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Saxidomus purpurata extract (SPE) is a highly consumable seafood worldwide with known health-related benefits. However, there are no reports of its' anti-obesity effect. This study explores the potential of SPE for anti-obesity effects by modulating adipogenesis and lipolysis. SPE reduced intracellular lipid and triglyceride accumulation while increasing free glycerol release in adipocytes. SPE inhibited lipogenesis protein expressions and increased the phosphorylation of hormone-sensitive lipase and Adenosine monophosphate-activated protein kinase (AMPK) to promote lipolysis. In addition, SPE suppressed adipogenesis by downregulating protein expression of key adipogenic markers, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) via Wnt/β-catenin signaling. SPE augmented the heme oxygenase-1 (HO-1) expression. Thus, pharmacological intervention with Zinc protoporphyrin (ZnPP-HO-1 antagonist) was employed to validate the HO-1 role. The presence of ZnPP increased the lipid accumulation and reduced the free glycerol release. At the molecular level, adipogenic transcription factors (PPARγ, C/EBPα, and SREBP1) expressions were restored in the presence of ZnPP. GC-MS analysis revealed that SPE was comprised of several fatty acids, contributing to its anti-obesity activity. SPE is an effective nutraceutical that can be used to reduce the progression of obesity. HO-1 expression during adipogenesis might be the mechanism of action for the anti-obesity effect of SPE.

Mutations in CCAAT enhancer binding protein α (CEBPA) occur in approximately 10% of patients with de novo acute myeloid leukemia (AML). Emerging evidence supports that in-frame mutations in the basic leucine zipper domain of CEBPA (CEBPA bZIP-inf ) confer a survival benefit, and CEBPA bZIP-inf replaced CEBPA double mutations (CEBPA dm ) as a unique entity in the 2022 World Health Organization (WHO-2022) classification and International Consensus Classification (ICC). However, challenges remain in daily clinical practice since more than 30% patients with CEBPA bZIP-inf die of AML despite intensive treatment. This review aims to provide a comprehensive summary of the heterogeneities observed in AML with CEBPA dm and CEBPA bZIP-inf , and will discuss the prognostic implications of concurrent mutations and novel mechanistic targets that may inform future drug development. The ultimate goal is to optimize clinical management and to provide precision medicine for this category of patients., (© 2024. Japanese Society of Hematology.)

Sparassis latifolia (SL) has been reported to exhibit anti-obesity effects in high-fat diet animal models, yet research into its mechanisms of action remains limited. Therefore, this study aimed to elucidate the mechanisms behind the anti-obesity activity of SL's 30% ethanol extract (SL30E) using 3T3-L1 cells in an in vitro setting. SL30E effectively mitigated the accumulation of lipid droplets and triacylglycerol. SL30E downregulated PPARγ and CEBPα protein levels. The diminishment of PPARγ and C/EBPα, facilitated by SL30E, was impeded by the knockdown of β-catenin using β-catenin-specific siRNA. Furthermore, SL30E was observed to increase the protein levels of ATGL and p-HSL, while it concurrently decreased the protein levels of perilipin-1. SL30E downregulated p62/SQSTM1 protein level and upregulated LC3-II protein level. Moreover, SL30E was demonstrated to elevate the protein levels of p-AMPK and PGC-1α. The results indicate that SL30E inhibits lipid accumulation by suppressing adipogenesis and inducing lipolysis, lipophagy, and thermogenesis in 3T3-L1 cells. These observations provide potential insights into the mechanisms underlying the anti-obesity effects of SL, contributing valuable information to the existing body of knowledge.

Loss of terminal differentiation is a hallmark of cancer and offers a potential mechanism for differentiation therapy. Polycomb repressive complex 2 (PRC2) serves as the methyltransferase for K27 of histone H3 that is crucial in development. While PRC2 inhibitors show promise in treating various cancers, the underlying mechanisms remain incompletely understood. Here, we demonstrated that the inhibition or depletion of PRC2 enhanced adipocyte differentiation in malignant rhabdoid tumors and mesenchymal stem cells, through upregulation of peroxisome proliferator-activated receptor gamma (PPARG) and CEBPA. Mechanistically, PRC2 directly represses their transcription through H3K27 methylation, as both genes exhibit a bivalent state in mesenchymal stem cells. KO of PPARG compromised C/EBPα expression and impeded the PRC2 inhibitor-induced differentiation into adipocytes. Furthermore, the combination of the PPARγ agonist rosiglitazone and the PRC2 inhibitor MAK683 exhibited a higher inhibition on Ki67 positivity in tumor xenograft compared to MAK683 alone. High CEBPA, PLIN1, and FABP4 levels positively correlated with favorable prognosis in sarcoma patients in The Cancer Genome Atlas cohort. Together, these findings unveil an epigenetic regulatory mechanism for PPARG and highlight the essential role of PPARγ and C/EBPα in the adipocyte differentiation of malignant rhabdoid tumors and sarcomas with a potential clinical implication., Competing Interests: Conflict of interest W. Q., L. C., and Y. A. are inventors on a pending patent application describing compositions and methods for treating or characterizing cancer. The remaining authors declare no potential conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The regulation of iron (Fe) levels is essential to maintain an adequate supply for erythropoiesis, among other processes, and to avoid possible toxicity. The liver-produced peptide hepcidin is regarded as the main regulator of Fe absorption in enterocytes and release from hepatocytes and macrophages, as it impairs Fe export through ferroportin. The glycoprotein erythropoietin (Epo) drives erythroid progenitor survival and differentiation in the bone marrow, and has been linked to the mobilization of Fe reserves necessary for hemoglobin production. Herein we show that Epo inhibits hepcidin expression directly in the HepG2 hepatic cell line, thus leading to a decrease in intracellular Fe levels. Such inhibition was dependent on the Epo receptor-associated kinase JAK2, as well as on the PI3K/AKT/mTOR pathway, which regulates nutrient homeostasis. Epo was also found to decrease binding of the C/EBP-α transcription factor to the hepcidin promoter, which could be attributed to an increased expression of its inhibitor CHOP. Epo did not only hinder the stimulating effect of C/EBP-α on hepcidin transcription, but also favored hepcidin inhibition by HIF-1α, by increasing is nuclear translocation as well as its protein levels. Moreover, in assays with the inhibitor genistein, this transcription factor was found necessary for Epo-induced hepcidin suppression. Our findings support the involvement of the PI3K/AKT/mTOR pathway in the regulation of Fe levels by Epo, and highlight the contrasting roles of the C/EBP-α and HIF-1α transcription factors as downstream effectors of the cytokine in this process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The antiallergic effects of gut microbiota have been attracting attention in recent years, but the underlying cellular and molecular mechanisms have not yet been fully understood. In this study, we aimed to investigate these mechanisms specifically focusing on mast cells. Mast cells retain intracellular granules containing various inflammatory mediators such as histamine, which are released outside the cells upon IgE and allergen stimulation. We previously reported that increased expression of the transcription factor, CCAAT/enhancer-binding protein α (C/EBPα), suppresses granule formation in mast cells and that Lacticaseibacillus casei JCM1134 T (LC) upregulates C/EBPα levels. Here, granule formation in mouse bone marrow-derived mast cells was suppressed in a MyD88-dependent manner after LC treatment due to C/EBPα-dependent downregulation of the genes encoding serglycin (SRGN) and mast cell protease 4 (Mcpt4). Furthermore, C/EBPα expression was regulated by DNA methylation in the 5' region far upstream of the transcription start site. LC suppressed DNA methylation of specific CpG motifs in the 5' region of the C/EBPα gene. These results conclude that specific gut microbial components, such as those from LC, suppress granule formation in mast cells by inhibiting SRGN and Mcpt4 expression via reduced C/EBPα gene methylation., (© 2024 Wiley‐VCH GmbH.)

