ATP1B3 Restricts Hepatitis B Virus Replication Via Reducing the Expression of the Envelope Proteins

Our recent study reported that ATP1B3 inhibits hepatitis B virus(HBV) replication via inducing NF-κB activation.However, ATP1B3 mutants which were defective in NF-κB activation still maintained the moderate degree of suppression on HBV replication, suggesting that another uncharacterized mechanism is also responsible for ATP1B3-mediated HBV suppression. Here, we demonstrated that ATP1B3 reduced the expression of HBV envelope proteins LHBs, MHBs and SHBs, but had no effect on intracellular HBV DNA, RNA levels as well as HBV promoter activities. Further investigation showed that proteasome inhibitor MG132 rescued ATP1B3-mediated envelope proteins degradation, demonstrating that proteasome-dependent pathway is involved in ATP1B3-induced degradation of envelope proteins. Co-IP showed that ATP1B3 interacts with LHBs and MHBs and induces LHBs and MHBs polyubiquitination. Immunofluorescence colocalization analysis confirmed LHBs and MHBs colocalized with ATP1B3 together. Our work provides important information for targeting ATP1B3 as a potential therapeutic molecule for HBV infection.     

Hepatitis B virus large and middle glycoproteins are degraded by a proteasome pathway in glucosidase-inhibited cells but not in cells with functional glucosidase enzyme

The secretion of hepatitis B virus (HBV) large (LHBs) and middle (MHBs) envelope polypeptides from tissue cultures requires proper protein folding and is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase. Using competitive inhibitors of the ER glucosidase, here it is shown that the amounts of glycosylated and unglycosylated forms of LHBs and MHBs proteins are all greatly reduced in tissue cultures producing HBV envelope glycoproteins. In contrast, the HBV small (SHBs) protein was not affected. The reduction in secretion of LHBs and MHBs proteins appears to be mediated by proteasomal degradation pathways, since it is prevented by either lactacystin or epoxomicin, two inhibitors of proteasomal degradation. Although there is no detectable proteasomal degradation of LHBs and MHBs in cells with functional glucosidase, the implications of the nearly quantitative sensitivity of glycosylated and unglycosylated forms of LHBs and MHBs proteins, with selective sparing of SHBs protein, in cells in which glucosidase is inhibited is surprising, and its implications are discussed.     

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.     

The hepatitis B virus protein MHBs(t) sensitizes hepatoma cells to TRAIL-induced apoptosis through ERK2

The TNF-related apoptosis-inducing ligand (TRAIL) has recently been implicated in the death of hepatocytes under infectious but not normal conditions. Infectious agents, such as hepatitis B virus (HBV), may play important roles in regulating the sensitivity of hepatocytes to TRAIL. Our previous studies showed that HBx, a protein encoded by the HBV genome, enhanced TRAIL-induced apoptosis through upregulating Bax. We report here that another HBV protein called MHBs(t) (C-terminally truncated middle hepatitis B surface protein) is also a potent regulator of TRAIL-induced apoptosis. Overexpressing MHBs(t) in hepatoma cells enhanced TRAIL-induced apoptosis. Mechanistic studies reveal that MHBs(t) had no effect on Bax or TRAIL receptor expression or procaspase-8 activation, but selectively enhanced the activation of ERK2 (extracellular signal-regulated kinase 2) and the degradation of procaspases-3 and 9. ERK2 activation is required for the MHBs(t) effect because ERK2 inhibition by its inhibitor PD98059 significantly reversed TRAIL-induced apoptosis of MHBs(t)-transfected cells. These results establish that unlike HBx, MHBs(t) enhances TRAIL-induced hepatocyte apoptosis through a novel mechanism that involves ERK2. Therefore, manipulating the ERK2 signaling pathway may provide new therapeutic opportunities to contain hepatic cell death during HBV infection. Xiaohong Liang and Juan Du contributed equally to this work.     

G-CSF stimulates Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation

Granulocyte colony-stimulating factor (G-CSF), the major cytokine regulator of neutrophilic granulopoiesis, stimulates both the proliferation and differentiation of myeloid precursors. A variety of signaling proteins have been identified as mediators of G-CSF signaling, but understanding of their specific interactions and organization into signaling pathways for particular cellular effects is incomplete. The present study examined the role of the scaffolding protein Grb2-associated binding protein-2 (Gab2) in G-CSF signaling. We found that a chemical inhibitor of Janus kinases inhibited G-CSF-stimulated Gab2 phosphorylation. Transfection with Jak2 antisense and dominant negative constructs also inhibited Gab2 phosphorylation in response to G-CSF. In addition, G-CSF enhanced the association of Jak2 with Gab2. In vitro, activated Jak2 directly phosphorylated specific Gab2 tyrosine residues. Mutagenesis studies revealed that Gab2 tyrosine 643 (Y643) was a major target of Jak2 in vitro, and a key residue for Jak2-dependent phosphorylation in intact cells. Mutation of Gab2 Y643 inhibited G-CSF-stimulated Erk1/2 activation and Shp2 binding to Gab2. Loss of Y643 also inhibited Gab2-mediated G-CSF-stimulated cell proliferation. Together, these results identify a novel signaling pathway involving Jak2-dependent Gab2 phosphorylation leading to Erk1/2 activation and cell proliferation in response to G-CSF.     

RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-Ras signaling

The RasGRPs are a family of Ras activators that possess diacylglycerol-binding C1 domains. In T cells, RasGRP1 links TCR signaling to Ras. B cells coexpress RasGRP1 and RasGRP3. Using Rasgrp1 and Rasgrp3 single and double null mutant mice, we analyzed the role of these proteins in signaling to Ras and Erk in B cells. RasGRP1 and RasGRP3 both contribute to BCR-induced Ras activation, although RasGRP3 alone is responsible for maintaining basal Ras-GTP levels in unstimulated cells. Surprisingly, RasGRP-mediated Ras activation is not essential for B cell development because this process occurs normally in double-mutant mice. However, RasGRP-deficient mice do exhibit humoral defects. Loss of RasGRP3 led to isotype-specific deficiencies in Ab induction in immunized young mice. As reported previously, older Rasgrp1-/- mice develop splenomegaly and antinuclear Abs as a result of a T cell defect. We find that such mice have elevated serum Ig levels of several isotypes. In contrast, Rasgrp3-/- mice exhibit hypogammaglobulinemia and show no signs of splenomegaly or autoimmunity. Double-mutant mice exhibit intermediate serum Ab titers, albeit higher than wild-type mice. Remarkably, double-mutant mice exhibit no signs of autoimmunity or splenomegaly. B cell proliferation induced by BCR ligation with or without IL-4 was found to be RasGRP1- and RasGRP3-dependent. However, the RasGRPs are not required for B cell proliferation per se, because LPS-induced proliferation is unaffected in double-mutant mice.     

TCR-mediated Erk activation does not depend on Sos and Grb2 in peripheral human T cells

Sos proteins are ubiquitously expressed activators of Ras. Lymphoid cells also express RasGRP1, another Ras activator. Sos and RasGRP1 are thought to cooperatively control full Ras activation upon T-cell receptor triggering. Using RNA interference, we evaluated whether this mechanism operates in primary human T cells. We found that T-cell antigen receptor (TCR)-mediated Erk activation requires RasGRP1, but not Grb2/Sos. Conversely, Grb2/Sos—but not RasGRP1—are required for IL2-mediated Erk activation. Thus, RasGRP1 and Grb2/Sos are insulators of signals that lead to Ras activation induced by different stimuli, rather than cooperating downstream of the TCR.     

Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta

Activation of mitogen-activated protein kinase (Erk/MAPK) is a critical signal transduction event for estrogen (E(2))-mediated cell proliferation. Recent studies from our group and others have shown that persistent activation of Erk plays a major role in cell migration and tumor progression. The signaling mechanism(s) responsible for persistent Erk activation are not fully characterized, however. In this study, we have shown that E(2) induces a slow but persistent activation of Erk in MCF-7 breast carcinoma cells. The E(2)-induced Erk activation is dependent on new protein synthesis, suggesting that E(2)-induced growth factors play a major role in Erk activation. When MCF-7 cells were treated with E(2) in the presence of an anti-HER-2 monoclonal antibody (herceptin), 60-70% of E(2)-induced Erk activation is blocked. In addition, when untreated MCF-7 cells were exposed to conditioned medium from E(2)-treated cells, Erk activity was significantly enhanced. Furthermore Erk activity was blocked by an antibody against HER-2 or by heregulin (HRG) depletion from the conditioned medium through immunoprecipitation. In contrast, epidermal growth factor receptor (Ab528) antibody only blocked 10-20% of E(2)-induced Erk activation, suggesting that E(2)-induced Erk activation is predominantly mediated through the secretion of HRG and activation of HER-2 by an autoctine/paracrine mechanism. Inhibition of PKC-delta-mediated signaling by a dominant negative mutant or the relatively specific PKC-delta inhibitor rottlerin blocked most of the E(2)-induced Erk activation but had no effect on TGF alpha-induced Erk activation. By contrast inhibition of Ras, by inhibition of farnesyl transferase (Ftase-1) or dominant negative (N17)-Ras, significantly inhibited both E(2)- and TGF alpha-induced Erk activation. This evaluation of downstream signaling revealed that E(2)-induced Erk activation is mediated by a HRG/HER-2/PKC-delta/Ras pathway that could be crucial for E(2)-dependent growth-promoting effects in early stages of tumor progression.     

