Expression of the Proteins Ski and Akradia in the Retinal Tissue of Diabetic Rats

The aim of this study was to observe the effects of Ski and Arkadia protein expression in the retina of diabetic rats, as well as to explore the relationships between Ski, Arkadia, and diabetic retinopathy (DR) to provide theoretical insights into its pathogenesis. Forty healthy male Wistar rats were randomly divided into two groups: the normal control group, and the DM (diabetes mellitus, DM) group. A DM rat model was established through a single intraperitoneal injection of 50 mg · kg–1 STZ. Ten rats in each group were sacrificed at the 8th and 12th weeks after model generation; the left eyeball of each rat was removed completely and made into eye cups. Immunohistochemical methods were used to detect the expression of Ski and Arkadia in the retina of each rat. In the normal control group, Ski was highly expressed, while Arkadia was either not expressed or weakly expressed. At weeks 8 and 12, the expression of Arkadia in the retina of the rats in the DM group was significantly higher than in those of the normal control group (P <0.01), whereas the expression of Ski was significantly lower than in normal controls (P <0.01). In retinal tissue of diabetic rats, the ubiquitin proteasome pathway can degrade the expression of the Ski protein and the E3 ligase Arkadia is involved in the ubiquitination of Ski proteins.

Identification of the Oncogene SKI As a New Target Gene of the Myeloid Transcription Factor c-MYB in AML

Background: Acute myeloid leukemia (AML) accounts for 80% of acute leukemias and arises through clonal expansion and arrest of differentiation of hematopoietic progenitor cells in the bone marrow. Depending on the AML subtype and age of the patient, AML patients have a 5-year survival rate of about 25%. AML is a genetically heterogenous disease, where chromosomal aberrations, point mutations in critical oncogenes as well as aberrant expression of key regulatory factors of hematopoiesis collude during transformation. c-MYB is an important hematopoietic transcription factor involved in proliferation and differentiation of progenitor cells of the myeloid and lymphoid lineages. It was first described as a viral oncogene of avian leukemia viruses and is upregulated or mutated in many leukemic subtypes as well as solid tumors. c-MYB interacts with other transcription factors or co-factors, which are essential for its transcriptional activity. In this regard, c-MYB transactivation ability is inhibited by a histone deacetylases recruiting corepressor complex containing SKI, TIF1BETA, NCOR and mSIN3A. c-SKI is a proto-oncogene and an inhibitor of TGFβ signalling. However, it acts not only as a transcriptional co-repressor but also as a transcriptional co-activator. Like c-MYB, c-SKI is upregulated in different solid tumors and leukemias. Though SKI activity is well described, transcriptional regulation of the SKI gene itself still remains unknown. Here, we deliver insight into the transcriptional regulation of the human SKI gene via the transcription factor c-MYB. Methods: In silico analyses were performed to identify potential MYB binding sites in the SKI regulatory region. Chromatin immunoprecipitation (ChIP) assays were used to validate the interaction between MYB and the potential SKI regulatory regions. Reporter gene assays were engaged to analyze MYB regulatory potential regarding SKI expression. RNAi experiments were performed to further examine transcriptional regulation of SKI by MYB. Since MYB is known to be downregulated by the histone deacetylase inhibitor (HDACi) valproic acid (VPA), MYB and SKI protein levels were analyzed in AML cell lines treated with VPA via Western Blot. Correlated protein levels of MYB and SKI were examined in the myeloid leukemia cell lines HL60, U937, THP1, NB4 and K562 as well as in primary cells of AML patients (n=27). Correlation of MYB and SKI transcript levels were performed in three different data sets of primary AML samples. The first cohort (n=7) was analyzed via RT-qPCR. Cohort 2 consists of cDNA microarray data of AML patients (n=17). Furthermore, principal component analysis (PCA) of the gene expression profile of MYB and SKI of AML patients (n=542) were performed with the leukemia gene atlas (LGA). Results: In silico analyses revealed four putative MYB binding sites (MBS1-4) in the SKI regulatory region. Direct binding of MYB to the regulatory sites MBS2-4 of the SKI gene could be confirmed via ChIP experiments. Dual luciferase reporter gene assays comprising c-MYB binding sites present in the SKI gene locus further show c-MYB-dependent transcriptional activation of the reporter. RNAi experiments depleting c-MYB in leukemic cell lines resulted in the decrease of SKI protein levels and thereby reveal that c-MYB is essential for the induction of SKI gene expression. Accordingly, treatment of the AML cell lines with the HDACi VPA led to a decrease of MYB and consequently SKI protein levels. Consistently, we observed a positive correlation of MYB and SKI protein expression in leukemic cell lines and in samples of AML patients. Moreover, a highly positive correlation of MYB and SKI transcript levels could be observed in three different cohorts of AML patients, further confirming regulation of SKI expression by the transcription factor MYB. Conclusion: Our findings provide new insights in the transcriptional regulation of the proto-oncogene c-SKI by the oncogenic transcription factor c-MYB during leukemogenesis. c-MYB and c-SKI expression and functions are highly positively correlated in human AML suggesting that c-SKI is a mediator of c-MYB oncogenic potential. So far, various therapeutic approaches targeting MYB failed to be transferred to patients. In this regard, c-MYB and c-SKI represent promising marker and target proteins for novel HDACi-based therapeutic approaches in AML. Disclosures No relevant conflicts of interest to declare.

