C/EBPα alleviates hepatic ischemia-reperfusion injury by inhibiting endoplasmic reticulum stress via HDAC1-mediated deacetylation of ATF4.

Hepatic ischemia-reperfusion (IR) injury is a complex systemic process causing a series clinical problem. C/EBPα is a key transcription factor for hepatocyte function, but its role and mechanism in regulating hepatic IR injury are largely unknown. Occluding portal vein and hepatic artery was used to establish a mouse model of hepatic IR injury. C/EBPα expression was decreased in IR-injured liver compared with the sham, accompanied by increased contents of serum alanine transaminase (ALT), aspartate transaminase (AST), high mobility group box-1, and proportion of hepatic cells. Oxygen and glucose deprivation/recovery (OGD/R) was used to establish a cellular hepatic IR model in WRL-68 hepatocytes in vitro, and C/EBPα was overexpressed in the hepatocytes to evaluate its effect on hepatic IR injury. OGD/R promoted oxidative stress, cell apoptosis and endoplasmic reticulum (ER) stress in hepatocytes, which was reversed by C/EBPα overexpression. Then, we found that C/EBPα promoted histone deacetylase 1 (HDAC1) transcription through binding to HDAC1 promoter. Moreover, HDAC1 deacetylated the activating transcription factor 4 (ATF4), a key positive regulator of ER stress. Trichostatin-A (an HDAC inhibitor) or ATF4 overexpression reversed the improvement of C/EBPα on OGD/R-induced ER stress and hepatocyte dysfunction. 4-Phenylbutyric acid (an endoplasmic reticulum stress inhibitor) also reversed the hepatic IR injury induced by ATF4 overexpression. Finally, lentivirus-mediated C/EBPα overexpression vector was applied to administrate hepatic IR mice, and the results showed that C/EBPα overexpression ameliorated IR-induced hepatic injury, manifesting with reduced ALT/AST, oxidative stress and ER stress. Altogether, our findings suggested that C/EBPα ameliorated hepatic IR injury by inhibiting ER stress via HDAC1-mediated deacetylation of ATF4 promoter.

C/EBPα aggravates renal fibrosis in CKD through the NOX4-ROS-apoptosis pathway in tubular epithelial cells.

BACKGROUND: Chronic kidney disease (CKD) is a prevalent renal disorder with various risk factors. Emerging evidence indicates that the transcriptional factor CCAAT/enhancer binding protein alpha (C/EBPα) may be associated with renal fibrosis. However, the precise role of C/EBPα in CKD progression remains unexplored. METHODS: We investigated the involvement of C/EBPα in CKD using two distinct mouse models induced by folic acid (FA) and unilateral ureteral obstruction (UUO). Additionally, we used RNA sequencing and KEGG analysis to identify potential downstream pathways governed by C/EBPα. FINDINGS: Cebpa knockout significantly shielded mice from renal fibrosis and reduced reactive oxygen species (ROS) levels in both the FA and UUO models. Primary tubular epithelial cells (PTECs) lacking Cebpa exhibited reduced apoptosis and ROS accumulation following treatment with TGF-ß. RNA sequencing analysis suggested that apoptosis is among the primary pathways regulated by C/EBPα, and identified NADPH oxidoreductase 4 (NOX4) as a key protein upregulated upon C/EBPα induction (ICCB280). Treatment with l-Theanine, a potential NOX4 inhibitor, mitigated renal fibrosis and inflammation in both the FA and UUO mouse models. INTERPRETATION: Our study unveils a role for C/EBPα in suppressing renal fibrosis, mitigating ROS accumulation, and reducing cell apoptosis. Furthermore, we investigate whether these protective effects are mediated by C/EBPα's regulation of NOX4 expression. These findings present a promising therapeutic target for modulating ROS and apoptosis in renal tubular cells, potentially offering an approach to treating CKD and other fibrotic diseases.

Antioxidant and Anti-Obesity Effects of Juglans mandshurica in 3T3-L1 Cells and High-Fat Diet Obese Rats.

