Anticancer Activity of ST101, A Novel Antagonist of CCAAT/Enhancer Binding Protein ß.

CCAAT/enhancer binding protein ß (C/EBPß) is a basic leucine zipper (bZIP) family transcription factor, which is upregulated or overactivated in many cancers, resulting in a gene expression profile that drives oncogenesis. C/EBPß dimerization regulates binding to DNA at the canonical TTGCGCAA motif and subsequent transcriptional activity, suggesting that disruption of dimerization represents a powerful approach to inhibit this previously "undruggable" oncogenic target. Here we describe the mechanism of action and antitumor activity of ST101, a novel and selective peptide antagonist of C/EBPß that is currently in clinical evaluation in patients with advanced solid tumors. ST101 binds the leucine zipper domain of C/EBPß, preventing its dimerization and enhancing ubiquitin-proteasome dependent C/EBPß degradation. ST101 exposure attenuates transcription of C/EBPß target genes, including a significant decrease in expression of survival, transcription factors, and cell-cycle-related proteins. The result of ST101 exposure is potent, tumor-specific in vitro cytotoxic activity in cancer cell lines including glioblastoma, breast, melanoma, prostate, and lung cancer, whereas normal human immune and epithelial cells are not impacted. Further, in mouse xenograft models ST101 exposure results in potent tumor growth inhibition or regression, both as a single agent and in combination studies. These data provide the First Disclosure of ST101, and support continued clinical development of ST101 as a novel strategy for targeting C/EBPß-dependent cancers.

Stabilization of C/EBPß through direct interaction with STAT3 in H-Ras transformed human mammary epithelial cells.

Signal transducer and activator of transcription 3 (STAT3) plays important roles in cancer-associated inflammation by controlling expression of proinflammatory cytokines and chemokines. Recent studies suggest that C/EBPß (CCAAT-enhancer binding protein beta) and STAT3 synergistically stimulate cancer cell proliferation and epithelial-mesenchymal transition. C/EBPß is a leucine-zipper transcription factor that regulates expression of a variety of inflammatory cytokines or chemokines, such as IL-8, G-CSF (granulocyte colony stimulating factor), and GM-CSF (granulocyte macrophage colony stimulating factor) which induce neutrophil infiltration and differentiation. However, molecular mechanisms by which STAT3 and C/EBPß cooperatively interact had not been fully elucidated. In this study, we found that the level of C/EBPß protein, but not that of its mRNA transcript, was decreased in the absence of STAT3 in H-Ras transformed human mammary epithelial (H-Ras MCF10A) cells. In addition, silencing STAT3 dramatically induced ubiquitination of C/EBPß for proteasomal degradation. Furthermore, direct interaction between STAT3 and C/EBPß was confirmed by immunoprecipitation and proximity ligation assays. Taken together, these results suggest that STAT3 stabilizes C/EBPß, thereby promoting cancer-associated inflammation.

Sakuranetin downregulates inducible nitric oxide synthase expression by affecting interleukin-1 receptor and CCAAT/enhancer-binding protein ß.

Pruni Cortex is a herbal drug from the bark of the Japanese flowering cherries, Prunus jamasakura or Prunus verecunda, and is included in the traditional Japanese herbal (Kampo) formula Jumihaidokuto, which is administered orally to patients suffering from inflammatory skin diseases. The flavanones contained in Pruni Cortex (e.g., sakuranetin and naringenin) have potent anti-inflammatory, anti-allergic, and anti-microbial activities. Although the effects of Pruni Cortex on skin disease have been well studied, reports regarding its pharmacological effects on the liver are limited. In this study, we extracted the bark of Prunus jamasakura and purified it to isolate the pharmacologically active constituents by monitoring nitric oxide (NO) production in rat hepatocytes that were treated with the pro-inflammatory cytokine, interleukin (IL)-1ß. Sakuranetin and (-)-naringenin, which were present in an ethyl acetate-soluble fraction of the bark extract, significantly inhibited NO induction and inducible nitric oxide synthase (iNOS) expression. These two flavanones decreased the expression of type 1 IL-1 receptor gene and phosphorylation of Akt, also known as protein kinase B, which is regulated by phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Furthermore, sakuranetin decreased the phosphorylation of the activator isoforms of CCAAT/enhancer-binding protein ß (C/EBPß), which synergistically activates the transcription of the iNOS gene with nuclear factor κB (NF-κB). Therefore, sakuranetin inhibited the co-activating activity of C/EBPß with NF-κB, leading to the suppression of iNOS gene expression in hepatocytes. Taken together, sakuranetin in Pruni Cortex downregulated the iNOS gene by inhibiting PI3K/Akt signal transduction and the phosphorylation of C/EBPß. These results imply that sakuranetin may be primarily responsible for the anti-inflammatory effects of Pruni Cortex in the liver.

