA damage agent adrmycin (ADR) was both p53- and time-dependent. Following incubation with miR-34a, cellular growth inhibition was exhibited differently in the three cell lines harbored with different p53 backgrounds. Furthermore, the MTT assay demonstrated an miR-34a-related cytotoxic effect in cell growth. Senescence-associated β-galactosidase (SA-β-Gal) staining was used to examine senescence-like phenotypes induced by miR-34a. Mechanistic investigation suggested that the down-regulation of Sirtuin1 (SIRT1) and up-regulation of p53/p21 contributed to the anti-tumor mechanism of miR-34a in wild-type p53 ECa-109 cells, while neither of the apoptosis-related proteins PARP and caspase-3 caused significant changes. In summary, our findings indicated that the intrinsic expression of miR-34a was relatively low and was expressed differently among different p53 backgrounds and ADR treatment times. The anti-tumor effect of miR-34a was primarily dependent on the regulation of SIRT1 and p53/p21 protein, not apoptosis-associated proteins. PMID:26523671

  6. Rosiglitazone ameliorates diffuse axonal injury by reducing loss of tau and up-regulating caveolin-1 expression

    PubMed Central

    Zhao, Yong-lin; Song, Jin-ning; Ma, Xu-dong; Zhang, Bin-fei; Li, Dan-dong; Pang, Hong-gang

    2016-01-01

    Rosiglitazone up-regulates caveolin-1 levels and has neuroprotective effects in both chronic and acute brain injury. Therefore, we postulated that rosiglitazone may ameliorate diffuse axonal injury via its ability to up-regulate caveolin-1, inhibit expression of amyloid-beta precursor protein, and reduce the loss and abnormal phosphorylation of tau. In the present study, intraperitoneal injection of rosiglitazone significantly reduced the levels of amyloid-beta precursor protein and hyperphosphorylated tau (phosphorylated at Ser404(p-tau (S404)), and it increased the expression of total tau and caveolin-1 in the rat cortex. Our results show that rosiglitazone inhibits the expression of amyloid-beta precursor protein and lowers p-tau (S404) levels, and it reduces the loss of total tau, possibly by up-regulating caveolin-1. These actions of rosiglitazone may underlie its neuroprotective effects in the treatment of diffuse axonal injury. PMID:27482223

  7. Triazophos up-regulated gene expression in the female brown planthopper, Nilaparvata lugens.

    PubMed

    Bao, Yan-Yuan; Li, Bao-Ling; Liu, Zhao-Bu; Xue, Jian; Zhu, Zeng-Rong; Cheng, Jia-An; Zhang, Chuan-Xi

    2010-09-01

    The widespread use of insecticides has caused the resurgence of the brown planthopper, Nilaparvata lugens, in Asia. In this study, we investigated an organo-phosphorous insecticide, triazophos, and its ability to induce gene expression variation in female N. lugens nymphs just before emergence. By using the suppression subtractive hybridization method, a triazophos-induced cDNA library was constructed. In total, 402 differentially expressed cDNA clones were obtained. Real-time qPCR analysis confirmed that triazophos up-regulated the expression of six candidate genes at the transcript level in nymphs on day 3 of the 5th instar. These genes encode N. lugens vitellogenin, bystin, multidrug resistance protein (MRP), purine nucleoside phosphorylase (PNP), pyrroline-5-carboxylate reductase (P5CR) and carboxylesterase. Our results imply that the up-regulation of these genes may be involved in the induction of N. lugens female reproduction or resistance to insecticides. PMID:20223245

  8. Geometry optimization for peptides and proteins: comparison of Cartesian and internal coordinates.

    PubMed

    Koslover, Elena F; Wales, David J

    2007-12-21

    We present the results of several benchmarks comparing the relative efficiency of different coordinate systems in optimizing polypeptide geometries. Cartesian, natural internal, and primitive internal coordinates are employed in quasi-Newton minimization for a variety of biomolecules. The peptides and proteins used in these benchmarks range in size from 16 to 999 residues. They vary in complexity from polyalanine helices to a beta-barrel enzyme. We find that the relative performance of the different coordinate systems depends on the parameters of the optimization method, the starting point for the optimization, and the size of the system studied. In general, internal coordinates were found to be advantageous for small peptides. For larger structures, Cartesians appear to be more efficient for empirical potentials where the energy and gradient can be evaluated relatively quickly compared to the cost of the coordinate transformations. PMID:18154373

  9. Geometry optimization for peptides and proteins: Comparison of Cartesian and internal coordinates

    NASA Astrophysics Data System (ADS)

    Koslover, Elena F.; Wales, David J.

    2007-12-01

    We present the results of several benchmarks comparing the relative efficiency of different coordinate systems in optimizing polypeptide geometries. Cartesian, natural internal, and primitive internal coordinates are employed in quasi-Newton minimization for a variety of biomolecules. The peptides and proteins used in these benchmarks range in size from 16 to 999 residues. They vary in complexity from polyalanine helices to a β-barrel enzyme. We find that the relative performance of the different coordinate systems depends on the parameters of the optimization method, the starting point for the optimization, and the size of the system studied. In general, internal coordinates were found to be advantageous for small peptides. For larger structures, Cartesians appear to be more efficient for empirical potentials where the energy and gradient can be evaluated relatively quickly compared to the cost of the coordinate transformations.

  10. Mg2+ coordinating dynamics in Mg:ATP fueled motor proteins

    NASA Astrophysics Data System (ADS)

    Bojovschi, A.; Liu, Ming S.; Sadus, Richard J.

    2014-03-01

    The coordination of Mg2+ with the triphosphate group of adenosine triphosphate (ATP) in motor proteins is investigated using data mining and molecular dynamics. The possible coordination structures available from crystal data for actin, myosin, RNA polymerase, DNA polymerase, DNA helicase, and F1-ATPase are verified and investigated further by molecular dynamics. Coordination states are evaluated using structural analysis and quantified by radial distribution functions, coordination numbers, and pair interaction energy calculations. The results reveal a diverse range of both transitory and stable coordination arrangements between Mg2+ and ATP. The two most stable coordinating states occur when Mg2+ coordinates two or three oxygens from the triphosphate group of ATP. Evidence for five-site coordination is also reported involving water in addition to the triphosphate group. The stable states correspond to a pair interaction energy of either ˜-2750 kJ/mol or -3500 kJ/mol. The role of water molecules in the hydration shell surrounding Mg2+ is also reported.

  11. Up-Regulation of the Cardiac Lipid Metabolism at the Onset of Heart Failure

    PubMed Central

    AbdAlla, Said; Fu, Xuebin; Elzahwy, Sherif S; Klaetschke, Kristin; Streichert, Thomas; Quitterer, Ursula

    2011-01-01

    Chronic pressure overload and atherosclerosis are primary etiologic factors for cardiac hypertrophy and failure. However, mechanisms underlying the transition from hypertrophy to heart failure are incompletely understood. We analyzed the development of heart failure in mice with chronic pressure overload induced by aortic constriction and compared the results with aged apolipoprotein E-deficient mice suffering from advanced atherosclerosis. We combined cardiac function analysis by echocardiography and invasive hemodynamics with a comprehensive microarray gene expression study (GSE25765-8). The microarray data showed that the onset of heart failure induced by pressure overload or advanced atherosclerosis was accompanied by a strong up-regulation of key lipid metabolizing enzymes involved in fat synthesis, storage and oxidation. Cardiac lipid overload may be involved in the progression of heart failure by enhancing cardiomyocyte death. Up-regulation of the cardiac lipid metabolism was related to oxygen and ATP depletion of failing hearts because anti-ischemic treatment with ranolazine normalized the cardiac lipid metabolism and improved cardiac function. Vice versa, inhibition of cellular respiration and ATP generation by mild thiol-blocking with cystamine triggered the cardiac lipid metabolism and caused signs of heart failure. Cardiac tissue specimens of patients with heart failure also showed high protein levels of key fat metabolizing enzymes as well as lipid accumulation. Taken together, our data strongly indicate that up-regulation of the cardiac lipid metabolism and myocardial lipid overload are underlying the development of heart failure. PMID:21711241

  12. Up-regulation of miR-98 and unraveling regulatory mechanisms in gestational diabetes mellitus.

    PubMed

    Cao, Jing-Li; Zhang, Lu; Li, Jian; Tian, Shi; Lv, Xiao-Dan; Wang, Xue-Qin; Su, Xing; Li, Ying; Hu, Yi; Ma, Xu; Xia, Hong-Fei

    2016-01-01

    MiR-98 expression was up-regulated in kidney in response to early diabetic nephropathy in mouse and down-regulated in muscle in type 2 diabetes in human. However, the expression prolife and functional role of miR-98 in human gestational diabetes mellitus (GDM) remained unclear. Here, we investigated its expression and function in placental tissues from GDM patients and the possible molecular mechanisms. The results showed that miR-98 was up-regulated in placentas from GDM patients compared with normal placentas. MiR-98 over-expression increased global DNA methylational level and miR-98 knockdown reduced global DNA methylational level. Further investigation revealed that miR-98 could inhibit Mecp2 expression by binding the 3'-untranslated region (UTR) of methyl CpG binding protein 2 (Mecp2), and then led to the expression dysregulation of canonical transient receptor potential 3 (Trpc3), a glucose uptake related gene. More importantly, in vivo analysis found that the expression level of Mecp2 and Trpc3 in placental tissues from GDM patients, relative to the increase of miR-98, was diminished, especially for GDM patients over the age of 35 years. Collectively, up-regulation of miR-98 in the placental tissues of human GDM is linked to the global DNA methylation via targeting Mecp2, which may imply a novel regulatory mechanism in GDM. PMID:27573367

  13. Up-regulation of miR-98 and unraveling regulatory mechanisms in gestational diabetes mellitus

    PubMed Central

    Cao, Jing-Li; Zhang, Lu; Li, Jian; Tian, Shi; Lv, Xiao-Dan; Wang, Xue-Qin; Su, Xing; Li, Ying; Hu, Yi; Ma, Xu; Xia, Hong-Fei

    2016-01-01

    MiR-98 expression was up-regulated in kidney in response to early diabetic nephropathy in mouse and down-regulated in muscle in type 2 diabetes in human. However, the expression prolife and functional role of miR-98 in human gestational diabetes mellitus (GDM) remained unclear. Here, we investigated its expression and function in placental tissues from GDM patients and the possible molecular mechanisms. The results showed that miR-98 was up-regulated in placentas from GDM patients compared with normal placentas. MiR-98 over-expression increased global DNA methylational level and miR-98 knockdown reduced global DNA methylational level. Further investigation revealed that miR-98 could inhibit Mecp2 expression by binding the 3′-untranslated region (UTR) of methyl CpG binding protein 2 (Mecp2), and then led to the expression dysregulation of canonical transient receptor potential 3 (Trpc3), a glucose uptake related gene. More importantly, in vivo analysis found that the expression level of Mecp2 and Trpc3 in placental tissues from GDM patients, relative to the increase of miR-98, was diminished, especially for GDM patients over the age of 35 years. Collectively, up-regulation of miR-98 in the placental tissues of human GDM is linked to the global DNA methylation via targeting Mecp2, which may imply a novel regulatory mechanism in GDM. PMID:27573367

  14. Nitric oxide up-regulates endothelial expression of angiotensin II type 2 receptors.

    PubMed

    Dao, Vu Thao-Vi; Medini, Sawsan; Bisha, Marion; Balz, Vera; Suvorava, Tatsiana; Bas, Murat; Kojda, Georg

    2016-07-15

    Increasing vascular NO levels following up-regulation of endothelial nitric oxide synthase (eNOS) is considered beneficial in cardiovascular disease. Whether such beneficial effects exerted by increased NO-levels include the vascular renin-angiotensin system remains elucidated. Exposure of endothelial cells originated from porcine aorta, mouse brain and human umbilical veins to different NO-donors showed that expression of the angiotensin-II-type-2-receptor (AT2) mRNA and protein is up-regulated by activation of soluble guanylyl cyclase, protein kinase G and p38 mitogen-activated protein kinase without changing AT2 mRNA stability. In mice, endothelial-specific overexpression of eNOS stimulated, while chronic treatment with the NOS-blocker l-nitroarginine inhibited AT2 expression. The NO-induced AT2 up-regulation was associated with a profound inhibition of angiotensin-converting enzyme (ACE)-activity. In endothelial cells this reduction of ACE-activity was reversed by either the AT2 antagonist PD 123119 or by inhibition of transcription with actinomycin D. Furthermore, in C57Bl/6 mice an acute i.v. bolus of l-nitroarginine did not change AT2-expression and ACE-activity suggesting that inhibition of ACE-activity by endogenous NO is crucially dependent on AT2 protein level. Likewise, three weeks of either voluntary or forced exercise training increased AT2 expression and reduced ACE-activity in C57Bl/6 but not in mice lacking eNOS suggesting significance of this signaling interaction for vascular physiology. Finally, aortic AT2 expression is about 5 times greater in female as compared to male C57Bl/6 and at the same time aortic ACE activity is reduced in females by more than 50%. Together these findings imply that endothelial NO regulates AT2 expression and that AT2 may regulate ACE-activity. PMID:27235748

  15. Drug-induced up-regulation of dopamine D2 receptors on cultured cells.

    PubMed

    Starr, S; Kozell, L B; Neve, K A

    1995-08-01

    Ligand-induced up-regulation of recombinant dopamine D2 receptors was assessed using C6 glioma cells stably expressing the short (415-amino-acid; D2s) and long (444-amino-acid; D2L) forms of the receptor. Overnight treatment of C6-D2L cells with N-propylnorapomorphine (NPA) caused a time- and concentration-dependent increase in the density of receptors, as assessed by the binding of radioligand to membranes prepared from the cells, with no change in the affinity of the receptors for the radioligand. The effect of 10 microM NPA was maximal after 10 h, at which time the density of D2L receptors was more than doubled. The agonists dopamine and quinpirole also increased the density of D2L receptors. The receptor up-regulation was not specific for agonists, because the antagonists epidepride, sulpiride, and domperidone caused smaller (30-60%) increases in receptor density. Prolonged treatment with 10 microM NPA desensitized D2L receptors, as evidenced by a reduced ability of dopamine to inhibit adenylyl cyclase, whereas treatment with sulpiride was associated with an enhanced responsiveness to dopamine. The magnitude of NPA-induced receptor up-regulation in each of four clonal lines of C6-D2L cells (mean increase, 80%) was greater than in all four lines of C6-D2S cells (33%). Inactivation of pertussis toxin-sensitive G proteins had no effect on the basal density of D2L receptors or on the NPA-induced receptor up-regulation. Treatment with 5 micrograms/ml of cycloheximide, on the other hand, decreased the basal density of receptors and attenuated, but did not prevent, the NPA-induced increase.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7616211

  16. [HIV-1 infection up-regulating expression of interferon-stimulated gene 15 in cell lines].

    PubMed

    Wu, Huan-mei; Sun, Jun; Meng, Zhe-feng; Zhang, Xiao-yan; Xu, Jian-qing

    2013-09-01

    To investigate whether HIV-1 infection affects expression of interferon-stimulated gene 15 (ISG15) and determine the antiviral effect of ISG15 in vitro, ISG15 expression at transcription and protein level and supernatant p24 of HIV-1 was detected in various HIV-1 infected or transfected cell lines, respec tively. HIV-1 molecular clone pNL4-3 was used to transfect 293T, TZM-bl and HeLa cells while HIV-1 pseudo-typed virus was used to infect Jurkat, MT-1 and THP-1 cells. After twenty-four hours post infection or transfection, cells were harvested for extraction of total RNAs and subsequently used in real time PCR for quantification of ISG15 transcriptional expression. After forty-eight hours post infection or transfection, cells were harvested for extraction of total proteins to detect ISG15 protein expression. A significant up-regulation of ISG15 at transcription level was observed in HIV-1 infected or transfected cell lines, particulaly in THP-1 and TZM-bl cells. Up-regulation of ISG15 protein was observed only in TZM-bl cell. Cotransfection of ISG15 and HIV-1 indicated that ISG15 inhibited production of HIV-1 progeny virus in a dose and time depend manner in 293T cell but not TZM-bl cell. These results revealed upregulating ISG15 expression in transcriptional level and potential antagonistic mechanism against ISG15 by HIV-1 infection, simultanelusly. PMID:24386835

  17. Maternal obesity is associated with ovarian inflammation and up-regulation of early growth response factor 1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Obesity impairs reproductive functions through multiple mechanisms, possibly through disruption of ovarian function. We hypothesized that increased adiposity will lead to a pro-inflammatory gene signature and up-regulation of Egr-1 protein in ovaries from obese (OB, n=7) compared to lean (LN, n=10) ...

  18. Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

    PubMed Central

    Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

    2015-01-01

    We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934

  19. Coordination contributions to protein stability in metal-substituted carbonic anhydrase.

    PubMed

    Lisi, George P; Hughes, Russell P; Wilcox, Dean E

    2016-09-01

    Contributions of the active site metal to the stability of carbonic anhydrase (CA) were quantified by differential scanning calorimetry and complementary unfolding measurements of CA substituted with Co(2+), Cd(2+), Cu(2+), Ni(2+) and Mn(2+). The metal ions stabilize the protein to different extent, with the highest stability provided by the native Zn(2+). This additional stability does not correlate with the enthalpy of the three metal-imidazole (His) bonds at the active site or other properties of the metal ions (charge density, hydration enthalpy). However, DFT calculations reveal an energetic penalty associated with metal coordination at the active site, and the magnitude of this penalty correlates inversely with metal contributions to the stability of the protein. While the affinity of CA for metal ions generally reflects the Irving-Williams series, the additional thermal stability provided by metal ions is modulated by the rigid His3 coordination that is imposed at the protein site. PMID:27350155

  20. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy.

    PubMed

    Wang, Hao; Sun, Ruo-Qiong; Camera, Daria; Zeng, Xiao-Yi; Jo, Eunjung; Chan, Stanley M H; Herbert, Terence P; Molero, Juan C; Ye, Ji-Ming

    2016-07-01

    The accumulation of unfolded proteins within the endoplasmic reticulum (ER) causes ER stress and activation of unfolded protein response (UPR). This response can trigger ER-associated degradation and autophagy, which clear unfolded proteins and restore protein homeostasis. Recently, it has become clear that ubiquitination plays an important role in the regulation of autophagy. In the present study, we investigated how the E3 ubiquitin ligase neural precursor cell-expressed, developmentally down-regulated protein 4-2 (Nedd4-2) interacts with ER stress and autophagy. In mice, we found that an increase in the expression of Nedd4-2, which was concomitant with the activation of the UPR and autophagy, was caused by a prolonged high-fructose and high-fat diet that induces ER stress in the liver. Pharmacologic induction of ER stress also led to an increase in Nedd4-2 expression in cultured cells, which was coincident with UPR and autophagy activation. The inhibition of inositol-requiring enzyme 1 significantly suppressed Nedd4-2 expression. Moreover, increased Nedd4-2 expression in vivo was closely associated with the activation of inositol-requiring enzyme 1 and increased expression of the spliced form of X-box binding protein 1. Furthermore, knockdown of Nedd4-2 in cultured cells suppressed both basal autophagy and ER stress-induced autophagy, whereas overexpression of Nedd4-2-induced autophagy. Taken together, our findings provide evidence that Nedd4-2 is up-regulated in response to ER stress by the spliced form of X-box binding protein 1 and that this is important in the induction of an appropriate autophagic response.-Wang, H. Sun, R.-Q., Camera, D., Zeng, X.-Y., Jo, E., Chan, S. M. H., Herbert, T. P., Molero, J. C., Ye, J.-M. Endoplasmic reticulum stress up-regulates Nedd4-2 to induce autophagy. PMID:27022162

  1. Erbb2 up-regulation of ADAM12 expression accelerates skin cancer progression.

    PubMed

    Rao, Velidi H; Vogel, Kristen; Yanagida, Jodi K; Marwaha, Nitin; Kandel, Amrit; Trempus, Carol; Repertinger, Susan K; Hansen, Laura A

    2015-10-01

    Solar ultraviolet (UV) radiation can cause severe damage to the skin and is the primary cause of most skin cancer. UV radiation causes DNA damage leading to mutations and also activates the Erbb2/HER2 receptor through indirect mechanisms involving reactive oxygen species. We hypothesized that Erbb2 activation accelerates the malignant progression of UV-induced skin cancer. Following the induction of benign squamous papillomas by UV exposure of v-ras(Ha) transgenic Tg.AC mice, mice were treated topically with the Erbb2 inhibitor AG825 and tumor progression monitored. AG825 treatment reduced tumor volume, increased tumor regression, and delayed the development of malignant squamous cell carcinoma (SCC). Progression to malignancy was associated with increased Erbb2 and ADAM12 (A Disintegin And Metalloproteinase 12) transcripts and protein, while inhibition of Erbb2 blocked the increase in ADAM12 message upon malignant progression. Similarly, human SCC and SCC cell lines had increased ADAM12 protein and transcripts when compared to normal controls. To determine whether Erbb2 up-regulation of ADAM12 contributed to malignant progression of skin cancer, Erbb2 expression was modulated in cultured SCC cells using forced over-expression or siRNA targeting, demonstrating up-regulation of ADAM12 by Erbb2. Furthermore, ADAM12 transfection or siRNA targeting revealed that ADAM12 increased both the migration and invasion of cutaneous SCC cells. Collectively, these results suggest Erbb2 up-regulation of ADAM12 as a novel mechanism contributing to the malignant progression of UV-induced skin cancer. Inhibition of Erbb2/HER2 reduced tumor burden, increased tumor regression, and delayed the progression of benign skin tumors to malignant SCC in UV-exposed mice. Inhibition of Erbb2 suppressed the increase in metalloproteinase ADAM12 expression in skin tumors, which in turn increased migration and tumor cell invasiveness. PMID:24798404

  2. HBx sensitizes hepatocellular carcinoma cells to lapatinib by up-regulating ErbB3

    PubMed Central

    Yen, Chia-Jui; Chen, Wen-Shu; Huang, Wei-Chien

    2016-01-01

    Poor prognosis of hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) involves HBV X protein (HBx)-induced tumor progression. HBx also contributes to chemo-resistance via inducing the expressions of anti-apoptosis and multiple drug resistance genes. However, the impact of HBx expression on the therapeutic efficacy of various receptor tyrosine kinase inhibitors remains unknown. In this study, our data showed that HBx overexpression did not alter the cellular sensitivity of HCC cell lines to sorafenib but unexpectedly enhanced the cell death induced by EGFR family inhibitors, including gefitinib, erlotinib, and lapatinib due to ErbB3 up-regulation. Mechanistically, HBx transcriptionally up-regulates ErbB3 expression in a NF-κB dependent manner. In addition, HBx also physically interacts with ErbB2 and ErbB3 proteins and enhances the formation of ErbB2/ErbB3 heterodimeric complex. The cell viability of HBx-overexpressing cells was decreased by silencing ErbB3 expression, further revealing the pivotal role of ErbB3 in HBx-mediated cell survival. Our data suggest that HBx shifts the oncogenic addiction of HCC cells to ErbB2/ErbB3 signaling pathway via inducing ErbB3 expression and thereby enhances their sensitivity to EGFR/ErbB2 inhibitors. PMID:26595522

  3. Up-regulation of the homophilic adhesion molecule sidekick-1 in podocytes contributes to glomerulosclerosis.

    PubMed

    Kaufman, Lewis; Potla, Uma; Coleman, Sarah; Dikiy, Stanislav; Hata, Yutaka; Kurihara, Hidetake; He, John C; D'Agati, Vivette D; Klotman, Paul E

    2010-08-13

    Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, alpha-actinin-4, nephrin, JAM4, and beta-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function. PMID:20562105

  4. Midazolam inhibits the hypoxia-induced up-regulation of erythropoietin in the central nervous system.

    PubMed

    Matsuyama, Tomonori; Tanaka, Tomoharu; Tatsumi, Kenichiro; Daijo, Hiroki; Kai, Shinichi; Harada, Hiroshi; Fukuda, Kazuhiko

    2015-08-15

    Erythropoietin (EPO), a regulator of red blood cell production, is endogenously expressed in the central nervous system. It is mainly produced by astrocytes under hypoxic conditions and has proven to have neuroprotective and neurotrophic effects. In the present study, we investigated the effect of midazolam on EPO expression in primary cultured astrocytes and the mouse brain. Midazolam was administered to 6-week-old BALB/c male mice under hypoxic conditions and pregnant C57BL/6N mice under normoxic conditions. Primary cultured astrocytes were also treated with midazolam under hypoxic conditions. The expression of EPO mRNA in mice brains and cultured astrocytes was studied. In addition, the expression of hypoxia-inducible factor (HIF), known as the main regulator of EPO, was evaluated. Midazolam significantly reduced the hypoxia-induced up-regulation of EPO in BALB/c mice brains and primary cultured astrocytes and suppressed EPO expression in the fetal brain. Midazolam did not affect the total amount of HIF proteins but significantly inhibited the nuclear expression of HIF-1α and HIF-2α proteins. These results demonstrated the suppressive effects of midazolam on the hypoxia-induced up-regulation of EPO both in vivo and in vitro. PMID:26001375

  5. Utrophin Up-Regulation by an Artificial Transcription Factor in Transgenic Mice

    PubMed Central

    Mattei, Elisabetta; Corbi, Nicoletta; Di Certo, Maria Grazia; Strimpakos, Georgios; Severini, Cinzia; Onori, Annalisa; Desantis, Agata; Libri, Valentina; Buontempo, Serena; Floridi, Aristide; Fanciulli, Maurizio; Baban, Dilair; Davies, Kay E.; Passananti, Claudio

    2007-01-01

    Duchenne Muscular Dystrophy (DMD) is a severe muscle degenerative disease, due to absence of dystrophin. There is currently no effective treatment for DMD. Our aim is to up-regulate the expression level of the dystrophin related gene utrophin in DMD, complementing in this way the lack of dystrophin functions. To this end we designed and engineered several synthetic zinc finger based transcription factors. In particular, we have previously shown that the artificial three zinc finger protein named Jazz, fused with the appropriate effector domain, is able to drive the transcription of a test gene from the utrophin promoter “A”. Here we report on the characterization of Vp16-Jazz-transgenic mice that specifically over-express the utrophin gene at the muscular level. A Chromatin Immunoprecipitation assay (ChIP) demonstrated the effective access/binding of the Jazz protein to active chromatin in mouse muscle and Vp16-Jazz was shown to be able to up-regulate endogenous utrophin gene expression by immunohistochemistry, western blot analyses and real-time PCR. To our knowledge, this is the first example of a transgenic mouse expressing an artificial gene coding for a zinc finger based transcription factor. The achievement of Vp16-Jazz transgenic mice validates the strategy of transcriptional targeting of endogenous genes and could represent an exclusive animal model for use in drug discovery and therapeutics. PMID:17712422

  6. Interleukin-20 Promotes Migration of Bladder Cancer Cells through Extracellular Signal-regulated Kinase (ERK)-mediated MMP-9 Protein Expression Leading to Nuclear Factor (NF-κB) Activation by Inducing the Up-regulation of p21WAF1 Protein Expression*

    PubMed Central

    Lee, Se-Jung; Cho, Seok-Cheol; Lee, Eo-Jin; Kim, Sangtae; Lee, Soo-Bok; Lim, Jung-Hyurk; Choi, Yung Hyun; Kim, Wun-Jae; Moon, Sung-Kwon

    2013-01-01

    The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21WAF1 was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21WAF1 function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21WAF1 is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer. PMID:23271730

  7. Up-regulation of FGF23 release by aldosterone.

    PubMed

    Zhang, Bingbing; Umbach, Anja T; Chen, Hong; Yan, Jing; Fakhri, Hajar; Fajol, Abul; Salker, Madhuri S; Spichtig, Daniela; Daryadel, Arezoo; Wagner, Carsten A; Föller, Michael; Lang, Florian

    2016-02-01

    The fibroblast growth factor (FGF23) plasma level is high in cardiac and renal failure and is associated with poor clinical prognosis of these disorders. Both diseases are paralleled by hyperaldosteronism. Excessive FGF23 levels and hyperaldosteronism are further observed in Klotho-deficient mice. The present study explored a putative aldosterone sensitivity of Fgf23 transcription and secretion the putative involvement of the aldosterone sensitive serum & glucocorticoid inducible kinase SGK1, SGK1 sensitive transcription factor NFκB and store operated Ca(2+) entry (SOCE). Serum FGF23 levels were determined by ELISA in mice following sham treatment or exposure to deoxycorticosterone acetate (DOCA) or salt depletion. In osteoblastic UMR106 cells transcript levels were quantified by qRT-PCR, cytosolic Ca(2+) concentration utilizing Fura-2-fluorescence, and SOCE from Ca(2+) entry following store depletion by thapsigargin. As a result, DOCA treatment and salt depletion of mice elevated the serum C-terminal FGF23 concentration. In UMR106 cells aldosterone enhanced and spironolactone decreased SOCE. Aldosterone further increased Fgf23 transcript levels in UMR106 cells, an effect reversed by mineralocorticoid receptor blockers spironolactone and eplerenone, SGK1 inhibitor EMD638683, NFκB-inhibitor withaferin A, and Ca(2+) channel blocker YM58483. In conclusion, Fgf23 expression is up-regulated by aldosterone, an effect sensitive to SGK1, NFκB and store-operated Ca(2+) entry. PMID:26773502

  8. Conformational dependence of collagenase (matrix metalloproteinase-1) up-regulation by elastin peptides in cultured fibroblasts.

    PubMed

    Brassart, B; Fuchs, P; Huet, E; Alix, A J; Wallach, J; Tamburro, A M; Delacoux, F; Haye, B; Emonard, H; Hornebeck, W; Debelle, L

    2001-02-16

    We have established that treatment of cultured human skin fibroblasts with tropoelastin or with heterogenic peptides, obtained after organo-alkaline or leukocyte elastase hydrolysis of insoluble elastin, induces a high expression of pro-collagenase-1 (pro-matrix metalloproteinase-1 (pro-MMP-1)). The identical effect was achieved after stimulation with a VGVAPG synthetic peptide, reflecting the elastin-derived domain known to bind to the 67-kDa elastin-binding protein. This clearly indicated involvement of this receptor in the described phenomenon. This notion was further reinforced by the fact that elastin peptides-dependent MMP-1 up-regulation has not been demonstrated in cultures preincubated with 1 mm lactose, which causes shedding of the elastin-binding protein and with pertussis toxin, which blocks the elastin-binding protein-dependent signaling pathway involving G protein, phospholipase C, and protein kinase C. Moreover, we demonstrated that diverse peptides maintaining GXXPG sequences can also induce similar cellular effects as a "principal" VGVAPG ligand of the elastin receptor. Results of our biophysical studies suggest that this peculiar consensus sequence stabilizes a type VIII beta-turn in several similar, but not identical, peptides that maintain a sufficient conformation to be recognized by the elastin receptor. We have also established that GXXPG elastin-derived peptides, in addition to pro-MMP-1, cause up-regulation of pro-matrix metalloproteinase-3 (pro-stromelysin 1). Furthermore, we found that the presence of plasmin in the culture medium activated these MMP proenzymes, leading to a consequent degradation of collagen substrate. Our results may be, therefore, relevant to pathobiology of inflammation, in which elastin-derived peptides bearing the GXXPG conformation (created after leukocyte-dependent proteolysis) bind to the elastin receptor of local fibroblasts and trigger signals leading to expression and activation of MMP-1 and MMP-3, which in

  9. Ankyrin Repeat Domain 1 is Up-regulated During Hepatitis C Virus Infection and Regulates Hepatitis C Virus Entry.

    PubMed

    Than, Thoa T; Tran, Giao V Q; Son, Kidong; Park, Eun-Mee; Kim, Seungtaek; Lim, Yun-Sook; Hwang, Soon B

    2016-01-01

    Hepatitis C virus (HCV) is highly dependent on host proteins for its own propagation. By transcriptome sequencing (RNA-Seq) analysis, we identified 30 host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected and characterized ankyrin repeat domain 1 (ANKRD1). Here, we showed that protein expression of ANKRD1 was up-regulated in HCVcc-infected cells. We further showed that protein expression level of ANKRD1 was increased by nonstructural 5A (NS5A) protein. ANKRD1 specifically interacted with NS5A both in vitro and coimmunoprecipitation assays. Protein interaction was mediated through the domain II of NS5A and the C-terminal region of ANKRD1. Promoter activity of ANKRD1 was also increased by NS5A protein. Moreover, up-regulation of ANKRD1 expression was mediated through alteration in intracellular calcium homeostasis and ER stress in HCVcc-infected cells. We showed that silencing of ANKRD1 impaired HCV propagation without affecting HCV replication. By using HCV-like infectious particle (HCV-LP), we demonstrated that HCV single-cycle infection was drastically impaired in ANKRD1 knockdown cells. Finally, we verified that ANKRD1 was required for HCV entry. These data suggest that HCV coopts ANKRD1 for its own propagation and up-regulation of ANKRD1 may contribute to HCV-mediated liver pathogenesis. PMID:26860204

  10. Ankyrin Repeat Domain 1 is Up-regulated During Hepatitis C Virus Infection and Regulates Hepatitis C Virus Entry

    PubMed Central

    Than, Thoa T.; Tran, Giao V. Q.; Son, Kidong; Park, Eun-Mee; Kim, Seungtaek; Lim, Yun-Sook; Hwang, Soon B.

    2016-01-01

    Hepatitis C virus (HCV) is highly dependent on host proteins for its own propagation. By transcriptome sequencing (RNA-Seq) analysis, we identified 30 host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected and characterized ankyrin repeat domain 1 (ANKRD1). Here, we showed that protein expression of ANKRD1 was up-regulated in HCVcc-infected cells. We further showed that protein expression level of ANKRD1 was increased by nonstructural 5A (NS5A) protein. ANKRD1 specifically interacted with NS5A both in vitro and coimmunoprecipitation assays. Protein interaction was mediated through the domain II of NS5A and the C-terminal region of ANKRD1. Promoter activity of ANKRD1 was also increased by NS5A protein. Moreover, up-regulation of ANKRD1 expression was mediated through alteration in intracellular calcium homeostasis and ER stress in HCVcc-infected cells. We showed that silencing of ANKRD1 impaired HCV propagation without affecting HCV replication. By using HCV-like infectious particle (HCV-LP), we demonstrated that HCV single-cycle infection was drastically impaired in ANKRD1 knockdown cells. Finally, we verified that ANKRD1 was required for HCV entry. These data suggest that HCV coopts ANKRD1 for its own propagation and up-regulation of ANKRD1 may contribute to HCV-mediated liver pathogenesis. PMID:26860204

  11. φXANES: In vivo imaging of metal-protein coordination environments

    PubMed Central

    James, Simon A.; Hare, Dominic J.; Jenkins, Nicole L.; de Jonge, Martin D.; Bush, Ashley I.; McColl, Gawain

    2016-01-01

    We have developed an X-ray absorption near edge structure spectroscopy method using fluorescence detection for visualizing in vivo coordination environments of metals in biological specimens. This approach, which we term fluorescence imaging XANES (φXANES), allows us to spatially depict metal-protein associations in a native, hydrated state whilst avoiding intrinsic chemical damage from radiation. This method was validated using iron-challenged Caenorhabditis elegans to observe marked alterations in redox environment. PMID:26861174

  12. φXANES: In vivo imaging of metal-protein coordination environments.

    PubMed

    James, Simon A; Hare, Dominic J; Jenkins, Nicole L; de Jonge, Martin D; Bush, Ashley I; McColl, Gawain

    2016-01-01

    We have developed an X-ray absorption near edge structure spectroscopy method using fluorescence detection for visualizing in vivo coordination environments of metals in biological specimens. This approach, which we term fluorescence imaging XANES (φXANES), allows us to spatially depict metal-protein associations in a native, hydrated state whilst avoiding intrinsic chemical damage from radiation. This method was validated using iron-challenged Caenorhabditis elegans to observe marked alterations in redox environment. PMID:26861174

  13. φXANES: In vivo imaging of metal-protein coordination environments

    NASA Astrophysics Data System (ADS)

    James, Simon A.; Hare, Dominic J.; Jenkins, Nicole L.; de Jonge, Martin D.; Bush, Ashley I.; McColl, Gawain

    2016-02-01

    We have developed an X-ray absorption near edge structure spectroscopy method using fluorescence detection for visualizing in vivo coordination environments of metals in biological specimens. This approach, which we term fluorescence imaging XANES (φXANES), allows us to spatially depict metal-protein associations in a native, hydrated state whilst avoiding intrinsic chemical damage from radiation. This method was validated using iron-challenged Caenorhabditis elegans to observe marked alterations in redox environment.

  14. Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes.

    PubMed

    Mares, V; Malík, R; Lisá, V; Sedo, A

    2005-05-20

    Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2'7'dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity. PMID:15893589

  15. Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation.

    PubMed

    Li, Shih-Wen; Yang, Tsuey-Ching; Lai, Chien-Chen; Huang, Su-Hua; Liao, Jun-Ming; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2014-09-01

    Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05 μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2'5',-OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2'5',-oligoadenylate synthetase (2'5',-OAS) expression. Treatment with aloe-emodin could control influenza infection in humans. PMID:24877694

  16. The Absence of Core Fucose Up-regulates GnT-III and Wnt Target Genes

    PubMed Central

    Kurimoto, Ayako; Kitazume, Shinobu; Kizuka, Yasuhiko; Nakajima, Kazuki; Oka, Ritsuko; Fujinawa, Reiko; Korekane, Hiroaki; Yamaguchi, Yoshiki; Wada, Yoshinao; Taniguchi, Naoyuki

    2014-01-01

    Glycans play key roles in a variety of protein functions under normal and pathological conditions, but several glycosyltransferase-deficient mice exhibit no or only mild phenotypes due to redundancy or compensation of glycan functions. However, we have only a limited understanding of the underlying mechanism for these observations. Our previous studies indicated that 70% of Fut8-deficient (Fut8−/−) mice that lack core fucose structure die within 3 days after birth, but the remainder survive for up to several weeks although they show growth retardation as well as emphysema. In this study, we show that, in mouse embryonic fibroblasts (MEFs) from Fut8−/− mice, another N-glycan branching structure, bisecting GlcNAc, is specifically up-regulated by enhanced gene expression of the responsible enzyme N-acetylglucosaminyltransferase III (GnT-III). As candidate target glycoproteins for bisecting GlcNAc modification, we confirmed that level of bisecting GlcNAc on β1-integrin and N-cadherin was increased in Fut8−/− MEFs. Moreover using mass spectrometry, glycan analysis of IgG1 in Fut8−/− mouse serum demonstrated that bisecting GlcNAc contents were also increased by Fut8 deficiency in vivo. As an underlying mechanism, we found that in Fut8−/− MEFs Wnt/β-catenin signaling is up-regulated, and an inhibitor against Wnt signaling was found to abrogate GnT-III expression, indicating that Wnt/β-catenin is involved in GnT-III up-regulation. Furthermore, various oxidative stress-related genes were also increased in Fut8−/− MEFs. These data suggest that Fut8−/− mice adapted to oxidative stress, both ex vivo and in vivo, by inducing various genes including GnT-III, which may compensate for the loss of core fucose functions. PMID:24619415

  17. Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P

    PubMed Central

    Kovrigina, Elizaveta; Wesolowski, Donna; Altman, Sidney

    2003-01-01

    The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited. PMID:12552092

  18. A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins.

    PubMed

    Barraud, Pierre; Schubert, Mario; Allain, Frédéric H-T

    2012-06-01

    Zinc is the second most abundant metal ion incorporated in proteins, and is in many cases a crucial component of protein three-dimensional structures. Zinc ions are frequently coordinated by cysteine and histidine residues. Whereas cysteines bind to zinc via their unique S(γ) atom, histidines can coordinate zinc with two different coordination modes, either N(δ1) or N(ε2) is coordinating the zinc ion. The determination of this coordination mode is crucial for the accurate structure determination of a histidine-containing zinc-binding site by NMR. NMR chemical shifts contain a vast amount of information on local electronic and structural environments and surprisingly their utilization for the determination of the coordination mode of zinc-ligated histidines has been limited so far to (15)N nuclei. In the present report, we observed that the (13)C chemical shifts of aromatic carbons in zinc-ligated histidines represent a reliable signature of their coordination mode. Using a statistical analysis of (13)C chemical shifts, we show that (13)C(δ2) chemical shift is sensitive to the histidine coordination mode and that the chemical shift difference δ{(13)C(ε1)} - δ{(13)C(δ2)} provides a reference-independent marker of this coordination mode. The present approach allows the direct determination of the coordination mode of zinc-ligated histidines even with non-isotopically enriched protein samples and without any prior structural information. PMID:22528293

  19. Up-regulated expression of Ran reveals its potential role to deltamethrin stress in Kc cells.

    PubMed

    Liu, Wei; Xu, Qin; Chi, Qingping; Hu, Junli; Li, Fengliang; Cheng, Luogen

    2016-05-25

    The GTP-binding nuclear protein Ran has mostly been reported to be an essential player in nuclear transport, chromosome alignment, microtubule dynamics, centrosome duplication, kinetochore attachment of microtubules, nuclear-envelope dynamics, and phagocytosis. However, until now, there has been no report showing the involvement of Ran in DM stress. In this paper, two-dimensional electrophoresis analysis showed that the expression level of Ran in Kc cells in response to DM was higher than that in the control group. In addition, quantitative analysis using real-time PCR revealed that the expression of Ran was obviously up-regulated at various concentrations of DM. Western blot analysis showed that Ran was up-regulated 2.27-fold over the control at 48h. Because we still could not pinpoint whether Ran was actually involved in DM stress reaction, to further verify the role of Ran in stress reaction, RNA interference and cell transfection were utilized. Overexpression of Ran in cells conferred a degree of protection against DM after 72h. Furthermore, interference with Ran significantly decrease cell viability. All of the above findings strongly imply that Ran may participate in the development of stress reaction to DM. Therefore, investigating the possible role of Ran in DM stress will broaden our limited knowledge regarding DM stress inducible genes. PMID:26924245

  20. Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells.

    PubMed

    Spitzner, Melanie; Martins, Joana Raquel; Soria, René Barro; Ousingsawat, Jiraporn; Scheidt, Kerstin; Schreiber, Rainer; Kunzelmann, Karl

    2008-03-21

    Ion channels like voltage-gated ether-á-go-go (Eag1) K(+) channels or Ca(2+)-activated Cl(-) channels have been shown to support cell proliferation. Bestrophin 1 (Best1) has been proposed to form Ca(2+)-activated Cl(-) channels in epithelial cells. Here we show that original T(84) colonic carcinoma cells grow slowly (T(84)-slow) and express low amounts of Eag1 and Best1, whereas spontaneously transformed T(84) cells grow fast (T(84)-fast) and express high levels of both proteins. Both Eag1 and Best1 currents are up-regulated in T(84)-fast cells. Eag1 currents were cell cycle-dependent with up-regulation during G(1)/S transition. T(84)-slow, but not T(84)-fast, cells formed tight monolayers when grown on permeable supports. RNA interference inhibition of Eag1 and Best1 reduced proliferation of T(84)-fast cells, whereas overexpression of Best1 turned T(84)-slow into fast-growing cells. Eag1 and Best1 improve intracellular Ca(2+) signaling and cell volume regulation. These results establish a novel role for bestrophins in cell proliferation. PMID:18222922

  1. Up-regulation of glycolytic metabolism is required for HIF1α-driven bone formation.

    PubMed

    Regan, Jenna N; Lim, Joohyun; Shi, Yu; Joeng, Kyu Sang; Arbeit, Jeffrey M; Shohet, Ralph V; Long, Fanxin

    2014-06-10

    The bone marrow environment is among the most hypoxic in the body, but how hypoxia affects bone formation is not known. Because low oxygen tension stabilizes hypoxia-inducible factor alpha (HIFα) proteins, we have investigated the effect of expressing a stabilized form of HIF1α in osteoblast precursors. Brief stabilization of HIF1α in SP7-positive cells in postnatal mice dramatically stimulated cancellous bone formation via marked expansion of the osteoblast population. Remarkably, concomitant deletion of vascular endothelial growth factor A (VEGFA) in the mouse did not diminish bone accrual caused by HIF1α stabilization. Thus, HIF1α-driven bone formation is independent of VEGFA up-regulation and increased angiogenesis. On the other hand, HIF1α stabilization stimulated glycolysis in bone through up-regulation of key glycolytic enzymes including pyruvate dehydrogenase kinase 1 (PDK1). Pharmacological inhibition of PDK1 completely reversed HIF1α-driven bone formation in vivo. Thus, HIF1α stimulates osteoblast formation through direct activation of glycolysis, and alterations in cellular metabolism may be a broadly applicable mechanism for regulating cell differentiation. PMID:24912186

  2. Up-regulation of podoplanin involves in neuronal apoptosis in LPS-induced neuroinflammation.

    PubMed

    Song, Yan; Shen, Jianhong; Lin, Yuchang; Shen, Jiabing; Wu, Xinming; Yan, Yaohua; Zhou, Li; Zhang, Haiyan; Zhou, Ying; Cao, Maohong; Liu, Yonghua

    2014-08-01

    Podoplanin (PDPN) is a mucin-type transmembrane sialoglycoprotein expressed in multiple tissues in adult animals, including the brain, lungs, kidney, and lymphoid organs. Studies of this molecule have demonstrated its great importance in tumor metastasis, platelet aggregation, and lymphatic vessel formation. However, information regarding its regulation and possible function in the central nervous system is still limited. In this study, we performed a neuroinflammatory model by lipopolysaccharide (LPS) lateral ventral injection in adult rats and detected increased expression of PDPN in the brain cortex. Immunofluorescence indicated that PDPN was located in the neurons, but not astrocytes. Moreover, there was a concomitant up-regulation of active caspase-3, cyclin D1, and CDK4 in vivo and vitro studies. In addition, the expression of these three proteins in cortical primary neurons was decreased after knocking down PDPN by siRNA. Collectively, all these results suggested that the up-regulation of PDPN might be involved in neuronal apoptosis in neuroinflammation after LPS injection. PMID:24821010

  3. Modeling Structural Coordination and Ligand Binding in Zinc Proteins with a Polarizable Potential

    PubMed Central

    Zhang, Jiajing; Yang, Wei; Piquemal, Jean-Philip; Ren, Pengyu

    2012-01-01

    As the second most abundant cation in human body, zinc is vital for the structures and functions of many proteins. Zinc-containing matrix metalloproteinases (MMPs) have been widely investigated as potential drug targets in a range of diseases ranging from cardiovascular disorders to cancers. However, it remains a challenge in theoretical studies to treat zinc in proteins with classical mechanics. In this study, we examined Zn2+ coordination with organic compounds and protein side chains using a polarizable atomic multipole based electrostatic model. We find that polarization effect plays a determining role in Zn2+ coordination geometry in both matrix metalloproteinase (MMP) complexes and in zinc-finger proteins. In addition, the relative binding free energies of selected inhibitors binding with MMP13 have been estimated and compared with experimental results. While not directly interacting with the small molecule inhibitors, the permanent and polarizing field of Zn2+ exerts a strong influence on the relative affinities of the ligands. The simulation results also reveal the polarization effect on binding is ligand dependent and thus difficult to be incorporated into fixed-charge models implicitly. PMID:22754403

  4. Lipid activation of the signal recognition particle receptor provides spatial coordination of protein targeting

    PubMed Central

    Lam, Vinh Q.; Akopian, David; Rome, Michael; Henningsen, Doug

    2010-01-01

    The signal recognition particle (SRP) and SRP receptor comprise the major cellular machinery that mediates the cotranslational targeting of proteins to cellular membranes. It remains unclear how the delivery of cargos to the target membrane is spatially coordinated. We show here that phospholipid binding drives important conformational rearrangements that activate the bacterial SRP receptor FtsY and the SRP–FtsY complex. This leads to accelerated SRP–FtsY complex assembly, and allows the SRP–FtsY complex to more efficiently unload cargo proteins. Likewise, formation of an active SRP–FtsY GTPase complex exposes FtsY’s lipid-binding helix and enables stable membrane association of the targeting complex. Thus, membrane binding, complex assembly with SRP, and cargo unloading are inextricably linked to each other via conformational changes in FtsY. These allosteric communications allow the membrane delivery of cargo proteins to be efficiently coupled to their subsequent unloading and translocation, thus providing spatial coordination during protein targeting. PMID:20733058

  5. Spatial coordination between chromosomes and cell division proteins in Escherichia coli.

    PubMed

    Männik, Jaan; Bailey, Matthew W

    2015-01-01

    To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness. PMID:25926826

  6. Spatial coordination between chromosomes and cell division proteins in Escherichia coli

    PubMed Central

    Männik, Jaan; Bailey, Matthew W.

