Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Eukaryotic initiation factor 5A hypusine as a negative regulator of adenosine 2B receptor (A2bAR) through interaction with stem loop sequences within the A2bAR 3'-untranslated region.

PURPOSE: The aim of this study was to investigate whether eIF5A hypusine (eIF5AHyp) reduces adenosine 2b receptor (A2bAR) gene expression through interaction with highly structured stem-loop sequences within the A2bAR 3'UTR. METHODS AND RESULTS: Based on real-time PCR and western blotting, expression of A2bAR mRNA was significantly decreased upon treatment with eIF5AHyp in mouse embryonic fibroblasts of eIF5A (eIF5A-MEF) and 3T3-L1 cells. Target Scan software and RNAfold web server predicted two different structures formed by stem-loop sequences with overlapping microRNA 27 seed sequences and mutations. The EMSA results showed significantly impaired formation of the wild type (WT) biotin-labeled A2bAR probe (27 base) containing stem loop sequences-eIF5AHyp complex by mutation of stem-loop sequences or by eIF5A non-hypusine (eIF5ALys). The luciferase reporter assay showed that GC7-induced eIF5ALys accumulation increased the activity of pMIR-A2bAR WT containing the same stem-loop sequence in 3T3-L1 cells, whereas the activity with pMIR-A2bAR Mut was increased compared to WT control without dependence on GC7. Oil Red O staining showed that suppression of A2bAR expression (A2bAR siRNA and eIF5AHyp) increased the amount of lipid droplet formation and the mRNA levels of lipid droplet-related genes (C/EBP-ß, PPAR-γ, FABP4, SREBP-1, and Perilipin). In contrast, overexpression of A2bAR (A2bAR vector, eIF5ALys vector, and GC7) significantly decreased the expression of lipid droplet-associated genes and lipid droplet formation. CONCLUSIONS: eIF5AHyp acts as a negative regulator of A2bAR gene expression through stem loop sequences in A2bAR 3'UTR, allowing differentiation of adipocytes.

Hepatitis C Virus Evades Interferon Signaling by Suppressing Long Noncoding RNA Linc-Pint Involving C/EBP-ß.

Hepatitis C virus (HCV) regulates many cellular genes in modulating the host immune system for benefit of viral replication and long-term persistence in a host for chronic infection. Long noncoding RNAs (lncRNAs) play an important role in the regulation of many important cellular processes, including immune responses. We recently reported that HCV infection downregulates lncRNA Linc-Pint (long intergenic non-protein-coding RNA p53-induced transcript) expression, although the mechanism of repression and functional consequences are not well understood. In this study, we demonstrate that HCV infection of hepatocytes transcriptionally reduces Linc-Pint expression through CCAAT/enhancer binding protein ß (C/EBP-ß). Subsequently, we observed that the overexpression of Linc-Pint significantly upregulates interferon alpha (IFN-α) and IFN-ß expression in HCV-replicating hepatocytes. Using unbiased proteomics, we identified that Linc-Pint associates with DDX24, which enables RIP1 to interact with IFN-regulatory factor 7 (IRF7) of the IFN signaling pathway. We furthermore observed that IFN-α14 promoter activity was enhanced in the presence of Linc-Pint. Together, these results demonstrated that Linc-Pint acts as a positive regulator of host innate immune responses, especially IFN signaling. HCV-mediated downregulation of Linc-Pint expression appears to be one of the mechanisms by which HCV may evade innate immunity for long-term persistence and chronicity. IMPORTANCE The mechanism by which lncRNA regulates the host immune response during HCV infection is poorly understood. We observed that Linc-Pint was transcriptionally downregulated by HCV. Using a chromatin immunoprecipitation (ChIP) assay, we showed inhibition of transcription factor C/EBP-ß binding to the Linc-Pint promoter in the presence of HCV infection. We further identified that Linc-Pint associates with DDX24 for immunomodulatory function. The overexpression of Linc-Pint reduces DDX24 expression, which in turn results in the disruption of DDX24-RIP1 complex formation and the activation of IRF7. The induction of IFN-α14 promoter activity in the presence of Linc-Pint further confirms our observation. Together, our results suggest that Linc-Pint acts as a positive regulator of host innate immune responses. Downregulation of Linc-Pint expression by HCV helps in escaping the innate immune system for the development of chronicity.

