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Downstream Targets (downstream + target)
Terms modified by Downstream Targets Selected AbstractsIdentification of Tgf,1i4 as a downstream target of Foxc1DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2006Paula Sommer Craniofacial development is severely affected by null mutations in Foxc1, indicating a multifunctional role for Foxc1 in ocular, maxilla and mandible, skull and facial gland development. To delineate signaling pathways in which Foxc1 is involved we compared the transcriptomes of whole heads of Foxc1+/+ and Foxc1,/, embryos using a candidate cDNA array comprising genes expressed in the head mesenchyme and ocular region, and a 7K oligo array. Absence of Foxc1 led to downregulation of Stat1 and Galnt4, and upregulation of Tgf,1i4 at embryonic day 13.5 in the developing head mesenchyme. Comparative analyses revealed differences in the expression pattern of Tgf,1i4 in the head mesenchyme of Foxc1,/, and Foxc1+/+ embryos. In the ocular regions of Foxc1,/, embryos, Tgf,1i4 was expressed in higher levels in the conjunctival epithelium and in the condensing mesenchyme on the nasal aspect of the developing eye while in wild-type embryos more intense expression was seen in the mesenchyme on the temporal aspect of the eye. Such data indicate that Foxc1 regulation of Tgf,1i4 is complex and may be cell-type dependent. Analysis of the regulation of Tgf,1i4 by Foxc1 in a more homogenous cell population, mesenchymal cells isolated from the periocular region revealed that, in these cells, Foxc1 negatively regulated Tgf,1i4 expression, presumably via secreted factors such as TGF-,1. Since Foxc1 expression is essential for normal craniofacial development, it is possible that its downstream targets play a role in the development of the phenotypes associated with null mutations in Foxc1. [source] Fjx1: A notch-inducible secreted ligand with specific binding sites in developing mouse embryos and adult brainDEVELOPMENTAL DYNAMICS, Issue 3 2005Rebecca Rock Abstract The mouse fjx1 gene was identified as a homologue to the Drosophila gene four-jointed (fj). Fj encodes a transmembrane type II glycoprotein that is partially secreted. The gene was found to be a downstream target of the Notch signaling pathway in leg segmentation and planar cell polarity processes during eye development of Drosophila. Here, we show that fjx1 is not only conserved in vertebrates, but we also identified the murine fjx1 gene as a direct target of Notch signaling. In addition to the previously described expression of fjx1 in mouse brain, we show here that fjx1 is expressed in the peripheral nervous system, epithelial cells of multiple organs, and during limb development. The protein is processed and secreted as a presumptive ligand. Through the use of an fjx1-AP fusion protein, we could visualize fjx1 binding sites at complementary locations, supporting the notion that fjx1 may function as a novel signaling molecule. Developmental Dynamics 234:602,612, 2005. © 2005 Wiley-Liss, Inc. [source] ,-catenin mediates insulin-like growth factor-I actions to promote cyclin D1 mRNA expression, cell proliferation and survival in oligodendroglial culturesGLIA, Issue 9 2010Ping Ye Abstract By promoting cell proliferation, survival and maturation insulin-like growth factor (IGF)-I is essential to the normal growth and development of the central nervous system. It is clear that IGF-I actions are primarily mediated by the type I IGF receptor (IGF1R), and that phosphoinositide 3 (PI3)-Akt kinases and MAP kinases signal many of IGF-I-IGF1R actions in neural cells, including oligodendrocyte lineage cells. The precise downstream targets of these signaling pathways, however, remain to be defined. We studied oligodendroglial cells to determine whether ,-catenin, a molecule that is a downstream target of glycogen synthase kinase-3, (GSK3,) and plays a key role in the Wnt canonical signaling pathway, mediates IGF-I actions. We found that IGF-I increases ,-catenin protein abundance within an hour after IGF-I-induced phosphorylation of Akt and GSK3,. Inhibiting the PI3-Akt pathway suppressed IGF-I-induced increases in ,-catenin and cyclin D1 mRNA, while suppression of GSK3, activity simulated IGF-I actions. Knocking-down ,-catenin mRNA by RNA interference suppressed IGF-I-stimulated increases in the abundance of cyclin D1 mRNA, cell proliferation, and cell survival. Our data suggest that ,-catenin is an important downstream molecule in the PI3-Akt-GSK3, pathway, and as such it mediates IGF-I upregulation of cyclin D1 mRNA and promotion of cell proliferation and survival in oligodendroglial cells. © 2010 Wiley-Liss, Inc. [source] Phosphorylation and lipid raft association of fibroblast growth factor receptor-2 in oligodendrocytes,GLIA, Issue 9 2009M. R. Bryant Abstract Fibroblast growth factors (FGFs) and their receptors (FGFRs) initiate diverse cellular responses that contribute to the regulation of oligodendrocyte (OL) function. To understand the mechanisms by which FGFRs elicit these cellular responses, we investigated the phosphorylation of signal transduction proteins and the role of cholesterol-glycosphingolipid-enriched "lipid raft" microdomains in differentiated OLs. Surprisingly, we found that the most abundant tyrosine-phosphorylated protein in OLs was the 120-kd isoform of FGFR2 and that it was phosphorylated even in the absence of FGF2, suggesting a potential ligand-independent function for this receptor. Furthermore, FGFR2, but not FGFR1, was associated with lipid raft microdomains in OLs and myelin (but not in astrocytes). This provides the first evidence for the association of FGFR with TX-100-insoluble lipid raft fractions. FGFR2 phosphorylated the key downstream target, FRS2 in OLs. Raft disruption resulted in loss of phosphorylated FRS2 from lipid rafts, coupled with the loss of Akt but not of Mek or Erk phosphorylation. This suggests that FGFR2-FRS2 signaling in lipid