CCAAT/enhancer-binding protein α (C/EBPα), a key myeloid transcription factor, drives myeloid differentiation from blast cells by regulating the expression of granulocyte colony stimulating factor receptor and C/EBPε as required for promoting granulocyte differentiation. Here, we show that serine/threonine-protein kinase NLK, also known as Nemo-like kinase, physically associates with C/EBPα and phosphorylates it at multiple sites, including Ser21, Thr226, Thr230 and S234, leading to its ubiquitin-mediated degradation. Individual phospho-point mutants of C/EBPα could be phosphorylated by NLK, but a mutant with all phosphorylatable residues replaced by alanine resisted phosphorylation and degradation by NLK, as did the single point mutants. Furthermore, although ectopic expression of NLK enhanced phosphorylation of C/EBPα levels, it markedly inhibited total C/EBPα protein levels. Conversely, NLK depletion inhibited endogenous C/EBPα phosphorylation but enhanced its total protein levels in several acute myeloid leukemia (AML) cell lines and in peripheral blood mononuclear cells isolated from number of AML patient samples. Importantly, NLK depletion in peripheral blood mononuclear cells from primary AML patients not only restored C/EBPα protein levels, but also induced myeloid differentiation, suggesting that NLK could be therapeutically targeted to restore C/EBPα to resolve differentiation arrest in AML., (© 2024 Federation of European Biochemical Societies.)

Nucleostemin (NS) plays a role in liver regeneration, and aging reduces its expression in the baseline and regenerating livers following 70% partial hepatectomy (PHx). Here we interrogate the mechanism controlling NS expression during liver regeneration and aging. The NS promoter was analyzed by TRANSFAC. Functional studies were performed using cell-based luciferase assay, endogenous NS expression in Hep3B cells, mouse livers with a gain-of-function mutation of C/EBPα (S193D), and mouse livers with C/EBPα knockdown. We found a CAAT box with four C/EBPα binding sites (-1216 to -735) and a GC box with consensus binding sites for c-Myc, E2F1, and p300-associated protein complex (-633 to -1). Age-related changes in NS expression correlated positively with the expression of c-Myc, E2F1, and p300, and negatively with that of C/EBPα and C/EBPβ. PHx upregulated NS expression at 1d, coinciding with an increase in E2F1 and a decrease in C/EBPα. C/EBPα bound to the consensus sequences found in the NS promoter in vitro and in vivo, inhibited its transactivational activity in a binding site-dependent manner, and decreased the expression of endogenous NS in Hep3B cells. In vivo activation of C/EBPα by the S193D mutation resulted in a 4th-day post-PHx reduction of NS, a feature shared by 16-m/o livers. Finally, C/EBPα knockdown increased its expression in aged (24-m/o) livers under both baseline and regeneration conditions. This study reports the C/EBPα suppression of NS expression in aged livers, providing a new perspective on the mechanistic orchestration of tissue homeostasis in aging., Competing Interests: he authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Stem cells demonstrate differentiation and regulatory functions. In this discussion, we will explore the impacts of cell culture density on stem cell proliferation, adipogenesis, and regulatory abilities. This study aimed to investigate the impact of the initial culture density of human periodontal ligament stem cells (hPDLSCs) on the adipogenic differentiation of autologous cells. Our findings indicate that the proliferation rate of hPDLSCs increased with increasing initial cell density (0.5-8 × 10 4 cells/cm 2 ). After adipogenic differentiation induced by different initial cell densities of hPDLSC, we found that the mean adipose concentration and the expression levels of lipoprotein lipase (LPL), CCAAT/enhancer binding protein α (CEBPα), and peroxisome proliferator-activated receptor γ (PPAR-γ) genes all increased with increasing cell density. To investigate the regulatory role of hPDLSCs in the adipogenic differentiation of other cells, we used secreted exocrine vesicles derived from hPDLSCs cultivated at different initial cell densities of 50 μg/mL to induce the adipogenic differentiation of human bone marrow stromal cells. We also found that the mean adipose concentration and expression of LPL, CEBPα, and PPARγ genes increased with increasing cell density, with an optimal culture density of 8 × 10 4 cells/cm 2 . This study provides a foundation for the application of adipogenic differentiation in stem cells., (© 2024 John Wiley & Sons Ltd.)

Citrus fruits offer a range of health benefits due to their rich nutritional profile, including vitamin C, flavonoids, carotenoids, and fiber. It is known that unripe citrus has higher levels of vitamin C, dietary fiber, polyphenols, and flavonoids compared to mature fruits. In this study, we assessed the nutritional components of unripe citrus peel and pressed juices, as well as their anti-obesity potential through the modulation of adipocyte differentiation and the expression of adipogenesis-related genes, specifically PPARγ and C/EBPα, in 3T3-L1 preadipocytes. Our analysis revealed that unripe citrus peel exhibited elevated levels of fiber and protein compared to pressed juice, with markedly low levels of free sugar, particularly sucrose. The content of hesperidin, a representative flavonoid in citrus fruits, was 3,157.6 mg/kg in unripe citrus peel and 455.5 mg/kg in pressed juice, indicating that it was approximately seven times higher in unripe citrus peel compared to pressed juice. Moreover, we observed that the peel had a dose-dependently inhibitory effect on adipocyte differentiation, which was linked to a significant downregulation of adipogenesis-related gene expression. Thus, our findings suggest that unripe citrus possesses anti-obesity effects by impeding adipogenesis and adipocyte differentiation, with the peel demonstrating a more pronounced effect compared to pressed juice.

Obesity requires treatment to mitigate the potential development of further metabolic disorders, including diabetes, hyperlipidemia, tumor growth, and non-alcoholic fatty liver disease. We investigated the anti-obesity effect of a 30% ethanol extract of Eisenia bicyclis (Kjellman) Setchell (EEB) on 3T3-L1 preadipocytes and high-fat diet (HFD)-induced obese C57BL/6 mice. Adipogenesis transcription factors including peroxisome proliferator-activated receptor (PPAR)γ, CCAAT/enhancer-binding protein-alpha (C/EBPα), and sterol regulatory element-binding protein-1 (SREBP-1) were ameliorated through the AMP-activated protein kinase (AMPK) pathway by EEB treatment in differentiated 3T3-L1 cells. EEB attenuated mitotic clonal expansion by upregulating cyclin-dependent kinase inhibitors (CDKIs) while downregulating cyclins and CDKs. In HFD-fed mice, EEB significantly decreased the total body weight, fat tissue weight, and fat in the tissue. The protein expression of PPARγ, C/EBPα, and SREBP-1 was increased in the subcutaneous fat and liver tissues, while EEB decreased the expression levels of these transcription factors. EEB also inhibited lipogenesis by downregulating acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) expression in the subcutaneous fat and liver tissues. Moreover, the phosphorylation of AMPK and ACC was downregulated in the HFD-induced mouse group, whereas the administration of EEB improved AMPK and ACC phosphorylation; thus, EEB treatment may be related to the AMPK pathway. Histological analysis showed that EEB reduced the adipocyte size and fat accumulation in subcutaneous fat and liver tissues, respectively. EEB promotes thermogenesis in brown adipose tissue and improves insulin and leptin levels and blood lipid profiles. Our results suggest that EEB could be used as a potential agent to prevent obesity.

