Hepatitis B virus inhibits liver regeneration via epigenetic regulation of urokinase-type plasminogen activator.

UNLABELLED: Liver regeneration after liver damage caused by toxins and pathogens is critical for liver homeostasis. Retardation of liver proliferation was reported in hepatitis B virus (HBV) X protein (HBx)-transgenic mice. However, the underlying mechanism of the HBx-mediated disturbance of liver regeneration is unknown. We investigated the molecular mechanism of the inhibition of liver regeneration using liver cell lines and a mouse model. The mouse model of acute HBV infection was established by hydrodynamic injection of viral DNA. Liver regeneration after partial hepatectomy was significantly inhibited in the HBV DNA-treated mice. Mechanism studies have revealed that the expression of urokinase-type plasminogen activator (uPA), which regulates the activation of hepatocyte growth factor (HGF), was significantly decreased in the liver tissues of HBV or HBx-expressing mice. The down-regulation of uPA was further confirmed using liver cell lines transiently or stably transfected with HBx and the HBV genome. HBx suppressed uPA expression through the epigenetic regulation of the uPA promoter in mouse liver tissues and human liver cell lines. Expression of HBx strongly induced hypermethylation of the uPA promoter by recruiting DNA methyltransferase (DNMT) 3A2. CONCLUSION: Taken together, these results suggest that infection of HBV impairs liver regeneration through the epigenetic dysregulation of liver regeneration signals by HBx.

Synergistic activation of lipopolysaccharide-stimulated glial cells by propofol.

Despite the extensive use of propofol in general anesthetic procedures, the effects of propofol on glial cell were not completely understood. In lipopolysaccharide (LPS)-stimulated rat primary astrocytes and BV2 microglial cell lines, co-treatment of propofol synergistically induced inflammatory activation as evidenced by the increased production of NO, ROS and expression of iNOS, MMP-9 and several cytokines. Propofol augmented the activation of JNK and p38 MAPKs induced by LPS and the synergistic activation of glial cells by propofol was prevented by pretreatment of JNK and p38 inhibitors. When we treated BV2 cell culture supernatants treated with LPS plus propofol on cultured rat primary neuron, it induced a significant neuronal cell death. The results suggest that the repeated use of propofol in immunologically challenged situation may induce glial activation in brain.

A role of CPEB1 in the modulation of proliferation and neuronal maturation of rat primary neural progenitor cells.

Cytoplasmic polyadenylation binding protein 1 (CPEB1) is a RNA binding protein, which regulates translation of target mRNAs by regulating polyadenylation status. CPEB1 plays important roles in the regulation of germline cell development by modulating cell cycle progression through the polyadenylation of target mRNAs such as cyclin B1. Similar mechanism is reported in proliferating astrocytes by us, although CPEB1 is involved in the transport of target mRNAs as well as local translation at dendritic spines. In this study, we found the expression of CPEB1 in cultured rat primary neural progenitor cells (NPCs). EGF stimulation of cultured NPCs induced rapid phosphorylation of CPEB1, a hallmark of CPEB1-dependent translational control along with cyclin B1 polyadenylation and translation. EGF-induced activation of ERK1/2 and Aurora A kinase was responsible for CPEB1 phosphorylation. Pharmacological inhibition studies suggested that ERK1/2 is involved in the activation of Aurora A kinase and regulation of CPEB1 phosphorylation in cultured NPCs. Long-term incubation in EGF resulted in the down-regulation of CPEB1 expression, which further increased expression of cyclin B1 and cell cycle progression. When we down-regulated the expression of CPEB1 in NPCs by siRNA transfection, the proliferation of NPCs was increased. Increased NPCs proliferation by down-regulation of CPEB1 resulted in eventual up-regulation of neuronal differentiation with increase in both pre- and post-synaptic proteins. The results from the present study may suggest the importance of translational control in the regulation of neuronal development, an emerging concept in many neurodevelopmental and psychiatric disorders such as autism spectrum disorder.

Positive feedback regulation of Akt-FMRP pathway protects neurons from cell death.