    2015-01-01

    To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness. PMID:25926826

  7. Identification of genes up-regulated in response to Cd exposure in Brassica juncea L.

    PubMed

    Minglin, Lang; Yuxiu, Zhang; Tuanyao, Chai

    2005-12-19

    In this paper, the fluorescent mRNA differential display (DD) technique was applied to analyze transcriptional regulation in response to Cd treatment in a heavy-metal accumulator, Brassica juncea. 154 DD bands were identified, of which fragments corresponding to 15 and 13 cDNAs were successfully cloned from leaves and roots, respectively. Many of the genes were confirmed to have a 2-5 fold increase in expression in both roots and leaves after 48 h Cd exposure (approximately 22.4 ppm). However, several isolated genes, e.g., DD2, DD21, DD22, showed a reversed mRNA expression pattern. Sequencing revealed those Cd-induced up-regulated genes displayed mRNAs corresponding to 19 different genes, 18 of which had a clear identity to Arabidopsis thaliana sequences and a putative function was assigned to 15 of them, including the auxin-responsive GH3, ARF-like small GTPases/ARFs, ARD/ARD', APS reductase, Nop, catalase, zinc finger (C3HC4-type RING finger), diacylglycerol kinase, and haloacid dehalogenase-like hydrolase families. Three cDNAs corresponded to predicted membrane proteins (KOG3491) or a ribosome-associated membrane protein RAMP4. One other clone, DD26, did not show significant identities to any translated sequence in the GenBank database, suggesting it may either encode unidentified proteins, or correspond to un-translated, non-conserved regions of mRNA molecules. These Cd-responsive up-regulated genes are mostly also regulated by abiotic or biotic stresses, e.g., dehydration, chilling, high salt, auxin, heat and infection, in other plants. The present study leads to an increased understanding of genes and/or the biochemical pathways involved in heavy-metal resistance and accumulation in plants. PMID:16226851

  8. Up-Regulation of Nerve Growth Factor in Cholestatic Livers and Its Hepatoprotective Role against Oxidative Stress

    PubMed Central

    Tsai, Ming-Shian; Lin, Yu-Chun; Sun, Cheuk-Kwan; Huang, Shih-Che; Lee, Po-Huang; Kao, Ying-Hsien

    2014-01-01

    The role of nerve growth factor (NGF) in liver injury induced by bile duct ligation (BDL) remains elusive. This study aimed to investigate the relationship between inflammation and hepatic NGF expression, to explore the possible upstream molecules up-regulating NGF, and to determine whether NGF could protect hepatocytes from oxidative liver injury. Biochemical and molecular detection showed that NGF was up-regulated in cholestatic livers and plasma, and well correlated with systemic and hepatic inflammation. Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice. Immunohistochemistry showed that the up-regulated NGF was mainly localized in parenchymal hepatocytes. In vitro mechanistic study further demonstrated that TGF-β1 up-regulated NGF expression in clone-9 and primary rat hepatocytes. Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG. TUNEL staining confirmed the involvement of anti-apoptosis in the NGF-exhibited hepatoprotection. Moreover, NGF potently induced Akt phosphorylation and increased Bcl-2 to Bax ratios, whereas these molecular alterations by NGF were only seen in the H2O2-, but not TGF-β1-treated hepatocytes. In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases. PMID:25397406

  9. Up-regulation of nerve growth factor in cholestatic livers and its hepatoprotective role against oxidative stress.

    PubMed

    Tsai, Ming-Shian; Lin, Yu-Chun; Sun, Cheuk-Kwan; Huang, Shih-Che; Lee, Po-Huang; Kao, Ying-Hsien

    2014-01-01

    The role of nerve growth factor (NGF) in liver injury induced by bile duct ligation (BDL) remains elusive. This study aimed to investigate the relationship between inflammation and hepatic NGF expression, to explore the possible upstream molecules up-regulating NGF, and to determine whether NGF could protect hepatocytes from oxidative liver injury. Biochemical and molecular detection showed that NGF was up-regulated in cholestatic livers and plasma, and well correlated with systemic and hepatic inflammation. Conversely, systemic immunosuppression reduced serum NGF levels and resulted in higher mortality in BDL-treated mice. Immunohistochemistry showed that the up-regulated NGF was mainly localized in parenchymal hepatocytes. In vitro mechanistic study further demonstrated that TGF-β1 up-regulated NGF expression in clone-9 and primary rat hepatocytes. Exogenous NGF supplementation and endogenous NGF overexpression effectively protected hepatocytes against TGF-β1- and oxidative stress-induced cell death in vitro, along with reduced formation of oxidative adducted proteins modified by 4-HNE and 8-OHdG. TUNEL staining confirmed the involvement of anti-apoptosis in the NGF-exhibited hepatoprotection. Moreover, NGF potently induced Akt phosphorylation and increased Bcl-2 to Bax ratios, whereas these molecular alterations by NGF were only seen in the H2O2-, but not TGF-β1-treated hepatocytes. In conclusion, NGF exhibits anti-oxidative and hepatoprotective effects and is suggested to be therapeutically applicable in treating cholestatic liver diseases. PMID:25397406

  10. Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps muscle after exercise.

    PubMed

    Miller, Benjamin F; Olesen, Jens L; Hansen, Mette; Døssing, Simon; Crameri, Regina M; Welling, Rasmus J; Langberg, Henning; Flyvbjerg, Allan; Kjaer, Michael; Babraj, John A; Smith, Kenneth; Rennie, Michael J

    2005-09-15

    We hypothesized that an acute bout of strenuous, non-damaging exercise would increase rates of protein synthesis of collagen in tendon and skeletal muscle but these would be less than those of muscle myofibrillar and sarcoplasmic proteins. Two groups (n = 8 and 6) of healthy young men were studied over 72 h after 1 h of one-legged kicking exercise at 67% of maximum workload (W(max)). To label tissue proteins in muscle and tendon primed, constant infusions of [1-(13)C]leucine or [1-(13)C]valine and flooding doses of [(15)N] or [(13)C]proline were given intravenously, with estimation of labelling in target proteins by gas chromatography-mass spectrometry. Patellar tendon and quadriceps biopsies were taken in exercised and rested legs at 6, 24, 42 or 48 and 72 h after exercise. The fractional synthetic rates of all proteins were elevated at 6 h and rose rapidly to peak at 24 h post exercise (tendon collagen (0.077% h(-1)), muscle collagen (0.054% h(-1)), myofibrillar protein (0.121% h(-1)), and sarcoplasmic protein (0.134% h(-1))). The rates decreased toward basal values by 72 h although rates of tendon collagen and myofibrillar protein synthesis remained elevated. There was no tissue damage of muscle visible on histological evaluation. Neither tissue microdialysate nor serum concentrations of IGF-I and IGF binding proteins (IGFBP-3 and IGFBP-4) or procollagen type I N-terminal propeptide changed from resting values. Thus, there is a rapid increase in collagen synthesis after strenuous exercise in human tendon and muscle. The similar time course of changes of protein synthetic rates in different cell types supports the idea of coordinated musculotendinous adaptation. PMID:16002437

  11. Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

    PubMed

    Arumugam, Sridhar; Hoerauf, Achim; Pfarr, Kenneth M

    2014-03-01

    Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link

  12. Producing high-accuracy lattice models from protein atomic coordinates including side chains.

    PubMed

    Mann, Martin; Saunders, Rhodri; Smith, Cameron; Backofen, Rolf; Deane, Charlotte M

    2012-01-01

    Lattice models are a common abstraction used in the study of protein structure, folding, and refinement. They are advantageous because the discretisation of space can make extensive protein evaluations computationally feasible. Various approaches to the protein chain lattice fitting problem have been suggested but only a single backbone-only tool is available currently. We introduce LatFit, a new tool to produce high-accuracy lattice protein models. It generates both backbone-only and backbone-side-chain models in any user defined lattice. LatFit implements a new distance RMSD-optimisation fitting procedure in addition to the known coordinate RMSD method. We tested LatFit's accuracy and speed using a large nonredundant set of high resolution proteins (SCOP database) on three commonly used lattices: 3D cubic, face-centred cubic, and knight's walk. Fitting speed compared favourably to other methods and both backbone-only and backbone-side-chain models show low deviation from the original data (~1.5 Å RMSD in the FCC lattice). To our knowledge this represents the first comprehensive study of lattice quality for on-lattice protein models including side chains while LatFit is the only available tool for such models. PMID:22934109

  13. Producing High-Accuracy Lattice Models from Protein Atomic Coordinates Including Side Chains

    PubMed Central

    Mann, Martin; Saunders, Rhodri; Smith, Cameron; Backofen, Rolf; Deane, Charlotte M.

    2012-01-01

    Lattice models are a common abstraction used in the study of protein structure, folding, and refinement. They are advantageous because the discretisation of space can make extensive protein evaluations computationally feasible. Various approaches to the protein chain lattice fitting problem have been suggested but only a single backbone-only tool is available currently. We introduce LatFit, a new tool to produce high-accuracy lattice protein models. It generates both backbone-only and backbone-side-chain models in any user defined lattice. LatFit implements a new distance RMSD-optimisation fitting procedure in addition to the known coordinate RMSD method. We tested LatFit's accuracy and speed using a large nonredundant set of high resolution proteins (SCOP database) on three commonly used lattices: 3D cubic, face-centred cubic, and knight's walk. Fitting speed compared favourably to other methods and both backbone-only and backbone-side-chain models show low deviation from the original data (~1.5 Å RMSD in the FCC lattice). To our knowledge this represents the first comprehensive study of lattice quality for on-lattice protein models including side chains while LatFit is the only available tool for such models. PMID:22934109

  14. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication.

    PubMed

    Gu, Haidong

    2016-02-12

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced "conquer and compromise" strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  15. Infected cell protein 0 functional domains and their coordination in herpes simplex virus replication

    PubMed Central

    Gu, Haidong

    2016-01-01

    Herpes simplex virus 1 (HSV-1) is a ubiquitous human pathogen that establishes latent infection in ganglia neurons. Its unique life cycle requires a balanced “conquer and compromise” strategy to deal with the host anti-viral defenses. One of HSV-1 α (immediate early) gene products, infected cell protein 0 (ICP0), is a multifunctional protein that interacts with and modulates a wide range of cellular defensive pathways. These pathways may locate in different cell compartments, which then migrate or exchange factors upon stimulation, for the purpose of a concerted and effective defense. ICP0 is able to simultaneously attack multiple host pathways by either degrading key restrictive factors or modifying repressive complexes. This is a viral protein that contains an E3 ubiquitin ligase, translocates among different cell compartments and interacts with major defensive complexes. The multiple functional domains of ICP0 can work independently and at the same time coordinate with each other. Dissecting the functional domains of ICP0 and delineating the coordination of these domains will help us understand HSV-1 pathogenicity as well as host defense mechanisms. This article focuses on describing individual ICP0 domains, their biochemical properties and their implication in HSV-1 infection. By putting individual domain functions back into the picture of host anti-viral defense network, this review seeks to elaborate the complex interactions between HSV-1 and its host. PMID:26870669

  16. Lipopolysaccharide-induced Pulpitis Up-regulates TRPV1 in Trigeminal Ganglia

    PubMed Central

    Chung, M.-K.; Lee, J.; Duraes, G.; Ro, J.Y.

    2011-01-01

    Tooth pain often accompanies pulpitis. Accumulation of lipopolysaccharides (LPS), a product of Gram-negative bacteria, is associated with painful clinical symptoms. However, the mechanisms underlying LPS-induced tooth pain are not clearly understood. TRPV1 is a capsaicin- and heat-gated nociceptive ion channel implicated in thermosensation and hyperalgesia under inflammation or injury. Although TRPV1 is expressed in pulpal afferents, it is not known whether the application of LPS to teeth modulates TRPV1 in trigeminal nociceptors. By assessing the levels of protein and transcript of TRPV1 in mouse trigeminal ganglia, we demonstrate that dentinal application of LPS increases the expression of TRPV1. Our results suggest that the up-regulation of TRPV1 in trigeminal nociceptors following bacterial infection could contribute to hyperalgesia under pulpitis conditions. PMID:21712529

  17. The juxtamembrane domain in ETV6/FLT3 is critical for PIM-1 up-regulation and cell proliferation

    SciTech Connect

    Vu, Hoang Anh; Xinh, Phan Thi; Kano, Yasuhiko; Tokunaga, Katsushi; Sato, Yuko

    2009-06-05

    We recently reported that the ETV6/FLT3 fusion protein conferred interleukin-3-independent growth on Ba/F3 cells. The present study has been conducted to assess role of the juxtamembrane domain of FLT3 for signal transduction and cell transformation. The wild-type ETV6/FLT3 fusion protein in transfected cells was a constitutively activated tyrosine kinase that led to up-regulation of PIM-1 and activations of STAT5, AKT, and MAPK. Deletion of the juxtamembrane domain abrogated interleukin-3-independent growth of the transfected cells and PIM-1 up-regulation, whereas it retained compatible levels of phosphorylations of STAT5, AKT, and MAPK. Further deletion of N-terminal region of the tyrosine kinase I domain of FLT3 completely abolished these phosphorylations. Our data indicate that the juxtamembrane domain of FLT3 in ETV6/FLT3 fusion protein is critical for cell proliferation and PIM-1 up-regulation that might be independent of a requirement for signaling through STAT5, MAPK, and AKT pathways.

  18. A model for protocellular coordination of nucleic acid and protein syntheses

    NASA Technical Reports Server (NTRS)

    Fox, S. W.

    1981-01-01

    The proteinoid model for the coordination of protein synthesis with nucleic acid coding within the evolving protocell is discussed. Evidence for the self-ordering of amino acid chains, which would enhance the catalytic activity of a lysine-rich proteinoid, is presented, along with that for the preferential formation of microparticles, particularly proteinoid microparticles, in various solutions. Demonstrations of the catalytic activity of lysine-rich proteinoids in the synthesis of peptide and internucleotide bonds are pointed out. The view of evolution as a two stage sequence in which the geological synthesis of peptides evolved to the protocellular synthesis of peptides and oligonucleotides is discussed, and contrasted with the alternative view, in accord with the central dogma, that nucleic acids arose first then governed the production of proteins and protocells.

  19. The SH3-SAM adaptor HACS1 is up-regulated in B cell activation signaling cascades.

    PubMed

    Zhu, Yuan Xiao; Benn, Sally; Li, Zhi Hua; Wei, Ellen; Masih-Khan, Esther; Trieu, Young; Bali, Meenakshi; McGlade, C Jane; Claudio, Jaime O; Stewart, A Keith

    2004-09-20

    HACS1 is a Src homology 3 and sterile alpha motif domain-containing adaptor that is preferentially expressed in normal hematopoietic tissues and malignancies including myeloid leukemia, lymphoma, and myeloma. Microarray data showed HACS1 expression is up-regulated in activated human B cells treated with interleukin (IL)-4, CD40L, and anti-immunoglobulin (Ig)M and clustered with genes involved in signaling, including TNF receptor-associated protein 1, signaling lymphocytic activation molecule, IL-6, and DEC205. Immunoblot analysis demonstrated that HACS1 is up-regulated by IL-4, IL-13, anti-IgM, and anti-CD40 in human peripheral blood B cells. In murine spleen B cells, Hacs1 can also be up-regulated by lipopolysaccharide but not IL-13. Induction of Hacs1 by IL-4 is dependent on Stat6 signaling and can also be impaired by inhibitors of phosphatidylinositol 3-kinase, protein kinase C, and nuclear factor kappaB. HACS1 associates with tyrosine-phosphorylated proteins after B cell activation and binds in vitro to the inhibitory molecule paired Ig-like receptor B. Overexpression of HACS1 in murine spleen B cells resulted in a down-regulation of the activation marker CD23 and enhancement of CD138 expression, IgM secretion, and Xbp-1 expression. Knock down of HACS1 in a human B lymphoma cell line by small interfering ribonucleic acid did not significantly change IL-4-stimulated B cell proliferation. Our study demonstrates that HACS1 is up-regulated by B cell activation signals and is a participant in B cell activation and differentiation. PMID:15381729

  20. Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion.

    PubMed

    Fleissner, André; Leeder, Abigail C; Roca, M Gabriela; Read, Nick D; Glass, N Louise

    2009-11-17

    Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion. PMID:19884508

  1. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

    PubMed Central

    Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

    2016-01-01

    Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

  2. The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

    PubMed Central

    Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

    2016-01-01

    Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

  3. Up-regulation of the embryonic self-renewal network through reversible polyploidy in irradiated p53-mutant tumour cells

    SciTech Connect

    Salmina, Kristine; Jankevics, Eriks; Huna, Anda; Perminov, Dmitry; Radovica, Ilze; Klymenko, Tetyana; Ivanov, Andrey; Jascenko, Elina; Scherthan, Harry; Cragg, Mark; Erenpreisa, Jekaterina

    2010-08-01

    We have previously documented that transient polyploidy is a potential cell survival strategy underlying the clonogenic re-growth of tumour cells after genotoxic treatment. In an attempt to better define this mechanism, we recently documented the key role of meiotic genes in regulating the DNA repair and return of the endopolyploid tumour cells (ETC) to diploidy through reduction divisions after irradiation. Here, we studied the role of the pluripotency and self-renewal stem cell genes NANOG, OCT4 and SOX2 in this polyploidy-dependent survival mechanism. In irradiation-resistant p53-mutated lymphoma cell-lines (Namalwa and WI-L2-NS) but not sensitive p53 wild-type counterparts (TK6), low background expression of OCT4 and NANOG was up-regulated by ionising radiation with protein accumulation evident in ETC as detected by OCT4/DNA flow cytometry and immunofluorescence (IF). IF analysis also showed that the ETC generate PML bodies that appear to concentrate OCT4, NANOG and SOX2 proteins, which extend into complex nuclear networks. These polyploid tumour cells resist apoptosis, overcome cellular senescence and undergo bi- and multi-polar divisions transmitting the up-regulated OCT4, NANOG and SOX2 self-renewal cassette to their descendents. Altogether, our observations indicate that irradiation-induced ETC up-regulate key components of germ-line cells, which potentially facilitate survival and propagation of the tumour cell population.

  4. Isoform-Specific Up-Regulation of Plasma Membrane Ca2+ATPase Expression During Colon and Gastric Cancer Cell Differentiation

    PubMed Central

    Ribiczey, Polett; Tordai, Attila; Andrikovics, Hajnalka; Filoteo, Adelaida G.; Penniston, John T.; Enouf, Jocelyne; Enyedi, Ágnes; Papp, Béla; Kovács, Tünde

    2007-01-01

    Summary In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype. PMID:17433436

  5. Up-regulation of SNCA gene expression: implications to synucleinopathies.

    PubMed

    Tagliafierro, L; Chiba-Falek, O

    2016-07-01

    Synucleinopathies are a group of neurodegenerative diseases that share a common pathological lesion of intracellular protein inclusions largely composed by aggregates of alpha-synuclein protein. Accumulating evidence, including genome wide association studies, has implicated alpha-synuclein (SNCA) gene in the etiology of synucleinopathies. However, the precise variants within SNCA gene that contribute to the sporadic forms of Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and other synucleinopathies and their molecular mechanisms of action remain elusive. It has been suggested that SNCA expression levels are critical for the development of these diseases. Here, we review several model systems that have been developed to advance the understanding of the role of SNCA expression levels in the etiology of synucleinopathies. We also describe different molecular mechanisms that regulate SNCA gene expression and discuss possible strategies for SNCA down-regulation as means for therapeutic approaches. Finally, we highlight some examples that underscore the relationships between the genetic association findings and the regulatory mechanisms of SNCA expression, which suggest that genetic variability in SNCA locus is directly responsible, at least in part, to the changes in gene expression and explain the reported associations of SNCA with synucleinopathies. Future studies utilizing induced pluripotent stem cells (iPSCs)-derived neuronal lines and genome editing by CRISPR/Cas9, will allow us to validate, characterize, and manipulate the effects of particular cis-genetic variants on SNCA expression. Moreover, this model system will enable us to compare different neuronal and glial lineages involved in synucleinopathies representing an attractive strategy to elucidate-common and specific-SNCA-genetic variants, regulatory mechanisms, and vulnerable expression levels underlying synucleinopathy spectrum disorders. This forthcoming

  6. Up-regulation of placental leptin by human chorionic gonadotropin.

    PubMed

    Maymó, Julieta L; Pérez Pérez, Antonio; Sánchez-Margalet, Víctor; Dueñas, José L; Calvo, Juan Carlos; Varone, Cecilia L

    2009-01-01

    Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling molecule for the central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 microM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 microm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction

  7. Neurotrophin-4 is up-regulated in ragged-red fibers associated with pathogenic mitochondrial DNA mutations.

    PubMed

    Walker, U A; Schon, E A

    1998-04-01

    Ragged-red fibers (RRFs) are found more frequently in highly oxidative type I fibers than in glycolytic type II fibers in the muscle of many patients with mitochondrial myopathies. Neurotrophin-4 (NT-4), a neuronal signaling molecule, is also expressed in skeletal muscle, predominantly in type I fibers. We found that NT-4 protein and mRNA were present in both type I and type II fibers but were up-regulated in RRFs of patients with mitochondrial encephalomyopathies; it is noteworthy that NT-4 was not up-regulated in muscle fibers from healthy aerobically trained athletes. Thus, NT-4 might represent a member of a new class of candidate molecules involved in the compensatory adjustments of muscle fibers to oxidative dysfunction, and may even play a role as a signaling molecule for mitochondrial proliferation. PMID:9546339

  8. Ischemic postconditioning protects against ischemic brain injury by up-regulation of acid-sensing ion channel 2a

    PubMed Central

    Duanmu, Wang-sheng; Cao, Liu; Chen, Jing-yu; Ge, Hong-fei; Hu, Rong; Feng, Hua

    2016-01-01

    Ischemic postconditioning renders brain tissue tolerant to brain ischemia, thereby alleviating ischemic brain injury. However, the exact mechanism of action is still unclear. In this study, a rat model of global brain ischemia was subjected to ischemic postconditioning treatment using the vessel occlusion method. After 2 hours of ischemia, the bilateral common carotid arteries were blocked immediately for 10 seconds and then perfused for 10 seconds. This procedure was repeated six times. Ischemic postconditioning was found to mitigate hippocampal CA1 neuronal damage in rats with brain ischemia, and up-regulate acid-sensing ion channel 2a expression at the mRNA and protein level. These findings suggest that ischemic postconditioning up-regulates acid-sensing ion channel 2a expression in the rat hippocampus after global brain ischemia, which promotes neuronal tolerance to ischemic brain injury. PMID:27212927

  9. Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90)

    NASA Technical Reports Server (NTRS)

    Kano, Mihoko; Kitano, Takako; Ikemoto, Madoka; Hirasaka, Katsuya; Asanoma, Yuki; Ogawa, Takayuki; Takeda, Shinichi; Nonaka, Ikuya; Adams, Gregory R.; Baldwin, Kenneth M.; Oarada, Motoko; Kishi, Kyoichi; Nikawa, Takeshi

    2003-01-01

    We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

  10. Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90).

    PubMed

    Kano, Mihoko; Kitano, Takako; Ikemoto, Madoka; Hirasaka, Katsuya; Asanoma, Yuki; Ogawa, Takayuki; Takeda, Shinichi; Nonaka, Ikuya; Adams, Gregory R; Baldwin, Kenneth M; Oarada, Motoko; Kishi, Kyoichi; Nikawa, Takeshi

    2003-02-01

    We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle. PMID:12630567

  11. Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation

    PubMed Central

    Kim, Ji Eun; Yoo, Hyun Ju; Gu, Ja Yoon; Kim, Hyun Kyung

    2016-01-01

    The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment. PMID:27258428

  12. NFAT5 Is Up-Regulated by Hypoxia: Possible Implications in Preeclampsia and Intrauterine Growth Restriction.

    PubMed

    Dobierzewska, Aneta; Palominos, Macarena; Irarrazabal, Carlos E; Sanchez, Marianela; Lozano, Mauricio; Perez-Sepulveda, Alejandra; Monteiro, Lara J; Burmeister, Yara; Figueroa-Diesel, Horacio; Rice, Gregory E; Illanes, Sebastian E

    2015-07-01

    During gestation, low oxygen environment is a major determinant of early placentation process, while persistent placental hypoxia leads to pregnancy-related complications such as preeclampsia (PE) and intrauterine growth restriction (IUGR). PE affects 5%-8% of all pregnancies worldwide and is a cause of maternal and fetal morbidity and mortality. During placental development, persistent hypoxia due to poor trophoblast invasion and reduced uteroplacental perfusion leads to maternal endothelial dysfunction and clinical manifestation of PE. Here we hypothesized that nuclear factor of activated T cells-5 (NFAT5), a well-known osmosensitive renal factor and recently characterized hypoxia-inducible protein, is also activated in vivo in placentas of PE and IUGR complications as well as in the in vitro model of trophoblast hypoxia. In JAR cells, low oxygen tension (1% O2) induced NFAT5 mRNA and increased its nuclear abundance, peaking at 16 h. This increase did not occur in parallel with the earlier HIF1A induction. Real-time PCR and Western blot analysis confirmed up-regulation of NFAT5 mRNA and NFAT5 nuclear content in human preeclamptic placentas and in rabbit placentas of an experimentally induced IUGR model, as compared with the control groups. In vitro lambda protein phosphatase (lambda PPase) treatment revealed that increased abundance of NFAT5 protein in nuclei of either JAR cells (16 h of hypoxia) or PE and IUGR placentas is at least partially due to NFAT5 phosphorylation. NFAT5 downstream targets aldose reductase (AR) and sodium-myo-inositol cotransporter (SMIT; official symbol SLC5A3) were not significantly up-regulated either in JAR cells exposed to hypoxia or in placentas of PE- and IUGR-complicated pregnancies, suggesting that hypoxia-dependent activation of NFAT5 serves as a separate function to its tonicity-dependent stimulation. In conclusion, we propose that NFAT5 may serve as a novel marker of placental hypoxia and ischemia independently of HIF1A. PMID

  13. Gα12 Drives Invasion of Oral Squamous Cell Carcinoma through Up-Regulation of Proinflammatory Cytokines

    PubMed Central

    Jian, Shiou-Ling; Hsieh, Hsin-Yi; Liao, Chun-Ta; Yen, Tzu-Chen; Nien, Shu-Wei; Cheng, Ann-Joy; Juang, Jyh-Lyh

    2013-01-01

    Oral squamous cell carcinoma (OSCC) ranks among the top ten most prevalent cancers worldwide. Like most head and neck squamous cell carcinomas (HNSCCs), OSCC is highly inflammatory and aggressive. However, the signaling pathways triggering the activation of its inflammatory processes remain elusive. G protein-coupled receptor signaling regulates the inflammatory response and invasiveness of cancers, but it remains unclear whether Gα12 is a critical player in the inflammatory cytokine pathway during the tumorigenesis of OSCC. This study was undertaken to determine the role of Gα12 signaling in the regulation of proinflammatory cytokines in their mediation of OSCC invasion. We found that both the transcription and protein levels of Gα12 are up-regulated in OSCC tumors. The elevated Gα12 expressions in OSCC patients also correlated with extra-capsular spread, an indicator of tumor invasiveness in HNSCCs. This clinical finding was supported by the studies of overexpression and RNAi knockdown of Gα12 in OSCC cells, which demonstrated that Gα12 promoted tumor cell migration and invasion. To understand how Gα12 modulates OSCC invasiveness, we analyzed key biological processes in microarray data upon depletion of Gα12 and found that cytokine- and other immune-related pathways were severely impaired. Importantly, the mRNA levels of IL-6 and IL-8 proinflammatory cytokines in clinical samples were found to be significantly correlated with the increased Gα12 levels, suggesting a potential role of Gα12 in modulating the IL-6 and IL-8 expressions. Supporting this hypothesis, overexpression or RNAi knockdown of Gα12 in OSCC cell lines both showed that Gα12 positively regulated the mRNA and protein levels of IL-6 and IL-8. Finally, we demonstrated that the Gα12 promotion of tumor cell invasiveness was suppressed by the neutralization of IL-6 and IL-8 in OSCC cells. Together, these findings suggest that Gα12 drives OSCC invasion through the up-regulation of IL-6 and

  14. Up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis in human papillary thyroid cancer cells.

    PubMed

    Mo, Xiao-Mei; Li, Li; Zhu, Ping; Dai, Yu-Jie; Zhao, Ting-Ting; Liao, Ling-Yao; Chen, George G; Liu, Zhi-Min

    2016-08-15

    17β-estradiol (E2) has been suggested to play a role in the development and progression of papillary thyroid cancer. Heat shock protein 27 (Hsp27) is a member of the Hsp family that is responsible for cell survival under stressful conditions. Previous studies have shown that the 5'-promoter region of Hsp27 gene contains a specificity protein-1 (Spl) and estrogen response element half-site (ERE-half), which contributes to Hsp27 induction by E2 in breast cancer cells. However, it is unclear whether Hsp27 can be up-regulated by E2 and which estrogen receptor (ER) isoform and tethered transcription factor are involved in this regulation in papillary thyroid cancer cells. In the present study, we demonstrated that Hsp27 can be effectively up-regulated by E2 at mRNA and protein levels in human K1 and BCPAP papillary thyroid cancer cells which have more than two times higher level of ERα than that of ERβ. The up-regulation of Hsp27 by E2 is mediated by ERα/Sp1 and ERβ has repressive effect on this ERα/Sp1-mediated up-regulation of Hsp27. Moreover, we showed that the up-regulation of Hsp27 by ERα/Sp1 facilitates proliferation and confers resistance to apoptosis through interaction with procaspase-3. Targeting this pathway may be a potential strategy for therapy of papillary thyroid cancer. PMID:27179757

  15. Predicting the reaction coordinates of millisecond light-induced conformational changes in photoactive yellow protein

    PubMed Central

    Vreede, Jocelyne; Juraszek, Jarek; Bolhuis, Peter G.

    2010-01-01

    Understanding the dynamics of large-scale conformational changes in proteins still poses a challenge for molecular simulations. We employ transition path sampling of explicit solvent molecular dynamics trajectories to obtain atomistic insight in the reaction network of the millisecond timescale partial unfolding transition in the photocycle of the bacterial sensor photoactive yellow protein. Likelihood maximization analysis predicts the best model for the reaction coordinates of each substep as well as tentative transition states, without further simulation. We find that the unfolding of the α-helical region 43–51 is followed by sequential solvent exposure of both Glu46 and the chromophore. Which of these two residues is exposed first is correlated with the presence of a salt bridge that is part of the N-terminal domain. Additional molecular dynamics simulations indicate that the exposure of the chromophore does not result in a productive pathway. We discuss several possibilities for experimental validation of these predictions. Our results open the way for studying millisecond conformational changes in other medium-sized (signaling) proteins. PMID:20133754

  16. A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

    PubMed Central

    Tian, Tian; Salis, Howard M.

    2015-01-01

    Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

  17. Regulator of G Protein Signaling 6 (RGS6) Protein Ensures Coordination of Motor Movement by Modulating GABAB Receptor Signaling*

    PubMed Central

    Maity, Biswanath; Stewart, Adele; Yang, Jianqi; Loo, Lipin; Sheff, David; Shepherd, Andrew J.; Mohapatra, Durga P.; Fisher, Rory A.

    2012-01-01

    γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABAB receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABABR signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ5 and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABABR antagonist. RGS6−/− mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABABR, and GIRK channel subunits, and cerebellar granule neurons from RGS6−/− mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABABR signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin. PMID:22179605

  18. Regulator of G protein signaling 6 (RGS6) protein ensures coordination of motor movement by modulating GABAB receptor signaling.

    PubMed

    Maity, Biswanath; Stewart, Adele; Yang, Jianqi; Loo, Lipin; Sheff, David; Shepherd, Andrew J; Mohapatra, Durga P; Fisher, Rory A

    2012-02-10

    γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin. PMID:22179605

  19. GPR55, a G-protein coupled receptor for lysophosphatidylinositol, plays a role in motor coordination.

    PubMed

    Wu, Chia-Shan; Chen, Hongmei; Sun, Hao; Zhu, Jie; Jew, Chris P; Wager-Miller, James; Straiker, Alex; Spencer, Corinne; Bradshaw, Heather; Mackie, Ken; Lu, Hui-Chen

    2013-01-01

    The G-protein coupled receptor 55 (GPR55) is activated by lysophosphatidylinositols and some cannabinoids. Recent studies found prominent roles for GPR55 in neuropathic/inflammatory pain, cancer and bone physiology. However, little is known about the role of GPR55 in CNS development and function. To address this question, we performed a detailed characterization of GPR55 knockout mice using molecular, anatomical, electrophysiological, and behavioral assays. Quantitative PCR studies found that GPR55 mRNA was expressed (in order of decreasing abundance) in the striatum, hippocampus, forebrain, cortex, and cerebellum. GPR55 deficiency did not affect the concentrations of endocannabinoids and related lipids or mRNA levels for several components of the endocannabinoid system in the hippocampus. Normal synaptic transmission and short-term as well as long-term synaptic plasticity were found in GPR55 knockout CA1 pyramidal neurons. Deleting GPR55 function did not affect behavioral assays assessing muscle strength, gross motor skills, sensory-motor integration, motor learning, anxiety or depressive behaviors. In addition, GPR55 null mutant mice exhibited normal contextual and auditory-cue conditioned fear learning and memory in a Pavlovian conditioned fear test. In contrast, when presented with tasks requiring more challenging motor responses, GPR55 knockout mice showed impaired movement coordination. Taken together, these results suggest that GPR55 plays a role in motor coordination, but does not strongly regulate CNS development, gross motor movement or several types of learned behavior. PMID:23565223

  20. Schisandra polysaccharide increased glucose consumption by up-regulating the expression of GLUT-4.

    PubMed

    Jin, Dun; Zhao, Ting; Feng, Wei-Wei; Mao, Guang-Hua; Zou, Ye; Wang, Wei; Li, Qian; Chen, Yao; Wang, Xin-Tong; Yang, Liu-Qing; Wu, Xiang-Yang

    2016-06-01

    In our previous study, a polysaccharide was extracted from Schisandra Chinensis (Trucz.) Baill and found with anti-diabetic effects. The aim of this study was to investigate the anti-diabetic effects of the low weight molecular polysaccharide (SCPP11) purified from crude Schisandra polysaccharide and illustrate the underlying mechanism in buffalo rat liver cells. The insulin resistance model of BRL cells was established by incubating with insulin solution for 24h. The effects of SCPP11 on regulating related protein and mRNA expression in an insulin and AMPK signal pathway were investigated by western blot and RT-PCR analysis. SCPP11 showed no cytotoxicity to BRL cells and could improve the glucose consumption in BRL cells. SCPP11 increased the protein expression of Akt, p-AMPK and GLUT-4 in BRL cells. Moreover, SCPP11 could enhance the mRNA expression levels of IRS-1, PI3K, Akt, GLUT-4, AMPKα and PPAR-γ in BRL cells at the same time. In conclusion, SCPP11 possessed effects in improving glucose consumption by up-regulating the expression of GLUT-4 which might occur via insulin and AMPK signal pathway and could be a potential functional food to prevent and mitigate the insulin resistance condition. PMID:26993529

  1. PPM1D controls nucleolar formation by up-regulating phosphorylation of nucleophosmin.

    PubMed

    Kozakai, Yuuki; Kamada, Rui; Furuta, Junya; Kiyota, Yuhei; Chuman, Yoshiro; Sakaguchi, Kazuyasu

    2016-01-01

    An increase of nucleolar number and size has made nucleoli essential markers for cytology and tumour development. However, the underlying basis for their structural integrity and abundance remains unclear. Protein phosphatase PPM1D was found to be up-regulated in different carcinomas including breast cancers. Here, we demonstrate for the first time that PPM1D regulates nucleolar formation via inducing an increased phosphorylation of the nucleolar protein NPM. We show that PPM1D overexpression induces an increase in the nucleolar number regardless of p53 status. We also demonstrated that specific sequential phosphorylation of NPM is important for nucleolar formation and that PPM1D is a novel upstream regulator of this phosphorylation pathway. These results enhance our understanding of the molecular mechanisms that govern nucleoli formation by demonstrating that PPM1D regulates nucleolar formation by regulating NPM phosphorylation status through a novel signalling pathway, PPM1D-CDC25C-CDK1-PLK1. PMID:27619510

  2. Transient ribosomal attenuation coordinates protein synthesis and co-translational folding.

    PubMed

    Zhang, Gong; Hubalewska, Magdalena; Ignatova, Zoya

    2009-03-01

    Clustered codons that pair to low-abundance tRNA isoacceptors can form slow-translating regions in the mRNA and cause transient ribosomal arrest. We report that folding efficiency of the Escherichia coli multidomain protein SufI can be severely perturbed by alterations in ribosome-mediated translational attenuation. Such alterations were achieved by global acceleration of the translation rate with tRNA excess in vitro or by synonymous substitutions to codons with highly abundant tRNAs both in vitro and in vivo. Conversely, the global slow-down of the translation rate modulated by low temperature suppresses the deleterious effect of the altered translational attenuation pattern. We propose that local discontinuous translation temporally separates the translation of segments of the peptide chain and actively coordinates their co-translational folding. PMID:19198590

  3. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis

    PubMed Central

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4’-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. DOI: http://dx.doi.org/10.7554/eLife.17828.001 PMID:27540631

  4. The mitochondrial acyl carrier protein (ACP) coordinates mitochondrial fatty acid synthesis with iron sulfur cluster biogenesis.

    PubMed

    Van Vranken, Jonathan G; Jeong, Mi-Young; Wei, Peng; Chen, Yu-Chan; Gygi, Steven P; Winge, Dennis R; Rutter, Jared

    2016-01-01

    Mitochondrial fatty acid synthesis (FASII) and iron sulfur cluster (FeS) biogenesis are both vital biosynthetic processes within mitochondria. In this study, we demonstrate that the mitochondrial acyl carrier protein (ACP), which has a well-known role in FASII, plays an unexpected and evolutionarily conserved role in FeS biogenesis. ACP is a stable and essential subunit of the eukaryotic FeS biogenesis complex. In the absence of ACP, the complex is destabilized resulting in a profound depletion of FeS throughout the cell. This role of ACP depends upon its covalently bound 4'-phosphopantetheine (4-PP)-conjugated acyl chain to support maximal cysteine desulfurase activity. Thus, it is likely that ACP is not simply an obligate subunit but also exploits the 4-PP-conjugated acyl chain to coordinate mitochondrial fatty acid and FeS biogenesis. PMID:27540631

  5. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

    PubMed

    Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-07-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  6. The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

    PubMed Central

    Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

    2012-01-01

    Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

  7. Acquired resistance to ABT-737 in lymphoma cells that up-regulate MCL-1 and BFL-1

    PubMed Central

    Yecies, Derek; Carlson, Nicole E.; Deng, Jing

    2010-01-01

    ABT-737 is a small-molecule antagonist of BCL-2 currently under evaluation in clinical trials in the oral form of ABT-263. We anticipate that acquired resistance to this promising drug will inevitably arise. To study potential mechanisms of resistance to ABT-737, we derived resistant lines from initially sensitive OCI-Ly1 and SU-DHL-4 lymphoma cell lines via long-term exposure. Resistance was based in the mitochondria and not due to an inability of the drug to bind BCL-2. Resistant cells had increased levels of BFL-1 and/or MCL-1 proteins, which are not targeted by ABT-737. Proapoptotic BIM was displaced from BCL-2 by ABT-737 in both parental and resistant cells, but in resistant cells, BIM was sequestered by the additional BFL-1 and/or MCL-1. Decreasing MCL-1 levels with flavopiridol, PHA 767491, or shRNA restored sensitivity to ABT-737 resistant cells. MCL-1 was up-regulated not by protein stabilization but rather by increased transcript levels. Surprisingly, in addition to stable increases in MCL-1 transcript and protein in resistant cells, there was a dynamic increase within hours after ABT-737 treatment. BFL-1 protein and transcript levels in resistant cells were similarly dynamically up-regulated. This dynamic increase suggests a novel mechanism whereby modulation of antiapoptotic protein function communicates with nuclear transcriptional machinery. PMID:20197552

  8. Hypoxia up-regulates vascular endothelial growth factor in U-87 MG cells: involvement of TRPC1.

    PubMed

    Wang, Bin; Li, Wenjun; Meng, Xiaojing; Zou, Fei

    2009-08-14

    Canonical Transient Receptor Potential (TRPC) channels play important roles in diverse physiological processes. The contribution of TRPC channels to up-regulate VEGF expression under hypoxic conditions was studied in a malignant glioma cell line, U-87 MG cells. Up-regulation of VEGF gene expression by hypoxia was markedly suppressed by a TRPC channel blocker. RT-PCR showed that U-87 MG cells expressed four TRPC isoforms in normoxia: TRPC1, 3, 4, and 5. In addition, the expression of TRPC3, 4, and 5 decreased greatly under hypoxia exposure in U-87 MG cells. In contrast, TRPC1 expression was unchanged. These results suggest TRPC channels were involved in hypoxia-induced VEGF expression, and compared with other TRPC isoforms, TRPC1 might play a different role in this process. Furthermore, we determined the function of TRPC1 by RNAi. Two different siRNAs against TRPC1 largely inhibited hypoxia-induced up-regulation of VEGF mRNA and protein levels. However, overexpression of TRPC3 or 5 neither enhanced hypoxia-induced VEGF expression, nor prevented it. Taken together, our present data suggest that TRPC1, but not TRPC3 or 5, is involved in hypoxia-induced VEGF expression in U-87 MG cells. PMID:19446005

  9. Andrographolide up-regulates cellular-reduced glutathione level and protects cardiomyocytes against hypoxia/reoxygenation injury.

