Transfection Of Mutant Research Articles

Mild Electrical Stimulation at 0.1-ms Pulse Width Induces p53 Protein Phosphorylation and G2 Arrest in Human Epithelial Cells

Exogenous low-intensity electrical stimulation has been used for treatment of various intractable diseases despite the dearth of information on the molecular underpinnings of its effects. Our work and that of others have demonstrated that applied electrical stimulation at physiological strength or mild electrical stimulation (MES) activates the PI3K-Akt pathway, but whether MES activates other molecules remains unknown. Considering that MES is a form of physiological stress, we hypothesized that it can activate the tumor suppressor p53, which is a key modulator of the cell cycle and apoptosis in response to cell stresses. The potential response of p53 to an applied electrical current of low intensity has not been investigated. Here, we show that p53 was transiently phosphorylated at Ser-15 in epithelial cells treated with an imperceptible voltage (1 V/cm) and a 0.1-ms pulse width. MES-induced p53 phosphorylation was inhibited by pretreatment with a p38 MAPK inhibitor and transfection of dominant-negative mutants of p38, MKK3b, and MKK6b, implying the involvement of the p38 MAPK signaling pathway. Furthermore, MES treatment enhanced p53 transcriptional function and increased the expression of p53 target genes p21, BAX, PUMA, NOXA, and IRF9. Importantly, MES treatment triggered G2 cell cycle arrest, but not cell apoptosis. MES treatment had no effect on the cell cycle in HCT116 p53(-/-) cells, suggesting a dependence on p53. These findings identify some molecular targets of electrical stimulation and incorporate the p38-p53 signaling pathway among the transduction pathways that MES affects.

Paraquat Modulates Alternative Pre-mRNA Splicing by Modifying the Intracellular Distribution of SRPK2

Paraquat (PQ) is a neurotoxic herbicide that induces superoxide formation. Although it is known that its toxic properties are linked to ROS production, the cellular response to PQ is still poorly understood. We reported previously that treatment with PQ induced genome-wide changes in pre-mRNA splicing. Here, we investigated the molecular mechanism underlying PQ-induced pre-mRNA splicing alterations. We show that PQ treatment leads to the phosphorylation and nuclear accumulation of SRPK2, a member of the family of serine/arginine (SR) protein-specific kinases. Concomitantly, we observed increased phosphorylation of SR proteins. Site-specific mutagenesis identified a single serine residue that is necessary and sufficient for nuclear localization of SRPK2. Transfection of a phosphomimetic mutant modified splice site selection of the E1A minigene splicing reporter similar to PQ-treatment. Finally, we found that PQ induces DNA damage and vice versa that genotoxic treatments are also able to promote SRPK2 phosphorylation and nuclear localization. Consistent with these observations, treatment with PQ, cisplatin or γ-radiation promote changes in the splicing pattern of genes involved in DNA repair, cell cycle control, and apoptosis. Altogether, our findings reveal a novel regulatory mechanism that connects PQ to the DNA damage response and to the modulation of alternative splicing via SRPK2 phosphorylation.

Abstract LB-333: IDH1 modulates the immune response to glioblastoma.

