C/EBPß promotes the expression of atrophy-inducing factors by tumours and is a central regulator of cancer cachexia.

BACKGROUND: CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor whose high expression in human cancers is associated with tumour aggressiveness and poor outcomes. Most advanced cancer patients will develop cachexia, characterized by loss of skeletal muscle mass. In response to secreted factors from cachexia-inducing tumours, C/EBPß is stimulated in muscle, leading to both myofibre atrophy and the inhibition of muscle regeneration. Involved in the regulation of immune responses, C/EBPß induces the expression of many secreted factors, including cytokines. Because tumour-secreted factors drive cachexia and aggressive tumours have higher expression of C/EBPß, we examined a potential role for C/EBPß in the expression of tumour-derived cachexia-inducing factors. METHODS: We used gain-of-function and loss-of-function approaches in vitro and in vivo to evaluate the role of tumour C/EBPß expression on the secretion of cachexia-inducing factors. RESULTS: We report that C/EBPß overexpression up-regulates the expression of 260 secreted protein genes, resulting in a secretome that inhibits myogenic differentiation (-31%, P < 0.05) and myotube maturation [-38% (fusion index) and -25% (myotube diameter), P < 0.05]. We find that knockdown of C/EBPß in cachexia-inducing Lewis lung carcinoma cells restores myogenic differentiation (+25%, P < 0.0001) and myotube diameter (+90%, P < 0.0001) in conditioned medium experiments and, in vivo, prevents muscle wasting (-51% for small myofibres vs. controls, P < 0.01; +140% for large myofibres, P < 0.01). Conversely, overexpression of C/EBPß in non-cachectic tumours converts their secretome into a cachexia-inducing one, resulting in reduced myotube diameter (-41%, P < 0.0001, EL4 model) and inhibition of differentiation in culture (-26%, P < 0.01, EL4 model) and muscle wasting in vivo (+98% small fibres, P < 0.001; -76% large fibres, P < 0.001). Comparison of the differently expressed transcripts coding for secreted proteins in C/EBPß-overexpressing myoblasts with the secretome from 27 different types of human cancers revealed ~18% similarity between C/EBPß-regulated secreted proteins and those secreted by highly cachectic tumours (brain, pancreatic, and stomach cancers). At the protein level, we identified 16 novel secreted factors that are present in human cancer secretomes and are up-regulated by C/EBPß. Of these, we tested the effect of three factors (SERPINF1, TNFRSF11B, and CD93) on myotubes and found that all had atrophic potential (-33 to -36% for myotube diameter, P < 0.01). CONCLUSIONS: We find that C/EBPß is necessary and sufficient to induce the secretion of cachexia-inducing factors by cancer cells and loss of C/EBPß in tumours attenuates muscle atrophy in an animal model of cancer cachexia. Our findings establish C/EBPß as a central regulator of cancer cachexia and an important therapeutic target.

Insulin induces expression of the hepatic vaspin gene.

Visceral adipose tissue-derived serine protease inhibitor (vaspin), initially identified in the visceral adipose tissue, is an adipokine that improves endoplasmic reticulum stress in obesity or insulin sensitivity and glucose tolerance. However, the transcriptional regulation of the hepatic vaspin gene remains elusive. We have previously shown that CCAAT-enhancer-binding protein α, a transcription factor of the basic leucine zipper class, positively regulates the vaspin gene. The present study aimed to investigate the nutritional or hormonal regulators of vaspin expression in the liver. For the fasting and refeeding study, mice in the fasting group were subjected to fasting for 24 h and then sacrificed. Mice in the refeeding group were subjected to fasting for 24 h and then refed with a 50% (w/w) sucrose/MF diet for further 24 h and then sacrificed. For the streptozotocin (STZ) study, STZ (50 mg/kg) was intraperitoneally injected into C57BL/6JJc1 mice for 5 d. Hepatic vaspin was repressed due to fasting for 24 h and was induced upon refeeding with a high-sucrose diet. In studies on liver-specific C/EBPα-deficient mice, C/EBPα was not involved in the induction of hepatic vaspin upon refeeding. In addition, the depletion of insulin by streptozotocin treatment markedly decreased hepatic vaspin expression. Finally, fasting-repressed vaspin expression in the liver was significantly increased by direct injection of insulin into fasting mice. In conclusion, our results suggest that insulin is a positive regulator of hepatic vaspin expression.

