S6K1 deficiency in tumor stroma impairs lung metastasis of melanoma in mice.

Accumulating data suggest that ribosomal protein S6 kinase 1 (S6K1), an effector in the mammalian target of rapamycin (mTOR) pathway, plays pleiotropic roles in tumor progression. However, to date, while the tumorigenic function of S6K1 in tumor cells has been well elucidated, its role in the tumor stroma remains poorly understood. We recently showed that S6K1 mediates vascular endothelial growth factor A (VEGF-A) production in macrophages, thereby supporting tumor angiogenesis and growth. As macrophage-derived VEGF-A is crucial for both tumor cell intravasation and extravasation across the vascular endothelium, our previous findings suggest that stromal S6K1 signaling is required for tumor metastatic spread. Therefore, we aimed to determine the impact of host S6K1 depletion on tumor metastasis using a murine model of pulmonary metastasis (S6k1-/- mice implanted with B16F10 melanoma). The ablation of S6K1 in the host microenvironment significantly reduced the metastasized B16F10 melanoma cells on the lung surface in both spontaneous and intravenous lung metastasis mouse models without affecting the incidence of metastasis to distant lymph nodes. In addition, stromal S6K1 loss decreased the number of tumor cells circulating in the peripheral blood of mice bearing B16F10 xenografts without affecting the vascular leakage induced by VEGF-A in vivo. These observations demonstrate that S6K1 signaling in host cells other than endothelial cells is required to modulate the host microenvironment to facilitate the metastatic spread of tumors via blood circulation, thus revealing its novel role in the tumor stroma during tumor progression.

S6K1 Phosphorylation of H2B Mediates EZH2 Trimethylation of H3: A Determinant of Early Adipogenesis.

S6K1 has been implicated in a number of key metabolic responses, which contribute to obesity. Critical among these is the control of a transcriptional program required for the commitment of mesenchymal stem cells to the adipocytic lineage. However, in contrast to its role in the cytosol, the functions and targets of nuclear S6K1 are unknown. Here, we show that adipogenic stimuli trigger nuclear translocation of S6K1, leading to H2BS36 phosphorylation and recruitment of EZH2 to H3, which mediates H3K27 trimethylation. This blocks Wnt gene expression, inducing the upregulation of PPARγ and Cebpa and driving increased adipogenesis. Consistent with this finding, white adipose tissue from S6K1-deficient mice exhibits no detectable H2BS36 phosphorylation or H3K27 trimethylation, whereas both responses are highly elevated in obese humans or in mice fed a high-fat diet. These findings define an S6K1-dependent mechanism in early adipogenesis, contributing to the promotion of obesity.

Comparison of the Liverpool Causality Assessment Tool vs. the Naranjo Scale for predicting the likelihood of an adverse drug reaction: A retrospective cohort study.

AIMS: The aim of this study is to compare the Liverpool Causality Assessment Tool vs. Naranjo Scale for screening suspected adverse drug reaction (ADR) cases. METHODS: We retrospectively reviewed patient charts with a history of suspected ADR, scored using both tools, and determined how each correlates with laboratory and other investigations. A total of 924 charts from the Clinical Pharmacology Clinic at the London Health Sciences Centre were reviewed, and 529 charts contained objective findings to support or against the diagnosis of ADR. The participant age ranged from 1 month to 93 years. We determined that the sensitivity (SN) and specificity (SP) of Liverpool and Naranjo tools for predicting ADRs with scores ranging from Possible to Definite were considered positive and Unlikely/Doubtful as negative for ADR. These results were confirmed by laboratory or clinical (re-challenge) testing in 529 cases. RESULTS: Liverpool causality tool had SN of 97.2 ± 2.4% and SP of 2.3 ± 1.57%. The positive (PPV) and negative predictive values (NPV) were 34.1 and 61.5%, respectively. The Naranjo Scale had SN of 81.2 ± 5.69% and SP of 13.2 ± 3.56%. PPV and NPV were 32.7 and 57.5%, respectively. CONCLUSION: The Liverpool Causality Assessment Tool is a more sensitive tool than the Naranjo Scale in the assessment of possible ADRs, but both tools have poor SP. The Liverpool Tool can be a useful screening tool in settings where other tests may not be readily available. However, the low PPV and NPV of both tools suggest that to pursue further testing is needed to confirm or deny an ADR.