rafts operates via the PI3-Kinase/Akt pathway rather than the Ras/Mek/Erk pathway, emphasizing the importance of microenvironments within the cell membrane. Also present in lipid rafts in OLs and myelin, but not in astrocytes, was a novel 52-kd isoform of FGFR2 that lacked the extracellular ligand-binding region. These results demonstrate that FGFR2 in OLs and myelin possess unique characteristics that are specific both to receptor type and to OLs and provide a novel mechanism to elicit distinct cellular responses that mediate both FGF-dependent and -independent functions. © 2008 Wiley-Liss, Inc. [source] Synergistic induction of cyclin D1 in oligodendrocyte progenitor cells by IGF-I and FGF-2 requires differential stimulation of multiple signaling pathwaysGLIA, Issue 10 2007Terra J. Frederick Abstract D-type cyclins are direct targets of extracellular signals and critical regulators of G1 progression. Our previous data demonstrated that IGF-I and FGF-2 synergize to enhance cyclin D1 expression, cyclin E/cdk2 complex activation, and S-phase entry in OP cells. Here, we provide a mechanistic explanation for how two growth factor signaling pathways converge on a major cell cycle regulator. IGF-I and FGF-2 differentially activate signaling pathways to coordinately promote cyclin D1 expression. We show that the p44/p42 MAPK signaling pathway is essential for FGF-2 induction of cyclin D1 mRNA. In contrast, blocking the PI3-Kinase pathway results in loss of IGF-I/FGF-2 synergistic induction of cyclin D1 protein levels. Moreover, the presence of IGF-I significantly enhances nuclear localization of cyclin D1, which also requires PI3K signaling. GSK-3,, a downstream target of the PI3K/Akt pathway, is phosphorylated in the presence of IGF-I in OPs. Consistent with a known role for GSK-3, in cyclin D1 degradation, we show that proteasome inhibition in OPs exposed to FGF-2 increased cyclin D1 levels, equivalent to levels seen in IGF-I/FGF-2 treated cells. Thus, we provide a model for cyclin D1 coordinate regulation where FGF-2 stimulation of the MAPK pathway promotes cyclin D1 mRNA expression while IGF-I activation of the PI3K pathway inhibits proteasome degradation of cyclin D1 and enhances nuclear localization of cyclin D1. © 2007 Wiley-Liss, Inc. [source] Nmi (N-Myc interactor) inhibits Wnt/,-catenin signaling and retards tumor growthINTERNATIONAL JOURNAL OF CANCER, Issue 3 2009Rebecca A. Fillmore Abstract We found that the expression levels of N-Myc interactor (Nmi) were low in aggressive breast cancer cell lines when compared with less aggressive cell lines. However, the lower levels in the aggressive lines were inducible by interferon-, (IFN-,). Because Nmi has been reported to be a transcription cofactor that augments IFN-, induced transcription activity, we decided to test whether Nmi regulates expression of Dkk1, which is also inducible by IFN-,. We established stable clones constitutively expressing Nmi in MDA-MB-231 (breast) and MDA-MB-435 (melanoma) cell lines. Dkk1 was significantly up-regulated in the Nmi expressing clones concurrent with reduced levels of the critical transcription cofactor of Wnt pathway, ,-catenin. Treatment of the Nmi expressors with blocking antibody to Dkk1 restored ,-catenin protein levels. c-Myc is a known downstream target of activated ,-catenin signaling. Treatment of Nmi expressors with the proteosome inhibitor MG132, resulted in elevated ,-catenin levels with concomitant elevation of c-Myc levels. Our functional studies showed that constitutive expression of Nmi reduced the ability of tumor cells for the invasion, anchorage independent growth and tumor growth in vivo. Collectively, the data suggest that overexpression of Nmi inhibits the Wnt/,-catenin signaling via up-regulation of Dkk1 and retards tumor growth. © 2009 UICC [source] Craniosynostosis-Associated Gene Nell-1 Is Regulated by Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2007Thien Truong Abstract We studied the transcriptional regulation of NELL-1, a craniosynostosis-related gene. We identitifed three OSE2 elements in the NELL-1 promoter that are directly bound and transactivated by Runx2. Forced expression of Runx2 induces NELL-1 expression in rat calvarial cells. Introduction: We previously reported the upregulation of NELL-1 in human craniosynostosis and the overexpression of Nell-1 in transgenic animals that induced premature suture closure associated with increased osteoblast differentiation. To study the transcriptional regulation of NELL-1, we analyzed the 5, flanking region of the human NELL-1 gene. We identified three osteoblast specific binding elements 2 (OSE2) sites (A, B, and C) within 2.2 kb upstream of the transcription start site and further studied the functionality of these sites. Materials and Methods: An area of 2.2 kb and a truncated 325 bp, which lacked the three OSE sites, were cloned into a luciferase reporter gene, and co-transfected with Runx2 expression plasmid. The three OSE2 sites were individually mutated and co-transfected with Runx2 expression plasmid into Saos2 cells. Gel shifts and supershifts with Runx2 antibodies were used to determine specific binding to OSE2 sites. CHIP assays were used to study in vivo binding of Runx2 to the Nell-1 promoter. Runx2 expression plasmid was transfected into wildtype and Runx2,/, calvarial cells. Nell-1, osteocalcin, and Runx2 expression levels were measured using RT-PCR. Results: Addition of Runx2 dose-dependently increased the luciferase activity in the human NELL-1 promoter-luciferase p2213. The p325 truncated NELL-1 construct showed significantly lower basal level of activity. Nuclear extract from Saos2 cells formed complexes with site A, B, and C probes and were supershifted with Runx2 antibody. Mutation of sites A, B, and C significantly decreased basal promoter activity. Furthermore, mutation of sites B and C had a blunted response to Runx2, whereas mutation of site A had a lesser effect. Runx2 bound to