Troxerutin downregulates C/EBP-ß gene expression via modulating the IFNγ-ERK1/2 signaling pathway to ameliorate rotenone-induced retinal neurodegeneration.

Troxerutin, a natural flavonoid guards against oxidative stress and apoptosis with a high capability of passing through the blood-brain barrier. Our aim was to investigate the role of troxerutin in experimentally induced retinal neurodegeneration by modulating the interferon-gamma (IFNγ)-extracellular signal-regulated kinases 1/2 (ERK1/2)-CCAAT enhancer-binding protein ß (C/EBP-ß) signaling pathway. Three groups of rats (10 each group) were included. Group I (control group), group II (rotenone treated group): the rats were injected subcutaneously with a single rotenone dosage of 3 mg/kg repeated every 48 hours for 60 days to trigger retinal neurodegeneration. Group III (troxerutin-treated group): rats received troxerutin (150 mg/kg/day) by oral gavage 1 hour before rotenone administration. A real-time polymerase chain reaction technique was applied to measure messenger RNA (mRNA) levels of retinal C/EBP-ß. Enzyme-linked immunosorbent assay technique was utilized to assay tumor necrosis factor-α (TNF-α), IFNγ, and ERK1/2 levels. Finally, reactive oxygen species (ROS), as well as carbonylated protein (CP) levels, were assessed spectrophotometrically. Improved retinal neurodegeneration by downregulation of C/EBP-ß mRNA gene expression, also caused a significant reduction of TNF-α, IFNγ, ERK1/2 as well as ROS and CP levels compared with the diseased group. These findings could hold promise for the usage of troxerutin as a protective agent against rotenone-induced retinal neurodegeneration.

PRMT7 deficiency enhances adipogenesis through modulation of C/EBP-ß.

Obesity that is critically correlated with the initiation and development of metabolic syndrome and cardiovascular diseases has increased worldwide. Adipogenesis is coordinated through multi-steps involving adipogenic commitment, mitotic clonal expansion (MCE) and differentiation. Recently, protein arginine methyltransferase 4 (PRMT4) and PRMT5 have been implicated in modulation of adipogenesis via regulation of C/EBP-ß activity or PPAR-γ2 expression. In the current study, we demonstrate a suppressive role of PRMT7 in adipogenesis. PRMT7-depleted preadipocytes or PRMT7-/- mouse embryonic fibroblasts (MEFs) displayed increased adipogenesis while PRMT7 overexpression attenuated it. PRMT7 depletion in preadipocytes promoted MCE, an initial step of adipogenesis. Furthermore, we found that PRMT7 interacted with and methylated a key adipogenic factor C/EBP-ß upon adipogenic induction and modulated the accumulation of C/EBP-ß at its target sites in the PPAR-γ2 promoter. Taken together, our data suggest that PRMT7 suppresses adipogenesis through modulation of C/EBP-ß activity.

Molecular cloning, characterisation, and expression patterns of pigeon CCAAT/enhancer binding protein-α and -ß genes.

1. CCAAT/enhancer binding proteins (C/EBPs), as a family of transcription factors, consists of six functionally and structurally related proteins which share a conserved basic leucine zipper (bZIP) DNA-binding domain. The aim of this study was to clone the full-length coding sequences (CDS) of C/EBP-α and -ß genes, and determine the abundance of these two genes in various tissues of white king pigeon (C. livia). 2. The complete cDNA sequences of C/EBP-α and -ß genes were cloned from pigeons by using PCR combined with rapid amplification of cDNA ends (RACE). The sequences were bioinformatically analysed, and the tissue distribution determined by quantitative real-time RT-PCR (qRT-PCR). 3. The results showed that the full-length cDNA sequences of pigeon C/EBP-α and -ß genes were 2,807bp and 1,778bp, respectively. The open reading frames of C/EBP-α (978 bp) and -ß (987bp) encoded 325 amino acids and 328 amino acids, respectively. The pigeon C/EBP-α and C/EBP-ß proteins were predicted to have a conserved basic leucine zipper (bZIP) domain, which is a common structure feature of the C/EBP family. Multiple sequence alignments indicated that pigeon C/EBP-α and -ß shared more than 90% amino-acid identity with their corresponding homologues in other avian species. Phylogenetic analysis revealed that these two proteins were highly conserved across different species and evolutionary processes. QRT-PCR results indicated that the pigeon C/EBP-α and -ß mRNA transcripts were expressed in all investigated organs. The mRNA expression levels of pigeon C/EBP-α in descending order, were in spleen, heart, liver, lung, kidney and muscle. The pigeon C/EBP-ß gene had the most abundant expression in lung, followed by the kidney, with minimal expression detected in muscle. 4. This study investigated the full-length cDNA sequences, genetic characteristics and tissue distribution of pigeon C/EBP-α and -ß genes and found that they may have functions in various tissues of pigeon. This provides a foundation for further study for regulatory mechanisms of these two genes in birds.