Bioactive Phytochemicals from Salix pseudolasiogyne Twigs: Anti-Adipogenic Effect of 2'-O-Acetylsalicortin in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae) is a willow tree and has been used as a medicinal herb in Korea to treat pain and fever. As a part of an ongoing study to identify bioactive natural products, potential anti-adipogenic compounds were investigated using the ethanol (EtOH) extract of S. pseudolasiogyne twigs. Phytochemical investigation of the EtOH extracts using liquid chromatography-mass spectrometry (LC/MS) led to the separation of two compounds, oregonin (1) and 2'-O-acetylsalicortin (2). The structures of the isolates were identified using nuclear magnetic resonance spectroscopy and LC/MS analysis. To the best of our knowledge, it is the first report identifying oregonin (1) in twigs of S. pseudolasiogyne. Here, we found that the isolated compounds, oregonin (1) and 2'-O-acetylsalicortin (2), showed anti-adipogenic effects during 3T3-L1 cell differentiation. Notably, 2'-O-acetylsalicortin (2), at a concentration of 50 µM, significantly suppressed lipid accumulation. Moreover, the mRNA and protein levels of lipogenic and adipogenic transcription factors were reduced in 2'-O-acetylsalicortin (2)-treated 3T3-L1 cells. Taken together, these results indicate that 2'-O-acetylsalicortin (2), isolated from S. pseudolasiogyne twigs, has the potential to be applied as a therapeutic agent to effectively control adipocyte differentiation, a critical stage in the progression of obesity.

Anti-Adipogenic Effects of Salicortin from the Twigs of Weeping Willow (Salix pseudolasiogyne) in 3T3-L1 Cells.

Salix pseudolasiogyne (Salicaceae), the "weeping willow," has been used in traditional Korean medicine to treat pain and fever due to its high concentrations of salicylic acid and salicin. The present study investigated bioactive compounds from S. pseudolasiogyne twigs to discover bioactive natural products. Phytochemical investigation of the ethanol (EtOH) extract of S. pseudolasiogyne twigs followed by liquid chromatography-mass spectrometry (LC/MS)-based analysis led to the isolation of two salicin derivatives, salicortinol and salicortin, the structures of which were determined by interpretation of their NMR spectra and data from the LC/MS analysis. To the best of our knowledge, this is the first report of salicortinol isolated from S. pseudolasiogyne. The isolated compounds were evaluated for their anti-adipogenic effects in 3T3-L1 cells. Both salicortinol and salicortin were found to significantly inhibit adipocyte differentiation in 3T3-L1 cells. In particular, salicortin exhibited a strong inhibitory effect on lipid accumulation. Furthermore, salicortin inhibited the expression of lipogenic and adipogenic transcription factors, including FASN, FABP4, C/EBPα, C/EBPß, and PPARγ, without inducing cytotoxicity. These results suggest that salicortin could be a potential therapeutic compound for the prevention or treatment of metabolic disorders such as obesity.

Growth differentiation factor 15 predicts advanced fibrosis in biopsy-proven non-alcoholic fatty liver disease.

BACKGROUND & AIMS: We explored whether growth differentiation factor 15 (GDF15) affects the histological severity of non-alcoholic fatty liver disease (NAFLD) independent of insulin resistance. METHODS: In a biopsy-proven NAFLD cohort, we measured serum GDF15 levels using enzyme-linked immunosorbent assays. RESULTS: Among 190 subjects (mean age, 53 ± 14 years; men, 52.1%), 72 (men, 65.3%) and 78 (men, 44.9%) were diagnosed with biopsy-proven non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH) respectively. GDF15 levels were significantly higher in NASH patients than in controls (P = .010) or NAFL patients (P = .001). Subjects with advanced fibrosis (≥F3) also showed higher GDF15 levels compared to the others (F0-2; P < .001). Among NAFLD patients, the highest quartile of GDF15 levels was significantly associated with a risk of advanced fibrosis even after adjustment for age, gender, body mass index, smoking status, hypertension, diabetes, aspartate aminotransferase, platelet, albumin, insulin resistance and low skeletal muscle mass (odds ratio, 4.27; 95% confidence interval, 1.04-17.63), but not with NASH risk. GDF15 levels showed a significant positive correlation with liver stiffness (Spearman's ρ, .525; P < .001). Palmitate treatment increased the GDF15 mRNA expression level significantly in Kupffer cells, but not in hepatocytes. In LX-2 cells, GDF15 treatment resulted in enhanced expression of α-smooth muscle actin and collagen I, as well as phosphorylation of SMAD2 and SMAD3. CONCLUSIONS: Our findings suggest that GDF15 may serve as a novel biomarker of advanced fibrosis in NAFLD, thereby indicating the need for urgent anti-fibrotic pharmacotherapy.