Antiretroviral therapy-naive people living with HIV possess less fat than people without HIV. Previously, we found that HIV-1 transactivator of transcription (TAT) decreases fat in ob/ob mice. The TAT38 (a.a. 20-57) is important in the inhibition of adipogenesis and contains three functional domains: Cys-ZF domain (a.a. 20-35 TACTNCYCAKCCFQVC), core-domain (a.a. 36-46, FITKALGISYG), and protein transduction domain (PTD)(a.a. 47-57, RAKRRQRRR). Interestingly, the TAT38 region interacts with the Cyclin T1 of the P-TEFb complex, of which expression increases during adipogenesis. The X-ray crystallographic structure of the complex showed that the Cys-ZF and the core domain bind to the Cyclin T1 via hydrophobic interactions. To prepare TAT38 mimics with structural and functional similarities to TAT38, we replaced the core domain with a hydrophobic aliphatic amino acid (from carbon numbers 5 to 8). The TAT38 mimics with 6-hexanoic amino acid (TAT38 Ahx (C6)) and 7-heptanoic amino acid (TAT38 Ahp (C7)) inhibited adipogenesis of 3T3-L1 potently, reduced cellular triglyceride content, and decreased body weight of diet-induced obese (DIO) mice by 10.4-11 % in two weeks. The TAT38 and the TAT38 mimics potently repressed the adipogenic transcription factors genes, C/EBPα, PPARγ, and SREBP1. Also, they inhibit the phosphorylation of PPARγ. The TAT peptides may be promising candidates for development into a drug against obesity or diabetes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

E3 ubiquitin ligase, ring finger protein 138 (RNF138) is involved in several biological processes; however, its role in myeloid differentiation or tumorigenesis remains unclear. RNAseq data from TNMplot showed that RNF138 mRNA levels are highly elevated in acute myeloid leukemia (AML) bone marrow samples as compared with bone marrow of normal volunteers. Here, we show that RNF138 serves as an E3 ligase for the tumor suppressor CCAAT/enhancer binding protein (C/EBPα) and promotes its degradation leading to myeloid differentiation arrest in AML. Wild-type RNF138 physically interacts with C/EBPα and promotes its ubiquitin-dependent proteasome degradation while a mutant RNF-138 deficient in ligase activity though interacts with C/EBPα, fails to down-regulate it. We show that RNF138 depletion enhances endogenous C/EBPα levels in peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers. Our data further shows that RNF138-mediated degradation of C/EBPα negatively affects its transactivation potential on its target genes. Furthermore, RNF138 overexpression inhibits all-trans-retinoic acid-induced differentiation of HL-60 cells whereas RNF138 RNAi enhances. In line with RNF138 inhibiting C/EBPα protein turnover, we also observed that RNF138 overexpression inhibited β-estradiol (E2)-induced C/EBPα driven granulocytic differentiation in C/EBPα inducible K562-p42C/EBPα-estrogen receptor cells. Furthermore, we also recapitulated these findings in PBMCs isolated from AML patients where depletion of RNF138 increased the expression of myeloid differentiation marker CD11b. These results suggest that RNF138 inhibits myeloid differentiation by targeting C/EBPα for proteasomal degradation and may provide a plausible mechanism for loss of C/EBPα expression often observed in myeloid leukemia. Also, targeting RNF138 may resolve differentiation arrest by restoring C/EBPα expression in AML., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Translation initiation at alternative start sites can dynamically control the synthesis of two or more functionally distinct protein isoforms from a single mRNA. Alternate isoforms of the developmental transcription factor CCAAT/enhancer-binding protein α (C/EBPα) produced from different start sites exert opposing effects during myeloid cell development. This choice between alternative start sites depends on sequence features of the CEBPA transcript, including a regulatory uORF, but the molecular basis is not fully understood. Here, we identify the factors that affect C/EBPα isoform choice using a sensitive and quantitative two-color fluorescent reporter coupled with CRISPRi screening. Our screen uncovered a role of the ribosome rescue factor PELOTA (PELO) in promoting the expression of the longer C/EBPα isoform by directly removing inhibitory unrecycled ribosomes and through indirect effects mediated by the mechanistic target of rapamycin kinase. Our work uncovers further links between ribosome recycling and translation reinitiation that regulate a key transcription factor, with implications for normal hematopoiesis and leukemogenesis., (© 2024 Fernandez et al.)

" Ganghwal " is a widely used herbal medicine in Republic of Korea, but it has not been reported as a treatment strategy for obesity and diabetes within adipocytes. In this study, we determined that Ostericum koreanum extract (OKE) exerts an anti-obesity effect by inhibiting adipogenesis and an anti-diabetic effect by increasing the expression of genes related to glucose uptake in adipocytes and inhibiting α-glucosidase activity. 3T3-L1 preadipocytes were differentiated for 8 days in methylisobutylxanthine, dexamethasone, and insulin medium, and the effect of OKE was confirmed by the addition of 50 and 100 µg/mL of OKE during the differentiation process. This resulted in a reduction in lipid accumulation and the expression of PPARγ (Peroxisome proliferator-activated receptor γ) and C/EBPα (CCAAT enhancer binding protein α). Significant activation of AMPK (AMP-activated protein kinase), increased expression of GLUT4 (Glucose Transporter Type 4), and inhibition of α-glucosidase activity were also observed. These findings provide the basis for the anti-obesity and anti-diabetic effects of OKE. In addition, OKE has a significant antioxidant effect. This study presents OKE as a potential natural product-derived material for the treatment of patients with metabolic diseases such as obesity- and obesity-induced diabetes., Competing Interests: The authors declare no conflicts of interest.

Carbon nanodots (CDs) are commonly found in food products and have attracted significant attention from food scientists. There is a high probability of CD exposure in humans, but its impacts on health are unclear. Therefore, health effects associated with CD consumption should be investigated. In this study, we attempted to create a model system of the Maillard reaction between cystine and glucose using a simple cooking approach. The CDs (CG-CDs) were isolated from cystine-glucose-based Maillard reaction products and characterized using fluorescence spectroscopy, X-ray diffractometer (XRD), and transmission electron microscope (TEM). Furthermore, human mesenchymal stem cells (hMCs) were used as a model to unravel the CDs' cytotoxic properties. The physiochemical assessment revealed that CG-CDs emit excitation-dependent fluorescence and possess a circular shape with sizes ranging from 2 to 13 nm. CG-CDs are predominantly composed of carbon, oxygen, and sulfur. The results of the cytotoxicity evaluation indicate good biocompatibility, where no severe toxicity was observed in hMCs up to 400 μg/mL. The DPPH assay demonstrated that CDs exert potent antioxidant abilities. The qPCR analysis revealed that CDs promote the downregulation of the key regulatory genes, PPARγ, C/EBPα, SREBP-1, and HMGCR, coupled with the upregulation of anti-inflammatory genes. Our findings suggested that, along with their excellent biocompatibility, CG-CDs may offer positive health outcomes by modulating critical genes involved in lipogenesis, homeostasis, and obesity pathogenesis.