Pattern analysis uncovers a chronic ethanol-induced disruption of the switch-like dynamics of C/EBP-ß and C/EBP-α genome-wide binding during liver regeneration.

Chronic ethanol intake impairs liver regeneration through a system-wide alteration in the regulatory networks driving the response to injury. Our study focused on the initial phase of response to 2/3rd partial hepatectomy (PHx) to investigate how adaptation to chronic ethanol intake affects the genome-wide binding profiles of the transcription factors C/EBP-ß and C/EBP-α. These factors participate in complementary and often opposing functions for maintaining cellular differentiation, regulating metabolism, and governing cell growth during liver regeneration. We analyzed ChIP-seq data with a comparative pattern count (COMPACT) analysis, which exhaustively enumerates temporal patterns of discretized binding profiles to identify dominant as well as subtle patterns that may not be apparent from conventional clustering analyses. We found that adaptation to chronic ethanol intake significantly alters the genome-wide binding profile of C/EBP-ß and C/EBP-α before and following PHx. A subset of these ethanol-induced changes include C/EBP-ß binding to promoters of genes involved in the profibrogenic transforming growth factor-ß pathway, and both C/EBP-ß and C/EBP-α binding to promoters of genes involved in the cell cycle, apoptosis, homeostasis, and metabolic processes. The shift in C/EBP binding loci, coupled with an ethanol-induced increase in C/EBP-ß binding at 6 h post-resection, indicates that ethanol adaptation may change both the amount and nature of C/EBP binding postresection. Taken together, our results suggest that chronic ethanol consumption leads to a spatially and temporally reorganized activity at many genomic loci, resulting in a shift in the dynamic balance and coordination of cellular processes underlying regenerative response.

Endogenous prostaglandin E2 potentiates anti-inflammatory phenotype of macrophage through the CREB-C/EBP-ß cascade.

Macrophages have important functions in tissue homeostasis, but the exact mechanisms regarding wide spectrum of macrophage phenotype remain unresolved. In this study, we report that mouse bone marrow derived naïve macrophages produce prostaglandin E2 (PGE2 ) endogenously, resulting in anti-inflammatory gene expression upon differentiation induced by macrophage colony stimulating factor (M-CSF). Cyclooxygenase (COX) inhibition by indomethacin reduced endogenous PGE2 production of macrophages and subsequently reduced arg1, IL10 and Mrc1, YmI and FizzI gene expressions. Of note, PGE2 phosphorylates CREB via EP2 and EP4 receptor ligation, thereby transcriptionally increasing C/EBP-ß expression in BALB/c bone marrow derived macrophages. Activated CREB directly binds to the CREB-responsive element of the C/EBP-ß promoter, such that PGE2 ultimately reinforces arg1, IL10 and Mrc1 gene expression. Cyclic AMP activator forskolin also phosphorylated CREB and induced the C/EBP-ß cascade, but this was completely blocked by the PKA inhibitor, H89. Consequently, M-CSF grown macrophages inhibited T-cell proliferation but the inhibition ability was reduced when the COX is inhibited by indomethacin or macrophage C/EBP-ß expression was decreased by siRNA transduction. Our results collectively describe the molecular basis for homeostatic macrophage differentiation by endogenous PGE2 .