Juglans mandshurica Maxim. walnut (JMW) is well-known for the treatment of dermatosis, cancer, gastritis, diarrhea, and leukorrhea in Korea. However, the molecular mechanism underlying its anti-obesity activity remains unknown. In the current study, we aimed to determine whether JMW can influence adipogenesis in 3T3-L1 preadipocytes and high-fat diet rats and determine the antioxidant activity. The 20% ethanol extract of JMW (JMWE) had a total polyphenol content of 133.33 ± 2.60 mg GAE/g. Considering the antioxidant capacity, the ABTS and DPPH values of 200 µg/ml of JMWE were 95.69 ± 0.94 and 79.38 ± 1.55%, respectively. To assess the anti-obesity activity of JMWE, we analyzed the cell viability, fat accumulation, and adipogenesis-related factors, including CCAAT-enhancer-binding protein alpha (C/EBPα), sterol regulatory element-binding protein-1c (SREBP1c), peroxisome proliferator-activated receptor-gamma (PPARγ), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). We found that total lipid accumulation and triglyceride levels were reduced, and the fat accumulation rate decreased in a dose-dependent manner. Furthermore, JMWE suppressed adipogenesis-related factors C/EBPα, PPARγ, and SREBP1c, as well as FAS and ACC, both related to lipogenesis. Moreover, animal experiments revealed that JMWE could be employed to prevent and treat obesity-related diseases. Hence, JMWE could be developed as a healthy functional food and further explored as an anti-obesity drug.

Methylation of lncSHGL promotes adipocyte differentiation by regulating miR-149/Mospd3 axis.

Obesity poses significant health risks and can negatively impact an individual's quality of life. The human obesity phenotype results from the differentiation of pre-adipocytes into adipocytes, which leads to hypertrophy and hyperplasia in adipose tissue. The molecular mechanisms by which long non-coding RNAs (lncRNAs) modulate adipocyte differentiation, a process implicated in obesity development, remain poorly characterized. A lncRNA which suppressed the hepatic gluconeogenesis and lipogenesis (lncSHGL) was newly identified. Our research aims to elucidate the functional role and mechanistic underpinnings of suppressor of lncSHGL in adipocyte differentiation. We observed that lncSHGL expression progressively diminished during 3T3-L1 differentiation and was downregulated in the liver and perirenal adipose tissue of ob/ob mice. lncSHGL acts as a molecular sponge for miR-149, with Mospd3 identified as a target of miR-149.Overexpression of lncSHGL and inhibition of miR-149 led to suppressed 3T3-L1 proliferation, decreased lipid droplet accumulation, and attenuated promoter activity of PPARγ2 and C/EBPα. These changes consequently resulted in reduced expression of Cyclin D1, LPL, PPARγ2, AP2, and C/EBPα, as well as inhibited the PI3K/AKT/mTOR signaling pathway. In contrast, lncSHGL suppression yielded opposing outcomes. Moreover, the effects of lncSHGL overexpression and miR-149 inhibition on reduced expression of Cyclin D1, LPL, PPARγ2, AP2, and C/EBPα were reversible upon miR-149 overexpression and Mospd3 suppression. These findings were further validated in vivo. We also discovered a significant increase in methylation levels during 3T3-L1 differentiation, with lncSHGL highly expressed in the presence of a methylation inhibitor. In conclusion. lncSHGL methylation facilitates adipocyte differentiation by modulating the miR-149/Mospd3 axis. Targeting lncSHGL expression may represent a promising therapeutic strategy for obesity-associated adipogenesis, particularly in the context of fatty liver disease.

Xiao-Gao-Jiang-Zhuo-containing serum inhibits adipogenesis through SIRT1-IGF-1 crosstalk in 3T3-L1 preadipocytes.

