Jan Jong Hung

An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

ABSTRACT Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

Differential Activation of p38 Mitogen-activated Protein Kinase and Extracellular Signal-regulated Protein Kinases Confers Cadmium-induced HSP70 Expression in 9L Rat Brain Tumor Cells

We have reported that treatment with CdCl2 at 40–100 μm induces the heat shock proteins (HSPs) in 9L rat brain tumor cells, during which the activation of heat shock factor (HSF) is essentially involved. By exploiting protein kinase inhibitors, we further analyzed the possible participation of specific protein kinases in the above processes. It was found that induction of HSP70 in cells treated with a high concentration of cadmium (i.e. 100 μm) is preceded by the phosphorylation and activation of p38 mitogen-activated protein kinase (p38MAPK), while that in cells treated with a low concentration (60 μm) is accompanied by the phosphorylation and activation of extracellular-regulated protein kinases 1 and 2 (ERK1/2). In 100 μm cadmium-treated cells, both HSP70 induction and HSF1 activation are eliminated in the presence of SB203580, a specific inhibitor of p38MAPK. By contrast, in 60 μm cadmium-treated cells, the processes are not affected by SB203580 but are significantly suppressed by PD98059, which indirectly inhibits ERK1/2 by acting on MAPK-ERK kinase. Taken together, we demonstrate that p38MAPK and ERK1/2 can be simultaneously or independently activated under different concentrations of cadmium and that the signaling pathways participate in the induction of HSP70 by acting on the inducible phosphorylation of HSF1. We thus provide the first evidence that both p38MAPKand ERK signaling pathways can differentially participate in the activation of HSF1, which leads to the induction of HSP70 by cadmium.

Sp1 deacetylation induced by phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription

ABSTRACT We previous reported that Sp1 recruits c-Jun to the promoter of the 12(S)-lipoxygenase gene in 12-myristate 13-acetate-treated cells. We now show that Sp1 that recruited HDAC1 to the Sp1/cJun complex was constitutively acetylated when cells were exposed to phorbol 12-myristate 13-acetate (PMA) (3 h). Prolonged stimulation of the cells with PMA (9 h), however, caused the dissociation of histone deacetylase 1 (HDAC1) and the deacetylation of Sp1, with the latter being able to recruit p300 that in turn caused the acetylation and dissociation of histone 3, thus enhancing the expression of 12(S)-lipoxygenase. We also overexpressed an Sp1 mutant (K703/A, lacking acetylation sites) in the cell and found that cells recruited more p300 and expressed more 12(S)-lipoxygenase. Taken together, our results indicated that Sp1 recruits HDAC1 together with c-Jun to the gene promoter, followed by deacetylation of Sp1 upon PMA treatment. p300 is then recruited to the gene promoter through the interaction with deacetylated Sp1 to acetylate histone 3, leading to the enhancement of the expression of 12(S)-lipoxygenase.

Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells

ABSTRACT Molecular chaperones assist protein folding, and some chaperones are induced by heat, nutrient depletion, or pathogen invasion. This study investigates the role played by Hsp90 in the life cycle of vaccinia virus. The titer of vaccinia intracellular mature virions (IMV) was reduced by 2 orders of magnitude in RK13 cells treated with geldanamycin (GA), which blocks the ATPase activity of Hsp90. GA does not affect expression from the viral early promoter, but treatment with GA delays DNA replication and intermediate gene transcription and reduces expression from the viral late promoter. Vaccinia virus infection does not induce Hsp90 expression; however, intracellular distribution of Hsp90 is altered in virus-infected cells. Hsp90 is restricted to the cytoplasm of mock-infected cells; in contrast, Hsp90 is transiently associated with virosomes in virus-infected cells although it is not incorporated into IMV. In addition, Hsp90 interacts with viral core protein 4a, the mature form of the A10L gene product, in virus-infected cells. In conclusion, these results suggest that a cellular chaperone protein, Hsp90, is important for vaccinia virus growth in cultured cells and that viral core protein 4a associates with Hsp90-containing complexes in the infected cells.

Sp1 expression regulates lung tumor progression

The role of specificity protein 1 (Sp1) in controlling gene expression in lung tumor development and metastasis is not well understood. In this study, we showed that the Sp1 level was highly increased and required for lung tumor growth in transgenic mice bearing Kras-induced lung tumors under the control of doxycycline. Furthermore, the Sp1 level was highly upregulated in lung adenocarcinoma cells with low invasiveness and in patients with stage I lung cancer. We also demonstrated that Sp1 was downregulated in lung adenocarcinoma cells with high invasiveness and in patients with stage IV lung adenocarcinoma. Moreover, Sp1 inversely regulated migration, invasion and metastasis of lung adenocarcinoma cells in vivo. In addition, a decrease in the Sp1 level in highly invasive lung adenocarcinoma cells resulted from instability of the Sp1 protein. Furthermore, overexpression of Sp1 in highly invasive lung adenocarcinoma cells increased expression of E-cadherin, a suppressor of metastasis, and attenuated the translocation of β-catenin into the cellular nucleus that leads to tumor malignancy. Taken together, Sp1 level accumulated strongly in early stage and then declined in late stage, which is important for lung cancer cell proliferation and metastasis during tumorigenesis.

