Decreased expression of dual-specificity phosphatase 9 is associated with poor prognosis in clear cell renal cell carcinoma

Background: The molecular mechanisms involved in the development and progression of clear cell renal cell carcinomas (ccRCCs) are poorly understood. The objective of this study was to analyze the expression of dual-specificity phosphatase 9 (DUSP-9) and determine its clinical significance in human ccRCCs. Methods: The expression of DUSP-9 mRNA was determined in 46 paired samples of ccRCCs and adjacent normal tissues by using real-time qPCR. The expression of the DUSP-9 was determined in 211 samples of ccRCCs and 107 paired samples of adjacent normal tissues by immunohistochemical analysis. Statistical analysis was performed to define the relationship between the expression of DUSP-9 and the clinical features of ccRCC. Results: The mRNA level of DUSP-9, which was determined by real-time RT-PCR, was found to be significantly lower in tumorous tissues than in the adjacent non-tumorous tissues (p < 0.001). An immunohistochemical analysis of 107 paired tissue specimens showed that the DUSP-9 expression was lower in tumorous tissues than in the adjacent non-tumorous tissues (p < 0.001). Moreover, there was a significant correlation between the DUSP-9 expression in ccRCCs and gender (p = 0.031), tumor size (p = 0.001), pathologic stage (p = 0.001), Fuhrman grade (p = 0.002), T stage (p = 0.001), N classification (p = 0.012), metastasis (p = 0.005), and recurrence (p < 0.001). Patients with lower DUSP-9 expression had shorter overall survival time than those with higher DUSP-9 expression (p < 0.001). Multivariate analysis indicated that low expression of the DUSP-9 was an independent predictor for poor survival of ccRCC patients. Conclusion: To our knowledge, this is the first study that determines the relationship between DUSP-9 expression and prognosis in ccRCC. We found that decreased expression of DUSP-9 is associated with poor prognosis in ccRCC. DUSP-9 may represent a novel and useful prognostic marker for ccRCC

Carbon Monoxide Promotes Respiratory Hemoproteins Iron Reduction Using Peroxides as Electron Donors

The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O2 via its binding to their ferrous (FeII) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (FeIII, met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage

MAP Kinase Phosphatase-2 Plays a Critical Role in Response to Infection by Leishmania mexicana

In this study we generated a novel dual specific phosphatase 4 (DUSP4) deletion mouse using a targeted deletion strategy in order to examine the role of MAP kinase phosphatase-2 (MKP-2) in immune responses. Lipopolysaccharide (LPS) induced a rapid, time and concentration-dependent increase in MKP-2 protein expression in bone marrow-derived macrophages from MKP-2+/+ but not from MKP-2−/− mice. LPS-induced JNK and p38 MAP kinase phosphorylation was significantly increased and prolonged in MKP-2−/− macrophages whilst ERK phosphorylation was unaffected. MKP-2 deletion also potentiated LPS-stimulated induction of the inflammatory cytokines, IL-6, IL-12p40, TNF-α, and also COX-2 derived PGE2 production. However surprisingly, in MKP-2−/− macrophages, there was a marked reduction in LPS or IFNγ-induced iNOS and nitric oxide release and enhanced basal expression of arginase-1, suggesting that MKP-2 may have an additional regulatory function significant in pathogen-mediated immunity. Indeed, following infection with the intracellular parasite Leishmania mexicana, MKP-2−/− mice displayed increased lesion size and parasite burden, and a significantly modified Th1/Th2 bias compared with wild-type counterparts. However, there was no intrinsic defect in MKP-2−/− T cell function as measured by anti-CD3 induced IFN-γ production. Rather, MKP-2−/− bone marrow-derived macrophages were found to be inherently more susceptible to infection with Leishmania mexicana, an effect reversed following treatment with the arginase inhibitor nor-NOHA. These findings show for the first time a role for MKP-2 in vivo and demonstrate that MKP-2 may be essential in orchestrating protection against intracellular infection at the level of the macrophage