Regulation of Expression of CEBP Genes by Variably Expressed Vitamin D Receptor and Retinoic Acid Receptor α in Human Acute Myeloid Leukemia Cell Lines.

All-trans-retinoic acid (ATRA) and 1α,25-dihydroxyvitamin D (1,25D) are potent inducers of differentiation of myeloid leukemia cells. During myeloid differentiation specific transcription factors are expressed at crucial developmental stages. However, precise mechanism controlling the diversification of myeloid progenitors is largely unknown, CCAAT/enhancer-binding protein (C/EBP) transcription factors have been characterized as key regulators of the development and function of the myeloid system. Past data point at functional redundancy among C/EBP family members during myeloid differentiation. In this study, we show that in acute myeloid leukemia (AML) cells, high expression of vitamin D receptor gene (VDR) is needed for strong and sustained upregulation of CEBPB gene, while the moderate expression of VDR is sufficient for upregulation of CEBPD in response to 1,25D. The high expression level of the gene encoding for retinoic acid receptor α (RARA) allows for high and sustained expression of CEBPB, which becomes decreased along with a decrease of RARA expression. Expression of CEBPB induced by ATRA is accompanied by upregulated expression of CEBPE with similar kinetics. Our results suggest that CEBPB is the major VDR and RARA-responsive gene among the CEBP family, necessary for expression of genes connected with myeloid functions.

Theophylline suppresses interleukin-6 expression by inhibiting glucocorticoid receptor signaling in pre-adipocytes.

Adipose tissues in obese individuals are characterized by a state of chronic low-grade inflammation. Pre-adipocytes and adipocytes in this state secrete pro-inflammatory adipokines, such as interleukin 6 (IL-6), which induce insulin resistance and hyperglycemia. Theophylline (1,3-dimethylxanthine) exerts anti-inflammatory effects, but its effects on pro-inflammatory adipokine secretion by pre-adipocytes and adipocytes have not been examined. In this study, we found that theophylline decreased IL-6 secretion by 3T3-L1 pre-adipocytes and mouse-derived primary pre-adipocytes. The synthetic glucocorticoid dexamethasone (DEX) induced IL-6 expression in 3T3-L1 pre-adipocytes, and this effect was suppressed by theophylline at the mRNA level. Knockdown of CCAAT/enhancer binding protein (C/EBP) δ inhibited DEX-induced IL-6 expression, and theophylline suppressed C/EBPδ expression. Furthermore, theophylline suppressed transcriptional activity of the glucocorticoid receptor (GR) through suppression of nuclear localization of GR. In vivo, glucocorticoid corticosterone treatment (100 µg/mL) increased fasting blood glucose and plasma IL-6 levels in C57BL/6 N mice. Theophylline administration (0.1% diet) reduced corticosterone-increased fasting blood glucose, plasma IL-6 levels, and Il6 gene expression in adipose tissues. These results show that theophylline administration attenuated glucocorticoid-induced hyperglycemia and IL-6 production by inhibiting GR activity. The present findings indicate the potential of theophylline as a candidate therapeutic agent to treat insulin resistance and hyperglycemia.

Effect of neurotrophic factor-3 on the proliferation and apoptosis of bone marrow mesenchymal stem cells and mechanisms / 中华行为医学与脑科学杂志

Objective To investigate the effect of neurotrophin-3(NT-3)on the proliferation and apoptosis of bone marrow mesenchymal stem cells(BMSCs)in rats and possible mechanisms. Methods The NT-3 overexpression and lentiviral transfection of BMSCs were co-cultured with neuronal cells respectively and then they were divided into overexpression control group,NT-3 transfection group and shRNA-NT-3 transfection group(NT-3 silencing group).MTT assay was used to detect the cell culture for 24 h,48 h and 72 h. Cell cycle and apoptosis were detected by flow cytometry for 48 h. Real-time quantitative PCR was used to detect the expression of C/EBPβmRNA.The expression of C/EBPβprotein was detected by Western blot. Results MTT results showed that the proliferation ability of BMSCs in the NT-3 overexpres-sion group was significantly higher than that in the control group(0.650±0.042,0.826±0.074)at 48 h and 72 h(P<0.05).Compared with the control group(P<0.05),the cell cycle and apoptosis of BMSCs in NT-3 silencing group were significantly decreased at 48 h and 72 h(P<0.05). The results of 48 h cell cycle and apoptosis showed that the percentage of G1 phase in BMSCs was decreased,G2 and S were increased and the apoptosis was decreased. The percentage of G1 phase in G2-S phase and the increase of apoptosis were in-creased in NT-3 silencing group. The results of Western Blot showed that C/EBPβ mRNA and protein levels were significantly up-regulated in BMSCs of NT-3 overexpression group and significantly decreased in NT-3 silencing group(P<0.05).Conclusion NT-3 may promote the expression of C/EBP beta and affect the ex-pression of its downstream target genes,which can inhibit the apoptosis of BMSCs cells.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity.