Isoeugenol (IEG), a natural component of clove oil, possesses antioxidant, anti-inflammatory, and antibacterial properties. However, the effects of IEG on adipogenesis have not yet been elucidated. Here, we showed that IEG blocks adipogenesis in 3T3-L1 cells at an early stage. IEG inhibits lipid accumulation in adipocytes in a concentration-dependent manner and reduces the expression of mature adipocyte-related factors including PPARγ, C/EBPα, and FABP4. IEG treatment at different stages of adipogenesis showed that IEG inhibited adipocyte differentiation by suppressing the early stage, as confirmed by lipid accumulation and adipocyte-related biomarkers. The early stage stimulates growth-arrested preadipocytes to enter mitotic clonal expansion (MCE) and initiates their differentiation into adipocytes by regulating cell cycle-related factors. IEG arrested 3T3-L1 preadipocytes in the G 0 /G 1 phase of the cell cycle and attenuated cell cycle-related factors including cyclinD1, CDK6, CDK2, and cyclinB1 during the MCE stage. Furthermore, IEG suppresses reactive oxygen species (ROS) production during MCE and inhibits ROS-related antioxidant enzymes, including superoxide dismutase1 (SOD1) and catalase. The expression of cell proliferation-related biomarkers, including pAKT and pERK1/2, was attenuated by the IEG treatment of 3T3-L1 preadipocytes. These findings suggest that it is a potential therapeutic agent for the treatment of obesity.

Objective: To systematically screen and identify long noncoding RNA (lncRNA) associated with bone marrow adiposity changes in aplastic anemia (AA)., Methods: The PPARγ and C/EBPα ChIP-Seq data in ChIPBase was analyzed by bioinformatics and the potential lncRNA co-transcriptionally regulated by PPARγ and C/EBPα was screened. The expression of candidate lncRNA was verified by qRT-PCR in the in vitro adipogenic differentiation model of BM-MSC, BM-MSC infected with lenti-shPPARγ and lenti-shC/EBPα as well as clinical BM-MSC samples derived from AA and controls., Results: PPARγ and C/EBPα were significantly highly expressed in AA BM-MSC, and knock-down of PPARγ and C/EBPα impaired the adipogenic capacity of AA BM-MSC. PPARγ and C/EBPα cotranscriptionally activate LINC01230 promoter activity in binding sites dependant manner. The LINC01230 was also aberrantly highly expressed in AA BM-MSC compared with controls., Conclusion: PPARγ and C/EBPα are aberrantly expressed in AA BM-MSC and may promote the adipogenic differentiation of AA BM-MSC, and to a certain extent mediate the bone marrow adiposity alteration by transcriptionally activating LINC01230 expression.

Pioneer transcription factors are vital for cell fate changes. PU.1 and C/EBPα work together to regulate hematopoietic stem cell differentiation. However, how they recognize in vivo nucleosomal DNA targets remains elusive. Here we report the structures of the nucleosome containing the mouse genomic CX3CR1 enhancer DNA and its complexes with PU.1 alone and with both PU.1 and the C/EBPα DNA binding domain. Our structures reveal that PU.1 binds the DNA motif at the exit linker, shifting 17 bp of DNA into the core region through interactions with H2A, unwrapping ~20 bp of nucleosomal DNA. C/EBPα binding, aided by PU.1's repositioning, unwraps ~25 bp of entry DNA. The PU.1 Q218H mutation, linked to acute myeloid leukemia, disrupts PU.1-H2A interactions. PU.1 and C/EBPα jointly displace linker histone H1 and open the H1-condensed nucleosome array. Our study unveils how two pioneer factors can work cooperatively to open closed chromatin by altering DNA positioning in the nucleosome., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

CCAAT/enhancer binding protein zeta (C/EBPZ) was differentially expressed in abdominal adipose tissues of fat and lean broilers and regulated adipogenesis in chicken. The objective of this study was to elucidate the transcriptional regulation of C/EBPZ gene in chicken adipose tissue. A 2,031-base pair (bp) chicken C/EBPZ sequence (2,025 nucleotides upstream to 6 nucleotides downstream from the initiator codon, -2,025/+6) was studied. The sequence exhibited a significant promoter activity (P < 0.05) and had some cis-acting elements, notably, a core promoter was identified in nucleotides -94 to +6. Additionally, DNA pull-down assay showed that proteins interacted with chicken C/EBPZ promoter (-173/+6) in preadipocytes were implicated in transcription, post-transcriptional regulation and translation. In addition, KLF2 facilitated the activities of chicken C/EBPZ promoter (-2,025/+6, -1,409/+6, -793/+6, -485/+6, -173/+6, and -94/+6) in preadipocytes (P < 0.05). The expression levels of KLF2 and C/EBPZ in chicken abdominal adipose tissue were substantially associated (r = 0.5978278, P < 0.0001), and KLF2 increased C/EBPZ expression in vitro (P < 0.05). Additionally, chromatin immunoprecipitation (ChIP)-PCR analysis revealed that KLF2 has the ability to interact with the chicken C/EBPZ promoter regions at least at the positions -1,245/-1,048 and -571/-397. Mutation analysis showed that the CGCAGCGCCCG motif located in the chicken C/EBPZ promoter at positions -45 to -35 is involved in regulating transcription and facilitates trans activation by KLF2. These results provided some information of transcription control of C/EBPZ in chicken adipose tissue., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The feed-forward loop between the transcription factors Ppar-gamma and C/ebp-alpha is critical for lineage commitment during adipocytic differentiation. Ppar-gamma interacts with epigenetic cofactors to activate C/ebp-alpha and the downstream adipocytic gene expression program. Therefore, knowledge of the epigenetic cofactors associated with Ppar-gamma, is central to understanding adipocyte differentiation in normal differentiation and disease. We found that Prmt6 is present with Ppar-gamma on the Ppar-gamma and C/ebp-alpha promoter. It contributes to the repression of C/ebp-alpha expression, in part through its ability to induce H3R2me2a. During adipocyte differentiation, Prmt6 expression is reduced and the methyltransferase leaves the promoters. As a result, the expression of Ppar-gamma and C/ebp-alpha is upregulated and the adipocytic gene expression program is established. Inhibition of Prmt6 by a small molecule enhances adipogenesis, opening up the possibility of epigenetic manipulation of differentiation. Our data provide detailed information on the molecular mechanism controlling the Ppar-gamma-C/ebp-alpha feed-forward loop. Thus, they advance our understanding of adipogenesis in normal and aberrant adipogenesis., (© 2024. The Author(s).)

Background: Acute myeloid leukemia (AML) with biallelic (CEBPA bi ) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs., Methods: Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents., Results: We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo., Conclusion: Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPA bi ) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers., (© 2024. The Author(s).)