Association between nonalcoholic fatty liver disease and bone mineral density in postmenopausal women.

OBJECTIVE: Growing evidence suggests that nonalcoholic fatty liver disease (NAFLD) is associated with reduced bone mineral density (BMD). This study examined the association between NAFLD and BMD in postmenopausal Korean women. METHODS: This retrospective cross-sectional study analyzed 3739 postmenopausal women aged 50-59 years who visited a health promotion center for a routine checkup from 2009 to 2014. Menopause was defined as absence of menstruation for ≥12 months and an elevated follicle-stimulating hormone serum level (>20 IU/L). Women were excluded if they had a history of disease or use of medication that might affect bone metabolism. NAFLD was diagnosed by ultrasonography; BMD was measured by dual-energy X-ray absorptiometry. BMDs were compared according to the presence of NAFLD, and associations between NAFLD and BMD were analyzed after adjustment. RESULTS: Among 3739 postmenopausal women, 605 (16.2%) had NAFLD. Mean BMD at the lumbar spine (P = 0.017) and femur neck (P < 0.0001) was significantly lower in women with NAFLD than in those without NAFLD. Associations between NAFLD and BMD were significantly negative at both sites after adjusting for potential clinical confounders and factors which were significantly associated with BMD. CONCLUSIONS: NAFLD is negatively associated with BMD in postmenopausal women. A longitudinal study is recommended.

Bilateral blood pressure differential as a clinical marker for acute aortic dissection in the emergency department.

INTRODUCTION: Interarm systolic BP differential is a classic sign of acute aortic dissection (AAD). All previous studies looking at the association of BP differential with AAD combine systolic BP differential >20 mm Hg with pulse deficit. Our aim was to assess the association of systolic BP differential with AAD, and its role in predicting AAD in the context of other signs and symptoms. METHODS: Historical matched case-control study using patient data collected between 2002 and 2014: participants were adults >18 years old with a bilateral BP measurement presenting to two tertiary care EDs or one regional cardiac referral centre in Canada. Cases were patients with diagnosed AAD; controls were those with a triage diagnosis of truncal pain (<14 days) and an absence of a clear diagnosis on basic investigation. Cases and controls were matched in a 1:1 ratio by sex and age. Bilateral BP differential and pulse deficit were compared between groups. RESULTS: A total of 222 patients (111 cases and 111 controls) were analysed. On univariate analysis systolic BP differential >20 mm Hg (OR 2.7, 95% CI 1.39 to 5.25) was significantly associated with AAD. Pulse deficit (diagnostic OR (DOR) 28.9) in isolation had better diagnostic accuracy than systolic BP differential >20 mm Hg in isolation (DOR 2.71) or combined with systolic BP differential >20 mm Hg (DOR 4.2). CONCLUSION: Bilateral systolic BP differentials >20 mm Hg are associated with non-traumatic AAD. However, the poor diagnostic accuracy and potential variability in measurement limits its clinical usefulness.

Interferon-alpha inhibits adipogenesis via regulation of JAK/STAT1 signaling.

BACKGROUND INFORMATION: Adipose tissue regulates energy metabolism by means of adipocyte hypertrophy and/or the differentiation of pre-existing adipocytes. Excessive production of some cytokines in adipose tissue is known to be a negative regulator of adipocyte differentiation, and the resulting impaired adipogenesis contributes to disorders like insulin resistance. IFN-α is a key immunoregulatory cytokine in the development of type 1 diabetes, lipid disorders and insulin resistance; however, its effect on adipogenesis remains unknown. METHOD: We examined the effect of IFN-α on adipocyte differentiation and its mechanisms. The effect of IFN-α on adipogenesis was evaluated by Western blotting, qRT-PCR, flow cytometric analysis and Oil Red O staining. We also investigated the role of STAT1 in adipogenesis using gene silencing analysis. RESULTS: IFN-α inhibited the accumulation of lipid droplets and the expression of adipogenesis related genes. The inhibition of adipocyte differentiation by IFN-α occurred in the early stages of differentiation. IFN-α arrested the cell cycle at the G0/G1 phase and regulated the expression of CDK2 and p21. These results were confirmed in MEF cells. Treatment with IFN-α increased STAT1 phosphorylation, and STAT1 siRNA or inhibitor prevented IFN-α from inhibiting the expression of PPARγ and C/EBPα as well as cell cycle progression in 3T3-L1 cells. CONCLUSION: We suggest that IFN-α inhibits adipocyte differentiation during the early stage of adipogenesis by regulating the expression of PPARγ and C/EBPα as well as the cell cycle through JAK/STAT1 signaling pathways. GENERAL SIGNIFICANCE: Our study provides new insights into possible mechanisms of the anti-adipogenetic effects of IFN-α.