2-O Heparan Sulfate Sulfation by Hs2st Is Required for Erk/Mapk Signalling Activation at the Mid-Gestational Mouse Telencephalic Midline

Heparan sulfate (HS) is a linear carbohydrate composed of polymerized uronate-glucosamine disaccharide units that decorates cell surface and secreted glycoproteins in the extracellular matrix. In mammals HS is subjected to differential sulfation by fifteen different heparan sulfotransferase (HST) enzymes of which Hs2st uniquely catalyzes the sulfation of the 2-O position of the uronate in HS. HS sulfation is postulated to be important for regulation of signaling pathways by facilitating the interaction of HS with signaling proteins including those of the Fibroblast Growth Factor (Fgf) family which signal through phosphorylation of extracellular signal-regulated kinases Erk1/2. In the developing mouse telencephalon Fgf2 signaling regulates proliferation and neurogenesis. Loss of Hs2st function phenocopies the thinned cerebral cortex of mutant mice in which Fgf2 or Erk1/2 function are abrogated, suggesting the hypothesis that 2-O-sulfated HS structures play a specific role in Fgf2/Erk signaling pathway in this context in vivo. This study investigated the molecular role of 2-O sulfation in Fgf2/Erk signaling in the developing telencephalic midline midway through mouse embryogenesis at E12.5. We examined the expression of Hs2st, Fgf2, and Erk1/2 activity in wild-type and Hs2st^-/- mice. We found that Hs2st is expressed at high levels at the midline correlating with high levels of Erk1/2 activation and Erk1/2 activation was drastically reduced in the Hs2st^-/- mutant at the rostral telencephalic midline. We also found that 2-O sulfation is specifically required for the binding of Fgf2 protein to Fgfr1, its major cell-surface receptor at the rostral telencephalic midline. We conclude that 2-O sulfated HS structures generated by Hs2st are needed to form productive signaling complexes between HS, Fgf2 and Fgfr1 that activate Erk1/2 at the midline. Overall, our data suggest the interesting possibility that differential expression of Hs2st targets the rostral telencephalic midline for high levels of Erk signaling by increasing the sensitivity of cells to an Fgf2 signal that is rather more widespread.     

Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways

Prolactin (PRL) stimulates breast cancer cell proliferation; however, the involvement of PRL-activated signaling molecules in cell proliferation is not fully established. Here we studied the role of c-Src on PRL-stimulated proliferation of T47D and MCF7 breast cancer cells. We initially observed that PRL-dependent activation of focal adhesion kinase (Fak), Erk1/2, and cell proliferation was mediated by c-Src in T47D cells, because expression of a dominant-negative form of c-Src (SrcDM, K295A/Y527F) blocked the PRL-dependent effects. The Src inhibitor PP1 abrogated PRL-dependent in vivo activation of Fak, Erk1/2, p70S6K, and Akt and the proliferation of T47D and MCF7 cells; Janus kinase 2 (Jak2) activation was not affected. However, in vitro, Fak and Jak2 kinases were not directly inhibited by PP1, demonstrating the effect of PP1 on c-Src kinase as an upstream activator of Fak. Expression of Fak mutant Y397F abrogated PRL-dependent activation of Fak, Erk1/2, and thymidine incorporation, but had no effect on p70S6K and Akt kinases. MAPK kinase 1/2 (Mek1/2) inhibitor PD184352 blocked PRL-induced stimulation of Erk1/2 and cell proliferation; however, p70S6K and Akt activation were unaffected. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished cell proliferation and activation of p70S6K and Akt; however, PRL-dependent activation of Erk1/2 was not modified. Moreover, we show that both c-Src/PI3K and c-Src/Fak/Erk1/2 pathways are involved in the up-regulation of c-myc and cyclin d1 expression mediated by PRL. The previous findings suggest the existence of two PRL-dependent signaling cascades, initiated by the c-Src-mediated activation of Fak/Erk1/2 and PI3K pathways that, subsequently, control the expression of c-Myc and cyclin D1 and the proliferation of T47D and MCF7 breast cancer cells.     