    PubMed

    Woo, Anthony Y H; Waye, Mary M Y; Tsui, Stephen K W; Yeung, Sandy T W; Cheng, Christopher H K

    2008-04-01

    Recent studies revealed that the herb Andrographis paniculata possesses cardioprotective activities. Using neonatal rat cardiomyocytes, the cardioprotective actions of several diterpene lactones derived from A. paniculata including andrographolide, 14-deoxyandrographolide, 14-deoxy-11,12-didehydroandrographolide, and sodium 14-deoxyandrographolide-12-sulfonate were investigated. Pretreatment with andrographolide but not with the other compounds protected the cardiomyocytes against hypoxia/ reoxygenation injury and up-regulated the cellular-reduced glutathione (GSH) level and antioxidant enzyme activities. The cardioprotective action of andrographolide was found to coincide in a time-dependent manner with the up-regulation of GSH, indicating the important role of GSH. The cardioprotective action of andrographolide was also completely abolished by buthionine sulfoximine, which acts as a specific gamma-glutamate cysteine ligase (GCL) inhibitor to deplete cellular GSH level. It was subsequently found that the mRNA and protein levels of the GCL catalytic subunit (GCLC) and modifier subunit (GCLM) were up-regulated by andrographolide. Luciferase reporter assay also demonstrated that andrographolide activated both the GCLC and the GCLM promoters in the transfected rat H9C2 cardiomyocyte cell line. The 12-O-tetradecanoylphorbo-13-acetate response element or the antioxidant response element may be involved in the transactivating actions of andrographolide on the GCLC and GCLM promoters. The present study pinpoints andrographolide as a cardioprotective principle in A. paniculata and reveals its cytoprotective mechanism. PMID:18174384

  10. Catalpol increases hippocampal neuroplasticity and up-regulates PKC and BDNF in the aged rats.

    PubMed

    Liu, Jing; He, Qiao-Jie; Zou, Wei; Wang, Hong-Xia; Bao, Yong-Ming; Liu, Yu-Xin; An, Li-Jia

    2006-12-01

    Rehmannia, a traditional Chinese medical herb, has a long history in age-related disease therapy. Previous work has indicated that catalpol is a main active ingredient performing neuroprotective effect in rehmannia, while the mechanism underlying the effect remains poorly understood. In this study, we attempt to investigate the effect of catalpol on presynaptic proteins and explore a potential mechanism. The hippocampal levels of GAP-43 and synaptophysin in 3 groups of 4 months (young group), 22-24 months (aged group) and catalpol-treated 22-24 months (catalpol-treated group) rats were evaluated by western blotting. Results clearly showed a significant decrease in synaptophysin (46.6%) and GAP-43 (61.4%) levels in the aged group against the young animals and an increase (45.0% and 31.8% respectively) in the catalpol-treated aged rats in comparison with the untreated aged group. In particular, synaptophysin immunoreactivity (OD) in the dentate granule layer of the hippocampus was increased 0.0251 in the catalpol-treated group as compared with the aged group. The study also revealed a catalpol-associated increase of PKC and BDNF in the hippocampus of the catalpol-treated group in comparison with the aged rats and highly correlated with synaptophysin and GAP-43. Such positive correlations between presynaptic proteins and signaling molecules also existed in the young group. These results suggested that catalpol could increase presynaptic proteins and up-regulate relative signaling molecules in the hippocampus of the aged rats. Consequently, it seemed to indicate that catalpol might ameliorate age-related neuroplasticity loss by "normalizing" presynaptic proteins and their relative signaling pathways in the aged rats. PMID:17078935

  11. Orphan receptor GPR15/BOB is up-regulated in rheumatoid arthritis

    PubMed Central

    Cartwright, Alison; Schmutz, Caroline; Askari, Ayman; Kuiper, Jan-Herman; Middleton, Jim

    2014-01-01

    Chemokine receptors on leukocytes mediate the recruitment and accumulation of these cells within affected joints in chronic inflammatory diseases such as rheumatoid arthritis (RA). Identification of involved receptors offers potential for development of therapeutic interventions. The objective of this study was to investigate the expression of orphan receptor GPR15/BOB in the synovium of RA and non-RA patients and in peripheral blood of RA patients and healthy donors. GPR15/BOB protein and messenger RNA expression were examined in RA and non-RA synovium by immunofluorescence and reverse-transcription polymerase chain reaction (RT-PCR) respectively. GPR15/BOB expression on peripheral blood leukocytes was analysed by flow cytometry and GPR15/BOB messenger RNA was examined in peripheral blood monocytes by RT-PCR. GPR15/BOB protein was observed in CD68+ and CD14+ macrophages in synovia, with greater expression in RA synovia. GPR15/BOB protein was expressed in all patient synovia whereas in non-RA synovia expression was low or absent. Similarly GPR15/BOB messenger RNA was detected in all RA and a minority of non-RA synovia. GPR15/BOB protein was expressed on peripheral blood leukocytes from RA and healthy individuals with increased expression by monocytes and neutrophils in RA. GPR15/BOB messenger RNA expression was confirmed in peripheral blood monocytes. In conclusion GPR15/BOB is expressed by macrophages in synovial tissue and on monocytes and neutrophils in peripheral blood, and expression is up-regulated in RA patients compared to non-RA controls. This orphan receptor on monocytes/macrophages and neutrophils may play a role in RA pathophysiology. PMID:24725539

  12. The Aspergillus fumigatus Damage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

    PubMed Central

    Song, Jinxing; Zhai, Pengfei; Zhang, Yuanwei; Zhang, Caiyun; Sang, Hong; Han, Guanzhu; Keller, Nancy P.

    2016-01-01

    ABSTRACT Ergosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogen Aspergillus fumigatus has developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochrome b5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapA strains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme. In vivo data demonstrate that inactivated DapA combined with activated DapB yields an A. fumigatus mutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli. PMID:26908577

  13. Up-regulation of the Sirtuin 1 (Sirt1) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) genes in white adipose tissue of Id1 protein-deficient mice: implications in the protection against diet and age-induced glucose intolerance.

    PubMed

    Zhao, Ying; Ling, Flora; Griffin, Timothy M; He, Ting; Towner, Rheal; Ruan, Hong; Sun, Xiao-Hong

    2014-10-17

    Id1, a helix-loop-helix (HLH) protein that inhibits the function of basic HLH E protein transcription factors in lymphoid cells, has been implicated in diet- and age-induced obesity by unknown mechanisms. Here we show that Id1-deficient mice are resistant to a high fat diet- and age-induced obesity, as revealed by reduced weight gain and body fat, increased lipid oxidation, attenuated hepatosteatosis, lower levels of lipid droplets in brown adipose tissue, and smaller white adipocytes after a high fat diet feeding or in aged animals. Id1 deficiency improves glucose tolerance, lowers serum insulin levels, and reduces TNFα gene expression in white adipose tissue. Id1 deficiency also increased expression of Sirtuin 1 and peroxisome proliferator-activated receptor γ coactivator 1α, regulators of mitochondrial biogenesis and energy expenditure, in the white adipose tissue. This effect was accompanied by the elevation of several genes encoding proteins involved in oxidative phosphorylation and fatty acid oxidation, such as cytochrome c, medium-chain acyl-CoA dehydrogenase, and adipocyte protein 2. Moreover, the phenotype for Id1 deficiency was similar to that of mice expressing an E protein dominant-positive construct, ET2, suggesting that the balance between Id and E proteins plays a role in regulating lipid metabolism and insulin sensitivity. PMID:25190816

  14. Sesamin induces melanogenesis by microphthalmia-associated transcription factor and tyrosinase up-regulation via cAMP signaling pathway.

    PubMed

    Jiang, Zequn; Li, Shasha; Liu, Yunyi; Deng, Pengyi; Huang, Jianguo; He, Guangyuan

    2011-10-01

    In this study, we confirmed that sesamin, an active lignan isolated from sesame seed and oil, is a novel skin-tanning compound. The melanin content and tyrosinase activity were increased by sesamin in a dose-dependent manner in B16 melanoma cells. The mRNA and protein levels of tyrosinase were also enhanced after the treatment with sesamin. Western blot analysis revealed that sesamin induced and sustained up-regulation of microphthalmia-associated transcription factor (MITF). Sesamin could activate cAMP response element (CRE) binding protein (CREB), but it had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK) or Akt. Moreover, sesamin activated protein kinase A (PKA) via a cAMP-dependent pathway. Consistent with these results, sesamin-mediated increase of melanin synthesis was reduced significantly by H-89, a PKA inhibitor, but not by SB203580, a p38 MAPK inhibitor or by LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor. Sesamin-mediated phosphorylation of CREB and induction of MITF and tyrosinase expression were also inhibited by H-89. These findings indicated that sesamin could stimulate melanogenesis in B16 cells via the up-regulation of MITF and tyrosinase, which was, in turn, due to the activation of cAMP signaling. PMID:21896570

  15. Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine.

    PubMed

    Cordova, Juan Carlos; Olivares, Adrian O; Shin, Yongdae; Stinson, Benjamin M; Calmat, Stephane; Schmitz, Karl R; Aubin-Tam, Marie-Eve; Baker, Tania A; Lang, Matthew J; Sauer, Robert T

    2014-07-31

    ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring. PMID:25083874

  16. Hox Proteins Coordinate Motor Neuron Differentiation and Connectivity Programs through Ret/Gfrα Genes.

    PubMed

    Catela, Catarina; Shin, Maggie M; Lee, David H; Liu, Jeh-Ping; Dasen, Jeremy S

    2016-03-01

    The accuracy of neural circuit assembly relies on the precise spatial and temporal control of synaptic specificity determinants during development. Hox transcription factors govern key aspects of motor neuron (MN) differentiation; however, the terminal effectors of their actions are largely unknown. We show that Hox/Hox cofactor interactions coordinate MN subtype diversification and connectivity through Ret/Gfrα receptor genes. Hox and Meis proteins determine the levels of Ret in MNs and define the intrasegmental profiles of Gfrα1 and Gfrα3 expression. Loss of Ret or Gfrα3 leads to MN specification and innervation defects similar to those observed in Hox mutants, while expression of Ret and Gfrα1 can bypass the requirement for Hox genes during MN pool differentiation. These studies indicate that Hox proteins contribute to neuronal fate and muscle connectivity through controlling the levels and pattern of cell surface receptor expression, consequently gating the ability of MNs to respond to limb-derived instructive cues. PMID:26904955

  17. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  18. Hypoxia promotes Mycobacterium tuberculosis-specific up-regulation of granulysin in human T cells.

    PubMed

    Zenk, Sebastian F; Vollmer, Michael; Schercher, Esra; Kallert, Stephanie; Kubis, Jan; Stenger, Steffen

    2016-06-01

    Oxygen tension affects local immune responses in inflammation and infection. In tuberculosis mycobacteria avoid hypoxic areas and preferentially persist and reactivate in the oxygen-rich apex of the lung. Oxygen restriction activates antimicrobial effector mechanisms in macrophages and restricts growth of intracellular Mycobacterium tuberculosis (M.Tb). The effect of oxygen restriction on T cell-mediated antimicrobial effector mechanisms is unknown. Therefore we determined the influence of hypoxia on the expression of granulysin, an antimicrobial peptide of lymphocytes. Hypoxia increased the antigen-specific up-regulation of granulysin mRNA and protein in human CD4(+) and CD8(+) T lymphocytes. This observation was functionally relevant, because oxygen restriction supported the growth-limiting effect of antigen-specific T cells against virulent M.Tb residing in primary human macrophages. Our results provide evidence that oxygen restriction promotes the expression of granulysin and suggest that this effect-in conjunction with additional T cell-mediated immune responses-supports protection against mycobacteria. The therapeutic modulation of oxygen availability may offer a new strategy for the host-directed therapy of infectious diseases with intracellular pathogens. PMID:26613797

  19. Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii.

    PubMed

    Miozzi, Laura; Balestrini, Raffaella; Bolchi, Angelo; Novero, Mara; Ottonello, Simone; Bonfante, Paola

    2005-07-01

    TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation. PMID:15948845

  20. Neuregulin 1 Controls Glutamate Uptake by Up-regulating Excitatory Amino Acid Carrier 1 (EAAC1).

    PubMed

    Yu, Ha-Nul; Park, Woo-Kyu; Nam, Ki-Hoan; Song, Dae-Yong; Kim, Hye-Sun; Baik, Tai-Kyoung; Woo, Ran-Sook

    2015-08-14

    Neuregulin 1 (NRG1) is a trophic factor that is thought to have important roles in the regulating brain circuitry. Recent studies suggest that NRG1 regulates synaptic transmission, although the precise mechanisms remain unknown. Here we report that NRG1 influences glutamate uptake by increasing the protein level of excitatory amino acid carrier (EAAC1). Our data indicate that NRG1 induced the up-regulation of EAAC1 in primary cortical neurons with an increase in glutamate uptake. These in vitro results were corroborated in the prefrontal cortex (PFC) of mice given NRG1. The stimulatory effect of NRG1 was blocked by inhibition of the NRG1 receptor ErbB4. The suppressed expression of ErbB4 by siRNA led to a decrease in the expression of EAAC1. In addition, the ablation of ErbB4 in parvalbumin (PV)-positive neurons in PV-ErbB4(-/-) mice suppressed EAAC1 expression. Taken together, our results show that NRG1 signaling through ErbB4 modulates EAAC1. These findings link proposed effectors in schizophrenia: NRG1/ErbB4 signaling perturbation, EAAC1 deficit, and neurotransmission dysfunction. PMID:26092725

  1. RECK Is Up-Regulated and Involved in Chondrocyte Cloning in Human Osteoarthritic Cartilage

    PubMed Central

    Kimura, Tokuhiro; Okada, Aiko; Yatabe, Taku; Okubo, Masashi; Toyama, Yoshiaki; Noda, Makoto; Okada, Yasunori

    2010-01-01

    Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is a membrane-anchored matrix metalloproteinase regulator, but its functions in cartilage are not fully understood. The aim of the present study was to examine the expression and functions of RECK in human osteoarthritic (OA) cartilage. Quantitative RT-PCR indicated that the expression level of RECK is significantly higher in OA cartilage than in normal cartilage. By immunohistochemical analysis, RECK was localized to chondrocytes in OA cartilage, and the immunoreactivity directly correlated with the Mankin score and degree of chondrocyte cloning and proliferation. In cultured OA chondrocytes, RECK was expressed on the cell surface by glycosylphosphatidylinositol anchoring. The expression was stimulated by insulin-like growth factor-1 and suppressed by interleukin-1 and tumor necrosis factor-α. Down-regulation of RECK by small interfering RNA showed reduced spreading and smaller focal adhesions in the chondrocytes. Chondrocyte migration in a monolayer wounding assay was increased by down-regulation of RECK and inhibited by RECK overexpression in an matrix metalloproteinase activity-dependent manner. On the other hand, chondrocyte proliferation was suppressed by RECK silencing, and this was associated with reduced phosphorylation of focal adhesion kinase and extracellular signal-regulated kinase, whereas the proliferation was enhanced by RECK overexpression. These data are the first to demonstrate that RECK is up-regulated in human OA cartilage and suggest that RECK plays a role in chondrocyte cloning probably through suppression and promotion of chondrocyte migration and proliferation, respectively. PMID:20395433

  2. Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

    PubMed Central

    Tata, J. R.

    1970-01-01

    hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis. PMID:4314393

  3. Ultraviolet B radiation up-regulates the expression of IL-15 in human skin

    SciTech Connect

    Mohamadzadeh, M.; Takashima, Akira; Dougherty, I.

    1995-11-01

    Ultraviolet B (UVB) radiation is a potent modulator of skin-related immune responses, particularly those involving the synthesis and the secretion of cytokines. The discovery of a new T cell mitogen, IL-15, prompted us to investigate its expression in skin and to examine the effects of UVB radiation on such expression. RNA from unirradiated and UVB-irradiated epidermal and dermal sheets derived from human foreskin as well as from unirradiated and UVB-irradiated skin cell populations were assayed for IL-15 expression by semiquantitative RT-PCR. Constitutive levels of IL-15 mRNA were detected in dermal sheets, but not in epidermal sheets. Following UVB treatment, IL-15 mRNA was induced in epidermal sheets and enhanced in dermal sheets. UVB-inducible epidermal expression of IL-15 mRNA was traced to HLA-DR{sup -} cells (presumably keratinocytes) and not to HLA-DR{sup +} cells (Langerhans cells). Cultured keratinocytes and dermal fibroblasts displayed basal levels of IL-15 mRNA that were also up-regulated following UVB exposure. Immunoblot analysis revealed secretion of IL-15 protein by keratinocytes that enhanced following UVB treatment. These results constitute the first report of IL-15 mRNA expression and protein production in human skin. In addition to expanding the known influence of UVB radiation on the capacity of keratinocytes and dermal fibroblasts to express immunomodulatory cytokines, these findings suggest a new mechanism by which UVB can promote Ag-independent T cell responses via elaboration of IL-15. 51 refs., 6 figs.

  4. Hypoxia Suppresses Spontaneous Mineralization and Osteogenic Differentiation of Mesenchymal Stem Cells via IGFBP3 Up-Regulation.

    PubMed

    Kim, Ji Hye; Yoon, Sei Mee; Song, Sun U; Park, Sang Gyu; Kim, Won-Serk; Park, In Guk; Lee, Jinu; Sung, Jong-Hyuk

    2016-01-01

    Hypoxia has diverse stimulatory effects on human adipose-derived stem cells (ASCs). In the present study, we investigated whether hypoxic culture conditions (2% O₂) suppress spontaneous mineralization and osteogenic differentiation of ASCs. We also investigated signaling pathways and molecular mechanisms involved in this process. We found that hypoxia suppressed spontaneous mineralization and osteogenic differentiation of ASCs, and up-regulated mRNA and protein expression of Insulin-like growth factor binding proteins (IGFBPs) in ASCs. Although treatment with recombinant IGFBPs did not affect osteogenic differentiation of ASCs, siRNA-mediated inhibition of IGFBP3 attenuated hypoxia-suppressed osteogenic differentiation of ASCs. In contrast, overexpression of IGFBP3 via lentiviral vectors inhibited ASC osteogenic differentiation. These results indicate that hypoxia suppresses spontaneous mineralization and osteogenic differentiation of ASCs via intracellular IGFBP3 up-regulation. We determined that reactive oxygen species (ROS) generation followed by activation of the MAPK and PI3K/Akt pathways play pivotal roles in IGFBP3 expression under hypoxia. For example, ROS scavengers and inhibitors for MAPK and PI3K/Akt pathways attenuated the hypoxia-induced IGFBP3 expression. Inhibition of Elk1 and NF-κB through siRNA transfection also led to down-regulation of IGFBP3 mRNA expression. We next addressed the proliferative potential of ASCs with overexpressed IGFBP3, but IGFBP3 overexpression reduced the proliferation of ASCs. In addition, hypoxia reduced the osteogenic differentiation of bone marrow-derived clonal mesenchymal stem cells. Collectively, our results indicate that hypoxia suppresses the osteogenic differentiation of mesenchymal stem cells via IGFBP3 up-regulation. PMID:27563882

  5. Lipopolysaccharide augments the uptake of oxidized LDL by up-regulating lectin-like oxidized LDL receptor-1 in macrophages.

    PubMed

    Hossain, Ekhtear; Ota, Akinobu; Karnan, Sivasundaram; Takahashi, Miyuki; Mannan, Shahnewaj B; Konishi, Hiroyuki; Hosokawa, Yoshitaka

    2015-02-01

    There is a growing body of evidence supporting an intimate association of immune activation with the pathogenesis of cardiovascular diseases, including atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) through scavenging receptors promotes the formation of mature lipid-laden macrophages, which subsequently leads to exacerbation of regional inflammation and atherosclerotic plaque formation. In this study, we first examined changes in the mRNA level of the lectin-like oxLDL receptor-1 (LOX-1) in the mouse macrophage cell line RAW264.7 and the human PMA-induced macrophage cell line THP-1 after LPS stimulation. LPS significantly up-regulated LOX-1 mRNA in RAW264.7 cells; LOX-1 cell-surface protein expression was also increased. Flow cytometry and fluorescence microscopy analyses showed that cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with LPS stimulation. The augmented uptake of Dil-oxLDL was almost completely abrogated by treatment with an anti-LOX-1 antibody. Of note, knockdown of Erk1/2 resulted in a significant reduction of LPS-induced LOX-1 up-regulation. Treatment with U0126, a specific inhibitor of MEK, significantly suppressed LPS-induced expression of LOX-1 at both the mRNA and protein levels. Furthermore, LOX-1 promoter activity was significantly augmented by LPS stimulation; this augmentation was prevented by U0126 treatment. Similar results were also observed in human PMA-induced THP-1 macrophages. Taken together, our results indicate that LPS up-regulates LOX-1, at least in part through activation of the Erk1/2 signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of TLR4-mediated aberrant LOX-1 signaling in the pathogenesis of atherosclerosis. PMID:25348362

  6. Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

    PubMed Central

    Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

    2012-01-01

    Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

  7. Signal pathways in up-regulation of chemokines by tyrosine kinase MER/NYK in prostate cancer cells.

    PubMed

    Wu, Yi-Mi; Robinson, Dan R; Kung, Hsing-Jien

    2004-10-15

    The AXL/UFO family of tyrosine kinases is characterized by a common N-CAM (neural adhesion molecule)-related extracellular domain and a common ligand, GAS6 (growth arrest-specific protein 6). Family members are prone to transcriptional regulation and carry out diverse functions including the regulation of cell adhesion, migration, phagocytosis, and survival. In this report, we describe a new role of MER/N-CAM-related kinase (NYK), a member of the AXL family of kinases, in the up-regulation of chemokines in prostate cancer cells. We show that NYK has elevated expression in a subset of tumor specimens and prostate cancer cell lines. Activation of NYK in the prostate cancer cell line DU145 does not cause a mitogenic effect; instead, it causes a differentiation phenotype. Microarray analysis revealed that NYK is a strong inducer of endocrine factors including interleukin (IL)-8 and several other angiogenic CXC chemokines as well as bone morphogenic factors. The dramatic increase of IL-8 expression is seen at both transcriptional and posttranscriptional levels. The downstream signals engaged by NYK were characterized, and those responsible for the up-regulation of IL-8 transcription were defined. In contrast to IL-1alpha, NYK-induced up-regulation of IL-8 in DU145 depends on the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase/Jun/Fos pathway, but not phosphoinositide 3'-kinase/nuclear factor-kappaB. These data define a new function of the AXL family of kinases and suggest a potential role of NYK in prostate cancer progression. PMID:15492251

  8. Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

    PubMed

    Peng, Lei; Utesch, Tillmann; Yarman, Aysu; Jeoung, Jae-Hun; Steinborn, Silke; Dobbek, Holger; Mroginski, Maria Andrea; Tanne, Johannes; Wollenberger, Ulla; Scheller, Frieder W

    2015-05-11

    Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\\prime }}\\hfill \\atop {\\rm app}\\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH. PMID:25825040

  9. Ischemia-Reperfusion Injury and Pregnancy Initiate Time-Dependent and Robust Signs of Up-Regulation of Cardiac Progenitor Cells

    PubMed Central

    Genead, Rami; Fischer, Helene; Hussain, Alamdar; Jaksch, Marie; Andersson, Agneta B.; Ljung, Karin; Bulatovic, Ivana; Franco-Cereceda, Anders; Elsheikh, Elzafir; Corbascio, Matthias; Smith, C. I. Edvard; Sylvén, Christer; Grinnemo, Karl-Henrik

    2012-01-01

    To explore how cardiac regeneration and cell turnover adapts to disease, different forms of stress were studied for their effects on the cardiac progenitor cell markers c-Kit and Isl1, the early cardiomyocyte marker Nkx2.5, and mast cells. Adult female rats were examined during pregnancy, after myocardial infarction and ischemia-reperfusion injury with/out insulin like growth factor-1(IGF-1) and hepatocyte growth factor (HGF). Different cardiac sub-domains were analyzed at one and two weeks post-intervention, both at the mRNA and protein levels. While pregnancy and myocardial infarction up-regulated Nkx2.5 and c-Kit (adjusted for mast cell activation), ischemia-reperfusion injury induced the strongest up-regulation which occurred globally throughout the entire heart and not just around the site of injury. This response seems to be partly mediated by increased endogenous production of IGF-1 and HGF. Contrary to c-Kit, Isl1 was not up-regulated by pregnancy or myocardial infarction while ischemia-reperfusion injury induced not a global but a focal up-regulation in the outflow tract and also in the peri-ischemic region, correlating with the up-regulation of endogenous IGF-1. The addition of IGF-1 and HGF did boost the endogenous expression of IGF and HGF correlating to focal up-regulation of Isl1. c-Kit expression was not further influenced by the exogenous growth factors. This indicates that there is a spatial mismatch between on one hand c-Kit and Nkx2.5 expression and on the other hand Isl1 expression. In conclusion, ischemia-reperfusion injury was the strongest stimulus with both global and focal cardiomyocyte progenitor cell marker up-regulations, correlating to the endogenous up-regulation of the growth factors IGF-1 and HGF. Also pregnancy induced a general up-regulation of c-Kit and early Nkx2.5+ cardiomyocytes throughout the heart. Utilization of these pathways could provide new strategies for the treatment of cardiac disease. PMID:22590612

  10. AGEs-Induced IL-6 Synthesis Precedes RAGE Up-Regulation in HEK 293 Cells: An Alternative Inflammatory Mechanism?

    PubMed

    Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Dinischiotu, Anca

    2015-01-01

    Advanced glycation end products (AGEs) can activate the inflammatory pathways involved in diabetic nephropathy. Understanding these molecular pathways could contribute to therapeutic strategies for diabetes complications. We evaluated the modulation of inflammatory and oxidative markers, as well as the protective mechanisms employed by human embryonic kidney cells (HEK 293) upon exposure to 200 μg/mL bovine serum albumine (BSA) or AGEs-BSA for 12, 24 and 48 h. The mRNA and protein expression levels of AGEs receptor (RAGE) and heat shock proteins (HSPs) 27, 60 and 70, the activity of antioxidant enzymes and the expression levels of eight cytokines were analysed. Cell damage via oxidative mechanisms was evaluated by glutathione and malondialdehyde levels. The data revealed two different time scale responses. First, the up-regulation of interleukin-6 (IL-6), HSP 27 and high catalase activity were detected as early as 12 h after exposure to AGEs-BSA, while the second response, after 24 h, consisted of NF-κB p65, RAGE, HSP 70 and inflammatory cytokine up-regulation, glutathione depletion, malondialdehyde increase and the activation of antioxidant enzymes. IL-6 might be important in the early ignition of inflammatory responses, while the cellular redox imbalance, RAGE activation and NF-κB p65 increased expression further enhance inflammatory signals in HEK 293 cells. PMID:26307981

  11. AGEs-Induced IL-6 Synthesis Precedes RAGE Up-Regulation in HEK 293 Cells: An Alternative Inflammatory Mechanism?

    PubMed Central

    Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Dinischiotu, Anca

    2015-01-01

    Advanced glycation end products (AGEs) can activate the inflammatory pathways involved in diabetic nephropathy. Understanding these molecular pathways could contribute to therapeutic strategies for diabetes complications. We evaluated the modulation of inflammatory and oxidative markers, as well as the protective mechanisms employed by human embryonic kidney cells (HEK 293) upon exposure to 200 μg/mL bovine serum albumine (BSA) or AGEs–BSA for 12, 24 and 48 h. The mRNA and protein expression levels of AGEs receptor (RAGE) and heat shock proteins (HSPs) 27, 60 and 70, the activity of antioxidant enzymes and the expression levels of eight cytokines were analysed. Cell damage via oxidative mechanisms was evaluated by glutathione and malondialdehyde levels. The data revealed two different time scale responses. First, the up-regulation of interleukin-6 (IL-6), HSP 27 and high catalase activity were detected as early as 12 h after exposure to AGEs–BSA, while the second response, after 24 h, consisted of NF-κB p65, RAGE, HSP 70 and inflammatory cytokine up-regulation, glutathione depletion, malondialdehyde increase and the activation of antioxidant enzymes. IL-6 might be important in the early ignition of inflammatory responses, while the cellular redox imbalance, RAGE activation and NF-κB p65 increased expression further enhance inflammatory signals in HEK 293 cells. PMID:26307981

  12. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14

    PubMed Central

    Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC. PMID:27494117

  13. Modified AS1411 Aptamer Suppresses Hepatocellular Carcinoma by Up-Regulating Galectin-14.

    PubMed

    Cho, Yuri; Lee, Yun Bin; Lee, Jeong-Hoon; Lee, Dong Hyeon; Cho, Eun Ju; Yu, Su Jong; Kim, Yoon Jun; Kim, Jong In; Im, Jong Hun; Lee, Jung Hwan; Oh, Eun Ju; Yoon, Jung-Hwan

    2016-01-01

    Aptamers are small synthetic oligonucleotides that bind to target proteins with high specificity and affinity. AS1411 is an aptamer that binds to nucleolin, which is overexpressed in the cytoplasm and occurs on the surface of cancer cells. We investigated the therapeutic potential of aptamers in hepatocellular carcinoma (HCC) by evaluating anti-tumor effects and confirming the affinity and specificity of AS1411- and modified AS1411-aptamers in HCC cells. Cell growth was assessed using the MTS assay, and cell death signaling was explored by immunoblot analysis. Fluorescence-activated cell sorting was performed to evaluate the affinity and specificity of AS1411-aptamers in SNU-761 HCC cells. We investigated the in vivo effects of the AS1411-aptamer using BALB/c nude mice in a subcutaneous xenograft model with SNU-761 cells. Treatment with a modified AS1411-aptamer significantly decreased in vitro (under normoxic [P = 0.035] and hypoxic [P = 0.018] conditions) and in vivo (under normoxic conditions, P = 0.041) HCC cell proliferation compared to control aptamers. AS1411- and control aptamers failed to control HCC cell proliferation. However, AS1411- and the modified AS1411-aptamer did not induce caspase activation. Decrease in cell growth by AS1411 or modified AS1411 was not prevented by caspase or necrosis inhibitors. In a microarray, AS1411 significantly enhanced galectin-14 expression. Suppression of HCC cell proliferation by the modified AS1411-aptamer was attenuated by galectin-14 siRNA transfection. Modified AS1411-aptamer suppressed HCC cell growth in vitro and in vivo by up-regulating galectin-14 expressions. Modified AS1411-aptamers may have therapeutic potential as a novel targeted therapy for HCC. PMID:27494117

  14. The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

    PubMed

    Spatz, Jordan M; Wein, Marc N; Gooi, Jonathan H; Qu, Yili; Garr, Jenna L; Liu, Shawn; Barry, Kevin J; Uda, Yuhei; Lai, Forest; Dedic, Christopher; Balcells-Camps, Mercedes; Kronenberg, Henry M; Babij, Philip; Pajevic, Paola Divieti

    2015-07-01

    Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone. PMID:25953900

  15. Pannexin-1 Up-regulation in the Dorsal Root Ganglion Contributes to Neuropathic Pain Development.

    PubMed

    Zhang, Yuhao; Laumet, Geoffroy; Chen, Shao-Rui; Hittelman, Walter N; Pan, Hui-Lin

    2015-06-01

    Pannexin-1 (Panx1) is a large-pore membrane channel involved in the release of ATP and other signaling mediators. Little is known about the expression and functional role of Panx1 in the dorsal root ganglion (DRG) in the development of chronic neuropathic pain. In this study, we determined the epigenetic mechanism involved in increased Panx1 expression in the DRG after nerve injury. Spinal nerve ligation in rats significantly increased the mRNA and protein levels of Panx1 in the DRG but not in the spinal cord. Immunocytochemical labeling showed that Panx1 was primarily expressed in a subset of medium and large DRG neurons in control rats and that nerve injury markedly increased the number of Panx1-immunoreactive DRG neurons. Nerve injury significantly increased the enrichment of two activating histone marks (H3K4me2 and H3K9ac) and decreased the occupancy of two repressive histone marks (H3K9me2 and H3K27me3) around the promoter region of Panx1 in the DRG. However, nerve injury had no effect on the DNA methylation level around the Panx1 promoter in the DRG. Furthermore, intrathecal injection of the Panx1 blockers or Panx1-specific siRNA significantly reduced pain hypersensitivity induced by nerve injury. In addition, siRNA knockdown of Panx1 expression in a DRG cell line significantly reduced caspase-1 release induced by neuronal depolarization. Our findings suggest that nerve injury increases Panx1 expression levels in the DRG through altered histone modifications. Panx1 up-regulation contributes to the development of neuropathic pain and stimulation of inflammasome signaling. PMID:25925949

  16. Pannexin-1 Up-regulation in the Dorsal Root Ganglion Contributes to Neuropathic Pain Development*

    PubMed Central

    Zhang, Yuhao; Laumet, Geoffroy; Chen, Shao-Rui; Hittelman, Walter N.; Pan, Hui-Lin

    2015-01-01

    Pannexin-1 (Panx1) is a large-pore membrane channel involved in the release of ATP and other signaling mediators. Little is known about the expression and functional role of Panx1 in the dorsal root ganglion (DRG) in the development of chronic neuropathic pain. In this study, we determined the epigenetic mechanism involved in increased Panx1 expression in the DRG after nerve injury. Spinal nerve ligation in rats significantly increased the mRNA and protein levels of Panx1 in the DRG but not in the spinal cord. Immunocytochemical labeling showed that Panx1 was primarily expressed in a subset of medium and large DRG neurons in control rats and that nerve injury markedly increased the number of Panx1-immunoreactive DRG neurons. Nerve injury significantly increased the enrichment of two activating histone marks (H3K4me2 and H3K9ac) and decreased the occupancy of two repressive histone marks (H3K9me2 and H3K27me3) around the promoter region of Panx1 in the DRG. However, nerve injury had no effect on the DNA methylation level around the Panx1 promoter in the DRG. Furthermore, intrathecal injection of the Panx1 blockers or Panx1-specific siRNA significantly reduced pain hypersensitivity induced by nerve injury. In addition, siRNA knockdown of Panx1 expression in a DRG cell line significantly reduced caspase-1 release induced by neuronal depolarization. Our findings suggest that nerve injury increases Panx1 expression levels in the DRG through altered histone modifications. Panx1 up-regulation contributes to the development of neuropathic pain and stimulation of inflammasome signaling. PMID:25925949

  17. The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro*

    PubMed Central

    Spatz, Jordan M.; Wein, Marc N.; Gooi, Jonathan H.; Qu, Yili; Garr, Jenna L.; Liu, Shawn; Barry, Kevin J.; Uda, Yuhei; Lai, Forest; Dedic, Christopher; Balcells-Camps, Mercedes; Kronenberg, Henry M.; Babij, Philip; Pajevic, Paola Divieti

    2015-01-01

    Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone. PMID:25953900

  18. Quantitative Proteomic Analysis Revealed 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone-induced Up-regulation of 20S Proteasome in Cultured Human Fibroblast Cells

    PubMed Central

    Prins, John M.; Wang, Yinsheng

    2012-01-01

    The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90 and MCF-7 cells upon NNK treatment. Furthermore, the β-adrenergic receptor (β-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the β-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK. PMID:22369695

  19. The prevention and treatment of hypoadiponectinemia-associated human diseases by up-regulation of plasma adiponectin.

    PubMed

    Hossain, Md Murad; Mukheem, Abdul; Kamarul, Tunku

    2015-08-15

    Hypoadiponectinemia is characterized by low plasma adiponectin levels that can be caused by genetic factors, such as single nucleotide polymorphisms (SNPs) and mutations in the adiponectin gene or by visceral fat deposition/obesity. Reports have suggested that hypoadiponectinemia is associated with dyslipidemia, hypertension, hyperuricemia, metabolic syndrome, atherosclerosis, type 2 diabetes mellitus and various cardiovascular diseases. Previous studies have highlighted several potential strategies to up-regulate adiponectin secretion and function, including visceral fat reduction through diet therapy and exercise, administration of exogenous adiponectin, treatment with peroxisome proliferator-activating receptor gamma (PPARγ) agonists (e.g., thiazolidinediones (TZDs)) and ligands (e.g., bezafibrate and fenofibrate) or the blocking of the renin-angiotensin system. Likewise, the up-regulation of the expression and stimulation of adiponectin receptors by using adiponectin receptor agonists would be an effective method to treat obesity-related conditions. Notably, adiponectin is an abundantly expressed bioactive protein that also exhibits a wide spectrum of biological properties, such as insulin-sensitizing, anti-diabetic, anti-inflammatory and anti-atherosclerotic activities. Although targeting adiponectin and its receptors has been useful for treating diabetes and other metabolic-related diseases in experimental studies, current drug development based on adiponectin/adiponectin receptors for clinical applications is scarce, and there is a lack of available clinical trial data. This comprehensive review discusses the strategies that are presently being pursued to harness the potential of adiponectin up-regulation. In addition, we examined the current status of drug development and its potential for clinical applications. PMID:25818192

  20. Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

    PubMed Central

    Krtková, Jana; Thomas, Elizabeth B.; Alas, Germain C. M.; Schraner, Elisabeth M.; Behjatnia, Habib R.; Hehl, Adrian B.

    2016-01-01

    ABSTRACT Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-RacCA) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis. PMID:27555307

  1. Up-regulation of emotional responses to reward-predicting stimuli: an ERP study.

    PubMed

    Langeslag, Sandra J E; van Strien, Jan W

    2013-09-01

    Altered reward processing is a hallmark symptom of many psychiatric disorders. It has recently been shown that people are capable of down-regulating reward processing. Here, we examined whether people are capable of up-regulating emotional responses to reward-predicting stimuli. Participants passively viewed colored squares that predicted a reward or no reward, and up- or down-regulated their emotional responses to these reward-predicting stimuli by focusing on the reward meaning or the color of the squares respectively. The amplitude of the late positive potential (LPP) was taken as an objective index of regulation success. The LPP in response to reward-predicting squares was enhanced by up-regulation, suggesting that up-regulation of emotional responses to reward-predicting stimuli using a cognitive strategy is feasible. These results are highly relevant for the treatment of disorders characterized by diminished motivation, and for reward-based decision making in daily life. PMID:23770414

  2. Representation of protein 3D structures in spherical (ρ, ϕ, θ) coordinates and two of its potential applications.

    PubMed

    Reyes, Vicente M

    2011-09-01

    Three-dimensional objects can be represented using cartesian, spherical or cylindrical coordinate systems, among many others. Currently all protein 3D structures in the PDB are in cartesian coordinates. We wanted to explore the possibility that protein 3D structures, especially the globular type (spheroproteins), when represented in spherical coordinates might find useful novel applications. A Fortran program was written to transform protein 3D structure files in cartesian coordinates (x,y,z) to spherical coordinates (ρ, ϕ, θ), with the centroid of the protein molecule as origin. We present here two applications, namely, (1) separation of the protein outer layer (OL) from the inner core (IC); and (2) identifying protrusions and invaginations on the protein surface. In the first application, ϕ and θ were partitioned into suitable intervals and the point with maximum ρ in each such 'ϕ-θ bin' was determined. A suitable cutoff value for ρ is adopted, and for each ϕ-θ bin, all points with ρ values less than the cutoff are considered part of the IC, and those with ρ values equal to or greater than the cutoff are considered part of the OL. We show that this separation procedure is successful as it gives rise to an OL that is significantly more enriched in hydrophilic amino acid residues, and an IC that is significantly more enriched in hydrophobic amino acid residues, as expected. In the second application, the point with maximum ρ in each ϕ-θ bin are sequestered and their frequency distribution constructed (i.e., maximum ρ's sorted from lowest to highest, collected into 1.50Å-intervals, and the frequency in each interval plotted). We show in such plots that invaginations on the protein surface give rise to subpeaks or shoulders on the lagging side of the main peak, while protrusions give rise to similar subpeaks or shoulders, but on the leading side of the main peak. We used the dataset of Laskowski et al. (1996) to demonstrate both applications. PMID

  3. Metformin Inhibits Androgen-Induced IGF-IR Up-Regulation in Prostate Cancer Cells by Disrupting Membrane-Initiated Androgen Signaling

    PubMed Central

    Malaguarnera, Roberta; Sacco, Antonella; Morcavallo, Alaide; Squatrito, Sebastiano; Migliaccio, Antimo; Morrione, Andrea; Maggiolini, Marcello

    2014-01-01

    We have previously demonstrated that, in prostate cancer cells, androgens up-regulate IGF-I receptor (IGF-IR) by inducing cAMP-response element-binding protein (CREB) activation and CREB-dependent IGF-IR gene transcription through androgen receptor (AR)-dependent membrane-initiated effects. This IGF-IR up-regulation is not blocked by classical antiandrogens and sensitizes cells to IGF-I-induced biological effects. Metformin exerts complex antitumoral functions in various models and may inhibit CREB activation in hepatocytes. We, therefore, evaluated whether metformin may affect androgen-dependent IGF-IR up-regulation. In the AR+ LNCaP prostate cancer cells, we found that metformin inhibits androgen-induced CRE activity and IGF-IR gene transcription. CRE activity requires the formation of a CREB-CREB binding protein-CREB regulated transcription coactivator 2 (CRTC2) complex, which follows Ser133-CREB phosphorylation. Metformin inhibited Ser133-CREB phosphorylation and induced nuclear exclusion of CREB cofactor CRTC2, thus dissociating the CREB-CREB binding protein-CRTC2 complex and blocking its transcriptional activity. Similarly to metformin action, CRTC2 silencing inhibited IGF-IR promoter activity. Moreover, metformin blocked membrane-initiated signals of AR to the mammalian target of rapamycin/p70S6Kinase pathway by inhibiting AR phosphorylation and its association with c-Src. AMPK signals were also involved to some extent. By inhibiting androgen-dependent IGF-IR up-regulation, metformin reduced IGF-I-mediated proliferation of LNCaP cells. These results indicate that, in prostate cancer cells, metformin inhibits IGF-I-mediated biological effects by disrupting membrane-initiated AR action responsible for IGF-IR up-regulation and suggest that metformin could represent a useful adjunct to the classical antiandrogen therapy. PMID:24437490

  4. Mu opioid receptor up-regulation and participation in excitability of hippocampal pyramidal cell electrophysiology

    SciTech Connect

    Moudy, A.M.

    1988-01-01

    Chronic administration of opiate antagonists to rats results in up-regulation of their brain opioid receptors. Using subcellular fractionation techniques, brain opioid receptors were resolved into two membrane populations, one associated with synaptic plasma membranes (SPM) and the other enriched in smooth endoplasmic reticulum and Golgi (microsomes). This study addressed in part the question of whether an antagonist induces up-regulation uniformly in these two populations. Rats were administered naltrexone by subcutaneously implanted osmotic minipumps. Forebrain mu receptor levels were determined by homologous displacement of ({sup 3}H)D-ala{sup 2}-mePhe{sup 4}-gly-ol{sup 5}-enkephalin (DAGO) followed by computer estimation of binding parameters. Receptor levels in crude membranes rose 77% after treatment. Microsomes displayed a 92% increase, a two-fold greater change than in SPMs (51%). These results establish that naltrexone induces up-regulation of both membrane populations; and that microsomal and SPM receptors represent discrete populations of intracellular and cell surface sites, respectively. Binding experiments on isolated hippocampi also demonstrated up-regulation (71%) of mu receptors. To demonstrate up-regulation of opioid receptors electrophysiologically, hippocampal slices were prepared from rats which had been chronically treated with naltrexone. After superfusion with DAGO, these slices showed a 42% greater population spike output than controls in response to the same EPSP input. Hippocampi from animals treated for two weeks showed an additional increase in sensitivity. The results support a disinhibitory role for opioids in pyramidal cell hyper-excitability. More importantly, they demonstrate a significant physiological correlate to opioid receptor up-regulation.

  5. Ursolic Acid, a Pentacyclin Triterpene, Potentiates TRAIL-induced Apoptosis through p53-independent Up-regulation of Death Receptors

    PubMed Central

    Prasad, Sahdeo; Yadav, Vivek R.; Kannappan, Ramaswamy; Aggarwal, Bharat B.

    2011-01-01

    Discovery of the molecular targets of traditional medicine and its chemical footprints can validate the use of such medicine. In the present report, we investigated the effect of ursolic acid (UA), a pentacyclic triterpenoid found in rosemary and holy basil, on apoptosis induced by TRAIL. We found that UA potentiated TRAIL-induced apoptosis in cancer cells. In addition, UA also sensitized TRAIL-resistant cancer cells to the cytokine. When we investigated the mechanism, we found that UA down-regulated cell survival proteins and induced the cell surface expression of both TRAIL receptors, death receptors 4 and 5 (DR4 and -5). Induction of receptors by UA occurred independently of cell type. Gene silencing of either receptor by small interfering RNA reduced the apoptosis induced by UA and the effect of TRAIL. In addition, UA also decreased the expression of decoy receptor 2 (DcR2) but not DcR1. Induction of DRs was independent of p53 because UA induced DR4 and DR5 in HCT116 p53−/− cells. Induction of DRs, however, was dependent on JNK because UA induced JNK, and its pharmacologic inhibition abolished the induction of the receptors. The down-regulation of survival proteins and up-regulation of the DRs required reactive oxygen species (ROS) because UA induced ROS, and its quenching abolished the effect of the terpene. Also, potentiation of TRAIL-induced apoptosis by UA was significantly reduced by both ROS quenchers and JNK inhibitor. In addition, UA was also found to induce the expression of DRs, down-regulate cell survival proteins, and activate JNK in orthotopically implanted human colorectal cancer in a nude mouse model. Overall, our results showed that UA potentiates TRAIL-induced apoptosis through activation of ROS and JNK-mediated up-regulation of DRs and down-regulation of DcR2 and cell survival proteins. PMID:21156789

  6. Up-Regulated Expression of LAMP2 and Autophagy Activity during Neuroendocrine Differentiation of Prostate Cancer LNCaP Cells.