Redox-Neutral Rh(III)-Catalyzed Olefination of Carboxamides with Trifluoromethyl Allylic Carbonate.

The rhodium(III)-catalyzed olefination of various carboxamides with α-CF3-substituted allylic carbonate is described. This reaction provides direct access to linear CF3-allyl frameworks with complete trans-selectivity. In particular, a rhodium catalyst provided Heck-type γ-CF3-allylation products via the ß-O-elimination of rhodacycle intermediate and subsequent olefin migration process.

Polarized macrophages treated with nonylphenol differently regulate lipopolysaccharide-induced sepsis.

Nonylphenol (NP) as well-known "endocrine disrupter" influences sexual and reproductive development. Here, we investigated the effect of NP on M1-/M2-type macrophages and their role in lipopolysaccharide (LPS)-induced sepsis. Polarized macrophages of M1- and M2-types were obtained by the treatment with LPS and interleukin-4 (IL-4) to bone marrow-derived macrophages (BMDM), respectively. Coincubation of M1-macrophages with NP decreased COX-2, iNOS, IL-6, and TNF-α expression but no changes were detected in the production of nitric oxide (NO). Survival probability of LPS-induced sepsis mice was enhanced by the injection of NP-treated BMDM as compared to the injection of NP-untreated control BMDM. In the meanwhile, the expression of arginase 1(Arg1), a marker for M2-polarized macrophages was increased by the stimulation with LPS in BMDM. Arg1 expression was also enhanced by the treatment with IL-4 in BMDM, which was reduced by the coincubation with NP. Survival probability of LPS-induced sepsis mice was decreased by the injection of BMDM treated with IL-4 and NP as compared to the injection of IL-4-treated BMDM. It suggests that NP might inhibit macrophage function and the polarization to M2-macrophages. Taken together, data demonstrate that NP could differently affect immune responses of polarized macrophages resulted in the modulation of LPS-induced sepsis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2081-2089, 2016.

CD10 expression is enhanced by Twist1 and associated with poor prognosis in esophageal squamous cell carcinoma with facilitating tumorigenicity in vitro and in vivo.

CD10 expression was identified as a contributor to cancer progression in several cancers; however, the exact biological significance and mechanism of CD10 expression remains unclear. In addition, CD10 expression in esophageal squamous cell carcinoma (ESCC) has not been studied. We investigated the relationship between CD10 and Twist1. Furthermore, we examined the effect of CD10 on tumorigenicity using in vivo and in vitro systems as well as establishing the clinical significance of CD10 expression in ESCC using large clinical samples. CD10 expression was upregulated by Twist1 and there was a strong correlation between mRNA and protein expression. Twist1 can specifically upregulate CD10 at the transcriptional level via an interaction with the promoter region of CD10 and the proximal E-box CAGGTG in the CD10 promoter was identified as a binding site for Twist1. CD10 is frequently expressed in ESCC cell lines and silencing CD10 suppresses migration/invasion and anchorage-independent tumor growth of ESCC cells. Knockdown of CD10 inhibits the growth of ESCC xenograft in nude mice, suggesting that CD10 plays a role in enhancing the tumorigenesis of ESCC. From among 153 ESCC samples, 46 (30.0%) showed varying degrees of CD10 expression in cancer cells. In addition, stromal fibroblasts also showed varying amounts of CD10 expression in 92 (60.9%) tumor samples. CD10 overexpression in cancer cells as well as in stromal fibroblasts was an independent poor prognostic factor in both overall survival and disease-free survival. CD10 could be a promising target for the treatment of ESCC.

Fisetin induces Sirt1 expression while inhibiting early adipogenesis in 3T3-L1 cells.