Abstract Dendritic cell (DC) vaccines for glioblastoma (GBM) can effectively extend overall patient survival, but they are not curative. Younger patients responded poorly to vaccination in ours and other trials. Mutations in isocitrate dehydrogenase 1 (IDH1) are commonly found in younger patients, encouraging the hypothesis that IDH1 mutation may subvert the clinical benefits of DC vaccination. Sequencing IDH1-mutated human GBM samples revealed an increase in IDH1 mutated tumor tissue after vaccination, consistent with this hypothesis. This led us to consider whether CTL recognition might be functionally altered by IDH1 and/or its mutation. Indeed, alloreactive CD8+ hybridoma cells exhibited significantly delayed lysis of GL26 cells expressing mutant IDH1 relative to untransfected GL26, but significantly increased lysis of GL26 transfected with unmutated IDH1. In contrast, wild-type and mutant IDH1 transfectants of GL26 exhibited identical sensitivity to imidazole-induced cell death, as well as identical MHC I expression. Moreover, preliminary studies suggest that wild-type and mutant IDH1 transfectants of GL26 are differentially sensitive to DC vaccination, with mutant IDH1 abrogating the longer survival conferred by such treatment. We therefore examined the possibility that these observed differences were mediated by a novel, immune-specific property of IDH1. Generalized sialidase activity can de-sialylate the CD8 coreceptor on T cells, and such modification enhances CD8 binding to MHC I, thereby modulating T cell responsiveness to MHC I-bound peptides. As the IDH1 catalytic site is structurally similar to microbial sialidase, we hypothesized that IDH1 possesses CD8-directed sialidase activity capable of enhancing CTL lysis, and that IDH1mutation critically diminishes this activity. Accordingly, IDH1 treatment increased binding to tumor pHLA multimers and to PNA lectin by human CD8 T cells, with the pHLA binding inhibited by either free sialic acid prior to treatment, or by anti-CD3 antibody after treatment. In addition, IDH1 treatment increased binding to several tumor pMHCI multimers by mouse CD8 T cells, and markedly increased IFN-γ production in response to stimulation by such multimers. Finally, labeled IDH1 bound to the surface of spleen cells, and physiologic concentrations of IDH1 specifically associated with a putative surface heterodimer resembling the CD8 coreceptor in murine T cell lysates. Together, these results suggest that wild-type IDH1possesses immune-specific sialidase function that enhances CTL lysis. In contrast, mutant IDH1 appears to delay immune lysis of tumor cells expressing it, and abrogates vaccine benefits, potentially by diminishing its immune-specific sialidase function. Citation Format: Ryan Cordner, Michelle Jhun, Akanksha Panwar, HongQiang Wang, Nicole Yeager, Joseph McAbee, Armen Mardiros, Akane Takei, Xuemo Fan, Emmaunel Jouanneau, Keith Black, Christopher Wheeler. IDH1 modulates the immune response to glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-333. doi:10.1158/1538-7445.AM2013-LB-333

Abstract 3758: RIG-I plays a critical role in negatively regulating stemness properties in hepatoma cell lines.

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of death from cancer worldwide due to its high rate of recurrence. Cancer stem cells are considered to generate tumors through the stem cell processes including self-renewal and differentiation into multiple cell types. And these distinct populations of cells are suggested to cause relapse and metastasis by eliciting new tumors. Retinoic acid-inducible gene I (RIG-I) has been identified as a pattern recognition receptor in innate immune defense for sensing intracellular viral RNAs, like Hepatits C virus which is one of the major risks of HCC, and inducing type I interferons production. In previous studies, RIG-I-like receptors recognizing some potentially oncogenic agents may have functional consequences that can lead to association of such single nucleotide polymorphisms with risk of some diseases or even cancers. Thus these gene polymorphisms may affect cancer risk indirectly. On the other hand, a previous study have demonstrated that RIG-I is an essential regulator of physiological myelopoiesis suggesting RIG-I possesses the potential of modulating stem cell differentiation. In our present study, we examined Huh7 and its RIG-I-mutant cell line by sphere formation assay and found solely the mutated cells possessed the self-renewal ability. After transfection of dominant-negative mutant of RIG-I to Huh7, the self-renewal ability were rescued as expected. We also injected 1000 cells for each two cell lines into NOD/SCID mice subcutaneously and observed the mutated cells formed tumors whereas Huh7 didn't. Hence, our study reveals the potential of RIG-I to suppress the stemness properties in the HCC cell lines and provides a new possible remedy target of liver cancer. Citation Format: Ping-Yu Chen, Chao-Kuen Lai, Min-Linag Kuo. RIG-I plays a critical role in negatively regulating stemness properties in hepatoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3758. doi:10.1158/1538-7445.AM2013-3758