    PubMed

    Morell, Cecilia; Bort, Alicia; Vara-Ciruelos, Diana; Ramos-Torres, Ágata; Altamirano-Dimas, Manuel; Díaz-Laviada, Inés; Rodríguez-Henche, Nieves

    2016-01-01

    Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer. PMID:27627761

  7. Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina

    PubMed Central

    Huang, Pin-Chien; Hsiao, Yu-Tien; Kao, Shao-Yen; Chen, Ching-Feng; Chen, Yu-Chieh; Chiang, Chung-Wei; Lee, Chien-fei; Lu, Juu-Chin; Chern, Yijuang; Wang, Chih-Tien

    2014-01-01

    Background Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A2A receptor (A2AR) regulates retinal waves and whether A2AR regulation of retinal waves acts via presynaptic SACs. Methodology/Principal Findings We showed that A2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A2AR decreased the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR may up-regulate wave frequency. To investigate whether A2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca2+ transient frequency was increased by expressing wild-type A2AR (A2AR-WT) in SACs, suggesting that A2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A2AR mutant (A2AR-ΔC) in SACs, the wave frequency was reduced compared to the A2AR-WT, but was similar to the control, suggesting that the full-length A2AR in SACs is required for A2AR up-regulation of retinal waves. Conclusions/Significance A2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full

  8. Fulvestrant up regulates UGT1A4 and MRPs through ERα and c-Myb pathways: a possible primary drug disposition mechanism.

    PubMed

    Edavana, Vineetha K; Penney, Rosalind B; Yao-Borengasser, Aiwei; Williams, Suzanne; Rogers, Lora; Dhakal, Ishwori B; Kadlubar, Susan

    2013-01-01

    Fulvestrant (Faslodex™) is a pure antiestrogen that is effective in treating estrogen receptor-(ER) positive breast cancer tumors that are resistant to selective estrogen receptor modulators such as tamoxifen. Clinical trials investigating the utility of adding fulvestrant to other therapeutics have not been shown to affect cytochrome P450-mediated metabolism. Effects on phase II metabolism and drug resistance have not been explored. This study demonstrates that fulvestrant up regulates the expression of UDP glucuronosyltransferase 1A4 (UGT1A4) >2.5- and >3.5-fold in MCF7 and HepG2 cells, respectively. Up regulation occurred in a time- and concentration-dependent manner, and was inhibited by siRNA silencing of ERα. Fulvestrant also up regulates multidrug resistance-associated proteins (MRPs). There was an up regulation of MRP2 (1.5- and 3.5-fold), and MRP3 (5.5- and 4.5-fold) in MCF7 and HepG2 cell lines, respectively, and an up regulation of MRP1 (4-fold) in MCF7 cells. UGT1A4 mRNA up regulation was significantly correlated with UGT1A4 protein expression, anastrozole glucuronidation, ERα mRNA expression and MRP mRNA expression, but not with ERα protein expression. Genetic variants in the UGT1A4 promoter (-163A, -217G and -219T) reduced the basal activity of UGT1A4 by 40-60%. In silico analysis indicated that transcription factor c-Myb binding capacity may be affected by these variations. Luciferase activity assays demonstrate that silencing c-Myb abolished UGT1A4 up regulation by fulvestrant in promoters with the common genotype (-163G, -217 T and -219C) in MCF7 cells. These data indicate that fulvestrant can influence the disposition of other UGT1A4 substrates. These findings suggest a clinically significant role for UGT1A4 and MRPs in drug efficacy. PMID:24298433

  9. Transcutaneous Electrical Nerve Stimulation (TENS) Improves the Diabetic Cytopathy (DCP) via Up-Regulation of CGRP and cAMP

    PubMed Central

    Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n = 15), DM group (n = 15) and control group (n = 15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  10. Transcutaneous electrical nerve stimulation (TENS) improves the diabetic cytopathy (DCP) via up-regulation of CGRP and cAMP.

    PubMed

    Ding, Liucheng; Song, Tao; Yi, Chaoran; Huang, Yi; Yu, Wen; Ling, Lin; Dai, Yutian; Wei, Zhongqing

    2013-01-01

    The objective of this study was to investigate the effects and mechanism of Transcutaneous Electrical Nerve Stimulation (TENS) on the diabetic cytopathy (DCP) in the diabetic bladder. A total of 45 rats were randomly divided into diabetes mellitus (DM)/TENS group (n=15), DM group (n=15) and control group (n=15). The rats in the DM/TENS and TENS groups were electronically stimulated (stimulating parameters: intensity-31 V, frequency-31 Hz, and duration of stimulation of 15 min) for three weeks. Bladder histology, urodynamics and contractile responses to field stimulation and carbachol were determined. The expression of calcitonin gene-related peptide (CGRP) was analyzed by RT-PCR and Western blotting. The results showed that contractile responses of the DM rats were ameliorated after 3 weeks of TENS. Furthermore, TENS significantly increased bladder wet weight, volume threshold for micturition and reduced PVR, V% and cAMP content of the bladder. The mRNA and protein levels of CGRP in dorsal root ganglion (DRG) in the DM/TENS group were higher than those in the DM group. TENS also significantly up-regulated the cAMP content in the bladder body and base compared with diabetic rats. We conclude that TENS can significantly improve the urine contractility and ameliorate the feeling of bladder fullness in DM rats possibly via up-regulation of cAMP and CGRP in DRG. PMID:23468996

  11. Angiostatin up-regulation in gastric cancer cell SGC7901 inhibits tumorigenesis in nude mice

    PubMed Central

    Wu, Jing; Shi, Yong-Quan; Wu, Kai-Chun; Zhang, De-Xin; Yang, Jing-Hua; Fan, Dai-Ming

    2003-01-01

    AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gastric cancer cells in vitro and in vivo, and the potential of angiostatin gene therapy in the treatment of human gastric cancer. METHODS: Mouse angiostatin cDNA was subcloned into the eukaryotic expression vector pcDNA3.1(+) and identified by restriction endonucleases digestion and sequencing. The recombinant vector pcDNA3.1(+)-angio was transfected into human gastric cancer cells SGC7901 with liposome and paralleled with the vector control and the mock control. Angiostatin transcription and protein expression were examined by RT-PCR and Western blot in the stable cell lines selected by G418. Cell proliferation and growth in vitro of the three groups were observed respectively under microscope, cell number counting and FACS. The cells overexpressing angiostatin, vector transfected and untreated were respectively implanted subcutaneously into nude mice. After 30 days the size of tumors formed was measured, and microvessel density count (MVD) in the tumor tissues was assessed by immunohistochemistry with the primary anti-vWF antibody. RESULTS: The recombinant vector pcDNA3.1(+)-angio was confirmed with the correct sequence of mouse angiostatin under the promoter CMV. After 30 d of transfection and selection with G418, macroscopic resistant cell clones were formed in the experimental group transfected with pcDNA 3.1(+)-angio and the vector control. But no untreated cells survived in the mock control. Angiostatin mRNA transcription and protein expression were detected in the experimental group. No significant differences were observed among the three groups in cell morphology, cell growth curves and cell cycle phase distributions in vitro. However, in nude mice model, markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experimental group as compared with the controls, which was paralleled with decreased microvessel density in and around tumor tissues (P

  12. Cotton benzoquinone reductase: up-regulation during early fiber development and heterologous expression and characterization in Pichia pastoris.

    PubMed

    Turley, Rickie B; Taliercio, Earl

    2008-01-01

    Benzoquinone reductase (BR; EC 1.6.5.7) is an enzyme which catalyzes the bivalent redox reactions of quinones without the production of free radical intermediates. Using 2D-PAGE comparisons, two proteins were found to be up-regulated in wild-type cotton ovules during the fiber initiation stage but not in the fiberless line SL 1-7-1. These proteins were excised from the gel, partially sequenced and identified to be BR isoforms. PCR was used to amplify both full length coding regions of 609bp and once cloned, the restriction enzyme HindIII was used to distinguish the clones encoding the BR1 (one site) and BR2 (two sites) isoforms. Both deduced protein sequences had 203 residues which differed at 14 residues. The molecular mass and pIs were similar between the measured protein (2D-PAGE) and the theoretical protein (deduced). Heterologous proteins BR1 and BR2 were produced for further study by ligating the BR1 and BR2 clones in frame into the alpha-factor secretion sequence in pPICZalphaA vector and expressed with Pichia pastoris. Both BR1 and BR2 were approximately 26.5kDa and did enzymatically reduce 2,6-dimethoxybenzoquinone similar to the fungal BR. PMID:18534861

  13. A novel copper(II) coordination at His186 in full-length murine prion protein

    SciTech Connect

    Watanabe, Yasuko; Hiraoka, Wakako; Igarashi, Manabu; Ito, Kimihito; Shimoyama, Yuhei; Horiuchi, Motohiro; Yamamori, Tohru; Yasui, Hironobu; Kuwabara, Mikinori; Inagaki, Fuyuhiko; Inanami, Osamu

    2010-04-09

    To explore Cu(II) ion coordination by His{sup 186} in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 A. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 A, 18.1 A, 10.7 A, and 8.4 A, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP{sup C}.

  14. Up-Regulation of Intestinal Vascular Endothelial Growth Factor by Afa/Dr Diffusely Adhering Escherichia coli

    PubMed Central

    Cane, Gaëlle; Moal, Vanessa Liévin-Le; Pagès, Gilles; Servin, Alain L.; Hofman, Paul; Vouret-Craviari, Valérie

    2007-01-01

    Background Angiogenesis has been recently described as a novel component of inflammatory bowel disease pathogenesis. The level of vascular endothelial growth factor (VEGF) has been found increased in Crohn's disease and ulcerative colitis mucosa. To question whether a pro-inflammatory Escherichia coli could regulate the expression of VEGF in human intestinal epithelial cells, we examine the response of cultured human colonic T84 cells to infection by E. coli strain C1845 that belongs to the typical Afa/Dr diffusely adhering E. coli family (Afa/Dr DAEC). Methodology VEGF mRNA expression was examined by Northern blotting and q-PCR. VEGF protein levels were assayed by ELISA and its bioactivity was analysed in endothelial cells. The bacterial factor involved in VEGF induction was identified using recombinant E. coli expressing Dr adhesin, purified Dr adhesin and lipopolysaccharide. The signaling pathway activated for the up-regulation of VEGF was identified using a blocking monoclonal anti-DAF antibody, Western blot analysis and specific pharmacological inhibitors. Principal Findings C1845 bacteria induce the production of VEGF protein which is bioactive. VEGF is induced by adhering C1845 in both a time- and bacteria concentration-dependent manner. This phenomenon is not cell line dependent since we reproduced this observation in intestinal LS174, Caco2/TC7 and INT407 cells. Up-regulation of VEGF production requires: (1) the interaction of the bacterial F1845 adhesin with the brush border-associated decay accelerating factor (DAF, CD55) acting as a bacterial receptor, and (2) the activation of a Src protein kinase upstream of the activation of the Erk and Akt signaling pathways. Conclusions Results demonstrate that a Afa/Dr DAEC strain induces an adhesin-dependent activation of DAF signaling that leads to the up-regulation of bioactive VEGF in cultured human intestinal cells. Thus, these results suggest a link between an entero-adherent, pro-inflammatory E. coli strain

  15. Methamphetamine and 3,4-methylenedioxymethamphetamine interact with central nicotinic receptors and induce their up-regulation

    SciTech Connect

    Garcia-Rates, Sara; Camarasa, Jordi; Escubedo, Elena; Pubill, David

    2007-09-15

    Previous work from our group indicated that {alpha}7 nicotinic acetylcholine receptors ({alpha}7 nAChR) potentially play a role in methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA) neurotoxicity. The aims of the present study were two-fold: (1) to demonstrate the interaction of METH and MDMA with homomeric {alpha}7 nAChR ([{sup 3}H]methyllycaconitine binding) and other heteromeric subtypes ([{sup 3}H]epibatidine binding); and (2) to show the effects of amphetamine derivative pretreatment on the density of binding sites. METH and MDMA displaced [{sup 3}H]methyllycaconitine and [{sup 3}H]epibatidine binding in membranes from NGF-differentiated PC 12 cells and mouse brain, with K{sub i} values in the micromolar range, MDMA revealing a greater affinity than METH. In addition, METH and MDMA induced a time- and concentration-dependent increase in [{sup 3}H]methyllycaconitine and [{sup 3}H]epibatidine binding; which had already been apparent after 6 h of pretreatment, and which peaked in differentiated PC 12 cells after 48 h. The highest increases were found in [{sup 3}H]epibatidine binding, with MDMA inducing higher increases than METH. Treatment with METH and MDMA increased B{sub max} of high-affinity sites for both radioligands without affecting K{sub d}. The heightened binding was inhibited by pretreatment with cycloheximide, suggesting the participation of newly synthesised proteins while inhibition of protein trafficking to plasma membrane did not block up-regulation. The effects of protein kinase and cyclophilin inhibitors on such up-regulation were explored, revealing a rapid, differential and complex regulation, similar to that described for nicotinic ligands. All of these results demonstrate that METH and MDMA have affinity for, and can interact with, nAChR, inducing their up-regulation, specially when higher doses are used. Such effects may have a role in METH- and MDMA-induced neurotoxicity, cholinergic neurotransmission, and in processes

  16. Identification of Anaplasma marginale proteins specifically up-regulated during colonization of the tick vector

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The transition between infection of the mammalian host and colonization of an arthropod vector is required for ongoing transmission of a broad array of pathogens, from viruses to protozoa. Understanding how this transition is mediated provides opportunities to disrupt transmission through either ch...

  17. Fibulin 2, a tyrosine O-sulfated protein, is up-regulated following retinal detachment.

    PubMed

    Kanan, Yogita; Brobst, Daniel; Han, Zongchao; Naash, Muna I; Al-Ubaidi, Muayyad R

    2014-05-01

    Retinal detachment is the physical separation of the retina from the retinal pigment epithelium. It occurs during aging, trauma, or during a variety of retinal disorders such as age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, or as a complication following cataract surgery. This report investigates the role of fibulin 2, an extracellular component, in retinal detachment. A major mechanism for detachment resolution is enhancement of cellular adhesion between the retina and the retinal pigment epithelium and prevention of its cellular migration. This report shows that fibulin 2 is mainly present in the retinal pigment epithelium, Bruch membrane, choriocapillary, and to a lesser degree in the retina. In vitro studies revealed the presence of two isoforms for fibulin 2. The small isoform is located inside the cell, and the large isoform is present inside and outside the cells. Furthermore, fibulin 2 is post-translationally modified by tyrosine sulfation, and the sulfated isoform is present outside the cell, whereas the unsulfated pool is internally located. Interestingly, sulfated fibulin 2 significantly reduced the rate of cellular growth and migration. Finally, levels of fibulin 2 dramatically increased in the retinal pigment epithelium following retinal detachment, suggesting a direct role for fibulin 2 in the re-attachment of the retina to the retinal pigment epithelium. Understanding the role of fibulin 2 in enhancing retinal attachment is likely to help improve the current therapies or allow the development of new strategies for the treatment of this sight-threatening condition. PMID:24692557

  18. Tissue specific up regulation of ACE2 in rabbit model of atherosclerosis by atorvastatin: role of epigenetic histone modifications.

    PubMed

    Tikoo, Kulbhushan; Patel, Gaurang; Kumar, Sandeep; Karpe, Pinakin Arun; Sanghavi, Maitri; Malek, Vajir; Srinivasan, K

    2015-02-01

    Growing body of evidence points out the crucial role of ACE2 in preventing atherosclerosis. However, data on how atherosclerosis affects ACE2 expression in heart and kidney remains unknown. Atherosclerosis was induced by feeding New Zealand White rabbits with high cholesterol diet (HCD - 2%) for 12 weeks and atorvastatin was administered (5mg/kg/day p.o) in last 3 weeks. ACE2 mRNA and protein expression was assessed by Western blotting and real time PCR. HCD fed rabbits developed atherosclerosis as confirmed by increase in plasma total cholesterol, LDL and triglycerides as well as formation atherosclerotic plaques in arch of aorta. The ACE2 protein but not mRNA expression was reduced in heart and kidney of HCD rabbits. Interestingly, atorvastatin increased the ACE2 protein expression in heart and kidney of HCD rabbits. However, atorvastatin increased ACE2 mRNA in heart but not in kidney of HCD rabbits. Atorvastatin increased the occupancy of histone H3 acetylation (H3-Ac) mark on ACE2 promoter region in heart of HCD rabbits indicating direct or indirect epigenetic up-regulation of ACE2 by atorvastatin. Further, atorvastatin suppressed Ang II-induced contractile responses and enhanced AT2 receptor mediated relaxant responses in atherosclerotic aorta. We propose that atherosclerosis is associated with reduced ACE2 expression in heart and kidney. We also show an unexplored potential of atorvastatin to up-regulate ACE2 via epigenetic histone modifications. Our data suggest a novel way of replenishing ACE2 expression for preventing not only atherosclerosis but also other cardiovascular disorders. PMID:25482567

  19. Heat shock up-regulates expression of Toll-like receptor-2 and Toll-like receptor-4 in human monocytes via p38 kinase signal pathway

    PubMed Central

    Zhou, Jun; An, Huazhang; Xu, Hongmei; Liu, Shuxun; Cao, Xuetao

    2005-01-01

    Heat stress can alert innate immunity by inducing stress proteins such as heat-shock proteins (HSPs). However, it remains unclear whether heat stress affects the activation of antigen-presenting cell (APC) in response to pathogen-associated molecule patterns (PAMPs) by directly regulating pathogen recognition receptors (PRRs). As an important kind of PRRs, Toll-like receptors (TLRs) play critical roles in the activation of immune system. In this study, we demonstrated that heat shock up-regulated the expression of HSP70 as well as TLR2 and TLR4 in monocytes. The induction of TLRs was prior to that of HSP70, which suggesting the up-regulation of TLR2 and TLR4 might be independent of the induction of HSP70. Heat shock activated p38 kinase, extracellular signal-related kinase (ERK) and nuclear factor-kappa B (NF-κB) signal pathways in monocytes. Pretreatment with specific inhibitor of p38 kinase, but not those of ERK and NF-κB, inhibited heat shock-induced up-regulation of TLR2 and TLR4. This indicates that p38 pathway takes part in heat shock-induced up-regulation of TLR2 and TLR4. Heat shock also increased lipoteichoic acid- or lipopolysaccharide-induced interleukin-6 production by monocytes. These results suggest that the p38 kinase-mediated up-regulation of TLR2 and TLR4 might be involved in the enhanced response to PAMP in human monocytes induced by heat shock. PMID:15804289

  20. AGING UP-REGULATES EXPRESSION OF INFLAMMATORY MEDIATORS IN MOUSE ADIPOSE TISSUE (AT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence of type 2 diabetes (T2D) increases with age. Low-grade inflammation in AT is implicated in development of insulin resistance and T2D. This study investigated if inflammatory responses are up-regulated with age in AT. Results showed that visceral AT from old mice have higher mRNA expres...

  1. Up-regulation of DRP-3 long isoform during the induction of neural progenitor cells by glutamate treatment in the ex vivo rat retina

    SciTech Connect

    Tokuda, Kazuhiro; Kuramitsu, Yasuhiro; Byron, Baron; Kitagawa, Takao; Tokuda, Nobuko; Kobayashi, Daiki; Nagayama, Megumi; Araki, Norie; Sonoda, Koh-Hei; Nakamura, Kazuyuki

    2015-08-07

    Glutamate has been shown to induce neural progenitor cells in the adult vertebrate retina. However, protein dynamics during progenitor cell induction by glutamate are not fully understood. To identify specific proteins involved in the process, we employed two-dimensional electrophoresis-based proteomics on glutamate untreated and treated retinal ex vivo sections. Rat retinal tissues were incubated with 1 mM glutamate for 1 h, followed by incubation in glutamate-free media for a total of 24 h. Consistent with prior reports, it was found that mitotic cells appeared in the outer nuclear layer without any histological damage. Immunohistological evaluations and immunoblotting confirmed the emergence of neuronal progenitor cells in the mature retina treated with glutamate. Proteomic analysis revealed the up-regulation of dihydropyrimidinase-related protein 3 (DRP-3), DRP-2 and stress-induced-phosphoprotein 1 (STIP1) during neural progenitor cell induction by glutamate. Moreover, mRNA expression of DRP-3, especially, its long isoform, robustly increased in the treated retina compared to that in the untreated retina. These results may indicate that glutamate induces neural progenitor cells in the mature rat retina by up-regulating the proteins which mediate cell mitosis and neurite growth. - Highlights: • Glutamate induced neuronal progenitor cells in the mature rat retina. • Proteomic analysis revealed the up-regulation of DRP-3, DRP-2 and STIP1. • mRNA expression of DRP-3, especially, its long isoform, robustly increased.

  2. Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes.

    PubMed

    Loison, Fabien; Debure, Laure; Nizard, Philippe; le Goff, Pascale; Michel, Denis; le Dréan, Yves

    2006-04-01

    Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity. PMID:16336210

  3. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation. PMID:25683686

  4. Green tea diet decreases PCB 126-induced oxidative stress in mice by up-regulating antioxidant enzymes.

    PubMed

    Newsome, Bradley J; Petriello, Michael C; Han, Sung Gu; Murphy, Margaret O; Eske, Katryn E; Sunkara, Manjula; Morris, Andrew J; Hennig, Bernhard

    2014-02-01

    Superfund chemicals such as polychlorinated biphenyls pose a serious human health risk due to their environmental persistence and link to multiple diseases. Selective bioactive food components such as flavonoids have been shown to ameliorate PCB toxicity, but primarily in an in vitro setting. Here, we show that mice fed a green tea-enriched diet and subsequently exposed to environmentally relevant doses of coplanar PCB exhibit decreased overall oxidative stress primarily due to the up-regulation of a battery of antioxidant enzymes. C57BL/6 mice were fed a low-fat diet supplemented with green tea extract (GTE) for 12 weeks and exposed to 5 μmol PCB 126/kg mouse weight (1.63 mg/kg-day) on weeks 10, 11 and 12 (total body burden: 4.9 mg/kg). F2-isoprostane and its metabolites, established markers of in vivo oxidative stress, measured in plasma via HPLC-MS/MS exhibited fivefold decreased levels in mice supplemented with GTE and subsequently exposed to PCB compared to animals on a control diet exposed to PCB. Livers were collected and harvested for both messenger RNA and protein analyses, and it was determined that many genes transcriptionally controlled by aryl hydrocarbon receptor and nuclear factor (erythroid-derived 2)-like 2 proteins were up-regulated in PCB-exposed mice fed the green tea-supplemented diet. An increased induction of genes such as SOD1, GSR, NQO1 and GST, key antioxidant enzymes, in these mice (green tea plus PCB) may explain the observed decrease in overall oxidative stress. A diet supplemented with green tea allows for an efficient antioxidant response in the presence of PCB 126, which supports the emerging paradigm that healthful nutrition may be able to bolster and buffer a physiological system against the toxicities of environmental pollutants. PMID:24378064

  5. Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

    PubMed Central

    Wu, Siva; Li, Xiaojin; Gunawardana, Manjula; Maguire, Kathleen; Guerrero-Given, Debbie; Schaudinn, Christoph; Wang, Charles; Baum, Marc M.; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended. PMID:25007395

  6. Ampelopsin Improves Insulin Resistance by Activating PPARγ and Subsequently Up-Regulating FGF21-AMPK Signaling Pathway

    PubMed Central

    Qin, Yu; Liu, Lei; Wan, Jing; Zou, Lingyun; Zhang, Qianyong; Zhu, Jundong; Mi, Mantian

    2016-01-01

    Ampelopsin (APL), a major bioactive constituent of Ampelopsis grossedentata, exerts a number of biological effects. Here, we explored the anti-diabetic activity of APL and elucidate the underlying mechanism of this action. In palmitate-induced insulin resistance of L6 myotubes, APL treatment markedly up- regulated phosphorylated insulin receptor substrate-1 and protein kinase B, along with a corresponding increase of glucose uptake capacity. APL treatment also increased expressions of fibroblast growth factor (FGF21) and phosphorylated adenosine 5’-monophosphate -activated protein kinase (p-AMPK), however inhibiting AMPK by Compound C or AMPK siRNA, or blockage of FGF21 by FGF21 siRNA, obviously weakened APL -induced increases of FGF21 and p-AMPK as well as glucose uptake capacity in palmitate -pretreated L6 myotubes. Furthermore, APL could activate PPAR γ resulting in increases of glucose uptake capacity and expressions of FGF21 and p-AMPK in palmitate -pretreated L6 myotubes, whereas all those effects were obviously abolished by addition of GW9662, a specific inhibitor of peroxisome proliferator- activated receptor –γ (PPARγ), and PPARγsiRNA. Using molecular modeling and the luciferase reporter assays, we observed that APL could dock with the catalytic domain of PPARγ and dose-dependently up-regulate PPARγ activity. In summary, APL maybe a potential agonist of PPARγ and promotes insulin sensitization by activating PPARγ and subsequently regulating FGF21- AMPK signaling pathway. These results provide new insights into the protective health effects of APL, especially for the treatment of Type 2 diabetes mellitus. PMID:27391974

  7. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier

    PubMed Central

    Hartz, Anika M. S.; Bauer, Björn; Block, Michelle L.; Hong, Jau-Shyong; Miller, David S.

    2008-01-01

    Here, we report that diesel exhaust particles (DEPs), a major constituent of urban air pollution, affect blood-brain barrier function at the tissue, cellular, and molecular levels. Isolated rat brain capillaries exposed to DEPs showed increased expression and transport activity of the key drug efflux transporter, P-glycoprotein (6 h EC50 was ∼5 μg/ml). Up-regulation of P-glycoprotein was abolished by blocking transcription or protein synthesis. Inhibition of NADPH oxidase or pretreatment of capillaries with radical scavengers ameliorated DEP-induced P-glycoprotein up-regulation, indicating a role for reactive oxygen species in signaling. DEP exposure also increased brain capillary tumor necrosis factor-α (TNF-α) levels. DEP-induced P-glycoprotein up-regulation was abolished when TNF-receptor 1 (TNF-R1) was blocked and was not evident in experiments with capillaries from TNF-R1 knockout mice. Inhibition of JNK, but not NF-κB, blocked DEP-induced P-glycoprotein up-regulation, indicating a role for AP-1 in the signaling pathway. Consistent with this, DEPs increased phosphorylation of c-jun. Together, our results show for the first time that a component of air pollution, DEPs, alters blood-brain barrier function through oxidative stress and proinflammatory cytokine production. These experiments disclose a novel blood-brain barrier signaling pathway, with clear implications for environmental toxicology, CNS pathology, and the pharmacotherapy of CNS disorders.—Hartz, A. M. S., Bauer, B., Block, M. L., Hong, J.-S., Miller, D.-S. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier. PMID:18474546

  8. Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors.

    PubMed

    Kwon, Hyeeun; Lee, Jung-Jin; Lee, Ji-Hye; Cho, Won-Kyung; Gu, Min Jung; Lee, Kwang Jin; Ma, Jin Yeul

    2015-01-01

    Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression. PMID:26119954

  9. HSP70 increases extracellular matrix production by human vascular smooth muscle through TGF-β1 up-regulation.

    PubMed

    González-Ramos, Marta; Calleros, Laura; López-Ongil, Susana; Raoch, Viviana; Griera, Mercedes; Rodríguez-Puyol, Manuel; de Frutos, Sergio; Rodríguez-Puyol, Diego

    2013-02-01

    The circulating levels of heat shock proteins (HSP) are increased in cardiovascular diseases; however, the implication of this for the fibrotic process typical of such diseases remains unclear. HSP70 can interact with the vascular smooth muscle cells (SMC), the major producer of extracellular matrix (ECM) proteins, through the Toll-like receptors 4 (TLR4). The transforming growth factor type-β1 (TGF-β1) is a well known vascular pro-fibrotic cytokine that is regulated in part by AP-1-dependent transcriptional mechanisms. We hypothesized that extracellular HSP70 could interact with SMCs, inducing TGF-β1 synthesis and subsequent changes in the vascular ECM. We demonstrate that extracellular HSP70 binds to human aorta SMC TLR4, which up-regulates the AP-1-dependent transcriptional activity of the TGF-β1 promoter. This is achieved through the mitogen activated protein kinases JNK and ERK, as demonstrated by the use of specific blockers and the knockdown of TLR4 with specific small interfering RNAs. The TGF-β1 upregulation increase the expression of the ECM proteins type I collagen and fibronectin. This novel observation may elucidate the mechanisms by which HSP70 contributes in the inflammation and fibrosis present in atherosclerosis and other fibrosis-related diseases. PMID:23084979

  10. The Expression of Porcine Prdx6 Gene Is Up-Regulated by C/EBPβ and CREB

    PubMed Central

    Wu, Xinyu; Ji, Panlong; Zhang, Liang; Bu, Guowei; Gu, Hao; Wang, Xiaojing; Xiong, Yuanzhu; Zuo, Bo

    2015-01-01

    Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5′ rapid-amplification of cDNA ends (5′ RACE). Several regulatory elements including CCAAT/enhancer-binding proteinβ (C/EBPβ), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPβ or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPβ and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPβ could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPβ and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function. PMID:26659441

  11. YAP is up-regulated in the bronchial airway smooth muscle of the chronic asthma mouse model.

    PubMed

    Zhou, Jing; Xu, Fei; Yu, Jing Jing; Zhang, Wei

    2015-01-01

    Asthma is characterized by leukocytic infiltration and tissue remodeling with structural changes including subepithelial fibrosis and ASM cells proliferation. The Hippo pathway is a key regulatory point involved in cell proliferation, fibroblasts, and smooth muscle cell differentiation. In order to disclose the relation between asthma and the Hippo pathway, expression of the Yes-associated protein (YAP), a key gene in the Hippo pathway, in the bronchial smooth muscle of chronic asthma model (CAM) was studied. 40 mice were randomly divided into control (wide type) and experimental group to construct CAM using chicken ovalbumin (OVA). Pathological changes of the lung tissues were observed in the CAM mice compared with the control using HE staining method. Immunohistochemistry (IHC) was used to detect if YAP protein is expressed in the lung tissues. The pathological changes of the CAM group showed that a large number of inflammatory cells infiltration including mainly lymphocytes and a small amount of eosinophilic, with the presence of certain airway smooth muscle hyperplasia, was observed in comparison with the control. IHC results showed that the YAP protein was significantly increased compared with the control groups (P < 0.01). This result was further confirmed by quantitative real-time PCR (qPCR) assay which detected the up-regulation of the YAP gene (P < 0.01) and Western blot. In conclusion, the YAP protein was significantly expressed in the bronchial airway tissues of the CAM mice, and could be used as an indicator for asthma. PMID:26617833

  12. Interferon β (IFN-β) Production during the Double-stranded RNA (dsRNA) Response in Hepatocytes Involves Coordinated and Feedforward Signaling through Toll-like Receptor 3 (TLR3), RNA-dependent Protein Kinase (PKR), Inducible Nitric Oxide Synthase (iNOS), and Src Protein.

    PubMed

    Zhang, Liyong; Xiang, Wenpei; Wang, Guoliang; Yan, Zhengzheng; Zhu, Zhaowei; Guo, Zhong; Sengupta, Rajib; Chen, Alex F; Loughran, Patricia A; Lu, Ben; Wang, Qingde; Billiar, Timothy R

    2016-07-15

    The sensing of double-stranded RNA (dsRNA) in the liver is important for antiviral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here we sought to establish the pathways involved in the production of type I interferons (IFN-I) in response to extracellular poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and, unlike most immune cells that readily die after activation with dsRNA, are not viewed as cells with robust antimicrobial capacity. We found that poly(I:C) leads to rapid up-regulation of inducible nitric oxide synthase (iNOS), double-stranded RNA-dependent protein kinase (PKR), and Src. The production of IFN-β was dependent on iNOS, PKR, and Src and partially dependent on TLR3/Trif. iNOS and Src up-regulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-β production in an iNOS- and Src-dependent manner, and Src was found to directly interact with TLR3 in the endosomal compartment of poly(I:C)-treated cells. Furthermore, we identified a robust NO/cGMP/PKG-dependent feedforward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-β production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR. PMID:27226571

  13. Up-regulation of cyclooxygenase-2 by product-prostaglandin E2

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Hughes-Fulford, M.

    1997-01-01

    The development of prostate cancer has been linked to high level of dietary fat intake. Our laboratory investigates the connection between cancer cell growth and fatty acid products. Studying human prostatic carcinoma PC-3 cells, we found that prostaglandin E2 (PGE2) increased cell growth and up-regulated the gene expression of its own synthesizing enzyme, cyclooxygenase-2 (COX-2). PGE2 increased COX-2 mRNA expression dose-dependently with the highest levels of stimulation seen at the 3-hour period following PGE2 addition. The NSAID flurbiprofen (5 microM), in the presence of exogenous PGE2, inhibited the up-regulation of COX-2 mRNA and cell growth. These data suggest that the levels of local intracellular PGE2 play a major role in the growth of prostate cancer cells through an activation of COX-2 gene expression.

  14. Rapid systemic up-regulation of genes after heat-wounding and electrical stimulation

    NASA Technical Reports Server (NTRS)

    Davies, E.; Vian, A.; Vian, C.; Stankovic, B.

    1997-01-01

    When one leaf of a tomato plant is electrically-stimulated or heat-wounded, proteinase inhibitor genes are rapidly up-regulated in distant leaves. The identity of the systemic wound signal(s) is not yet known, but major candidates include hormones transmitted via the phloem or the xylem, the electrically-stimulated self-propagating electrical signal in the phloem (the action potential, AP), or the heat-wound-induced surge in hydraulic pressure in the xylem evoking a local change in membrane potential in adjacent living cells (the variation potential, VP). In order to discriminate between these signals we have adopted two approaches. The first approach involves applying stimuli that evoke known signals and determining whether these signals have similar effects on the "model" transcripts for proteinase inhibitors (pin) and calmodulin (cal). Here we show that a heat wound almost invariably evokes a VP, while an electrical stimulation occasionally evokes an AP, and both of these signals induce accumulation of transcripts encoding proteinase inhibitors. The second approach involves identifying the array of genes turned on by heat-wounding. To this end, we have constructed a subtractive library for heat-wounded tissue, isolated over 800 putatively up-regulated clones, and shown that all but two of the fifty that we have analyzed by Northern hybridization are, indeed, up-regulated. Here we show the early kinetics of up-regulation of three of these transcripts in the terminal (4th) leaf in response to heat-wounding the 3rd leaf, about 5 cm away. Even though these transcripts show somewhat different time courses of induction, with one peaking at 30 min, another at 15 min, and another at 5 min after flaming of a distant leaf, they all exhibit a similar pattern, i.e., a transient period of transcript accumulation preceding a period of transcript decrease, followed by a second period of transcript accumulation.

  15. Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

    SciTech Connect

    Sun, Yue; Du, Chengli; Wang, Bo; Zhang, Yanling; Liu, Xiaoyan; Ren, Guoping

    2014-07-18

    Highlights: • The expression of eEF1A2 is up-regulated in prostate cancer tissues. • Suppression of eEF1A2 inhibits the proliferation and promotes apoptosis. • Inhibition of eEF1A2 enhances the expression of apoptotic relevant proteins. • The expressions of eEF1A2 and cleavage-caspase3 are inversely correlated. - Abstract: Background: eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development. Methods: We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis. Results: Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues. Conclusion: Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.

  16. Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium*

    PubMed Central

    Li, Yafeng; Song, Delu; Song, Ying; Zhao, Liangliang; Wolkow, Natalie; Tobias, John W.; Song, Wenchao; Dunaief, Joshua L.

    2015-01-01

    Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation. PMID:25802332

  17. Iron-induced Local Complement Component 3 (C3) Up-regulation via Non-canonical Transforming Growth Factor (TGF)-β Signaling in the Retinal Pigment Epithelium.

    PubMed

    Li, Yafeng; Song, Delu; Song, Ying; Zhao, Liangliang; Wolkow, Natalie; Tobias, John W; Song, Wenchao; Dunaief, Joshua L

    2015-05-01

    Dysregulation of iron homeostasis may be a pathogenic factor in age-related macular degeneration (AMD). Meanwhile, the formation of complement-containing deposits under the retinal pigment epithelial (RPE) cell layer is a pathognomonic feature of AMD. In this study, we investigated the molecular mechanisms by which complement component 3 (C3), a central protein in the complement cascade, is up-regulated by iron in RPE cells. Modulation of TGF-β signaling, involving ERK1/2, SMAD3, and CCAAT/enhancer-binding protein-δ, is responsible for iron-induced C3 expression. The differential effects of spatially distinct SMAD3 phosphorylation sites at the linker region and at the C terminus determined the up-regulation of C3. Pharmacologic inhibition of either ERK1/2 or SMAD3 phosphorylation decreased iron-induced C3 expression levels. Knockdown of SMAD3 blocked the iron-induced up-regulation and nuclear accumulation of CCAAT/enhancer-binding protein-δ, a transcription factor that has been shown previously to bind the basic leucine zipper 1 domain in the C3 promoter. We show herein that mutation of this domain reduced iron-induced C3 promoter activity. In vivo studies support our in vitro finding of iron-induced C3 up-regulation. Mice with a mosaic pattern of RPE-specific iron overload demonstrated co-localization of iron-induced ferritin and C3d deposits. Humans with aceruloplasminemia causing RPE iron overload had increased RPE C3d deposition. The molecular events in the iron-C3 pathway represent therapeutic targets for AMD or other diseases exacerbated by iron-induced local complement dysregulation. PMID:25802332

  18. Placental fractalkine is up-regulated in severe early-onset preeclampsia.

    PubMed

    Siwetz, Monika; Dieber-Rotheneder, Martina; Cervar-Zivkovic, Mila; Kummer, Daniel; Kremshofer, Julia; Weiss, Gregor; Herse, Florian; Huppertz, Berthold; Gauster, Martin

    2015-05-01

    The pathogenesis of preeclampsia (PE) includes the release of placental factors into the maternal circulation, inducing an inflammatory environment in the mother. One of the factors may be the proinflammatory chemokine fractalkine, which is expressed in the syncytiotrophoblast of human placenta, from where it is released into the maternal circulation by constitutive shedding. We examined whether placental fractalkine is up-regulated in severe early-onset PE and whether the proinflammatory cytokines tumor necrosis factor (TNF)-α and IL-6 are able to increase the expression of fractalkine. Gene expression analysis, enzyme-linked immunosorbent assay, and immunohistochemistry consistently showed increased fractalkine expression in placentas from severe early-onset PE, compared to gestational age-matched controls. Expression of a disintegrin and metalloproteinases (ADAMs) 10 and 17, which convert transmembrane fractalkine into the soluble form, was significantly increased in these cases. Incubation of first-trimester placental explants with TNF-α provoked a significant increase in fractalkine expression and release of the soluble form, whereas IL-6 had no effect. TNF-α-mediated up-regulation of placental fractalkine was reversed in the presence of the aspirin-derivative salicylate, which impaired activation of NF-κB p65 in TNF-α-treated explants. On the basis of data from placental explants, we suggest that increased maternal TNF-α may up-regulate the expression and release of placental fractalkine, which, in turn, may contribute to an exaggerated systemic inflammatory response in PE. PMID:25769431

  19. Placental fractalkine is up-regulated in severe early onset preeclampsia

    PubMed Central

    Siwetz, Monika; Dieber-Rotheneder, Martina; Cervar-Zivkovic, Mila; Kummer, Daniel; Kremshofer, Julia; Weiss, Gregor; Herse, Florian; Huppertz, Berthold; Gauster, Martin

    2015-01-01

    The pathogenesis of preeclampsia includes the release of placental factors into the maternal circulation inducing an inflammatory environment in the mother. One of the factors may be the pro-inflammatory chemokine fractalkine, which is expressed in the syncytiotrophoblast of human placenta, from where it is released into the maternal circulation by constitutive shedding. We examined whether placental fractalkine is up-regulated in severe early onset preeclampsia and whether the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin-6 are able to increase the expression of fractalkine. Gene expression analysis, ELISA, and immunohistochemistry consistently showed increased fractalkine expression in placentas from severe early onset preeclampsia, compared to gestational age-matched controls. Expression of the metalloproteinases ADAM10 and ADAM17, which convert transmembrane fractalkine into the soluble form, was significantly increased in these cases. Incubation of first trimester placental explants with TNF-α provoked a significant increase in fractalkine expression and release of the soluble form, whereas interleukin-6 had no effect. TNF-α-mediated up-regulation of placental fractalkine was reversed in the presence of the Aspirin-derivative salicylate, which impaired activation of NF-κB p65 in TNF-α-treated explants. Based on data from placental explants we suggest that increased maternal TNF-α may up-regulate the expression and release of placental fractalkine, which in turn may contribute to an exaggerated systemic inflammatory response in preeclampsia. PMID:25769431

  20. Up-regulation of annexin A2 in cholangiocarcinoma caused by Opisthorchis viverrini and its implication as a prognostic marker

    PubMed Central

    Yonglitthipagon, Ponlapat; Pairojkul, Chawalit; Chamgramol, Yaovalux; Mulvenna, Jason; Sripa, Banchob

    2013-01-01

    Cholangiocarcinoma (CCA), or cancer of the bile ducts, is primarily associated with infection with the liver fluke Opisthorchis viverrini in northeast Thailand. The disease . is associated with late presentation, poses challenges for diagnosis and has a high mortality rate – features that highlight the need for tumor markers. At present, there are no specific tumor markers that can indicate the early stages and status of CCA. Proteomic analysis of the proteins expressed on the surface of tumor cells is particularly difficult since proteome-wide analysis of surface membrane proteins has thus far been hampered by the lack of effective strategies to profile hydrophobic membrane proteins. In this study, a sequential protein extraction was utilized to overcome this problem. Membrane protein was extracted from four CCA cell lines with different tumor forming capabilities. The non-tumor H69 biliary cell line was used as a control. Two-dimensional-PAGE followed by MALDI-TOF-MS was used to identify differentially expressed proteins. Among 20 up-regulated membrane proteins identified in the CCA cell lines was ANXA2, a participant in tumor invasion and metastasis in other cancers. Accordingly, ANXA2 was verified in human subjects by probing, using a commercial anti-mouse monoclonal antibody and a tissue microarray of CCA (301 diagnosed cases), where it was found to associate with one of several tumor progression stages as reflected by lymphatic invasion (P = 0.014) and metastasis (P = 0.026). Patients with high expression of ANXA2 had a significantly shorter survival time (P = 0.011). ANXA2 expression in tumors may be useful for predicting the poor outcome of CCA patients. PMID:20493868

  1. Exposure to diesel exhaust up-regulates iNOS expression in ApoE knockout mice

    SciTech Connect

    Bai Ni; Kido, Takashi; Kavanagh, Terrance J.; Kaufman, Joel D.; Rosenfeld, Michael E.; Breemen, Cornelis van; Eeden, Stephan F. van

    2011-09-01

    blood vessels and heart. > DE exposure enhanced iNOS protein and mRNA expression in the aorta and heart. > iNOS activity was also increased after DE exposure. > This up-regulation of iNOS may contribute to vascular dysfunction and atherogenesis.

