Ke-Sheng Wang

Epigenetic modification induced by hepatitis B virus X protein via interaction with de novo DNA methyltransferase DNMT3A

BACKGROUND/AIMS The hepatitis B virus X protein (HBx) has been implicated as a potential trigger of the epigenetic deregulation of some genes, but the underlying mechanisms remain unknown. The aim of this study was to identify underlying mechanisms involved in HBx-mediated epigenetic modification. METHODS Interactions between HBx and DNA methyltransferase (DNMT) or histone deacetylase-1 (HDAC1) were assessed by co-immunoprecipitation. DNA methylation of gene promoters was detected by bisulfite sequencing, and HBx-mediated protein binding to gene regulatory elements was evaluated by chromatin immunoprecipitation. Target gene transcriptional activity was measured by real-time polymerase chain reaction. RESULTS HBx can interact directly with DNMT3A and HDAC1. HBx recruited DNMT3A to the regulatory promoters of interleukin-4 receptor and metallothionein-1F and subsequently silenced their transcription via de novo DNA methylation. By contrast, the transcription of CDH6 and IGFBP3 was triggered by HBx through the deprivation of DNMT3A from their promoters. Transcriptional levels of target genes in hepatocellular carcinoma (HCC) specimens were strongly correlated with the occurrence of HBx. CONCLUSIONS The interaction of HBx and DNMT3A facilitates cellular epigenetic modification (via regional hypermethylation or hypomethylation) at distinct genomic loci, providing an alternative mechanism within HBx-mediated transcriptional regulation, and a profound understanding of hepatitis and HCC pathogenesis.

ING4 induces G2/M cell cycle arrest and enhances the chemosensitivity to DNA-damage agents in HepG2 cells.

The known members of inhibitor of growth (ING) gene family are considered as candidate tumor suppressor genes. ING4, a novel member of ING family, is recently reported to interact with tumor suppressor p53, p300 (a major component of histone acetyl transferase complexes), and p65(RelA) subunit of NF‐κB. In this study, we investigated the cellular behaviors of HepG2 cells with exogenous ING4. Interestingly, the overexpression of ING4 negatively regulated the cell growth with significant G2/M arrest of cell cycle, and moreover, enhanced the cell apoptosis triggered by serum starvation in HepG2 cells. Furthermore, the exogenous ING4 could upregulate endogenous p21 and Bax in HepG2 cells, not in p53‐deficient Saos‐2 cells, suggesting that G2/M arrest induced by ING4 could be mediated by the increased p21 expression in a p53‐dependent manner, although there is no significant increase of p53 expression in HepG2 cells. Moreover, HepG2 cells with exogenous ING4 could significantly increase cell death, as exposed to some DNA‐damage agents, such as etoposide and doxorubicin, implying that ING4 could enhance chemosensitivity to certain DNA‐damage agents in HepG2 cells.

E2F8 Contributes to Human Hepatocellular Carcinoma via Regulating Cell Proliferation

The E2F family member of transcription factors includes the atypical member E2F8, which has been little studied in cancer. We report that E2F8 is strongly upregulated in human hepatocellular carcinoma (HCC), where it was evidenced to contribute to oncogenesis and progression. Ectopic overexpression of E2F8 promoted cell proliferation, colony formation, and tumorigenicity, whereas E2F8 knockdown inhibited these phenotypes, as documented in Huh-7, Focus, Hep3B, and YY-8103 HCC cell lines. Mechanistic analyses indicated that E2F8 could bind to regulatory elements of cyclin D1, regulating its transcription and promoting accumulation of S-phase cells. Together, our findings suggest that E2F8 contributes to the oncogenic potential of HCC and may constitute a potential therapeutic target in this disease.

