Chiung Yuan Ko

Epigenetic Silencing of CCAAT/Enhancer-binding Protein δ Activity by YY1/Polycomb Group/DNA Methyltransferase Complex

Human CCAAT/enhancer-binding protein δ (CEBPD) has been reported as a tumor suppressor because it both induces growth arrest involved in differentiation and plays a crucial role as a regulator of pro-apoptotic gene expression. In this study, CEBPD gene expression is down-regulated, and “loss of function” alterations in CEBPD gene expression are observed in cervical cancer and hepatocellular carcinoma. Suppressor of zeste 12 (SUZ12), a component of the polycomb repressive complex 2 (PRC2), silences CEBPD promoter activity, enhancing the methylation of exogenous CEBPD promoter through the proximal CpG islands. Moreover, this molecular approach is consistent with the opposite mRNA expression pattern between SUZ12 and CEBPD in cervical cancer and hepatocellular carcinoma patients. We further demonstrated that Yin-Yang-1 (YY1) physically interacts with SUZ12 and can act as a mediator to recruit the polycomb group proteins and DNA methyltransferases to participate in the CEBPD gene silencing process. Taking these results into consideration, we not only demonstrate the advantage of SUZ12-silenced CEBPD expression in tumor formation but also clarify an in vivo evidence for YY1-mediated silencing paths of SUZ12 and DNA methyltransferases on the CEBPD promoter.

Functional role of NF-IL6β and its sumoylation and acetylation modifications in promoter activation of cyclooxygenase 2 gene

NF-IL6β regulates gene expression and plays function roles in many tissues. The EGF-regulated cyclooxygenase-2 (cox-2) expression is mediated through p38MAPK signaling pathway and positively correlates with NF-IL6β expression in A431 cells. NF-IL6β coordinated with c-Jun on cox-2 transcriptional activation by reporter and small interfering RNA assays. NF-IL6β could directly bind to CCAAT/enhancer-binding protein (C/EBP) and cyclic AMP-response element (CRE) sites of the cox-2 promoter by in vitro-DNA binding assay. The C/EBP site was important for basal and, to a lesser extent, for EGF-regulated cox-2 transcription, while the CRE site was a more specific response to EGF inducibility of cox-2 gene. SUMO1 expression attenuated EGF- and NF-IL6β-induced cox-2 promoter activities. NF-IL6β was found to be sumoylated by in vivo- and in vitro-sumoylation assays, and the SUMO1-NF-IL6β (suNF-IL6β) lost its ability to interact with p300 in in vitro-binding assay. NF-IL6β was also acetylated by p300, and acetylation of NF-IL6β enhanced the cox-2 promoter activity stimulated by NF-IL6β itself. In vivo-DNA binding assay demonstrated that EGF stimulated the recruitment of p300 and NF-IL6β to the cox-2 promoter, yet promoted the dissociation of SUMO1-modificated proteins from the promoter. These results indicated that NF-IL6β plays a pivotal role in the regulation of basal and EGF-induced cox-2 transcription.

Glycogen synthase kinase-3β-mediated CCAAT/enhancer-binding protein delta phosphorylation in astrocytes promot es migration and activation of microglia/macrophages

Alzheimers disease is neuropathologically characterized by the accumulation of amyloid-β protein into senile plaques that are sites of chronic inflammation involving reactive microglia, astrocytes, and proinflammatory molecules, such as interleukin-1β and tumor necrosis factor-α. The human CCAAT/enhancer-binding protein (CEBP) delta (CEBPD) is known to be induced in many inflammation-related diseases. In Alzheimers disease, this protein is responsive to amyloid-β and proinflammatory cytokines in astrocytes. However, the functional role of CEBPD in astrocytes remains largely unclear. In this study, we show that CEBPD is upregulated by interleukin-1β through the mitogen-activated protein kinase p38 (MAPKp38) signaling pathway and phosphorylated by glycogen synthase kinase (GSK)-3β at Ser167 in astrocytes. CEBPD in astrocytes is associated with microglia activation and migration in amyloid precursor protein transgenic mice (AppTg) mice. We further identified that the monocyte chemotactic protein-1, a chemoattractive factor, and migration factors matrix metalloproteinase-1 and -3 are responsive to GSK3β-mediated CEBPD Ser167 phosphorylation. Our results revealed the novel regulation of LiCl on astrocytes and that GSK3β-mediated CEBPD phosphorylation in astrocytes plays an important role in the activation of microglia.

Biological roles of CCAAT/Enhancer-binding protein delta during inflammation.

