Endoplasmic reticulum-associated degradation potentiates the infectivity of influenza A virus by regulating the host redox state.

During influenza A virus (IAV) infection, significant effects of oxidative stress often emerge due to the disruption of the redox balance. Reactive oxygen species (ROS) generated during IAV infection have been known to exert various effects on both the virus and host tissue. However, the mechanisms underlying the accumulation of ROS and their physiological significance in IAV infection have been extensively studied but remain to be fully understood. Here, we show that the levels of Sp1, a key controller of Cu-Zn superoxide dismutase (SOD1) gene expression, and SOD1 are mainly dependent upon the activity of X-box-binding protein 1 (XBP1), which is a downstream factor of the endoplasmic reticulum (ER) transmembrane sensor inositol-requiring enzyme 1 (IRE1) during ER stress. In IRE1-deficient mouse embryo fibroblasts (MEFs) or A549 human lung cells treated with XBP1 siRNA, IAV-induced Sp1 loss was mitigated. However, overexpression of the spliced form of XBP1 in IRE1-deficient MEFs resulted in a further decrease in Sp1 levels, whereas the unspliced form showed no significant differences. Treatment with proteasome inhibitor MG132 markedly inhibited the IRE1/XBP1-mediated loss of Sp1 and SOD, suggesting the involvement of proteasome-dependent ER-associated degradation (ERAD). The increase in SOD1 levels with the expression of siRNA-targeting p97, a central component of the ubiquitin-proteasome system, supports the major role of the ERAD process in IAV-mediated SOD1 loss. In addition, ROS generation due to IAV infection was attenuated in cells lacking either IRE1 or JNK. These results reveal the important roles of both IRE1/XBP1-mediated ERAD and the JNK pathway in IAV infection. Interestingly, the increase in ROS due to IAV infection is correlated with the increase in the virus titer in vitro and in vivo. However, 4-phenylbutyrate (4-PBA), an inhibitor of ER stress signaling, weakened the effect of IAV infection on SOD1 loss in a dose-dependent manner. Furthermore, the treatment of mice with 4-PBA efficiently attenuated ROS generation and ER stress in lung tissue and eventually lowered the IAV titer. These results strongly suggest that the ERAD process plays a major role in IAV infection, thus making it a potential target for antiviral drug therapy.

ER stress attenuation by Aloe-derived polysaccharides in the protection of pancreatic ß-cells from free fatty acid-induced lipotoxicity.

Insulin resistance, a pathophysiology of type 2 diabetes, is associated with obesity. Lipotoxicity in obesity leads to the dysfunction and death of pancreatic ß-cells and inadequate insulin production, thereby aggravating type 2 diabetes. The present study was conducted to determine the effect of Aloe vera polysaccharides (APs) as an anti-hyperglycemic agent and their mechanisms of action. Gel polysaccharides from Aloe extracts were separated using ultrafiltration devices with molecular weight-cutoff membranes, and the protective effect of APs on pancreatic ß-cells in response to free fatty acids (FFAs) was determined. Hamster pancreatic ß-cell line HIT-T15 was treated with palmitate and APs to analyze cellular responses. We observed a large number of apoptotic ß-cell death after treatment with high levels of palmitate, but this was efficiently prevented by the addition of APs in a dose-dependent manner. It was found that the anti-apoptotic properties of APs were largely due to the relief of endoplasmic reticulum (ER) stress signaling. APs were effective in interfering with the FFA-induced activation of the PERK and IRE1 pathways as well as ROS generation, thereby protecting pancreatic ß-cells from lipotoxicity. Although variation in the chain length of APs can influence the activity of FFA-mediated ER stress signaling in different ways, polysaccharide mixtures with molecular weights higher than 50 kDa showed greater antiapoptotic and antioxidant activity in ß-cells. After oral administration of APs, markedly lowering fasting blood glucose levels were observed in db/db mice, providing evidence of the potential of APs as an alternative insulin sensitizer. Therefore, it was concluded that APs have a protective effect against type 2 diabetes by modulating obesity-induced ER stress in pancreatic ß-cells.

Influenza A virus-induced autophagy contributes to enhancement of virus infectivity by SOD1 downregulation in alveolar epithelial cells.