Constitutive association of c-N-Ras with c-Raf-1 and protein kinase C epsilon in latent signaling modules

Phorbol ester stimulation of the MAPK cascade is believed to be mediated through the protein kinase C (PKC)-dependent activation of Raf-1. Although several studies suggest that phorbol ester stimulation of MAPK is insensitive to dominant-negative Ras, a requirement for Ras in Raf-1 activation by PKC has been suggested recently. We now demonstrate that in normal, quiescent mouse fibroblasts, endogenous c-N-Ras is constitutively associated with both c-Raf-1 and PKC epsilon in a biochemically silent, but latent, signaling module. Chemical inhibition of novel PKCs blocks phorbol 12-myristate 13-acetate (PMA)-mediated activation of MAPKs. Down-regulation of PKC epsilon protein levels by antisense oligodeoxyribonucleotides blocks MAPK activation in response to PMA stimulation, demonstrating that PKC epsilon activity is required for MAPK activation by PMA. c-Raf-1 activity in immunoprecipitated c-N-Ras.c-Raf-1.PKC epsilon complexes is stimulated by PMA and is inhibited by GF109203X, thereby linking c-Raf-1 activation in this complex to PKC activation. These observations suggest that in quiescent cells Ras is organized into ordered, inactive signaling modules. Furthermore, the regulation of the MAPK cascade by both Ras and PKC is intimately linked, converging at the plasma membrane through their association with c-Raf-1.     

Bisindolylmaleimide I suppresses fibroblast growth factor-mediated activation of Erk MAP kinase in chondrocytes by preventing Shp2 association with the Frs2 and Gab1 adaptor proteins

Fibroblast growth factors (FGFs) inhibit chondrocyte proliferation via the Erk MAP kinase pathway. Here, we explored the role of protein kinase C in FGF signaling in chondrocytes. Erk activity in FGF2-treated RCS (rat chondrosarcoma) chondrocytes or human primary chondrocytes was abolished by the protein kinase C inhibitor bisindolylmaleimide I (Bis I). Bis I inhibited FGF2-induced activation of MEK, Raf-1, and Ras members of Erk signaling module but not the FGF2-induced tyrosine phosphorylation of Frs2 or the kinase activity of FGFR3, demonstrating that it targets the Erk cascade immediately upstream of Ras. Indeed, Bis I abolished the FGF2-mediated association of Shp2 tyrosine phosphatase with Frs2 and Gab1 adaptor proteins necessary for proper Ras activation. We also determined which PKC isoform is involved in FGF2-mediated activation of Erk. When both conventional and novel PKCs expressed by RCS chondrocytes (PKCalpha, -gamma, -delta, and -epsilon) were down-regulated by phorbol ester, cells remained responsive to FGF2 with Erk activation, and this activation was sensitive to Bis I. Moreover, treatment with PKClambda/zeta pseudosubstrate lead to significant reduction of FGF2-mediated activation of Erk, suggesting involvement of an atypical PKC.     

用Far-Western印迹技术筛选人肝组织中与乙肝病毒表面抗原PreS1相互作用的蛋白

目的:利用Far-Western印迹技术从正常人肝组织中筛选乙型肝炎病毒(HBV)表面抗原PreS1结合蛋白,为阐明HBV的感染致病机理提供依据。方法:提取正常人肝组织细胞膜蛋白,双向电泳展示后转膜,对表达纯化获得的PreS1的重要片段与GST的融合蛋白PreS/1-48myr-GST进行Far-Western印迹实验,对筛选获得的蛋白点切胶,质谱鉴定。结果:对PreS/1-48myr-GST融合蛋白进行Far-Western-2D筛选,共获得22个蛋白点,经质谱鉴定获得15个候选相互作用蛋白,其中膜蛋白Ezrin可能在HBV感染致病过程中具有重要作用。结论:Ezrin蛋白能够与乙肝病毒表面抗原PreS1结合,其在HBV感染致病过程中的重要作用值得探索。     

乙型肝炎病毒PreS2蛋白研究进展

乙型肝炎病毒(HBV)外膜蛋白包括S蛋白、PreS1蛋白和PreS2蛋白,它们是HBV包膜的主要成分,可诱导机体产生相应的抗体。PreS2蛋白在HBV侵入肝细胞的过程中起着非常重要的作用,对于防治HBV的感染有重要意义。我们简要综述了有关PreS2蛋白的研究,介绍了PreS2蛋白的各项功能。     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Insulin-like growth factor 1 stimulates KCl cotransport, which is necessary for invasion and proliferation of cervical cancer and ovarian cancer cells

The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral KCl cotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical and ovarian cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose- and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascades leading to the activation of Akt and extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2 mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling pathways are differentially required for IGF-1-stimulated biosyn-thesis of KCC polypeptides. Specific reduction of Erk1/2 protein levels with small interference RNA abolishes IGF-1-stimulated KCC activity. Pharmacological inhibition and genetic modification of KCC activity demonstrate that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n = 28) and ovarian cancer (n = 35), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and spread of gynecological cancers.