NELL-1 promoter in vivo. Transfection of Runx2 in rat osteoblasts upregulated Nell-1 and Ocn expression, and in Runx2 null calvarial cells, both Nell-1 and Ocn expression were rescued. Conclusions: Runx2 directly binds to the OSE2 elements and transactivates the human NELL-1 promoter. These results suggest that Nell-1 is likely a downstream target of Runx2. These findings may also extend our understanding of the molecular mechanisms governing the pathogenesis of craniosynostosis. [source] FGFR1/PI3K/AKT signaling pathway is a novel target for antiangiogenic effects of the cancer drug Fumagillin (TNP-470)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2007Gregory J. Chen Abstract Fibroblast growth factor-1 (FGF1), a prototypic member of the FGF family, is a potent angiogenic factor. Although FGF-stimulated angiogenesis has been extensively studied, the molecular mechanisms regulating FGF1-induced angiogenesis are poorly understood in vivo. Fumagillin, an antiangiogenic fungal metabolite, has the ability to inhibit FGF-stimulated angiogenesis in the chicken chorioallantoic membrane (CAM). In the current study, chicken CAMs were transfected with a signal peptide-containing version of the FGF1 gene construct (sp-FGF1). Transfected CAMs were then analyzed in the presence and absence of fumagillin treatment with respect to the mRNA expression levels and protein activity of the FGF1 receptor protein (FGFR1), phosphatidylinositol 3-kinase (PI3K), and its immediate downstream target, AKT-1 (protein kinase B). Treatment of sp-FGF1-transfected CAMs with fumagillin showed downregulation for both PI3K and AKT-1 proteins in mRNA expression and protein activity. In contrast, no major alterations in FGFR1 mRNA expression level were observed. Similar patterns of mRNA expression for the above three proteins were observed when the CAMs were treated with recombinant FGF1 protein in place of sp-FGF1 gene transfection. Investigation using biotin-labeled fumagillin showed that only the FGF1 receptor protein containing the cytoplasmic domain demonstrated binding to fumagillin. Furthermore, we demonstrated endothelial-specificity of the proposed antiangiogenic signaling cascade using an in vitro system. Based on these findings, we conclude that the binding of fumagillin to the cytoplasmic domain of the FGF1 receptor inhibited FGF1-stimulated angiogenesis both in vitro and in vivo. J. Cell. Biochem. 101: 1492,1504, 2007. © 2007 Wiley-Liss, Inc. [source] Rapamycin impairs trabecular bone acquisition from high-dose but not low-dose intermittent parathyroid hormone treatmentJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2009P.J. Niziolek The osteo-anabolic effects of intermittent parathyroid hormone (PTH) treatment require insulin-like growth factor (IGF) signaling through the IGF-I receptor. A major downstream target of the IGF-I receptor (via Akt) is the mammalian target of rapamycin (mTOR), a kinase involved in protein synthesis. We investigated whether the bone-building effects of intermittent PTH require functional mTOR signaling. Mice were treated with daily PTH 1,34 (0, 10, 30, or 90,µg/kg) for 6 weeks in the presence or absence of rapamycin, a selective inhibitor of mTOR. We found that all PTH doses were effective in enhancing bone mass, whether rapamycin was present or not. Rapamycin had little to no effect on the anabolic response at low (10,µg) PTH doses, small effects in a minority of anabolic measures at moderate doses (30,µg), but the anabolic effects of high-dose PTH (90,µg) were consistently and significantly suppressed by rapamycin (,4,36% reduction). Serum levels of Trap5b, a marker of resorption, were significantly enhanced by rapamycin, but these effects were observed whether PTH was absent or present. Our data suggest that intermittent PTH, particularly at lower doses, is effective in building bone mass in the presence of rapamycin. However, the full anabolic effects of higher doses of PTH are significantly suppressed by rapamycin, suggesting that PTH might normally activate additional pathways (including mTOR) for its enhanced high-dose anabolic effects. Clinical doses of intermittent PTH could be an effective treatment for maintaining or increasing bone mass among patients taking rapamycin analogs for unrelated health issues. J. Cell. Physiol. 221: 579,585, 2009. © 2009 Wiley-Liss, Inc. [source] The PVT-1 oncogene is a Myc protein target that is overexpressed in transformed cells,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2007Letizia Carramusa The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt's lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins. J. Cell. Physiol. 213: 511,518, 2007. © 2007 Wiley-Liss, Inc. [source] Expression of the Hypothalamic Transcription Factor Nhlh2 is Dependent on Energy AvailabilityJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2007K. R. Vella Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 colocalises with the hypothalamic thyrotrophin-releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) and pro-opiomelanocortin (POMC) neurones in the arcuate nucleus. We show that Nhlh2 expression is significantly reduced in response to 24-h food deprivation in the arcuate nucleus, PVN, lateral hypothalamus, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Food intake for 2 h following deprivation stimulates Nhlh2 expression in the arcuate nucleus and the PVN, and leptin injection following deprivation results in increased Nhlh2 expression in the arcuate nucleus, PVN, lateral hypothalamus, VMH, and DMH. Hypothalamic Nhlh2 expression in response to leptin injection is maximal by 2 h. Following leptin injection, Nhlh2 mRNA colocalises in POMC neurones in the arcuate nucleus and TRH neurones in the PVN. Nhlh2 mRNA expression in POMC neurones in the arcuate nucleus and TRH neurones in the PVN is reduced with energy deprivation and is stimulated with food intake and leptin injection. Modulation of POMC expression in response