Gene expression profile of AIDS-related Kaposi's sarcoma

BACKGROUND: Kaposi's Sarcoma (KS) is a proliferation of aberrant vascular structures lined by spindle cells, and is caused by a gammaherpes virus (HHV8/KSHV). Its course is aggravated by co-infection with HIV-1, where the timing of infection with HIV-1 and HHV8 is important for the clinical outcome. METHODS: In order to better understand the pathogenesis of KS, we have analysed tissue from two AIDS-KS lesions, and from normal skin by serial analysis of gene expression (SAGE). Semi-quantitative RT-PCR was then used to validate the results. RESULTS: The expression profile of AIDS-related KS (AIDS-KS) reflects an active process in the skin. Transcripts of HHV8 were found to be very low, and HIV-1 mRNA was not detected by SAGE, although it could be found using RT-PCR. Comparing the expression profile of AIDS-KS tissue with publicly available SAGE libraries suggested that AIDS-KS mRNA levels are most similar to those in an artificially mixed library of endothelial cells and leukocytes, in line with the description of KS lesions as containing spindle cells with endothelial characteristics, and an inflammatory infiltrate. At least 64 transcripts were found to be significantly elevated, and 28 were statistically downregulated in AIDS-KS compared to normal skin. Five of the upregulated mRNAs, including Tie 1 and sialoadhesin/CD169, were confirmed by semi-quantitative PCR to be elevated in additional AIDS-KS biopsies. Antibodies to sialoadhesin/CD169, a known marker of activated macrophages, were shown to specifically label tumour macrophages. CONCLUSION: The expression profile of AIDS-KS showed 64 genes to be significantly upregulated, and 28 genes downregulated, compared with normal skin. One of the genes with increased expression was sialoadhesin (CD169). Antibodies to sialoadhesin/CD169 specifically labelled tumour-associated macrophages, suggesting that macrophages present in AIDS-KS lesions belong to a subset of human CD169+ macrophages

Lipoic acid plays a role in scleroderma: insights obtained from scleroderma dermal fibroblasts

Abstract Introduction Systemic sclerosis (SSc) is a connective tissue disease characterized by fibrosis of the skin and organs. Increase in oxidative stress and platelet-derived growth factor receptor (PDGFR) activation promote type I collagen (Col I) production, leading to fibrosis in SSc. Lipoic acid (LA) and its active metabolite dihydrolipoic acid (DHLA) are naturally occurring thiols that act as cofactors and antioxidants and are produced by lipoic acid synthetase (LIAS). Our goals in this study were to examine whether LA and LIAS were deficient in SSc patients and to determine the effect of DHLA on the phenotype of SSc dermal fibroblasts. N-acetylcysteine (NAC), a commonly used thiol antioxidant, was included as a comparison. Methods Dermal fibroblasts were isolated from healthy subjects and patients with diffuse cutaneous SSc. Matrix metalloproteinase (MMPs), tissue inhibitors of MMPs (TIMP), plasminogen activator inhibitor 1 (PAI-1) and LIAS were measured by enzyme-linked immunosorbent assay. The expression of Col I was measured by immunofluorescence, hydroxyproline assay and quantitative PCR. PDGFR phosphorylation and α-smooth muscle actin (αSMA) were measured by Western blotting. Student’s t-tests were performed for statistical analysis, and P-values less than 0.05 with two-tailed analysis were considered statistically significant. Results The expression of LA and LIAS in SSc dermal fibroblasts was lower than normal fibroblasts; however, LIAS was significantly higher in SSc plasma and appeared to be released from monocytes. DHLA lowered cellular oxidative stress and decreased PDGFR phosphorylation, Col I, PAI-1 and αSMA expression in SSc dermal fibroblasts. It also restored the activities of phosphatases that inactivated the PDGFR. SSc fibroblasts produced lower levels of MMP-1 and MMP-3, and DHLA increased them. In contrast, TIMP-1 levels were higher in SSc, but DHLA had a minimal effect. Both DHLA and NAC increased MMP-1 activity when SSc cells were stimulated with PDGF. In general, DHLA showed better efficacy than NAC in most cases. Conclusions DHLA acts not only as an antioxidant but also as an antifibrotic because it has the ability to reverse the profibrotic phenotype of SSc dermal fibroblasts. Our study suggests that thiol antioxidants, including NAC, LA, or DHLA, could be beneficial for patients with SSc.http://deepblue.lib.umich.edu/bitstream/2027.42/112060/1/13075_2014_Article_411.pd