  2. Coordinate estrogen-regulated instability of serum protein-coding messenger RNAs in Xenopus laevis.

    PubMed

    Pastori, R L; Moskaitis, J E; Buzek, S W; Schoenberg, D R

    1991-04-01

    Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver. PMID:1922078

  3. Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice

    PubMed Central

    Zhang, Xuan; Wang, Jian-Mei; He, Ya-Ping; Shi, Yan; Sun, Zhao-Gui; Shi, Hui-Juan; Wang, Jian

    2016-01-01

    Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy. PMID:27175791

  4. Uterine Expression of NDRG4 Is Induced by Estrogen and Up-Regulated during Embryo Implantation Process in Mice.

    PubMed

    Yang, Qian; Gu, Yan; Zhang, Xuan; Wang, Jian-Mei; He, Ya-Ping; Shi, Yan; Sun, Zhao-Gui; Shi, Hui-Juan; Wang, Jian

    2016-01-01

    Embryo implantation is an essential step for the establishment of pregnancy and dynamically regulated by estrogen and progesterone. NDRG4 (N-myc down-regulated gene 4) is a tumor suppressor that participates in cell survival, tumor invasion and angiogenesis. The objective of this study was to preliminarily explore the role of NDRG4 in embryo implantation. By immunohistochemistry (IHC) and quantitive RT-PCR (qRT-PCR), we found that uterine expression of NDRG4 was increased along with puberal development, and its expression in adult females reached the peak at the estrus stage during the estrus cycle. Furthermore, uterine NDRG4 expression was significantly induced by the treatment of estradiol (E2) both in pre-puberty females and ovariectomized adult females. Uterine expression pattern of NDRG4 during the peri-implantation period in mice was determined by IHC, qRT-PCR and Western blot. It was observed that NDRG4 expression was up-regulated during the implantation process, and its expression level at the implantation sites was significantly higher than that at the inter-implantation sites. Meanwhile, an increased expression in NDRG4 was associated with artificial decidualization as well as the activation of delayed implantation. By qRT-PCR and Western blot, we found that the in vitro decidualization of endometrial stromal cells (ESCs) was accompanied by up-regulation of NDRG4 expression, whereas knockdown of its expression in these cells by siRNA inhibited the decidualization process. In addition, Western blot analysis showed that NDRG4 protein expression was decreased in human villus tissues of recurrent miscarriage (RM) patients compared to normal pregnant women. Collectively, these data suggested that uterine NDRG4 expression could be induced by estrogen, and NDRG4 might play an important role during early pregnancy. PMID:27175791

  5. Up-regulation of C1GALT1 promotes breast cancer cell growth through MUC1-C signaling pathway

    PubMed Central

    Chou, Chih-Hsing; Huang, Miao-Juei; Chen, Chi-Hau; Shyu, Ming-Kwang; Huang, John; Hung, Ji-Shiang; Huang, Chiun-Sheng; Huang, Min-Chuan

    2015-01-01

    Aberrant glycosylation is frequently observed in cancers. Core 1 β1,3-galactosyltransferase (C1GALT1) is an exclusive enzyme in humans that catalyzes the biosynthesis of core 1 O-glycan structure, Gal-GalNAc-O-Ser/Thr, whose expression is commonly up-regulated during tumorigenesis. Little is known about the function of C1GALT1 in breast cancer. This study aims to determine the correlation between C1GALT1 expression and breast cancer clinicopathological features and roles of C1GALT1 in breast cancer malignant phenotypes. Public databases and our data showed that C1GALT1 mRNA and C1GALT1 protein are frequently up-regulated in breast cancer; and increased C1GALT1 expression correlates with higher histological grade and advanced tumor stage. Overexpression of C1GALT1 enhanced breast cancer cell growth, migration, and invasion in vitro as well as tumor growth in vivo. Conversely, C1GALT1 knockdown suppressed these malignant phenotypes. Furthermore, C1GALT1 modulates O-glycan structures on Mucin (MUC) 1 and promotes MUC1-C/β-catenin signaling in breast cancer cells. These findings suggest that C1GALT1 enhances breast cancer malignant progression through promoting MUC1-C/β-catenin signaling pathway. Unveiling the function of C1GALT1 in breast cancer opens new insights to the roles of C1GALT1 and O-glycosylation in tumorigenesis and renders the potential of C1GALT1 as a target of novel therapeutic agent development. PMID:25762620

  6. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

    PubMed

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-08-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. PMID:27235552

  7. Astragalus membranaceus up-regulate Cosmc expression and reverse IgA dys-glycosylation in IgA nephropathy

    PubMed Central

    2014-01-01

    Background Decreased Core I β3-Gal-T-specific molecular chaperone (Cosmc) expression induced IgA1 aberrant glycosylation is the main characteristic of IgA nephropathy (IgAN). This study tried to elucidate the effect of Astragalus membranaceus on Cosmc expression and IgA O-glycosylation of peripheral B lymphocytes in IgAN patients. Methods Peripheral B lymphocytes of 21 IgAN patients and 10 normal controls were isolated and cultured with or without lipopolysaccharide (LPS) and Astragalus membranaceus injection (AMI). Cosmc mRNA and protein expression levels were measured by real-time RT-PCR and Western blot. IgA1 and glycosylation level were determined by enzyme-linked immunosorbent assay (ELISA) and VV lectin-binding method. Results Cosmc mRNA expression and IgA1 O-glycosylation level in IgAN patients was significantly lower than normal controls at baseline. Treatment of LPS could obviously inhibit Cosmc expression and increase the IgA1 secretion in peripheral B lymphocytes of IgAN patients, which resulted in a significantly increase in IgA1 aberrant glycosylation level. Addition of AMI could remarkably up regulated Cosmc expression, decrease IgA1 secretion, and reverse glycosylation level in a dose related manner. Conclusion AMI can up-regulate Cosmc expression of peripheral B lymphocytes and reverse IgA1 aberrant O-glycosylation level, which might be the underlying mechanism of AMI therapy in treating IgAN. Trial registration TCTR20140515001 (Registration Date: 2014-05-15) PMID:24942185

  8. Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation

    PubMed Central

    Cheng, Wen-Han; Ho, Wen-Yu; Chang, Chien-Feng; Lu, Pei-Jung; Cheng, Pei-Wen; Yeh, Tung-Chen; Hong, Ling-Zong; Sun, Gwo-Ching; Hsiao, Michael; Tseng, Ching-Jiunn

    2013-01-01

    BACKGROUND AND PURPOSE Clinical studies indicate that statins have a BP-lowering effect in hypercholesterolemic individuals with hypertension. Specifically, statins modulate BP through the up-regulation of endothelial NOS (eNOS) activation in the brain. However, the signalling mechanisms through which statins enhance eNOS activation remain unclear. Therefore, we examined the possible signalling pathways involved in statin-mediated BP regulation in the nucleus tractus solitarii (NTS). EXPERIMENTAL APPROACH To investigate the involvement of Ras and other signalling pathways in simvastatin-induced effects on BP, BP and renal sympathetic nerve activity (RSNA) were determined in spontaneously hypertensive rats (SHRs) before and after i.c.v. administration of simvastatin in the absence and presence of a Ras-specific inhibitor (farnesyl thiosalicylic acid, FTS), a geranylgeranyltransferase inhibitor (GGTI-2133), a PI3K inhibitor (LY294002) or a MAPK-ERK kinase (MEK) inhibitor (PD98059). KEY RESULTS FTS significantly attenuated the decrease in BP and increased NO evoked by simvastatin and reversed the decrease in basal RSNA induced by simvastatin. Immunoblotting and pharmacological studies showed that inhibition of Ras activity by FTS significantly abolished simvastatin-induced phosphorylation of ERK1/2, ribosomal protein S6 kinase (RSK), Akt and decreased eNOS phosphorylation. Likewise, administration of Akt and ERK1/2 signalling inhibitors, LY294002 and PD98059, attenuated the reduction in BP evoked by simvastatin. Furthermore, i.c.v. simvastatin decreased Rac1 activation and the number of ROS-positive cells in the NTS. CONCLUSIONS AND IMPLICATIONS Simvastatin modulates central BP control in the NTS of SHRs by increasing Ras-mediated activation of the PI3K-Akt and ERK1/2-RSK signalling pathways, which then up-regulates eNOS activation. PMID:23889671

  9. Sex-dependent up-regulation of two splicing factors, Psf and Srp20, during hippocampal memory formation.

    PubMed

    Antunes-Martins, Ana; Mizuno, Keiko; Irvine, Elaine E; Lepicard, Eve M; Giese, K Peter

    2007-10-01

    Gene transcription is required for long-term memory (LTM) formation. LTM formation is impaired in a male-specific manner in mice lacking either of the two Ca(2+)/calmodulin-dependent kinase kinase (Camkk) genes. Since altered transcription was suggested to cause these impairments in LTM formation, we used microarrays to screen for CaMKKbeta-dependent gene expression changes. Here we show that the hippocampal mRNA expression of two splicing factors, splicing factor arginine/serine-rich 3 (Sfrs3/Srp20) and polypyrimidine tract-binding protein-associated splicing factor (Psf), is altered in CaMKKbeta-deficient males. In wild-type (WT) mice, the basal expression level in the hippocampus is higher in males than in females, and the sex difference in Srp20 expression is detectable before puberty. Training in two hippocampus-dependent learning tasks, the spatial version of the Morris water maze (MWM) and background contextual fear conditioning, increases the hippocampal mRNA expression of both splicing factors in WT males. However, the increase in Srp20 mRNA expression occurs only in males and not in females, whereas the up-regulation of Psf expression occurs in both sexes. Importantly, control experiments demonstrate that the up-regulation of both splicing factors is specific for the learned associations after contextual fear conditioning. In summary, we provide the first evidence for a regulation of splicing factors during LTM formation and we suggest that alternative splicing contributes to sex differences in LTM formation. PMID:17911373

  10. Lipid-induced up-regulation of human acyl-CoA synthetase 5 promotes hepatocellular apoptosis.

    PubMed

    Reinartz, Andrea; Ehling, Josef; Leue, Andrea; Liedtke, Christian; Schneider, Ursula; Kopitz, Jürgen; Weiss, Thomas; Hellerbrand, Claus; Weiskirchen, Ralf; Knüchel, Ruth; Gassler, Nikolaus

    2010-09-01

    In the pathogenesis of nonalcoholic fatty liver disease, accumulation of lipids in hepatocytes and hepatocyte apoptosis are strongly implicated in disease progression from the potentially reversible condition of steatosis to severe acute and chronic liver injury. Acyl-CoA synthetase 5, a member of the ACSL gene family that catalyzes the activation of long-chain fatty acids for lipid biosynthesis, is the only ACSL isoform that is both, located on mitochondria and functionally involved in enterocyte apoptosis. In this study, the regulation of human ACSL5 in hepatocellular fatty acid degeneration and its involvement in hepatocyte apoptosis was investigated using models of in vitro and in vivo steatosis as well as plasmid-mediated stable gene transfer and RNAi-mediated gene silencing. ACSL5 mRNA and protein were strongly increased by uptake of dietary derived fatty acids in primary human hepatocytes, HepG2 cells and human steatotic liver. Over-expression of ACSL5 decreased HepG2 cell viability and increased susceptibility to TRAIL- and TNFalpha-, but not FAS- induced apoptosis, whereas knock down of ACSL5 reduced apoptosis susceptibility. High ACSL5 activity resulted in enhanced caspase-3/7 activity, but was not accompanied by up-regulation of death receptors, DR4, DR5 or TNF-R1. This study gives evidence that hepatocyte steatosis is associated with ACSL5 up-regulation resulting in increased susceptibility to hepatic cell death. We propose that ACSL5 could play a role in promoting fatty acid-induced lipoapoptosis in hepatocytes as important mechanism in fatty liver-related disorders. PMID:20470896

  11. Berberine metabolites could induce low density lipoprotein receptor up-regulation to exert lipid-lowering effects in human hepatoma cells.

    PubMed

    Zhou, Yan; Cao, Shijie; Wang, Ying; Xu, Peixiang; Yan, Jiankun; Bin, Wen; Qiu, Feng; Kang, Ning

    2014-01-01

    Berberine (BBR) is an isoquinoline alkaloid isolated from several Chinese herbal medicines, such as Coptis chinensis, Berberis aristata, and Coptis japonica. It exhibits a lipid-lowering effect by up-regulating the hepatic low density lipoprotein receptor (LDLR) expression. However, the plasma concentration of BBR is very low after oral administration for the reason that BBR is poorly absorbed and rapidly metabolized. Therefore, it is hard to explain the pharmacological effects of BBR in vivo. Here, RT-PCR, Western blotting and Oil Red O staining were used to investigate the effects of four BBR metabolites on LDLR expression and lipid accumulation in human hepatoma Hep G2 cells. Our results suggested that BBR increased the LDLR mRNA and protein levels in a time- and dose-dependent manner. Four metabolites of BBR, jatrorrhizine, columbamine, berberrubine and demethyleneberberine, were found to be able to up-regulate LDLR mRNA and protein expression. Moreover, almost all the metabolites had potent effects on inhibiting cellular lipid accumulation. These results suggest that both BBR and its metabolites exhibit lipid-lowering effects by up-regulating LDLR expression, and BBR and its metabolites might be the in vivo active forms of BBR produced after oral administration. This study provides information to help us understand the mechanisms underlying the hypolipidemic effects of BBR in vivo. PMID:24321576

  12. Wedelolactone Regulates Lipid Metabolism and Improves Hepatic Steatosis Partly by AMPK Activation and Up-Regulation of Expression of PPARα/LPL and LDLR

    PubMed Central

    Yang, Li-chao; Xu, Xu-dong; Li, Wei-jie; Luo, Xiu-mei; Jin, Xin

    2015-01-01

    Hyperlipidemia is considered one of the greatest risk factors of cardiovascular diseases. We investigated the anti-hyperlipidemic effect and the underlying mechanism of wedelolactone, a plant-derived coumestan, in HepG2 cells and high-fat diet (HFD)−induced hyperlipidemic hamsters. We showed that in cultured HepG2 cells, wedelolactone up-regulated protein levels of adenosine monophosphate activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-alpha (PPARα) as well as the gene expression of AMPK, PPARα, lipoprotein lipase (LPL), and the low-density lipoprotein receptor (LDLR). Meanwhile, administration of wedelolactone for 4 weeks decreased the lipid profiles of plasma and liver in HFD−induced hyperlipidemic hamsters, including total cholesterol (TC), triglycerides (TG), and low-density lipoprotein-cholesterol (LDL-C). The activation of AMPK and up-regulation of PPARα was also observed with wedelolactone treatment. Furthermore, wedelolactone also increased the activities of superoxidase dismutase (SOD) and glutathione peroxidase (GSH-Px) and decreased the level of the lipid peroxidation product malondialdehyde (MDA) in the liver, therefore decreasing the activity of alanine aminotransferase (ALT). In conclusion, we provide novel experimental evidence that wedelolactone possesses lipid-lowering and steatosis-improving effects, and the underlying mechanism is, at least in part, mediated by the activation of AMPK and the up-regulation of PPARα/LPL and LDLR. PMID:26168156

  13. Epstein-Barr Virus nuclear antigen 1 (EBNA1) confers resistance to apoptosis in EBV-positive B-lymphoma cells through up-regulation of survivin

    SciTech Connect

    Lu Jie; Murakami, Masanao; Verma, Subhash C.; Cai Qiliang; Haldar, Sabyasachi; Kaul, Rajeev; Wasik, Mariusz A.; Middeldorp, Jaap; Robertson, Erle S.

    2011-02-05

    Resistance to apoptosis is an important component of the overall mechanism which drives the tumorigenic process. EBV is a ubiquitous human gamma-herpesvirus which preferentially establishes latent infection in viral infected B-lymphocytes. EBNA1 is typically expressed in most forms of EBV-positive malignancies and is important for replication of the latent episome in concert with replication of the host cells. Here, we investigate the effects of EBNA1 on survivin up-regulation in EBV-infected human B-lymphoma cells. We present evidence which demonstrates that EBNA1 forms a complex with Sp1 or Sp1-like proteins bound to their cis-element at the survivin promoter. This enhances the activity of the complex and up-regulates survivin. Knockdown of survivin and EBNA1 showed enhanced apoptosis in infected cells and thus supports a role for EBNA1 in suppressing apoptosis in EBV-infected cells. Here, we suggest that EBV encoded EBNA1 can contribute to the oncogenic process by up-regulating the apoptosis suppressor protein, survivin in EBV-associated B-lymphoma cells.

  14. Up-Regulation of Oligodendrocyte Lineage Markers in the Cerebellum of Autistic Patients: Evidence from Network Analysis of Gene Expression.

    PubMed

    Zeidán-Chuliá, Fares; de Oliveira, Ben-Hur Neves; Casanova, Manuel F; Casanova, Emily L; Noda, Mami; Salmina, Alla B; Verkhratsky, Alexei

    2016-08-01

    Autism is a neurodevelopmental disorder manifested by impaired social interaction, deficits in communication skills, restricted interests, and repetitive behaviors. In neurodevelopmental, neurodegenerative, and psychiatric disorders, glial cells undergo morphological, biochemical, and functional rearrangements, which are critical for neuronal development, neurotransmission, and synaptic connectivity. Cerebellar function is not limited to motor coordination but also contributes to cognition and may be affected in autism. Oligodendrocytes and specifically oligodendroglial precursors are highly susceptible to oxidative stress and excitotoxic insult. In the present study, we searched for evidence for developmental oligodendropathy in the context of autism by performing a network analysis of gene expression of cerebellar tissue. We created an in silico network model (OLIGO) showing the landscape of interactions between oligodendrocyte markers and demonstrated that more than 50 % (16 out of 30) of the genes within this model displayed significant changes of expression (corrected p value <0.05) in the cerebellum of autistic patients. In particular, we found up-regulation of OLIG2-, MBP-, OLIG1-, and MAG-specific oligodendrocyte markers. We postulate that aberrant expression of oligodendrocyte-specific genes, potentially related to changes in oligodendrogenesis, may contribute to abnormal cerebellar development, impaired myelination, and anomalous synaptic connectivity in autism spectrum disorders (ASD). PMID:26189831

  15. A-Kinase Anchoring Protein 79/150 Coordinates Metabotropic Glutamate Receptor Sensitization of Peripheral Sensory Neurons

    PubMed Central

    Szteyn, Kalina; Rowan, Matthew P.; Gomez, Ruben; Du, Junhui; Carlton, Susan M.; Jeske, Nathaniel A.

    2016-01-01

    Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-Kinase Anchoring Protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity following group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature. PMID:26172554

  16. Tax-interacting protein 1 coordinates the spatiotemporal activation of Rho GTPases and regulates the infiltrative growth of human glioblastoma

    PubMed Central

    Wang, Hailun; Han, Miaojun; Whetsell, William; Wang, Jialiang; Rich, Jeremy; Hallahan, Dennis; Han, Zhaozhong

    2014-01-01

    PDZ domains represent one group of the major structural units that mediate protein interactions in intercellular contact, signal transduction and assembly of biological machineries. TIP-1 protein is composed of a single PDZ domain that distinguishes TIP-1 from other PDZ domain proteins that more often contain multiple protein domains and function as scaffolds for protein complex assembly. However, the biological functions of TIP-1, especially in cell transformation and tumor progression, are still controversial as observed in a variety of cell types. In this study, we have identified ARHGEF7, a guanine nucleotide exchange factor (GEF) for Rho GTPases, as one novel TIP-1 interacting protein in human glioblastoma cells. We found that the presence of TIP-1 protein is essential to the intracellular redistribution of ARHGEF7 and rhotekin, one Rho effector, and the spatiotemporally coordinated activation of Rho GTPases (RhoA, Cdc42 and Rac1) in migrating glioblastoma cells. TIP-1 knockdown resulted in both aberrant localization of ARHGEF7 and rhotekin, as well as abnormal activation of Rho GTPases that was accompanied with impaired motility of glioblastoma cells. Furthermore, TIP-1 knockdown suppressed tumor cell dispersal in orthotopic glioblastoma murine models. We also observed high levels of TIP-1 expression in human glioblastoma specimens, and the elevated TIP-1 levels are associated with advanced staging and poor prognosis in glioma patients. Although more studies are needed to further dissect the mechanism(s) by which TIP-1 modulates the intracellular redistribution and activation of Rho GTPases, this study suggests that TIP-1 holds potential as both a prognostic biomarker and a therapeutic target of malignant gliomas. PMID:23563176

  17. Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia

    PubMed Central

    Imanishi, Hisayoshi; Tsuruta, Daisuke; Tateishi, Chiharu; Sugawara, Koji; Paus, Ralf; Tsuji, Tsutomu; Ishii, Masamitsu; Ikeda, Kazuo; Kunimoto, Hiroyuki; Nakajima, Koichi; Jones, Jonathan C.R.; Kobayashi, Hiromi

    2010-01-01

    Background Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth. Objective In this study, we examined the role and localization of laminin-332 and -511 in CIA. Methods We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment. Results Our data indicate that both laminin-332 and its receptor α6β4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of α3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the α3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA. Conclusions We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels. PMID:20211547

  18. Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5.

    PubMed

    Huang, Jiqing; Kast, Juergen

    2015-08-01

    Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin-PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin-PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification. PMID:26159767

  19. Database algorithm for generating protein backbone and side-chain co-ordinates from a C alpha trace application to model building and detection of co-ordinate errors.

    PubMed

    Holm, L; Sander, C

    1991-03-01

    The problem of constructing all-atom model co-ordinates of a protein from an outline of the polypeptide chain is encountered in protein structure determination by crystallography or nuclear magnetic resonance spectroscopy, in model building by homology and in protein design. Here, we present an automatic procedure for generating full protein co-ordinates (backbone and, optionally, side-chains) given the C alpha trace and amino acid sequence. To construct backbones, a protein structure database is first scanned for fragments that locally fit the chain trace according to distance criteria. A best path algorithm then sifts through these segments and selects an optimal path with minimal mismatch at fragment joints. In blind tests, using fully known protein structures, backbones (C alpha, C, N, O) can be reconstructed with a reliability of 0.4 to 0.6 A root-mean-square position deviation and not more than 0 to 5% peptide flips. This accuracy is sufficient to identify possible errors in protein co-ordinate sets. To construct full co-ordinates, side-chains are added from a library of frequently occurring rotamers using a simple and fast Monte Carlo procedure with simulated annealing. In tests on X-ray structures determined at better than 2.5 A resolution, the positions of side-chain atoms in the protein core (less than 20% relative accessibility) have an accuracy of 1.6 A (r.m.s. deviation) and 70% of chi 1 angles are within 30 degrees of the X-ray structure. The computer program MaxSprout is available on request. PMID:2002501

  20. Optogenetic Control of Motor Coordination by Gi/o Protein-coupled Vertebrate Rhodopsin in Cerebellar Purkinje Cells*

    PubMed Central

    Gutierrez, Davina V.; Mark, Melanie D.; Masseck, Olivia; Maejima, Takashi; Kuckelsberg, Denise; Hyde, Robert A.; Krause, Martin; Kruse, Wolfgang; Herlitze, Stefan

    2011-01-01

    G protein-coupled receptors are involved in the modulation of complex neuronal networks in the brain. To investigate the impact of a cell-specific Gi/o protein-mediated signaling pathway on brain function, we created a new optogenetic mouse model in which the Gi/o protein-coupled receptor vertebrate rhodopsin can be cell-specifically expressed with the aid of Cre recombinase. Here we use this mouse model to study the functional impact of Gi/o modulation in cerebellar Purkinje cells (PCs). We show that in vivo light activation of vertebrate rhodopsin specifically expressed in PCs reduces simple spike firing that is comparable with the reduction in firing observed for the activation of cerebellar Gi/o-coupled GABAB receptors. Notably, the light exposure of the cerebellar vermis in freely moving mice changes the motor behavior. Thus, our studies directly demonstrate that spike modulation via Gi/o-mediated signaling in cerebellar PCs affects motor coordination and show a new promising approach for studying the physiological function of G protein-coupled receptor-mediated signaling in a cell type-specific manner. PMID:21628464

  1. N-acetylcysteine inhibits the up-regulation of mitochondrial biogenesis genes in livers from rats fed ethanol chronically

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Chronic ethanol (EtOH) administration to experimental animals induces hepatic oxidative stress and up-regulates mitochondrial biogenesis. The mechanisms by which chronic EtOH up-regulates mitochondrial biogenesis have not been fully explored. In this work, we hypothesized that oxidative ...

  2. Urban Air Pollution Produces Up-Regulation of Myocardial Inflammatory Genes and Dark Chocolate Provides Cardioprotection

    PubMed Central

    Villarreal-Calderon, Rodolfo; Reed, William; Palacios-Moreno, Juan; Keefe, Sheyla; Herritt, Lou; Brooks, Diane; Torres-Jardón, Ricardo; Calderón-Garcidueñas, Lilian

    2010-01-01

    Air pollution is a serious environmental problem. Elderly subjects show increased cardiac morbidity and mortality associated with air pollution exposure. Mexico City (MC) residents are chronically exposed to high concentrations of fine particulate matter (PM2.5) and PM-associated lipopolysaccharides (PM-LPS). To test the hypothesis that chronic exposure to urban pollution produces myocardial inflammation, female Balb-c mice age 4 weeks were exposed for 16 months to two distinctly different polluted areas within MC: Southwest (SW) and Northwest (NW). SW mice were given either no treatment or chocolate 2g/9.5 mg polyphenols/3 times per week. Results were compared to mice kept in clean air. Key inflammatory mediator genes: cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the LPS receptor CD14 (cluster of differentiation antigen 14) were measured by real time polymerase chain reaction. Also explored were target NFκB (Nuclear Factor κ B), oxidative stress and antioxidant defense genes. TNF-α, IL-6, and COX-2 were significantly increased in both NW and SWMC mice (p=0.0001). CD14 was up-regulated in SW mice in keeping with the high exposures to particulate matter associated endotoxin. Chocolate administration resulted in a significant down-regulation of TNF-α (p<0.0001), IL-6 (p=0.01), and IL-1β (p=0.02). The up-regulation of antioxidant enzymes and the down-regulation of potent oxidases, toll-like receptors, and pro-apoptotic signaling genes completed the protective profile. Exposure to air pollution produces up-regulation of inflammatory myocardial genes and endotoxin plays a key role in the inflammatory response. Regular consumption of dark chocolate may reduce myocardial inflammation and have cardioprotective properties in the setting of air pollution exposures. PMID:20932730

  3. The Natural Antimicrobial Enzyme Lysozyme is Up-Regulated in Gastrointestinal Inflammatory Conditions.

    PubMed

    Rubio, Carlos A

    2014-01-01

    The cells that line the mucosa of the human gastrointestinal tract (GI, that is, oral cavity, oesophagus, stomach, small intestine, large intestine, and rectum) are constantly challenged by adverse micro-environmental factors, such as different pH, enzymes, and bacterial flora. With exception of the oral cavity, these microenvironments also contain remnant cocktails of secreted enzymes and bacteria from upper organs along the tract. The density of the GI bacteria varies, from 103/mL near the gastric outlet, to 1010/mL at the ileocecal valve, to 1011 to 1012/mL in the colon. The total microbial population (ca. 1014) exceeds the total number of cells in the tract. It is, therefore, remarkable that despite the prima facie inauspicious mixture of harmful secretions and bacteria, the normal GI mucosa retains a healthy state of cell renewal. To counteract the hostile microenvironment, the GI epithelia react by speeding cell exfoliation (the GI mucosa has a turnover time of two to three days), by increasing peristalsis, by eliminating bacteria through secretion of plasma cell-immunoglobulins and by increasing production of natural antibacterial compounds, such as defensin-5 and lysozyme. Only recently, lysozyme was found up-regulated in Barrett's oesophagitis, chronic gastritis, gluten-induced atrophic duodenitis (coeliac disease), collagenous colitis, lymphocytic colitis, and Crohn's colitis. This up-regulation is a response directed to the special types of bacteria recently detected in these diseases. The aim of lysozyme up-regulation is to protect individual mucosal segments to chronic inflammation. The molecular mechanisms connected to the crosstalk between the intraluminal bacterial flora and the production of lysozyme released by the GI mucosae, are discussed. Bacterial resistance continues to exhaust our supply of commercial antibiotics. The potential use of lysozyme to treat infectious diseases is receiving much attention. PMID:25437608

  4. Up-regulation of 2-oxoglutarate dehydrogenase as a stress response.

    PubMed

    Graf, Anastasia; Trofimova, Lidia; Loshinskaja, Alexandra; Mkrtchyan, Garik; Strokina, Anastasiia; Lovat, Maxim; Tylicky, Adam; Strumilo, Slawomir; Bettendorff, Lucien; Bunik, Victoria I

    2013-01-01

    2-Oxoglutarate dehydrogenase multienzyme complex (OGDHC) operates at a metabolic cross-road, mediating Ca(2+)- and ADP-dependent signals in mitochondria. Here, we test our hypothesis that OGDHC plays a major role in the neurotransmitter metabolism and associated stress response. This possibility was assessed using succinyl phosphonate (SP), a highly specific and efficient in vivo inhibitor of OGDHC. Animals exposed to toxicants (SP, ethanol or MnCl(2)), trauma or acute hypoxia showed intrinsic up-regulation of OGDHC in brain and heart. The known mechanism of the SP action as OGDHC inhibitor pointed to the up-regulation triggered by the enzyme impairment. The animal behavior and skeletal muscle or heart performance were tested to correlate physiology with the OGDHC regulation and associated changes in the glutamate and cellular energy status. The SP-treated animals exhibited interdependent changes in the brain OGDHC activity, glutamate level and cardiac autonomic balance, suggesting the neurotransmitter role of glutamate to be involved in the changed heart performance. Energy insufficiency after OGDHC inhibition was detectable neither in animals up to 25 mg/kg SP, nor in cell culture during 24 h incubation with 0.1 mM SP. However, in animals subjected to acute ethanol intoxication SP did evoke energy deficit, decreasing muscular strength and locomotion and increasing the narcotic sleep duration. This correlated with the SP-induced decrease in NAD(P)H levels of the ethanol-exposed neurons. Thus, we show the existence of natural mechanisms to up-regulate mammalian OGDHC in response to stress, with both the glutamate neurotransmission and energy production potentially involved in the OGDHC impact on physiological performance. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy. PMID:22814169

  5. Evidence That Up-Regulation of MicroRNA-29 Contributes to Postnatal Body Growth Deceleration

    PubMed Central

    Kamran, Fariha; Andrade, Anenisia C.; Nella, Aikaterini A.; Clokie, Samuel J.; Rezvani, Geoffrey; Nilsson, Ola; Baron, Jeffrey

    2015-01-01

    Body growth is rapid in infancy but subsequently slows and eventually ceases due to a progressive decline in cell proliferation that occurs simultaneously in multiple organs. We previously showed that this decline in proliferation is driven in part by postnatal down-regulation of a large set of growth-promoting genes in multiple organs. We hypothesized that this growth-limiting genetic program is orchestrated by microRNAs (miRNAs). Bioinformatic analysis identified target sequences of the miR-29 family of miRNAs to be overrepresented in age–down-regulated genes. Concomitantly, expression microarray analysis in mouse kidney and lung showed that all members of the miR-29 family, miR-29a, -b, and -c, were strongly up-regulated from 1 to 6 weeks of age. Real-time PCR confirmed that miR-29a, -b, and -c were up-regulated with age in liver, kidney, lung, and heart, and their expression levels were higher in hepatocytes isolated from 5-week-old mice than in hepatocytes from embryonic mouse liver at embryonic day 16.5. We next focused on 3 predicted miR-29 target genes (Igf1, Imp1, and Mest), all of which are growth-promoting. A 3′-untranslated region containing the predicted target sequences from each gene was placed individually in a luciferase reporter construct. Transfection of miR-29 mimics suppressed luciferase gene activity for all 3 genes, and this suppression was diminished by mutating the target sequences, suggesting that these genes are indeed regulated by miR-29. Taken together, the findings suggest that up-regulation of miR-29 during juvenile life drives the down-regulation of multiple growth-promoting genes, thus contributing to physiological slowing and eventual cessation of body growth. PMID:25866874

  6. Urban air pollution produces up-regulation of myocardial inflammatory genes and dark chocolate provides cardioprotection.

    PubMed

    Villarreal-Calderon, Rodolfo; Reed, William; Palacios-Moreno, Juan; Keefe, Sheyla; Herritt, Lou; Brooks, Diane; Torres-Jardón, Ricardo; Calderón-Garcidueñas, Lilian

    2012-05-01

    Air pollution is a serious environmental problem. Elderly subjects show increased cardiac morbidity and mortality associated with air pollution exposure. Mexico City (MC) residents are chronically exposed to high concentrations of fine particulate matter (PM(2.5)) and PM-associated lipopolysaccharides (PM-LPS). To test the hypothesis that chronic exposure to urban pollution produces myocardial inflammation, female Balb-c mice age 4 weeks were exposed for 16 months to two distinctly different polluted areas within MC: southwest (SW) and northwest (NW). SW mice were given either no treatment or chocolate 2g/9.5 mg polyphenols/3 times per week. Results were compared to mice kept in clean air. Key inflammatory mediator genes: cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the LPS receptor CD14 (cluster of differentiation antigen 14) were measured by real-time polymerase chain reaction. Also explored were target NFκB (nuclear factor κB), oxidative stress and antioxidant defense genes. TNF-α, IL-6, and COX-2 were significantly increased in both NW and SWMC mice (p=0.0001). CD14 was up-regulated in SW mice in keeping with the high exposures to particulate matter associated endotoxin. Chocolate administration resulted in a significant down-regulation of TNF-α (p<0.0001), IL-6 (p=0.01), and IL-1β (p=0.02). The up-regulation of antioxidant enzymes and the down-regulation of potent oxidases, toll-like receptors, and pro-apoptotic signaling genes completed the protective profile. Exposure to air pollution produces up-regulation of inflammatory myocardial genes and endotoxin plays a key role in the inflammatory response. Regular consumption of dark chocolate may reduce myocardial inflammation and have cardioprotective properties in the setting of air pollution exposures. PMID:20932730

  7. Inhibition of the ERK phosphorylation plays a role in terbinafine-induced p21 up-regulation and DNA synthesis inhibition in human vascular endothelial cells

    SciTech Connect

    Ho, P.-Y.; Hsu, S.-P.; Liang, Y.-C.; Kuo, M.-L.; Ho, Y.-S.; Lee, W.-S.

    2008-05-15

    Previously, we showed that terbinafine (TB) induces cell-cycle arrest in cultured human umbilical vein endothelial cells (HUVEC) through an up-regulation of the p21 protein. The aim of this study is to delineate the molecular mechanisms underlying TB-induced increase of p21 protein. RT-PCR analysis demonstrated that the mRNA levels of p21 and p53 were increased in the TB-treated HUVEC. The p21 promoter activity was also increased by TB treatment. Transfection of HUVEC with p53 dominant negative (DN) abolished the TB-induced increases of p21 promoter activity and protein level, suggesting that the TB-induced increase of p21 is p53-dependent. Western blot analysis demonstrated that TB decreased the levels of phosphorylated extracellular signal-regulated kinase (ERK). Over-expression of mitogen-activated protein kinase (MEK)-1, the immediate upstream activator kinase of ERK, abolished the TB-induced increases of p21 and p53 protein and decrease of thymidine incorporation. The ERK inhibitor (PD98059) enhanced the TB-induced inhibition of thymidine incorporation into HUVEC. Taken together, these data suggest that the decrease of ERK activity plays a role in the TB-induced up-regulation of p21 in HUVEC. On the other hand, pretreatment of the cells with geranylgeraniol (GGOH), farnesol (FOH), or Ras inhibitor peptide did not affect the TB-induced decrease of thymidine incorporation. Taken together, our results suggest that TB might cause a decrease of MEK, which in turn up-regulates p53 through the inhibition of ERK phosphorylation, and finally causes an increase of p21 expression and cell-cycle arrest.

  8. Vitamin D Up-Regulates the Vitamin D Receptor by Protecting It from Proteasomal Degradation in Human CD4+ T Cells

    PubMed Central

    Kongsbak, Martin; von Essen, Marina R.; Boding, Lasse; Levring, Trine B.; Schjerling, Peter; Lauritsen, Jens P. H.; Woetmann, Anders; Ødum, Niels; Bonefeld, Charlotte M.; Geisler, Carsten

    2014-01-01

    The active form of vitamin D3, 1,25(OH)2D3, has significant immunomodulatory properties and is an important determinant in the differentiation of CD4+ effector T cells. The biological actions of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and are believed to correlate with the VDR protein expression level in a given cell. The aim of this study was to determine if and how 1,25(OH)2D3 by itself regulates VDR expression in human CD4+ T cells. We found that activated CD4+ T cells have the capacity to convert the inactive 25(OH)D3 to the active 1,25(OH)2D3 that subsequently up-regulates VDR protein expression approximately 2-fold. 1,25(OH)2D3 does not increase VDR mRNA expression but increases the half-life of the VDR protein in activated CD4+ T cells. Furthermore, 1,25(OH)2D3 induces a significant intracellular redistribution of the VDR. We show that 1,25(OH)2D3 stabilizes the VDR by protecting it from proteasomal degradation. Finally, we demonstrate that proteasome inhibition leads to up-regulation of VDR protein expression and increases 1,25(OH)2D3-induced gene activation. In conclusion, our study shows that activated CD4+ T cells can produce 1,25(OH)2D3, and that 1,25(OH)2D3 induces a 2-fold up-regulation of the VDR protein expression in activated CD4+ T cells by protecting the VDR against proteasomal degradation. PMID:24792400

  9. Up-Regulation of S100A11 in Lung Adenocarcinoma – Its Potential Relationship with Cancer Progression

    PubMed Central

    Woo, Tetsukan; Okudela, Koji; Mitsui, Hideaki; Tajiri, Michihiko; Rino, Yasushi; Ohashi, Kenichi; Masuda, Munetaka

    2015-01-01

    We previously reported that patients with lung adenocarcinomas with KRAS gene mutations and strong proliferating activity had poorer outcomes, even in the early stage of the disease. The aim of the present study was to elucidate the potential molecular basis of these highly malignant lung tumors by focusing on S100 proteins (S100A2, S100A7, and S100A11), which are downstream targets of oncogenic KRAS and promoters of tumor progression. The immunohistochemical expression of S100 proteins was examined in 179 primary lung adenocarcinomas, and the potential relationships between their levels and clinicopathologic factors were analyzed. Among the three subtypes, S100A11 levels were significantly higher in adenocarcinomas with KRAS mutations and strong proliferating activity. They were also higher in adenocarcinomas with poorly differentiated tumors. Furthermore, higher levels of S100A11 were associated with shorter disease-free survival. These results suggest that the up-regulation of S100A11 plays a role in tumor progression, particularly in KRAS-mutated lung adenocarcinomas. PMID:26544866

  10. Transcript profiling reveals that cysteine protease inhibitors are up-regulated in tuber sprouts after extended darkness.

    PubMed

    Grandellis, Carolina; Giammaria, Veronica; Fantino, Elisa; Cerrudo, Ignacio; Bachmann, Sandra; Santin, Franco; Ulloa, Rita M

    2016-07-01

    Potato (Solanum tuberosum L.) tubers are an excellent staple food due to its high nutritional value. When the tuber reaches physiological competence, sprouting proceeds accompanied by changes at mRNA and protein levels. Potato tubers become a source of carbon and energy until sprouts are capable of independent growth. Transcript profiling of sprouts grown under continuous light or dark conditions was performed using the TIGR 10K EST Solanaceae microarray. The profiles analyzed show a core of highly expressed transcripts that are associated to the reactivation of growth. Under light conditions, the photosynthetic machinery was fully activated; the highest up-regulation was observed for the Rubisco activase (RCA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the Photosystem II 22 kDa protein (CP22) genes, among others. On the other hand, sprouts exposed to continuous darkness elongate longer, and after extended darkness, synthesis of chloroplast components was repressed, the expression of proteases was reduced while genes encoding cysteine protease inhibitors (CPIs) and metallocarboxypeptidase inhibitors (MPIs) were strongly induced. Northern blot and RT-PCR analysis confirmed that MPI levels correlated with the length of the dark period; however, CPI expression was strong only after longer periods of darkness, suggesting a feedback loop (regulation mechanism) in response to dark-induced senescence. Prevention of cysteine protease activity in etiolated sprouts exposed to extended darkness could delay senescence until they emerge to light. PMID:27075731

  11. Folic acid protects against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1

    PubMed Central

    Ma, Yan; Zhang, Chen; Gao, Xiao-Bo; Luo, Hai-Yan; Chen, Yang; Li, Hui-hua; Ma, Xu; Lu, Cai-Ling

    2015-01-01

    As a nutritional factor, folic acid can prevent cardiac and neural defects during embryo development. Our previous study showed that arsenic impairs embryo development by down-regulating Dvr1/GDF1 expression in zebrafish. Here, we investigated whether folic acid could protect against arsenic-mediated embryo toxicity. We found that folic acid supplementation increases hatching and survival rates, decreases malformation rate and ameliorates abnormal cardiac and neural development of zebrafish embryos exposed to arsenite. Both real-time PCR analysis and whole in-mount hybridization showed that folic acid significantly rescued the decrease in Dvr1 expression caused by arsenite. Subsequently, our data demonstrated that arsenite significantly decreased cell viability and GDF1 mRNA and protein levels in HEK293ET cells, while folic acid reversed these effects. Folic acid attenuated the increase in subcellular reactive oxygen species (ROS) levels and oxidative adaptor p66Shc protein expression in parallel with the changes in GDF1 expression and cell viability. P66Shc knockdown significantly inhibited the production of ROS and the down-regulation of GDF1 induced by arsenite. Our data demonstrated that folic acid supplementation protected against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1/GDF1, and folic acid enhanced the expression of GDF1 by decreasing p66Shc expression and subcellular ROS levels. PMID:26537450

  12. The long noncoding RNA MALAT1 promotes tumor-driven angiogenesis by up-regulating pro-angiogenic gene expression

    PubMed Central

    Tee, Andrew E.; Liu, Bing; Song, Renhua; Li, Jinyan; Pasquier, Eddy; Cheung, Belamy B.; Jiang, Cizhong; Marshall, Glenn M.; Haber, Michelle; Norris, Murray D.; Fletcher, Jamie I.; Dinger, Marcel E.; Liu, Tao

    2016-01-01

    Neuroblastoma is the most common solid tumor during early childhood. One of the key features of neuroblastoma is extensive tumor-driven angiogenesis due to hypoxia. However, the mechanism through which neuroblastoma cells drive angiogenesis is poorly understood. Here we show that the long noncoding RNA MALAT1 was upregulated in human neuroblastoma cell lines under hypoxic conditions. Conditioned media from neuroblastoma cells transfected with small interfering RNAs (siRNA) targeting MALAT1, compared with conditioned media from neuroblastoma cells transfected with control siRNAs, induced significantly less endothelial cell migration, invasion and vasculature formation. Microarray-based differential gene expression analysis showed that one of the genes most significantly down-regulated following MALAT1 suppression in human neuroblastoma cells under hypoxic conditions was fibroblast growth factor 2 (FGF2). RT-PCR and immunoblot analyses confirmed that MALAT1 suppression reduced FGF2 expression, and Enzyme-Linked Immunosorbent Assays revealed that transfection with MALAT1 siRNAs reduced FGF2 protein secretion from neuroblastoma cells. Importantly, addition of recombinant FGF2 protein to the cell culture media reversed the effects of MALAT1 siRNA on vasculature formation. Taken together, our data suggest that up-regulation of MALAT1 expression in human neuroblastoma cells under hypoxic conditions increases FGF2 expression and promotes vasculature formation, and therefore plays an important role in tumor-driven angiogenesis. PMID:26848616

  13. Prolonged Starvation Causes Up-Regulation of AQP1 in Adipose Tissue Capillaries of AQP7 Knock-Out Mice

    PubMed Central

    Skowronski, Mariusz T.; Skowronska, Agnieszka; Rojek, Aleksandra; Oklinski, Michal K.; Nielsen, Søren

    2016-01-01

    Aquaporins (AQPs) are membrane proteins involved in the regulation of cellular transport and the balance of water and glycerol and cell volume in the white adipose tissue (WAT). In our previous study, we found the co-expression of the AQP1 water channel and AQP7 in the mouse WAT. In our present study, we aimed to find out whether prolonged starvation influences the AQP1 expression of AQP7 knock-out mice (AQP7 KO) in the WAT. To resolve this hypothesis, immunoperoxidase, immunoblot and immunogold microscopy were used. AQP1 expression was found with the use of immunohistochemistry and was confirmed by immunogold microscopy in the vessels of mouse WAT of all studied groups. Semi-quantitative immunoblot and quantitative immunogold microscopy showed a significant increase (by 2.5- to 3-fold) in the abundance of AQP1 protein expression in WAT in the 72 h starved AQP7 KO mice as compared to AQP7+/+ (p < 0.05) and AQP7−/− (p < 0.01) controls, respectively. In conclusion, the AQP1 water channel located in the vessels of WAT is up-regulated in response to prolonged starvation in the WAT of AQP7 KO mice. The present data suggest that an interaction of different AQP isoforms is required for maintaining proper water homeostasis within the mice WAT. PMID:27455244

  14. Prolonged Starvation Causes Up-Regulation of AQP1 in Adipose Tissue Capillaries of AQP7 Knock-Out Mice.

    PubMed

    Skowronski, Mariusz T; Skowronska, Agnieszka; Rojek, Aleksandra; Oklinski, Michal K; Nielsen, Søren

    2016-01-01

    Aquaporins (AQPs) are membrane proteins involved in the regulation of cellular transport and the balance of water and glycerol and cell volume in the white adipose tissue (WAT). In our previous study, we found the co-expression of the AQP1 water channel and AQP7 in the mouse WAT. In our present study, we aimed to find out whether prolonged starvation influences the AQP1 expression of AQP7 knock-out mice (AQP7 KO) in the WAT. To resolve this hypothesis, immunoperoxidase, immunoblot and immunogold microscopy were used. AQP1 expression was found with the use of immunohistochemistry and was confirmed by immunogold microscopy in the vessels of mouse WAT of all studied groups. Semi-quantitative immunoblot and quantitative immunogold microscopy showed a significant increase (by 2.5- to 3-fold) in the abundance of AQP1 protein expression in WAT in the 72 h starved AQP7 KO mice as compared to AQP7+/+ (p < 0.05) and AQP7-/- (p < 0.01) controls, respectively. In conclusion, the AQP1 water channel located in the vessels of WAT is up-regulated in response to prolonged starvation in the WAT of AQP7 KO mice. The present data suggest that an interaction of different AQP isoforms is required for maintaining proper water homeostasis within the mice WAT. PMID:27455244

  15. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle

    PubMed Central

    Heijink, Irene H.; Holtzer, Laura J.; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J.; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-01-01

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  16. A-kinase-anchoring proteins coordinate inflammatory responses to cigarette smoke in airway smooth muscle.

    PubMed

    Poppinga, Wilfred J; Heijink, Irene H; Holtzer, Laura J; Skroblin, Philipp; Klussmann, Enno; Halayko, Andrew J; Timens, Wim; Maarsingh, Harm; Schmidt, Martina

    2015-04-15

    β2-Agonist inhibitors can relieve chronic obstructive pulmonary disease (COPD) symptoms by stimulating cyclic AMP (cAMP) signaling. A-kinase-anchoring proteins (AKAPs) compartmentalize cAMP signaling by establishing protein complexes. We previously reported that the β2-agonist fenoterol, direct activation of protein kinase A (PKA), and exchange factor directly activated by cAMP decrease cigarette smoke extract (CSE)-induced release of neutrophil attractant interleukin-8 (IL-8) from human airway smooth muscle (ASM) cells. In the present study, we tested the role of AKAPs in CSE-induced IL-8 release from ASM cells and assessed the effect of CSE on the expression levels of different AKAPs. We also studied mRNA and protein expression of AKAPs in lung tissue from patients with COPD. Our data show that CSE exposure of ASM cells decreases AKAP5 and AKAP12, both capable of interacting with β2-adrenoceptors. In lung tissue of patients with COPD, mRNA levels of AKAP5 and AKAP12 were decreased compared with lung tissue from controls. Using immunohistochemistry, we detected less AKAP5 protein in ASM of patients with COPD Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage II compared with control subjects. St-Ht31, which disrupts AKAP-PKA interactions, augmented CSE-induced IL-8 release from ASM cells and diminished its suppression by fenoterol, an effect mediated by disturbed ERK signaling. The modulatory role of AKAP-PKA interactions in the anti-inflammatory effects of fenoterol in ASM cells and the decrease in expression of AKAP5 and AKAP12 in response to cigarette smoke and in lungs of patients with COPD suggest that cigarette smoke-induced changes in AKAP5 and AKAP12 in patients with COPD may affect efficacy of pharmacotherapy. PMID:25637608

  17. Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt.