17β‐Estradiol (E2) induces sulfotransferase (SULT) 2A1 expression through estrogen receptor (ER) α

Our recent microarray study showed that E2 increases mRNA levels of SULT2A1 in human hepatocytes. SULT2A1 sulfonates dehydroepiandrosterone and oxysterol, playing an important role in maintenance of steroid and lipid homeostasis. The objective of this study is to elucidate regulatory mechanisms underlying SULT2A1 induction by E2. In HepG2‐ER cells (HepG2 stably expressing ERα) and human hepatocytes, E2 induced SULT2A1 expression at both mRNA and protein levels (1.8–2.7‐fold). Co‐treatment of ERα‐degrading antiestrogen ICI182,780 abrogated the induction, indicating that ERα is required for SULT2A1 induction by E2. Results from luciferase reporter assays in HepG2 cells showed that E2 increased SULT2A1 promoter activity by 13‐fold. Transient transfection of ERα mutants harboring point mutations in the DNA‐binding domain significantly decreased the induction, suggesting involvement of the classical mechanism of ERα action in SULT2A1 regulation. Results from deletion assays revealed that the cis‐element responsible for the ER action is located in −256/+140 of SULT2A1. Together, the results demonstrated that E2 induces transcription of SULT2A1 via ERα, potentially through direct binding of ERα to the promoter region. The induction of SULT2A1, which may alter the intracellular concentration of oxysterol, potentially provides insights into the known lipid‐lowing effects of estrogen.This work was supported by the National Institute of Child Health and Human Development (HD065532).

The Hepatitis E Virus Capsid C-Terminal Region Is Essential for the Viral Life Cycle: Implication for Viral Genome Encapsidation and Particle Stabilization

Although the C-terminal 52 amino acids (C52aa) of hepatitis E virus (HEV) capsid are not essential for morphology, the C52aa-encoding region is required for replication. Transfection of a C52aa knockdown mutant showed transient growth, and the earliest population included a majority of noninfectious (possibly empty) particles and a minority of infectious particles with C-terminal capsid degradation. Finally, the complete revertant was generated reproducibly. C52aa is essential for the viral life cycle, promoting accurate encapsidation and stabilizing encapsidated particles.

Increase of oxidative stress by a novel PINK1 mutation, P209A

Mutation in the human PTEN-induced protein kinase 1 (PINK1) gene is responsible for the second most common form of recessive Parkinson disease (PD). We have identified a single heterozygous PINK1 mutation, P209A, from a cohort of 68 patients with early onset PD. From age 31, this patient developed an asymmetric bradykinesia with rigidity that was L-DOPA responsive. An [18F]-fluorodopa PET scan showed reduced DOPA uptake in the bilateral basal ganglia. The H2O2-induced cell death, ROS production, and caspase-3 activation in SH-SY5Y cells were enhanced by the transfection of the PINK1 P209A mutant. The heme oxygenase-1 (HO-1) induction in response to H2O2 and MPP+ treatment was impaired by the overexpression of the PINK1 P209A mutant. In addition, SOD2 induction after TNFα treatment was also inhibited by the PINK1 P209A mutation. Akt and ERK are involved in HO-1 induction after oxidative stress. The phosphorylation of Akt and ERK after exposure to H2O2 or MPP+ was also inhibited in PINK1 P209A mutant cells compared with empty-vector-transfected cells. These results indicate a novel pathway by which the P209A defect in the PINK1 kinase domain inhibits oxidative stress-induced HO-1 and SOD2 induction, which may accelerate the neurodegeneration in PD with PINK1 defect.

