Dongmeng Qian

p300-Dependent ATF5 Acetylation Is Essential for Egr-1 Gene Activation and Cell Proliferation and Survival

ABSTRACT ATF5 has been shown to be a critical regulator of cell proliferation and survival; however, the underlying mechanism remains largely unknown. We demonstrate here that ATF5 interacts with the transcriptional coactivator p300, which acetylates ATF5 at lysine-29 (K29), which in turn enhances the interaction between ATF5 and p300 and binding of the ATF5/p300 complex to the ATF5 response element (ARE) region of the Egr-1 promoter. ARE-bound ATF5/p300 acetylates lysine-14 (K14) of nucleosomal histone H3 at both the ARE and serum response element (SRE) of the Egr-1 promoter, which facilitates binding of extracellular signal-regulated kinase (ERK)-phosphorylated Elk-1 to the SRE, activating the Egr-1 promoter. Interference of p300-dependent acetylation of ATF5 or nucleosomal histone H3 or blockade of ERK-dependent Elk-1 phosphorylation abrogates ATF5-dependent Egr-1 activation and cell proliferation and survival. These findings assign a central role for the ATF5/p300 complex in ATF5 function and suggest that coordinated actions by ATF5, p300, Elk-1, and ERK/mitogen-activated protein kinase (MAPK) are essential for ATF5-dependent Egr-1 activation and cell proliferation and survival.

Nucleophosmin (NPM1/B23) Interacts with Activating Transcription Factor 5 (ATF5) Protein and Promotes Proteasome- and Caspase-dependent ATF5 Degradation in Hepatocellular Carcinoma Cells

Background: NPM1 promotes whereas ATF5 inhibits HCC proliferation; NPM1 and ATF5 are regulated in an opposite manner in normal hepatocytes and HCC. Results: NPM1 competes against HSP70 for ATF5 binding and promotes proteasome- and caspase-dependent ATF5 protein degradation. Conclusion: NPM1 is a novel ATF5-interacting protein and abrogates ATF5 function in HCC. Significance: We reveal a mechanism by which NPM1 promotes HCC proliferation and survival via regulation of ATF5. Nucleophosmin (NPM1/B23) and the activating transcription factor 5 (ATF5) are both known to subject to cell type-dependent regulation. NPM1 is expressed weakly in hepatocytes and highly expressed in hepatocellular carcinomas (HCC) with a clear correlation between enhanced NPM1 expression and increased tumor grading and poor prognosis, whereas in contrast, ATF5 is expressed abundantly in hepatocytes and down-regulated in HCC. Re-expression of ATF5 in HCC inhibits cell proliferation. We report here that using an unbiased approach, tandem affinity purification (TAP) followed with mass spectrometry (MS), we identified NPM1 as a novel ATF5-interacting protein. Unlike many other NPM1-interacting proteins that interact with the N-terminal oligomerization domain of NPM1, ATF5 binds via its basic leucine zipper to the C-terminal region of NPM1 where its nucleolar localization signal is located. NPM1 association with ATF5, whose staining patterns partially overlap in the nucleoli, promotes ATF5 protein degradation through proteasome-dependent and caspase-dependent pathways. NPM1-c, a mutant NPM1 that is defective in nucleolar localization, failed to stimulate ATF5 polyubiquitination and was unable to down-regulate ATF5. NPM1 interaction with ATF5 displaces HSP70, a known ATF5-interacting protein, from ATF5 protein complexes and antagonizes its role in stabilization of ATF5 protein. NPM1-promoted ATF5 down-regulation diminished ATF5-mediated repression of cAMP-responsive element-dependent gene transcription and abrogates ATF5-induced G2/M cell cycle blockade and inhibition of cell proliferation in HCC cells. Our study establishes a mechanistic link between elevated NPM1 expression and depressed ATF5 in HCC and suggests that regulation of ATF5 by NPM1 plays an important role in the proliferation and survival of HCC.

HCMV Induces Dysregulation of Glutamate Uptake and Transporter Expression in Human Fetal Astrocytes

Human cytomegalovirus (HCMV) infections are the leading cause of viral induced birth defects, affecting the central nervous system (CNS) primarily. Fetal CNS is especially vulnerable to CMV induced injury. As HCMV permissive cells, astrocytes are responsible for major glutamate transport and regulate extracellular levels of glutamate avoiding its accumulation which is implicated in neurodegenerative disorders. In this study, highly purified astrocytes isolated from human first trimester aborted fetal brain were infected with HCMV AD169, glutamate uptake function was detected by 3H labeling technic, and the expression level alterations of glutamate transporters (GLAST, GLT-1), glutamine synthetase (GS) and its activity were also investigated. Protein kinases C (PKC) inhibitor treatment was to identify whether PKC signalling involved in regulating glutamate uptake, protein expression of GLAST, GLT-1, GS and GS activity. Results indicated HCMV AD169 infection could modulate glutamate uptake, expression levels of GLAST, GLT-1, GS and it activity through PKC signalling, suggesting a great susceptibility of human fetal astrocytes to HCMV infection, which significantly alters the uptake and metabolism of an important excitatory amino acid, glutamate, may be a potential mechanism for HCMV associated neurological disease, and an effective therapeutic target in neural diseases.