Fisetin (3,7,3',4'-tetrahydroxyflavone) is a naturally found flavonol in many fruits and vegetables and is known to have anti-aging, anti-cancer and anti-viral effects. However, the effects of fisetin on early adipocyte differentiation and the epigenetic regulator controlling adipogenic transcription factors remain unclear. Here, we show that fisetin inhibits lipid accumulation and suppresses the expression of PPARγ in 3T3-L1 cells. Fisetin suppressed early stages of preadipocyte differentiation, and induced expression of Sirt1. Depletion of Sirt1 abolished the inhibitory effects of fisetin on intracellular lipid accumulation and on PPARγ expression. Mechanistically, fisetin facilitated Sirt1-mediated deacetylation of PPARγ and FoxO1, and enhanced the association of Sirt1 with the PPARγ promoter, leading to suppression of PPARγ transcriptional activity, thereby repressing adipogenesis. Lowering Sirt1 levels reversed the effects of fisetin on deacetylation of PPARγ and increased PPARγ transactivation. Collectively, our results suggest the effects of fisetin in increasing Sirt1 expression and in epigenetic control of early adipogenesis.

Vaccinia virus outperforms a panel of other poxviruses as a potent oncolytic agent for the control of head and neck squamous cell carcinoma cell lines.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer worldwide. Existing therapies for advanced tumors have high failure rates and can have severe consequences in terms of pain, disfigurement, and poor speech and swallowing function. New treatment strategies are needed to improve outcomes for patients suffering with this disease and oncolytic viruses represent a promising approach. METHODS: We infected six well-characterized HNSCC cell lines (Cal27, Detroit562, FaDu, SCC4, SCC15, SCC25), with increasing doses of a panel of poxviruses (including myxoma, vaccinia, raccoonpox and tanapox viruses) modified to express green fluorescence protein to determine which virus was the most effective oncolytic agent in cell-based assays. RESULTS: While myxoma, raccoonpox and tanapox displayed differing efficacy in the panel of cell lines, vaccinia virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all cell lines. CONCLUSION: Oncolytic poxviruses, particularly vaccinia virus, were effective in killing HNSCC in vitro and hold promise as potential treatments for patients with HNSCC.

Cardiac genetic test yields and genotype-phenotype correlations from large cohort investigated by medical examiner's office.

BACKGROUND: Few reports describe the yield of postmortem genetic testing from medical examiners' offices or correlate genetic test results with autopsy-confirmed phenotypes from a large cohort. OBJECTIVES: To report results from cardiomyopathy- and cardiac arrhythmia-associated genetic testing in conjunction with autopsy findings of cases investigated at the United States' largest medical examiner office. METHODS: Postmortem cases tested from 2015 to 2022 with a cardiomyopathy- and cardiac arrhythmia-associated gene panel were reviewed. American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines were used to classify variant pathogenicity. Correlations of pathogenic/likely pathogenic variants (P/LPVs) with cardiac pathology were evaluated. RESULTS: The cohort included 1107 decedents of diverse ages and ethnicities. P/LPVs were detected in 87 (7.9%) cases, with 73 and 14 variants in cardiomyopathy and cardiac arrhythmia genes, respectively. Variants of uncertain significance were detected in 437 (39.5%) cases. The diagnostic yield (percentage of P/LPV) in decedents with cardiomyopathy (26.1%) was significantly higher than those without (P<.0001). The diagnostic yield was significantly lower in infants (0.7%) than older age groups (ranging from 1 to 74 years old, 5.7%-25.9%), which had no statistical difference between their yields. The diagnostic yields by cardiac autopsy findings were 54.0% for hypertrophic cardiomyopathy, 47.1% for arrhythmogenic cardiomyopathy, 20.0% for myocardial fibrosis, 19.0% for dilated cardiomyopathy, and 11.3% for myocarditis. Most P/LPVs were in MYBPC3, TTN, PKP2, SCN5A, MYH7, and FLNC. Ten P/LPVs were novel. CONCLUSIONS: Our results support the importance of performing postmortem genetic testing on decedents of all ages with cardiomyopathy, cardiac lesions insufficient to diagnosis a specific cardiomyopathy (e.g., myocardial fibrosis), and myocarditis. Combined postmortem cardiac examination and genetic analysis are advantageous in accurately determining the underlying cause of death and informing effective clinical care of family members.

Prognostic significance and function of phosphorylated ribosomal protein S6 in esophageal squamous cell carcinoma.