J. Neurochem. (2012) 123, 226-238. ABSTRACT: Fragile X syndrome (FXS), the most common single genetic cause of mental retardation and autistic spectrum disease, occurs when FMR1 gene is mutated. FMR1 encodes fragile X mental retardation protein (FMRP) which regulates translation of mRNAs playing important roles in the development of neurons as well as formation and maintenance of synapses. To examine whether FMRP regulates cell viability, we induced apoptosis in rat primary cortical neurons with glutamate in vitro and with middle cerebral artery occlusion (MCAO) in striatal neurons in vivo. Both conditions elicited a rapid, but transient FMRP expression in neurons. This up-regulated FMRP expression was abolished by pre-treatment with PI3K and Protein Kinase B (Akt) inhibitors: LY294002, Akt inhibitor IV, and VIII. Reduced FMRP expression in vitro or in vivo using small hairpin Fmr1 virus exacerbated cell death by glutamate or MCAO, presumably via hypophosphorylation of Akt and reduced expression of B-cell lymphoma-extra large (Bcl-xL). However, over-expression of FMRP using enhanced green fluorescent protein (eGFP)-FMRP constructs alleviated cell death, increased Akt activity, and enhanced Bcl-xL production. The pro-survival role of Akt-dependent up-regulation of FMRP in glutamate-stimulated cultured neuron as well as in ischemic brain may have a clinical importance in FXS as well as in neurodegenerative disorders and traumatic brain injury.

Cellular stress-induced up-regulation of FMRP promotes cell survival by modulating PI3K-Akt phosphorylation cascades.

BACKGROUND: Fragile X syndrome (FXS), the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP) is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival. METHODS: Apoptotic cell death was induced by etoposide treatment on Hela cells. After we transiently modulated FMRP expression (silencing or enhancing) by using molecular biotechnological methods such as small hairpin RNA virus-induced knock down and overexpression using transfection with FMRP expression vectors, cellular viability was measured using propidium iodide staining, TUNEL staining, and FACS analysis along with the level of activation of PI3K-Akt pathway by Western blot. Expression level of FMRP and apoptotic regulator BcL-xL was analyzed by Western blot, RT-PCR and immunocytochemistry. RESULTS: An increased FMRP expression was measured in etoposide-treated HeLa cells, which was induced by PI3K-Akt activation. Without FMRP expression, cellular defence mechanism via PI3K-Akt-Bcl-xL was weakened and resulted in an augmented cell death by etoposide. In addition, FMRP over-expression lead to the activation of PI3K-Akt signalling pathway as well as increased FMRP and BcL-xL expression, which culminates with the increased cell survival in etoposide-treated HeLa cells. CONCLUSIONS: Taken together, these results suggest that FMRP expression is an essential part of cellular survival mechanisms through the modulation of PI3K, Akt, and Bcl-xL signal pathways.

Activation of adenosine A2A receptor up-regulates BDNF expression in rat primary cortical neurons.

As a member of neurotrophin family, brain derived neurotrophic factor (BDNF) plays critical roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. There have been reported that adenosine A2(A) receptor subtype is widely distributed in the brain regions, such as hippocampus, striatum, and cortex. Adenosine A2(A) receptor is colocalized with BDNF in brain regions and the functional interaction between A2(A) receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that the activation of A2(A) receptor modulates BDNF production in rat primary cortical neuron. CGS21680, an adenosine A2(A) receptor agonist, induced BDNF expression and release. An antagonist against A2(A) receptor, ZM241385, prevented CGS21680-induced increase in BDNF production. A2(A) receptor stimulation induced the activation of Akt-GSK-3ß signaling pathway and the blockade of the signaling pathway with specific inhibitors abolished the increase in BDNF production, possibly via modulation of ERK1/2-CREB pathway. The physiological roles of A2(A) receptor-induced BDNF production was demonstrated by the protection of neurons from the excitotoxicity and increased neurite extension as well as synapse formation from immature and mature neurons. Taken together, activation of A2(A) receptor regulates BDNF production in rat cortical neuron, which provides neuro-protective action.