Infection with influenza A virus (IAV) A/WSN/1933 (H1N1) causes oxidative stress and severe lung injury. We have demonstrated that the generation of reactive oxygen species (ROS) during IAV infection is tightly regulated by superoxide dismutase 1 (SOD1) and correlated with viral replication in alveolar epithelial cells. However, the molecular mechanism underlying SOD1 reduction during IAV infection is uncertain. Here we demonstrate that the autophagy pathway is activated by IAV infection and involved in enhanced ROS generation in the early phase of infection. We observed that IAV infection induced autophagic vacuolation, leading to autophagic degradation of cellular proteins, including the protease sensitive antioxidant SOD1. Silencing of the microtubule-associated protein 1A/1B-light chain 3 (LC3) gene in A549 cells supported the critical role of autophagy in the ROS increase. The decrease in viral titer and viral polymerase activity caused by LC3 silencing or the autophagy inhibitor clearly evidenced the involvement of autophagy in the control of ROS generation and viral infectivity. Therefore, we concluded that early stage IAV infection induces autophagic degradation of antioxidant enzyme SOD1, thereby contributing to increased ROS generation and viral infectivity in alveolar epithelial cells.

Influenza A virus PB1-F2 is involved in regulation of cellular redox state in alveolar epithelial cells.

Occurrence of oxidative stress is common in influenza, and renders the host more susceptible to pathogenic effects including cell death. We previously reported that down-regulation of superoxide anion dismutase 1 (SOD1) by influenza A virus (IAV) resulted in a significant increase in the levels of reactive oxygen species (ROS) and viral PB1 polymerase gene product in the early stage of infection. However, the precise molecular mechanism of IAV-mediated ROS generation is not yet fully understood. In this study, we investigated the possible involvement of the key virulence factor PB1-F2 in ROS generation and its contribution to the viral propagation and cell death. The key virulence factor PB1-F2 was found to be responsible, at least in part, for the ROS generation through lowering the SOD1 level in alveolar epithelial A549 cells. PB1-F2 overexpression resulted in SOD1 diminishment and ROS enhancement, while another virulent factor, NS1, did not show significant changes. Inversely, we examined the effects of the absence of PB1-F2 using mutant IAV lacking PB1-F2 expression (mutantΔF2). Infection with mutantΔF2 virus did not significantly lower the SOD1 level, and thus generated moderately low levels of ROS. In addition, the oxidative activity of PB1-F2 was directly reflected by cell viability and death. Infection with the mutant virus reduced the percentage of apoptotic cells more than two-fold compared to the wild-type IAV in A549 cells. Furthermore, expression of exogenous SOD1 gene abrogated a large portion of the PB1-F2-induced apoptosis of cells infected with wild-type IAV, but affected much less of the mutantΔF2 virus-infected cells. These results suggest that the PB1-F2 is directly implicated in virus-induced oxidative stress, thereby contributing to the early stages of IAV replication cycle and ultimately to disease severity.

RNA polymerase III control elements are required for trans-activation by the murine retroviral long terminal repeat sequences.

RNA leukemia viruses induce T-cell lymphoblastic lymphomas or myeloid leukemias. Infection of cells with Moloney murine leukemia virus (M-MuLV) up-regulates the expression of a number of cellular genes, including those involved in T-lymphocyte activation. Previously, we demonstrated that this up-regulation occurs via the trans-activation activity of the M-MuLV long terminal repeat (LTR) sequences which produce an LTR-encoded transcript. Sequence analysis of the LTR revealed a potential transcription unit for RNA polymerase III (Pol III) within the U3 region that is actively occupied by Pol II factors. Here, we provide the direct evidence of involvement of Pol III in the trans-activation process and demonstrate the precise localization of the intragenic control elements for accurate and active Pol III transcription. Deletions of a copy of the directed repeats and further immediate upstream sequences significantly abrogated the generation of LTR-encoded transcript and abolished the trans-activational activity, whereas the deletion of a copy of directed repeats alone proportionally reduced the transcript size, but still retained moderately high trans-activational activity. In electrophoretic mobility shift assay, the fraction containing a multiple transcription factor TFIIIC complex strongly bound to the LTR-U3 probe containing the essential control elements. The specificity of the DNA-TFIIIC interaction was confirmed by conducting competition assays with DNA fragments containing a genuine Pol III-transcribed gene, VA1, and by vaccinia virus infection which stimulates the expression of Pol III factors. However, a deletion mutant lacking an essential control element bound to the TFIIIC complex poorly, consequently resulting in weak Pol III transcription as assessed by an IRES-GFP reporter system. This correlation strongly supports the possibility that the generation of LTR-encoded transcript is directed by Pol III. Therefore, this finding suggests the involvement of Pol III transcription in the retrovirus-induced activation of cellular genes, potentially contributing to leukemogenesis.