The Novel Lipopolysaccharide-Binding Protein CRISPLD2 Is a Critical Serum Protein to Regulate Endotoxin Function

LPS is an immunostimulatory component of Gram-negative bacteria. Acting on the immune system in a systemic fashion, LPS exposes the body to the hazard of septic shock. In this study we report that cysteine-rich secretory protein LCCL domain containing 2 (CRISPLD2/Crispld2; human and mouse/rat versions, respectively), expressed by multitissues and leukocytes, is a novel LPS-binding protein. As a serum protein, median CRISPLD2 concentrations in health volunteers and umbilical cord blood samples are 607 μg/ml and 290 μg/ml, respectively. Human peripheral blood granulocytes and mononuclear cells including monocytes, NK cells, and T cells spontaneously release CRISPLD2 (range, 0.2–0.9 μg/ml) and enhance CRISPLD2 secretion (range, 1.5–4.2 μg/ml) in response to stimulation of both LPS and humanized anti-human TLR4-IgA Ab in vitro. CRISPLD2 exhibits significant LPS binding affinity similar to that of soluble CD14, prevents LPS binding to target cells, reduces LPS-induced TNF-α and IL-6 production, and protects mice against endotoxin shock. In in vivo experiments, serum Crispld2 concentrations increased in response to a nontoxic dose of LPS and correlated negatively with LPS lethality, suggesting that CRISPLD2 serum concentrations not only are indicators of the degree of a body’s exposure to LPS but also reflect an individual’s LPS sensitivity.

hBolA, novel non-classical secreted proteins, belonging to different BolA family with functional divergence

This study reported that all three human BolA proteins (hBolA1, hBolA2, and hBolA3) are novel non-classical secreted proteins identified with bioinformatics and molecular biology experiments. The three BolA fusion proteins with c-Myc tag could be secreted into the culture medium of the transfected Cos-7 cells, although they could not be colocalized with Golgi apparatus. And the secretion of three BolA proteins could not be inhibited after BFA treatment. Furthermore, the secretion was not dependent on its predicted signal peptide. All the experiment results suggested that the secretion was a non-classical export. Phylogenetic analysis showed that the human BolAs belong to three different groups with functional divergence of BolA subfamily, where the different helix-turn-helix motif among hBolA1, hBolA2, and hBolA3 could be responsible for their functional divergence. Our data provided a basis for functional studies of BolA protein family.

Insulin receptor tyrosine kinase substrate enhances low levels of MDM2-mediated p53 ubiquitination.

The tumor suppressor p53 controls multiple cellular functions including DNA repair, cell cycle arrest and apoptosis. MDM2-mediated p53 ubiquitination affects both degradation and cytoplasmic localization of p53. Several cofactors are known to modulate MDM2-mediated p53 ubiquitination and proteasomal degradation. Here we show that IRTKS, a novel IRSp53-like protein inhibited p53-induced apoptosis and depressed its transcription activity. IRTKS bound directly to p53 and increased p53 ubiquitination and cytoplasmic localization. Further studies revealed that IRTKS interacted with MDM2 and promoted low levels of MDM2-mediated p53 ubiquitination in vitro and in vivo. In unstressed cells with low levels of MDM2, IRTKS was found to stabilize the interaction of p53 and MDM2. In stressed cells, IRTKS dissociated from p53, and high levels of MDM2 induced by p53 activation mediate IRTKS poly-ubiquitination and subsequent proteasomal degradation. These data suggest that IRTKS is a novel regulator of p53, modulating low level of MDM2-mediated p53 ubiquitination in unstressed cells.