Akt regulates the expression of MafK, synaptotagmin I, and syntenin-1, which play roles in neuronal function.

BACKGROUND: Akt regulates various cellular processes, including cell growth, survival, and metabolism. Recently, Akt's role in neurite outgrowth has also emerged. We thus aimed to identify neuronal function-related genes that are regulated by Akt. METHODS: We performed suppression subtractive hybridization on two previously established PC12 sublines, one of which overexpresses the wild-type (WT) form and the other, the dominant-negative (DN) form of Akt. These sublines respond differently to NGF's neuronal differentiation effect. RESULTS: A variety of genes was identified and could be classified into several functional groups, one of which was developmental processes. Two genes involved in neuronal differentiation and function were found in this group. v-Maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) induces the neuronal differentiation of PC12 cells and immature telencephalon neurons, and synaptotagmin I (SytI) is essential for neurotransmitter release. Another gene, syntenin-1 (Syn-1) was also recognized in the same functional group into which MafK and SytI were classified. Syn-1 has been reported to promote the formation of membrane varicosities in neurons. Quantitative reverse transcription polymerase chain reaction analyses show that the transcript levels of these three genes were lower in PC12 (WT-Akt) cells than in parental PC12 and PC12 (DN-Akt) cells. Furthermore, treatment of PC12 (WT-Akt) cells with an Akt inhibitor resulted in the increase of the expression of these genes and the improvement of neurite outgrowth. These results indicate that dominant-negative or pharmacological inhibition of Akt increases the expression of MafK, SytI, and Syn-1 genes. Using lentiviral shRNA to knock down endogenous Syn-1 expression, we demonstrated that Syn-1 promotes an increase in the numbers of neurites and branches. CONCLUSIONS: Taken together, these results indicate that Akt negatively regulates the expression of MafK, SytI, and Syn-1 genes that all participate in regulating neuronal integrity in some way or another.

Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.

Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.

Inhibition of GSK-3beta mediates expression of MMP-9 through ERK1/2 activation and translocation of NF-kappaB in rat primary astrocyte.

Glycogen synthase kinase (GSK)-3beta and extracellular signal-regulated kinase (ERK) regulate several cellular signaling pathways in common, including embryonic development, cell differentiation and apoptosis. In this study, we investigated whether GSK-3beta inhibition is involved in ERK activation, which affects the activation of NF-kappaB and induction of MMP-9 in cultured rat primary astrocytes. Here, we found that GSK-3beta inhibition using GSK-3beta inhibitor TDZD-8 increased the phosphorylation of GSK-3beta at Ser9 site as well as the phosphorylation of ERK1/2 and Akt at Ser473 site. In this condition, GSK-3beta inhibition increased MMP-9 but not MMP-2 activity in a concentration-dependent manner. In RT-PCR analysis, MMP-9 mRNA level was increased by GSK-3beta inhibition in a concentration-dependent manner. MMP-9 promoter reporter activity was similarly increased by GSK-3beta inhibition. Pretreatment of U-0126 (MEK1/2 inhibitor) completely abolished the GSK-3beta inhibition-induced phosphorylation of ERK1/2. U-0126 prevented GSK-3beta inhibition-mediated induction of MMP-9 reporter activity as well as the MMP-9 gene expression. The transcriptional activity of NF-kappaB was significantly increased by GSK-3beta inhibition, which was determined by nuclear translocation of NF-kappaB. Inhibition of ERK1/2 activity by U-0126 also completely blocked the nuclear translocation of NF-kappaB. Transfection of dominant negative plasmid (S9A) of GSK-3beta significantly decreased phosphorylation of ERK, MMP-9 expression and nuclear translocation of NF-kappaB by GSK-3beta inhibition as compared to wild type GSK-3beta. These data suggest that GSK-3beta inhibition mediates ERK1/2 activation followed by NF-kappaB activation, which directly regulates the induction of MMP-9 in rat primary astrocytes.

Identification and characterization of four novel peptide motifs that recognize distinct regions of the transcription factor CP2.