Relationship between Sloan-Kettering virus expression and mammalian follicular development-RETRACTION

Zygotewishes to inform its readers that its Editor-in-Chief has decided to retract the above article after an investigation carried out in compliance with the Committee on Publication Ethics guidelines found that the authors duplicated substantial parts of the following two articles:1.Kim H, Yamanouchi K, Nishihara M. (2006) Expression of ski in the granulosa cells of atretic follicles in the rat ovary.J. Reprod. Dev.52, 715–7212.Kim H, Yamanouchi K, Matsuwaki T, Nishihara M. (2012) Induction of Ski protein expression upon luteinization in rat granulosa cells without a change in its mRNA expression.J. Reprod. Dev.58, 254–259

Protective Effect of Triptolide against Glomerular Mesangial Cell Proliferation and Glomerular Fibrosis in Rats Involves the TGF-β1/Smad Signaling Pathway

Triptolide as a main active ingredient ofTripterygium wilfordiiis known to be exerting anti-inflammatory, marked immunosuppressive, and podocyte-protective effects. In this study, we investigated the protective effect of triptolide in kidney disease. Rat glomerular mesangial cells were randomly divided into three groups: (1) control group, (2) TGF-β1 (10 μg/mL) group, and (3) triptolide group (triptolide 10 μg/L + TGF-β1 10 μg/L). Sixty male Sprague-Dawley rats were randomly divided into three groups: (1) control group, (2) chronic serum sickness glomerulonephritis model group, and (3) triptolide (0.2 mg/kg·d) group. Reverse transcription PCR was used to assess Ski and Smad3 mRNA expression in the mesangial cells and renal tissues. Western blotting was used to determine Ski and Smad3 protein expressions. Laser confocal fluorescence microscopy was used to observe the subcellular localization of Smad3 and Ski proteins in the mesangial cells. Triptolide inhibited the TGF-β1-induced proliferation of mesangial cells. It significantly upregulated Ski protein expression and downregulated Smad3 mRNA and protein expressions in a time-dependent manner. Laser confocal fluorescence microscopy detected high Smad3 fluorescence intensity in the cytoplasm and low Smad3 and high Ski fluorescence intensity in the nucleus. By upregulating Ski protein expression triptolide decreased the extent of fibrosis by affecting the TGF-β1/Smad3 signaling pathway.

Expression of the Nuclear Corepressor Ski Predicts Response to Histone Deacetylase Inhibitor Valproic Acid in Cells of Acute Myeloid Leukemia.

Abstract 2410 Acute myeloid leukemia (AML) with deletion of chromosome 7 (−7) or 7q (del7q) has a poor prognosis. Using gene expression analysis, we previously identified the nuclear oncogene Ski as being up-regulated in AML, especially in AML with −7/del7q. We demonstrated that the transcriptional corepressor Ski acts as an inhibitor of vitamin A induced myeloid differentiation through interaction with N-CoR recruiting histone deacetylases (HDAC) (Ritter et al., Leukemia 2006). HDAC inhibitors such as valproic acid (VPA) promote histone acetylation, induce apoptosis and cell growth arrest in tumor cells (e.g. Kraemer et al., Trends Endocrinol Metab 2001). As Ski interacts with HDACs the aim of our investigation was to test the effect of HDAC inhibitors to cellular differentiation, apoptosis and Ski expression in primary AML cells. Treatment of the AML cell line HL60 expressing Ski with the HDAC inhibitor VPA enhances expression of the myeloid differentiation markers CD11b and CD11c as well as apoptosis. To address whether this effect is also observed in primary AML cells, we isolated mononuclear cells from blood or bone marrow of 12 AML patients (first diagnosis or relapse) and treated these cells with the HDAC inhibitor VPA. After harvesting, Ski protein expression was determined by Western blot. Flow cytometry was used to analyse expression of the differentiation markers CD11b and CD11c and apoptosis after propidium iodide staining. Of six AMLs with Ski protein expression, four responded either with differentiation or apoptosis, whereas none of six primary AMLs without Ski expression showed an effect to VPA compared to untreated control cells. To test whether other HDAC inhibitors would also reveal this effect we treated primary AML cells with further HDAC inhibitors (TSA, SAHA, LBH589) and confirmed our observation that HDAC inhibitors renders AML cells expressing Ski sensitive to differentiation. In parallel, we observed that VPA down regulates Ski in AML cells as well as in melanoma cell lines with high Ski protein levels. Our goal was to elucidate the molecular background of Ski reduction by VPA. Treatment of melanoma cells expressing Ski with VPA and/or the proteasomal inhibitor MG132 revealed that decrease of Ski depends on proteasomal degradation. The ring finger protein Arkadia (RNF111) is an E3-ligase of Ski (Nagano et al., J Biol Chem 2007) and we tested whether Arkadia is involved in Ski reduction after VPA addition. First we demonstrated that Arkadia expression is inversely associated with Ski expression in several AML cell lines. Furthermore the expression of Arkadia is induced by VPA on protein and RNA level while Ski protein is down regulated in melanoma cells. We also found that knockdown of Arkadia using RNAi impairs reduction of Ski by VPA in melanoma cells. Taken together our data suggest that high Ski expression in AML cells could be a molecular marker for VPA therapy. Thereby, VPA reduces the expression of the oncogene Ski as VPA induces expression of the E3-ligase Arkadia which abolish Ski by proteasomal degradation. Disclosures: No relevant conflicts of interest to declare.