Mild endothelial dysfunction in Sirt3 knockout mice fed a high-cholesterol diet: protective role of a novel C/EBP-ß-dependent feedback regulation of SOD2.

Sirtuin 3 (Sirt3) is an NAD(+)-dependent mitochondrial deacetylase associated with superoxide dismutase 2 (SOD2)-mediated protection from oxidative stress. We have reported accelerated weight gain and impaired metabolic flexibility in atherosclerotic Sirt3 (-/-) mice. Oxidative stress is a hallmark of endothelial dysfunction. Yet, the role of Sirt3 in this context remains unknown. Thus, we aimed to unravel the effects of endogenous Sirt3 on endothelial function and oxidative stress. Knockdown of Sirt3 in human aortic endothelial cells (HAEC) increased intracellular mitochondrial superoxide accumulation, as assessed by electron spin resonance spectroscopy and fluorescence imaging. Endothelium-dependent relaxation of aortic rings from Sirt3 (-/-) mice exposed to a normal diet did not differ from wild-type controls. However, following 12 weeks of high-cholesterol diet and increasing oxidative stress, endothelial function of Sirt3 (-/-) mice was mildly impaired compared with wild-type controls. Relaxation was restored upon enhanced superoxide scavenging using pegylated superoxide dismutase. Knockdown of Sirt3 in cultured HAEC diminished SOD2 specific activity, which was compensated for by a CCAAT/enhancer binding protein beta (C/EBP-ß)-dependent transcriptional induction of SOD2. Abrogation of this feedback regulation by simultaneous knockdown of C/EBP-ß and Sirt3 exacerbated mitochondrial superoxide accumulation and culminated into endothelial cell death upon prolonged culture. Taken together, Sirt3 deficiency induces a mild, superoxide-dependent endothelial dysfunction in mice fed a high-cholesterol diet. In cultured endothelial cells, a novel C/EBP-ß-dependent rescue mechanism maintains net SOD2 activity upon transient knockdown of Sirt3.

Phorbol-12-myristate-13-acetate (PMA) mediated transcriptional regulation of Oncostatin-M.

Oncostatin-M (OSM), an IL-6 family cytokine, exhibits varied roles in different patho-physiological conditions. Differential expression of OSM in response to varying stimuli indicates importance of its regulation of expression. The present study illustrated transcriptional induction of osm on treatment with chemical inducer, phorbol-12-myristate-13-acetate (PMA). Following initial hours of PMA treatment, a nuclear protein C/EBP-ß binds specifically to the CCAAT consensus sequence at the proximal end of the OSM promoter. Genistein (a specific Tyr phosphorylation inhibitor) leads to the interaction of CHOP (C/EBP Homologous Protein) with C/EBP-ß, thus negatively regulating it. Knockdown of C/EBP-ß also leads to inhibition of PMA-mediated OSM induction.

Neonatal Persistent Exposure to 6-Propyl-2-thiouracil, a Thyroid-Disrupting Chemical, Differentially Modulates Expression of Hepatic Catalase and C/EBP-ß in Adult Rats.

Persistent exposure of rats to 6-propyl-2-thiouracil (PTU) from birth resulted in decreases in plasma thyroid hormone (TH) levels and hepatic expression of catalase and CCAAT enhancer binding protein ß (C/EBP-ß). Catalase promoter region (-185 to +52) that contains binding sites for C/EBP-ß showed an augmentation in the methylation level along with a change in methylation pattern of CpG islands in response to PTU treatment. PTU withdrawal on 30 days of birth restored TH levels and C/EBP-ß to control rats in adulthood. Although catalase expression was restored to some extent in adult rats in response to PTU withdrawal, a permanent change in its promoter CpG methylation pattern was recorded. The results suggest that downregulation of adult hepatic catalase gene in response to persistent neonatal PTU exposure may not solely be attributed to thyroid-disrupting properties of PTU. It is possible that besides thyroid-disrupting behavior, PTU may impair expression of hepatic catalase by altering methylation pattern of its promoter.

Expression of microRNA-155 correlates positively with the expression of Toll-like receptor 7 and modulates hepatitis B virus via C/EBP-ß in hepatocytes.