14-Deoxy-11,12-didehydroandrographolide suppresses adipogenesis of 3 T3-L1 preadipocytes by inhibiting CCAAT/enhancer-binding protein ß activation and AMPK-mediated mitotic clonal expansion.

Obesity is highly correlated with several metabolic disorders. Adipocyte differentiation is a key process in determining obesogenesis. 14-Deoxy-11,12-didehydroandrographolide (deAND) is a diterpenoid rich in Andrographis paniculata (Burm.f.) Nees., a herbal medicine commonly used to treat colds, infections, and liver diseases. We investigated whether deAND inhibits the adipogenesis of 3T3-L1 cells and the underlying mechanisms. We found that deAND (0-15 µM) dose-dependently inhibits the mRNA and protein expression of peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein 1c, fatty acid synthase, and stearoyl-CoA desaturase-1. Cellular lipid accumulation was decreased by deAND, and the early phase of adipocyte differentiation was critical for this inhibition. Immunoblotting revealed that deAND attenuated differentiation medium-induced protein kinase A (PKA) and cAMP response element-binding protein (CREB) activation, which leads to down-regulating C/EBPß transcription. Moreover, deAND inhibited ERK- and GSK3ß-mediated C/EBPß transcriptional activity. Flow cytometry analysis showed that deAND impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the G0/G1 phase, while the expression of cyclin D1, cyclin E, CDK6, and CDK2 was attenuated. deAND increased the phosphorylation of AMPK and raptor, an mTOR-interacting partner, which inhibited the mTOR-driven phosphorylation of P70S6K and eukaryotic translation initiation factor 4E binding protein. In the presence of compound C, deAND modulation of AMPK-mTOR signaling and inhibition of cell cycle regulator expression were reversed. Our results reveal that the anti-adipogenic effect of deAND is likely through inhibition of the PKA-CREB-C/EBPß and AMPK/mTOR pathways, which leads to down-regulating C/EBPß-driven lipogenic protein expression and halting MCE progression.

C/EBPß Acts Upstream of NF-κB P65 Subunit in Ox-LDL-Induced IL-1ß Production by Macrophages.

BACKGROUND/AIMS: Interleukin-1ß (IL-1ß) is one of the critical inflammatory factors during atherogenesis. CCAAT/enhancer binding proteins ß (C/EBPß), a regulator of IL-1ß production, recently been evidenced as a key player in the development of atherosclerosis. However, the mechanisms of how C/EBPß regulates the production of IL-1ß are unclear. In this study, we aimed to explore the role of C/EBPß in regulating IL-1ß production in macrophages after oxidized low-density lipoprotein (ox-LDL) exposure and the underlying mechanisms. METHODS: RAW264.7 macrophages were treated with 0, 25, 50 or 100 µg/ml ox-LDL for 12, 24 or 48 h. Small interfering RNAs were used to silence related proteins. The gene and protein expression levels were determined by quantitative real-time polymerase chain reaction or western blot (WB). IL-1ß secretion was assessed by enzyme-linked immunosorbent assay. The cytoplasmic and nuclear proteins were evaluated by nuclear fractionation followed by WB. Localization of p65 was observed by immunofluorescence. The binding activity of p65 to IL-1ß was tested by dual-luciferase reporter assay. RESULTS: Ox-LDL increased IL-1ß production, accompanied with increasing C/EBPß and p65 expression in a dose- and time-dependent manner. Moreover, C/EBPß deficiency in macrophages blocked ox-LDL-induced increases in IL-1ß expression, maturation as well as p65 activation. However, p65 deficiency inhibited the increase in IL-1ß production, but not C/EBPß expression. Dual-luciferase reporter results showed that overexpression of C/EBPß significantly enhanced binding activity of p65 to IL-1ß promoter. In addition, C/EBP 1ß deficiency in macrophages abolished the ox-LDL-induced gene transcription increases of IL-1ß, IL-6, p65 and caspase-1. CONCLUSIONS: Our results demonstrate that C/EBPß acts upstream of NF-κB p65 subunit in ox-LDL-induced IL-1ß production in macrophages and may regulate IL-1ß maturation by promoting caspase-1. C/EBPß may be a promising candidate for the prevention and treatment of atherosclerosis.