    PubMed

    Chen, Jialin; Chen, Peng; Backman, Ludvig J; Zhou, Qingjun; Danielson, Patrik

    2016-01-01

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway. PMID:27174608

  18. Inositol 1,4,5-Trisphosphate (IP3) Receptor Up-regulation in Hypertension Is Associated with Sensitization of Ca2+ Release and Vascular Smooth Muscle Contractility*

    PubMed Central

    Abou-Saleh, Haissam; Pathan, Asif R.; Daalis, Arwa; Hubrack, Satanay; Abou-Jassoum, Hamda; Al-Naeimi, Hamda; Rusch, Nancy J.; Machaca, Khaled

    2013-01-01

    Resistance arteries show accentuated responsiveness to vasoconstrictor agonists in hypertension, and this abnormality relies partly on enhanced Ca2+ signaling in vascular smooth muscle (VSM). Although inositol 1,4,5-triphosphate receptors (IP3Rs) are abundant in VSM, their role in the molecular remodeling of the Ca2+ signaling machinery during hypertension has not been addressed. Therefore, we compared IP3R expression and function between mesenteric arteries of normotensive and hypertensive animals. Levels of IP3R transcript and protein were significantly increased in mesenteric arteries of hypertensive animals, and pharmacological inhibition of the IP3R revealed a higher contribution of IP3-dependent Ca2+ release to vascular contraction in these arteries. Subsequently, we established cultured aortic VSM A7r5 cells as a cellular model that replicates IP3R up-regulation during hypertension by depolarizing the VSM cell membrane. IP3R up-regulation requires Ca2+ influx through L-type Ca2+ channels, followed by activation of the calcineurin-NFAT axis, resulting in IP3R transcription. Functionally, IP3R up-regulation in VSM is associated with enhancement and sensitization of IP3-dependent Ca2+ release, resulting in increased VSM contraction in response to agonist stimulation. PMID:24097979

  19. Ciliary Neurotrophic Factor Promotes the Migration of Corneal Epithelial Stem/progenitor Cells by Up-regulation of MMPs through the Phosphorylation of Akt

    PubMed Central

    Chen, Jialin; Chen, Peng; Backman, Ludvig J.; Zhou, Qingjun; Danielson, Patrik

    2016-01-01

    The migration of limbal epithelial stem cells is important for the homeostasis and regeneration of corneal epithelium. Ciliary neurotrophic factor (CNTF) has been found to promote corneal epithelial wound healing by activating corneal epithelial stem/progenitor cells. However, the possible effect of CNTF on the migration of corneal epithelial stem/progenitor cells is not clear. This study found the expression of CNTF in mouse corneal epithelial stem/progenitor cells (TKE2) to be up-regulated after injury, on both gene and protein level. CNTF promoted migration of TKE2 in a dose-dependent manner and the peak was seen at 10 ng/ml. The phosphorylation level of Akt (p-Akt), and the expression of MMP3 and MMP14, were up-regulated after CNTF treatment both in vitro and in vivo. Akt and MMP3 inhibitor treatment delayed the migration effect by CNTF. Finally, a decreased expression of MMP3 and MMP14 was observed when Akt inhibitor was applied both in vitro and in vivo. This study provides new insights into the role of CNTF on the migration of corneal epithelial stem/progenitor cells and its inherent mechanism of Up-regulation of matrix metalloproteinases through the Akt signalling pathway. PMID:27174608

  20. Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera.

    PubMed

    Mao, Wenfu; Schuler, Mary A; Berenbaum, May R

    2013-05-28

    As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses. PMID:23630255

  1. Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera

    PubMed Central

    Mao, Wenfu; Schuler, Mary A.; Berenbaum, May R.

    2013-01-01

    As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p-coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p-coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p-coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p-coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p-coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses. PMID:23630255

  2. Glutamate Transporter EAAT2 Expression is Up-Regulated in Reactive Astrocytes in Human Periventricular Leukomalacia

    PubMed Central

    DESILVA, TARA M.; BILLIARDS, SARAID S.; BORENSTEIN, NATALIA S.; TRACHTENBERG, FELICIA L.; VOLPE, JOSEPH J.; KINNEY, HANNAH C.; ROSENBERG, PAUL A.

    2010-01-01

    The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% ± 5.6%) compared with control white matter (21.4% ± 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation. PMID:18314905

  3. Transcriptional up-regulation of the human androgen receptor by androgen in bone cells.

    PubMed

    Wiren, K M; Zhang, X; Chang, C; Keenan, E; Orwoll, E S

    1997-06-01

    Androgen regulation of androgen receptor (AR) expression has been observed in a variety of tissues, generally as inhibition, and is thought to attenuate cellular responses to androgen. AR is expressed in osteoblasts, the bone-forming cell, suggesting direct actions of androgens on bone. Here we characterized the effect of androgen exposure on AR gene expression in human osteoblastic SaOS-2 and U-2 OS cells. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone increased AR steady state messenger RNA levels in a time- and dose-dependent fashion. Reporter assays with 2.3 kilobases of the proximal 5'-flanking region of the human AR promoter linked to the chloramphenicol acetyltransferase gene in transfected cultures showed that up-regulation of AR promoter activity by androgen was time and dose dependent. Treatment with other steroid hormones, including progesterone, 17beta-estradiol, and dexamethasone, was without effect. The antiandrogen hydroxyflutamide completely antagonized androgen up-regulation. Thus, in contrast to many other androgen target tissues, androgen exposure increases steady state AR messenger RNA levels in osteoblasts. This regulation occurs at least partially at the level of transcription, is mediated by the 5'-promoter region of the AR gene, and is dependent on functional AR. These results suggest that physiological concentrations of androgens have significant effects on AR expression in skeletal tissue. PMID:9165014

  4. 17-β-estradiol up-regulates apolipoprotein genes expression during osteoblast differentiation in vitro.

    PubMed

    Gui, Yuyan; Chu, Nan; Qiu, Xuemin; Tang, Wei; Gober, Hans-Jürgen; Li, Dajin; Wang, Ling

    2016-05-23

    Apolipoproteins are of great physiological importance and are associated with different diseases. Many independent studies of patterns of gene expression during osteoblast differentiation have been described, and some apolipoproteins have been induced during this process. 17-β-estradiol (E2) may enhance osteoblast physiological function. However, no studies have indicated whether E2 can modulate the expression of apolipoproteins during osteoblast differentiation in vitro. The aim of the current study was to observe the regulation of apolipoprotein mRNA expression by E2 during this process. Primary osteoblasts were collected from the calvaria of newborn mice and were subjected to osteoblast differentiation in vitro with serial concentrations of E2. RNA was isolated on days 0, 5, and 25 of differentiation. Real-time PCR was performed to analyze the levels of apolipoprotein mRNA. Results showed that during osteoblast differentiation all of the apolipoprotein genes were up-regulated by E2 in a dose-dependent manner. Moreover, only ApoE was strongly induced during the mineralization of cultured osteoblasts. This result suggests that ApoE might be involved in osteoblast differentiation. The hypothesis is that E2 promotes osteoblast differentiation by up-regulating ApoE gene expression, though further study is needed to confirm this hypothesis. PMID:27074899

  5. N-glycoprotein analysis discovers new up-regulated glycoproteins in colorectal cancer tissue.

    PubMed

    Nicastri, Annalisa; Gaspari, Marco; Sacco, Rosario; Elia, Laura; Gabriele, Caterina; Romano, Roberto; Rizzuto, Antonia; Cuda, Giovanni

    2014-11-01

    Colorectal cancer is one of the leading causes of death due to cancer worldwide. Therefore, the identification of high-specificity and -sensitivity biomarkers for the early detection of colorectal cancer is urgently needed. Post-translational modifications, such as glycosylation, are known to play an important role in cancer progression. In the present work, we used a quantitative proteomic technique based on (18)O stable isotope labeling to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared with healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. We identified 54 up-regulated glycoproteins in colorectal cancer samples, therefore potentially involved in the biological processes of tumorigenesis. In particular, nine of these (PLOD2, DPEP1, SE1L1, CD82, PAR1, PLOD3, S12A2, LAMP3, OLFM4) were found to be up-regulated in the great majority of the cohort, and, interestingly, the association with colorectal cancer of four (PLOD2, S12A2, PLOD3, CD82) has not been hitherto described. PMID:25247386

  6. Poncirin Induces Apoptosis in AGS Human Gastric Cancer Cells through Extrinsic Apoptotic Pathway by up-Regulation of Fas Ligand

    PubMed Central

    Venkatarame Gowda Saralamma, Venu; Nagappan, Arulkumar; Hong, Gyeong Eun; Lee, Ho Jeong; Yumnam, Silvia; Raha, Suchismita; Heo, Jeong Doo; Lee, Sang Joon; Lee, Won Sup; Kim, Eun Hee; Kim, Gon Sup

    2015-01-01

    Poncirin, a natural bitter flavanone glycoside abundantly present in many species of citrus fruits, has various biological benefits such as anti-oxidant, anti-microbial, anti-inflammatory and anti-cancer activities. The anti-cancer mechanism of Poncirin remains elusive to date. In this study, we investigated the anti-cancer effects of Poncirin in AGS human gastric cancer cells (gastric adenocarcinoma). The results revealed that Poncirin could inhibit the proliferation of AGS cells in a dose-dependent manner. It was observed Poncirin induced accumulation of sub-G1 DNA content, apoptotic cell population, apoptotic bodies, chromatin condensation, and DNA fragmentation in a dose-dependent manner in AGS cells. The expression of Fas Ligand (FasL) protein was up-regulated dose dependently in Poncirin-treated AGS cells Moreover, Poncirin in AGS cells induced activation of Caspase-8 and -3, and subsequent cleavage of poly(ADP-ribose) polymerase (PARP). Inhibitor studies’ results confirm that the induction of caspase-dependent apoptotic cell death in Poncirin-treated AGS cells was led by the Fas death receptor. Interestingly, Poncirin did not show any effect on mitochondrial membrane potential (ΔΨm), pro-apoptotic proteins (Bax and Bak) and anti-apoptotic protein (Bcl-xL) in AGS-treated cells followed by no activation in the mitochondrial apoptotic protein caspase-9. This result suggests that the mitochondrial-mediated pathway is not involved in Poncirin-induced cell death in gastric cancer. These findings suggest that Poncirin has a potential anti-cancer effect via extrinsic pathway-mediated apoptosis, possibly making it a strong therapeutic agent for human gastric cancer. PMID:26393583

  7. C5a anaphylatoxin as a product of complement activation up-regulates the complement inhibitory factor H in rat Kupffer cells.

    PubMed

    Schlaf, Gerald; Nitzki, Frauke; Heine, Ines; Hardeland, Rüdiger; Schieferdecker, Henrike L; Götze, Otto

    2004-11-01

    The 155-kDa complement regulator factor H (FH) is the predominant soluble regulatory protein of the complement system. It acts as a cofactor for the factor I-mediated conversion of the component C3b to iC3b, competes with factor B for a binding site on C3b and C3(H2O) and promotes the dissociation of the C3bBb complex. The primary site of synthesis is the liver, i.e. FH-specific mRNA and protein were identified in both hepatocytes (HC) and Kupffer cells (KC). Previous studies in rat primary HC and KC had shown that the proinflammatory cytokine IFN-gamma influences the balance between activation and inhibition of the complement system through up-regulation of the inhibitory FH. In this study we show that C5a, as a product of complement activation, stimulates the expression of FH-specific mRNA and protein in KC and thus induces a negative feedback. Quantitative-competitive RT-PCR showed an approximate threefold C5a-induced up-regulation of FH. ELISA analyses revealed a corresponding increase in FH protein in the supernatants of KC. The up-regulation of FH was completely inhibited by the C5a-blocking monoclonal antibody 6-9F. Furthermore, an involvement of LPS and IFN-gamma was excluded, which strongly indicates a direct effect of C5a on the expression of FH in KC. PMID:15376195

  8. Developmentally regulated GTP-binding protein 2 coordinates Rab5 activity and transferrin recycling

    PubMed Central

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Kim, Hyo Jeong; Ko, Myoung Seok; Seol, Wongi; Joe, Yeonsoo; Chung, Hun Taeg; Lee, Byung Ju; Moon, Chang Hoon; Cho, Wha Ja; Park, Jeong Woo

    2016-01-01

    The small GTPase Rab5 regulates the early endocytic pathway of transferrin (Tfn), and Rab5 deactivation is required for Tfn recycling. Rab5 deactivation is achieved by RabGAP5, a GTPase-activating protein, on the endosomes. Here we report that recruitment of RabGAP5 is insufficient to deactivate Rab5 and that developmentally regulated GTP-binding protein 2 (DRG2) is required for Rab5 deactivation and Tfn recycling. DRG2 was associated with phosphatidylinositol 3-phosphate–containing endosomes. It colocalized and interacted with EEA1 and Rab5 on endosomes in a phosphatidylinositol 3-kinase–dependent manner. DRG2 depletion did not affect Tfn uptake and recruitment of RabGAP5 and Rac1 to Rab5 endosomes. However, it resulted in impairment of interaction between Rab5 and RabGAP5, Rab5 deactivation on endosomes, and Tfn recycling. Ectopic expression of shRNA-resistant DRG2 rescued Tfn recycling in DRG2-depleted cells. Our results demonstrate that DRG2 is an endosomal protein and a key regulator of Rab5 deactivation and Tfn recycling. PMID:26582392

  9. Canonical and noncanonical g-protein signaling helps coordinate actin dynamics to promote macrophage phagocytosis of zymosan.

    PubMed

    Huang, Ning-Na; Becker, Steven; Boularan, Cedric; Kamenyeva, Olena; Vural, Ali; Hwang, Il-Young; Shi, Chong-Shan; Kehrl, John H

    2014-11-15

    Both chemotaxis and phagocytosis depend upon actin-driven cell protrusions and cell membrane remodeling. While chemoattractant receptors rely upon canonical G-protein signaling to activate downstream effectors, whether such signaling pathways affect phagocytosis is contentious. Here, we report that Gαi nucleotide exchange and signaling helps macrophages coordinate the recognition, capture, and engulfment of zymosan bioparticles. We show that zymosan exposure recruits F-actin, Gαi proteins, and Elmo1 to phagocytic cups and early phagosomes. Zymosan triggered an increase in intracellular Ca(2+) that was partially sensitive to Gαi nucleotide exchange inhibition and expression of GTP-bound Gαi recruited Elmo1 to the plasma membrane. Reducing GDP-Gαi nucleotide exchange, decreasing Gαi expression, pharmacologically interrupting Gβγ signaling, or reducing Elmo1 expression all impaired phagocytosis, while favoring the duration that Gαi remained GTP bound promoted it. Our studies demonstrate that targeting heterotrimeric G-protein signaling offers opportunities to enhance or retard macrophage engulfment of phagocytic targets such as zymosan. PMID:25225330

  10. Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

    PubMed

    Marques, M do V; Borges, A C; de Oliveira, J C; Gomes, S L

    1992-02-01

    The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella. PMID:1309711

  11. Human p38{delta} MAP kinase mediates UV irradiation induced up-regulation of the gene expression of chemokine BRAK/CXCL14

    SciTech Connect

    Ozawa, Shigeyuki; Ito, Shin; Kato, Yasumasa; Kubota, Eiro; Hata, Ryu-Ichiro

    2010-06-11

    The mitogen-activated protein kinase (MAPK) family comprises ERK, JNK, p38 and ERK5 (big-MAPK, BMK1). UV irradiation of squamous cell carcinoma cells induced up-regulation of gene expression of chemokine BRAK/CXCL14, stimulated p38 phosphorylation, and down-regulated the phosphorylation of ERK. Human p38 MAPKs exist in 4 isoforms: p38{alpha}, {beta}, {gamma} and {delta}. The UV stimulation of p38 phosphorylation was not inhibited by the presence of SB203580 or PD169316, inhibitors of p38{alpha} and {beta}, suggesting p38 phosphorylation was not dependent on these 2 isoforms and that p38{gamma} and/or {delta} was responsible for the phosphorylation. In fact, inhibition of each of these 4 p38 isoforms by the introduction of short hairpin (sh) RNAs for respective isoforms revealed that only shRNA for p38{delta} attenuated the UV-induced up-regulation of BRAK/CXCL14 gene expression. In addition, over-expression of p38 isoforms in the cells showed the association of p38{delta} with ERK1 and 2, concomitant with down-regulation of ERK phosphorylation. The usage of p38{delta} isoform by UV irradiation is not merely due to the abundance of this p38 isoform in the cells. Because serum deprivation of the cells also induced an increase in BRAK/CXCL14 gene expression, and in this case p38{alpha} and/or {beta} isoform is responsible for up-regulation of BRAK/CXCL14 gene expression. Taken together, the data indicate that the respective stress-dependent action of p38 isoforms is responsible for the up-regulation of the gene expression of the chemokine BRAK/CXCL14.

  12. Intranasal deferoxamine attenuates synapse loss via up-regulating the P38/HIF-1α pathway on the brain of APP/PS1 transgenic mice

    PubMed Central

    Guo, Chuang; Zhang, Yu-Xin; Wang, Tao; Zhong, Man-Li; Yang, Zhao-Hui; Hao, Li-Juan; Chai, Rui; Zhang, Shuai

    2015-01-01

    The widely recognized neuroprotective effect of iron chelators is contributed by their ability to prevent reactive oxygen species (ROS) generation via the Fenton reaction, which sequesters redox-active Fe. An additional neuroprotective mechanism of iron-chelating compounds is to regulate the transcriptional activator hypoxia-inducible factor 1α (HIF-1α). In the present study, we observed that intranasal administration of deferoxamine decreased beta-amyloid (Aβ) deposition and rescued synapse loss in the brain of Aβ precursor protein and presenilin-1 (APP/PS1) double transgenic mice. We found that deferoxamine (DFO) up-regulated HIF-1α mRNA expression and its protein level, and further induced the proteins that are encoded from HIF-1-adaptive genes, including transferrin receptor (TFR), divalent metal transporter 1 (DMT1), and brain-derived neurotrophic factor (BDNF). The effects of DFO on the induction and stabilization of HIF-1α were further confirmed in vitro. This was accompanied by a decrease of Fe in the CA3 region of the hippocampus. Western blotting studies revealed that DFO differentially enhanced the phosphorylation of mitogen-activated protein kinase (MAPK)/P38 kinase in vitro and in vivo. The results suggest that the DFO may up-regulate several HIF-1-dependent neuroprotective-adaptive genes in AD via activating P38/HIF-1α pathway, which may serve as important therapeutic targets to the disease. PMID:26082716

  13. [Hydroxysafflor yellow A up-regulates HIF-1alpha via inhibition of VHL and p53 in Eahy 926 cell line exposed to hypoxia].

    PubMed

    Lian, Ze-Qin; Zhao, Da-Long; Zhu, Hai-Bo

    2008-05-01

    In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression. PMID:18717335

  14. Coordinated regulation of mesenchymal stem cell differentiation on microstructured titanium surfaces by endogenous bone morphogenetic proteins.

    PubMed

    Olivares-Navarrete, Rene; Hyzy, Sharon L; Haithcock, David A; Cundiff, Caitlin A; Schwartz, Zvi; Boyan, Barbara D

    2015-04-01

    Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4 μm); sandblasted/acid-etched Ti (SLA, Ra=3.2 μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment. PMID:25554602

  15. Coordinated Regulation of Mesenchymal Stem Cell Differentiation on Microstructured Titanium Surfaces by Endogenous Bone Morphogenetic Proteins

    PubMed Central

    Olivares-Navarrete, Rene; Hyzy, Sharon L.; Haithcock, David A.; Cundiff, Caitlin A.; Schwartz, Zvi; Boyan, Barbara D.

    2015-01-01

    Human mesenchymal stem cells (MSCs) differentiate into osteoblasts on microstructured titanium (Ti) surfaces without addition of medium supplements, suggesting that surface-dependent endogenous mechanisms are involved. They produce bone morphogenetic proteins (BMPs), which regulate MSC differentiation and bone formation via autocrine/paracrine mechanisms that are modulated by changes in BMP mRNA and protein, receptors, and inhibitors (Noggin, Cerberus, Gremlin 1, and Chordin). We examined expression of BMPs, their receptors and their inhibitors over time and used BMP2-silenced cells to determine how modulating endogenous BMP signaling can affect the process. MSCs were cultured on tissue culture polystyrene or Ti [PT (Ra<0.4μm); sandblasted/acid-etched Ti (SLA, Ra=3.2μm); or hydrophilic-SLA (modSLA)]. BMP mRNAs and proteins increased by day 4 of culture. Exogenous BMP2 increased differentiation whereas differentiation was decreased in BMP2-silenced cells. Noggin was regulated by day 2 whereas Gremlin 1 and Cerberus were regulated after 6 days. Osteoblastic differentiation increased in cells cultured with blocking antibodies against Noggin, Gremlin 1, and Cerberus. Endogenous BMPs enhance an osteogenic microenvironment whereas exogenous BMPs are inhibitory. Antibody blocking of the BMP2 inhibitor Cerberus resulted in IL-6 and IL-8 levels that were similar to those observed when treating cells with exogenous BMP2, while antibodies targeting the inhibitors Gremlin or Noggin did not. These results suggest that microstructured titanium implants supporting therapeutic stem cells may be treated with appropriately selected agents antagonistic to extracellular BMP inhibitors in order to enhance BMP2 mediated bone repair while avoiding undesirable inflammatory side effects observed with exogenous BMP2 treatment. PMID:25554602

  16. Polyvalent Display of Heme on Hepatitis B Virus Capsid Protein through Coordination to Hexahistidine Tags

    PubMed Central

    Prasuhn, Duane E.; Kuzelka, Jane; Strable, Erica; Udit, Andrew K.; Cho, So-Hye; Lander, Gabriel C.; Quispe, Joel D.; Diers, James R.; Bocian, David F.; Potter, Clint; Carragher, Bridget; Finn, M.G.

    2009-01-01

    SUMMARY The addition of a hexahistidine tag to the N terminus of the hepatitis B capsid protein gives rise to a self-assembled particle with 80 sites of high local density of histidine side chains. Iron protoporphyrin IX has been found to bind tightly at each of these sites, making a polyvalent system of well-defined spacing between metalloporphyrin complexes. The spectroscopic and redox properties of the resulting particle are consistent with the presence of 80 site-isolated bis(histidine)-bound heme centers, comprising a polyvalent b-type cytochrome mimic. PMID:18482703

  17. Kinetic Definition of Protein Folding Transition State Ensembles and Reaction Coordinates

    PubMed Central

    Snow, Christopher D.; Rhee, Young Min; Pande, Vijay S.

    2006-01-01

    Using distributed molecular dynamics simulations we located four distinct folding transitions for a 39-residue ββαβ protein fold. To characterize the nature of each room temperature transition, we calculated the probability of transmission for 500 points along each free energy barrier. We introduced a method for determining transition states by employing the transmission probability, Ptrans, and determined which conformations were transition state ensemble members (Ptrans ≈ 0.5). The transmission probability may be used to characterize the barrier in several ways. For example, we ran simulations at 82°C, determined the change in Ptrans with temperature for all 2,000 conformations, and quantified Hammond behavior directly using Ptrans correlation. Additionally, we propose that diffusion along Ptrans may provide the configurational diffusion rate at the top of the barrier. Specifically, given a transition state conformation x0 with estimated Ptrans = 0.5, we selected a large set of subsequent conformations from independent trajectories, each exactly a small time δt after x0 (250 ps). Calculating Ptrans for the new trial conformations, we generated the P(Ptrans|δt = 250 ps) distribution that reflected diffusion. This approach provides a novel perspective on the diffusive nature of a protein folding transition and provides a framework for a quantitative study of activated relaxation kinetics. PMID:16617068

  18. Latent membrane protein 1 of Epstein-Barr virus coordinately regulates proliferation with control of apoptosis.

    PubMed

    Dirmeier, Ulrike; Hoffmann, Reinhard; Kilger, Ellen; Schultheiss, Ute; Briseño, Cinthia; Gires, Olivier; Kieser, Arnd; Eick, Dirk; Sugden, Bill; Hammerschmidt, Wolfgang

    2005-03-01

    Latent membrane protein 1 (LMP1), an oncoprotein encoded by Epstein-Barr virus (EBV), is an integral membrane protein, which acts like a constitutively active receptor. LMP1 is critical for some facet of EBV's induction and maintenance of proliferation of infected B cells. It, in part, mimics signaling by the CD40 receptor and has been implicated in regulating proliferation, survival, or both properties of EBV-infected cells. We established a conditional LMP1 allele in the context of the intact EBV genome to define the immediate-early cellular target genes regulated by LMP1 in order to assess its contributions to infected human B cells. The functional analysis of this conditional system indicated that LMP1 specifically induces mitogenic B-cell activation through c-myc and Jun/AP1 family members and confirms its direct role in upregulating expression of multiple genes with opposing activities involved in cell survival. LMP1's signals were found to be essential for the G1/S transition in human B cells; cells lacking LMP1's signals are cell cycle arrested and survive quiescently. LMP1's activities are therefore not required to maintain survival in nonproliferating cells. LMP1 does induce both pro- and antiapoptotic genes whose balance seems to permit survival during LMP1's induction and maintenance of proliferation. PMID:15674340

  19. Translation initiation factor 5A in Picrorhiza is up-regulated during leaf senescence and in response to abscisic acid.

    PubMed

    Parkash, Jai; Vaidya, Tanmay; Kirti, Shruti; Dutt, Som

    2014-05-25

    Translation initiation, the first step of protein synthesis process is the principal regulatory step controlling translation and involves a pool of translation initiation factors. In plants, from recent studies it is becoming evident that these translation initiation factors impact various aspects of plant growth and development in addition to their role in protein synthesis. Eukaryotic translation initiation factor eIF5A is one such factor which functions in start site selection for the eIF2-GTP-tRNAi ternary complex within the ribosomal-bound preinitiation complex and also stabilizes the binding of GDP to eIF2. In the present study we have cloned and analysed a gene (eIF5a) encoding eIF5A from Picrorhiza (Picrorhiza kurrooa Royle ex Benth.) a medicinal plant of the western Himalayan region. The full length eIF5a cDNA consisted of 838 bp with an open reading frame of 480 bp, 88 bp 5' untranslated region and 270 bp 3' untranslated region. The deduced eIF5A protein contained 159 amino acids with a molecular weight of 17.359 kDa and an isoelectric point of 5.59. Secondary structure analysis revealed eIF5A having 24.53% α-helices, 8.81% β-turns, 23.27% extended strands and 43.40% random coils. pk-eIF5a transcript was found to be expressing during the active growth phase as well as during leaf senescence stage, however, highest expression was observed during leaf senescence stage. Further, its expression was up-regulated in response to exogenous application of abscisic acid. Both high intensity as well as low intensity light decreased the expression of pk-eIF5a. The findings suggest eIF5a to be an important candidate to develop genetic engineering based strategies for delaying leaf senescence. PMID:24656625

  20. Up-regulation of BMP2/4 signaling increases both osteoblast-specific marker expression and bone marrow adipogenesis in Gja1Jrt/+ stromal cell cultures

    PubMed Central

    Zappitelli, Tanya; Chen, Frieda; Aubin, Jane E.

    2015-01-01

    Gja1Jrt/+ mice carry a mutation in one allele of the gap junction protein α1 gene (Gja1), resulting in a G60S connexin 43 (Cx43) mutant protein that is dominant negative for Cx43 protein production of <50% of wild-type (WT) levels and significantly reduced gap junction formation and function in osteoblasts and other Cx43-expressing cells. Previously we reported that Gja1Jrt/+ mice exhibited early-onset osteopenia caused by activation of osteoclasts secondary to activation of osteoblast lineage cells, which expressed increased RANKL and produced an abnormal resorption-stimulating bone matrix high in BSP content. Gja1Jrt/+ mice also displayed early and progressive bone marrow atrophy, with a significant increase in bone marrow adiposity versus WT littermates but no increase in adipose tissues elsewhere in the body. BMP2/4 production and signaling were increased in Gja1Jrt/+ trabecular bone and osteogenic stromal cell cultures, which contributed to the up-regulated expression of osteoblast-specific markers (e.g., Bsp and Ocn) in Gja1Jrt/+ osteoblasts and increased Pparg2 expression in bone marrow–derived adipoprogenitors in vitro. The elevated levels of BMP2/4 signaling in G60S Cx43-containing cells resulted at least in part from elevated levels of cAMP. We conclude that up-regulation of BMP2/4 signaling in trabecular bone and/or stromal cells increases osteoblast-specific marker expression in hyperactive Gja1Jrt/+ osteoblasts and may also increase bone marrow adipogenesis by up-regulation of Pparg2 in the Cx43-deficient Gja1Jrt/+ mouse model. PMID:25568340

  1. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function

    PubMed Central

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  2. Ribosomal protein uS19 mutants reveal its role in coordinating ribosome structure and function.

    PubMed

    Bowen, Alicia M; Musalgaonkar, Sharmishtha; Moomau, Christine A; Gulay, Suna P; Mirvis, Mary; Dinman, Jonathan D

    2015-01-01

    Prior studies identified allosteric information pathways connecting functional centers in the large ribosomal subunit to the decoding center in the small subunit through the B1a and B1b/c intersubunit bridges in yeast. In prokaryotes a single SSU protein, uS13, partners with H38 (the A-site finger) and uL5 to form the B1a and B1b/c bridges respectively. In eukaryotes, the SSU component was split into 2 separate proteins during the course of evolution. One, also known as uS13, participates in B1b/c bridge with uL5 in eukaryotes. The other, called uS19 is the SSU partner in the B1a bridge with H38. Here, polyalanine mutants of uS19 involved in the uS19/uS13 and the uS19/H38 interfaces were used to elucidate the important amino acid residues involved in these intersubunit communication pathways. Two key clusters of amino acids were identified: one located at the junction between uS19 and uS13, and a second that appears to interact with the distal tip of H38. Biochemical analyses reveal that these mutations shift the ribosomal rotational equilibrium toward the unrotated state, increasing ribosomal affinity for tRNAs in the P-site and for ternary complex in the A-site, and inhibit binding of the translocase, eEF2. These defects in turn affect specific aspects of translational fidelity. These findings suggest that uS19 plays a critical role as a conduit of information exchange between the large and small ribosomal subunits directly through the B1a, and indirectly through the B1b/c bridges. PMID:26824029

  3. Chelidonic acid evokes antidepressant-like effect through the up-regulation of BDNF in forced swimming test.

    PubMed

    Jeong, Hyun-Ja; Yang, Shi-Young; Kim, Hee-Yun; Kim, Na-Rae; Jang, Jae-Bum; Kim, Hyung-Min

    2016-08-01

    Depression is usually accompanied by neuro-inflammatory reactions. Chelidonic acid, in particular, has shown anti-inflammatory effects. The objective of this study was to evaluate the anti-depressant effects of chelidonic acid and to discuss the potential mechanisms of a forced swimming test. Chelidonic acid was administered orally once a day for 14 days. On the 14th day, chelidonic acid resulted in a significant decrease in immobility time during the forced swimming test without alteration of locomotor activity, in an open field test. Chelidonic acid also increased the number of nissl bodies in the hippocampus. Brain-derived neurotrophic factor expression and extracellular signal-regulated protein kinase phosphorylation in the hippocampus were up-regulated by the administration of chelidonic acid. Chelidonic acid administration significantly increased the mRNA expression of hippocampal estrogen receptor-β. The levels of hippocampal interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were effectively attenuated by the administration of chelidonic acid. In addition, chelidonic acid significantly increased the levels of 5-hydroxytryptamine (serotonin), dopamine, and norepinephrine compared with those levels for the mice that were administered distilled water in the hippocampus. These results suggest that chelidonic acid might serve as a new therapeutic strategy for the regulation of depression associated with inflammation. PMID:27037280

  4. HBsAg blocks TYPE I IFN induced up-regulation of A3G through inhibition of STAT3.

    PubMed

    Xu, Fengchao; Song, Hongxiao; Li, Na; Tan, Guangyun

    2016-04-22

    Interferon (IFN) is a regularly utilized therapeutic for the treatment of chronic hepatitis B and appears to induce superior HBeAg seroconversion comparing nucleos/tide analogs. However, the mechanisms underlying IFN inhibition of HBV replication, as well as poor responses to IFN are unclear. Apobec3G has been reported to be involved in regulating HBV replication. In this study, we investigated Apobec3G expression and regulatory pathways during HBV infection. We show that over-expression of A3G leads to inhibition of HBV replication. We also show that IFN induces a significant increase in A3G protein expression, which is associated with STAT3 activation. We further show that A3G expression in HBV patients is lower compared to non-infected controls, possibly by HBsAg which inhibits IFN induced A3G up-regulation in a dose dependent manner. This process is likely mediated through inhibition of STAT3-Ser727 phosphorylation. The results presented in this study indicate that STAT3 plays an important role in IFN-induced A3G production, and HBsAg may correlated with poor response to IFN treatment. PMID:27003258

  5. Apocynin improving cardiac remodeling in chronic renal failure disease is associated with up-regulation of epoxyeicosatrienoic acids

    PubMed Central

    Chen, Jie; Cai, Qingqing; Wang, Jingfeng; Huang, Hui

    2015-01-01

    Cardiac remodeling is one of the most common cardiac abnormalities and associated with a high mortality in chronic renal failure (CRF) patients. Apocynin, a nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor, has been showed cardio-protective effects. However, whether apocynin can improve cardiac remodeling in CRF and what is the underlying mechanism are unclear. In the present study, we enrolled 94 participants. In addition, we used 5/6 nephrectomized rats to mimic cardiac remodeling in CRF. Serum levels of epoxyeicosatrienoic acids (EETs) and its mainly metabolic enzyme-soluble epoxide hydrolase (sEH) were measured. The results showed that the serum levels of EETs were significantly decreased in renocardiac syndrome participants (P < 0.05). In 5/6 nephrectomized CRF model, the ratio of left ventricular weight /body weight, left ventricular posterior wall thickness, and cardiac interstitial fibrosis were significantly increased while ejection fraction significantly decreased (P < 0.05). All these effects could partly be reversed by apocynin. Meanwhile, we found during the process of cardiac remodeling in CRF, apocynin significantly increased the reduced serum levels of EETs and decreased the mRNA and protein expressions of sEH in the heart (P < 0.05). Our findings indicated that the protective effect of apocynin on cardiac remodeling in CRF was associated with the up-regulation of EETs. EETs may be a new mediator for the injury of kidney-heart interactions. PMID:26322503

  6. Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity

    PubMed Central

    Serobyan, Vahan; Xiao, Hua; Namdeo, Suryesh; Rödelsperger, Christian; Sieriebriennikov, Bogdan; Witte, Hanh; Röseler, Waltraud; Sommer, Ralf J.

    2016-01-01

    Phenotypic plasticity has been suggested to act through developmental switches, but little is known about associated molecular mechanisms. In the nematode Pristionchus pacificus, the sulfatase eud-1 was identified as part of a developmental switch controlling mouth-form plasticity governing a predatory versus bacteriovorous mouth-form decision. Here we show that mutations in the conserved histone-acetyltransferase Ppa-lsy-12 and the methyl-binding-protein Ppa-mbd-2 mimic the eud-1 phenotype, resulting in the absence of one mouth-form. Mutations in both genes cause histone modification defects and reduced eud-1 expression. Surprisingly, Ppa-lsy-12 mutants also result in the down-regulation of an antisense-eud-1 RNA. eud-1 and antisense-eud-1 are co-expressed and further experiments suggest that antisense-eud-1 acts through eud-1 itself. Indeed, overexpression of the antisense-eud-1 RNA increases the eud-1-sensitive mouth-form and extends eud-1 expression. In contrast, this effect is absent in eud-1 mutants indicating that antisense-eud-1 positively regulates eud-1. Thus, chromatin remodelling and antisense-mediated up-regulation of eud-1 control feeding plasticity in Pristionchus. PMID:27487725

  7. Up-regulation of trypsin and mesenchymal MMP-8 during development of hyperoxic lung injury in the rat.

    PubMed

    Cederqvist, Katariina; Janer, Joakim; Tervahartiala, Taina; Sorsa, Timo; Haglund, Caj; Salmenkivi, Kaisa; Stenman, Ulf-Håkan; Andersson, Sture

    2006-10-01

    Acute lung injury is marked by damage to alveolar-capillary barrier. High pulmonary levels of matrix-degrading serine proteinase trypsin and matrix metalloproteinases (MMP)-2, -8, and -9 have been shown in preterm infants with respiratory distress syndrome (RDS). We studied expression of trypsin and MMP-2, -8, and -9 in rats exposed to >95% oxygen for 24, 48, or 60 h. As demonstrated by zymography and Western immunoblotting, levels of trypsin and MMP-2, -8, and -9 in bronchoalveolar lavage fluid (BALF) sharply increased after 48 h of hyperoxia relative to normoxia controls. This coincided with increase in alveolar-capillary permeability, as indicated by increased protein concentration in BALF. Both neutrophil-derived 80-kD and mesenchymal cell-derived 60-kD MMP-8 isoforms were detected in BALF. Of them, mesenchymal-type MMP-8 predominated. In immunohistochemistry, alveolar epithelium showed strong trypsin expression at 48 and 60 h of hyperoxia, whereas it was predominantly negative in controls. MMP-8 was mostly expressed in macrophages. Marked up-regulation of trypsin and MMP-8 early during hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury and may therefore be potential targets for therapy of lung injury. PMID:16940237

  8. Apocynin improving cardiac remodeling in chronic renal failure disease is associated with up-regulation of epoxyeicosatrienoic acids.

    PubMed

    Zhang, Kun; Liu, Yu; Liu, Xiaoqiang; Chen, Jie; Cai, Qingqing; Wang, Jingfeng; Huang, Hui

    2015-09-22

    Cardiac remodeling is one of the most common cardiac abnormalities and associated with a high mortality in chronic renal failure (CRF) patients. Apocynin, a nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase inhibitor, has been showed cardio-protective effects. However, whether apocynin can improve cardiac remodeling in CRF and what is the underlying mechanism are unclear. In the present study, we enrolled 94 participants. In addition, we used 5/6 nephrectomized rats to mimic cardiac remodeling in CRF. Serum levels of epoxyeicosatrienoic acids (EETs) and its mainly metabolic enzyme-soluble epoxide hydrolase (sEH) were measured. The results showed that the serum levels of EETs were significantly decreased in renocardiac syndrome participants (P < 0.05). In 5/6 nephrectomized CRF model, the ratio of left ventricular weight / body weight, left ventricular posterior wall thickness, and cardiac interstitial fibrosis were significantly increased while ejection fraction significantly decreased (P < 0.05). All these effects could partly be reversed by apocynin. Meanwhile, we found during the process of cardiac remodeling in CRF, apocynin significantly increased the reduced serum levels of EETs and decreased the mRNA and protein expressions of sEH in the heart (P < 0.05). Our findings indicated that the protective effect of apocynin on cardiac remodeling in CRF was associated with the up-regulation of EETs. EETs may be a new mediator for the injury of kidney-heart interactions. PMID:26322503

  9. Hemokinin-1(4-11)-Induced Analgesia Selectively Up-Regulates δ-Opioid Receptor Expression in Mice

    PubMed Central

    Fu, Cai-Yun; Xia, Rui-Long; Zhang, Teng-Fei; Lu, Yan; Zhang, Shi-Fu; Yu, Zhi-Qiang; Jin, Tao; Mou, Xiao-Zhou

    2014-01-01

    Our previous studies have shown that an active fragment of human tachykinins (hHK-1(4-11)) produced an opioid-independent analgesia after intracerebroventricular (i.c.v.) injection in mice, which has been markedly enhanced by a δ OR antagonist, naltrindole hydrochloride (NTI). In this study, we have further characterized the in vivo analgesia after i.c.v. injection of hHK-1(4-11) in mouse model. Our qRT-PCR results showed that the mRNA levels of several ligands and receptors (e.g. PPT-A, PPT-C, KOR, PDYN and PENK) have not changed significantly. Furthermore, neither transcription nor expression of NK1 receptor, MOR and POMC have changed noticeably. In contrast, both mRNA and protein levels of DOR have been up-regulated significantly, indicating that the enhanced expression of δ opioid receptor negatively modulates the analgesia induced by i.c.v. injection of hHK-1(4-11). Additionally, the combinatorial data from our previous and present experiments strongly suggest that the discriminable distribution sites in the central nervous system between hHK-1(4-11) and r/mHK-1 may be attributed to their discriminable analgesic effects. Altogether, our findings will not only contribute to the understanding of the complicated mechanisms regarding the nociceptive modulation of hemokinin-1 as well as its active fragments at supraspinal level, but may also lead to novel pharmacological interventions. PMID:24587368

  10. Chromatin remodelling and antisense-mediated up-regulation of the developmental switch gene eud-1 control predatory feeding plasticity.

    PubMed

    Serobyan, Vahan; Xiao, Hua; Namdeo, Suryesh; Rödelsperger, Christian; Sieriebriennikov, Bogdan; Witte, Hanh; Röseler, Waltraud; Sommer, Ralf J

    2016-01-01

    Phenotypic plasticity has been suggested to act through developmental switches, but little is known about associated molecular mechanisms. In the nematode Pristionchus pacificus, the sulfatase eud-1 was identified as part of a developmental switch controlling mouth-form plasticity governing a predatory versus bacteriovorous mouth-form decision. Here we show that mutations in the conserved histone-acetyltransferase Ppa-lsy-12 and the methyl-binding-protein Ppa-mbd-2 mimic the eud-1 phenotype, resulting in the absence of one mouth-form. Mutations in both genes cause histone modification defects and reduced eud-1 expression. Surprisingly, Ppa-lsy-12 mutants also result in the down-regulation of an antisense-eud-1 RNA. eud-1 and antisense-eud-1 are co-expressed and further experiments suggest that antisense-eud-1 acts through eud-1 itself. Indeed, overexpression of the antisense-eud-1 RNA increases the eud-1-sensitive mouth-form and extends eud-1 expression. In contrast, this effect is absent in eud-1 mutants indicating that antisense-eud-1 positively regulates eud-1. Thus, chromatin remodelling and antisense-mediated up-regulation of eud-1 control feeding plasticity in Pristionchus. PMID:27487725

  11. Hepatic and Nephric NRF2 Pathway Up-Regulation, an Early Antioxidant Response, in Acute Arsenic-Exposed Mice

    PubMed Central

    Li, Jinlong; Duan, Xiaoxu; Dong, Dandan; Zhang, Yang; Li, Wei; Zhao, Lu; Nie, Huifang; Sun, Guifan; Li, Bing

    2015-01-01

    Inorganic arsenic (iAs), a proven human carcinogen, damages biological systems through multiple mechanisms, one of them being reactive oxygen species (ROS) production. NRF2 is a redox-sensitive transcription factor that positively regulates the genes of encoding antioxidant and detoxification enzymes to neutralize ROS. Although NRF2 pathway activation by iAs has been reported in various cell types, however, the experimental data in vivo are very limited and not fully elucidated in humans. The present investigation aimed to explore the hepatic and nephric NRF2 pathway upregulation in acute arsenic-exposed mice in vivo. Our results showed 10 mg/kg NaAsO2 elevated the NRF2 protein and increased the transcription of Nrf2 mRNA, as well as up-regulated NRF2 downstream targets HO-1, GST and GCLC time- and dose-dependently both in the liver and kidney. Acute NaAsO2 exposure also resulted in obvious imbalance of oxidative redox status represented by the increase of GSH and MDA, and the decrease of T-AOC. The present investigation reveals that hepatic and nephric NRF2 pathway expression is an early antioxidant defensive response upon iAs exposure. A better knowledge about the NRF2 pathway involvment in the cellular response against arsenic could help improve the strategies for reducing the cellular toxicity related to this metalloid. PMID:26473898

  12. Growth differentiation factor 8 suppresses cell proliferation by up-regulating CTGF expression in human granulosa cells.