PPARγ, CEBP/α, and SREBP1C are the major transcriptional factors participating in adipogenesis and lipogenesis. SIRT1 and IGF-1 signaling pathways are important pathways involved in body endocrine and metabolism. Our unique Chinese herbal medicine Xiao-Gao-Jiang-Zhuo (XGJZ) has a remarkable clinical effect on obesity. However, the molecular basis remains unknown. XGJZ-containing serum was treated in the incubation of 3T3-L1 preadipocytes to observe its function in the 3T3-L1 cell differentiation. Oil Red O staining was used to monitor the lipid droplets accumulated after 8 days of incubation. RT-qPCR and western blotting were used to investigate the regulatory effects of XGJZ-containing serum on adipogenesis-related factors. The protein levels of main molecules in SIRT1 and IGF-1 signaling pathways were also detected by western blotting. XGJZ-containing serum notably suppressed the lipid accumulation in differentiated adipocytes through SIRT1/IGF-1 pathway. XGJZ-containing serum activated the SIRT1/IGF-1 pathway and reduced the expression levels of PPARγ, CEBP/α, and SREBP1C through this pathway. Additionally, XGJZ-containing serum enhanced the phosphorylation of ATGL and HSL and then induced lipolysis. XGJZ-containing serum has inhibitory effects on adipogenesis in 3T3-L1 preadipocytes through SIRT1/IGF-1 signaling pathway. Our study affirmed the effect of XGJZ-containing serum in the treatment of obesity. It provides a basis for the mechanism of obesity.

IL-35 inhibits adipogenesis via PPARγ-Wnt/ß-catenin signaling pathway by targeting Axin2.

Interleukin (IL)-35, a member of the IL-12 family, functions as an immunosuppressive cytokine that plays a crucial role in the regulation of immune-related disorders and inflammatory diseases. Adipose tissue, which is now recognized as an immune organ, is regulated by immunocytes through various signaling pathways, including the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) pathway and the Wnt/ß-actin pathway. However, there is limited research regarding the effects of IL-35 on adipogenesis. Our current findings indicated that IL-35 impedes the proliferation and promotes the cytotoxicity of 3T3-L1 preadipocytes. Furthermore, IL-35 inhibited the adipogenic differentiation, as well as suppressed triglyceride and lipid accumulation. Additionally, the expression of PPARγ and C/EBPα, two key regulators of adipogenesis, were both down-regulated with IL-35 treatment. In order to explicate the mechanisms underlying the effects of IL-35, we conducted an investigation into the expression of Axin2, an intracellular inhibitor of Wnt/ß-catenin signaling, in 3T3-L1 preadipocyte cells. Gene silencing of Axin2 through small interfering RNAs (siRNAs) enhanced PPARγ and C/EBPα expression while decreasing nuclear ß-catenin levels in the presence of IL-35. Furthermore, in IL-35-treated cells, Axin2 knockdown boosted adipogenic differentiation (as measured by increased Oil Red O staining). These findings imply that IL-35 regulates Axin2 expression and thereby plays an important role in adipocyte development.

Serine Threonine-Protein Kinase-Derived IW13 Improves Lipid Metabolism via C/EBP-α/SREBP1/FAS Signaling Pathways in HFD-Induced Zebrafish In Vivo Larval Model.

Obesity is linked to the development of major metabolic disorders such as type 2 diabetes, cardiovascular disease, and cancer. Recent research has focused on the molecular link between obesity and oxidative stress. Obesity impairs antioxidant function, resulting in dramatically increased reactive oxygen levels and apoptosis. In this study, we investigated the effect of IW13 peptide on inhibiting lipid accumulation and regulating the antioxidant mechanism to normalize the lipid metabolism in HFD induced zebrafish larvae. Our results showed that co-treatment with IW13 peptide showed a protective effect in HFD zebra fish larvae by increasing the survival and heart rate. However, IW13 peptide co-treatment reduced triglycerides and cholesterol levels while also restoring the SOD and CAT antioxidant enzymes. In addition, IW13 co-treatment inhibited the formation of lipid peroxidation and superoxide anion by regulating the glutathione level. Also, the results showed that IW13 specifically downregulated the expression of the lipogenic-specific genes (C/EBP-α, SREBP1, and FAS). The findings exhibited that the IW13 peptide with effective antioxidant and anti-obesity activity could act as a futuristic drug to treat obesity and oxidative stress-related diseases.

Methionine sulfoxide suppresses adipogenic differentiation by regulating the mitogen-activated protein kinase signaling pathway.