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) ß was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPß and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPß, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

CEBP Epigenetic Dysregulation as a Drug Target for the Treatment of Hematologic and Gynecologic Malignancies.

BACKGROUND: The CCAAT/enhancer binding proteins (C/EBPs) form a family of transcription factors regulating many genes' expression in a variety of cells/tissues/organs at different developmental stages. With their capability of binding to their cognate DNA elements and through protein-protein interactions, C/EBPs modulate diverse functions including cell differentiation, metabolism, and immune response, under both physiological and pathological conditions such as the establishment of hematological lineages, the maintenance of normal reproductive function, and the development of malignancies. OBJECTIVES: This review concentrates on the role(s) and epigenetic alterations of C/EBP genes in hematologic malignancies and gynecologic organs and disorders. New research findings on molecular pathways involved in C/EBP function and regulation are reviewed and analyzed. The potential therapeutic values of these findings are also discussed. CONCLUSION: Unlike in hematologic malignancies in which C/EBP mutations and their disruption of wild type C/EBP tumor suppressive activities have been well documented, mutation of C/EBP does not appear to be a common event in gynecologic cancers, raising some doubt if C/EBPs may have tumor suppressor activity in gynecologic cancers. However, this notion could not exclude the possibility that downregulation or DNA methylation-meditated epigenetic silencing of C/EBPs may contribute to the development of gynecologic malignancies.

Insights into specificity, redundancy and new cellular functions of C/EBPa and C/EBPb transcription factors through interactome network analysis.

BACKGROUND: C/EBPa and C/EBPb are transcription factors with tissue specific expression regulating several important cellular processes. They work by recruiting protein complexes to a common DNA recognition motif and both are able to compensate each other's absence in many cell types, thus showing functional redundancy. They also play distinct roles in specific cellular pathways and their abnormal functioning gives raise to different human pathologies. METHODS: To investigate the molecular basis of C/EBPa and C/EBPb specificity and redundancy we characterized their in vivo protein-protein interaction networks by Tandem Affinity Purification (TAP) and Mass Spectrometry (MS). To unravel the functional features of C/EBPa and C/EBPb proteomes we studied the statistical enrichment of binding partners related to Gene Ontology (GO) terms and KEGG pathways. RESULTS: Our data confirmed that the C/EBPa and C/EBPb regulate biological processes like cell proliferation, apoptosis and transformation. We found that both C/EBPa and C/EBPb are involved in other cellular pathways such as RNA maturation, RNA splicing and DNA repair. Specific interactions of C/EBPa with MRE11, RUVBL1 and RUVBL2 components of DNA repair system were confirmed by co-immunoprecipitation assays. CONCLUSIONS: Our comparative analysis of the C/EBPa and C/EBPb proteomes provides an insight for understanding both their redundant and specific roles in cells indicating their involvement in new pathways. Such novel predicted functions are relevant to normal cellular processes and disease phenotypes controlled by these transcription factors. GENERAL SIGNIFICANCE: Functional characterization of C/EBPa and C/EBPb proteomes suggests they can regulate novel pathways and indicate potential molecular targets for therapeutic intervention.

Transcription Factors Regulating Inflammatory Cytokine Production Are Differentially Expressed in Peripheral Blood Mononuclear Cells of Behçet Disease Depending on Disease Activity