Inhibitor of growth 4 (ING4) is up-regulated by a low K intake and suppresses renal outer medullary K channels (ROMK) by MAPK stimulation

Dietary K intake plays an important role in the regulation of renal K secretion: a high K intake stimulates whereas low K intake suppresses renal K secretion. Our previous studies demonstrated that the Src family protein-tyrosine kinase and mitogen-activated protein kinase (MAPK) are involved in mediating the effect of low K intake on renal K channels and K secretion. However, the molecular mechanism by which low K intake stimulates MAPK is not completely understood. Here we show that inhibitor of growth 4 (ING4), a protein with a highly conserved plant homeodomain finger motif, is involved in mediating the effect of low K intake on MAPK. K restriction stimulates the expression of ING4 in the kidney and superoxide anions, and its related products are involved in mediating the effect of low K intake on ING4 expression. We used HEK293 cells to express ING4 and observed that expression of ING4 increased the phosphorylation of p38 and ERK MAPK, whereas down-regulation of ING4 with small interfering RNA decreased the phosphorylation of p38 and ERK. Immunocytochemistry showed that ING4 was expressed in the renal outer medullary potassium (ROMK)-positive tubules. Moreover, ING4 decreased K currents in Xenopus oocytes injected with ROMK channel cRNA. This inhibitory effect was reversed by blocking p38 and ERK MAPK. These data provide evidence for the role of ING4 in mediating the effect of low K intake on ROMK channel activity by stimulation of p38 and ERK MAPK.

Abstract 1008: IRTKS suppresses p53 activity through promoting MDM2 mediated p53 monoubiquitination

The ubiquitination of p53, including poly- and mono- ubiqitination, is important for the regulation of p53 function. The E3 ubiquitin ligase MDM2 mediates both monoubiquitination and polyubiquitination on p53 in a dose-dependent manner. Low levels of MDM2 induce monoubiquitination and nuclear export of p53, while high levels promote its polyubiquitination and degradation. The regulator of MDM2 mediated p53 monoubiquitination has been rarely identified, whereas several cofactors have been defined to regulate MDM2 induced p53 degradation. Insulin Receptor Tyrosine Kinase Substrate (IRTKS), an widespread expressed IRSP53/MIM homology domain(IMD) containing protein, has been reported as a scaffold protein in actin dynamics and membrane protrusions by associating with RAC1. As a protein located ubiquitously in cells, the function of IRTKS in nucleus remains to be elucidated. Here, we showed that ectopic IRTKS reduced and knockdown of IRTKS increased the apoptosis induced by UV irradiation in HT1080 cells in a p53-dependent manner. IRTKS inhibited the apoptosis and the expression of p53 target genes, BAX, PIG3 and MDM2, in p53 null SOAS-2 cells with force expressed p53. We found that IRTKS could directly bind to p53 in cellular nucleus in unstressed cells by using reciprocal co-immunoprecipitation analysis and fluorescence resonance energy transfer (FRET) assay. In vitro binding experiment indicated that IMD and the central region of IRTKS, DNA bind domain and C-terminal regulating domain of p53 were responsible for the interaction. Interestingly, over-expression of IRTKS induced p53 ubiquitination and nuclear export without affecting its degradation. Because IRTKS alone could not mediate p53 ubiquitination in vitro, we examined the association of IRTKS and MDM2 by using the co-immunoprecipitation analysis and in vitro binding experiment. The directly interaction through IMD of IRTKS and the central region of MDM2 has been detected. Both in vitro and in vivo ubiquitin experiment showed that IRTKS only enhanced p53 monoubiquitination mediated by low levels of MDM2, but not affected its polyubiquitination. In H1299 cells cotransfected with IRTKS, p53 and different amount MDM2, the co-immunoprecipitation experiment indicated that the association of MDM2 and p53 was significantly enhanced by ectopic IRTKS at low levels of MDM2. In IRTKS siRNA-treated H1299 cells, the p53-MDM2 interaction was reduced at low levels of MDM2. Interestingly, overexpression of MDM2 reduced the protein levels of IRTKS in HT1080 and H1299 cells. Additional in vivo and in vitro ubiquitin assays unraveled that MDM2 is an E3 ubiquitin ligase of IRTKS and mediated IRTKS polyubiquitination at high levels. Taking together, we demonstrated that IRTKS, as a scaffold protein, may play an important role in fine tune of p53 functions in an MDM2 dependent manner during normal homeostasis of the unstressed cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1008.