Familial acute myeloid leukemia (AML) pedigrees with germline CCAAT/enhancer-binding protein-α (CEBPA) mutation have been rarely reported due to insufficient knowledge of their clinical features. Here, we report two Chinese families with multiple AML cases carrying germline CEBPA mutations, one of which had 11 cases spanning four consecutive generations. Additionally, we collected clinical data of 57 AML patients from 22 families with germline CEBPA mutations, with 58.3% of them harboring double CEBPA mutations. The first mutation frequently occurred at the N-terminal of CEBP/α (78.6%), resulting in an exclusive expression of p30 of CEBPA (CEBPA p30 ). The second mutation was mostly found at the C-terminal of CEBP/α (CEBPA others ). Germline CEBPA p30 carriers had higher incidences of AML (80.36% vs. 42.86%) and earlier onset of AML (18 vs. 38.5 years old) compared to germline CEBPA others carriers. Despite the high rates of relapse, most familial AML cases exhibited favorable overall survival (OS), with germline CEBPA p30 carriers having better survival outcomes (>25 years vs. 11 years for CEBPA others carriers). Among the 27 healthy germline CEBPA-mutated carriers, we detected a pre-leukemia clone harboring a pathogenic IDH2 variant (R140Q)in one individual. These findings should aid in the genetic counseling and management of AML patients and healthy carriers with germline CEBPA mutations., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cornelian cherry ( Cornus mas L.) fruits, abundant in iridoids and anthocyanins, are natural products with proven beneficial impacts on the functions of the cardiovascular system and the liver. This study aims to assess and compare whether and to what extent two different doses of resin-purified cornelian cherry extract (10 mg/kg b.w. or 50 mg/kg b.w.) applied in a cholesterol-rich diet rabbit model affect the levels of sterol regulatory element-binding protein 1c (SREBP-1c) and CCAAT/enhancer binding protein α (C/EBPα), and various liver X receptor-α (LXR-α), peroxisome proliferator-activated receptor-α (PPAR-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) target genes. Moreover, the aim is to evaluate the resistive index (RI) of common carotid arteries (CCAs) and aortas, and histopathological changes in CCAs. For this purpose, the levels of SREBP-1c, C/EBPα, ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), fatty acid synthase (FAS), endothelial lipase (LIPG), carnitine palmitoyltransferase 1A (CPT1A), and adiponectin receptor 2 (AdipoR2) in liver tissue were measured. Also, the levels of lipoprotein lipase (LPL), visceral adipose tissue-derived serine protease inhibitor (Vaspin), and retinol-binding protein 4 (RBP4) in visceral adipose tissue were measured. The RI of CCAs and aortas, and histopathological changes in CCAs, were indicated. The oral administration of the cornelian cherry extract decreased the SREBP-1c and C/EBPα in both doses. The dose of 10 mg/kg b.w. increased ABCA1 and decreased FAS, CPT1A, and RBP4, and the dose of 50 mg/kg b.w. enhanced ABCG1 and AdipoR2. Mitigations in atheromatous changes in rabbits' CCAs were also observed. The obtained outcomes were compared to the results of our previous works. The beneficial results confirm that cornelian cherry fruit extract may constitute a potentially effective product in the prevention and treatment of obesity-related disorders.

Intramuscular fat (IMF) is a crucial determinant of meat quality and is influenced by various regulatory factors. Despite the growing recognition of the important role of long noncoding RNAs (lncRNAs) in IMF deposition, the mechanisms underlying buffalo IMF deposition remain poorly understood. In this study, we identified and characterized a lncRNA, lncFABP4, which is transcribed from the antisense strand of fatty acid-binding protein 4 (FABP4). lncFABP4 inhibited cell proliferation in buffalo intramuscular preadipocytes. Moreover, lncFABP4 significantly increased intramuscular preadipocyte differentiation, as indicated by an increase in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARG), CCAAT enhancer binding protein alpha (C/EBPα), and FABP4. Mechanistically, lncFABP4 was found to have the potential to regulate downstream gene expression by participating in protein-protein interaction pathways. These findings contribute to further understanding of the intricate mechanisms through which lncRNAs modulate intramuscular adipogenesis in buffaloes., (© 2024 Japanese Society of Animal Science.)

Background & Aims: The obesity-associated nonalcoholic fatty liver disease represents a common cause of pediatric liver diseases, including the pediatric liver cancer hepatoblastoma. The mechanisms behind the development of fatty liver in children are not yet known. We examined the role of the C/EBPα-p300 pathway in the development of maternal obesity-associated fatty liver phenotype in offspring., Methods: Because the ability of C/EBPα to promote fatty liver phenotype is enhanced by CDK4-mediated phosphorylation of C/EBPα at Ser193 and subsequent formation of C/EBPα-p300 complexes, we used wild-type (WT) and C/EBPα-S193D and C/EBPα-S193A mutant mice to study the effects of maternal high-fat diet (HFD) on the liver health of offspring. The females of these mouse lines were fed an HFD before mating, and the pups were further subjected to either an HFD or a normal diet for 12 weeks., Results: WT female mice on the HFD before and during pregnancy and their subsequent offspring on the HFD had severe fatty liver, fibrosis, and an increased rate of liver proliferation. However, the HFD in C/EBPα-S193A mice did not cause development of these disorders. In HFD-HFD treated WT mice, C/EBPα is phosphorylated at Ser193 and forms complexes with p300, which activate expression of genes involved in development of fatty liver, fibrosis, and proliferation. However, S193A-C/EBPα mice do not have complexes of C/EBPα-S193A with p300, leading to a lack of activation of genes of fatty liver, fibrosis, and proliferation. The mutant C/EBPα-S193D mice have accelerated cdk4-dependent pathway and have developed steatosis at early stages., Conclusions: These studies identified the epigenetic cause of obese pregnancy-associated liver diseases and suggest a potential therapy based on inhibition of cdk4-ph-S193-C/EBPα-p300 pathway., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Progesterone synthesized in the placenta is essential for pregnancy maintenance. CYP11A1 is a key enzyme in progesterone synthesis, and its expression increases greatly during trophoblast syncytialization. However, the underlying mechanism remains elusive. Here, we demonstrated that passive demethylation of CYP11A1 promoter accounted for the upregulation of CYP11A1 expression during syncytialization with the participation of the transcription factor C/EBPα. We found that the methylation rate of a CpG locus in the CYP11A1 promoter was significantly reduced along with decreased DNA methyltransferase 1 (DNMT1) expression and its enrichment at the CYP11A1 promoter during syncytialization. DNMT1 overexpression not only increased the methylation of this CpG locus in the CYP11A1 promoter, but also decreased CYP11A1 expression and progesterone production. In silico analysis disclosed multiple C/EBPα binding sites in both CYP11A1 and DNMT1 promoters. C/EBPα expression and its enrichments at both the DNMT1 and CYP11A1 promoters were significantly increased during syncytialization. Knocking-down C/EBPα expression increased DNMT1 while it decreased CYP11A1 expression during syncytialization. Conclusively, C/EBPα plays a dual role in the regulation of CYP11A1 during syncytialization. C/EBPα not only drives CYP11A1 expression directly, but also indirectly through downregulation of DNMT1, which leads to decreased methylation in the CpG locus of the CYP11A1 promoter, resulting in increased progesterone production during syncytialization., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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.

CCAAT/enhancer binding protein α (C/EBPα) regulates myeloid differentiation, and its dysregulation contributes to acute myeloid leukaemia (AML) progress. Clarifying its functional implementation mechanism is of great significance for its further clinical application. Here, we show that C/EBPα regulates AML cell differentiation through liquid-liquid phase separation (LLPS), which can be disrupted by C/EBPα-p30. Considering that C/EBPα-p30 inhibits the functions of C/EBPα through the LZ region, a small peptide TAT-LZ that could instantaneously interfere with the homodimerization of C/EBPα-p42 was constructed, and dynamic inhibition of C/EBPα phase separation was observed, demonstrating the importance of C/EBPα-p42 homodimers for its LLPS. Mechanistically, homodimerization of C/EBPα-p42 mediated its phosphorylation at the novel phosphorylation site S16, which promoted LLPS and subsequent AML cell differentiation. Finally, decreasing the endogenous C/EBPα-p30/C/EBPα-p42 ratio rescued the phase separation of C/EBPα in AML cells, which provided a new insight for the treatment of the AML., (© 2023. The Author(s).)