High-efficiency lentiviral transduction of primary human CD34⁺ hematopoietic cells with low-dose viral inocula.

Lentivirus-based vectors have the potential to transduce non-dividing primary stem cells. However, primary cells tend to be less susceptible to manipulation and require a high concentration of virus inoculum. Furthermore, increasing the concentration of the lentivirus inoculum may raise safety risks. Therefore, to develop a technique that allows high transduction efficiency at low multiplicities of infection (MOIs), we optimized a lentivirus-based system for cell lines and primary cells by determining the best condition using various parameters. When progenitor cell assays were conducted using human CD34(+) bone marrow and mononuclear cells, the transduction condition yielded a great number of eGFP(+) colonies with lower-dose viral inocula compared to that of current lentivirus-based transduction technologies. In conclusion, this system is anticipated to produce stable expression of a gene introduced into primary cells for preclinical studies with lower safety risks.

Alteration of copper-zinc superoxide dismutase 1 expression by influenza A virus is correlated with virus replication.

Viruses have evolved mechanisms designated to potentiate virus replication by modulating the physiological condition of host cells. The generation of reactive oxygen species (ROS) during infection with influenza virus A (IAV) is a well-established mechanism in animals, but little is known about the generation of ROS in in vitro cell culture models and about its role in virus replication. We show here that IAV H1N1 infected human alveolar cells increased superoxide anion level mainly by suppressing the copper-zinc superoxide dismutase 1 (SOD1) gene, and that the SOD1-controlled generation of ROS was tightly correlated with virus replication. The transcription factor Sp1, which is a major element of the proximal region of the sod1 promoter, was slightly downregulated at the transcriptional level during IAV infection, and subsequently modulated by post-translational control. A gradual reduction of whole Sp1 was largely responsible for the repression of sod1 transcription with increasing time post-infection, and their rescue by the proteasome inhibitor, MG132, proved the involvement of proteasomal degradation in Sp1 regulation during IAV infection. Furthermore, we observed that expression of viral polymerase PB1 was inversely proportional to SOD1 level. The antioxidant N-acetyl-cysteine (NAC) neutralized IAV-mediated oxidative stress, and either NAC treatment or sod1 transfection considerably diminished viral polymerase activity. These data indicate that IAV-induced SOD1 repression, which may cause impaired redox balance in host cells, can be attributed, at least in part, to enhance viral replication.

Activation of TOPK by lipopolysaccharide promotes induction of inducible nitric oxide synthase through NF-κB activity in leukemia cells.

T-LAK cell-originated protein kinase (TOPK) is known to be involved in tumorigenesis or cancer progression. However, the role of TOPK in inflammatory response remains elusive. Here we show that TOPK positively regulates inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production in response to lipopolysaccharide (LPS). In TOPK-depleted cells, the iNOS expression was shown to be greatly abolished. Also, we revealed that LPS treatment augmented the expression and activity of TOPK, the interaction of TOPK with IκBα, and promoted TOPK kinase activity against IκBα. Moreover, NF-κB or iNOS promoter-driven transcriptional activity in response to LPS was markedly reduced by knocking down of TOPK or deletion of NF-κB sites. On the other hand, endogenous TOPK level was expressed very lowly in bone marrow-derived macrophage (BMDM) prepared from Toll-like receptor 4 (TLR4) knockout mice, compared to BMDM from wild type (WT) mice. Collectively, these findings demonstrate that TOPK upregulates iNOS gene expression in T cell leukemia Jurkat cells or macrophage leukemic Raw 264.7 cells via NF-κB activation in response to LPS, and might act as a critical effector in LPS/TLR4-mediated signaling cascade, suggesting a possible role of TOPK in inflammatory response or inflammation-related diseases.

Oxidative stress-induced cyclin D1 depletion and its role in cell cycle processing.