CCAAT/enhancer-binding protein delta (CEBPD) belongs to the CCAAT/enhancer-binding protein family, and these proteins function as transcription factors in many biological processes, including cell differentiation, motility, growth arrest, proliferation, cell death, metabolism and immune responses. The functional diversity of CEBPD depends, in part, on the cell type and cellular context, which indicates that CEBPD could interpret a variety of cues to adjust cellular responses in specific situations. Here, we review the regulation of the CEBPD gene and its function in response to inflammatory stimuli. We also address its effects in inflammation-related diseases through a discussion of its recently discovered downstream targets. Regarding to the previous discoveries and new insights in inflammation-associated diseases, suggesting CEBPD could also be a central gene in inflammation. Importantly, the results of this study indicate that the investigation of CEBPD could open a new avenue to help better understand the inflammatory response.

Increase of Zinc Finger Protein 179 in Response to CCAAT/Enhancer Binding Protein Delta Conferring an Antiapoptotic Effect in Astrocytes of Alzheimer’s Disease

Reactive astrogliosis is a cellular manifestation of neuroinflammation and occurs in response to all forms and severities of the central nervous system (CNS)’s injury and disease. Both astroglial proliferation and antiapoptotic processes are aspects of astrogliosis. However, the underlying mechanism of this response remains poorly understood. In addition, little is known about why activated astrocytes are more resistant to stress and inflammation. CCAAT/enhancer binding protein delta (CEBPD) is a transcription factor found in activated astrocytes that surround β-amyloid plaques. In this study, we found that astrocytes activation was attenuated in the cortex and hippocampus of APPswe/PS1 E9 (AppTg)/Cebpd−/−mice. Furthermore, an increase in apoptotic astrocytes was observed in AppTg/Cebpd−/−mice, suggesting that CEBPD plays a functional role in enhancing the antiapoptotic ability of astrocytes. We found that Zinc Finger Protein 179 (ZNF179) was a CEBPD-regulated gene that played an antiapoptotic, but not proliferative, role in astrocytes. The transcriptions of the proapoptotic genes, insulin-like growth factor binding protein 3 (IGFBP3) and BCL2-interacting killer (BIK), were suppressed by ZNF179 via its interaction with the promyelocytic leukemia zinc finger (PLZF) protein in astrocytes. This study provides the first evidence that ZNF179, PLZF, IGFBP3, and BIK contributed to the novel CEBPD-induced antiapoptotic feature of astrocytes.

Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a

The progression of Alzheimer’s disease (AD) has been associated with astrocytes-induced neuroinflammation. However, the detailed mechanism of astrocytes associated with learning impairments and neuronal loss in AD is poorly defined. Here, we provide novel evidences that astrocytic miR-135a is critical for neuronal viability and spatial learning ability in vivo. The AppTg/Cebpd−/− mice showed a spatial learning improvement compared with the APPswe/PS1/E9 bigenic (AppTg) mice. miR-135a was found to be a CCAAT/enhancer binding protein δ (CEBPD) responsive miRNA and can repress the transcription of thrombospondin 1 (THBS1) / Thbs1 (mouse) via its 3′-untranslated region (3′UTR). We used different experimental approaches to attenuate the expression of CEBPD/Cebpd (mouse) or miR-135a in astrocytes and found the following results: increase in THBS1/Thbs1 expression, decrease in neuronal apoptosis, and increase in growth of neurites. Importantly, injection of miR-135a antagonist (AM135a) into the brain of AppTg mice was found to prevent neuronal apoptosis and improved the spatial learning ability. Together, our findings demonstrate a critical function for the astrocytic CEBPD, and point to miR-135a antagonist as an attractive therapeutic target for the treatment of Alzheimer’s disease.

HMDB and 5-AzadC Combination Reverses Tumor Suppressor CCAAT/Enhancer-Binding Protein Delta to Strengthen the Death of Liver Cancer Cells

Hepatocellular carcinoma (HCC) can arise from chronic inflammation due to viral infection, organ damage, drug toxicity, or alcohol abuse. Moreover, gene desensitization via aberrant CpG island methylation is a frequent epigenetic defect in HCC. However, the details of how inflammation is linked with epigenetic-mediated desensitization of tumor suppressor genes remains less investigated. In this study, we found that loss of CEBPD enhances the growth of liver cancer cells and is associated with the occurrence of liver cancers, as determined by the assessment of clinical specimens and in vivo animal models. Moreover, E2F1-regulated epigenetic axis attenuated CEBPD expression in liver cancer cells. CEBPD is responsive to the hydroxymethyldibenzoylmethane (HMDB)-induced p38/CREB pathway and plays an important role in the HMDB-induced apoptosis of cancer cells. Regarding depression of epigenetic effects to enhance HMDB-induced CEBPD expression, the combination of HMDB and 5-Aza-2′-deoxycytidine (5-AzadC) could enhance the death of liver cancer cells and reduce the tumor formation of Huh7 xenograft mice. In conclusion, these results suggest that CEBPD could be a useful diagnostic marker and therapeutic target in HCC. The results also reveal the therapeutic potential for low-dose 5-AzadC to enhance the HMDB-induced death of HCC cells. Mol Cancer Ther; 14(11); 2623–33. ©2015 AACR.