BACKGROUND: Behçet disease (BD) is a relapsing inflammatory disease with increased production of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs); however, the underlying molecular mechanisms are not well known. OBJECTIVE: To analyze whether the differential expression of transcription factors is involved in the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6 production by PBMCs of BD patients compared to healthy controls (HCs). METHODS: Expression of transcription factors was examined by real-time reverse transcriptase-polymerase chain reaction and western blotting. Cytokine production by CD11b+ cells transfected with siRNAs against transcription factors was measured by enzyme-linked immunosorbent assay. RESULTS: In the absence of lipopolysaccharide stimulation, the transcript level of CCAAT-enhancer-binding proteins (C/EBP) β was increased in PBMCs from patients with active BD compared to that in PBMCs from patients with stable BD. The C/EBPδ transcript level was higher in PBMCs from patients with active BD than in those from HCs. The activating transcription factor 3 (ATF3) transcript level was increased in PBMCs from patients with stable BD compared to that in PBMCs from HCs. siRNAs targeting C/EBPβ and C/EBPδ significantly reduced the production of IL-6 and TNF-α in lipopolysaccharide-stimulated CD11b+ cells from patients with BD as well as from HCs. CONCLUSION: We found differential expression of C/EBPβ, C/EBPδ, and ATF3 in PBMCs from patients with BD depending on disease activity, indicating the involvement of these molecules in BD pathogenesis.

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation.

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPß, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.

A study of the lipoprotein lipase inhibitory mechanism of Poncirus trifoliata water extracts / 한국영양학회지

PURPOSE: Poncirus trifoliata has been reported to have anti-inflammatory, antioxidant, and immune activities. However, its anti-obesity activity and the mechanism by which the water extract of dried, immature fruit of Poncirus trifoliata (PF-W) acts are not clear. This study suggests a potential mechanism associated with the anti-obesity activity of PF-W. METHODS: We measured the effect of PF-W on lipoprotein lipase (LPL) regulation using enzyme-linked immunosorbent assay (ELISA) and an activity assay. The LPL regulation mechanism was examined by reverse transcription polymerase chain reaction (RT-PCR) to measure the mRNA expression of biomarkers related to protein transport and by western blot for analysis of the protein expression of the transcription factor CCAAT-enhancer-binding protein (C/EBPbeta) RESULTS: The total polyphenol and flavonoid content of PF-W was 52.15 +/- 4.02 and 6.56 +/- 0.47 mg/g, respectively. PF-W treatment decreased LPL content in media to 58 +/- 5% of that in control adipocyte media, and increased LPL content to 117 +/- 3.5% of that in control adipocytes, but did not affect the mRNA expression of LPL. PF-W also increased the mRNA expression of sortilin-related receptor (SorLA), a receptor that induces endocytosis and intracellular trafficking of LPL, in a concentration- and time-dependent manner. Finally, cell fractionation revealed that PF-W treatment induced the expression of C/EBPbeta, a SorLA transcription factor, in the nuclei of 3T3-L1 adipocytes. CONCLUSION: The LPL secretion and activity assay showed PF-W to be an LPL secretion inhibitor, and these results suggest the potential mechanism of PF-W involving inhibition of LPL secretion through C/EBPbeta-mediated induction of SorLA expression.

The chalcones cardamonin and flavokawain B inhibit the differentiation of preadipocytes to adipocytes by activating ERK.

AIM: We searched for polyphenols capable of inhibiting the lipid accumulation in 3T3-L1 cells, and investigated the mechanisms of two effective chalcones cardamonin and flavokawain B on differentiation of preadipocytes. METHOD AND RESULTS: We treated 3T3-L1 cells with a panel of 46 polyphenols and measured intracellular lipid accumulation by Sudan II staining. Four of them, including cardamonin and flavokawain B, inhibited lipid accumulation. In the further study, cardamonin and flavokawain B inhibited lipid accumulation by downregulating the expression of CCAAT/enhancer binding protein (C/EBP)-ß, C/EBPα, and peroxisome proliferator-activated receptor-γ (PPARγ) at both mRNA and protein levels. Cardamonin and flavokawain B also increased phosphorylation of extracellular signal-regulated kinase (ERK) in the early phase of adipocyte differentiation. PD98059, an ERK inhibitor, restored C/EBPß, PPARγ expression and intracellular lipid accumulation in adipocytes. Moreover, cardamonin and flavokawain B also modulated the secretion of C-reactive protein, dipeptidyl peptidase IV, interleukin-6, tumor necrosis factor-α and fibroblast growth factor-21 in mature adipocytes. CONCLUSIONS: These results indicate that ERK activation and consequent downregulation of adipocyte-specific transcription factors are involved in the inhibitory effects of the chalcones cardamonin and flavokawain B on adipocyte differentiation. Moreover, cardamonin and flavokawain B are able to modulate secretion of adipokines in mature adipocytes.

The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65.