Effective recognition of viral infection and successive activation of antiviral innate immune responses are vital for host antiviral defence, which largely depends on multiple regulators, including Toll-like receptors (TLRs) and microRNAs. Several early reports suggest that specific TLR-mediated immune responses can control hepatitis B virus (HBV) replication and express differentially with disease outcome. Considering the versatile function of miR-155 in the TLR-mediated innate immune response, we aimed to study the association between miR-155 and TLRs and their subsequent impact on HBV replication using both a HBV-replicating stable cell line (HepG2.2.15) and HBV-infected liver biopsy and serum samples. Our results showed that miR-155 was suppressed during HBV infection and a subsequent positive correlation of miR-155 with TLR7 activation was noted. Further, ectopic expression of miR-155 in vitro reduced HBV load as evidenced from reduced viral DNA, mRNA and subsequently reduced level of secreted viral antigens (HBsAg and HBeAg). Our results further suggested that CCAAT/enhancer-binding protein-ß (C/EBP-ß), a positive regulator of HBV transcription, was inhibited by miR-155. Taken together, our study established a correlation between miR-155 and TLR7 during HBV infection and also demonstrated in vitro that increased miR-155 level could help to reduce HBV viral load by targeting C/EBP-ß.

Nonstructural protein 14 of PDCoV promotes complement C3 expression via the activation of p38-MAPK-C/EBP pathway.

Porcine deltacoronavirus (PDCoV) is an emergent enteric coronavirus, primarily inducing diarrhea in swine, particularly in nursing piglets, with the additional potential for zoonotic transmission to humans. Despite the significant impact of PDCoV on swine populations, its pathogenic mechanisms remain incompletely understood. Complement component 3 (C3) plays a pivotal role in the prevention of viral infections, however, there are no reports concerning the influence of C3 on the proliferation of PDCoV. In this study, we initially demonstrated that PDCoV is capable of activating the C3 and eliciting inflammatory responses. The overexpression of C3 significantly suppressed PDCoV replication, while inhibition of C3 expression facilitated PDCoV replication. We discovered that nonstructural proteins Nsp7, Nsp14, and M, considerably stimulated C3 expression, particularly Nsp14, through activation of the p38-MAPK-C/EBP-ß pathway. The N7-MTase constitutes a significant functional domain of the non-structural protein Nsp14, which is more obvious to upregulate C3. Furthermore, functional mutants of the N7-MTase domain suggested that the D44 and T135 of N7-Mtase constituted a pivotal amino acid site to promote C3 expression. This provides fresh insights into comprehending how the virus manipulates the host immune response and suggests potential antiviral strategies against PDCoV.

Excess Iodine Consumption Induces Oxidative Stress and Pancreatic Damage Independently of Chemical Form in Male Wistar Rats: Participation of PPAR-γ and C/EBP-ß.

BACKGROUND: Human beings consume different chemical forms of iodine in their diet. These are transported by different mechanisms in the cell. The forms of iodine can be part of thyroid hormones, bind to lipids, be an antioxidant, or be an oxidant, depending on their chemical form. The excessive consumption of iodine has been associated with pancreatic damage and diabetes mellitus type 2, but the association between disease and the chemical form consumed in the diet is unknown. This research analyzes the effect of excessive iodine consumption as Lugol (molecular iodine/potassium iodide solution) and iodate on parameters of pancreatic function, thyroid and lipid profiles, antioxidant and oxidant status, the expression of IR/Akt/P-Akt/GLUT4, and transcription factors PPAR-γ and CEBP-ß. METHODS: Three groups of Wistar rats were treated with 300 µg/L of iodine in drinking water: (1) control, (2) KIO3, and (3) Lugol. RESULTS: Lugol and KIO3 consumption increased total iodine levels. Only KIO3 increased TSH levels. Both induced high serum glucose levels and increased oxidative stress and pancreatic alpha-amylase activity. Insulin levels and antioxidant status decreased significantly. PPAR-γ and C/EBP-ß mRNA expression increased. CONCLUSION: The pancreatic damage, hypertriglyceridemia, and oxidative stress were independent of the chemical form of iodine consumed. These effects depended on PPAR-γ, C/EBP-ß, GLUT-4, and IR.