The transcription factor CCAAT-enhancer binding factor alpha (C/ebpα) is a master controller of myeloid differentiation that is expressed as long (p42) and short (p30) isoform. Mutations within the CEBPA gene selectively deleting p42 are frequent in human acute myeloid leukemia. Here we investigated the individual genomics and transcriptomics of p42 and p30. Both proteins bound to identical sites across the genome. For most targets, they induced a highly similar transcriptional response with the exception of a few isoform specific genes. Amongst those we identified early growth response 1 (Egr1) and tribbles1 (Trib1) as key targets selectively induced by p42 that are also underrepresented in CEBPA-mutated AML. Egr1 executed a program of myeloid differentiation and growth arrest. Oppositely, Trib1 established a negative feedback loop through activation of Erk1/2 kinase thus placing differentiation under control of signaling. Unexpectedly, differentiation elicited either by removal of an oncogenic input or by G-CSF did not peruse C/ebpα as mediator but rather directly affected the cell cycle core by upregulation of p21/p27 inhibitors. This points to functions downstream of C/ebpα as intersection point where transforming and differentiation stimuli converge and this finding offers a new perspective for therapeutic intervention., (© 2023. The Author(s).)

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/β-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/β-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 β-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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Osteosarcoma (OS) is characterized by TP53 mutations in humans. In mice, loss of p53 triggers OS development, and osteoprogenitor-specific p53-deleted mice are widely used to study the process of osteosarcomagenesis. However, the molecular mechanisms underlying the initiation or progression of OS following or parallel to p53 inactivation remain largely unknown. Here, we examined the role of transcription factors involved in adipogenesis (adipo-TFs) in p53-deficient OS and identified a novel tumor suppressive molecular mechanism mediated by C/ebpα. C/ebpα specifically interacts with Runx3, a p53 deficiency-dependent oncogene, and, in the same manner as p53, decreases the activity of the oncogenic axis of OS, Runx3-Myc, by inhibiting Runx3 DNA binding. The identification of a novel molecular role for C/ebpα in p53-deficient osteosarcomagenesis underscores the importance of the Runx-Myc oncogenic axis as a therapeutic target for OS., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Although transcription factor CCAAT-enhancer binding protein α (C/EBPα) is critical for normal and leukemic differentiation, its role in cell and metabolic homeostasis is largely unknown in cancer. Here, multiomics analyses uncovered a coordinated activation of C/EBPα and Fms-like tyrosine kinase 3 (FLT3) that increased lipid anabolism in vivo and in patients with FLT3-mutant acute myeloid leukemia (AML). Mechanistically, C/EBPα regulated the fatty acid synthase (FASN)-stearoyl-CoA desaturase (SCD) axis to promote fatty acid (FA) biosynthesis and desaturation. We further demonstrated that FLT3 or C/EBPα inactivation decreased monounsaturated FA incorporation to membrane phospholipids through SCD downregulation. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress that was exploited by combining FLT3 and glutathione peroxidase 4 inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells. Altogether, our study reveals a C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML to ferroptosis with promising therapeutic application., Significance: FLT3 mutations are found in 30% of AML cases and are actionable by tyrosine kinase inhibitors. Here, we discovered that C/EBPα regulates FA biosynthesis and protection from lipid redox stress downstream mutant-FLT3 signaling, which confers a vulnerability to ferroptosis upon FLT3 inhibition with therapeutic potential in AML. This article is highlighted in the In This Issue feature, p. 1501., (©2023 American Association for Cancer Research.)

Adipogenesis, that is, the formation of terminally differentiated adipocytes is intricately regulated by transcription factors where CCAAT/enhancer binding protein alpha (C/EBPα) plays a key role. In the current study, we demonstrate that E3 ubiquitin ligase AIP4 negatively regulates C/EBPα protein stability leading to reduced adipogenesis. While AIP4 overexpression in 3T3-L1 cells preadipocytes inhibited lipid accumulation when treated with differentiation inducing media (MDI), AIP4 depletion was sufficient to partially promote lipid accumulation even in the absence of MDI. Mechanistically, overexpression of AIP4 inhibited protein levels of both ectopically expressed as well as endogenous C/EBPα while catalytically inactive AIP4 failed. On the contrary, AIP4 depletion profoundly enhanced endogenous C/EBPα protein levels. The observation that AIP4 levels decrease with concomitant increase in C/EBPα levels during adipocyte differentiation further indicated that AIP4 negatively regulates C/EBPα levels. We further show that AIP4 physically interacts with C/EBPα and ubiquitinates it leading to its proteasomal degradation. AIP4 promoted K48-linked ubiquitination of C/EBPα while catalytically inactive AIP4-C830A failed. Taken together, our data demonstrate that AIP4 inhibits adipogenesis by targeting C/EBPα for ubiquitin-mediated proteasome degradation., (© 2023 Wiley Periodicals LLC.)

Hydroxylated polymethoxyflavones (HPMFs) have been shown to possess various anti-disease effects, including against obesity. This study investigates the anti-obesity effects of HPMFs in further detail, aiming to gain understanding of their mechanism of action in this context. The current study demonstrates that two HPMFs; 3'-hydroxy-5,7,4',5'-tetramethoxyflavone (3'OH-TetMF) and 4'-hydroxy-5,7,3',5'-tetramethoxyflavone (4'OH-TetMF) possess anti-obesity effects. They both significantly reduced pancreatic lipase activity in a competitive manner as demonstrated by molecular docking and kinetic studies. In cell studies, it was revealed that both of the HPMFs suppress differentiation of 3T3-L1 mouse embryonic fibroblast cells during the early stages of adipogenesis. They also reduced expression of key adipogenic and lipogenic marker genes, namely peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), adipocyte binding protein 2 (aP2), fatty acid synthase (FASN), and sterol regulatory element-binding protein 1 (SREBF 1). They also enhanced the expression of cell cycle genes, i.e., cyclin D1 (CCND1) and C-Myc, and reduced cyclin A2 expression. When further investigated, it was also observed that these HPMFs accelerate lipid breakdown (lipolysis) and enhance lipolytic genes expression. Moreover, they also reduced the secretion of proteins (adipokines), including pro-inflammatory cytokines, from mature adipocytes. Taken together, this study concludes that these HPMFs have anti-obesity effects, which are worthy of further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: We evaluated the characteristics of CCAAT/enhancer-binding protein α (CEBPA) mutations and the significance of a basic leucine zipper in-frame mutation (bZIPin-f) of CEBPA in patients with acute myeloid leukemia with a normal karyotype., Materials and Methods: Based on updated knowledge of CEBPA mutations, we conducted next-generation sequencing analyses in a previously established real-world cohort., Results: Among 78 of a total of 395 patients (19.7%), 50 had bZIPin-f CEBPA, and 28 had non-bZIPin-f CEBPA. In the multivariate analysis, patients with NPM1mut, those with bZIPin-f CEBPA, and those who underwent allogeneic hematopoietic cell transplantation (allo-HCT) had favorable overall survival (OS), but FLT3-ITDmut was a poor prognostic indicator. For relapse-free survival (RFS) and cumulative incidence of relapse, bZIPin-f CEBPA, and allo-HCT were associated with favorable outcomes; FLT3-ITDpos was associated with worse outcomes. In the CEBPA double-mutated group (CEBPAdm), bZIPin-f CEBPA was associated with superior outcomes in terms of OS (p=0.007) and RFS (p=0.007) compared with non-bZIPin-f CEBPA. Of 50 patients with bZIPin-f CEBPA, 36 patients had at least one mutation. When grouped by the presence of mutations in chromatic/DNA modifiers (C), cohesion complex (C), and splicing genes (S) (CCS mutations), CCS-mutated bZIPin-f CEBPA was associated with poor OS (p=0.044; hazard ratio [HR], 2.419) and a trend in inferior RFS (p=0.186; HR, 1.838)., Conclusion: Only bZIPin-f CEBPA was associated with favorable outcomes in patients with CEBPAdm. However, some mutations accompanying bZIPin-f CEBPA showed inferior OS; thus, further studies with larger numbers of patients are required for clear conclusions of the significance of bZIPin-f CEBPA.