Interleukin-8 (IL8) is an immediate-early chemokine that has been well characterized in several fish species. Ten IL8 gene variants have already been described in rainbow trout, but none of their promoters has structurally been defined or functionally characterized in teleost fish. To uncover key factors regulating IL8 expression, we intended to functionally characterize an IL8 promoter from rainbow trout. Incidentally, we isolated a novel IL8 gene variant (IL8-G). It is structurally highly similar to the other trout IL8 gene variants and its mRNA concentration increased significantly in secondary lymphoid tissues after infecting healthy fish with Aeromonas salmonicida. The proximal promoter sequence of the IL8-G-encoding gene features in close proximity two consensus elements for CEBP attachment. The proximal site overlaps with a NF-κB-binding site. Cotransfection of an IL8-G promoter-driven reporter gene together with vectors expressing various mammalian CEBP or NF-κB factors revealed in human HEK-293 cells that CEBPA and NF-κB p50, but not NF-κB p65 activate this promoter. The stimulatory effect of NF-κB p50 is likely conveyed by synergizing with CEBPA. Deletion or mutation of either the distal or the proximal CEBP-binding site, respectively, caused a significant decrease in IL8-G promoter activation. We confirmed the significance of the CEBPA factor for IL8-G expression by comparing the stimulatory capacity of the trout CEBPA and -B factors, thereby reducing the evolutionary distance in the inter-species expression assays. Similar promoter induction potential and intracellular localization of the mammalian and teleostean CEBPA and -B factors suggests their functional conservation throughout evolution.

MiR-27 orchestrates the transcriptional regulation of brown adipogenesis.

OBJECTIVE: Brown adipose tissue (BAT) produces heat using chemical energy of lipids and glucose, a function induced by cold exposure or diet. The brown adipogenesis is tightly controlled in a coordinated interplay between several transcriptional factors. It is not known what enables and coordinates this robust program of concerted cooperation between the transcriptional factors and co-regulators necessary for the brown adipogenesis. MATERIALS/METHODS: A. In vivo studies--we investigated the expression levels of miR-27a and b in mice after cold exposure. B. Using gene expression and functional studies together with high throughput imaging in primary preadipocytes, and cell culture models, we investigated the role of miR-27 in beige and brown adipogenesis. C. Using gene silencing and rescue experiments we dissected the molecular mechanisms of the miR-27 action. RESULTS: After cold exposure, miR-27 is downregulated in BAT and subcutaneous white adipose tissue (SAT). MiR-27 is also downregulated during brown adipogenesis of primary preadipocytes in vitro, and it directly targets and negatively regulates the essential components of the brown transcriptional network: Prdm16, Pparα, Creb, and in part Pgc1ß. Together with its direct effect on Pparγ, and indirect on Pgc1α, mir-27 decreases brown differentiation of cultured cells and of primary SAT preadipocytes. CONCLUSIONS: Our results point to miR-27 as a central upstream regulator of the transcriptional network involved in beige and brown adipogenesis after cold exposure, and suggest miR-27 inhibition as a novel therapeutic approach for metabolic diseases aiming at increasing the beige/brown fat mass.

Therapeutic effects of CCAAT/enhancer-binding protein α gene on liver fibrosis in mice / 中华消化杂志