to changes in energy availability is not affected in mice with a targeted deletion of Nhlh2. However, deletion of Nhlh2 does result in loss of normal TRH mRNA expression in mice exposed to food deprivation and leptin stimulation. These data implicate Nhlh2 as a regulatory target of the leptin-mediated energy availability network of the hypothalamus, and TRH as a putative downstream target of Nhlh2. [source] Deletion of tau attenuates heat shock-induced injury in cultured cortical neuronsJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2010Yanying Miao Abstract The microtubule-associated protein tau has been implicated in ,-amyloid- and glutamate-induced neurotoxicity. However, the potential role of tau in response to other insults to neurons remains unclear. In this study, we examined whether deletion of tau would change cell injury induced by heat shock in primary cultures of cortical neurons. After 30 min of a 45°C heat shock, lactate dehydrogenase (LDH) release increased, reaching a peak at 6 hr in wild-type (WT) neurons. A significantly lower LDH release, with a peak delayed by 24 hr, was detected in tau knockout (TKO) neurons. After heat shock treatment, MAP-2 and tubulin staining of the processes of WT neurons revealed more dramatic abnormalities than in TKO neurons. Both WT and TKO neurons exhibited a similar elevation of HSP70 level but different time courses of Akt phosphorylation. In contrast to an early, brief response in WT neurons, TKO neurons displayed a late, but long-lasting increase in phosphorylation of Akt and its downstream target, glycogen synthase kinase 3,. Additionally, inhibition of Akt activity aggravated the cell morbidity caused by heat shock exposure in both WT and TKO neurons, indicating a protective role of Akt against cell injury. In conclusion, our results demonstrate that deletion of tau attenuated heat shock-induced neuronal injury. Enhanced Akt response in the absence of endogenous tau is suggested to represent a compensatory mechanism for regulating cell reactions to stress stimuli. © 2009 Wiley-Liss, Inc. [source] ,-Amino-3-hydroxy-5-methyl-4-isoxazole propionate attenuates glutamate-induced caspase-3 cleavage via regulation of glycogen synthase kinase 3,JOURNAL OF NEUROSCIENCE RESEARCH, Issue 5 2008Takaaki Nishimoto Abstract Preconditioning of sublethal ischemia exhibits neuroprotection against subsequent ischemia-induced neuronal death. It has been indicated that glutamate, an excitatory amino acid, is involved in the pathogenesis of ischemia-induced neuronal death or neurodegeneration. To elucidate whether prestimulation of glutamate receptor could counter ischemia-induced neuronal death or neurodegeneration, we examined the effect of ,-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), an ionotropic subtype of glutamate receptor, on excess glutamate-induced excitotoxicity using primary cortical neuronal cultures. We found that AMPA exerted a neuroprotective effect in a time- and concentration-dependent manner. A blocker of phosphatidylinositol-3 kinase (PI3K), LY294002 (10 ,M), significantly attenuated AMPA-induced protection. In addition, Ser473 of Akt/PKB, a downstream target of PI3K, was phosphorylated by AMPA administration (10 ,M). Glycogen synthase kinase 3, (GSK3,), which has been reported to be inactivated by Akt, was phosphorylated at Ser9 by AMPA. Ser9-phosphorylated GSK3, or inactivated form would be a key molecule for neuroprotection, insofar as lithium chloride (100 ,M) and SB216763 (10 ,M), inhibitors of GSK3,, also induced phosphorylation of GSK3, at Ser9 and exerted neuroprotection, respectively. Glutamate (100 ,M) increased cleaved caspase-3, an apoptosis-related cysteine protease, and caspase-3 inhibitor (Ac-DEVD-CHO; 1 ,M) blocked glutamate-induced excitotoxicity in our culture. AMPA (10 ,M, 24 hr) and SB216763 (10 ,M) prominently decreased glutamate-induced caspase-3 cleavage. These findings suggest that AMPA activates PI3K-Akt and subsequently inhibits GSK3, and that inactivated GSK3, attenuates glutamate-induced caspase-3 cleavage and neurotoxicity. © 2007 Wiley-Liss, Inc. [source] Vertical merger: monopolization for downstream quasi-rentsMANAGERIAL AND DECISION ECONOMICS, Issue 3 2009Richard S. Higgins This paper provides a welfare analysis of vertical merger between an input monopolist and downstream firms that compete perfectly in a homogeneous product market. The distinguishing feature of the present model is that the downstream firms face capacity constraints. As a result of downstream quasi-rents, vertical merger,the extent of merger is gauged by the capacity share of the acquired downstream firm,may either raise or lower final output. An analytical criterion for distinguishing pro- and anti-competitive mergers is derived, which relies entirely on pre-merger market quantities and the capacity share of the downstream target. A common result is that vertical merger is output-increasing even when unaffiliated downstream rivals are completely foreclosed. Copyright © 2008 John Wiley & Sons, Ltd. [source] Interferon-inducible protein IFIX, inhibits cell invasion by upregulating the metastasis suppressor maspin,MOLECULAR CARCINOGENESIS, Issue 10 2008Hirohito Yamaguchi Abstract IFIX,, a member of the interferon-inducible HIN-200 family, has been identified as a putative tumor suppressor. However, the molecular mechanisms underlying IFIX,-mediated tumor suppression are poorly understood. In the present study, we demonstrated that the metastasis suppressor maspin acts as the downstream target of IFIX,. IFIX, suppressed the invasion activity of MDA-MB-468 breast cancer cells, and its inhibitory effect was reversed by the knockdown of maspin. Both Maspin mRNA and protein were upregulated by IFIX,. Histone deacetylase (HDAC) inhibitors, but not DNA methyltransferase inhibitor upregulated maspin, and HDAC1 inhibited the transactivation of maspin promoter. Although the HDAC1 protein was downregulated in IFIX,-expressing