Anti-obesity effects of Laminaria japonica fermentation on 3T3-L1 adipocytes are mediated by the inhibition of C/EBP-α/ß and PPAR-γ.

Obesity is global problem that contributes to disease, and is partly caused by fast-food, high-fat diets. Much attention has been focused on developing anti-obesity foods and chemical materials from natural sources. Seaweed has bioactive properties that influence immune activity and have anti-cancer and anti-obesity effects. Laminaria japonica is a widely consumed seaweed, and has been promoted as a health food in Korea. The bioactive properties of L. japonica include anti-cancer, anti-diabetic, and anti-inflammation effects. Most Laminaria japonica are distributed in a simple processing form such as drying, and their availability is very low. Therefore, various types of functional products can be developed if they can be applied to foods through functionalization using fermentation techniques. It is a structural problem that is the most problematic in seaweed processing. In this study, we used fermented Laminaria japonica. To increase physiological activity, fermentation treatment was performed to loosen the structure, thereby increasing the activity of the glycoprotein. First, we screened the anti-obesity potential of an L. japonica fermentation extract (LJF) using 3T3-L1 adipocyte cells. We determined cytotoxicity using an MTS assay and measured LJF for its ability to affect adipogenesis through glucose uptake, triglyceride levels, and Oil Red O staining. We confirmed that LJF inhibited adipocyte differentiation. CCAAT/enhancer-binding proteins α/ß (C/EBP-α/ß) and peroxisome proliferator-activated receptor-γ (PPAR-γ) are involved in the early and late stages of adipocyte differentiation. LJF significantly reduced the expression levels of C/EBP-α/ß and PPAR-γ and decreased the concentration of adiponectin. Thus, our results suggest that LJF inhibits adipogenesis in 3T3-L1 cells, and may be valuable for its anti-obesity effects.

CCAAT/enhancer-binding protein ß overexpression alleviates myocardial remodelling by regulating angiotensin-converting enzyme-2 expression in diabetes.

Diabetic cardiomyopathy, a major cardiac complication, contributes to heart remodelling and heart failure. Our previous study discovered that CCAAT/enhancer-binding protein ß (C/EBPß), a transcription factor that belongs to a family of basic leucine zipper transcription factors, interacts with the angiotensin-converting enzyme 2 (ACE2) promoter sequence in other disease models. Here, we aimed to determine the role of C/EBPß in diabetes and whether ACE2 expression is regulated by C/EBPß. A type 1 diabetic mouse model was generated by an intraperitoneal injection of streptozotocin. Diabetic mice were injected with a lentivirus expressing either C/EBPß or sh-C/EBPß or treated with valsartan after 12 weeks to observe the effects of C/EBPß. In vitro, cardiac fibroblasts and cardiomyocytes were treated with high glucose (HG) to investigate the anti-fibrosis, anti-apoptosis and regulatory mechanisms of C/EBPß. C/EBPß expression was down-regulated in diabetic mice and HG-induced cardiac neonatal cells. C/EBPß overexpression significantly attenuated collagen deposition and cardiomyocyte apoptosis by up-regulating ACE2 expression. The molecular mechanism involved the binding of C/EBPß to the ACE2 promoter sequence. Although valsartan, a classic angiotensin receptor blocker, relieved diabetic complications, the up-regulation of ACE2 expression by C/EBPß overexpression may exert greater beneficial effects on patients with diabetic cardiomyopathy.

CCAAT/Enhancer binding protein ß silencing mitigates glial activation and neurodegeneration in a rat model of Parkinson's disease.