ATF5 is overexpressed in epithelial ovarian carcinomas and interference with its function increases apoptosis through the downregulation of Bcl-2 in SKOV-3 cells.

The activating transcription factor 5 (ATF5) is highly expressed in many kinds of tumors including glioblastoma and breast cancers, but its expression in epithelial ovarian neoplasms has not been investigated. Here, we show that ATF5 is highly expressed in the majority of epithelial ovarian cancer samples (43/60) as compared with benign ovarian tumor tissues (4/13) and normal ovarian tissues (1/10). Furthermore, we found that ATF5 expression significantly correlated with advanced clinical stage (P<0.05) and poor differentiation of epithelial ovarian carcinomas (P<0.05). Previous studies suggested that ATF5 is required for the survival of cancer cells, but the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Our data additionally demonstrated that interference with the function of ATF5 could markedly increase the apoptosis of ovarian cancer cells and identified B-cell leukemia lymphoma-2 as an ATF5-targeted apoptosis-related gene. These findings may provide potential therapeutic application in epithelial ovarian cancer.

Upregulation of Toll-like receptor 2 expression in colorectal cancer infected by human cytomegalovirus.

The aim of the present study was to investigate the association between Toll-like receptor 2 (TLR2) expression and human cytomegalovirus (HCMV) in colorectal carcinoma by detecting the expression of IE1–72, TLR2, TLR4 and tumor necrosis factor (TNF)-α in colorectal carcinoma and colon adenoma samples, as well as by analyzing the mRNA levels of the proteins in colon cancer cell lines, following HCMV infection. For this study, 56 colorectal cancer and 36 colon adenoma samples were collected, and normal mucosal tissue adjacent to the tumor was used as the control. The expression of the IE1–72, TLR2, TLR4, nuclear factor (NF)-κB and TNF-α protein was detected by immunohistochemistry. Cells from the SW480 human colon carcinoma cell line were infected with HCMV. The expression of IE1–72, TLR2, TLR4, NF-κB and TNF-α mRNA was quantified at different time points prior to and following infection. The positive expression rate of IE1–72 was 44.6% (25/56) in colorectal cancer and 41.7% (15/36) in colon adenoma. These rates were significantly higher when compared with the 12.5% (7/56) observed in the normal tissues adjacent to the cancer tissues (P<0.05). The expression levels of TLR2, TLR4, NF-κB and TNF-α in colorectal cancer and adenoma were also higher than those in the control tissues. Furthermore, the expression of IE1–72 in colorectal cancer tissues was found to correlate with TLR2 and TLR4, and the correlation coefficients were 0.515 and 0.462, respectively. Following the infection of SW480 cells, the mRNA levels of TLR2 and TNF-α increased gradually from 6 h, peaked at 48 h, and then decreased gradually. No significant differences in TLR4 and NF-κB expression were identified. The results of the present study indicated that there is a specific association between HCMV and the occurrence and development of colorectal cancer, which may be facilitated by the TLR2 signaling pathway.

Human cytomegalovirus inhibits apoptosis by regulating the activating transcription factor 5 signaling pathway in human malignant glioma cells

The activating transcription factor 5 (ATF5), also termed ATFx, is a member of the ATF/cAMP response element-binding protein (CREB) family of basic zipper proteins. ATF5 is an anti-apoptotic protein that is highly expressed in malignant glioma and is essential for glioma cell survival. Accumulating evidence indicates that human malignant gliomas are universally infected with human cytomegalovirus (HCMV). Recent studies have shown that HCMV may be resistant to the induction of apoptosis by disrupting cellular pathways in glioblastoma. To investigate the potential anti-apoptotic function of HCMV in glioma, malignant U87 glioma cells were infected with HCMV. The present study showed that HCMV infection suppressed apoptosis in glioblastoma U87 cells by regulating the expression of ATF5. Furthermore, in glioblastoma U87 cells, HCMV infection induced cellular proliferation in parallel with an increase in the expression level of ATF5 and B-cell lymphoma/leukemia-2 to Bcl-2-associated X protein ratio. Loss of ATF5 function was achieved using a dominant-negative form of ATF5 in U87 cells, whereby cells appeared to grow marginally following HCMV infection when compared with the control. However, the anti-apoptotic ability was appeared to decline in the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. These results indicate that ATF5 signaling pathways may be important in the anti-apoptotic activity of HCMV-infected glioblastoma cells; therefore, the anti-apoptotic molecular mechanisms of HCMV in human glioblastoma cells were investigated in the current study. Prevention of HCMV infection may present a potential and promising approach for the treatment of malignant gliomas.