BACKGROUND: Cyclin D1 is immediately down-regulated in response to reactive oxygen species (ROS) and implicated in the induction of cell cycle arrest in G2 phase by an unknown mechanism. Either treatment with a protease inhibitor alone or expression of protease-resistant cyclin D1 T286A resulted in only a partial relief from the ROS-induced cell cycle arrest, indicating the presence of an additional control mechanism. METHODS: Cells were exposed to hydrogen peroxide (H2O2), and analyzed to assess the changes in cyclin D1 level and its effects on cell cycle processing by kinase assay, de novo synthesis, gene silencing, and polysomal analysis, etc. RESULTS: Exposure of cells to excessive H2O2 induced ubiquitin-dependent proteasomal degradation of cyclin D1, which was subsequently followed by translational repression. This dual control mechanism was found to contribute to the induction of cell cycle arrest in G2 phase under oxidative stress. Silencing of an eIF2α kinase PERK significantly retarded cyclin D1 depletion, and contributed largely to rescuing cells from G2 arrest. Also the cyclin D1 level was found to be correlated with Chk1 activity. CONCLUSIONS: In addition to an immediate removal of the pre-existing cyclin D1 under oxidative stress, the following translational repression appear to be required for ensuring full depletion of cyclin D1 and cell cycle arrest. Oxidative stress-induced cyclin D1 depletion is linked to the regulation of G2/M transit via the Chk1-Cdc2 DNA damage checkpoint pathway. GENERAL SIGNIFICANCE: The control of cyclin D1 is a gate keeping program to protect cells from severe oxidative damages.

Human lactoferrin suppresses TNF-α-induced intercellular adhesion molecule-1 expression via competition with NF-κB in endothelial cells.

Lactoferrin (Lf) is known to have anti-inflammatory activity, but the mechanisms of action by Lf remain to be elucidated. Here, we demonstrated that TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1) was down-regulated by Lf in a DNA-binding dependent manner at transcriptional level in endothelial cells. Our results showed that Lf bound to a DNA region in the ICAM-1 promoter in vitro as well as in chromatin context. Lf inhibited binding of NF-κB to a proximal NF-κB site in ICAM-1 promoter. This type of repression represents an additional mechanism for the action of Lf in regulation of gene expression.

Metabolite profiling of Angelica gigas from different geographical origins using 1H NMR and UPLC-MS analyses.

Angelica gigas obtained from different geographical regions was characterized using (1)H nuclear magnetic resonance (NMR) spectroscopy and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) followed by multivariate data analyses. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) score plots from (1)H NMR and UPLC-MS data sets showed a clear distinction among A. gigas from three different regions in Korea. The major metabolites that contributed to the discrimination factor were primary metabolites including acetate, choline, citrate, 1,3-dimethylurate, fumarate, glucose, histamine, lactose, malate, N-acetylglutamate, succinate, and valine and secondary metabolites including decursin, decursinol, nodakenin, marmesin, 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-ethyl)coumarin in A. gigas roots. The results demonstrate that (1)H NMR and UPLC-MS-based metabolic profiling coupled with chemometric analysis can be used to discriminate the geographical origins of various herbal medicines and to identify primary and secondary metabolites responsible for discrimination.

Establishment of novel cell lines latently infected with human immunodeficiency virus 1.

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.01 cells infected with HIV-1. Analysis of the flanking sequence of HIV-1 proviral DNA integrated into chromosomal cellular DNA revealed that proviral DNA was inserted into different sites of different chromosomes in the two examined cell lines. In these lines, virus reactivation could be induced by a phorbol 12-myristate 13-acetate (PMA) treatment that resulted in a marked increase of the production HIV-1 p24 antigen and appearance of the infectious virus. The novel cell lines latently infected with HIV-1 represent further tool for the study of molecular mechanisms of viral latency and development of anti-reservoir therapy of HIV-1 infection.

Gene silencing in HIV-1 latency by polycomb repressive group.

BACKGROUND: The persistence of latently human immunodeficiency virus-1 (HIV-1) infected cellular reservoirs in resting CD4+ T cells is a major obstacle to HIV-1 eradication. The detailed mechanism of HIV-1 latency remains unclear. We investigated histones and their post-translational modification associated with HIV-1 latency in novel HIV-1 latently infected cell lines established previously, NCHA cells. METHODS: To examine histones and their modification linked with HIV-1 latency, the expression profiles for core histone proteins and histone deacetylases (HDACs) in NCHA cells were characterized by RT-PCR, ELISA, and western blot. The levels of histone acetylation and methylation at histone H3 Lys9 (H3K9) and Lys27 (H3K27) in HIV-1 latently infected cells were analyzed by western blot and chromatin immunoprecipitation-sequencing (ChIP-seq). RESULTS: The expression levels for four core histone proteins (H2A, H2B, H3 and H4) and HDACs (HDAC1-8) in NCHA cells were not significantly different from those in their parental cells. Histone H3K9 and H3K27 acetylations in NCHA cells showed no difference in parental and NCHA cells, whereas the levels of di- and tri-methylation were increased in NCHA cells. The expression of EED which is a component of polycomb repressive complex 2 (PRC2), and BMI1 and RING2 which are constituents of PRC1, were upregulated in NCHA cells. In addition, more ubiquitylation at histone H2A was detected in NCHA cells. CONCLUSIONS: Our results suggest that tri-methylation of histone H3K27 and H2A ubiquitylation via polycomb group protein may play a crucial role in epigenetic silencing accounting for HIV-1 latency in NCHA cells.