Negative feedback regulation of the ERK1/2 MAPK pathway

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3l isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-offunction mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.This work was supported by the Spanish MICINN (Grants BFU2009-09771, EUI2009-04009 to PV), Generalitat Valenciana (Prometeo2010/020 to PV) and the German DFG (SCHE 235/15-1 to DS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Carrasco Jiménez, JL.; Castelló Llopis, MJ.; Naumann, K.; Lassowskat, I.; Navarrete Gomez, ML.; Scheel, D.; Vera Vera, P. (2014). Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense. PLoS ONE. 9:1-10. https://doi.org/10.1371/journal.pone.0090734S1109Carrasco, J. L. (2003). A novel transcription factor involved in plant defense endowed with protein phosphatase activity. The EMBO Journal, 22(13), 3376-3384. doi:10.1093/emboj/cdg323Carrasco, J. L., Ancillo, G., Castelló, M. J., & Vera, P. (2005). A Novel DNA-Binding Motif, Hallmark of a New Family of Plant Transcription Factors. Plant Physiology, 137(2), 602-606. doi:10.1104/pp.104.056002Castelló, M. J., Carrasco, J. L., & Vera, P. (2010). DNA-Binding Protein Phosphatase AtDBP1 Mediates Susceptibility to Two Potyviruses in Arabidopsis. Plant Physiology, 153(4), 1521-1525. doi:10.1104/pp.110.158923Castelló, M. J., Carrasco, J. L., Navarrete-Gómez, M., Daniel, J., Granot, D., & Vera, P. (2011). A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis. Plant Physiology, 157(4), 2206-2215. doi:10.1104/pp.111.188953Denison, F. C., Paul, A.-L., Zupanska, A. K., & Ferl, R. J. (2011). 14-3-3 proteins in plant physiology. Seminars in Cell & Developmental Biology, 22(7), 720-727. doi:10.1016/j.semcdb.2011.08.006Carrasco, J. L., Castelló, M. J., & Vera, P. (2006). 14-3-3 Mediates Transcriptional Regulation by Modulating Nucleocytoplasmic Shuttling of Tobacco DNA-binding Protein Phosphatase-1. Journal of Biological Chemistry, 281(32), 22875-22881. doi:10.1074/jbc.m512611200Colcombet, J., & Hirt, H. (2008). ArabidopsisMAPKs: a complex signalling network involved in multiple biological processes. Biochemical Journal, 413(2), 217-226. doi:10.1042/bj20080625Kiegerl, S., Cardinale, F., Siligan, C., Gross, A., Baudouin, E., Liwosz, A., … Meskiene, I. (2000). SIMKK, a Mitogen-Activated Protein Kinase (MAPK) Kinase, Is a Specific Activator of the Salt Stress–Induced MAPK, SIMK. The Plant Cell, 12(11), 2247-2258. doi:10.1105/tpc.12.11.2247CAMPS, M., NICHOLS, A., & ARKINSTALL, S. (2000). Dual specificity phosphatases: a gene family for control of MAP kinase function. The FASEB Journal, 14(1), 6-16. doi:10.1096/fasebj.14.1.6Bethke, G., Pecher, P., Eschen-Lippold, L., Tsuda, K., Katagiri, F., Glazebrook, J., … Lee, J. (2012). Activation of the Arabidopsis thaliana Mitogen-Activated Protein Kinase MPK11 by the Flagellin-Derived Elicitor Peptide, flg22. Molecular Plant-Microbe Interactions, 25(4), 471-480. doi:10.1094/mpmi-11-11-0281Wolschin, F., Wienkoop, S., & Weckwerth, W. (2005). Enrichment of phosphorylated proteins and peptides from complex mixtures using metal oxide/hydroxide affinity chromatography (MOAC). PROTEOMICS, 5(17), 4389-4397. doi:10.1002/pmic.200402049Petersen, M., Brodersen, P., Naested, H., Andreasson, E., Lindhart, U., Johansen, B., … Mundy, J. (2000). Arabidopsis MAP Kinase 4 Negatively Regulates Systemic Acquired Resistance. Cell, 103(7), 1111-1120. doi:10.1016/s0092-8674(00)00213-0Asai, T., Tena, G., Plotnikova, J., Willmann, M. R., Chiu, W.