cells, IFIX, did not affect HDAC1 mRNA levels. Conversely, a proteasome inhibitor restored the level of HDAC1 protein in IFIX,-expressing cells, and the polyubiqutination of HDAC1 was promoted by IFIX,, suggesting that HDAC1 is regulated by IFIX, through a ubiquitin-proteasome pathway. Together, these data provide novel insights into the tumor-suppressive function of IFIX,. © 2008 Wiley-Liss, Inc. [source] Pediatric ALK+ anaplastic large cell lymphoma with t(3;8)(q26.2;q24) translocation and c-myc rearrangement terminating in a leukemic phaseAMERICAN JOURNAL OF HEMATOLOGY, Issue 1 2007Sara Monaco Abstract Pediatric ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) is usually associated with a favorable prognosis. ALK+ ALCL associated with a leukemic phase is uncommon, but has been associated with an aggressive clinical course and unfavorable prognosis. Overexpression of c-myc has been shown to be a consistent finding in ALK+, but not ALK-negative ALCL (ALK, ALCL), and the c-myc gene is considered a downstream target of deregulated ALK signaling. We describe a pediatric ALK+ ALCL with a leukemic phase at relapse. Similar to other rare cases described in the literature, it followed an aggressive clinical course despite multiple regimens of chemotherapy and bone marrow transplantation. Lymphoma cells showed aberrant ALK expression and c-myc overexpression. In addition to the characteristic t(2;5)(p23;q35) translocation, a t(3;8)(q26.2;q24) translocation was also present, and c-myc gene rearrangement was confirmed by FISH analysis. The findings in this case demonstrate the association of peripheral blood leukemic involvement and aggressive clinical course, and suggest that other factors, such as c-myc rearrangement, may be responsible for the aggressive clinical behavior in ALK+ ALCL. Am. J. Hematol., 2006. © 2006 Wiley-Liss, Inc. [source] AKT2 is a downstream target of metabotropic glutamate receptor 1 (Grm1)PIGMENT CELL & MELANOMA RESEARCH, Issue 1 2010Seung-Shick Shin Summary We reported earlier on the oncogenic properties of Grm1 by demonstrating that stable Grm1 -mouse-melanocytic clones proliferate in the absence of growth supplement and anchorage in vitro. In addition, these clones also exhibit aggressive tumorigenic phenotypes in vivo with short latency in tumor formation in both immunodeficient and syngeneic mice. We also detected strong activation of AKT in allograft tumors specifically AKT2 as the predominant isoform involved. In parallel, we assessed several human melanoma biopsy samples and found again that AKT2 was the predominantly activated AKT in these human melanoma biopsies. In cultured stable Grm1 -mouse-melanocytic clones, as well as an metabotropic glutamate receptor 1 (Grm1) expressing human melanoma cell line, C8161, stimulation of Grm1 by its agonist led to the activation of AKT, while preincubation with Grm1-antagonist abolished Grm1-agonist-induced AKT activation. In addition, a reduction in tumor volume of Grm1 -mouse-melanocytic-allografts was detected in the presence of small interfering AKT2 RNA (siAKT2). Taken together, these results showed that, in addition to the MAPK pathway previously reported being a downstream target of stimulated Grm1, AKT2 is another downstream target in Grm1 mediated melanocyte transformation. [source] Proteomic analysis of EZH2 downstream target proteins in hepatocellular carcinomaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 17 2007Yangchao Chen Abstract Enhancer of zeste homolog 2 (EZH2) is suggested to be a potential therapeutic target and a diagnostic marker for cancer. Our previous study also showed the critical role of EZH2 in hepatocellular carcinoma (HCC) tumorigenesis. The present study is aimed at revealing the comprehensive downstream pathways of EZH2 by functional proteomic profiling. Lentivirus mediated RNA interference (RNAi) was employed to knockdown EZH2 in HCC cells. The 2-DE was employed to compare the expression profile difference between parental and EZH2-knockdown HCC cells. In total, 28 spots were differentially expressed during EZH2 inhibition. Among all, 18 proteins were identified by PMF with MALDI-TOF MS. Western blotting further validated upregulation of 60S acidic ribosomal protein P0 (L10E), and downregulation of two proteins with EZH2 inhibition: stathmin1 and probable protein disulfide isomerase (PDI) ER-60 precursor (ERp57). Moreover, L10E was downregulated with overexpression of EZH2 in hepatocytes, and L10E reversed the effect of EZH2 on cell proliferation, suggesting it a downstream target of EZH2. The comprehensive and comparative analyses of proteins associated with EZH2 could further our understanding on the downstream signal cascade of EZH2 leading to tumorigenesis. [source] Quantitative effects of vernalization on FLC and SOC1 expressionTHE PLANT JOURNAL, Issue 6 2006Candice C. Sheldon Summary Prolonged exposure to cold results in early flowering in Arabidopsis winter annual ecotypes, with longer exposures resulting in a greater promotion of flowering than shorter exposures. The promotion of flowering is mediated through an epigenetic down-regulation of the floral repressor FLOWERING LOCUS C (FLC). We present results that provide an insight into the quantitative regulation of FLC by vernalization. Analysis of the effect of seed or plant cold treatment on FLC expression indicates that the time-dependent nature of vernalization on FLC expression is mediated through the extent of the initial repression of FLC and not by affecting the ability to maintain the repressed state. In the over-expression mutant flc-11, the time-dependent repression of FLC correlates with the proportional deacetylation of histone H3. Our results indicate that sequences within intron 1 and the activities of both VERNALIZATION1 (VRN1) and VERNALIZATION2 (VRN2) are required for efficient establishment of FLC repression; however, VRN1 and VRN2 are not required for maintenance of the repressed state