    PubMed

    Chang, Hsun-Ming; Pan, Hui-Hui; Cheng, Jung-Chien; Zhu, Yi-Min; Leung, Peter C K

    2016-02-15

    Connective tissue growth factor (CTGF) is a matricellular protein that plays a critical role in the development of ovarian follicles. Growth differentiation factor 8 (GDF8) is mainly, but not exclusively, expressed in the mammalian musculoskeletal system and is a potent negative regulator of skeletal muscle growth. The aim of this study was to investigate the effects of GDF8 and CTGF on the regulation of cell proliferation in human granulosa cells and to examine its underlying molecular determinants. Using dual inhibition approaches (inhibitors and small interfering RNAs), we have demonstrated that GDF8 induces the up-regulation of CTGF expression through the activin receptor-like kinase (ALK)4/5-mediated SMAD2/3-dependent signaling pathways. In addition, the increase in CTGF expression contributes to the GDF8-induced suppressive effect on granulosa cell proliferation. Our findings suggest that GDF8 and CTGF may play critical roles in the regulation of proliferative events in human granulosa cells. PMID:26577677

  13. Maggot debridement therapy promotes diabetic foot wound healing by up-regulating endothelial cell activity.

    PubMed

    Sun, Xinjuan; Chen, Jin'an; Zhang, Jie; Wang, Wei; Sun, Jinshan; Wang, Aiping

    2016-03-01

    To determine the role of maggot debridement therapy (MDT) on diabetic foot wound healing, we compared growth related factors in wounds before and after treatment. Furthermore, we utilized human umbilical vein endothelial cells (HUVECs) to explore responses to maggot excretions/secretions on markers of angiogenesis and proliferation. The results showed that there was neo-granulation and angiogenesis in diabetic foot wounds after MDT. Moreover, significant elevation in CD34 and CD68 levels was also observed in treated wounds. In vitro, ES increased HUVEC proliferation, improved tube formation, and increased expression of vascular endothelial growth factor receptor 2 in a dose dependent manner. These results demonstrate that MDT and maggot ES can promote diabetic foot wound healing by up-regulating endothelial cell activity. PMID:26782021

  14. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. )

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  15. Physiological effects of major up-regulated Alnus glutinosa peptides on Frankia sp. ACN14a.

    PubMed

    Carro, Lorena; Pujic, Petar; Alloisio, Nicole; Fournier, Pascale; Boubakri, Hasna; Poly, Franck; Rey, Marjolaine; Heddi, Abdelaziz; Normand, Philippe

    2016-07-01

    Alnus glutinosa has been shown previously to synthesize, in response to nodulation by Frankia sp. ACN14a, an array of peptides called Alnus symbiotic up-regulated peptides (ASUPs). In a previous study one peptide (Ag5) was shown to bind to Frankia nitrogen-fixing vesicles and to modify their porosity. Here we analyse four other ASUPs, alongside Ag5, to determine whether they have different physiological effects on in vitro grown Frankia sp. ACN14a. The five studied peptides were shown to have different effects on nitrogen fixation, respiration, growth, the release of ions and amino acids, as well as on cell clumping and cell lysis. The mRNA abundance for all five peptides was quantified in symbiotic nodules and one (Ag11) was found to be more abundant in the meristem part of the nodule. These findings point to some peptides having complementary effects on Frankia cells. PMID:27082768

  16. Water deprivation up-regulates urine osmolality and renal aquaporin 2 in Mongolian gerbils (Meriones unguiculatus).

    PubMed

    Xu, Meng-Meng; Wang, De-Hua

    2016-04-01

    To better understand how desert rodents adapt to water scarcity, we examined urine osmolality, renal distribution and expression of aquaporins (AQPs) in Mongolian gerbils (Meriones unguiculatus) during 7 days of water deprivation (WD). Urine osmolality of the gerbils during WD averaged 7503 mOsm kg(-1). Renal distributions of AQP1, AQP2, and AQP3 were similar to that described in other rodents. After the 7 day WD, renal AQP2 was up-regulated, while resting metabolic rate and total evaporative water loss decreased by 43% and 36%, respectively. Our data demonstrated that Mongolian gerbils showed high urine concentration, renal AQPs expression and body water conservation to cope with limited water availability, which may be critical for their survival during dry seasons in cold deserts. PMID:26806059

  17. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis

    PubMed Central

    Einarsson, Elin; Troell, Karin; Hoeppner, Marc P.; Grabherr, Manfred; Ribacke, Ulf; Svärd, Staffan G.

    2016-01-01

    Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes. PMID:27015092

  18. Coordinated Changes in Gene Expression Throughout Encystation of Giardia intestinalis.

    PubMed

    Einarsson, Elin; Troell, Karin; Hoeppner, Marc P; Grabherr, Manfred; Ribacke, Ulf; Svärd, Staffan G

    2016-03-01

    Differentiation into infectious cysts through the process of encystation is crucial for transmission and survival of the intestinal protozoan parasite Giardia intestinalis. Hitherto the majority of studies have focused on the early events, leaving late encystation poorly defined. In order to further study encystation, focusing on the later events, we developed a new encystation protocol that generates a higher yield of mature cysts compared to standard methods. Transcriptome changes during the entire differentiation from trophozoites to cysts were thereafter studied using RNA sequencing (RNA-seq). A high level of periodicity was observed for up- and down-regulated genes, both at the level of the entire transcriptome and putative regulators. This suggests the trajectory of differentiation to be coordinated through developmentally linked gene regulatory activities. Our study identifies a core of 13 genes that are consistently up-regulated during initial encystation. Of these, two constitute previously uncharacterized proteins that we were able to localize to a new type of encystation-specific vesicles. Interestingly, the largest transcriptional changes were seen in the late phase of encystation with the majority of the highly up-regulated genes encoding hypothetical proteins. Several of these were epitope-tagged and localized to further characterize these previously unknown genetic components of encystation and possibly excystation. Finally, we also detected a switch of variant specific surface proteins (VSPs) in the late phase of encystation. This occurred at the same time as nuclear division and DNA replication, suggesting a potential link between the processes. PMID:27015092

  19. Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells

    PubMed Central

    Bansal, Nitu; Mishra, Prasun J.; Stein, Mark; DiPaola, Robert S.; Bertino, Joseph R.

    2015-01-01

    Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl. PMID:26036314

  20. Up-regulation of stromal versican expression in advanced stage serous ovarian cancer

    PubMed Central

    Ghosh, Sue; Albitar, Lina; LeBaron, Richard; Welch, William R.; Samimi, Goli; Birrer, Michael J.; Berkowitz, Ross S.; Mok, Samuel C.

    2010-01-01

    Objective The purpose of this study is to examine the role of versican (VCAN) in advanced stage serous ovarian cancer by investigating its expression, its function, and its correlation with clinical outcomes. Methods Microarray analysis was performed on RNA isolated from tumor and stromal components of advanced stage serous ovarian cancer and normal ovarian epithelial tissue to identify genes up-regulated in ovarian tumor stroma. Validation studies using immunohistochemistry and quantitative real-time PCR (Q-RT-PCR) was performed on one of the up-regulated genes, VCAN. Immunolocalization of VCAN (n=111) and CD31 (n= 56) were done on serous ovarian tumors. CD31 staining was performed to examine microvessel density (MVD). Q-RT-PCR was performed on 65 samples to evaluate the differential expression of VCAN isoforms. Cell proliferation and invasion assays were performed to examine how V1-treated ovarian cancer cell lines and an endothelial cell line would differ from controls. Univariate survival analyses were done with VCAN expression. Correlation analysis was done with CD31, platinum resistance, and clinical data. Results Validation studies using Q-RT-PCR and immunohistochemistry showed significantly higher VCAN V1 isoform expression in ovarian cancer stroma compared with normal ovarian stroma and ovarian cancer cells. Correlation studies showed stromal VCAN expression was associated with poorer overall and progression free survival, platinum resistance, and increased MVD. VCAN-treated ovarian cancer and endothelial cells showed increased invasion potential. Conclusions VCAN overexpression is associated with increased MVD and invasion potential, which may lead to poorer overall and progression free survival and platinum resistance. PMID:20619446

  1. A novel action mechanism for MPT0G013, a derivative of arylsulfonamide, inhibits tumor angiogenesis through up-regulation of TIMP3 expression

    PubMed Central

    Wang, Chih-Ya; Liou, Jing-Ping; Tsai, An-Chi; Lai, Mei-Jung; Liu, Yi-Min; Lee, Hsueh-Yun; Wang, Jing-Chi; Pan, Shiow-Lin; Teng, Che-Ming

    2014-01-01

    Tissue inhibitors of metalloproteinases 3 (TIMP3) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), acting as potent antiangiogenic proteins. In this study, we demonstrated that the arylsulfonamide derivative MPT0G013 has potent antiangiogenic activities in vitro and in vivo via inducing TIMP3 expression. Treatments with MPT0G013 significantly inhibited endothelial cell functions, such as cell proliferation, migration, and tube formation, as well as induced p21 and cell cycle arrest at the G0/G1 phase. Subsequent microarray analysis showed significant induction of TIMP3 gene expression by MPT0G013, and siRNA-mediated blockage of TIMP3 up-regulation abrogated the antiangiogenic activities of MPT0G013 and prevented inhibition of p-AKT and p-ERK proteins. Importantly, MPT0G013 exhibited antiangiogenic activities in in vivo Matrigel plug assays, inhibited tumor growth and up-regulated TIMP3 and p21 proteins in HCT116 mouse xenograft models. These data suggest potential therapeutic application of MPT0G013 for angiogenesis-related diseases such as cancer. PMID:25226613

  2. High-fat diet before and during pregnancy causes marked up-regulation of placental nutrient transport and fetal overgrowth in C57/BL6 mice.

    PubMed

    Jones, Helen N; Woollett, Laura A; Barbour, Nicolette; Prasad, Puttur D; Powell, Theresa L; Jansson, Thomas

    2009-01-01

    Maternal overweight and obesity in pregnancy often result in fetal overgrowth, which increases the risk for the baby to develop metabolic syndrome later in life. However, the mechanisms underlying fetal overgrowth are not established. We developed a mouse model and hypothesized that a maternal high-fat (HF) diet causes up-regulation of placental nutrient transport, resulting in fetal overgrowth. C57BL/6J female mice were fed a control (11% energy from fat) or HF (32% energy from fat) diet for 8 wk before mating and throughout gestation and were studied at embryonic day 18.5. The HF diet increased maternal adiposity, as assessed by fat pad weight, and circulating maternal leptin, decreased serum adiponectin concentrations, and caused a marked increase in fetal growth (+43%). The HF diet also increased transplacental transport of glucose (5-fold) and neutral amino acids (10-fold) in vivo. In microvillous plasma membranes (MVMs) isolated from placentas of HF-fed animals, protein expression of glucose transporter 1 (GLUT1) was increased 5-fold, and protein expression of sodium-coupled neutral amino acid transporter (SNAT) 2 was elevated 9-fold. In contrast, MVM protein expression of GLUT 3 or SNAT4 was unaltered. These data suggest that up-regulation of specific placental nutrient transporter isoforms constitute a mechanism linking maternal high-fat diet and obesity to fetal overgrowth. PMID:18827021

  3. Insecticide-Mediated Up-Regulation of Cytochrome P450 Genes in the Red Flour Beetle (Tribolium castaneum)

    PubMed Central

    Liang, Xiao; Xiao, Da; He, Yanping; Yao, Jianxiu; Zhu, Guonian; Zhu, Kun Yan

    2015-01-01

    Some cytochrome P450 (CYP) genes are known for their rapid up-regulation in response to insecticide exposures in insects. To date, however, limited information is available with respect to the relationships among the insecticide type, insecticide concentration, exposure duration and the up-regulated CYP genes. In this study, we examined the transcriptional response of eight selected CYP genes, including CYP4G7, CYP4Q4, CYP4BR3, CYP12H1, CYP6BK11, CYP9D4, CYP9Z5 and CYP345A1, to each of four insecticides in the red flour beetle, Tribolium castaneum. Reverse transcription quantitative PCR (RT-qPCR) revealed that CYP4G7 and CYP345A1 can be significantly up-regulated by cypermethrin (1.97- and 2.06-fold, respectively), permethrin (2.00- and 2.03-fold) and lambda-cyhalothrin (1.73- and 1.81-fold), whereas CYP4BR3 and CYP345A1 can be significantly up-regulated by imidacloprid (1.99- and 1.83-fold) when 20-day larvae were exposed to each of these insecticides at the concentration of LC20 for 24 h. Our studies also showed that similar levels of up-regulation can be achieved for CYP4G7, CYP4BR3 and CYP345A1 by cypermethrin, permethrin, lambda-cyhalothrin or imidacloprid with approximately one fourth of LC20 in 6 h. Our study demonstrated that up-regulation of these CYP genes was rapid and only required low concentrations of insecticides, and the up-regulation not only depended on the CYP genes but also the type of insecticides. Our results along with those from previous studies also indicated that there were no specific patterns for predicting the up-regulation of specific CYP gene families based on the insecticide classification. PMID:25607733

  4. Cyclic adenosine 5'-monophosphate and calcium induce CD152 (CTLA-4) up-regulation in resting CD4+ T lymphocytes.

    PubMed

    Vendetti, Silvia; Riccomi, Antonella; Sacchi, Alessandra; Gatta, Lucia; Pioli, Claudio; De Magistris, Maria Teresa

    2002-12-01

    The CTLA-4 (CD152) molecule is up-regulated upon T cell activation and proliferation, and plays a critical role in the inhibition of immune responses. We show in this study that cAMP induces up-regulation of CD152 in human CD4(+) T lymphocytes. This effect occurs in the absence of the up-regulation of CD69 and CD25 activation markers and T cell proliferation. In addition, we found that the Ca(2+) ionophore ionomycin also up-regulates CD152, and that the combination of a cAMP analog or cAMP inducers with ionomycin further enhances the expression of CD152 in resting CD4(+) T lymphocytes. However, cyclosporin A, which inhibits Ca(2+)/calcineurin signaling pathway, fully prevented the ionomycin- but not the cAMP-induced up-regulation of CD152. The effects of cAMP and ionomycin involve increase of both CD152 mRNA transcripts, coding for the membrane and the soluble forms of CD152. Furthermore, we show that CD152 molecules are translocated to the membrane and are functional, as their engagement by specific mAbs prevented NF-kappaB activation by anti-CD3/CD28 stimulation. These findings demonstrate that at least two novel signal pathways regulate CTLA-4 gene expression and CD152 molecule up-regulation in human CD4(+) T lymphocytes, in the absence of full T cell activation. PMID:12444128

  5. Integrin αDβ2, an adhesion receptor up-regulated on macrophage foam cells, exhibits multiligand-binding properties

    PubMed Central

    Yakubenko, Valentin P.; Yadav, Satya P.; Ugarova, Tatiana P.

    2006-01-01

    Integrin αDβ2, the most recently discovered member of the β2 subfamily of integrin adhesion receptors, is up-regulated on macrophage foam cells. Although other members of the subfamily have been subjects of extensive research, the recognition specificity and the molecular basis for αDβ2 ligand binding remain unknown. Based on the high extent of structural homology between αDβ2 and the major myeloid-cell-specific integrin αMβ2 (Mac-1), noted for its capacity to bind multiple ligands, we considered that the 2 integrins have similar recognition specificity. In this study, using recombinant and natural αDβ2-expressing cells, we demonstrate that αDβ2 supports adhesion and migration to many extracellular matrix proteins in a fashion similar to αMβ2. Consistent with these data, the recombinant αDI-domain of the receptor bound selected ligands. The binding was activation-dependent because the αDI-domain with its C-terminal α7 helix truncated, but not the form with the C-terminal part extended, bound ligands. When the αDI-domain segment Lys244-Lys260 (highly homologous to its αMI-domain counterpart Lys245-Arg261 responsible for αMβ2 multiligand-binding properties) was inserted into the mono-specific αLI-domain, the chimeric protein bound many ligands with affinities similar to those of wild-type αDI-domain. These results establish integrin αDβ2 as a multiligand receptor and indicate that the mechanism whereby αDβ2 exhibits broad ligand specificity resembles that used by αMβ2, the most promiscuous member of the integrin family. PMID:16239428

  6. Electrical stimulation induces calcium-dependent up-regulation of neuregulin-1β in dystrophic skeletal muscle cell lines.

    PubMed

    Juretić, Nevenka; Jorquera, Gonzalo; Caviedes, Pablo; Jaimovich, Enrique; Riveros, Nora

    2012-01-01

    Duchenne muscular dystrophy (DMD) is a neuromuscular disease originated by reduced or no expression of dystrophin, a cytoskeletal protein that provides structural integrity to muscle fibres. A promising pharmacological treatment for DMD aims to increase the level of a structural dystrophin homolog called utrophin. Neuregulin-1 (NRG-1), a growth factor that potentiates myogenesis, induces utrophin expression in skeletal muscle cells. Microarray analysis of total gene expression allowed us to determine that neuregulin-1β (NRG-1β) is one of 150 differentially expressed genes in electrically stimulated (400 pulses, 1 ms, 45 Hz) dystrophic human skeletal muscle cells (RCDMD). We investigated the effect of depolarization, and the involvement of intracellular Ca(2+) and PKC isoforms on NRG-1β expression in dystrophic myotubes. Electrical stimulation of RCDMD increased NRG-1β mRNA and protein levels, and mRNA enhancement was abolished by actinomycin D. NRG-1β transcription was inhibited by BAPTA-AM, an intracellular Ca(2+) chelator, and by inhibitors of IP(3)-dependent slow Ca(2+) transients, like 2-APB, Ly 294002 and Xestospongin B. Ryanodine, a fast Ca(2+) signal inhibitor, had no effect on electrical stimulation-induced expression. BIM VI (general inhibitor of PKC isoforms) and Gö 6976 (specific inhibitor of Ca(2+)-dependent PKC isoforms) abolished NRG-1β mRNA induction. Our results suggest that depolarization induced slow Ca(2+) signals stimulate NRG-1β transcription in RCDMD cells, and that Ca(2+)-dependent PKC isoforms are involved in this process. Based on utrophin's ability to partially compensate dystrophin disfunction, knowledge on the mechanism involved on NRG-1 up-regulation could be important for new therapeutic strategies design. PMID:22613991

  7. Isoflurane Preconditioning Induces Neuroprotection by Up-Regulation of TREK1 in a Rat Model of Spinal Cord Ischemic Injury

    PubMed Central

    Wang, Kun; Kong, Xiangang

    2016-01-01

    This study aimed to explore the neuroprotection and mechanism of isoflurane on rats with spinal cord ischemic injury. Total 40 adult male Sprague-Dawley rats were divided into the four groups (n=10). Group A was sham-operation group; group B was ischemia group; group C was isoflurane preconditioning group; group D was isoflurane preconditioning followed by ischemia treatment group. Then the expressions of TWIK-related K+ channel 1 (TREK1) in the four groups were detected by immunofluorescent assay, real time-polymerase chain reactions (RT-PCR) and western blot. The primary neurons of rats were isolated and cultured under normal and hypoxic conditions. Besides, the neurons under two conditions were transfected with green fluorescent protein (GFP)-TREK1 and lentivirual to overexpress and silence TREK1. Additionally, the neurons were treated with isoflurane or not. Then caspase-3 activity and cell cycle of neurons under normal and hypoxic conditions were detected. Furthermore, nicotinamide adenine dinucleotide hydrate (NADH) was detected using NAD+/NADH quantification colorimetric kit. Results showed that the mRNA and protein expressions of TREK1 increased significantly in group C and D. In neurons, when TREK1 silenced, isoflurane treatment improved the caspase-3 activity. In hypoxic condition, the caspase-3 activity and sub-G1 cell percentage significantly increased, however, when TREK1 overexpressed the caspase-3 activity and sub-G1 cell percentage decreased significantly. Furthermore, both isoflurane treatment and overexpression of TREK1 significantly decreased NADH. In conclusion, isoflurane-induced neuroprotection in spinal cord ischemic injury may be associated with the up-regulation of TREK1. PMID:27469140

  8. Recurrent MALAT1-GLI1 oncogenic fusion and GLI1 up-regulation define a subset of plexiform fibromyxoma.

    PubMed

    Spans, Lien; Fletcher, Christopher Dm; Antonescu, Cristina R; Rouquette, Alexandre; Coindre, Jean-Michel; Sciot, Raf; Debiec-Rychter, Maria

    2016-07-01

    Plexiform fibromyxomas are rare neoplasms, being officially recognized as a distinct entity among benign mesenchymal gastric tumours in the 2010 WHO Classification of Tumours of the Digestive System. Characteristically, these tumours have a multinodular/plexiform growth pattern, and histologically contain variably cellular areas of bland myofibroblastic-type spindle cells embedded in an abundant myxoid matrix, rich in capillary-type vessels. As yet, the molecular and/or genetic features of these tumours are unknown. Here we describe a recurrent translocation, t(11;12)(q11;q13), involving the long non-coding gene metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and the gene glioma-associated oncogene homologue 1 (GLI1) in a subgroup of these tumours. The presence of the fusion transcript in our index case was confirmed using polymerase chain reaction (PCR) on genomic DNA, followed by Sanger sequencing. We showed that the truncated GLI1 protein is overexpressed and retains its capacity to transcriptionally activate its target genes. A specific FISH assay was developed to detect the novel MALAT1-GLI1 translocation in formalin-fixed, paraffin-embedded (FFPE) material. This resulted in the identification of two additional cases with this fusion and two cases with polysomy of the GLI1 gene. Finally, immunohistochemistry revealed that the GLI1 protein is exclusively overexpressed in those cases that harbour GLI1/12q13 genomic alterations. In conclusion, overexpression of GLI1 through a recurrent MALAT1-GLI1 translocation or GLI1 up-regulation delineates a pathogenically distinct subgroup of plexiform fibromyxomas with activation of the Sonic Hedgehog signalling pathway. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:27101025

  9. Substance P primes lipoteichoic acid- and Pam3CysSerLys4-mediated activation of human mast cells by up-regulating Toll-like receptor 2.

    PubMed

    Tancowny, Brian P; Karpov, Victor; Schleimer, Robert P; Kulka, Marianna

    2010-10-01

    Substance P (SP) is a neuropeptide with neuroimmunoregulatory activity that may play a role in susceptibility to infection. Human mast cells, which are important in innate immune responses, were analysed for their responses to pathogen-associated molecules via Toll-like receptors (TLRs) in the presence of SP. Human cultured mast cells (LAD2) were activated by SP and TLR ligands including lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4) and lipoteichoic acid (LTA), and mast cell leukotriene and chemokine production was assessed by enzyme-linked immunosorbent assay (ELISA) and gene expression by quantitative PCR (qPCR). Mast cell degranulation was determined using a β-hexosaminidase (β-hex) assay. SP treatment of LAD2 up-regulated mRNA for TLR2, TLR4, TLR8 and TLR9 while anti-immunoglobulin E (IgE) stimulation up-regulated expression of TLR4 only. Flow cytometry and western blot confirmed up-regulation of TLR2 and TLR8. Pretreatment of LAD2 with SP followed by stimulation with Pam3CSK4 or LTA increased production of leukotriene C4 (LTC(4) ) and interleukin (IL)-8 compared with treatment with Pam3CSK4 or LTA alone (>2-fold; P<0·01). SP alone activated 5-lipoxygenase (5-LO) nuclear translocation but also augmented Pam3CSK4 and LTA-mediated 5-LO translocation. Pam3CSK4, LPS and LTA did not induce LAD2 degranulation. SP primed LTA and Pam3CSK4-mediated activation of JNK, p38 and extracellular-signal-regulated kinase (ERK) and activated the nuclear translocation of c-Jun, nuclear factor (NF)-κB, activating transcription factor 2 (ATF-2) and cyclic-AMP-responsive element binding protein (CREB) transcription factors. Pretreatment with SP followed by LTA stimulation synergistically induced production of chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8, chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1), tumour necrosis factor (TNF) and IL-6 protein. SP primes TLR2-mediated activation of human mast cells by up-regulating TLR expression and

  10. Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.

    PubMed

    Santra, Manoranjan; Chopp, Michael; Santra, Sutapa; Nallani, Ankita; Vyas, Shivam; Zhang, Zheng Gang; Morris, Daniel C

    2016-01-01

    Thymosin beta 4 (Tβ4), a secreted 43 amino acid peptide, promotes oligodendrogenesis, and improves neurological outcome in rat models of neurologic injury. We demonstrated that exogenous Tβ4 treatment up-regulated the expression of the miR-200a in vitro in rat brain progenitor cells and in vivo in the peri-infarct area of rats subjected to middle cerebral artery occlusion (MCAO). The up-regulation of miR-200a down-regulated the expression of the following targets in vitro and in vivo models: (i) growth factor receptor-bound protein 2 (Grb2), an adaptor protein involved in epidermal growth factor receptor (EGFR)/Grb2/Ras/MEK/ERK1/c-Jun signaling pathway, which negatively regulates the expression of myelin basic protein (MBP), a marker of mature oligodendrocyte; (ii) ERRFI-1/Mig-6, an endogenous potent kinase inhibitor of EGFR, which resulted in activation/phosphorylation of EGFR; (iii) friend of GATA 2, and phosphatase and tensin homolog deleted in chromosome 10 (PTEN), which are potent inhibitors of the phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway, and resulted in marked activation of AKT; and (iv) transcription factor, p53, which induces pro-apoptotic genes, and possibly reduced apoptosis of the progenitor cells subjected to oxygen glucose deprivation (OGD). Anti-miR-200a transfection reversed all the effects of Tβ4 treatment in vitro. Thus, Tβ4 up-regulated MBP synthesis, and inhibited OGD-induced apoptosis in a novel miR-200a dependent EGFR signaling pathway. Our findings of miR-200a-mediated protection of progenitor cells may provide a new therapeutic importance for the treatment of neurologic injury. Tβ4-induced micro-RNA-200a (miR-200a) regulates EGFR signaling pathways for MBP synthesis and apoptosis: up-regulation of miR-200a after Tβ4 treatment, increases MBP synthesis after targeting Grb2 and thereby inactivating c-Jun from inhibition of MBP synthesis; and also inhibits OGD-mediated apoptosis after targeting EGFR inhibitor (Mig-6), PI3

  11. The mitochondrial uncoupler DNP triggers brain cell mTOR signaling network reprogramming and CREB pathway up-regulation.

    PubMed

    Liu, Dong; Zhang, Yongqing; Gharavi, Robert; Park, Hee Ra; Lee, Jaewon; Siddiqui, Sana; Telljohann, Richard; Nassar, Matthew R; Cutler, Roy G; Becker, Kevin G; Mattson, Mark P

    2015-08-01

    Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4-dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca(2+) levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP-treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin--PI3K - MAPK pathways, and up-regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up-regulated in response to DNP. CREB (cAMP-response element-binding protein) signaling, Arc and brain-derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up-regulated in response to DNP, and DNP-treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP-induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up-regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity. Physiological bioenergetic challenges such as exercise and fasting can enhance neuroplasticity and protect neurons against injury and neurodegeneration. Here, we show that the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) elicits adaptive signaling responses in the

  12. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

    SciTech Connect

    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A.

    2013-08-02

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

  13. Nitric Oxide Up-Regulates RUNX2 in LNCaP Prostate Tumours: Implications for Tumour Growth In Vitro and In Vivo.

    PubMed

    Nesbitt, Heather; Browne, Gillian; O'Donovan, Katie M; Byrne, Niall M; Worthington, Jenny; McKeown, Stephanie R; McKenna, Declan J

    2016-02-01

    Aberrant expression of the transcription factor RUNX2 in prostate cancer has a number of important consequences including increased resistance to apoptosis, invasion and metastasis to bone. We previously demonstrated that hypoxia up-regulated RUNX2 in tumour cells, which in turn up-regulated the anti-apoptotic factor Bcl-2. Here, we investigate the impact of nitric oxide (NO) on RUNX2 and Bcl-2 expression in prostate cancer and further, how RUNX2 over-expression can impact tumour growth, angiogenesis and oxygenation in vivo. The effect of NO levels on RUNX2 and thus Bcl-2 expression was examined in prostate cancer cells in vitro using methods including gene and protein expression analyses, nitrite quantitation, protein-DNA interaction assays (ChIP) and viability assays (XTT). The effect of RUNX2 over-expression on tumour physiology (growth, oxygenation and angiogenesis) was also assessed in vivo using LNCaP xenografts. A low (but not high) concentration of NO (10 μM) induced expression of RUNX2 and Bcl-2, conferring resistance to docetaxel. These effects were induced via the ERK and PI3K pathways and were dependent on intact AP-1 binding sites in the RUNX2 promoter. RUNX2 over-expression in LNCaP tumours in vivo decreased the time to tumour presentation and increased tumour growth. Moreover, these tumours exhibited improved tumour angiogenesis and oxygenation. Low levels of NO increase expression of RUNX2 and Bcl-2 in LNCaP prostate tumour cells, and in vivo up-regulation of RUNX2 created tumours with a more malignant phenotype. Collectively, our data reveals the importance of NO-regulation of key factors in prostate cancer disease progression. PMID:26189652

  14. One Octarepeate Expansion to the Human Prion Protein Alters Both the Zn2plus and Cu2plus Coordination Environments within the Octarepeate Domain

    SciTech Connect

    J Shearer; K Rosenkoetter; P Callan; C Pham

    2011-12-31

    The influence of a single octarepeat expansion on the Cu{sup II} and Zn{sup II} coordination environments within the octarepeat domain of the human prion protein is examined. Using X-ray absorption spectroscopy and diethyl pyrocarbonate labeling studies, we find that at low copper concentrations the 'normal' octarepeat domain (four PHGGGWGQ repeats) coordinates Zn{sup II} in an (N/O){sub 6} coordination environment with two histidine residues and Cu{sup II} in a redox-inactive (N/O){sub 4} coordination environment using one imidazole residue. Expansion of the octarepeat region by one repeat (five PHGGGWGQ repeats) yields a three-histidine (N/O){sub 6} coordination environment for Zn{sup II} and a two-histidine (N/O){sub 4} coordination environment for Cu{sup II} at low copper concentrations. This Cu{sup II}[(N/O){sub 2}-histidine{sub 2}] coordination motif is redox-active and capable of generating H{sub 2}O{sub 2} under reducing aerobic conditions.

  15. Up-regulation of MicroRNA 146b is Associated with Myelofibrosis in Myeloproliferative Neoplasms.

    PubMed

    Ha, Jung-Sook; Jung, Hye-Ra

    2015-01-01

    In this study, our goal was to evaluate whether the expressions of microRNA (miR)-150, miR-146b, miR-31 and miR-95 demonstrate primary myelofibrosis (PMF) specificity, associations with fibrosis grade, hematologic phenotypes, or myeloproliferative neoplasm (MPN)-associated mutations. A total of 51 formalin-fixed and paraffin-embedded bone marrow MPN samples, including 15 polycythemia vera (PV), 26 essential thrombocythemia (ET), and 10 PMF, and 24 normal controls were included. The expression of microRNA (miRNA) was detected by quantitative real-time polymerase chain reaction using miRNA specific primers. RNU6-2 was analyzed for all samples as endogenous control for relative quantification. Information for fibrosis, hematologic parameters, Janus kinase 2 (JAK2) V617F, and calreticulin (CALR) mutations was obtained from medical records. Significant increment of miR-146b was detected in PMF compared to normal controls (P=0.008). Moreover, expression of miR-146b tended to increase according to increment of fibrosis grade, and patients with myelofibrosis (MF) grade 3 showed significantly higher expression than patients with MF 0 to 2 (P=0.022, 0.001 and 0.013, respectively) or normal controls (P<0.001). The expression of miR-31 also showed tendency to increase following fibrosis and miR-150 showed up-regulated expression in ET (P=0.015) compared to normal control. There was no relationship between miRNA expression and hematologic indices except miR-95 showed negative correlation with platelet count (P=0.024). There was no significant correlation between miRNA expression and JAK2 V617F or CALR mutation. Up-regulation of miR-146b could be used as a fibrosis-indicating marker and might be helpful in the study of fibrotic mechanism in MPN, as well as other fibrotic diseases. PMID:26116595

  16. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms.

    PubMed

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  17. Proteomic Dissection of Endosperm Starch Granule Associated Proteins Reveals a Network Coordinating Starch Biosynthesis and Amino Acid Metabolism and Glycolysis in Rice Endosperms

    PubMed Central

    Yu, Huatao; Wang, Tai

    2016-01-01

    Starch biosynthesis and starch granule packaging in cereal endosperms involve a coordinated action of starch biosynthesis enzymes and coordination with other metabolisms. Because directly binding to starch granules, starch granule-associated proteins (SGAPs) are essential to understand the underlying mechanisms, however the information on SGAPs remains largely unknown. Here, we dissected developmentally changed SGAPs from developing rice endosperms from 10 to 20 days after flowering (DAF). Starch granule packaging was not completed at 10 DAF, and was finished in the central endosperm at 15 DAF and in the whole endosperm at 20 DAF. Proteomic analysis with two-dimensional differential in-gel electrophoresis and mass spectrometry revealed 115 developmentally changed SGAPs, representing 37 unique proteins. 65% of the unique proteins had isoforms. 39% of the identified SGAPs were involved in starch biosynthesis with main functions in polyglucan elongation and granule structure trimming. Almost all proteins involved in starch biosynthesis, amino acid biosynthesis, glycolysis, protein folding, and PPDK pathways increased abundance as the endosperm developed, and were predicted in an interaction network. The network represents an important mechanism to orchestrate carbon partitioning among starch biosynthesis, amino acid biosynthesis and glycolysis for efficient starch and protein storage. These results provide novel insights into mechanisms of starch biosynthesis and its coordination with amino acid metabolisms and glycolysis in cereal endosperms. PMID:27252723

  18. A few shared up-regulated genes may influence conidia to yeast transformation in dimorphic fungal pathogens.

    PubMed

    Kirkland, Theo N

    2016-08-01

    The small number of fungi that commonly cause disease in normal people share the capacity to grow as mycelia in the soil at 25°C and as yeast (or spherules) in mammals at 37°C. This remarkable conversion has long been a topic of interest in medical mycology. The conidia to yeast conversion has been studied by transcription profiling in several fungal species, including Histoplasma capsulatum, Paracoccidioides brasiliensis, Coccidioides spp., Blastomyces dermatitidis, and Talaromyces marneffei One limitation of transcriptional profiling is determining which genes are involved in the process of conversion to yeast as opposed to a result of conversion to yeast. If there are genes that are up-regulated in the yeast phase of more than one dimorphic, pathogenic fungus they might be required for conversion to yeast (or spherules). To address this issue, 24 up-regulated genes common to Coccidioides spp spherules and H. capsulatum yeasts were identified. Four homologs of these genes were also found in P. brasiliensis, B. dermatitidis or T. marneffei genes that were up-regulated in yeast. 4-hydroxyphenylpurvate dioxygenase, a gene involved in tyrosine metabolism and melanin synthesis that has been shown to be required for yeast conversion, is conserved and up-regulated in yeast in all five species. Another up-regulated gene that is conserved in all five species is a MFS sugar porter. These results suggest that a minority of up-regulated yeast (or spherule) genes are conserved across species and raises the possibility that conserved up-regulated genes may be of special interest for differentiation of mycelium into yeast. PMID:27118798

  19. Synthetic retinoid Am80 up-regulates apelin expression by promoting interaction of RARα with KLF5 and Sp1 in vascular smooth muscle cells.

    PubMed

    Lv, Xin-Rui; Zheng, Bin; Li, Shu-Ya; Han, Ai-Li; Wang, Chang; Shi, Jian-Hong; Zhang, Xin-Hua; Liu, Yan; Li, Yong-Hui; Wen, Jin-Kun

    2013-11-15

    Previous studies have demonstrated that both retinoids and apelin possess potent cardiovascular properties and that retinoids can mediate the expression of many genes in the cardiovascular system. However, it is not clear whether and how retinoids regulate apelin expression in rat VSMCs (vascular smooth muscle cells). In the present study, we investigated the molecular mechanism of apelin expression regulation by the synthetic retinoid Am80 in VSMCs. The results showed that Am80 markedly up-regulated apelin mRNA and protein levels in VSMCs. Furthermore, KLF5 (Krüppel-like factor 5) and Sp1 (stimulating protein-1) co-operatively mediated Am80-induced apelin expression through their direct binding to the TCE (transforming growth factor-β control element) on the apelin promoter. Interestingly, upon Am80 stimulation, the RARα (retinoic acid receptor α) was recruited to the apelin promoter by interacting with KLF5 and Sp1 prebound to the TCE site of the apelin promoter to form a transcriptional activation complex, subsequently leading to the up-regulation of apelin expression in VSMCs. An in vivo study indicated that Am80 increased apelin expression in balloon-injured arteries of rats, consistent with the results from the cultured VSMCs. Thus the results of the present study describe a novel mechanism of apelin regulation by Am80 and further expand the network of RARα in the retinoid pathway. PMID:23992409

  20. EBV-driven LMP1 and IFN-γ up-regulate PD-L1 in nasopharyngeal carcinoma: Implications for oncotargeted therapy

    PubMed Central

    Chen, Nan; Qin, Tao; Tang, Yanna; Zhang, Yaxiong; Kang, Shiyang; Zhou, Ting; Wu, Xuan; Liang, Wenhua; Hu, Zhihuang; Ma, Yuxiang; Zhao, Yuanyuan; Tian, Ying; Yang, Yunpeng; Xue, Cong; Yan, Yue; Hou, Xue; Huang, Peiyu; Huang, Yan; Zhao, Hongyun; Zhang, Li

    2014-01-01

    PD-L1 expression is a feature of Epstein-Barr virus (EBV) associated malignancies such as nasopharyngeal carcinoma (NPC). Here, we found that EBV-induced latent membrane protein 1 (LMP1) and IFN-γ pathways cooperate to regulate programmed cell death protein 1 ligand (PD-L1). Expression of PD-L1 was higher in EBV positive NPC cell lines compared with EBV negative cell lines. PD-L1 expression could be increased by exogenous and endogenous induction of LMP1 induced PD-L1. In agreement, expression of PD-L1 was suppressed by knocking down LMP1 in EBV positive cell lines. We further demonstrated that LMP1 up-regulated PD-L1 through STAT3, AP-1, and NF-κB pathways. Besides, IFN-γ was independent of but synergetic with LMP1 in up-regulating PD-L1 in NPC. Furthermore, we showed that PD-L1 was associated with worse disease-free survival in NPC patients. These results imply that blocking both the LMP1 oncogenic pathway and PD-1/PD-L1 checkpoints may be a promising therapeutic approach for EBV positive NPC patients. PMID:25361008

  1. TNF-alpha increases ubiquitin-conjugating activity in skeletal muscle by up-regulating UbcH2/E220k

    NASA Technical Reports Server (NTRS)

    Li, Yi-Ping; Lecker, Stewart H.; Chen, Yuling; Waddell, Ian D.; Goldberg, Alfred L.; Reid, Michael B.

    2003-01-01

    In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.

  2. Osmotic stress up-regulates aquaporin-3 gene expression in cultured human keratinocytes.

    PubMed

    Sugiyama, Y; Ota, Y; Hara, M; Inoue, S

    2001-12-01

    Of ten members of the aquaporin family (AQP), the mRNA expression and regulation of AQP1, AQP3, AQP4 and AQP9 in cultured human keratinocytes were examined by an RNase protection assay. AQP3 mRNA was expressed in growing and differentiating cells, while AQP9 mRNA was only detected in differentiating cells. The epidermis in skin-equivalent cultures expressed both AQP3 and AQP9 mRNA. However, neither AQP1 nor AQP4 mRNA was detectable in either monolayer or skin-equivalent cultures. Incubation of keratinocytes in sorbitol-added hypertonic medium increased AQP3 mRNA expression. This was confirmed using other solutes such as NaCl, mannitol, glucose and sucrose. The effect of sorbitol was reversible, dose-dependent and maximal at 24 h after addition. However, AQP1, AQP4 and AQP9 mRNA expression were unchanged under any of the hypertonic conditions examined. These findings indicated that osmotic stress up-regulates AQP3 gene expression in cultured keratinocytes. PMID:11750058

  3. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation.

    PubMed

    Fey, Theres; Schubert, Kai Michael; Schneider, Holger; Fein, Evelyn; Kleinert, Eike; Pohl, Ulrich; Dendorfer, Andreas

    2016-08-01

    Podosomes are dynamic cytoskeletal membrane structures with local adhesive and proteolytic activity. They are critically involved in angiogenesis and vascular adaptive growth. Here, we studied in HUVECs and murine small vessels whether shear stress controls podosome assembly and local proteolytic activity. Podosomes were characterized by immunohistochemistry, and their proteolytic activity was assessed as degradation imprints in fluorescent gelatin that was used as growth substrate. Compared with controls (10 dyn/cm(2)), the number of podosomes formed per time was doubled when cells were exposed to low shear stress (0.3 dyn/cm(2)) or even increased 5-fold under static conditions. This was a result of an enhanced expression of VEGF after reduction of shear stress. Consequently, enhanced podosome formation could be prevented by a VEGF receptor antagonist as well by interruption of VEGF signaling via inhibition of PI3K, Src, or p38. Increase of podosome assembly went along with significantly augmented cell motility. In vivo experiments in mouse arteries confirmed increased endothelial podosome numbers when shear stress was abolished by vessel occlusion. We conclude that shear stress, by reducing VEGF release, inhibits podosome assembly. Hence, endothelial cell-mediated matrix proteolysis and migratory activity are inhibited, thereby stabilizing the structure of the vessel wall.-Fey, T., Schubert, K. M., Schneider, H., Fein, E., Kleinert, E., Pohl, U., Dendorfer, A. Impaired endothelial shear stress induces podosome assembly via VEGF up-regulation. PMID:27103579

  4. MicroRNA-276 promotes egg-hatching synchrony by up-regulating brm in locusts.

    PubMed

    He, Jing; Chen, Qianquan; Wei, Yuanyuan; Jiang, Feng; Yang, Meiling; Hao, Shuguang; Guo, Xiaojiao; Chen, Dahua; Kang, Le

    2016-01-19

    Developmental synchrony, the basis of uniform swarming, migration, and sexual maturation, is an important strategy for social animals to adapt to variable environments. However, the molecular mechanisms underlying developmental synchrony are largely unexplored. The migratory locust exhibits polyphenism between gregarious and solitarious individuals, with the former displaying more synchronous sexual maturation and migration than the latter. Here, we found that the egg-hatching time of gregarious locusts was more uniform compared with solitarious locusts and that microRNA-276 (miR-276) was expressed significantly higher in both ovaries and eggs of gregarious locusts than in solitarious locusts. Interestingly, inhibiting miR-276 in gregarious females and overexpressing it in solitarious females, respectively, caused more heterochronic and synchronous hatching of progeny eggs. Moreover, miR-276 directly targeted a transcription coactivator gene, brahma (brm), resulting in its up-regulation. Knockdown of brm not only resulted in asynchronous egg hatching in gregarious locusts but also impaired the miR-276-induced synchronous egg hatching in solitarious locusts. Mechanistically, miR-276 mediated brm activation in a manner that depended on the secondary structure of brm, namely, a stem-loop around the binding site of miR-276. Collectively, our results unravel a mechanism by which miR-276 enhances brm expression to promote developmental synchrony and provide insight into regulation of developmental homeostasis and population sustaining that are closely related to biological synchrony. PMID:26729868

  5. Up-Regulation of Kin17 Is Essential for Proliferation of Breast Cancer

    PubMed Central

    Zeng, Tao; Gao, Hongyi; Yu, Pei; He, Heng; Ouyang, Xiaoming; Deng, Lijuan; Zhang, Yan

    2011-01-01

    Background Kin17 is ubiquitously expressed at low levels in human tissue and participates in DNA replication, DNA repair and cell cycle control. Breast cancer cells are characterized by enabling replicative immortality and accumulated DNA damage. However, whether kin17 contributes to breast carcinogenesis remains unknown. Methodology/Principal Findings In this study, we show for the first time that kin17 is an important molecule related to breast cancer. Our results show that kin17 expression was markedly increased in clinical breast tumors and was associated with tumor grade, Ki-67 expression, p53 mutation status and progesterone receptor expression, which were assessed in a clinicopathologic characteristics review. Knockdown of kin17 inhibited DNA replication and repair, blocked cell cycle progression and inhibited anchorage-independent growth, while increasing sensitivity to chemotherapy in breast cancer cells. Moreover, kin17 silencing decreased EGF-stimulated cell growth. Furthermore, overexpression of kin17 promoted DNA replication and cell proliferation in MCF-10A. Conclusions/Significance Our findings indicate that up-regulation of kin17 is strongly associated with cellular proliferation, DNA replication, DNA damage response and breast cancer development. The increased level of kin17 was not only a consequence of immortalization but also associated with tumorigenesis. Therefore, kin17 could be a novel therapeutic target for inhibiting cell growth in breast cancer. PMID:21980430

  6. Effects of IL-8 Up-Regulation on Cell Survival and Osteoclastogenesis in Multiple Myeloma.

    PubMed

    Herrero, Ana B; García-Gómez, Antonio; Garayoa, Mercedes; Corchete, Luis A; Hernández, José M; San Miguel, Jesús; Gutierrez, Norma C

    2016-08-01

    IL-8 promotes cancer cell growth, survival, angiogenesis, and metastasis in several tumors. Herein, we investigated the sources of IL-8 production in multiple myeloma (MM) and its potential roles in MM pathogenesis. We found that bone marrow cells from patients with MM secreted higher amounts of IL-8 than healthy donors. IL-8 production was detected in cultures of CD138(+) plasma cells and CD138(-) cells isolated from bone marrows of MM patients, and in three of seven human myeloma cell lines (HMCLs) analyzed. Interactions between MM and stromal cells increased IL-8 secretion by stromal cells through cell-cell adhesion and soluble factors. Interestingly, IL8 expression also increased in HMCLs, stromal cells, and osteoclasts after treatment with the antimyeloma drugs melphalan and bortezomib. In fact, the effect of bortezomib on IL-8 production was higher than that exerted by stromal-MM cell interactions. Addition of exogenous IL-8 did not affect growth of HMCLs, although it protected cells from death induced by serum starvation through a caspase-independent mechanism. Furthermore, IL-8 induced by stromal-MM cell interactions strongly contributed to osteoclast formation in vitro, because osteoclastogenesis was markedly reduced by IL-8-specific neutralizing antibodies. In conclusion, our results implicate IL-8 in myeloma bone disease and point to the potential utility of an anti-IL-8 therapy to prevent unwanted effects of IL-8 up-regulation on survival, angiogenesis, and osteolysis in MM. PMID:27301357

  7. Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis

    PubMed Central

    Barros, Rita; Gomes, Catarina; de Matos, Augusto J.; Reis, Celso A.; Rutteman, Gerard R.; Gärtner, Fátima

    2015-01-01

    The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness. PMID:26222311

  8. MicroRNA-276 promotes egg-hatching synchrony by up-regulating brm in locusts

    PubMed Central

    He, Jing; Chen, Qianquan; Wei, Yuanyuan; Jiang, Feng; Yang, Meiling; Hao, Shuguang; Guo, Xiaojiao; Chen, Dahua; Kang, Le

    2016-01-01

    Developmental synchrony, the basis of uniform swarming, migration, and sexual maturation, is an important strategy for social animals to adapt to variable environments. However, the molecular mechanisms underlying developmental synchrony are largely unexplored. The migratory locust exhibits polyphenism between gregarious and solitarious individuals, with the former displaying more synchronous sexual maturation and migration than the latter. Here, we found that the egg-hatching time of gregarious locusts was more uniform compared with solitarious locusts and that microRNA-276 (miR-276) was expressed significantly higher in both ovaries and eggs of gregarious locusts than in solitarious locusts. Interestingly, inhibiting miR-276 in gregarious females and overexpressing it in solitarious females, respectively, caused more heterochronic and synchronous hatching of progeny eggs. Moreover, miR-276 directly targeted a transcription coactivator gene, brahma (brm), resulting in its up-regulation. Knockdown of brm not only resulted in asynchronous egg hatching in gregarious locusts but also impaired the miR-276–induced synchronous egg hatching in solitarious locusts. Mechanistically, miR-276 mediated brm activation in a manner that depended on the secondary structure of brm, namely, a stem-loop around the binding site of miR-276. Collectively, our results unravel a mechanism by which miR-276 enhances brm expression to promote developmental synchrony and provide insight into regulation of developmental homeostasis and population sustaining that are closely related to biological synchrony. PMID:26729868

  9. Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion.