Cytoplasmic and Nuclear Anti-Apoptotic Roles of αB-Crystallin in Retinal Pigment Epithelial Cells

In addition to its well-characterized role in the lens, αB-crystallin performs other functions. Methylglyoxal (MGO) can alter the function of the basement membrane of retinal pigment epithelial (RPE) cells. Thus, if MGO is not efficiently detoxified, it can induce adverse reactions in RPE cells. In this study, we examined the mechanisms underlying the anti-apoptotic activity of αB-crystallin in the human retinal pigment epithelial cell line ARPE-19 following MGO treatment using various assays, including nuclear staining, flow cytometry, DNA electrophoresis, pulse field gel electrophoresis, western blot analysis, confocal microscopy and co-immunoprecipitation assays. To directly assess the role of phosphorylation of αB-crystallin, we used site-directed mutagenesis to convert relevant serine residues to alanine residues. Using these techniques, we demonstrated that MGO induces apoptosis in ARPE-19 cells. Silencing αB-crystallin sensitized ARPE-19 cells to MGO-induced apoptosis, indicating that αB-crystallin protects ARPE-19 cells from MGO-induced apoptosis. Furthermore, we found that αB-crystallin interacts with the caspase subtypes, caspase-2L, -2S, -3, -4, -7, -8, -9 and -12 in untreated control ARPE-19 cells and that MGO treatment caused the dissociation of these caspase subtypes from αB-crystallin; transfection of S19A, S45A or S59A mutants caused the depletion of αB-crystallin from the nuclei of untreated control RPE cells leading to the release of caspase subtypes. Additionally, transfection of these mutants enhanced MGO-induced apoptosis in ARPE-19 cells, indicating that phosphorylation of nuclear αB-crystallin on serine residues 19, 45 and 59 plays a pivotal role in preventing apoptosis in ARPE-19 cells. Taken together, these results suggest that αB-crystallin prevents caspase activation by physically interacting with caspase subtypes in the cytoplasm and nucleus, thereby protecting RPE cells from MGO-induced apoptosis.

Wnt activation by wild type and mutant myocilin in cultured human trabecular meshwork cells.

BackgroundMyocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro370 to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln368 stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin.Methodology/Principal FindingsWild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1), an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA.Conclusions/SignificanceThe present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology.

Osteoblast-specific transcription factor Osterix (Osx) and HIF-1α cooperatively regulate gene expression of vascular endothelial growth factor (VEGF)

Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific transcription factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

Promoter hypermethylation-induced transcriptional down-regulation of the gene MYCT1in laryngeal squamous cell carcinoma

BackgroundMYCT1, previously named MTLC, is a novel candidate tumor suppressor gene. MYCT1 was cloned from laryngeal squamous cell cancer (LSCC) and has been found to be down-regulated in LSCC; however, the regulatory details have not been fully elucidated.MethodsHere, we sought to investigate the methylation status of the CpG islands of MYCT1 and mRNA levels by bisulfite-specific PCR (BSP) based on sequencing restriction enzyme digestion, reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR). The function of specific sites in the proximal promoter of MYCT1 in LSCC was measured by transient transfection, luciferase assays, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP).ResultsThe results suggested hypermethylation of 12 CpG sites of the promoter in both laryngeal cancer tissues and the laryngeal cancer line Hep-2 cell. The hypermethylation of the site CGCG (−695 to −692), which has been identified as the c-Myc binding site, was identified in laryngeal cancer tissues (59/73) compared to paired mucosa (13/73); in addition, statistical analysis revealed that the methylation status of this site significantly correlated with cancer cell differentiation(p < 0.01). The mRNA level of MYCT1 increased in Hep-2 cells treated with 5-aza-C (p < 0.01). The luciferase activity from mutant transfectants pGL3-MYCT1m (−852/+12, mut-695-C > A, mut-693-C > G) was significantly reduced compared with the wild type pGL3-MYCT1 (−852/+12), while the luciferase activity from wild transfectants pGL3-MYCT1 (−852/+12) rose after 5-aza treatment in Hep-2 cells. Finally, EMSA and ChIP confirmed that the methylation of the CGCG (−695 to −692) site prevented c-Myc from binding of the site and demethylation treatment of the 5′ flanking region of MYCT1 by 5-aza induced the increased occupation of the core promoter by c-Myc (p < 0.01).ConclusionIn summary, this study concluded that hypermethylation contributed to the transcriptional down-regulation of MYCT1 and could inhibit cancer cell differentiation in LSCC. DNA methylation of the CGCG site (−695 to −692) of MYCT1 altered the promoter activity by interfering with its binding to c-Myc in LSCC. Epigenetic therapy of reactivating MYCT1 by 5-aza should be further evaluated in clinical trails of LSCC.