Although ubiquitously expressed, the transcriptional factor CP2 also exhibits some tissue- or stage-specific activation toward certain genes such as globin in red blood cells and interleukin-4 in T helper cells. Because this specificity may be achieved by interaction with other proteins, we screened a peptide display library and identified four consensus motifs in numerous CP2-binding peptides: HXPR, PHL, ASR and PXHXH. Protein-database searching revealed that RE-1 silencing factor (REST), Yin-Yang1 (YY1) and five other proteins have one or two of these CP2-binding motifs. Glutathione S-transferase pull-down and coimmunoprecipitation assays showed that two HXPR motif-containing proteins REST and YY1 indeed were able to bind CP2. Importantly, this binding to CP2 was almost abolished when a double amino acid substitution was made on the HXPR sequence of REST and YY1 proteins. The suppressing effect of YY1 on CP2's transcriptional activity was lost by this point mutation on the HXPR sequence of YY1 and reduced by an HXPR-containing peptide, further supporting the interaction between CP2 and YY1 via the HXPR sequence. Mapping the sites on CP2 for interaction with the four distinct CP2-binding motifs revealed at least three different regions on CP2. This suggests that CP2 recognizes several distinct binding motifs by virtue of employing different regions, thus being able to interact with and regulate many cellular partners.

Substitution at rt269 in Hepatitis B Virus Polymerase Is a Compensatory Mutation Associated with Multi-Drug Resistance.

The emergence of compensatory mutations in the polymerase gene of drug resistant hepatitis B virus (HBV) is associated with treatment failure. We previously identified a multi-drug resistant HBV mutant, which displayed resistance towards lamivudine (LMV), clevudine (CLV), and entecavir (ETV), along with a strong replication capacity. The aim of this study was to identify the previously unknown compensatory mutations, and to determine the clinical relevance of this mutation during antiviral therapy. In vitro mutagenesis, drug susceptibility assay, and molecular modeling studies were performed. The rtL269I substitution conferred 2- to 7-fold higher replication capacity in the wild-type (WT) or YMDD mutation backbone, regardless of drug treatment. The rtL269I substitution alone did not confer resistance to LMV, ETV, adefovir (ADV), or tenofovir (TDF). However, upon combination with YMDD mutation, the replication capacity under LMV or ETV treatment was enhanced by several folds. Molecular modeling studies suggested that the rtL269I substitution affects template binding, which may eventually lead to the enhanced activity of rtI269-HBV polymerase in both WT virus and YMDD mutant. The clinical relevance of the rtL269I substitution was validated by its emergence in association with YMDD mutation in chronic hepatitis B (CHB) patients with sub-optimal response or treatment failure to LMV or CLV. Our study suggests that substitution at rt269 in HBV polymerase is associated with multi-drug resistance, which may serve as a novel compensatory mutation for replication-defective multi-drug resistant HBV.

Transcription factor CP2 is involved in activating mBMP4 in mouse mesenchymal stem cells.

CP2 is a member of a family of transcription factors that regulate genes involved in events from early development to terminal differentiation. In an effort to understand how it selects its target genes we carried out a database search, and located several CP2 binding motifs in the promoter region of bone morphogenetic protein-4 (BMP4). BMP4 is a key regulator of cell fate and body patterning throughout development. For the CP2 binding motifs in BMP4 promoter region to be relevant in vivo, CP2 and BMP4 should be expressed together. We found that CP2b and CP2c, two potent transcriptional activators, are expressed in a manner similar to BMP4 during osteoblast differentiation of C3H10T1/2 cells. In in vitro assays, the CP2 proteins bound to two CP2 binding motifs (-715 to -676 and -147 to -118) in the BMP4 promoter, and luciferase reporter assays indicated that this binding was essential for transcription of BMP4 during osteoblast differentiation. Taken together, our data indicate that CP2b and CP2c play important roles during bone development by activating BMP4 transcription.

Synthesis and in vitro evaluation of 1,8-diazaanthraquinones bearing 3-dialkylaminomethyl or 3-(N-alkyl- or N-aryl)carbamoyloxymethyl substituent.