during growth after the cold exposure. SUPPRESSOR OF OVER-EXPRESSION OF CO 1 (SOC1), a downstream target of FLC, is quantitatively induced by vernalization in a reciprocal manner to FLC. In addition, we show that SOC1 undergoes an acute induction by both short and long cold exposures. [source] Mutant p53 and cyclin A1 protein expression in primary laryngeal squamous cell carcinomas do not correlate to second primary tumours of the head and neck,ANZ JOURNAL OF SURGERY, Issue 1-2 2009Ross D. Farhadieh Abstract Background:, Field cancerization is a feature of head and neck squamous cell carcinoma. No biological marker in the index tumour has been correlated to the development of second primary tumours (SPT). Cyclin A1 is a cell cycle regulator and a downstream target of p53. This study assessed predictive correlation of cyclin A1 and mut-p53 with clinicopathological parameters and occurrence of (SPT) 7in the head and neck. Methods:, Using immunohistochemistry 106 patients treated for primary laryngeal squamous cell carcinoma were investigated for expression of cyclin A1 and mut-p53. Results:, Expression of cyclin A1 and mut-p53 were noted in 83 of 106 (78.3%) and 25 of 106 (23.6%) patients. There was a weak but significant correlation between mut-p53 and cyclin A1 (r = 0.301, P = 0.002) expression. During the follow-up period (median 41.0 months (range 1,205 months)), 21 of 106 (19.8%) patients developed an SPT. There was no statistically significant correlation between the markers investigated and disease recurrence, SPT diagnosis or clinicopathological parameters. Conclusion:, Second primary tumours are an intriguing problem in treatment of HNSCC and a predictive marker identifying those greatest at risk would be a leap forward. [source] HLA,B27 misfolding and the unfolded protein response augment interleukin-23 production and are associated with Th17 activation in transgenic ratsARTHRITIS & RHEUMATISM, Issue 9 2009Monica L. DeLay Objective To determine whether HLA,B27 misfolding and the unfolded protein response (UPR) result in cytokine dysregulation and whether this is associated with Th1 and/or Th17 activation in HLA,B27/human ,2 -microglobulin (Hu,2m),transgenic rats, an animal model of spondylarthritis. Methods Cytokine expression in lipopolysaccharide (LPS),stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or by HLA,B27 up-regulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real-time reverse transcription,polymerase chain reaction analysis, and differences in Th1 and Th17 CD4+ T cell populations were quantified after intracellular cytokine staining. Results Interleukin-23 (IL-23) was found to be synergistically up-regulated by LPS in macrophages undergoing a UPR induced by pharmacologic agents or by HLA,B27 misfolding. IL-23 was also increased in the colon tissue from B27/Hu,2m-transgenic rats concurrently with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust up-regulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen-presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17,expressing T cells. Conclusion The IL-23/IL-17 axis is strongly activated in the colon of B27/Hu,2m-transgenic rats with spondylarthritis-like disease. HLA,B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine IL-23. These results suggest a possible link between HLA,B27 misfolding and immune dysregulation in this animal model, with implications for human disease. [source] Emergence of protein kinase CK2 as a key target in cancer therapyBIOFACTORS, Issue 3 2010Janeen H. Trembley Abstract Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death, and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, for example, NF-,B as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a "master regulator" in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub-50 nm (i.e., less than 50 nm size) nanocapsule in which an anti-CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer. [source] Yes-associated protein is an independent prognostic marker in hepatocellular carcinomaCANCER, Issue 19 2009Michelle Z. Xu MD Abstract BACKGROUND: Yes-associated protein (YAP), a downstream target of the Hippo signaling pathway, was recently linked to hepatocarcinogenesis in a mouse hepatocellular carcinoma (HCC) model. The objective of the current study was to investigate the clinical significance of YAP in HCC and its prognostic values in predicting survival and tumor recurrence. METHODS: The authors collected 177 pairs of tumor and adjacent nontumor tissue from HCC patients with definitive clinicopathologic and follow-up data. YAP expression was determined by immunohistochemistry, Western blot analysis, and quantitative polymerase chain reaction. Association of YAP with each clinicopathologic feature was analyzed by Pearson chi-square test, and HCC-specific disease-free survival and overall survival by Kaplan-Meier curves and log-rank test. Multivariate Cox regression analyses of YAP in HCC were also performed. RESULTS: YAP was expressed in the majority of HCC cases (approximately 62%) and mainly accumulated in the tumor nucleus. Overexpression of YAP in HCC was significantly associated with poorer tumor differentiation (Edmonson grade; P = .021) and high serum ,-fetoprotein (AFP) level (P < .001). Kaplan-Meier and Cox regression data indicated that YAP was an independent predictor for HCC-specific disease-free survival (hazards ratio [HR], 1.653; 95% confidence interval [95% CI], 1.081-2.528 [P = .02]) and overall survival (HR, 2.148; 95% CI, 1.255-3.677 [P = .005]). CONCLUSIONS: YAP is an independent prognostic marker for overall survival and disease-free survival times of HCC patients and clinicopathologically associated with tumor differentiation and serum AFP level. It is a potential therapeutic target for this aggressive malignancy. Cancer 2009. © 2009 American Cancer Society. [source] Lack of Evidence of Association of p21WAF1/CIP1 Polymorphism with Lung Cancer Susceptibility and Prognosis