Here, we describe how the speed of C/EBPα-induced B cell to macrophage transdifferentiation (BMT) can be regulated, using both mouse and human models. The identification of a mutant of C/EBPα (C/EBPα R35A ) that greatly accelerates BMT helped to illuminate the mechanism. Thus, incoming C/EBPα binds to PU.1, an obligate partner expressed in B cells, leading to the release of PU.1 from B cell enhancers, chromatin closing and silencing of the B cell program. Released PU.1 redistributes to macrophage enhancers newly occupied by C/EBPα, causing chromatin opening and activation of macrophage genes. All these steps are accelerated by C/EBPα R35A , initiated by its increased affinity for PU.1. Wild-type C/EBPα is methylated by Carm1 at arginine 35 and the enzyme's perturbations modulate BMT velocity as predicted from the observations with the mutant. Increasing the proportion of unmethylated C/EBPα in granulocyte/macrophage progenitors by inhibiting Carm1 biases the cell's differentiation toward macrophages, suggesting that cell fate decision velocity and lineage directionality are closely linked processes., Competing Interests: GT, EK, TT, AK, JL, LD, VS, CB, MC, MC, RB, MF, SP, PM, KZ, AL, TG No competing interests declared, (© 2023, Torcal Garcia, Kowenz-Leutz, Tian et al.)

Activating brown adipose tissue (BAT) and stimulating white adipose tissue (WAT) browning is a prospective obesity treatment method. Dietary components derived from plants are the most effective approach to activate BAT and promote WAT browning in rodents. This study investigated the synergistic effects of Panax ginseng (PG) and Diospyros kaki leaf (DKL) extract on adipocyte differentiation and browning, as well as the molecular mechanism underlying their beneficial effects. The administration of PG and DKL to HFD-induced obese mice significantly decreased body weight and epididymal and abdominal adipose tissue mass. In in vitro, PG inhibited the adipogenesis of 3T3-L1 adipocytes by regulating the expression of key adipogenic regulators, such as peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)-α. In contrast, DKL negligibly influenced the adipogenesis of 3T3-L1 adipocytes but greatly increased the protein expression of UCP-1, PGC-1α, and PPARα in BAT and/or WAT. Moreover, PG and DKL inhibited adipogenesis synergistically and activated white adipocyte browning via AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) pathways. These results suggest that a combination of PG and DKL regulates adipogenesis in white adipocytes and browning in brown adipocytes by activating AMPK/SIRT1 axis. The potential use of PG and DKL may represent an important strategy in obesity management that will be safer and more effective.

Energy balance and nutrient availability are key determinants of cellular decisions to remain quiescent, proliferate, or differentiate into a mature cell. After assessing its environmental state, the cell must rewire its metabolism to support distinct cellular outcomes. Mechanistically, how metabolites regulate cell fate decisions is poorly understood. We used adipogenesis as our model system to ascertain the role of metabolism in differentiation. We isolated adipose tissue stromal vascular fraction cells and profiled metabolites before and after adipogenic differentiation to identify metabolic signatures associated with these distinct cellular states. We found that differentiation alters nucleotide accumulation. Furthermore, inhibition of nucleotide biosynthesis prevented lipid storage within adipocytes and downregulated the expression of lipogenic factors. In contrast to proliferating cells, in which mechanistic target of rapamycin complex 1 is activated by purine accumulation, mechanistic target of rapamycin complex 1 signaling was unaffected by purine levels in differentiating adipocytes. Rather, our data indicated that purines regulate transcriptional activators of adipogenesis, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, to promote differentiation. Although de novo nucleotide biosynthesis has mainly been studied in proliferation, our study points to its requirement in adipocyte differentiation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

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., (© 2022 International Federation for Cell Biology.)

In this study, the anti-obesity effects of 5,7,3',4',5-pentamethoxyflavone (PMF) and 6,2',4'-trimethoxyflavone (TMF) were evaluated through two distinct mechanisms of action: inhibition of crude porcine pancreatic lipase (PL), and inhibition of adipogenesis in 3T3-L1 pre-adipocytes. Both flavones show dose dependent, competitive inhibition of PL activity. Molecular docking studies revealed binding of the flavones to the active site of PL. In 3T3-L1 adipocytes, both flavones reduced the accumulation of lipids and triglycerides. PMF and TMF also lowered the expression of adipogenic and lipogenic genes. They both reduced the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ), CCAAT/enhancer-binding protein α and β (C/EBP α and β), sterol regulatory element-binding protein 1 (SREBF 1), fatty acid synthase (FASN), adipocyte binding protein 2 (aP2), and leptin gene. In addition, these flavones enhanced adiponectin mRNA expression, increased lipolysis and enhanced the expression of lipolytic genes: adipose triglycerides lipase (ATGL), hormone sensitive lipase (HSL) and monoglycerides lipase (MAGL) in mature 3T3-L1 adipocytes. Overall, PMF was seen to be a more potent inhibitor of both PL activity and adipogenesis versus TMF. These results suggest that PMF and TMF possess anti-obesity activities and can be further evaluated for their anti-obesity effects., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

CCAAT/enhancer binding protein α (C/EBPα) is the key transcription factor involved in lipid metabolism, however, the role of C/EBPα in milk fat synthesis of dairy goats remains unknown. The objective of the present research was to clarify the function of C/EBPα in goat mammary epithelial cells (GMECs) and its impact on peroxisome proliferator-activated receptor gamma (PPARG) promoter activity. In this study, C/EBPα overexpression increased its mRNA and protein levels by 42-fold and 6-fold, respectively. In contrast, transfecting siRNA targeting C/EBPα decreased its mRNA level to 20% and protein abundance to 80% of the basal level. The contents of lipid droplets, triacylglycerol (TAG), and cholesterol were increased (P < 0.05) in C/EBPα-overexpressing GMECs, and knockdown of C/EBPα led to the opposite results. Overexpression of C/EBPα significantly increased the expression levels of genes involved in TAG synthesis (AGPAT6, DGAT2, P < 0.01), lipid droplet formation (PLIN2, P < 0.01), and fatty acid synthesis (FADS2, P < 0.05; ELOVL6, P < 0.01). Knockdown of C/EBPα decreased (P < 0.05) the expression levels of AGPAT6, DGAT1, DGAT2, PLIN2, FADS2, and ELOVL6. C/EBPα upregulated the expression level of PPARG (P < 0.05), and four C/EBPα binding regions were identified in the PPARG promoter at -1,112 to -1,102 bp, -734 to -724 bp, -248 to -238 bp, and -119 to -109 bp. Knockdown of C/EBPα reduced (P < 0.05) the PPARG promoter activity when the C/EBPα binding regions were mutated at -1,112 to -1,102 bp, -734 to -724 bp, and -248 to -238 bp locations of the promoter. However, the promoter activity did not change when the mutation was located at -119 bp. In conclusion, our results suggest that C/EBPα can promote TAG synthesis in GMECs through its effects on mRNA abundance of genes related to lipid metabolism and regulation of the PPARG promoter activity via C/EBPα binding regions., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with CEBPA-double mutant (CEBPA N/C ) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells., (© 2022. The Author(s).)