In this study, methionine sulfoxide (MetO) was identified as an active metabolite that suppresses adipogenesis after screening obese individuals versus the normal population. MetO suppressed the gene and protein expression of CCAAT/enhancer binding protein (C/EBP) α, adipocyte fatty acid binding protein 4 (FABP4), and the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) during human preadipocyte (HPA) differentiation. Adipogenesis decreased following MetO treatment; however, the preadipocyte number, proliferation, and apoptosis were unaffected. The activity of phosphorylated extracellular signal-related kinase (P-ERK) of the mitogen-activated protein kinase (MAPK) pathway was significantly inhibited in HPA after MetO treatment. Furthermore, treatment of preadipocytes with the selective P-ERK1/2 agonist Ro 67-7476 abolished the effect of MetO against adipogenesis suggesting that MetO function is dependent on the MAPK pathway. The mechanistic insights of adipogenesis suppression by MetO presented in this study shows its potential as an antiobesity drug.

Functional inhibition of MEF2 by C/EBP is a possible mechanism of leukemia development by CEBP-IGH fusion gene.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

C/EBPα-Mediated Transcriptional Activation of PIK3C2A Regulates Autophagy, Matrix Metalloproteinase Expression, and Phenotypic of Vascular Smooth Muscle Cells in Aortic Dissection.

Purpose: To investigate the function of C/EBPα in the development of aortic dissection (AD) and the underlying mechanism. Methods: Aortic vascular smooth muscle cells (VSMCs) were isolated, cultured, and identified from AD rats. Then, C/EBPα and PIK3C2A were knockdown or overexpressed by siRNA or plasmid transfection, respectively. Rapamycin or 3-MA was utilized to stimulate and restrain autophagy of VSMCs, respectively. Western blot was used to evaluate the expression levels of C/EBPα, PIK3C2A, LC3, Beclin-1, p62, MMP-2, MMP-9, α-SMA, SM-MHC, and OPN. The pathological status of aortic ring was evaluated by stretch stress, and ChIP assay was used to analyze the binding between C/EBPα and PIK3C2A. C/EBPα shRNA was injected into tail vein to observe the effect of C/EBPα knockdown in vivo on phenotype, autophagy of aortic vascular tissue by immunohistochemical staining and Western blot. Results: The protein levels of C/EBPα, PIK3C2A, MMP-2, MMP-9, and LC3 in the aorta of AD rats were all upregulated significantly. C/EBPα and rapamycin promoted notable upregulation of the synthesized proteins (OPN), PIK3C2A, matrix metalloproteinases, LC3, and Beclin-1 in VSMCs, while suppressed contractile proteins (α-SMA and SM-MHC) and p62. The opposite results were observed in the C/EBPα-knockdown VSMCs, PIK3C2A-knockdown VSMCs, or VSMCs treated with 3-MA. C/EBPα, PIK3C2A, and LC3 were dramatically upregulated by the stimulation of 3 g and 5 g stretch stress. The downregulated contractile proteins, upregulated synthetic proteins, activated autophagy, and aggravated pathological state in 5 g stretch stress-treated aortic rings were significantly reversed by the knockdown of C/EBPα. ChIP results indicated that there was a binding site for C/EBPα in the promoter of PIK3C2A. C/EBPα also downregulated α-SMA level and upregulated OPN levels in AD rats in vivo. Conclusion: Our data indicated that during the development of AD, C/EBPα regulated the transition of VSMC phenotype and extracellular matrix remodeling by activating autophagy through regulating the transcriptional activity of PIK3C2A promoter.

[Overexpression of ATF3 inhibits the differentiation of goat intramuscular preadipocytes].