Inhibitory effects of Ginsenoside Rb1 on apoptosis caused by HSV-1 in human glioma cells.

To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1(HSV-1) in Human Glioma Cells (U251), U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1, GRb1+HSV-1, HSV-1 and control groups. MTT and cell apoptosis assays were used to detect the inhibitory effects of GRb1 on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay. We found that in the 400 μg/mL GRb1 and 400 μg/mL GRb1+HSV-1 groups, MTT values were higher than control group at all times (P<0. 05). Moreover, the apoptosis rate in the 400 μg/mL GRb1+HSV-1 group was lower than the HSV-1 group (P<0. 05). These results confirmed that, at appropriate concentrations, GRb1 could inhibit nerve cell apoptosis in HSV-1 infections.

Association between human cytomegalovirus infection and histone acetylation level in various histological types of glioma

At present, glioma is the most common intracranial tumor and accounts for 40–60% of intracranial tumors. Glioma is highly anaplastic and demonstrates invasive growth. Although considerable progression has been achieved in the treatment of malignant glioma, the prognosis of this disease remains poor. Over the previous decade, several studies have confirmed that human cytomegalovirus (HCMV) enhances the growth or survival of tumors. This is likely to occur through mechanisms distinct from those of classic tumor viruses, which express transforming viral oncoproteins in the majority of tumor cells. The immediate-early 2 protein (IE86; 86 kDa) of HCMV is a key regulator for viral replication and host cell proliferation. The present study aimed to identify the association between the acetylation level and HCMV IE86 expression in various histological types of glioma. Tissue samples were obtained from 60 patients with glioma, consisting of 25 patients with glioblastoma multiforme (GBM), 16 patients with anaplastic glioma and 19 patients with low-grade glioma, in addition to 9 tissue samples obtained from the normal cortex, which were used as the control. The in situ protein expression of IE86, which is encoded by the IE2 gene, activating transcription factor 5 (ATF5), P300, acetyl-histone H3K9 and acetyl-histone H3K14 was detected by immunohistochemistry. The mRNA levels of ATF5, IE2 and P300 were measured by reverse transcription-quantitative polymerase chain reaction in GBM, anaplastic glioma, low-grade glioma and normal cortex tissue specimens. The protein levels of ATF5, IE86, P300, acetyl-histone H3K9 and acetyl-histone H3K14 were assessed by western blot analysis in high-grade glioma, low-grade glioma and normal cortex tissues. Analysis of the expression of the proteins revealed that the excessive expression of the HCMV IE86 protein is associated with the malignancy degree and acetylation level in glioma. IE86 expression is also associated with ATF5, which is an anti-apoptotic protein that is highly expressed in malignant glioma, but not in normal brain tissues. The expression level of IE86 may demonstrate considerable importance for the evaluation of the malignancy degree of human gliomas and extensive application in diagnostic and therapeutic medicine.

High-level expression, purification and study of bioactivity of fusion protein M-IL-2(88Arg, 125Ala) in Pichia pastoris

M-IL-2((88)Arg, (125)Ala) is a fusion protein comprising melittin genetically linked to a mutant human interleukin 2((88)Arg, (125)Ala). In this study, we constructed an expression system of M-IL-2((88)Arg, (125)Ala) in Pichia pastoris: GS115/pPICZα A/M-IL-2((88)Arg, (125)Ala), and achieved the high-level expression of the fusion protein. The maximum yield of the fusion protein M-IL-2((88)Arg, (125)Ala) reached up to 814.5mg/L, higher than the system in Escherichiacoli. The fusion protein was purified by means of ammonium sulfate fractionation, dialysis and nickel ion affinity chromatography. The molecular weight of the fusion protein is about 26kDa, conforming the theoretical value. And M-IL-2((88)Arg, (125)Ala) possesses strong antigen-specificity by Western blot detection. Bioassay results indicated that the fusion protein could directly inhibit the growth of human ovarian cancer SKOV3 cells and Hela cells in vitro. This study provides an alternative strategy for large-scale production of bioactive M-IL-2((88)Arg, (125)Ala) using P. pastoris as an expression host and paves the way to clinical practice.

Expression, Purification, and Characterization of a Functional Mutant Recombinant Human Interleukin-2

In the current study, a mutant recombinant human interleukin-2 (MhIL-2) was generated using site-directed mutagenesis. The bacteria transformed with plasmid pET15b-MhIL-2 were cultured in LB medium containing 0.6mM IPTG for 8 hours at 27°C. Approximately 90% of His-MhIL-2 was efficiently expressed in soluble form. Purification efficiency was optimized using a number of strategies, including nickel ion chelating chromatography, desalting chromatography, thrombin cleavage and Superdex 75 gel filtration chromatography. The final product had >95% purity. PBMCs, CD4+ and CD8+ T cell proliferation assays revealed that one such mutant has identical functional property to the wild-type hIL-2. In summary, we generated a mutant hIL-2 that is functionally identical to wild-type hIL-2.