A series of 1,8-diazaanthraquinone derivatives carrying a 3-dialkylaminomethyl or a 3-(N-alkyl or aryl)carbamoyloxymethyl substituent was synthesised and their in vitro cytotoxic activities were evaluated against eight human cancer cell lines (HOP62, SK-OV-3, HCT-15, SF295, MCF7, SNU-354, KB-3-1 and KB-V-1). A number of compounds including 8c, 8d and 11c showed cytotoxic activity comparable to that of doxorubicin against all human cancer cell lines tested. The compounds 8c and 8d were 2-100 times more potent than doxorubicin against HCT-15, MCF7 and SNU-354 cancer cell lines. Furthermore, these compounds retained considerable cytotoxic activity against the doxorubicin-resistant cell line KB-V-1, implying their therapeutic potential to treat doxorubicin-resistant tumours. These compounds inhibited topoisomerase II-mediated DNA relaxation in vitro, suggesting that this inhibitory effect be attributable to their cytotoxicity.

Design and synthesis of isoindoloquinoline derivatives as potential antitumor agents.

A series of isoindoloquinoline derivatives was synthesized and evaluated in vitro cytotoxicity against four human cancer cell lines (HCT15, SK-OV-3, MDA-MB-468 and T-47D).

Synthesis and in vitro cytotoxicity of- 1,3-dioxoindan-2-carboxylic acid arylamides.

A series of 1,3-dioxoindan-2-carboxylic acid arylamides were synthesized and evaluated for in vitro cytotoxicity against four human cancer cell lines (HOP62, SK-OV-3, MD-MB-468 and T-47D). The most active was compound 3e (1.2 microM against SK-OV-3 cell line) bearing a 4-methyl substituent.

Transcriptional Upregulation of Plasminogen Activator Inhibitor-1 in Rat Primary Astrocytes by a Proteasomal Inhibitor MG132.

Plasminogen activator inhibitor-1 (PAI-1) is a member of serine protease inhibitor family, which regulates the activity of tissue plasminogen activator (tPA). In CNS, tPA/PAI-1 activity is involved in the regulation of a variety of cellular processes such as neuronal development, synaptic plasticity and cell survival. To gain a more insights into the regulatory mechanism modulating tPA/PAI-1 activity in brain, we investigated the effects of proteasome inhibitors on tPA/PAI-1 expression and activity in rat primary astrocytes, the major cell type expressing both tPA and PAI-1. We found that submicromolar concentration of MG132, a cell permeable peptide-aldehyde inhibitor of ubiquitin proteasome pathway selectively upregulates PAI-1 expression. Upregulation of PAI-1 mRNA as well as increased PAI-1 promoter reporter activity suggested that MG132 transcriptionally increased PAI-1 expression. The induction of PAI-1 downregulated tPA activity in rat primary astrocytes. Another proteasome inhibitor lactacystin similarly increased the expression of PAI-1 in rat primary astrocytes. MG132 activated MAPK pathways as well as PI3K/Akt pathways. Inhibitors of these signaling pathways reduced MG132-mediated upregulation of PAI-1 in varying degrees and most prominent effects were observed with SB203580, a p38 MAPK pathway inhibitor. The regulation of tPA/PAI-1 activity by proteasome inhibitor in rat primary astrocytes may underlie the observed CNS effects of MG132 such as neuroprotection.

Valproic Acid Regulates α-Synuclein Expression through JNK Pathway in Rat Primary Astrocytes.

Although the role of α-synuclein aggregation on Parkinson's disease is relatively well known, the physiological role and the regulatory mechanism governing the expression of α-synuclein are unclear yet. We recently reported that α-synuclein is expressed and secreted from cultured astrocytes. In this study, we investigated the effect of valproic acid (VPA), which has been suggested to provide neuroprotection by increasing α-synuclein in neuron, on α-synuclein expression in rat primary astrocytes. VPA concentrationdependently increased the protein expression level of α-synuclein in cultured rat primary astrocytes with concomitant increase in mRNA expression level. Likewise, the level of secreted α-synuclein was also increased by VPA. VPA increased the phosphorylation of Erk1/2 and JNK and pretreatment of a JNK inhibitor SP600125 prevented the VPA-induced increase in α-synuclein. Whether the increased α-synuclein in astrocytes is involved in the reported neuroprotective effects of VPA awaits further investigation.