Objective To investigate the difference of CCAAT/enhancer-binding protein α (C/EBP-α) gene induced apoptosis between hepatocytes and hepatic stellate cells (HSC) in mice with liver fibrosis.Methods Sixty BALB/c mice were evenly divided into normal group,model group,treatment group,blank control group and negative control group,12 mice in each group.Except the mice of normal control group,the mice of other groups were treated with intraperitoneal injection of CCl4 to establish liver fibrosis mice model.Mice of treatment group,blank control group and negative control group were administrated with C/EBP-α carried adenovirus (Ad-C/EBP-α),phosphate buffered solution and empty vector of adenovirus (Ad-EGFP) respectively through tail vein for the first week.The expression of C/EBP-α and α-smooth muscle actin (α-SMA) was detected by immunohistochemistry method.Sinusoidal endothelial structure of peri-portal regions and far from portal regions was observed by transmission electron microscope (TEM).Terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was applied to detect apoptosis of cells in liver tissue.The degree of liver fibrosis in mice was determined with sirius red staining and hydroxyproline content measurement.Single factor variance analysis was performed for comparison among multiple groups,and t test was used for comparison between two groups.Results C/EBP-α was expressed in nucleus of hepatocyte in normal control group mice.The expression decreased in model group,blank control group and negative control group.However,the expression of C/EBP-α of treatment group increased,and mainly expressed in cells located in perisinusoidal and perivascular.Hepatic sinusoids was distorted,blood vessel wall thickened.Hepatocyte degeneration and lots of lipid droplets was found in model group,blank control group and negative control group.The thicken degree of endothelial layer of blood vessel of treatment group was lower than that of model group.The percentage of sirius red positive cells of normal group,model group,treatment group,blank control group and negative control group was (0.10±0.03)％,(5.81±0.32)％,(2.32±0.45)％,(6.34± 0.81)％ and (6.10± 0.92)％,respectively; content of hydroxyproline was (0.07±0.00) μg/mg,(0.69 ± 0.10) μg/mg,(0.19±0.06) μg/mg,(0.56±0.03) μg/mg and (0.64±0.08) μg/mg,respectively; the percentage of α-SMA positive cells was (0.50 ±0.03)％,(5.30 ± 0.52)％,(2.15 ± 0.29)％,(5.53 ± 0.43) ％ and (5.42 ± 0.25) ％,respectively; the number of TUNEL positive cells was (0.25 ± 0.08),(0.15±0.02),(7.10±1.53),(0.13±0.03) and (0.18±0.07),respectively.The differences between the groups were statistically significant (F=113.74,148.29,292.43 and 140.25,all P＜0.05).The difference between normal group and model group,between model group and treatment group,between treatment group and blank control group,between treatment group and negative control group were statistically significant (tarirus positive cell =-52.54,-16.20,-10.60 and-7.99,thydroxyproline content =-168.00,11.53,11.07 and 12.54,ta SMA pusitive cells-24.77,-13.82,15.94 and 18.37,tTUNEL positive cells =3.26,-11.91,-11.95 and-11.88,all P＜ 0.05),there was no statistically significant difference between model group and blank control group,between model group and negative control group (both P＞0.05).TUNEL positive cells mainly located in perisinusoidal and perivascular of liver in mice,which was consistent with the distribution of α-SMA-positive cells.Conclusion C/EBP-α could effectively relieve CCl4 induced liver fibrosis in mice mainly through inducing HSC apoptosis,however no apoptosis effect on hepatocytes.

4-Hydroxyderricin and xanthoangelol from Ashitaba (Angelica keiskei) suppress differentiation of preadiopocytes to adipocytes via AMPK and MAPK pathways.

SCOPE: Adipocytes differentiation is deeply involved in the onset of obesity. 4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are the chalcones that are derived from Ashitaba (Angelica keiskei). In this study, we demonstrated the inhibitory effects of these chalcones on adipocytes differentiation. METHODS AND RESULTS: 4HD and XAG suppressed intracellular lipid accumulation by Oil red O staining at 5 µM without cytotoxicity. They inhibited adipocytes differentiation accompanied by down-expression of adipocyte-specific transcription factors, CCAAT/enhancer-binding protein-ß (C/EBP-ß), C/EBP-α, and peroxisome proliferator-activated receptor gamma (PPAR-γ) using RT-PCR and Western blotting analysis. To obtain insights into the underlying mechanism, the activation of AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase pathways was investigated. These two chalcones promoted phosphorylation of AMPK and acetyl CoA carboxylase during differentiation of 3T3-L1 adipocytes accompanied by a decrease in glycerol-3-phosphate acyl transferase-1 and an increase in carnitine palmitoyltransferase-1 mRNA expression. These chalcones also promoted phosphorylation of extracellular signal-regulated kinases and Jun aminoterminal kinases, but not p38. Moreover, the inhibitors for AMPK and extracellular signal-regulated kinases abolished the chalcones-caused down-expression of C/EBP-ß, C/EBP-α, and PPAR-γ. Treatment with Jun aminoterminal kinases inhibitor abolished the down-expression of C/EBP-α and PPAR-γ, but not C/EBP-ß. CONCLUSION: 4HD and XAG inhibit adipocytes differentiation through AMPK and mitogen-activated protein kinase pathways, resulting in the down-expression of adipocyte-specific transcription factors.

Asthma and COPD - The C/EBP Connection.

Asthma and chronic obstructive pulmonary disease (COPD) are the two most prominent chronic inflammatory lung diseases with increasing prevalence. Both diseases are associated with mild or severe remodeling of the airways. In this review, we postulate that the pathologies of asthma and COPD may result from inadequate responses and/or a deregulated balance of a group of cell differentiation regulating factors, the CCAAT/Enhancer Binding Proteins (C/EBPs). In addition, we will argue that the exposure to environmental factors, such as house dust mite and cigarette smoke, changes the response of C/EBPs and are different in diseased cells. These novel insights may lead to a better understanding of the etiology of the diseases and may provide new aspects for therapies.