The aim of this study was to investigate the effect of activating transcription factor 3 (ATF3) on the differentiation of intramuscular preadipocytes in goat, and to elucidate its possible action pathway at the molecular level. In this study, the recombinant plasmid of goat pEGFP-N1-ATF3 was constructed, and the intramuscular preadipocytes were transfected with liposomes. The relative expression levels of adipocyte differentiation marker genes were detected by quantitative real-time PCR (qRT-PCR). After transfection of goat intramuscular preadipocytes with the goat pEGFP-N1-ATF3 overexpression vector, it was found that the accumulation of lipid droplets was inhibited, and the adipocyte differentiation markers PPARγ, C/EBPα and SREBP1 were extremely significantly down-regulated (P < 0.01), while C/EBPß and AP2 were significantly down-regulated (P < 0.05). The ATF3 binding sites were predicted to exist in the promoter regions of PPARγ, C/EBPα and AP2 by the ALGGEN PROMO program. The overexpression of goat ATF3 inhibits the accumulation of lipid droplets in intramuscular preadipocytes, and this effect may be achieved by down-regulating PPARγ, C/EBPα and AP2. These results may facilitate elucidation of the regulatory mechanism of ATF3 in regulating the differentiation of goat intramuscular preadipocytes.

C/EBPα regulates the fate of bone marrow mesenchymal stem cells and steroid-induced avascular necrosis of the femoral head by targeting the PPARγ signalling pathway.

BACKGROUND: The imbalance of osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is closely related to steroid-induced avascular necrosis of the femoral head (SANFH). We aimed to investigate the epigenetic mechanism of intramedullary fat accumulation and continuous osteonecrosis after glucocorticoid (GC) withdrawal in SANFH. METHODS: An SANFH model was established in SD rats, which received an intermittent high GC dose for the first 4 weeks followed by an additional 4 weeks without GC. We explored the synergistic effects and mechanisms of C/EBPα and PPARγ on the differentiation of BMSCs by lentivirus-mediated gene knockdown and overexpression assays. A chromatin immunoprecipitation assay was performed to identify epigenetic modification sites on PPARγ in vivo and in vitro. RESULTS: In the SANFH model, intramedullary fat was significantly increased, and the transcription factors C/EBPα and PPARγ were upregulated simultaneously in the femoral head. In vitro, C/EBPα promoted adipogenic differentiation of BMSCs by targeting the PPARγ signalling pathway, while overexpression of C/EBPα significantly impaired osteogenic differentiation. Further studies demonstrated that histone H3K27 acetylation of PPARγ played an important role in the epigenetic mechanism underlying SANFH. C/EBPα upregulates the histone H3K27 acetylation level in the PPARγ promoter region by inhibiting HDAC1. Additionally, inhibiting the histone acetylation level of PPARγ effectively prevented adipogenic differentiation, thus slowing the progression of SANFH. CONCLUSIONS: Our results demonstrate the molecular mechanism by which C/EBPα regulates PPARγ expression by acetylating histones and revealed the epigenetic phenomenon in SANFH for the first time.

Sinomenine alleviates glomerular endothelial permeability by activating the C/EBP-α/claudin-5 signaling pathway.

Diabetic nephropathy (DN) is one of the main complications of diabetes. It is closely associated with the dysfunction of glomerular endothelial cells (GECs) under hyperglycemia. Severe inflammation is an important inducer for the development of GECs dysfunction, and it contributes to the disruption of tight junctions in GECs and the increased endothelial permeability. Sinomenine, an alkaloid monomer extracted from the rhizome of Sinomenium acutum, is recognized for its multiple pharmacological functions, including an anti-DN property. The present study aimed to explore the potential functional mechanism of Sinomenine against DN. Animals were randomly divided into Sham, DN, DN + Sinomenine (20 mg/kg), and DN + Sinomenine (40 mg/kg) groups. The Sinomenine or vehicle was administered every day for 6 weeks, followed by collecting renal tissues for further detection. Increased body weights, elevated blood glucose levels and UAE values, aggravated renal tissue pathology, higher concentrations of IL-18 and IL-1ß in renal tissues, and reduced claudin-5 expression were observed in DN rats. However, the administration of Sinomenine significantly alleviated all these DN-related changes. Furthermore, human renal glomerular endothelial cells (HrGECs) were treated with high glucose (HG, 30 mM) with or without Sinomenine (50, 100 µM) for 24 h. We found that Sinomenine treatment ameliorated the elevated production of IL-18 and IL-1ß, increased fluorescence intensity of FITC-dextran, declined trans-endothelial electrical resistance (TEER) value, and reduction of claudin-5 and C/EBP-α in HG-treated HrGECs. Moreover, the regulatory effect of Sinomenine on endothelial monolayer permeability in HG-treated HrGECs was abolished by the knockdown of C/EBP-α, indicating C/EBP-α is required for the effect of Sinomenine. We concluded that Sinomenine alleviated diabetic nephropathy-induced renal glomerular endothelial dysfunction via activating the C/EBP-α/claudin-5 axis.