Lactoferrin inhibits the inflammatory and angiogenic activation of bovine aortic endothelial cells.

OBJECTIVE: Lactoferrin (Lf) is known to have anti-cancer and anti-inflammatory activities; however, its therapeutic mechanism has not been defined. In this study, to explain the therapeutic mechanism of Lf, we examined the effect of Lf on endothelial cell activation, leukocyte integration, and angiogenesis in vitro. METHODS: Endothelia-leukocyte adhesion assays were used to assess primary cultures of bovine aortic endothelial cells (BAECs) activation following LPS treatment. The mRNA expression of ICAM-1 and proinflammatory cytokines was measured using RT-PCR. Each step of angiogenesis was evaluated in vitro, including endothelial cell proliferation, migration, and tube formation. Proliferation was examined using WST-1 and BrdU incorporation assays, while wound migration assays were used to evaluate cell migration; capillary-like tube formation assays on Matrigel were used to assess tube formation. RESULTS: Lf reduced the adhesion of human monocyte-like THP-1 cells to BAECs by 45%. Lf also reduced mRNA expression of ICAM-1 and proinflammatory cytokines in BAECs. Lf significantly inhibited BAEC proliferation, migration, and tube formation. CONCLUSIONS: Lf exerted a potent effect on BAEC activation, suggesting that it might function via an endothelia-based mechanism in the treatment of various diseases, including rheumatoid arthritis and cancer.

The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency.

HIV-1 can establish a latent infection in memory CD4+T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56(Lck), ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56(Lck), ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new antireservoir therapy.

E2F1-directed activation of Bcl-2 is correlated with lactoferrin-induced apoptosis in Jurkat leukemia T lymphocytes.

Lactoferrin (Lf) has been shown to control the proliferation of a variety of mammalian cells. Recently, we reported that human Lf induces apoptosis via a c-Jun N-terminal kinases (JNK)-associated Bcl-2 pathway that stimulates programmed cell death. In order to gain insight into the mechanism underlying Lf-triggered apoptotic features, we attempted to determine the mechanisms whereby the Lf-induced Bcl-2 family proteins exert their pro- or anti-apoptotic effects in Jurkat leukemia T lymphocytes. Treatment of the cells with high concentrations of Lf resulted in a significant reduction in in vitro growth and cell viability. As the levels of Lf increased, greater quantities of CDK6 and hyper-phosphorylated retinoblastoma protein were produced, resulting in the induction of E2F1-dependent apoptosis. Simultaneously, PARP and caspases were efficiently cleaved during Lf-induced apoptosis. The E2F1-induced apoptotic process occurred preferentially in p53-deficient Jurkat leukemia cells. Therefore, we attempted to determine whether E2F1-regulated Bcl-2 family proteins involved in the apoptotic process were relevant to Lf-induced apoptosis. We found that Lf increased the interaction of Bcl-2 with the pro-apoptotic protein Bad, whereas the total protein levels did not change significantly. Our results, collectively, suggest that Lf exploits the control mechanism of E2F1-regulated target genes or Bcl-2 family gene networks involved in the apoptotic process in Jurkat human leukemia T lymphocytes.

Adiponectin may contribute to synovitis and joint destruction in rheumatoid arthritis by stimulating vascular endothelial growth factor, matrix metalloproteinase-1, and matrix metalloproteinase-13 expression in fibroblast-like synoviocytes more than proinflammatory mediators.