The CCAAT/Enhancer binding protein ß (C/EBPß) is a transcription factor involved in numerous physiological as well as pathological conditions in the brain. However, little is known regarding its possible role in neurodegenerative disorders. We have previously shown that C/EBPß regulates the expression of genes involved in inflammatory processes and brain injury. Here, we have analyzed the effects of C/EBPß interference in dopaminergic cell death and glial activation in the 6-hydroxydopamine model of Parkinson's disease. Our results showed that lentivirus-mediated C/EBPß deprivation conferred marked in vitro and in vivo neuroprotection of dopaminergic cells concomitant with a significant attenuation of the level of the inflammatory response and glial activation. Additionally, C/EBPß interference diminished the induction of α-synuclein in the substantia nigra pars compacta of animals injected with 6-hydroxydopamine. Taking together, these results reveal an essential function for C/EBPß in the pathways leading to inflammatory-mediated brain damage and suggest novel roles for C/EBPß in neurodegenerative diseases, specifically in Parkinson's disease, opening the door for new therapeutic interventions.

Suppression of adipocyte differentiation and lipid accumulation by stearidonic acid (SDA) in 3T3-L1 cells.

BACKGROUND: Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. METHODS: 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). RESULTS: 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPß), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. CONCLUSION: These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity.

Echinomycin inhibits adipogenesis in 3T3-L1 cells in a HIF-independent manner.

Obesity is a risk factor for many diseases including diabetes, cancer, cardiovascular disease, and chronic kidney disease. Obesity is characterized by the expansion of white adipose tissue (WAT). Hypertrophy and hyperplasia of adipocytes cause tissue hypoxia followed by inflammation and fibrosis. Its trigger, preadipocyte differentiation into mature adipocytes, is finely regulated by transcription factors, signal molecules, and cofactors. We found that echinomycin, a potent HIF-1 inhibitor, completely inhibited adipogenesis in 3T3-L1 WAT preadipocytes by affecting the early phase of mitotic clonal expansion. The dose required to exert the effect was surprisingly low and the time was short. Interestingly, its inhibitory effect was independent of HIF-1 pathways. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor, CCAAT/enhancer-protein ß, as a key target of echinomycin. Echinomycin also inhibited adipogenesis and body weight gain in high fat diet mice. These findings highlight a novel role of echinomycin in suppressing adipocyte differentiation and offer a new therapeutic strategy against obesity and diabetes.

Withaferin A, a natural compound with anti-tumor activity, is a potent inhibitor of transcription factor C/EBPß.

Recent work has shown that deregulation of the transcription factor Myb contributes to the development of leukemia and several other human cancers, making Myb and its cooperation partners attractive targets for drug development. By employing a myeloid Myb-reporter cell line we have identified Withaferin A (WFA), a natural compound that exhibits anti-tumor activities, as an inhibitor of Myb-dependent transcription. Analysis of the inhibitory mechanism of WFA showed that WFA is a significantly more potent inhibitor of C/EBPß, a transcription factor cooperating with Myb in myeloid cells, than of Myb itself. We show that WFA covalently modifies specific cysteine residues of C/EBPß, resulting in the disruption of the interaction of C/EBPß with the co-activator p300. Our work identifies C/EBPß as a novel direct target of WFA and highlights the role of p300 as a crucial co-activator of C/EBPß. The finding that WFA is a potent inhibitor of C/EBPß suggests that inhibition of C/EBPß might contribute to the biological activities of WFA.

Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1.

Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein ß (C/EBPß) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPß activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.

C/EBP-ß Is Differentially Affected by PPARα Agonists Fenofibric Acid and GW7647, But Does Not Change Apolipoprotein A-I Production During ER-Stress and Inflammation.

