Hepatitis B virus contains protein attached to the 5′ terminus of its complete DNA strand

Hepatitis B virus DNA contains a tightly bound protein which was not removed by heating to 60°C with 2% SDS, 2% mercaptoethanol. The protein was indirectly demonstrated by the extraction of the DNA-protein complex with phenol before but not after its digestion with proteinase K. The DNA-protein complex had a lower buoyant density than protease-treated or free DNA; it was bound to glass fiber filters; it migrated at a slower rate in gel electrophoresis; and it could be radiolabeled by oxidative iodination. The binding site of the protein was mapped by extraction of restriction endonuclease digests with phenol and analysis of the digests for missing DNA fragments. The protein was localized to a site near the 5′ end of the complete viral DNA strand. It remained attached to this strand after heating with SDS to 90°C or treatment with 0.1 N NaOH, suggesting a covalent linkage. The 5′ end of neither viral DNA strand could be phosphorylated in a reaction with polynucleotide kinase, consistent with attachment of protein to the 5′ ends. The incomplete DNA strand, however, which is the strand elongated by the virion DNA polymerase reaction, did not contain a detectable amount of polypeptide as did the complete strand. The reasons for the apparent block of the 5′ end of the incomplete DNA strand is thus not known. The protein bound covalently to HBV DNA could be involved in the replication of the complete viral DNA strand and/or endonucleolytic generation of linear unit-length DNA pieces from replicative intermediates, although its function and origin are not yet known.     

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.     

Hepatitis B virus X protein drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration

Hepatitis B virus X protein (HBx) plays an important role in the development of hepatocellular carcinoma (HCC). However, the mechanism remains unclear. Recently, we have reported that HBx promotes hepatoma cell migration through the upregulation of calpain small subunit 1 (Capn4). In addition, several reports have revealed that osteopontin (OPN) plays important roles in tumor cell migration. In this study, we investigated the signaling pathways involving the promotion of cell migration mediated by HBx. We report that HBx stimulates several factors in a network manner to promote hepatoma cell migration. We showed that HBx was able to upregulate the expression of osteopontin (OPN) through 5-lipoxygenase (5-LOX) in HepG2-X/H7402-X (stable HBx-transfected cells) cells. Furthermore, we identified that HBx could increase the expression of 5-LOX through nuclear factor-κB (NF-κB). We also found that OPN could upregulate Capn4 through NF-κB. Interestingly, we showed that Capn4 was able to upregulate OPN through NF-κB in a positive feedback manner, suggesting that the OPN and Capn4 proteins involving cell migration affect each other in a network through NF-κB. Importantly, NF-κB plays a crucial role in the regulation of 5-LOX, OPN and Capn4. Thus, we conclude that HBx drives multiple cross-talk cascade loops involving NF-κB, 5-LOX, OPN and Capn4 to promote cell migration. This finding provides new insight into the mechanism involving the promotion of cell migration by HBx.     

CpG oligodeoxynucleotides discriminately enhance binding capacity of human naïve B cells to Hepatitis B virus epitopes

CpG oligodeoxynucleotides (CpG ODN) have the potential to enhance the antigen-presenting cells function of human na?ve B cells. In this study, we aim to define the effect of CpG ODNs on the binding capacity of human na?ve B cells for different Hepatitis B virus (HBV) epitopes. Three HLA-A2 restricted epitopes were selected to incubate with CpG ODN-primed human na?ve B cells. Binding capacity for each epitope and expression of CD80, CD86, class I major histocompatibility complex (MHC), and class II MHC of na?ve B cells was tested, respectively, by flow cytometry. CpG ODNs, especially ODN 2216, enhanced the binding capacity of human na?ve B cells for HBV epitopes (p < 0.01), and induced markedly higher expression of CD80, CD86, class I MHC, and class II MHC. The binding capacity of CpG-treated naive B cells for each epitope was significantly different. In all the 3 subjects, CpG ODN 2216-primed na?ve B cells showed the highest binding ability for Env172-180 compared with the other epitopes with a high expression of co-stimulatory and MHC molecules. CpG ODN showed the potential to selectively enhance the binding capacity of human na?ve B cells for HBV epitopes. These results suggest new strategies for development of vaccine design.     