Background: Hepatocellular carcinoma (HCC) is the world's third leading cause of cancer-related death; due to the fast growth and high prevalence of tumor recurrence, the prognosis of HCC patients remains dismal. Long non-coding RNA CEBPA-DT, a divergent transcript of the CCAAT Enhancer Binding Protein Alpha (CEBPA) gene, has been shown to participate in multiple tumor progression. However, no research has established its cancer-promoting mechanism in HCC yet., Methods: CEBPA-DT was identified in human HCC tissues through RNA sequencing. The expression level of CEBPA-DT was assessed by quantitative real-time PCR. The biological effects of CEBPA-DT were evaluated in vitro and in vivo through gain or loss of function experiments. RNA fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down assays were applied to investigate the downstream target of CEBPA-DT. Immunofluorescence, subcellular protein fractionation, western blot, and co-immunoprecipitation were performed to analyze the subcellular location of β-catenin and its interaction with Discoidin domain-containing receptor 2 (DDR2)., Results: CEBPA-DT was upregulated in human HCC tissues with postoperative distant metastasis and intimately related to the worse prognosis of HCC patients. Silencing of CEBPA-DT inhibited the growth, migration and invasion of hepatoma cells in vitro and in vivo, while enhancement of CEBPA-DT played a contrasting role. Mechanistic investigations demonstrated that CEBPA-DT could bind to heterogeneous nuclear ribonucleoprotein C (hnRNPC), which facilitated cytoplasmic translocation of hnRNPC, enhanced the interaction between hnRNPC and DDR2 mRNA, subsequently promoted the expression of DDR2. Meanwhile, CEBPA-DT induced epithelial-mesenchymal transition (EMT) process through upregulation of Snail1 via facilitating nuclear translocation of β-catenin. Using DDR2 inhibitor, we revealed that the CEBPA-DT induced the interaction between DDR2 and β-catenin, thus promoting the nuclear translocation of β-catenin to activate transcription of Snail1, contributing to EMT and HCC metastasis., Conclusions: Our results suggested that CEBPA-DT promoted HCC metastasis through DDR2/β-catenin mediated activation of Snail1 via interaction with hnRNPC, indicating that the CEBPA-DT-hnRNPC-DDR2/β-catenin axis may be used as a potential therapeutic target for HCC treatment., (© 2022. The Author(s).)

Objective To investigate the effect of Achyranthes bidentata polysaccharides (ABPS) on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its mechanism. Methods Five SD rats were sacrificed, and the BMSCs were dissected and isolated. The BMSCs were adherently cultured to logarithmic growth phase after identification, and treated with different doses of ABPS for 48 hours. The cell survival rates were detected by MTT assay. The highest dose of ABPS without toxicity to BMSCs was selected for subsequent experiments. Cells were randomly divided into control group, ABPS group, rosiglitazone group and ABPS combined with rosiglitazone group. Cell survival rates were detected by MTT assay. Triglyceride (TG) levels in BMSCs were detected by spectrophotometry. Lipid droplet formation in BMSCs was observed by oil red O staining. The mRNA and protein expression of peroxisome proliferater-activated receptor γ (PPARγ), transient receptor potential vanilloid 4 (TRPV4) and CCAAT/enhancer binding protein α (C/EBPα) were detected by real time quantitative PCR and Western blot analysis. Results The dose of ABPS≤200 mg/L had no obvious toxic effect on the growth of BMSCs after 48 hours, and the cell survival rate of 400 mg/L ABPS group was lower. Compared with the control group, the ABPS group showed decreased levels in TG, decreased relative expression of PPARγ, TRPV4 and C/EBPα mRNA and protein, and the decreased number of cytoplasmic lipid droplets. In the rosiglitazone group, observation reported the decreased cell survival rate, increased TG level, increased relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein, along with the increased number of cytoplasmic lipid droplets. Compared with the ABPS group, the cell survival rate was decreased, TG level was increased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein increased, and the number of cytoplasmic lipid droplets increased in the ABPS combined with rosiglitazone group. Compared with rosiglitazone group, the survival rate was increased, TG level was decreased, the relative expression levels of PPARγ, TRPV4 and C/EBPα mRNA and protein were decreased, and the number of cytoplasmic lipid droplets was decreased in the ABPS combined with rosiglitazone group. Conclusion ABPS can inhibit adipogenic differentiation of BMSCs, and the mechanism may be related to the regulation of PPARγ/TRPV4 pathway.

Mulberry leaf (Mori Folium) extract (MLE) is known to have anti-obesity effects. In this study, the enhanced effects of MLE after bioconversion treatment using Pectinex (BMLE) on obesity were explored, and the underlying mechanisms were investigated using the active components, neochlorogenic acid (5-CQA) and cryptochlorogenic acid (4-CQA), whose amounts were increased by bioconversion of MLE. Both MLE and BMLE inhibited lipid accumulation in 3T3-L1 adipocytes without cytotoxicity and suppressed the expression of CCAAT/enhancer-binding protein alpha (C/EBPα). In addition, MLE and BMLE decreased high-fat diet-induced adipose tissue mass expansion. Notably, BMLE significantly increased antiadipogenic and anti-obesity effects compared to MLE in vitro and in vivo. The active ingredients increased by bioconversion, 5-CQA and 4-CQA, inhibited the protein levels of C/EBPα and the mRNA levels of stearoyl-CoA desaturase 1 (Scd1). These findings provide new insights into the therapeutic possibility of using bioconversion of MLE, by which upregulation of 5-CQA and 4-CQA potently inhibits adipogenesis., (© 2022. The Author(s).)

As a key adipogenic marker, C/EBPα (CCAAT/enhancer binding protein α) is also an important factor in regulating targets containing CCAAT element for transcription, whose products include coding and non-coding RNAs (ncRNAs). However, knowledge of the mechanism of C/EBPα affecting pre-adipocyte proliferation and adipogenesis through regulating ncRNA is still limited. In this study, we firstly conducted an investigation concerning the impact of C/EBPα knockdown on porcine pre-adipocytes by using RNA sequencing (RNA-Seq) to identify the role of key ncRNAs, especially lncRNAs and their correlated mRNAs in regulating proliferation and differentiation of porcine pre-adipocytes. 97 differentially expressed (DE) mRNAs and 4 DE lncRNAs were identified in si-C/EBPα groups compared with the si-NC groups. Meanwhile, we found C/EBPα directly target the promoter of a novel lncRNA, namely MSTRG.12568.2, which was trans-correlated with STYX (serine/threonine/tyrosine interacting protein), an important candidate gene for regulating cell proliferation. Moreover, FOXO3 (forkhead box O3) was identified as a co-regulator with MSTR.12568.2 for STYX. Overexpression and knockdown of any of the MSTRG.12568.2, STYX, and FOXO3 increased and decreased the levels of pre-adipocyte proliferation and differentiation, respectively, which demonstrated that they played a positive role in adipogenesis of pre-adipocytes. Furthermore, our results revealed that FOXO3 was necessary for MSTRG.12568.2 to trans-activate STYX. We revealed that C/EBPα regulated pre-adipocyte proliferation and differentiation through mediating trans-activation of MSTRG.12568.2-FOXO3 to STYX. These results provide a novel regulation signal for C/EBPα to influence porcine pre-adipocyte proliferation and differentiation and greatly benefit to our understanding of molecular mechanism regulating subcutaneous adipogenesis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6., Competing Interests: Conflicts of interest The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)