Ribosomal protein S6 is a key regulator of 40S ribosome biogenesis, and its phosphorylation is closely related to cell growth capacity. However, as a downstream target of S6 kinases, the clinical significance and the roles of S6 and S6 phosphorylation in cell viability and motility of esophageal squamous cell carcinoma remain unclear. Here, we show that high level of phosphorylated-ribosomal protein S6 (p-S6) (immunohistochemistry score ≥5) and an increased ratio of p-S6/S6 (immunohistochemistry score ≥0.75) were significantly associated with shortened disease-free survival in patients with esophageal squamous cell carcinoma in univariate analysis (P=0.049 and P<0.001, respectively). After adjusting for age, tumor-nodes-metastasis stage, chemotherapy, and radiation therapy in multivariate analysis, both p-S6 (hazard ratio 2.21, P=0.005) and p-S6/S6 (hazard ratio 2.40, P<0.001) remained independent adverse prognostic factors. In addition, S6 and S6 kinase 1 knockdown resulted in attenuation of viability by suppressing cyclin D1 expression in esophageal cancer cells. Furthermore, depletion of S6 and S6 kinase 1 resulted in a reduction in esophageal cancer cell migration and invasion. This was paralleled by a reduction in focal adhesion and by suppression of extracellular signal-regulated kinase and c-jun N-terminal kinase phosphorylation, which control cell motility. Collectively, these findings suggest that p-S6 and the ratio of p-S6/S6 are closely relevant to tumor progression and have prognostic significance in esophageal squamous cell carcinoma.

S6 kinase 2 deficiency enhances ketone body production and increases peroxisome proliferator-activated receptor alpha activity in the liver.

UNLABELLED: Nutrient homeostasis is tightly regulated by the balance between energy production and utilization. During fasting, production of ketone bodies as an alternative energy source is critical to maintain nutrient homeostasis. An important component in the nutrient-sensitive signaling pathway is S6 kinase 2 (S6K2), a downstream effector of mammalian target of rapamycin. Here, we show that mice lacking S6K2 exhibit elevated levels of ketone bodies and enhanced peroxisome proliferator-activated receptor alpha (PPARα) activity upon nutrient availability. Consistent with this, knockdown of S6K2 increases the transcriptional activity of PPARα. S6K2 suppresses PPARα by associating with its corepressor, nuclear receptor corepressor 1 (NCoR1), and by inducing the recruitment of NCoR1 to the nucleus. Moreover, ob/ob mice, a genetic model of obesity, have markedly elevated S6K2 activity, and S6K2 was strongly associated with NCoR1 in the nucleus of liver cells. CONCLUSION: Our findings suggest that S6K2 regulates hepatic energy homeostasis by repressing PPARα activity and point to its potential relevance for therapeutic strategies designed to modulate S6K2 activity as a treatment for deregulated ketone body production.

Ethanol induces cell cycle arrest and triggers apoptosis via Sp1-dependent p75NTR expression in human neuroblastoma cells.

Ethanol exposure has deleterious effects on the central nervous system. Although several mechanisms for ethanol-induced damage have been suggested, the precise mechanism underlying ethanol-induced neuronal cell death remains unclear. Recent studies indicate that the p75 neurotrophin receptor (p75NTR) has a critical role in the regulation of neuronal survival. This study was designed to examine the role of p75NTR in ethanol-induced apoptotic signaling in neuroblastoma cells. Ethanol caused highly increased level of p75NTR expression. The use of small interfering RNA to inhibit p75NTR expression markedly attenuated ethanol-induced cell cycle arrest and apoptosis. DNA binding activity of Sp1 was increased by ethanol, whereas inhibition of Sp1 activity by mithramycin, a Sp1 inhibitor, or short hairpin RNA suppressed ethanol-induced p75NTR expression. In addition, inhibitors of casein kinase 2 (CK2) and extracellular signal-regulated kinase (ERK) augmented ethanol-induced p75NTR expression. Our results also demonstrate that inhibition of ERK and CK2 caused a further increase in the activation of the p75NTR proximal promoter induced by ethanol. This increased activation was partially suppressed by the deletion of the Sp1 binding sites. These results suggest that Sp1-mediated p75NTR expression is regulated at least in part by ERK and CK2 pathways. The present study also showed that treatment with ethanol resulted in significant increases in the expression of p21, but not the levels of p53 and p53 target genes such as Bax, Puma, and Bcl-2. Furthermore, the inhibition of p75NTR expression or Sp1 activity suppressed ethanol-induced p21 expression, cell cycle arrest, and apoptosis. These data suggest that ethanol increases p75NTR expression, and CK2 and ERK signaling inversely regulate Sp1-mediated p75NTR expression in ethanol-treated neuroblastoma cells. Thus, our study provides more insight into the mechanisms underlying ethanol actions.

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization.

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.