The anti-fibrotic effect of betulinic acid is mediated through the inhibition of NF-κB nuclear protein translocation

The purpose of the study was to investigate the anti-fibrotic effect and the potential mechanisms of action of betulinic acid (BA) against hepatic fibrosis in vivo and in vitro. BA is an active compound isolated from the bark of the birch tree Betula spp. (Betulaceae). Liver fibrosis was induced by intraperitoneal injections of thioacetamide (TAA, 200mg/kg) twice weekly for 6weeks in Wistar rats. The administration of BA (20 or 50mg/kg) was started following TAA injections and was continued for 6 or 8weeks to evaluate both the preventive and the protective effects. BA demonstrated great efficacy in preventing and curing hepatic fibrosis via attenuating the TAA-mediated increases in liver tissue hydroxyproline and α-smooth muscle actin (α-SMA). In vitro, BA effectively decreased the HSC-T6 cell viability induced by TNF-α and showed low toxicity in normal human chang liver cells. Moreover, BA significantly attenuated the expression of α-SMA and tissue inhibitor of metalloproteinase-1 (TIMP-1) and increased the levels of matrix metalloprotease (MMP)-13. BA also inhibited the expression of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and the activation of nuclear factor-κB (NF-κB) in a time-dependent manner. This study provides evidence that BA exerts a significant anti-fibrosis effect by modulating the TLR4/MyD88/NF-κB signaling pathway.     

Hepatitis B X protein upregulates decoy receptor 3 expression via the PI3K/NF-κB pathway

Chronic hepatitis B (CHB) is associated with the development of hepatocellular carcinoma (HCC). Decoy receptor 3 (DcR3) is a tumor necrosis factor receptor that promotes tumor cell survival by inhibiting apoptosis and interfering with immune surveillance. Previous studies showed that DcR3 was overexpressed in HCC cells and that short hairpin RNA (shDcR3) sensitizes TRAIL-resistant HCC cells. However, the expression of DcR3 during hepatitis B virus (HBV) infection has not been investigated. Here, we demonstrated that DcR3 was overexpressed in CHB patients and that DcR3 upregulation was positively correlated with the HBV DNA load and liver injury (determined by histological activity index, serum alanine aminotransferase level, and aspartate aminotransferase level). We found that hepatitis B virus X protein (HBx) upregulated DcR3 expression in a dose-dependent manner, but this increase was blocked by NF-κB inhibitors. HBx also induced the activation of NF-κB, and the NF-κB subunits p65 and p50 upregulated DcR3 by directly binding to the DcR3 promoters. Inhibition of PI3K significantly downregulated DcR3 and inhibited the binding of NF-κB to the DcR3 promoters. Our results demonstrate that the HBx induced DcR3 expression via the PI3K/NF-κB pathway; this process may contribute to the development of HBV-mediated HCC.     

Bortezomib induces nuclear translocation of IκBα resulting in gene-specific suppression of NF-κB--dependent transcription and induction of apoptosis in CTCL

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB-responsive promoters.     

Regional susceptibility to TNF-α induction of murine brain inflammation via classical IKK/NF-κB signalling

It is becoming clear that inflammation plays a significant role in a number of neurological and psychiatric conditions. Post mortem brain samples in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, schizophrenia and most recently autism spectrum condition, all exhibit neuroglial activation and inflammatory markers within the CSF. Many questions remain about the underlying molecular mechanisms. By adding the pro-inflammatory cytokine, TNF-α, to mouse brain tissue we demonstrated that the frontal lobes and temporal region, areas involved in higher functions such as memory and learning, are most susceptible to cytokine-induced inflammation via the NF-κB signalling pathway. We observed direct correlations between the volumetric increase and molecular expression indicating that therapeutic targets in these lobes may require different approaches when treating conditions with a central neuroinflammatory component.     

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

DJ-1 activates the noncanonical NF-κB pathway via interaction with Cezanne to inhibit the apoptosis and promote the proliferation of Ishikawa cells

Molecular Biology Reports - Endometrial cancer is generally one of the most evident malignant tumours of the female reproductive system, and the mechanisms underlying its cell proliferation and...     

Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson''s disease, Alzheimer''s disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically, we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit an ∼13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions.     

KSP inhibitor SB743921 induces death of multiple myeloma cells via inhibition of the NF-κB signaling pathway

SB743921 is a potent inhibitor of the spindle protein kinesin and is being investigated in ongoing clinical trials for the treatment of myeloma. However, little is known about the molecular events underlying the induction of cell death in multiple myeloma (MM) by SB743921, alone or in combination treatment. Here, we report that SB743921 induces mitochondria-mediated cell death via inhibition of the NF-κB signaling pathway, but does not cause cell cycle arrest in KMS20 MM cells. SB743921-mediated inhibition of the NF-κB pathway results in reduced expression of SOD2 and Mcl-1, leading to mitochondrial dysfunction. We also found that combination treatment with SB743921 and bortezomib induces death in bortezomib-resistant KMS20 cells. Altogether, these data suggest that treatment with SB743921 alone or in combination with bortezomib offers excellent translational potential and promises to be a novel MM therapy. [BMB Reports 2015; 48(10): 571-576]     

Agonistic antibody to the α1-adrenergic receptor mobilizes intracellular calcium and induces phosphorylation of a cardiac 15-kDa protein

Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the α₁-adrenergic receptor (α₁-AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (anti-α₁) was generated against the second extracellular loop of the α₁-AR that bound to its peptide epitope with high affinity (K _D ~50 nM). We studied anti-α₁ effects on intracellular calcium (Ca_i), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-α₁ induced pronounced but transient increases in Ca_i in CHO cells expressing the human α₁-AR (CHO-α₁) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-α₁ on Ca_i. However, preincubation with the antibody attenuated the effect of the α₁-AR antagonist prazosin. In neonatal cardiomyocytes anti-α₁ induced a robust phosphorylation of a 15-kDa protein that is involved in α₁-AR signalling. Our data support the notion that elevation of Ca_i is a general feature of agonistic antibodies’ action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the α₁-AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action.     

Investigation of the eIF2α phosphorylation mechanism in response to proteasome inhibition in melanoma and breast cancer cells

The 26S proteasome is an ATP-dependent proteolytic complex found in all eukaryotes, archaebacteria, and some eubacteria. Inhibition of the 26S proteasome causes pleiotropic effects in cells, including cellular apoptosis, a fact that has led to the use of the 26S proteasome inhibitor, bortezomib, for treatment of the multiple myeloma cancer. We previously showed that in addition to the effects of proteolysis, inhibition of the 26S proteasome causes a rapid decrease in the protein synthesis rate due to phosphorylating alfa subunit of the eukaryotic translation initiation factor 2 (eIF2α) by the heme-regulated inhibitor kinase (HRI). In order to test whether inhibition of the 26S proteasome causes the same effect in cancer cells, we have investigated the influence of two commonly used proteasome inhibitors, bortezomib and MG132, on the phosphorylation status of eIF2α in B16F10 melanoma and 4T1 breast cancer cells. It was found that both of the inhibitors caused rapid phosphorylation of eIF2α. Taking into account that the Hsp70 is a critical component needed for the HRI activation and enzymatic activity, we have tested a possible participation of this protein in the eIF2α phosphorylation event. However, treatment of the cells with two structurally different Hsp70 inhibitors, quercetin and KNK437, in the presence of the proteasome inhibitors did not affect the eIF2α phosphorylation. In addition, neither protein kinase C (PKC) nor p38 mitogen-activated protein kinase (MAPK) was required for the proteasome inhibitor-induced eIF2α phosphorylation; furthermore, both the PKC inhibitor staurosporine and the p38 MAPK inhibitor SB203580 caused enchanced phosphorylation of eIF2α. Zinc(II) protoporphyrine IX (ZnPP), an inhibitor of the heme-oxygenase-1 (HO-1), which has also been previously reported to be involved in HRI activation, also failed to prevent the induction of eIF2α phosphorylation in the presence of the proteasome inhibitor bortezomib or MG132.