in TaiwanCANCER SCIENCE, Issue 1 2000Chuen-Ming Shih An association between the Arg allele of the p21WAF1/CIP1 codon 31 polymorphism and lung cancer has been reported. However, the genotype distribution of the p21 codon 31 polymorphism, as well as the association of this polymorphism with lung cancer risk and prognosis, remain undefined in the Taiwanese population. Therefore, we investigated the genotype distribution of the p21 codon 31 polymorphism in 155 lung cancer patients and 189 non-cancer controls. The genotype frequencies in the Taiwanese non-cancer controls were 0.51 (Ser) and 0.49 (Arg). ,2 analysis indicated significant differences in Taiwanese genotype distribution of p21 from those reported for Swedes (P=0.001), Caucasians (P=0.001), Indians (P=0.001), and African-Americans (P=0.001). However, our data did not demonstrate an association of the Arg allele of the p21 polymorphism with lung cancer risk in Taiwan. Lung cancer patients with Ser/Arg and Arg/Arg genotypes were at a nonsignificant 1.15-fold increased risk of lung cancer when compared to individuals with the Ser/Ser genotype (95%CI, 0.70,1.86). In addition, although p21 is a downstream target of p53, we found no significant correlation of the p21 polymorphism with the p53 polymorphism and p53 gene mutation in lung cancer patients. We further investigated the association of the p21 polymorphism with prognosis in 154 lung cancer patients. Patients with the Ser/Ser genotype tended to have a poorer prognosis than those with the Ser/Arg and Arg/Arg genotypes (P=0.097, by the log rank test). Our data suggest that the p21 codon 31 polymorphism may not play a significant role in cancer susceptibility and the prognosis of lung cancer patients in Taiwan. [source] Regioselective Carbon,Carbon Bond Formation in Proteins with Palladium Catalysis; New Protein Chemistry by Organometallic ChemistryCHEMBIOCHEM, Issue 1 2006Koichiro Kodama Abstract Palladium-catalyzed reactions have contributed to the advancement of many areas of organic chemistry, in particular, the synthesis of organic compounds such as natural products and polymeric materials. In this study, we have used a Mizoroki,Heck reaction for site-specific carbon,carbon bond formation in the Ras protein. This was performed by the following two steps: 1) the His6 -fused Ras protein containing 4-iodo- L -phenylalanine at position 32 (iF32-Ras-His) was prepared by genetic engineering and 2) the aryl iodide group on the iF32-Ras-His was coupled with vinylated biotin in the presence of a palladium catalyst. The biotinylation was confirmed by Western blotting and liquid chromatography,mass spectrometry (LC-MS). The regioselectivity of the Mizoroki,Heck reaction was furthermore confirmed by LC-MS/MS analysis. However, in addition to the biotinylated product (bF32-Ras-His), a dehalogenated product (F32-Ras-His) was detected by LC-MS/MS. This dehalogenation resulted from the undesired termination of the Mizoroki,Heck reaction due to steric and electrostatic hindrance around residue 32. The biotinylated Ras showed binding activity for the Ras-binding domain as its downstream target, Raf-1, with no sign of decomposition. This study is the first report of an application of organometallic chemistry in protein chemistry. [source] Androgen replacement therapy improves function in male rat muscles independently of hypertrophy and activation of the Akt/mTOR pathwayACTA PHYSIOLOGICA, Issue 4 2009C. Hourdé Abstract Aim:, We analysed the effect of physiological doses of androgens following orchidectomy on skeletal muscle and bone of male rats, as well as the relationships between muscle performance, hypertrophy and the Akt/mammalian target of rapamycin (mTOR) signalling pathway involved in the control of anabolic and catabolic muscle metabolism. Methods:, We studied the soleus muscle and tibia from intact rats (SHAM), orchidectomized rats treated for 3 months with vehicle (ORX), nandrolone decanoate (NAN) or dihydrotestosterone (DHT). Results:, Orchidectomy had very little effect on the soleus muscle. However, maximal force production by soleus muscle (+69%) and fatigue resistance (+35%) in NAN rats were both increased when compared with ORX rats. In contrast, DHT treatment did not improve muscle function. The relative number of muscle fibres expressing slow myosin heavy chain and citrate synthase activity were not different in NAN and ORX rats. Moreover, NAN and DHT treatments did not modify muscle weights and cross-sectional area of muscle fibres. Furthermore, phosphorylation levels of downstream targets of the Akt/mTOR signalling pathway, Akt, ribosomal protein S6 and eukaryotic initiation factor 4E-binding protein 1 were similar in muscles of NAN, DHT and ORX rats. In addition, trabecular tibia from NAN and DHT rats displayed higher bone mineral density and bone volume when compared with ORX rats. Only in NAN rats was this associated with increased bone resistance to fracture. Conclusion:, Physiological doses of androgens are beneficial to muscle performance in orchidectomized rats without relationship to muscle and fibre hypertrophy and activation of the Akt/mTOR signalling pathway. Taken together our data clearly indicate that the activity of androgens on muscle and bone could participate in the global improvement of musculoskeletal status in the context of androgen deprivation induced by ageing. [source] Maspin controls mammary tumor cell migration through inhibiting Rac1 and Cdc42, but not the RhoA GTPaseCYTOSKELETON, Issue 5 2007Heidi Y. Shi Abstract Rac1 and Cdc42 are members of the Rho family of small GTPases that play essential roles in diverse cellular functions, including cell migration. The activities of these Rho family proteins are controlled by growth factor receptor activation and cell-ECM interactions. Here, we show that maspin, a well-documented tumor suppressor gene, also controls cell motility through inhibiting Rac1/Cdc42 activity. Using the GST-PAK and GST-Rho