INTRODUCTION: The role of adiponectin in the pathogenesis of arthritis is still controversial. This study was performed to examine whether adiponectin is involved in joint inflammation and destruction in rheumatoid arthritis (RA) in relation to the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). METHODS: Synovial cells from RA patients were treated with adiponectin or interleukin (IL)-1beta for 24 hours. The culture supernatant was collected and analyzed for the levels of IL-6, IL-8, prostaglandin E2 (PGE2), VEGF, and MMPs by enzyme-linked immunosorbent assay. The levels of adiponectin, VEGF, MMP-1, and MMP-13 in the joint fluids from 30 RA or osteoarthritis (OA) patients were also measured. RESULTS: Adiponectin at the concentration of 10 microg/mL stimulated the production of IL-6, IL-8, and PGE2 in RA fibroblast-like synoviocytes (FLSs), although the level of these was much lower than with 1 ng/mL IL-1beta. However, adiponectin stimulated the production of VEGF, MMP-1, and MMP-13 at the same level as IL-1beta. In addition, the level of adiponectin and MMP-1 in the joint fluid of RA patients was significantly higher than in OA patients. Adiponectin was positively correlated with VEGF in RA patients but not in OA patients, while the level of MMPs in joint fluid was not correlated with adiponectin in either RA or OA patients. CONCLUSIONS: Adiponectin may play a significant role in the pathogenesis of RA by stimulating the production of VEGF and MMPs in FLSs, leading to joint inflammation and destruction, respectively.

Iron-saturated lactoferrin stimulates cell cycle progression through PI3K/Akt pathway.

Iron binding lactoferrin (Lf) is involved in the control of cell cycle progression. However, the molecular basis underlying the effects of Lf on cell cycle control, as well as its target genes, remains incompletely understood. In this study, we have demonstrated that a relatively low level of ironsaturated Lf, Lf(Fe(3+)), can stimulate S phase cell cycle entry, and requires Akt activation in MCF-7 cells. Lf(Fe(3+)) immediately induced Akt phosphorylation at Ser473, which subsequently induced the phosphorylation of two G1-checkpoint Cdk inhibitors, p21(Cip/WAF1) and p27(kip1). The Lf(Fe(3+))-induced phosphorylation of Cdk inhibitors impaired their nuclear import behavior, thereby inducing cell cycle progression. However, the treatment of cells with a PI3K inhibitor, LY294002, almost completely blocked Lf(Fe(3+))-stimulated cell cycle progression. LY294002 treatment abrogated Lf(Fe(3+))-induced Akt activation, and prevented the cytoplasmic localization of p27(kip1). Higher levels of p21(Cip/WAF1) were also detected in the cytoplasmic sub-cellular compartment as a measure of cellular response to Lf(Fe(3+)). Consequently, the degree of phosphorylation of retinoblastoma protein was enhanced in response to Lf(Fe(3+)). Therefore, we conclude that Lf(Fe(3+)), as a potential antagonist of Cdk inhibitors, can facilitate the functions of E2F during progression to S phase via the Akt signaling pathway.

PKR protein kinase is activated by hepatitis C virus and inhibits viral replication through translational control.

Hepatitis C virus (HCV) infection is currently treated with IFNalpha-based therapy but little is known how IFNalpha inhibits HCV replication. We show here that HCV JFH1 infection of human hepatoma Huh-7 cells leads to the activation of IFN-inducible protein kinase PKR and phosphorylation of the translation initiation factor eIF2alpha. Compared to a control cell HCV replication was significantly elevated in a PKR-knockdown cell, giving rise to a 10-fold higher viral titer, and was less sensitive to IFNalpha treatment. Conversely, transient expression of PKR inhibited HCV replication in a kinase-dependent manner with concomitant increase of eIF2alpha phosphorylation. Further, expression of a phospho-mimetic eIF2alpha mutant moderately inhibited HCV replication. Together, these results demonstrate that PKR is activated by HCV infection and plays a critical antiviral role through inhibition of viral protein translation.

Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line.

The cell proliferation of p53-deficient Jurkat T cells is controlled after prolonged exposure to human lactoferrin (Lf). However, the molecular mechanism by which Lf influences these cellular responses remains unclear. In this study, we demonstrate that Lf-induced apoptosis in Jurkat T cells occurs in a dose- and time-dependent manner via the regulation of c-Jun N-terminal kinase (JNK) activity. Jurkat cells exposed to Lf for 1 day, especially at concentrations in excess of 500 microg/ml, showed typical apoptosis, as indicated by decreased cell viability and increased Annexin V binding. Our results also showed that Lf induced the activation of caspase 9 and caspase 3 activation, as demonstrated by our detection of cleaved caspases and PARP. Lf-induced apoptosis did not influence Bcl-2 expression via an ERK1/2 phosphorylation pathway, but was rather associated with the level of Bcl-2 phosphorylation. The treatment of cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, revealed that the JNK-Bcl-2 signaling cascade is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. These findings indicate that Lf functions as a biological mediator of apoptosis in the human leukemia Jurkat T-cell line, via the JNK-associated Bcl-2 signaling pathway.