Specificity protein 1-modulated superoxide dismutase 2 enhances temozolomide resistance in glioblastoma, which is independent of O6-methylguanine-DNA methyltransferase

Acquisition of temozolomide (TMZ) resistance is a major factor leading to the failure of glioblastoma (GBM) treatment. The exact mechanism by which GBM evades TMZ toxicity is not always related to the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), and so remains unclear. In this study, TMZ-resistant variants derived from MGMT-negative GBM clinical samples and cell lines were studied, revealing there to be increased specificity protein 1 (Sp1) expression associated with reduced reactive oxygen species (ROS) accumulation following TMZ treatment. Analysis of gene expression databases along with cell studies identified the ROS scavenger superoxide dismutase 2 (SOD2) as being disease-related. SOD2 expression was also increased, and it was found to be co-expressed with Sp1 in TMZ-resistant cells. Investigation of the SOD2 promoter revealed Sp1 as a critical transcriptional activator that enhances SOD2 gene expression. Co-treatment with an Sp1 inhibitor restored the inhibitory effects of TMZ, and decreased SOD2 levels in TMZ-resistant cells. This treatment strategy restored susceptibility to TMZ in xenograft animals, leading to prolonged survival in an orthotopic model. Thus, our results suggest that Sp1 modulates ROS scavengers as a novel mechanism to increase cancer malignancy and resistance to chemotherapy. Inhibition of this pathway may represent a potential therapeutic target for restoring treatment susceptibility in GBM.

Distinct roles and differential expression levels of Wnt5a mRNA isoforms in colorectal cancer cells

The canonical Wnt/β-catenin pathway is constitutively activated in more than 90% of colorectal cancer (CRC) cases in which β-catenin contributes to CRC cell growth and survival. In contrast to the Wnt/β-catenin pathway, the non-canonical Wnt pathway can antagonize functions of the canonical Wnt/β-catenin pathway. Wnt5a is a key factor in the non-canonical Wnt pathway, and it plays diverse roles in different types of cancers. It was shown that reintroducing Wnt5a into CRC cells resulted in inhibited cell proliferation and impaired cell motility. However, contradictory results were reported describing increased Wnt5a expression being associated with a poor prognosis of CRC patients. Recently, it was shown that the diverse roles of Wnt5a are due to two distinct roles of Wnt5a isoforms. However, the exact roles and functions of the Wnt5a isoforms in CRC remain largely unclear. The present study for the first time showed the ambiguous role of Wnt5a in CRC was due to the encoding of distinct roles of the various Wnt5a mRNA isoforms. A relatively high expression level of the Wnt5a-short (S) isoform transcript and a low expression level of the Wnt5a-long (L) isoform transcript were detected in CRC cell lines and specimens. In addition, high expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with tumor depth of CRC patients. Furthermore, knockdown of the endogenous expression of the Wnt5a-S mRNA isoform in HCT116 cells drastically inhibited their growth ability by inducing apoptosis through induction of FASLG expression and reduction of TNFRSF11B expression. Moreover, reactivation of methylation inactivation of the Wnt5a-L mRNA isoform by treatment with 5-azacytidine (5-Aza) enhanced the siWnt5a-S isoforms ability to induce apoptosis. Finally, we showed that the simultaneous reactivation of Wnt5a-L mRNA isoform and knockdown of Wnt5a-S mRNA isoform expression enhanced siWnt5a-S isoform-induced apoptosis and siWnt5a-L isoform-regulated suppression of β-catenin expression in vitro. High expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with high mRNA levels of β-catenin detection in vivo. Altogether, our study showed that, for the first time, different Wnt5a mRNA isoforms play distinct roles in CRC and can be used as novel prognostic markers for CRC in the future.

Astrocytic CCAAT/Enhancer-binding protein delta contributes to reactive oxygen species formation in neuroinflammation

Excessive reactive oxygen species (ROS) can form an oxidative stress and an associated neuroinflammation. However, the contribution of astrocytes to ROS formation, the cause of the resistance of astrocytes to oxidative stress, and the consequences on neurons remain largely uninvestigated. The transcription factor CCAAT/enhancer-binding protein delta (CEBPD) is highly expressed in astrocytes and has been suggested to contribute to the progress of Alzheimers disease (AD). In this study, we found that ROS formation and expression of p47phox and p67phox, subunits of NADPH oxidase, were increased in AppTg mice but attenuated in AppTg/Cebpd-/- mice. Cebpd can up-regulate p47phox and p67phox transcription via a direct binding on their promoters, which results in an increase in intracellular oxidative stress. In addition, Cebpd also up-regulated Cu/Zn superoxide dismutase (Sod1) in astrocytes. Inactivation of Sod1 increased the sensitization to oxidative stress, which provides a reason for the resistance of astrocytes in an oxidative stress environment. Taken together, the study first revealed and dissected the involvement of astrocytic Cebpd in the promotion of oxidative stress and the contribution of CEBPD to the resistance of astrocytes in an oxidative stress environment.