Linagliptin attenuates thioacetamide-induced hepatic encephalopathy in rats: Modulation of C/EBP-ß and CX3CL1/Fractalkine, neuro-inflammation, oxidative stress and behavioral defects.

The degree of neuroinflammation is correlated mainly with cognitive and motor dysfunctions associated with hepatic encephalopathy (HE). The current study was conducted to explore the possible protective potential of the antidiabetic drug; linagliptin (LNG; 10 or 20 mg/kg) against HE induced by thioacetamide (TAA) in rats. Animals received two consecutive intraperitoneal injections of TAA (200 mg/kg) on alternate days. Neurobehavioral tests were performed 24 h after the last injection, and rats were sacrificed 24 h later (48 h). The higher LNG dose more effectively protected against TAA-induced changes. Administration of LNG for 15 days before TAA notably mitigated TAA-induced acute liver injury and HE, as verified by the marked improvement in motor coordination, locomotor activity, and cognition function. LNG maintained both brain and liver weight indices and retracted the hyperammonemia with a prominent suppression in liver transaminases. This was accompanied by an evident modulation of hepatic and hippocampal oxidative stress markers; GSH and MDA. LNG attenuated both liver and hippocampal pro-inflammatory cytokine; IL-1ß while augmented the anti-inflammatory one; IL-10. It noticeably reduced hepatic and hippocampal COX-2 and TNF-α and maintained hepatic and brain architectures. It also induced a marked decrease in the inflammation-regulated transcription factor, C/EBP-ß, with a profound increase in hippocampi's anti-inflammatory chemokine, CX3CL1/Fractalkine. LNG modulated TAA-induced disturbances in hippocampal amino acids; glutamate, and GABA with a significant increase in hippocampal BDNF. In conclusion, the regulatory effect of LNG on neuroinflammatory signaling underlines its neuroprotective effect against progressive encephalopathy accompanying acute liver injury.

A novel peptide RIFV suppresses human adipocyte differentiation through the inhibition of C/EBP-ß expression.

BACKGROUND: Obesity is a global epidemic disease that increases the risk of metabolic syndrome. However, therapeutic drugs for obesity are still scarce. In recent years, peptides have been identified as new biological regulators. RIFV (R-I-F-V-P-I-K-G-R-P-A-P), a novel active peptide from our peptide database. METHODS: We performed oil red O staining and triglyceride measurement to analyze the influence of RIFV on white preadipocytes differentiation. Then the effects of RIFV on cell proliferation, apoptosis and cell cycle were determined by using CCK-8 assay and flow cytometry. The mRNA and protein levels of adipogenesis-related genes were respectively detected by qRT-PCR and western blot. Rescue experiment was conducted to confirm whether RIFV could regulate adipocytes differentiation via targeting C/EBP-ß. Finally, the luciferase reporter gene assay was performed to verify the regulation of RIFV on C/EBP-ß gene. RESULTS: RIFV was revealed to inhibit the differentiation of human white adipocytes without affecting their proliferation. Additionally, RIFV could also suppress the differentiation of mouse primary white preadipocytes isolated from inguinal fat tissues. Furthermore, RIFV may have an inhibitory effect on adipogenesis by inhibiting the regulation of the adipogenic gene C/EBP-ß. CONCLUSIONS: Our results indicated that RIFV may be a novel essential regulator of adipocyte differentiation and represents a therapeutic strategy for obesity and related complications.

Role of C/EBP-ß in Methamphetamine-Mediated Microglial Apoptosis.

Methamphetamine (MA) is a widely abused psychoactive drug that primarily damages the nervous system. However, the involvement of MA in the survival of microglia remains poorly understood. CCAAT-enhancer binding protein (C/EBP-ß) is a transcription factor and an important regulator of cell apoptosis. Lipocalin2 (lcn2) is a known apoptosis inducer and is involved in many cell death processes. We hypothesized that C/EBP-ß is involved in MA-induced lcn2-mediated microglial apoptosis. To test this hypothesis, we measured the protein expression of C/EBP-ß after MA treatment and evaluated the effects of silencing C/EBP-ß or lcn2 on MA-induced apoptosis in BV-2 cells and the mouse striatum after intrastriatal MA injection. MA exposure increased the expression of C/EBP-ß and stimulated the lcn2-mediated modulation of apoptosis. Moreover, silencing the C/EBP-ß-dependent lcn2 upregulation reversed the MA-induced microglial apoptosis. The in vivo relevance of these findings was confirmed in mouse models, which demonstrated that the microinjection of anti-C/EBP-ß into the striatum ameliorated the MA-induced decrease survival of microglia. These findings provide a new insight regarding the specific contributions of C/EBP-ß-lcn2 to microglial survival in the context of MA abuse.