Anti-obesity Effects of Dark Tea Extracts by Down-regulation of C/EBPα and PPARγ.

BACKGROUND/AIM: Dark tea, made by fermentation of tea leaves using microorganisms, is well known for its antiobesity effect; however, studies to identify this effect have not been sufficiently conducted. Herein, the anti-obesity effects of post-fermented dark tea were studied in high-fat diet mouse. MATERIALS AND METHODS: Obesity was induced through a high-fat diet in C57BL/6 mice, and then dark tea extract powder (DTP) was orally administered daily for 12 weeks to evaluate the body and organ weights. Changes in the biochemical markers of obesity were evaluated to study the mechanism of the anti-obesity effects of DTP. RESULTS: When DTP was administered to obesity mice, the weight and food intake reduced, blood aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C) decreased, whereas high-density lipoprotein cholesterol (HDL-C) increased. Histopathology showed that steatosis and inflammation scores were reduced within the liver and adipocyte sizes were reduced within epididymal adipocyte. In addition, a significant decrease in blood insulin and hepatic TG and a significant increase in blood adiponectin were also confirmed. The results of western blot and qPCR in week 12, showed a significant decrease in the mRNA and protein levels of C/EBPα, and the mRNA levels of PPARγ in the liver. CONCLUSION: Dark tea extracts are thought to have an anti-obesity effect by reducing the levels of the main transcription factors that promote adipocyte differentiation, such as C/EBPα, and PPARγ. Therefore, diet products using dark tea extracts could be developed.

Preventive effect of salicin ether against type-2 diabetes mellitus through targeting PPARγ-regulated gene expression.

Diabetes mellitus is the syndrome associated with metabolism having complicated pathogenesis and its morbidity rate is rapidly increasing every year. The present study investigated the preventive effect of salicin ether against type-2 diabetes and explored the underlying mechanism. Salicin ether reduced PPARγ-LBD level and transcriptional property of RXRα-PPARγ in 293T cells. The rosiglitazone significantly (p<0.01) increased grease droplet accumulation in adipocytes in comparison to control adipocytes. Increased grease droplet accumulation by rosiglitazone in adipocytes was reversed on treatment with salicin ether in dose-dependent manner. Salicin ether treatment of the adipocytes effectively suppressed rosiglitazone induced expression of FAS, C/EBPα, aP2, and HMG-CoA genes. Treatment of the adipocytes with salicin ether led to a prominent decrease in rosiglitazone mediated increase in aP2, CHIP, and C/EBPα protein expression. The inhibitory effect of rosiglitazone on expression of p-Akt/t-Akt, PPARa, p-FoxO1/t-FoxO1, and p-AMPK/t-AMPK was significantly (p<0.01) alleviated in the adipocytes by salicin ether. In summary, the present study demonstrated that salicin ether suppressed PPARγ activity and adipocyte differentiation. Moreover, the activation of FoxO1/Akt/AMPK was up-regulated and FAS/EBPα/aP2/HMG-CoA level inhibited by salicin ether in the adipocytes. Thus, salicin ether may be studied further for possible role in the treatment of diabetes.

LncRNA CEBPA-AS1 knockdown prevents neuronal apoptosis against oxygen glucose deprivation/reoxygenation by regulating the miR-455/GPER1 axis.