Antitumor effect of enhancer binding protein C/EBPα on the leukemic BALB/c nude mice / 白血病·淋巴瘤

Objective To explore the role of tumor inhibition of enhancer binding protein C/EBPα in the leukemic mice. Methods BALB/c nude mice were randomly divided into three groups. Three kinds of cells including pEGFP-C/EBPα-K562 cells, pEGFP-K562 cells and K562 cells as the control were injected into mice separately through the subcutaneous and tail vein, and subcutaneous tumors and leukemic models were formed. The changes of tumors were observed and the apoptosis of cells was detected by TUNEL; The capacity of proliferation of leukemia cells was observed in the bone marrow and the peripheral blood by Wright-Giemsa staining. The expression of genes of related to proliferation was detected by RT-PCR. Results The quality and the max diameter of tumors in the pEGFP-C/EBPα-K562 group were smaller than that of pEGFP-K562 group and K562 control group [(2.4±0.1) g vs (5.1±0.3) g and (5.7±0.4) g, both P ＜0.05; (11+2)mm vs (19+3) mm and (23+3) mm, both P ＜0.05]. More apoptosis cells were found in the pEGFP-C/EBPα-K562 group leukemic cells were found in the peripheral blood of leukemic models, and the proliferation of leukemic cells in the pEGFP-C/EBPo-K562 group were lower than that of other groups, accompany by the conspicuous cell differentiation. p53 was significantly elevated by RT-PCR, while down-regulated of c-myc.Conclusion Enhancer binding protein C/EBPα promote the apoptosis of cells and inhibit the proliferation of leukemia cells in leukemia mice, and further induce the cell differentiation. The inhibition of enhancer binding protein C/EBPα in the leukemia may have effect through the regulation of related genes.

Ginsenoside Rb_1 facilitates adipocyte differentiation and inhibits lipolysis in 3T3-L1 adipocytes / 中华内分泌代谢杂志

Objective To observe the effect of ginsenoside Rb1,the most abundant ginsenoside in ginseng root,on differentiation and lipolysis of 3T3-L1 cells and to explore its anti-diabetic mechanism.Methods 3T3-L1 preadipoeytes were induced under standard differentiation process in the presence of 0.1,1,10,100?mol/L ginsenoside Rb_1 for 6 days.Oil red O staining,measurement of triglyceride contents and glucose uptake assay were performed.The expressions of mRNA and protein of PPAR?2,C/EBP?,ap2,glucose transporter (Glut) 1,and Glut4 were analysed with quantitative real time-PCR and Western blot.The binding affinity of Rb_1 to PPAR?-LBD was evaluated by Surface Plasmon Resonance (SPR).Lipolysis of adipocytes was examined by the measurement of glycerol released from adipoeytes treated with Rb_1 for 1 h.Results Ginsenoside Rb_1 facilitated differentiation of 3T3-L1 preadipoeytes in a dose-depondent manner.10?mol/L ginsenoside Rb_1 increased lipid accumulation by about 56%.Treatment of differentiating adipocytes with 10?mol/L ginsenoside Rb_1 increased the expressions of PPAR?2 and C/EBP?mRNA and protein,as well as mRNA expression of ap2,one of their target genes.After treatment of differentiating adipoeytes with Rb_1,basal and insulin-mediated glucose transport augmented significantly accompanied by up-regulations of mRNA and protein level of Glut4,but not of Glutl.SPR showed Rb_1 could bind to PPAR?which suggested Rb_1 was a ligand of PPAR?.Ginsenoside Rb_1 inhibited basal lipolysis in adipoeytos in a dose-dependent manner.However,it did not affect isoproterenol-stimulated lipolysis.Conclusion As a PPAR?ligand,ginsenoside Rb_1 promotes adipogenesis,inhibitas basal lipolysis and inereasos basal and insulin-mediated glucose transport in cultured adipoeytes.Therefore,anti-diabetic and insulin-sensitizing activity of ginsenosides is,at least in part,involved in the enhancing effect on PPAR?2 and C/EBP?expressions,hence promoting adipogenesis and glucose uptake,and inhibiting lipolysis in adipocytes.