Tweaking the cholesterol efflux capacity of reconstituted HDL

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.

Smurf1 Protein Negatively Regulates Interferon-γ Signaling through Promoting STAT1 Protein Ubiquitination and Degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.

Original Article. Hepatitis B Virus S Promoter Deletion in Hepatocellular Carcinoma

Abstract Objective To identify the difference and significance of dominant types of hepatitis B virus (HBV) pre-S mutation between liver tumor tissues and paired adjacent non-tumor tissues and to test if the mutations were tumor tissue specific. Methods HBV DNA isolated from 34 paired intratumoral and peritumoral tissues of hepatocellular carcinoma (HCC) patients were screened by PCR and direct sequencing. All patients carried HBV with genotype C, except for one B/C heterozygote. The expression, localization and excretion of LHBs mutant carrying pre-S deletions were characterized in vitro. The expression of endoplasmic reticulum (ER) GRP78 mRNA was assayed. Results Four patterns of pre-S mutations were identified: pre-S1 in-frame deletion involving the first start codon; pre-S2 in-frame deletion; pre-S2 start codon mutation with or without in-frame deletion; and S promoter in-frame deletion (ΔSP). The first two types were evenly found in both tumor and non-tumor tissues. They were rarely present as dominant strains. The last two types were frequently found in the dominant strains in tumor tissues. The overall prevalence of HBV carrying ΔSP was 17.64% (6/34) in tumor tissues, but none were dominant in nontumor tissues. HBV carrying ΔSP was unable to produce S protein in vitro. Immunocytofluorescence assay showed that the ΔSP LHBs mutant aggregated in the cytoplasm, accumulating mainly in the ER. Transient transfection and expression of ΔSP mutant caused GRP78 up-regulation in vitro. Conclusions HBV S promoter deletion was found dominantly in HCC tumor tissue. The aggregation of mutant large surface proteins in the ER possibly involved in HBV-related HCC.

Cyclooxygenase-2 Expression Is Up-regulated by 2-Aminobiphenyl in a ROS and MAPK-Dependent Signaling Pathway in a Bladder Cancer Cell Line

Overexposure to biphenyl amine compounds, which are found in smoke and azo-dyes, is linked to the occurrence of bladder cancer. However, the molecular mechanisms of biphenyl amine compound-induced bladder cancer are still unclear. Many studies have demonstrated that overexpression of cyclooxygenase-2 (COX-2) in neoplastic lesions is associated with carcinogenesis. In this study, we have demonstrated that 2-aminobiphenyl (2-ABP) up-regulated the expression of COX-2 in a dose- and time-dependent manner in TSGH-8301 bladder cancer cells. This 2-ABP-induced COX-2 expression was attenuated by ROS scavenger NAC and NADPH oxidase inhibitors apocynin and DPI. The p22phox subunit of NADPH oxidase, but not p67, and Nox2 was up-regulated by 2-ABP. Knocking down p22phox by siRNA significantly reduced 2-ABP-induced COX-2 expression. Furthermore, 2-ABP also activated the ERK/JNK-AP1 pathways, and this effect was also abolished by NADPH oxidase inhibitors. Blocking the ERK/JNK-AP1 signaling pathways by pharmacological inhibitors attenuated 2-ABP-induced COX-2 expression. Overexpression of the upstream ERK activator MEK1 significantly and consistently increased 2-ABP-mediated COX-2 expression. Transfection of a dominant negative c-Jun mutant, TAM-67, blocked 2-ABP-mediated COX-2 expression, demonstrating that c-Jun was responsible for the transcriptional activation. Taken together, these results demonstrate that 2-ABP induces the carcinogenic factor COX-2 and that this induction is mediated through NADPH oxidase-derived ROS-dependent JNK/ERK-AP-1 pathways.