Increasing apolipoproteinA-I (apoA-I) production may be anti-atherogenic. Thus, there is a need to identify regulatory factors involved. Transcription of apoA-I involves peroxisome-proliferator-activated-receptor-alpha (PPARα) activation, but endoplasmic reticulum (ER) -stress and inflammation also influence apoA-I production. To unravel why PPARα agonist GW7647 increased apoA-I production compared to PPARα agonist fenofibric acid (FeAc) in human hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (CaCo-2) cells, gene expression profiles were compared. Microarray analyses suggested CCAAT/enhancer-binding-protein-beta (C/EBP-ß) involvement in the FeAc condition. Therefore, C/EBP-ß silencing and isoform-specific overexpression experiments were performed under ER-stressed, inflammatory and non-inflammatory conditions. mRNA expression of C/EBP-ß, ATF3, NF-IL3 and GDF15 were upregulated by FeAc compared to GW7647 in both cell lines, while DDIT3 and DDIT4 mRNA were only upregulated in HepG2 cells. This ER-stress related signature was associated with decreased apoA-I secretion. After ER-stress induction by thapsigargin or FeAc addition, intracellular apoA-I concentrations decreased, while ER-stress marker expression (CHOP, XBP1s, C/EBP-ß) increased. Cytokine addition increased intracellular C/EBP-ß levels and lowered apoA-I concentrations. Although a C/EBP binding place is present in the apoA-I promoter, C/EBP-ß silencing or isoform-specific overexpression did not affect apoA-I production in inflammatory, non-inflammatory and ER-stressed conditions. Therefore, C/EBP-ß is not a target to influence hepatic apoA-I production. J. Cell. Biochem. 118: 754-763, 2017. © 2016 Wiley Periodicals, Inc.

Analyzing the Impact of Pan- and Class-Specific HDACi on Differentiation-Associated Factors.

The differentiation of hematopoietic stem cells into mature blood cells is a highly ordered process and dysregulation of this process can lead to leukemogenesis. Agents that are used to cure acute promyelocytic leukemia (APL) can induce differentiation and/or apoptosis. Here, we describe how effects of all-trans retinoic acid (ATRA) and histone deacetylase inhibitors (HDACi) on APL cell differentiation can be evaluated by immunoblotting and by flow cytometry. We show how the levels of differentiation-associated transcription factors of the CCAAT enhancer binding protein (C/EBP) family can be determined by Western blot and we explain how the cell surface expression of the leukocyte surface antigen CD11b can be measured by flow cytometry.

Helenalin Acetate, a Natural Sesquiterpene Lactone with Anti-inflammatory and Anti-cancer Activity, Disrupts the Cooperation of CCAAT Box/Enhancer-binding Protein ß (C/EBPß) and Co-activator p300.

Recent work has demonstrated pro-oncogenic functions of the transcription factor CCAAT box/enhancer-binding protein ß (C/EBPß) in various tumors, implicating C/EBPß as an interesting target for the development of small-molecule inhibitors. We have previously discovered that the sesquiterpene lactone helenalin acetate, a natural compound known to inhibit NF-κB, is a potent C/EBPß inhibitor. We have now examined the inhibitory mechanism of helenalin acetate in more detail. We demonstrate that helenalin acetate is a significantly more potent inhibitor of C/EBPß than of NF-κB. Our work shows that helenalin acetate inhibits C/EBPß by binding to the N-terminal part of C/EBPß, thereby disrupting the cooperation of C/EBPß with the co-activator p300. C/EBPß is expressed in several isoforms from alternative translational start codons. We have previously demonstrated that helenalin acetate selectively inhibits only the full-length (liver-enriched activating protein* (LAP*)) isoform but not the slightly shorter (LAP) isoform. Consistent with this, helenalin acetate binds to the LAP* but not to the LAP isoform, explaining why its inhibitory activity is selective for LAP*. Although helenalin acetate contains reactive groups that are able to interact covalently with cysteine residues, as exemplified by its effect on NF-κB, the inhibition of C/EBPß by helenalin acetate is not due to irreversible reaction with cysteine residues of C/EBPß. In summary, helenalin acetate is the first highly active small-molecule C/EBPß inhibitor that inhibits C/EBPß by a direct binding mechanism. Its selectivity for the LAP* isoform also makes helenalin acetate an interesting tool to dissect the functions of the LAP* and LAP isoforms.

C/EBPß regulates homeostatic and oncogenic gastric cell proliferation.

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPß is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBPß knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPß. Cross-species comparison of gene expression profiles between C/EBPß-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPß signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38 % of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPß and that cross-regulation between C/EBPß-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis. KEY MESSAGE: C/EBPß controls proliferation and differentiation balance in the stomach. Homeostatic differentiation/proliferation balance is altered in gastric cancer. RUNX1t1 is a C/EBPß-associated tumor suppressor. RUNX1t1 negatively regulates C/EBPß pro-oncogenic functions.

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPß expression and activation.