Ischemic stroke (IS) is a common nervous system disease, which is a major cause of disability and death in the world. In present study, we demonstrated a regulatory mechanism of CCAAT/enhancer binding protein-alpha antisense 1 (CEBPA-AS1) in oxygen glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cells, with a focus on neuronal apoptosis. CEBPA-AS1, miR-455, and GPER1 expressions were evaluated by using qRT-PCR and Western blotting. The binding relationship among CEBPA-AS1, miR-455, and GPER1 was determined by a dual luciferase reporter assay. Neuronal viability and apoptosis were examined using MTT and flow cytometry assays, followed by determination of apoptosis-related factors (caspase 3, caspase 8, caspase 9, Bax, and Bcl-2). CEBPA-AS1 and GPER1 levels were upregulated, and miR-455 level was downregulated in the cell model of OGD/R induced. CEBPA-AS1 knockdown increased SH-SY5Y viability and reduced OGD/R-induced apoptosis. CEBPA-AS1 could act as a sponge of miR-455, and CEBPA-AS1 knockdown was found to elevate miR-455 expression. miR-455 overexpression also promoted SH-SY5Y cell viability and rescued them from OGD/R-induced apoptosis by binding to GPER1. GPER1 overexpression or miR-455 inhibition reversed the anti-apoptotic effect of CEBPA-AS1 knockdown. These findings suggest a regulatory network of CEBPA-AS1/miR-455/GPER1 that mediates neuronal cell apoptosis in the OGD model, providing a better understanding of pathogenic mechanisms after IS.

Mesonosides A-H, primeverose derivatives from Mesona procumbens suppress adipogenesis by downregulating PPARγ and C/EBPα in 3T3-L1 cells.

Obesity is becoming a worldwide epidemic, especially in industrialized countries. We hereby report a methanolic extract of Mesona procumbens (known as Hsian-tsao in Taiwan) significantly inhibits lipid accumulation in 3T3-L1 adipocytes, and eight new primeverose derivatives, mesonosides A-H (1-8), were isolated from the methanolic extract of M. procumbens. Structural elucidation of 1-8 was established by spectroscopic methods, especially 2D NMR techniques (1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Anti-obesity evaluation revealed that isolates 1-5, 7, and 8 showed inhibitory effects on lipid accumulation and protein levels of adipogenic transcription factor, PPARγ and C/EBPα in 3T3-L1 cells. Our study suggests that M. procumbens extract including new primeverose isolates may be potentially used as a natural source to ameliorate fat accumulation and even obesity.

The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML.

BACKGROUND: CCAAT/enhancer-binding protein-alpha (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. METHODS: In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. RESULTS: In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. CONCLUSION: This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.

Transcriptional repression of c-Myb and GATA-2 is involved in the biologic effects of C/EBPalpha in p210BCR/ABL-expressing cells.

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation, inhibits proliferation, and suppresses leukemogenesis. To assess the underlying mechanisms, C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation, expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL, K562, and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha, whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells, alone or upon C/EBPalpha activation. In summary, the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend, in part, on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells, the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets.

Reintroduction of C/EBPalpha in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis.

The CCAAT/enhancer binding protein (C/EBP) alpha transcription factor is indispensable for myeloid differentiation. In various myeloid leukemias, C/EBPalpha is mutated or functionally impaired due to decreased C/EBPalpha expression or phosphorylation. In order to investigate the functional consequences of decreased C/EBPalpha function in AML, we reintroduced C/EBPalpha in primary CD34(+) sorted acute myeloid leukemia (AML) cells using a lentiviral approach. Self-renewal and differentiation of primary AML stem cells were studied on long-term MS5 cocultures. Activation of C/EBPalpha immediately led to a growth arrest in all AML cultures (N = 7), resulting in severely reduced expansion compared with control cultures. This growth arrest corresponded with enhanced myeloid differentiation as assessed by fluorescence-activated cell sorter (FACS) analysis for CD14, CD15, and CD11b. Myeloid differentiation was further confirmed by the up-regulation of neutrophil elastase and granulocyte colony-stimulating factor (G-CSF) receptor in C/EBPalpha transduced cells. C/EBPalpha-expressing AML CD34(+) cells failed to generate second and third leukemic cobblestone areas (L-CAs) in serial replating experiments, while control cultures could be sequentially passaged for more than 4 times, indicating that reintroduction of C/EBPalpha impaired the self-renewal capacity of the leukemic CD34(+) compartment. Together, our data indicate that low C/EBPalpha levels are necessary to maintain self-renewal and the immature character of AML stem cells.