Oroxylin A Induces BDNF Expression on Cortical Neurons through Adenosine A2A Receptor Stimulation: A Possible Role in Neuroprotection.

Oroxylin A is a flavone isolated from a medicinal herb reported to be effective in reducing the inflammatory and oxidative stresses. It also modulates the production of brain derived neurotrophic factor (BDNF) in cortical neurons by the transactivation of cAMP response element-binding protein (CREB). As a neurotrophin, BDNF plays roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. Adenosine A2A receptor colocalized with BDNF in brain and the functional interaction between A2A receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that oroxylin A modulates BDNF production in cortical neuron through the regulation of A2A receptor system. As ex-pected, CGS21680 (A2A receptor agonist) induced BDNF expression and release, however, an antagonist, ZM241385, prevented oroxylin A-induced increase in BDNF production. Oroxylin A activated the PI3K-Akt-GSK-3ß signaling pathway, which is inhibited by ZM241385 and the blockade of the signaling pathway abolished the increase in BDNF production. The physiological roles of oroxylin A-induced BDNF production were demonstrated by the increased neurite extension as well as synapse formation from neurons. Overall, oroxylin A might regulate BDNF production in cortical neuron through A2A receptor stimulation, which promotes cellular survival, synapse formation and neurite extension.

The critical period of valproate exposure to induce autistic symptoms in Sprague-Dawley rats.

Prenatal exposure to valproic acid (VPA) induces neural tube defects and impairment in social behaviors related to autistic spectrum disorder in newborns, which make it a useful animal model of autism. In this study, we compared the effects of different time window of prenatal valproic acid exposure for inducing the altered social behaviors relevant to autism from embryonic day 7 to embryonic day 15 in Sprague-Dawley rats to determine the critical periods for the impairment. Compared to E7, E9.5 and E15 exposure, VPA exposure at E12 showed most significant changes in behaviors over control animals with reduced sociability and social preference. E9.5 exposure to valproic acid showed strong reproductive toxicity including decrease in the number of live birth. In general, exposure at E15 showed only marginal effects on reproduction and social behaviors. Finally, VPA-exposed rats at E12 were more sensitive to electric shock than VPA-exposed rats at any other periods. These results suggested that E12 is the critical period in rats when valproate exposure has prominent effects for inducing the altered social behavior similar to human autistic behavior.

RPS3a over-expressed in HBV-associated hepatocellular carcinoma enhances the HBx-induced NF-κB signaling via its novel chaperoning function.

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC) development. Hepatitis B virus X protein (HBx) is known to play a key role in the development of hepatocellular carcinoma (HCC). Several cellular proteins have been reported to be over-expressed in HBV-associated HCC tissues, but their role in the HBV-mediated oncogenesis remains largely unknown. Here, we explored the effect of the over-expressed cellular protein, a ribosomal protein S3a (RPS3a), on the HBx-induced NF-κB signaling as a critical step for HCC development. The enhancement of HBx-induced NF-κB signaling by RPS3a was investigated by its ability to translocate NF-κB (p65) into the nucleus and the knock-down analysis of RPS3a. Notably, further study revealed that the enhancement of NF-κB by RPS3a is mediated by its novel chaperoning activity toward physiological HBx. The over-expression of RPS3a significantly increased the solubility of highly aggregation-prone HBx. This chaperoning function of RPS3a for HBx is closely correlated with the enhanced NF-κB activity by RPS3a. In addition, the mutational study of RPS3a showed that its N-terminal domain (1-50 amino acids) is important for the chaperoning function and interaction with HBx. The results suggest that RPS3a, via extra-ribosomal chaperoning function for HBx, contributes to virally induced oncogenesis by enhancing HBx-induced NF-κB signaling pathway.