Abstract 2387: Modulation Of P35/cyclin-dependent Kinase 5 Signaling Rectifies Defective Human Brain Endothelial Cell Migration Produced By In Vitro Simulation Of Ischemic Stroke

Uncontrolled activation of calcium-sensitive calpain pathways induces neuronal damage during ischemic stroke. Vascular remodelling plays a critical role in tissue recovery. The main molecular events underpinning angiogenesis in stroke are still imprecise. Recently, we highlighted a novel non-neuronal role of the cyclin-dependent kinase (Cdk)-5 operating through p35 activator, which modulates in vitro brain angiogenesis. Cdk5 activators p35 and p25 were recently shown to be involved in neuronal protection (p35) or death (p25) after brain ischemia. However, the impact of p35/Cdk5 on angiogenesis during hypoxic injury remains unclear. Here, using the calpain inhibitor MDL28170 and GFP-Cdk5 wild type (wt) or kinase mutant (D144N) transfectants in an in vitro model of stroke, we tested the hypothesis that activation of p35/Cdk5 pathway may positively modulate angiogenesis during hypoxia. Human brain microvascular endothelial cells (hCMEC/D3) were treated (24h) with MDL28170 (10μM) under conditions of hypoxia (1%O 2 ) or normoxia. Angiogenesis was investigated by wound-healing assay and capillary-tube formation on matrigel, using Cell-IQ® Imaging system (Chip-Man Technologies Ltd). Cell viability was evaluated by assay of MTT proliferation, propidium iodide nuclear inclusion (nPI) and heat shock protein 70 (Hsp70) level. p35, Cdk5 and activated pY(15)Cdk5 intracellular localization were analyzed by confocal microscopy. Protein levels were estimated by western blot. Inhibition of calpain activity increased angiogenesis (p&lt;0.01), augmenting p35 protein levels (30%). Hypoxia induced cell stress, as evidenced by increased Hsp70 (0.8 ±0.02 vs 0.5±0.01, ratio on GAPDH optical density) and nPIs, and hampered cell migration and tube formation (p&lt;0.01). This was associated with reduced p35 (56%, ratio on GAPDH optical density) contents. Blocking Cdk5 activity with D144N transfectant reduced migration (p&lt;0.05). Cdk5 (wt) overexpression enhanced migration, which was further augmented in hypoxia (p&lt;0.05). Hypoxia impaired reorganization of the cytoskeleton and reduced p35/actin co-localization, without affecting the distribution of Cdk5 or pY(15)Cdk5 in association with actin filaments and focal tips. MDL28170 significantly reversed the effects of hypoxia on cell migration and p35 localization, suggesting a role of p35 in cytoskeleton stability. MDL28170 further increased cell migration in Cdk5(wt) or D144N either in hypoxia or normoxia, and Cdk5 protein levels in hypoxia (0.2 ±0.01 vs 0.18±0.01, ratio on GAPDH optical density), implying that increased p35 may be protective during stroke. In conclusion, our data suggest that p35/Cdk5 signaling supports angiogenesis in stroke. p35 may be a signalling intermediate, involved in cytoskeleton organization and cell dynamics during angiogenesis.

Unusual Features of Chick VSP and its Potential Role in Regulation of Cell Structure

Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the PTEN-like region with phosphoinositide-phosphatase activities. VSP is conserved from sea squirt to human, but little is known about its physiological roles. Here we characterize the molecular properties of chick ortholog of VSP (designated as Gg-VSP). Gg-VSP shares basic properties with other VSP orthologs, including voltage sensing by the transmembrane segments and phosphoinositide phsophatase activities of the cytoplasmic region. Imaging of PI(3,4)P2-sensitive fluorescent protein in Xenopus oocyte showed that membrane depolarization triggers dephosphorylation of PI(3,4,5)P3 into PI(3,4)P2. Notably, about 10% of cell populations of DF-1 cells transiently expressing Gg-VSP exhibited morphological feature of cell process outgrowths. This was accompanied by the decrease of actin filaments as detected by phalloidin-staining. Transfection of the voltage sensor mutant, Gg-VSPR153Q, showing shift of voltage dependence to a lower voltage, facilitated cell process outgrowths. Coexpression of chick PTEN cDNA suppressed this effect of Gg-VSP. Visualization of PI(3,4)P2 with the Ds-Red fused with the FAPP1-derived pleckstrin homology (PH) domain showed more fluorescent clusters than luciferase-transfected control cells, suggesting that cell process outgrowths paralleled with the increase of PI(3,4)P2. Whole mount in situ hybridization and immunostaining of chicken brain showed that Gg-VSP is expressed in neurons. These findings suggest that one of the VSP’s functions is to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile.

Ebola Virus Enters Host Cells by Macropinocytosis and Clathrin-Mediated Endocytosis

Virus entry into host cells is the first step of infection and a crucial determinant of pathogenicity. Here we show that Ebola virus-like particles (EBOV-VLPs) composed of the glycoprotein GP(1,2) and the matrix protein VP40 use macropinocytosis and clathrin-mediated endocytosis to enter cells. EBOV-VLPs applied to host cells induced actin-driven ruffling and enhanced FITC-dextran uptake, which indicated macropinocytosis as the main entry mechanism. This was further supported by inhibition of entry through inhibitors of actin polymerization (latrunculin A), Na(+)/H(+)-exchanger (EIPA), and PI3-kinase (wortmannin). A fraction of EBOV-VLPs, however, colocalized with clathrin heavy chain (CHC), and VLP uptake was reduced by CHC small interfering RNA transfection and expression of the dominant negative dynamin II-K44A mutant. In contrast, we found no evidence that EBOV-VLPs enter cells via caveolae. This work identifies macropinocytosis as the major, and clathrin-dependent endocytosis as an alternative, entry route for EBOV particles. Therefore, EBOV seems to utilize different entry pathways depending on both cell type and virus particle size.

Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ~18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons.

Insulin receptor substrate 2 and FoxO3a signalling are involved in E‐cadherin expression and transforming growth factor‐β1‐induced repression in kidney epithelial cells

Insulin receptor substrate (IRS) proteins comprise a family of adaptor molecules that integrate extracellular signals from insulin and other ligands to intracellular effectors such as phosphoinositide 3-kinase and mitogen-activated protein kinase. The predominant forms of IRS protein in humans, IRS1 and IRS2, are widely expressed. Despite structural similarities, IRS1 and IRS2 display distinct signalling modalities, and mice lacking these proteins present with distinct phenotypes. Transforming growth factor (TGF)-β1 is the primary cytokine shown to induce epithelial-mesenchymal transition. Recent data have demonstrated a role for IRS1 in TGF-β1-induced epithelial-mesenchymal transition in lung epithelial cells. In the present study, we report data showing that TGF-β1 signals via IRS2 in kidney epithelial cells. Small interfering RNA (siRNA)-mediated targeting of IRS2 increased E-cadherin expression, although it did not alter TGF-β1-mediated E-cadherin repression. Phosphorylation of the downstream target of IRS2/Akt signalling, FoxO3a, was induced on Ser253 and, to a lesser extent, on Thr32. Transfection of FoxO3aThr32Ala mutant for 24 h greatly reduced FoxO3a phosphorylation on Ser253 but over-expression of FoxO3a Ser253Ala did not effect Thr32 phosphorylation, suggesting that a distinct order of phosphorylation of FoxO3a is required for physiological function in cells. Transfection of FoxO3a Ser253Ala mutant partially inhibited TGF-β1-mediated E-cadherin repression at 24 h. Taken together, these data highlight novel roles for IRS2 and FoxO3a in the regulation of kidney epithelial cells by E-cadherin.