Sumoylation of Specificity Protein 1 Augments Its Degradation by Changing the Localization and Increasing the Specificity Protein 1 Proteolytic Process

Although specificity protein 1 (Sp1) accumulation has been found in various tumor strains, its mechanism is still not very clear. Herein, we found that modification of Sp1 by SUMO-1 facilitates Sp1 degradation. Our findings revealed that, although the amounts of Sp1 and Sp1 mutant (K16R) [Sp1(K16R)] mRNA in cells were equal, the protein level of Sp1(K16R) was higher than that of wild-type Sp1. We also proved that this sumoylation site was not the residue at which ubiquitination occurred. Invitro and in vivo pull-down assays revealed that more sumoylated Sp1 was localized in the cytoplasm, and the interaction between SUMO-1-Sp1 and the proteasome subunit rpt6 in HeLa cells was enhanced. In addition, although Sp1 accumulated in the tumorous cervical tissue, it was not prone to sumoylation. Finally, by overexpression of HA (hemagglutinin)-SUMO-1-Sp1-myc, HA-Sp1-myc, and HA-Sp1(K16R), we found that modification of Sp1 by SUMO-1 was important for Sp1 proteolysis. In conclusion, modification of Sp1 by SUMO-1 altered its localization and then increased its interaction with rpt6. This interaction increased the efficiency of Sp1 proteolytic processing and ubiquitination and then resulted in Sp1 degradation. Therefore, sumoylation of Sp1 is attenuated during tumorigenesis in order to increase Sp1 stability.

Overexpression of Sp1 leads to p53-dependent apoptosis in cancer cells.

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro‐apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP‐Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub‐G1 fraction, caspase‐3 cleavage, and annexin‐V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53‐dependent apoptotic pathways in the wild‐type p53 cells but not in the p53‐mutated or p53‐deleted cell lines when these cells were infected with adeno‐GFP‐Sp1 virus. In addition, A549 (p53+/+) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53−/−) cell viability was significantly inhibited by adeno‐GFP‐Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression‐induced apoptotic cell death in transforming cells.

Functional role of post-translational modifications of Sp1 in tumorigenesis

Specific protein 1 (Sp1), the first transcription factor to be isolated, regulates the expression of numerous genes involved in cell proliferation, apoptosis, and differentiation. Recent studies found that an increase in Sp1 transcriptional activity is associated with the tumorigenesis. Moreover, post-translational modifications of Sp1, including glycosylation, phosphorylation, acetylation, sumoylation, ubiquitination, and methylation, regulate Sp1 transcriptional activity and modulate target gene expression by affecting its DNA binding activity, transactivation activity, or protein level. In addition, recent studies have investigated several compounds with anti-cancer activity that could inhibit Sp1 transcriptional activity. In this review, we describe the effect of various post-translational modifications on Sp1 transcriptional activity and discuss compounds that inhibit the activity of Sp1.

Selective Inhibition of Early—but Not Late—Expressed HIF-1α Is Neuroprotective in Rats after Focal Ischemic Brain Damage

The expression of hypoxia‐inducible factor‐1‐alpha (HIF‐1α) is upregulated in ischemic stroke, but its function is still unclear. In the present study, biphasic expression of HIF‐1α was observed during 1–12 h and after 48 h in neurons exposed to ischemic stress in vitro and in vivo. Treating neurons with 2‐methoxyestradiol (2ME2) 0.5 h after ischemic stress or pre‐silencing HIF‐1α with small interfering RNA (siRNA) decreased brain injury, brain edema and number of apoptotic cell, and downregulates Nip‐like protein X (Nix) expression. Conversely, applying 2ME2 to neurons 8 h after ischemic stress or silencing the HIF‐1α with siRNA 12 h after oxygen–glucose deprivation (OGD) increased neuron damage and decreased vascular endothelial growth factor (VEGF) expression. Taken together, these results demonstrate that HIF‐1α induced by ischemia in early and late times leads cellular apoptosis and survival, respectively, and provides a new insight into the divergent roles of HIF‐1α expression in neurons after ischemic stroke.