    PubMed

    Coulson-Thomas, Vivien J; Coulson-Thomas, Yvette M; Gesteira, Tarsis F; de Paula, Cláudia A A; Mader, Ana M; Waisberg, Jaques; Pinhal, Maria A; Friedl, Andreas; Toma, Leny; Nader, Helena B

    2011-11-01

    During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion. PMID:21987222

  10. Compassion-based emotion regulation up-regulates experienced positive affect and associated neural networks.

    PubMed

    Engen, Haakon G; Singer, Tania

    2015-09-01

    Emotion regulation research has primarily focused on techniques that attenuate or modulate the impact of emotional stimuli. Recent evidence suggests that this mode regulation can be problematic in the context of regulation of emotion elicited by the suffering of others, resulting in reduced emotional connectedness. Here, we investigated the effects of an alternative emotion regulation technique based on the up-regulation of positive affect via Compassion-meditation on experiential and neural affective responses to depictions of individuals in distress, and compared these with the established emotion regulation strategy of Reappraisal. Using fMRI, we scanned 15 expert practitioners of Compassion-meditation either passively viewing, or using Compassion-meditation or Reappraisal to modulate their emotional reactions to film clips depicting people in distress. Both strategies effectively, but differentially regulated experienced affect, with Compassion primarily increasing positive and Reappraisal primarily decreasing negative affect. Imaging results showed that Compassion, relative to both passive-viewing and Reappraisal increased activation in regions involved in affiliation, positive affect and reward processing including ventral striatum and medial orbitfrontal cortex. This network was shown to be active prior to stimulus presentation, suggesting that the regulatory mechanism of Compassion is the stimulus-independent endogenous generation of positive affect. PMID:25698699

  11. Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase

    PubMed Central

    Henriques, Alexandre; Croixmarie, Vincent; Priestman, David A.; Rosenbohm, Angela; Dirrig-Grosch, Sylvie; D'Ambra, Eleonora; Huebecker, Mylene; Hussain, Ghulam; Boursier-Neyret, Claire; Echaniz-Laguna, Andoni; Ludolph, Albert C.; Platt, Frances M.; Walther, Bernard; Spedding, Michael; Loeffler, Jean-Philippe; Gonzalez De Aguilar, Jose-Luis

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets. PMID:26483191

  12. Endurance training increases brain lactate uptake during hypoglycemia by up regulation of brain lactate transporters.

    PubMed

    Aveseh, Malihe; Nikooie, Rohollah; Sheibani, Vahid; Esmaeili-Mahani, Saeed

    2014-08-25

    The capacity of the brain to metabolize non-glucose substrates under hypoglycemic state maintains its energy requirements. We hypothesized that exercise-induced increase in capacity for brain utilization of lactate by up regulation of the monocarboxylate transporters (MCTs) may contribute metabolic substrates during hypoglycemia in diabetic rats induced by streptozotocin. The induced diabetes increased MCT1 and MCT2 expression in the cortex and the hippocampus in the sedentary diabetic animals. There were exercise-induced increases in MCT1 in the cortex and the hippocampus and MCT2 expression in the cortex in trained diabetic animals; whereas, no changes were found in the healthy trained animals. Both diabetic and healthy trained animals showed higher values for brain lactate uptake during insulin-induced hypoglycemia when animals were intraperitoneally injected by L(+)-lactic acid. However, the response of counterregulatory hormones during hypoglycemia were blunted in the diabetic trained animals which indicates to carefully monitoring of glycemic targets both during and following prolonged exercise. PMID:25004253

  13. Hypoxia Up-Regulates Galectin-3 in Mammary Tumor Progression and Metastasis.

    PubMed

    de Oliveira, Joana T; Ribeiro, Cláudia; Barros, Rita; Gomes, Catarina; de Matos, Augusto J; Reis, Celso A; Rutteman, Gerard R; Gärtner, Fátima

    2015-01-01

    The tumor microenvironment encompasses several stressful conditions for cancer cells such as hypoxia, oxidative stress and pH alterations. Galectin-3, a well-studied member of the beta-galactoside-binding animal family of lectins has been implicated in multiple steps of metastasis as cell-cell and cell-ECM adhesion, promotion of angiogenesis, cell proliferation and resistance to apoptosis. However, both its aberrantly up- and down-regulated expression was observed in several types of cancer. Thus, the mechanisms that regulate galectin-3 expression in neoplastic settings are not clear. In order to demonstrate the putative role of hypoxia in regulating galectin-3 expression in canine mammary tumors (CMT), in vitro and in vivo studies were performed. In malignant CMT cells, hypoxia was observed to induce expression of galectin-3, a phenomenon that was almost completely prevented by catalase treatment of CMT-U27 cells. Increased galectin-3 expression was confirmed at the mRNA level. Under hypoxic conditions the expression of galectin-3 shifts from a predominant nuclear location to cytoplasmic and membrane expressions. In in vivo studies, galectin-3 was overexpressed in hypoxic areas of primary tumors and well-established metastases. Tumor hypoxia thus up-regulates the expression of galectin-3, which may in turn increase tumor aggressiveness. PMID:26222311

  14. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver

    PubMed Central

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. PMID:27226149

  15. Up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver.

    PubMed

    Zhang, Shuai; Li, Tao-Sheng; Soyama, Akihiko; Tanaka, Takayuki; Yan, Chen; Sakai, Yusuke; Hidaka, Masaaki; Kinoshita, Ayaka; Natsuda, Koji; Fujii, Mio; Kugiyama, Tota; Baimakhanov, Zhassulan; Kuroki, Tamotsu; Gu, Weili; Eguchi, Susumu

    2016-01-01

    Although the healthy liver is known to have high regenerative potential, poor liver regeneration under pathological conditions remains a substantial problem. We investigated the key molecules that impair the regeneration of cholestatic liver. C57BL/6 mice were randomly subjected to partial hepatectomy and bile duct ligation (PH+BDL group, n = 16), partial hepatectomy only (PH group, n = 16), or sham operation (Sham group, n = 16). The liver sizes and histological findings were similar in the PH and sham groups 14 days after operation. However, compared with those in the sham group, the livers in mice in the PH+BDL group had a smaller size, a lower cell proliferative activity, and more fibrotic tissue 14 days after the operation, suggesting the insufficient regeneration of the cholestatic liver. Pathway-focused array analysis showed that many genes were up- or down-regulated over 1.5-fold in both PH+BDL and PH groups at 1, 3, 7, and 14 days after treatment. Interestingly, more genes that were functionally related to the extracellular matrix and inflammatory chemokines were found in the PH+BDL group than in the PH group at 7 and 14 days after treatment. Our data suggest that up-regulated extracellular matrix components and inflammatory chemokines may impair the regeneration of cholestatic liver. PMID:27226149

  16. Δ9-Tetrahydrocannabinol Disrupts Estrogen-Signaling through Up-Regulation of Estrogen Receptor β (ERβ)

    PubMed Central

    Takeda, Shuso; Yoshida, Kazutaka; Nishimura, Hajime; Harada, Mari; Okajima, Shunsuke; Miyoshi, Hiroko; Okamoto, Yoshiko; Amamoto, Toshiaki; Watanabe, Kazuhito; Omiecinski, Curtis J.; Aramaki, Hironori

    2014-01-01

    Δ9-Tetrahydrocannabinol (Δ9-THC) has been reported as possessing antiestrogenic activity, although the mechanisms underlying these effects are poorly delineated. In this study, we used the estrogen receptor α (ERα)-positive human breast cancer cell line, MCF-7, as an experimental model and showed that Δ9-THC exposures markedly suppresses 17β-estradiol (E2)- induced MCF-7 cell proliferation. We demonstrate that these effects result from Δ9-THC’s ability to inhibit E2-liganded ERα activation. Mechanistically, the data obtained from biochemical analyses revealed that (i) Δ9-THC up-regulates ERβ, a repressor of ERα, inhibiting the expression of E2/ERα-regulated genes that promote cell growth and that (ii) Δ9-THC induction of ERβ modulates E2/ERα signaling in the absence of direct interaction with the E2 ligand binding site. Therefore, the data presented support the concept that Δ9-THC’s antiestrogenic activities are mediated by the ERβ disruption of E2/ERα signaling. PMID:23718638

  17. Two-Dimensional Magnesium Phosphate Nanosheets Form Highly Thixotropic Gels That Up-Regulate Bone Formation.

    PubMed

    Laurenti, Marco; Al Subaie, Ahmed; Abdallah, Mohamed-Nur; Cortes, Arthur R G; Ackerman, Jerome L; Vali, Hojatollah; Basu, Kaustuv; Zhang, Yu Ling; Murshed, Monzur; Strandman, Satu; Zhu, Julian; Makhoul, Nicholas; Barralet, Jake E; Tamimi, Faleh

    2016-08-10

    Hydrogels composed of two-dimensional (2D) nanomaterials have become an important alternative to replace traditional inorganic scaffolds for tissue engineering. Here, we describe a novel nanocrystalline material with 2D morphology that was synthesized by tuning the crystallization of the sodium-magnesium-phosphate system. We discovered that the sodium ion can regulate the precipitation of magnesium phosphate by interacting with the crystal's surface causing a preferential crystal growth that results in 2D morphology. The 2D nanomaterial gave rise to a physical hydrogel that presented extreme thixotropy, injectability, biocompatibility, bioresorption, and long-term stability. The nanocrystalline material was characterized in vitro and in vivo and we discovered that it presented unique biological properties. Magnesium phosphate nanosheets accelerated bone healing and osseointegration by enhancing collagen formation, osteoblasts differentiation, and osteoclasts proliferation through up-regulation of COL1A1, RunX2, ALP, OCN, and OPN. In summary, the 2D magnesium phosphate nanosheets could bring a paradigm shift in the field of minimally invasive orthopedic and craniofacial interventions because it is the only material available that can be injected through high gauge needles into bone defects in order to accelerate bone healing and osseointegration. PMID:27280476

  18. Zonisamide up-regulated the mRNAs encoding astrocytic anti-oxidative and neurotrophic factors.

    PubMed

    Choudhury, M E; Sugimoto, K; Kubo, M; Iwaki, H; Tsujii, T; Kyaw, W T; Nishikawa, N; Nagai, M; Tanaka, J; Nomoto, M

    2012-08-15

    Zonisamide has been proven as an effective drug for the recovery of degenerating dopaminergic neurons in the animal models of Parkinson's disease. However, several lines of evidence have questioned the neuroprotective capacity of zonisamide in animal models of Parkinson's disease. Although it suppresses dopaminergic neurodegeneration in animal models, the cellular and molecular mechanisms underlying the effectiveness of zonisamide are not fully understood. The current study demonstrates the effects of zonisamide on astrocyte cultures and two 6-hydroxydopamine-induced models of Parkinson's disease. Using primary astrocyte cultures, we showed that zonisamide up-regulated the expression of mRNA encoding mesencephalic astrocyte-derived neurotrophic factor, vascular endothelial growth factor, proliferating cell nuclear antigen, metallothionein-2, copper/zinc superoxide dismutase, and manganese superoxide dismutase. Similar responses to zonisamide were found in substantia nigra where the rats were pre-treated with 6-hydroxydopamine. Notably, pharmacological inhibition of 6-hydroxydopamine-induced toxicity by zonisamide pre-treatment was also confirmed using rat mesencephalic organotypic slice cultures of substantia nigra. In addition to this, zonisamide post-treatment also attenuated the nigral tyrosine hydroxylase-positive neuronal loss induced by 6-hydroxydopamine. Taken together, these studies demonstrate that zonisamide protected dopamine neurons in two Parkinson's disease models through a novel mechanism, namely increasing the expression of some important astrocyte-mediated neurotrophic and anti-oxidative factors. PMID:22659113

  19. Bottom-up regulation of plant community structure in an aridland ecosystem.

    PubMed

    Báez, Selene; Collins, Scott L; Lightfoot, David; Koontz, Terri L

    2006-11-01

    We conducted a long-term rodent exclosure experiment in native grass- and shrub-dominated vegetation to evaluate the importance of top-down and bottom-up controls on plant community structure in a low-productivity aridland ecosystem. Using multiple regressions and analysis of covariance, we assessed how bottom-up precipitation pulses cascade through vegetation to affect rodent populations, how rodent populations affect plant community structure, and how rodents alter rates of plant community change over time. Our findings showed that bottom-up pulses cascade through the system, increasing the abundances of plants and rodents, and that rodents exerted no control on plant community structure and rate of change in grass-dominated vegetation, and only limited control in shrub-dominated vegetation. These results were discussed in the context of top-down effects on plant communities across broad gradients of primary productivity. We conclude that bottom-up regulation maintains this ecosystem in a state of low primary productivity that constrains the abundance of consumers such that they exert limited influence on plant community structure and dynamics. PMID:17168019

  20. Top-down and bottom-up regulation of macroalgal community structure on a Kenyan reef

    NASA Astrophysics Data System (ADS)

    Mörk, Erik; Sjöö, Gustaf Lilliesköld; Kautsky, Nils; McClanahan, Tim R.

    2009-09-01

    Top-down and bottom-up regulation in the form of grazing by herbivores and nutrient availability are important factors governing macroalgal communities in the coral reef ecosystem. Today, anthropogenic activities, such as over-harvesting of herbivorous fish and sea urchins and increased nutrient loading, are altering the interaction of these two structuring forces. The present study was conducted in Kenya and investigates the relative importance of herbivory and nutrient loading on macroalgal community dynamics, by looking at alterations in macroalgal functional groups, species diversity ( H') and biomass within experimental quadrats. The experiment was conducted in situ for 42 days during the dry season. Cages excluding large herbivorous fish and sea urchins were used in the study and nutrient addition was conducted using coated, slow-release fertilizer (nitrogen and phosphorous) at a site where herbivory is generally low and nutrient levels are relatively high for the region. Nutrient addition increased tissue nutrient content in the algae, and fertilized quadrats had 24% higher species diversity. Herbivore exclusion resulted in a 77% increase in algal biomass, mainly attributable to a >1000% increase in corticated forms. These results are in accordance with similar studies in other regions, but are unique in that they indicate that, even when prevailing nutrient levels are relatively high and herbivore pressure is relatively low, continued anthropogenic disturbance results in further ecological responses and increased reef degradation.

  1. Trop-2 is up-regulated in invasive prostate cancer and displaces FAK from focal contacts.

    PubMed

    Trerotola, Marco; Ganguly, Kirat K; Fazli, Ladan; Fedele, Carmine; Lu, Huimin; Dutta, Anindita; Liu, Qin; De Angelis, Tiziana; Riddell, Luke W; Riobo, Natalia A; Gleave, Martin E; Zoubeidi, Amina; Pestell, Richard G; Altieri, Dario C; Languino, Lucia R

    2015-06-10

    In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa. PMID:26015409

  2. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

    PubMed

    Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen; Sun, Jingxin; Morotomi-Yano, Keiko; Saha, Debabrata; Chen, Benjamin P C

    2015-04-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality. PMID:25925375

  3. Coordinate loss of a microRNA and protein-coding gene cooperate in the pathogenesis of 5q- syndrome.

    PubMed

    Kumar, Madhu S; Narla, Anupama; Nonami, Atsushi; Mullally, Ann; Dimitrova, Nadya; Ball, Brian; McAuley, J Randall; Poveromo, Luke; Kutok, Jeffrey L; Galili, Naomi; Raza, Azra; Attar, Eyal; Gilliland, D Gary; Jacks, Tyler; Ebert, Benjamin L

    2011-10-27

    Large chromosomal deletions are among the most common molecular abnormalities in cancer, yet the identification of relevant genes has proven difficult. The 5q- syndrome, a subtype of myelodysplastic syndrome (MDS), is a chromosomal deletion syndrome characterized by anemia and thrombocytosis. Although we have previously shown that hemizygous loss of RPS14 recapitulates the failed erythroid differentiation seen in 5q- syndrome, it does not affect thrombocytosis. Here we show that a microRNA located in the common deletion region of 5q- syndrome, miR-145, affects megakaryocyte and erythroid differentiation. We find that miR-145 functions through repression of Fli-1, a megakaryocyte and erythroid regulatory transcription factor. Patients with del(5q) MDS have decreased expression of miR-145 and increased expression of Fli-1. Overexpression of miR-145 or inhibition of Fli-1 decreases the production of megakaryocytic cells relative to erythroid cells, whereas inhibition of miR-145 or overexpression of Fli-1 has a reciprocal effect. Moreover, combined loss of miR-145 and RPS14 cooperates to alter erythroid-megakaryocytic differentiation in a manner similar to the 5q- syndrome. Taken together, these findings demonstrate that coordinate deletion of a miRNA and a protein-coding gene contributes to the phenotype of a human malignancy, the 5q- syndrome. PMID:21873545

  4. Phorbol 12-myristate 13-acetate up-regulates the transcription of MUC2 intestinal mucin via Ras, ERK, and NF-kappa B.

    PubMed

    Lee, Hae-Wan; Ahn, Dae-Ho; Crawley, Suzanne C; Li, Jian-Dong; Gum, James R; Basbaum, Carol B; Fan, Nancy Q; Szymkowski, David E; Han, Sang-Young; Lee, Bong H; Sleisenger, Marvin H; Kim, Young S

    2002-09-01

    MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Src tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 up-regulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer. PMID:12077118

  5. Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2*

    PubMed Central

    Huang, Chuangxin; Wang, Joshua J.; Ma, Jacey H.; Jin, Chenjin; Yu, Qiang; Zhang, Sarah X.

    2015-01-01

    Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage. PMID:25568320

  6. Caffeine Mediates Sustained Inactivation of Breast Cancer-Associated Myofibroblasts via Up-Regulation of Tumor Suppressor Genes

    PubMed Central

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2014-01-01

    Background Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. Methodology/Principal Findings We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/−migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. Conclusion/Significance The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts. PMID:24595168

  7. Desoxyrhapontigenin up-regulates Nrf2-mediated heme oxygenase-1 expression in macrophages and inflammatory lung injury

    PubMed Central

    Joo Choi, Ran; Cheng, Mao-sheng; Shik Kim, Yeong

    2014-01-01

    Heme oxygenase-1 (HO-1) is an important anti-inflammatory, antioxidative and cytoprotective enzyme that is regulated by the activation of the major transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In the present study, six stilbene derivatives isolated from Rheum undulatum L. were assessed for their antioxidative potential. In the tert-butylhydroperoxide (t-BHP)-induced RAW 264.7 macrophage cell line, desoxyrhapontigenin was the most potent component that reduced intracellular reactive oxygen species (ROS) and peroxynitrite. In response to desoxyrhapontigenin, the mRNA expression levels of antioxidant enzymes were up-regulated. An electrophoretic mobility shift assay (EMSA) confirmed that desoxyrhapontigenin promoted the DNA binding of Nrf2 and increased the expression of antioxidant proteins and enzymes regulated by Nrf2. Further investigation utilizing specific inhibitors of Akt, p38, JNK and ERK demonstrated that the phosphatidylinositol 3-kinase (PI3K)/Akt pathway mediates HO-1 expression. Moreover, the increase in Nrf2 expression mediated by treatment with desoxyrhapontigenin was reversed by Nrf2 or Akt gene knock-down. In the LPS-induced in vivo lung inflammation model, pretreatment with desoxyrhapontigenin markedly ameliorated LPS-induced lung inflammation and histological changes. Immunohistochemical analysis of Nrf2, HO-1 and p65 was conducted and confirmed that treatment with desoxyrhapontigenin induced Nrf2 and HO-1 expression but reduced p65 expression. These findings suggest that desoxyrhapontigenin may be a potential therapeutic candidate as an antioxidant or an anti-inflammatory agent. PMID:24624340

  8. Carnitine palmitoyltransferase-1 up-regulation by PPAR-β/δ prevents lipid-induced endothelial dysfunction.

    PubMed

    Toral, Marta; Romero, Miguel; Jiménez, Rosario; Mahmoud, Ayman Moawad; Barroso, Emma; Gómez-Guzmán, Manuel; Sánchez, Manuel; Cogolludo, Ángel; García-Redondo, Ana B; Briones, Ana M; Vázquez-Carrera, Manuel; Pérez-Vizcaíno, Francisco; Duarte, Juan

    2015-11-01

    Fatty acids cause endothelial dysfunction involving increased ROS (reactive oxygen species) and reduced NO (nitric oxide) bioavailability. We show that in MAECs (mouse aortic endothelial cells), the PPARβ/δ (peroxisome- proliferator-activated receptor β/δ) agonist GW0742 prevented the decreased A23187-stimulated NO production, phosphorylation of eNOS (endothelial nitric oxide synthase) at Ser1177 and increased intracellular ROS levels caused by exposure to palmitate in vitro. The impaired endothelium-dependent relaxation to acetylcholine in mouse aorta induced by palmitate was restored by GW0742. In vivo, GW0742 treatment prevented the reduced aortic relaxation, phosphorylation of eNOS at Ser1177, and increased ROS production and NADPH oxidase in mice fed on a high-fat diet. The PPARβ/δ antagonist GSK0660 abolished all of these protective effects induced by GW0742. This agonist enhanced the expression of CPT (carnitine palmitoyltransferase)-1. The effects of GW0742 on acetylcholine- induced relaxation in aorta and on NO and ROS production in MAECs exposed to palmitate were abolished by the CPT-1 inhibitor etomoxir or by siRNA targeting CPT-1. GW0742 also inhibited the increase in DAG (diacylglycerol), PKCα/βII (protein kinase Cα/βII) activation, and phosphorylation of eNOS at Thr495 induced by palmitate in MAECs, which were abolished by etomoxir. In conclusion, PPARβ/δ activation restored the lipid-induced endothelial dysfunction by up-regulation of CPT-1, thus reducing DAG accumulation and the subsequent PKC-mediated ROS production and eNOS inhibition. PMID:26253087

  9. Natriuretic Peptide Receptor-C is Up-Regulated in the Intima of Advanced Carotid Artery Atherosclerosis

    PubMed Central

    Zayed, Mohamed A; Harring, Scott D; Abendschein, Dana R; Vemuri, Chandu; Lu, Dongsi; Detering, Lisa; Liu, Yongjian; Woodard, Pamela K

    2016-01-01

    Objective Natriuretic peptide receptor-C (NPR-C/NPR-3) is a cell surface protein involved in vascular remodelling that is up-regulated in atherosclerosis. NPR-C expression has not been well characterized in human carotid artery occlusive lesions. We hypothesized that NPR-C expression correlates with intimal features of vulnerable atherosclerotic carotid artery plaque. Methods To test this hypothesis, we evaluated NPR-C expression by immunohistochemistry (IHC) in carotid endarterectomy (CEA) specimens isolated from 18 patients. The grade, location, and co-localization of NPR-C in CEA specimens were evaluated using two tissue analysis techniques. Results Relative to minimally diseased CEA specimens, we observed avid NPR-C tissue staining in the intima of maximally diseased CEA specimens (65%; p=0.06). Specifically, maximally diseased CEA specimens demonstrated increased NPR-C expression in the superficial intima (61%, p=0.17), and deep intima (138% increase; p=0.05). In the superficial intima, NPR-C expression significantly co-localized with vascular smooth muscle cells (VSMCs) and macrophages. The intensity of NPR-C expression was also higher in the superficial intima plaque shoulder and cap regions, and significantly correlated with atheroma and fibroatheroma vulnerable plaque regions (β=1.04, 95% CI=0.46, 1.64). Conclusion These findings demonstrate significant NPR-C expression in the intima of advanced carotid artery plaques. Furthermore, NPR-C expression was higher in vulnerable carotid plaque intimal regions, and correlate with features of advanced disease. Our findings suggest that NPR-C may serve as a potential biomarker for carotid plaque vulnerability and progression, in patients with advanced carotid artery occlusive disease. PMID:27547837

  10. BlyS is up-regulated by hypoxia and promotes migration of human breast cancer cells

    PubMed Central

    2012-01-01

    Background The role of B Lymphocyte Stimulator (BLyS) in the survival of malignant B cells and the maintenance of normal B cell development and homeostasis has been intensively studied in the literature. However, the influence of BLyS on breast cancer progression remains unclear. The study aimed to investigate the effect of hypoxia on BLyS regulation, cell migratory response to BLyS and the possible molecular mechanisms. Methods In this study, we examined the role of BLyS in the migration of human breast cancer cells by transwell assay. We also explored whether BLyS and its receptors expressed in human breast cancer cell lines by immunofluorescence and Western Blotting. Then we detected the expression level of BLyS in both normoxic and hypoxic conditions by real time-PCR and Western Blotting. Pathways involved were confirmed by Western Blotting, immunofluorescence, transwell assay and luciferase assay. Results According to our study, the expression level of BlyS was increased in human breast cancer cell lines in hypoxic conditions. Up-regulation of this protein led to activation and nuclear translocation of NF-kappa B p65. We also found that the number of migrated cells was increased in the presence of BLyS and inhibition of phosphorylation of Akt attenuated the enhanced migratory response. Conclusions It suggested that better understanding of BLyS, an immunopotentiator, may offer a potential therapeutic target for the treatment of human breast cancers. In addition, BLyS promoted breast cancer cells migration, underscoring the necessity of appropriate applications of immunopotentiators to cancer treatment. PMID:22463935

  11. Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya

    PubMed Central

    Fang, Jingping; Lin, Aiting; Qiu, Weijing; Cai, Hanyang; Umar, Muhammad; Chen, Rukai; Ming, Ray

    2016-01-01

    Papaya is a productive and nutritious tropical fruit. Papaya Ringspot Virus (PRSV) is the most devastating pathogen threatening papaya production worldwide. Development of transgenic resistant varieties is the most effective strategy to control this disease. However, little is known about the genome-wide functional changes induced by particle bombardment transformation. We conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV. In total, 20,700 transcripts were identified, and 842 differentially expressed genes (DEGs) randomly distributed among papaya chromosomes. Gene ontology (GO) category analysis revealed that microtubule-related categories were highly enriched among these DEGs. Numerous DEGs related to various transcription factors, transporters and hormone biosynthesis showed clear differences between the two cultivars, and most were up-regulated in transgenic papaya. Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp, including MYB, WRKY, ERF, NAC, nitrate and zinc transporters, and genes involved in the abscisic acid, salicylic acid, and ethylene signaling pathways. We also identified 67,686 alternative splicing (AS) events in Sunset and 68,455 AS events in SunUp, mapping to 10,994 and 10,995 papaya annotated genes, respectively. GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp. Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences, which increased PRSV-resistance in transgenic papaya. No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp. Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya. PMID:27379138

  12. Transcriptome Profiling Revealed Stress-Induced and Disease Resistance Genes Up-Regulated in PRSV Resistant Transgenic Papaya.

    PubMed

    Fang, Jingping; Lin, Aiting; Qiu, Weijing; Cai, Hanyang; Umar, Muhammad; Chen, Rukai; Ming, Ray

    2016-01-01

    Papaya is a productive and nutritious tropical fruit. Papaya Ringspot Virus (PRSV) is the most devastating pathogen threatening papaya production worldwide. Development of transgenic resistant varieties is the most effective strategy to control this disease. However, little is known about the genome-wide functional changes induced by particle bombardment transformation. We conducted transcriptome sequencing of PRSV resistant transgenic papaya SunUp and its PRSV susceptible progenitor Sunset to compare the transcriptional changes in young healthy leaves prior to infection with PRSV. In total, 20,700 transcripts were identified, and 842 differentially expressed genes (DEGs) randomly distributed among papaya chromosomes. Gene ontology (GO) category analysis revealed that microtubule-related categories were highly enriched among these DEGs. Numerous DEGs related to various transcription factors, transporters and hormone biosynthesis showed clear differences between the two cultivars, and most were up-regulated in transgenic papaya. Many known and novel stress-induced and disease-resistance genes were most highly expressed in SunUp, including MYB, WRKY, ERF, NAC, nitrate and zinc transporters, and genes involved in the abscisic acid, salicylic acid, and ethylene signaling pathways. We also identified 67,686 alternative splicing (AS) events in Sunset and 68,455 AS events in SunUp, mapping to 10,994 and 10,995 papaya annotated genes, respectively. GO enrichment for the genes displaying AS events exclusively in Sunset was significantly different from those in SunUp. Transcriptomes in Sunset and transgenic SunUp are very similar with noteworthy differences, which increased PRSV-resistance in transgenic papaya. No detrimental pathways and allergenic or toxic proteins were induced on a genome-wide scale in transgenic SunUp. Our results provide a foundation for unraveling the mechanism of PRSV resistance in transgenic papaya. PMID:27379138

  13. Up-regulation of galectin-9 induces cell migration in human dendritic cells infected with dengue virus.

    PubMed

    Hsu, Yu-Lin; Wang, Mei-Yi; Ho, Ling-Jun; Huang, Chuan-Yueh; Lai, Jenn-Haung

    2015-05-01

    Galectin-9 (Gal-9) exerts immunosuppressive effects by inducing apoptosis in T cells that produce interferon-γ and interleukin (IL)-17. However, Gal-9 can be pro-inflammatory in lipopolysaccharide-stimulated monocytes. Using microarray analysis, we observed that Gal-9 was up-regulated in human dendritic cells (DCs) after dengue virus (DV) infection. The investigation into the immunomodulatory effects and mechanisms of Gal-9 in DCs exposed to DV revealed that DV infection specifically increased mRNA and protein levels of Gal-9 but not those of Gal-1 or Gal-3. Blocking p38, but not c-Jun N-terminal kinase or extracellular signal-regulated kinase (ERK), inhibited DV-induced expression of Gal-9. Reduction in Gal-9 by small interference RNA treatment suppressed DV-stimulated migration of DCs towards the chemoattractants CCL19 and CCL21. In addition, DV-induced IL-12p40 production was reduced after knockdown of Gal-9 in DCs. Furthermore, Gal-9 deficiency suppressed DV-induced activation of nuclear factor-κB. Inhibition of DV-induced DC migration under conditions of Gal-9 deficiency was mediated through suppressing ERK activation but not by regulating the expression of CCR7, the receptor for CCL19 and CCL21. Both the reduction in IL-12 production and the suppression of ERK activity might account for the inhibition of DV-induced DC migration after knockdown of Gal-9. In summary, this study reveals the roles of Gal-9 in DV-induced migration of DCs. The findings indicate that Gal-9 might be a therapeutic target for preventing immunopathogenesis induced by DV infection. PMID:25754930

  14. Lysophosphatidic Acid Up-Regulates Hexokinase II and Glycolysis to Promote Proliferation of Ovarian Cancer Cells1

    PubMed Central

    Mukherjee, Abir; Ma, Yibao; Yuan, Fang; Gong, Yongling; Fang, Zhenyu; Mohamed, Esraa M.; Berrios, Erika; Shao, Huanjie; Fang, Xianjun

    2015-01-01

    Lysophosphatidic acid (LPA), a blood-borne lipid mediator, is present in elevated concentrations in ascites of ovarian cancer patients and other malignant effusions. LPA is a potent mitogen in cancer cells. The mechanism linking LPA signal to cancer cell proliferation is not well understood. Little is known about whether LPA affects glucose metabolism to accommodate rapid proliferation of cancer cells. Here we describe that in ovarian cancer cells, LPA enhances glycolytic rate and lactate efflux. A real time PCR-based miniarray showed that hexokinase II (HK2) was the most dramatically induced glycolytic gene to promote glycolysis in LPA-treated cells. Analysis of the human HK2 gene promoter identified the sterol regulatory element-binding protein as the primary mediator of LPA-induced HK2 transcription. The effects of LPA on HK2 and glycolysis rely on LPA2, an LPA receptor subtype overexpressed in ovarian cancer and many other malignancies. We further examined the general role of growth factor-induced glycolysis in cell proliferation. Like LPA, epidermal growth factor (EGF) elicited robust glycolytic and proliferative responses in ovarian cancer cells. Insulin-like growth factor 1 (IGF-1) and insulin, however, potently stimulated cell proliferation but only modestly induced glycolysis. Consistent with their differential effects on glycolysis, LPA and EGF-dependent cell proliferation was highly sensitive to glycolytic inhibition while the growth-promoting effect of IGF-1 or insulin was more resistant. These results indicate that LPA- and EGF-induced cell proliferation selectively involves up-regulation of HK2 and glycolytic metabolism. The work is the first to implicate LPA signaling in promotion of glucose metabolism in cancer cells. PMID:26476080

  15. Neurodegeneration in Autoimmune Optic Neuritis Is Associated with Altered APP Cleavage in Neurons and Up-Regulation of p53

    PubMed Central

    Wichert, Sven P.; Kretzschmar, Benedikt; Bähr, Mathias; Rossner, Moritz J.; Hein, Katharina

    2015-01-01

    Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS). Histopathological and radiological analysis revealed that neurodegeneration occurs early in the disease course. However, the pathological mechanisms involved in neurodegeneration are poorly understood. Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in Brown Norway rats (BN-rats) is a well-established animal model, especially of the neurodegenerative aspects of MS. Previous studies in this animal model indicated that loss of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, occurs in the preclinical phase of the disease and is in part independent of overt histopathological changes of the optic nerve. Therefore, the aim of this study was to identify genes which are involved in neuronal cell loss at different disease stages of EAE. Furthermore, genes that are highly specific for autoimmune-driven neurodegeneration were compared to those regulated in RGCs after optic nerve axotomy at corresponding time points. Using laser capture micro dissection we isolated RNA from unfixed RGCs and performed global transcriptome analysis of retinal neurons. In total, we detected 582 genes sequentially expressed in the preclinical phase and 1150 genes in the clinical manifest EAE (P < 0.05, fold-induction >1.5). Furthermore, using ingenuity pathway analysis (IPA), we identified amyloid precursor protein (APP) as a potential upstream regulator of changes in gene expression in the preclinical EAE but neither in clinical EAE, nor at any time point after optic nerve transection. Therefore, the gene pathway analysis lead to the hypothesis that altered cleavage of APP in neurons in the preclinical phase of EAE leads to the enhanced production of APP intracellular domain (AICD), which in turn acts as a transcriptional regulator and thereby initiates an apoptotic signaling cascade via up-regulation of the

  16. Expression of ADAM15 in rheumatoid synovium: up-regulation by vascular endothelial growth factor and possible implications for angiogenesis

    PubMed Central

    Komiya, Koichiro; Enomoto, Hiroyuki; Inoki, Isao; Okazaki, Satoko; Fujita, Yoshinari; Ikeda, Eiji; Ohuchi, Eiko; Matsumoto, Hideo; Toyama, Yoshiaki; Okada, Yasunori

    2005-01-01

    ADAMs (a disintegrin and metalloproteinases) comprise a new gene family of metalloproteinases, and may play roles in cell-cell interaction, cell migration, signal transduction, shedding of membrane-anchored proteins and degradation of extracellular matrix. We screened the mRNA expression of 10 different ADAMs with a putative metalloproteinase motif in synovial tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Reverse transcription PCR and real-time quantitative PCR analyses indicated that among the ADAMs, ADAM15 mRNA was more frequently expressed in the RA samples and its expression level was significantly 3.8-fold higher in RA than in OA (p < 0.01). In situ hybridization, immunohistochemistry and immunoblotting demonstrated that ADAM15 is expressed in active and precursor forms in the synovial lining cells, endothelial cells of blood vessels and macrophage-like cells in the sublining layer of RA synovium. There was a direct correlation between ADAM15 mRNA expression levels and vascular density in the synovial tissues (r = 0.907, p < 0.001; n = 20). ADAM15 was constitutively expressed in RA synovial fibroblasts and human umbilical vein endothelial cells (HUVECs), and the expression level was increased in HUVECs by treatment with vascular endothelial growth factor (VEGF)165. On the other hand, ADAM15 expression in RA synovial fibroblasts was enhanced with VEGF165 only if vascular endothelial growth factor receptor (VEGFR)-2 expression was induced by treatment with tumor necrosis factor-α, and the expression was blocked with SU1498, a specific inhibitor of VEGFR-2. These data demonstrate that ADAM15 is overexpressed in RA synovium and its expression is up-regulated by the action of VEGF165 through VEGFR-2, and suggest the possibility that ADAM15 is involved in angiogenesis in RA synovium. PMID:16277668

  17. Clusterin/Akt Up-Regulation Is Critical for GATA-4 Mediated Cytoprotection of Mesenchymal Stem Cells against Ischemia Injury

    PubMed Central

    Yu, Bin; Yang, Yueting; Liu, Huan; Gong, Min; Millard, Ronald W.; Wang, Yi-Gang; Ashraf, Muhammad; Xu, Meifeng

    2016-01-01

    Background Clusterin (Clu) is a stress-responding protein with multiple biological functions. Our preliminary microarray studies show that clusterin was prominently upregulated in mesenchymal stem cells (MSCs) overexpressing GATA-4 (MSCGATA-4). We hypothesized that the upregulation of clusterin is involved in overexpression of GATA-4 mediated cytoprotection. Methods MSCs harvested from bone marrow of rats were transduced with GATA-4. The expression of clusterin in MSCs was further confirmed by real-time PCR and western blotting. Simulation of ischemia was achieved by exposure of MSCs to a hypoxic environment. Lactate dehydrogenase (LDH) released from MSCs was served as a biomarker of cell injury and MTs uptake was used to estimate cell viability. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (ΔΨm) and caspase 3/7 activity. Results (1) Clusterin expression was up-regulated in MSCGATA-4 compared to control MSCs transfected with empty-vector (MSCNull). MSCGATA-4 were tolerant to 72 h hypoxia exposure as shown by reduced LDH release and higher MTs uptake. This protection was abrogated by transfecting Clu-siRNA into MSCGATA-4. (2) Exogenous clusterin significantly decreased LDH release and increased MSC survival in hypoxic environment. Moreover, ΔΨm was maintained and caspase 3/7 activity was reduced by clusterin in a concentration-dependent manner. (3) p-Akt expression in MSCs was upregulated following pre-treatment with clusterin, with no change in total Akt. Moreover, cytoprotection mediated by clusterin was partially abrogated by Akt inhibitor LY294002. Conclusions Clusterin/Akt signaling pathway is involved in GATA-4 mediated cytoprotection against hypoxia stress. It is suggested that clusterin may be therapeutically exploited in MSC based therapy for cardiovascular diseases. PMID:26962868

  18. Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

    PubMed

    Charvet-Candela, V; Hitchin, S; Reddy, M S; Cournoyer, B; Marmeisse, R; Gay, G

    2002-03-01

    As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation. PMID:11874719

  19. Deletion of Mir155 Prevents Fas-Induced Liver Injury through Up-Regulation of Mcl-1

    PubMed Central

    Chen, Weina; Han, Chang; Zhang, Jinqiang; Song, Kyoungsub; Wang, Ying; Wu, Tong

    2016-01-01

    Fas-induced apoptosis is involved in diverse liver diseases. Herein, we investigated the effect of Mir155 deletion on Fas-induced liver injury. Wild-type (WT) mice and Mir155 knockout (KO) mice were i.p. administered with the anti-Fas antibody (Jo2) to determine animal survival and the extent of liver injury. After Jo2 injection, the Mir155 KO mice exhibited prolonged survival versus the WT mice (P < 0.01). The Mir155 KO mice showed lower alanine aminotransferase and aspartate aminotransferase levels, less liver tissue damage, fewer apoptotic hepatocytes, and lower liver tissue caspase 3/7, 8, and 9 activities compared with the WT mice, indicating that Mir155 deletion prevents Fas-induced hepatocyte apoptosis and liver injury. Hepatocytes isolated from Mir155 KO mice also showed resistance to Fas-induced apoptosis, in vitro. Higher protein level of myeloid cell leukemia-1 (Mcl-1) was also observed in Mir155 KO hepatocytes compared to WT hepatocytes. A miR-155 binding site was identified in the 3′-untranslated region of Mcl-1 mRNA; Mcl1 was identified as a direct target of miR-155 in hepatocytes. Consistently, pretreatment with a siRNA specific for Mcl1 reversed Mir155 deletion–mediated protection against Jo2-induced liver tissue damage. Finally, restoration of Mir155 expression in Mir155 KO mice abolished the protection against Fas-induced hepatocyte apoptosis. Taken together, these findings demonstrate that deletion of Mir155 prevents Fas-induced hepatocyte apoptosis and liver injury through the up-regulation of Mcl1. PMID:25794705

  20. AMPK-mediated up-regulation of mTORC2 and MCL-1 compromises the anti-cancer effects of aspirin

    PubMed Central

    Hua, Hui; Yin, Yancun; Wang, Jiao; Luo, Ting; Jiang, Yangfu

    2016-01-01

    AMP-activated protein kinase (AMPK) is an important energy sensor that may inhibit cell proliferation or promote cell survival during stresses. Besides cyclooxygenase, AMPK is another target of the nonsteroid anti-inflammatory agent aspirin. Preclinical and clinical investigations demonstrate that aspirin can inhibit several types of cancer such as colorectal adenomas and hepatocellular carcinoma (HCC). However, little is known about the cellular response to aspirin that may lead to aspirin resistance. Here, we show that aspirin induces the expression of MCL-1 in HepG2 and SW480 cells through AMPK-mTOR-Akt/ERK axis. Treatment of HepG2 and SW480 cells with aspirin leads to increased MCL-1 expression, Akt and ERK1/2 phosphorylation. Inhibition of Akt/MEK abrogates the induction of MCL-1 by aspirin. Aspirin activates AMPK, which in turn up-regulates mTORC2 activity, Akt, ERK1/2 phosphorylation and MCL-1 expression. MCL-1 knockdown sensitizes cancer cells to aspirin-induced apoptosis. Combination of aspirin and AMPK, Akt or MEK inhibitor results in more significant inhibition of cell proliferation and induction of apoptosis than single agent. Moreover, sorafenib blocks aspirin-induced MCL-1 up-regulation. Combination of aspirin and sorafenib leads to much more cell death and less cell proliferation than each drug alone. Treatment of HCC and colon cancer xenografts with both aspirin and sorafenib results in more significant tumor suppression than single agent. These data demonstrate that AMPK-mediated up-regulation of mTORC2 and MCL-1 may compromise the anticancer effects of aspirin. Combination of aspirin and sorafenib may be an effective regimen to treat HCC and colon cancer. PMID:26918349

  1. ERβ-dependent neuroglobin up-regulation impairs 17β-estradiol-induced apoptosis in DLD-1 colon cancer cells upon oxidative stress injury.

    PubMed

    Fiocchetti, Marco; Camilli, Giulia; Acconcia, Filippo; Leone, Stefano; Ascenzi, Paolo; Marino, Maria

    2015-05-01

    Besides other mechanism(s) 17β-estradiol (E2) facilitates neuronal survival by increasing, via estrogen receptor β (ERβ), the levels of neuroglobin (NGB) an anti-apoptotic protein. In contrast, E2 could exert protective effects in cancer cells by activating apoptosis when the ERβ level prevails on that of ERα as in colon cancer cell lines. These apparently contrasting results raise the possibility that E2-induced NGB up-regulation could regulate the ERβ activities shunning this receptor subtype to trigger an apoptotic cascade in neurons but not in non-neuronal cells. Here, human colorectal adenocarcinoma cell line (DLD-1) that only expresses ERβ and HeLa cells transiently transfected with ERβ encoding vector has been used to verify this hypothesis. In addition, neuroblastoma SK-N-BE cells were used as positive control. Surprisingly, E2 also induced NGB up-regulation, in a dose- and time-dependent manner, in DLD-1 cells. The ERβ-mediated activation of p38/MAPK was necessary for this E2 ef