-L., Gomez-Gomez, L., … Sheen, J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature, 415(6875), 977-983. doi:10.1038/415977aKosetsu, K., Matsunaga, S., Nakagami, H., Colcombet, J., Sasabe, M., Soyano, T., … Machida, Y. (2010). The MAP Kinase MPK4 Is Required for Cytokinesis in Arabidopsis thaliana. The Plant Cell, 22(11), 3778-3790. doi:10.1105/tpc.110.077164Koroleva, O. A., Tomlinson, M. L., Leader, D., Shaw, P., & Doonan, J. H. (2004). High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions. The Plant Journal, 41(1), 162-174. doi:10.1111/j.1365-313x.2004.02281.xVierstra, R. D. (2009). The ubiquitin–26S proteasome system at the nexus of plant biology. Nature Reviews Molecular Cell Biology, 10(6), 385-397. doi:10.1038/nrm2688Gökirmak, T., Paul, A.-L., & Ferl, R. J. (2010). Plant phosphopeptide-binding proteins as signaling mediators. Current Opinion in Plant Biology, 13(5), 527-532. doi:10.1016/j.pbi.2010.06.001Keyse, S. M. (2000). Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Current Opinion in Cell Biology, 12(2), 186-192. doi:10.1016/s0955-0674(99)00075-7Gupta, R., & Luan, S. (2003). Redox Control of Protein Tyrosine Phosphatases and Mitogen-Activated Protein Kinases in Plants. Plant Physiology, 132(3), 1149-1152. doi:10.1104/pp.103.020792Katou, S., Karita, E., Yamakawa, H., Seo, S., Mitsuhara, I., Kuchitsu, K., & Ohashi, Y. (2005). Catalytic Activation of the Plant MAPK Phosphatase NtMKP1 by Its Physiological Substrate Salicylic Acid-induced Protein Kinase but Not by Calmodulins. Journal of Biological Chemistry, 280(47), 39569-39581. doi:10.1074/jbc.m508115200Schweighofer, A., Kazanaviciute, V., Scheikl, E., Teige, M., Doczi, R., Hirt, H., … Meskiene, I. (2007). The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in Arabidopsis. The Plant Cell, 19(7), 2213-2224. doi:10.1105/tpc.106.049585Ulm, R. (2001). Mitogen-activated protein kinase phosphatase is required for genotoxic stress relief in Arabidopsis. Genes & Development, 15(6), 699-709. doi:10.1101/gad.192601Yamakawa, H., Katou, S., Seo, S., Mitsuhara, I., Kamada, H., & Ohashi, Y. (2003). Plant MAPK Phosphatase Interacts with Calmodulins. Journal of Biological Chemistry, 279(2), 928-936. doi:10.1074/jbc.m310277200Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Gerstein, M., Snyder, M., & Dinesh-Kumar, S. P. (2008). MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes & Development, 23(1), 80-92. doi:10.1101/gad.1740009Sato, T., Maekawa, S., Yasuda, S., Domeki, Y., Sueyoshi, K., Fujiwara, M., … Yamaguchi, J. (2011). Identification of 14-3-3 proteins as a target of ATL31 ubiquitin ligase, a regulator of the C/N response in Arabidopsis. The Plant Journal, 68(1), 137-146. doi:10.1111/j.1365-313x.2011.04673.xHunter, T. (2007). The Age of Crosstalk: Phosphorylation, Ubiquitination, and Beyond. Molecular Cell, 28(5), 730-738. doi:10.1016/j.molcel.2007.11.01