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0-15µM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPß mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPß expression. In addition, ERK and GSK3ß-dependent C/EBPß phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPß expression as well as C/EBPß transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression.

Melatonin attenuated adipogenesis through reduction of the CCAAT/enhancer binding protein beta by regulating the glycogen synthase 3 beta in human mesenchymal stem cells.

Adipogenic differentiation is characterized by an increase in two major transcription factors: peroxisome proliferator-activated receptor gamma (PPARγ) and the CCAAT/enhancer binding protein alpha (C/EBPα). These two signals are influenced by C/EBPß and C/EBPδ and cross-regulate each other's expression during the initial stages of adipogenesis. Melatonin has been known to act as not only a direct scavenger of free radicals but also an inhibitor of glycogen synthase kinase 3ß (GSK-3ß). Here, we report that melatonin inhibits the adipogenic differentiation of human mesenchymal stem cells (hMSCs) which is due to the regulations of C/EBPß in the early stage of adipogenic differentiation. Melatonin reduced the lipid accumulation, adiponectin, and lipoprotein lipase (LPL) during the adipogenic differentiation of hMSCs. Since C/EBPß has been associated with the activation of PPARγ and the consensus site of ERK/GSK-3ß, PPARγ and ß-catenin were detected by immunofluorescence staining after pretreatment of melatonin. Melatonin blocked the activation of PPARγ which induced the degradation of ß-catenin. Melatonin also decreased the levels of cyclic adenosine-3,5-monophosphate (cAMP) and reactive oxygen species (ROS). The cAMP triggered the activity of C/EBPß which is a critical inducer of PPARγ and C/EBPα activation in the early stage of adipogenic differentiation, and this is further affected by ROS production. The adipogenic marker proteins such as PPARγ, C/EBPα, C/EBPß, and pERK were also decreased by melatonin. In summary, melatonin inhibited the cAMP synthesis through ROS reduction and the phosphorylation of the ERK/GSK-3ß site which is known to be responsible for C/EBPß activation for adipogenic differentiation in hMSCs.

XBP1-Independent UPR Pathways Suppress C/EBP-ß Mediated Chondrocyte Differentiation in ER-Stress Related Skeletal Disease.

Schmid metaphyseal chondrodysplasia (MCDS) involves dwarfism and growth plate cartilage hypertrophic zone expansion resulting from dominant mutations in the hypertrophic zone collagen, Col10a1. Mouse models phenocopying MCDS through the expression of an exogenous misfolding protein in the endoplasmic reticulum (ER) in hypertrophic chondrocytes have demonstrated the central importance of ER stress in the pathology of MCDS. The resultant unfolded protein response (UPR) in affected chondrocytes involved activation of canonical ER stress sensors, IRE1, ATF6, and PERK with the downstream effect of disrupted chondrocyte differentiation. Here, we investigated the role of the highly conserved IRE1/XBP1 pathway in the pathology of MCDS. Mice with a MCDS collagen X p.N617K knock-in mutation (ColXN617K) were crossed with mice in which Xbp1 was inactivated specifically in cartilage (Xbp1CartΔEx2), generating the compound mutant, C/X. The severity of dwarfism and hypertrophic zone expansion in C/X did not differ significantly from ColXN617K, revealing surprising redundancy for the IRE1/XBP1 UPR pathway in the pathology of MCDS. Transcriptomic analyses of hypertrophic zone cartilage identified differentially expressed gene cohorts in MCDS that are pathologically relevant (XBP1-independent) or pathologically redundant (XBP1-dependent). XBP1-independent gene expression changes included large-scale transcriptional attenuation of genes encoding secreted proteins and disrupted differentiation from proliferative to hypertrophic chondrocytes. Moreover, these changes were consistent with disruption of C/EBP-ß, a master regulator of chondrocyte differentiation, by CHOP, a transcription factor downstream of PERK that inhibits C/EBP proteins, and down-regulation of C/EBP-ß transcriptional co-factors, GADD45-ß and RUNX2. Thus we propose that the pathology of MCDS is underpinned by XBP1 independent UPR-induced dysregulation of C/EBP-ß-mediated chondrocyte differentiation. Our data suggest that modulation of C/EBP-ß activity in MCDS chondrocytes may offer therapeutic opportunities.