binding protein pull-down assays for GTP-bound Rac1, Cdc42, and RhoA, we showed that treatment of MDA-MB-231 tumor cells with recombinant maspin for a short time period significantly inhibited the activity of Rac1 and Cdc42, but not RhoA. The reactive site loop (RSL) within maspin protein is the functional domain involved in the inhibition. Maspin mutants with the RSL deleted or a point mutation in the RSL region lost their inhibitory activity. We further examined the ability of maspin to inhibit Rac1- and Cdc42-mediated signaling pathways and transcription factors. Treatment of MDA-MB-231 cells with maspin led to the inhibition of JNK kinase activity as assayed by immuno-kinase assays. In addition, the AP-1 transcription activity downstream of JNK kinase pathway was also reduced. Together, we have identified Rac1 and Cdc42 as the downstream targets that mediate the inhibition of mammary tumor cell migration by maspin. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Identification of Tgf,1i4 as a downstream target of Foxc1DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2006Paula Sommer Craniofacial development is severely affected by null mutations in Foxc1, indicating a multifunctional role for Foxc1 in ocular, maxilla and mandible, skull and facial gland development. To delineate signaling pathways in which Foxc1 is involved we compared the transcriptomes of whole heads of Foxc1+/+ and Foxc1,/, embryos using a candidate cDNA array comprising genes expressed in the head mesenchyme and ocular region, and a 7K oligo array. Absence of Foxc1 led to downregulation of Stat1 and Galnt4, and upregulation of Tgf,1i4 at embryonic day 13.5 in the developing head mesenchyme. Comparative analyses revealed differences in the expression pattern of Tgf,1i4 in the head mesenchyme of Foxc1,/, and Foxc1+/+ embryos. In the ocular regions of Foxc1,/, embryos, Tgf,1i4 was expressed in higher levels in the conjunctival epithelium and in the condensing mesenchyme on the nasal aspect of the developing eye while in wild-type embryos more intense expression was seen in the mesenchyme on the temporal aspect of the eye. Such data indicate that Foxc1 regulation of Tgf,1i4 is complex and may be cell-type dependent. Analysis of the regulation of Tgf,1i4 by Foxc1 in a more homogenous cell population, mesenchymal cells isolated from the periocular region revealed that, in these cells, Foxc1 negatively regulated Tgf,1i4 expression, presumably via secreted factors such as TGF-,1. Since Foxc1 expression is essential for normal craniofacial development, it is possible that its downstream targets play a role in the development of the phenotypes associated with null mutations in Foxc1. [source] Neuroprotective signal transduction in model motor neurons exposed to thrombin: G-protein modulation effects on neurite outgrowth, Ca2+ mobilization, and apoptosis ,DEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001Irina V. Smirnova Abstract Thrombin, the ultimate protease in the blood coagulation cascade, mediates its known cellular effects by unique proteolytic activation of G-protein-coupled protease-activated receptors (PARs), such as PAR1, PAR3, and PAR4, and a "tethered ligand" mechanism. PAR1 is variably expressed in subpopulations of neurons and largely determines thrombin's effects on morphology, calcium mobilization, and caspase-mediated apoptosis. In spinal cord motoneurons, PAR1 expression correlates with transient thrombin-mediated [Ca2+]i flux, receptor cleavage, and elevation of rest [Ca2+]i activating intracellular proteases. At nanomolar concentrations, thrombin retracts neurites via PAR1 activation of the monomeric, 21 kDa Ras G-protein RhoA, which is also involved in neuroprotection at lower thrombin concentrations. Such results suggest potential downstream targets for thrombin's injurious effects. Consequently, we employed several G-protein-specific modulators prior to thrombin exposure in an attempt to uncouple both heterotrimeric and monomeric G-proteins from motoneuronal PAR1. Cholera toxin, stimulating Gs, and lovastatin, which blocks isoprenylation of Rho, reduced thrombin-induced calcium mobilization. In contrast, pertussis toxin and mastoparan, inhibiting or stimulating Go/Gi, were found to exacerbate thrombin action. Effects on neuronal rounding and apoptosis were also detected, suggesting therapeutic utility may result from interference with downstream components of thrombin signaling pathways in human motor neuron disorders, and possibly other neurodegenerative diseases. Published 2001 John Wiley & Sons, Inc. J Neurobiol 48: 87,100, 2001 [source] MAP-kinase-activated protein kinase 2 expression and activity is induced after neuronal depolarizationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2008Tobias Thomas Abstract Mitogen-activated protein kinase-activated protein kinase (MK)2 is one of several downstream targets of p38 mitogen-activated protein kinase and has a well documented role in inflammation. Here, we describe a possible new function of MK2. We show that triggering depolarization by potassium chloride or increasing the cellular cAMP by forskolin treatment led to elevated levels of expression and activity of mouse MK2. In both treatments, the kinase inhibitor H89 completely prevented the up-regulation of MK2 at the transcript level. By the use of different cell lines we demonstrated that the induction of MK2 expression is characteristic of neuronal cells and is absent in fibroblasts, macrophages and kidney cells. In vivo, induction of a status epilepticus by systemic administration of the chemoconvulsant kainic acid resulted in markedly reduced neurodegeneration in the pyramidal layer of the hippocampus, dentate gyrus and hilus of MK2-deficient mice compared with wild-type mice. Together, our data suggest a possible role of MK2 in the cellular response after neuronal depolarization, in particular in excitotoxicity. [source] | |||||||||||||||||||||||||||||||||||||