IκB-Kinase-epsilon (IKKε) over-expression promotes the growth of prostate cancer through the C/EBP-ß dependent activation of IL-6 gene expression.

The inflammatory cytokine IL-6 has been shown to induce the nuclear translocation of androgen receptors in prostate cancer cells and to activate the androgen receptors in a ligand-independent manner, suggesting it may contribute to the development of a castrate-resistant phenotype. Elevated IL-6 serum levels have also been associated with metastasis-related morbidity in prostate cancer patients. We have previously established that over-expression of I-kappa-B-kinase-epsilon (IKKε also named IKKi or IκBKε) in hormone-sensitive prostate cancer cell lines induces IL-6 secretion. We have also reported that prostate cancer cell lines lacking androgen receptor expression exhibit high constitutive IKKε expression and IL-6 secretion. In the present study, we validated the impact of IKKε depletion on the in vitro proliferation of castrate-resistant prostate cancer cells, and characterized how IKKε depletion affects tumor growth and IL-6 tumor secretion in vivo through a mouse xenograft-based approach. We observed a significant growth delay in IKKε-silenced PC-3 cells injected in SCID mice fed with a doxycycline-supplemented diet in comparison with mice fed with a normal diet. We also found a decrease in IL-6 secretion levels that strongly correlated with tumor growth inhibition. Finally, using constructs with various IL-6-mutated promoters, we demonstrated that IKKε over-expression induces a NF-κB-independent stimulation of the IL-6 gene promoter through the activation and nuclear accumulation of the transcription factor C/EBP-ß. Our study demonstrates the pro-proliferative role of the oncogene IKKε in castrate-resistant prostate cancer cell lines, involving the phosphorylation and nuclear translocation of C/EBP-ß that initiates IL-6 gene expression.

Porcine epidemic diarrhea virus nucleoprotein contributes to HMGB1 transcription and release by interacting with C/EBP-ß.

Porcine epidemic diarrhea is a devastating swine enteric disease, which is caused by porcine epidemic diarrhea virus (PEDV) infection. Our studies demonstrated that PEDV infection resulted in the up-regulation of proinflammatory cytokines. Meanwhile, PEDV infection and overexpression of viral nucleoprotein resulted in the acetylation and release of high mobility group box 1 proteins in vitro, an important proinflammatory response mediator, which contributes to the pathogenesis of various inflammatory diseases. Our studies also showed that SIRT1, histone acetyltransferase, and NF-κB regulated the acetylation and release of HMGB1. Chromatin immunoprecipitation, dual-luciferase reporter gene assay, and co-immunoprecipitation experiments illustrated that PEDV-N could induce HMGB1 transcription by interacting with C/EBP-ß, which could bind to C/EBP motif in HMGB1 promotor region. Collectively, our data indicate PEDV-N contributes to HMGB1 transcription and the subsequent release/acetylation of HMGB1 during PEDV infection.

Unraveling the complexity of hepatitis B virus: from molecular understanding to therapeutic strategy in 50 years.

Hepatitis B virus (HBV) is a well-known hepadnavirus with a double-stranded circular DNA genome. Although HBV was first described approximately 50 years ago, the precise mechanisms of HBV infection and effective therapeutic strategies remain unclear. Here, we focus on summarizing the complicated mechanisms of HBV replication and infection, as well as genomic factors and epigenetic regulation. Additionally, we discuss in vivo models of HBV, as well as diagnosis, prevention and therapeutic drugs for HBV. Together, the data in this 50-year review may provide new clues to elucidate molecular mechanisms of HBV pathogenesis and shed new light on the future HBV therapies.