Ski Protein Research Articles

Combination of high anti-SKI and low anti-TMED5 antibody levels is preferable prognostic factor in esophageal carcinoma.

Given that esophageal cancer is highly malignant, the discovery of novel prognostic markers is eagerly awaited. We performed serological identification of antigens by recombinant cDNA expression cloning (SEREX) and identified SKI proto-oncogene protein and transmembrane p24 trafficking protein 5 (TMED5) as antigens recognized by serum IgG antibodies in patients with esophageal carcinoma. SKI and TMED5 proteins were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens. The serum anti-SKI antibody (s-SKI-Ab) and anti-TMED5 antibody (s-TMED5-Ab) levels were significantly higher in 192 patients with esophageal carcinoma than in 96 healthy donors. The presence of s-SKI-Abs and s-TMED5-Abs in the patients' sera was confirmed by western blotting. Immunohistochemical staining showed that the TMED5 protein was highly expressed in the cytoplasm and nuclear compartments of the esophageal squamous cell carcinoma tissues, whereas the SKI protein was localized predominantly in the nuclei. Regarding the overall survival in 91 patients who underwent radical surgery, the s-SKI-Ab-positive and s-TMED5-Ab-negative statuses were significantly associated with a favorable prognosis. Additionally, the combination of s-SKI-Ab-positive and s-TMED5-Ab-negative cases showed an even clearer difference in overall survival as compared with that of s-SKI-Ab-negative and s-TMED5-Ab-positive cases. The s-SKI-Ab and s-TMED5-Ab biomarkers are useful for diagnosing esophageal carcinoma and distinguishing between favorable and poor prognoses.

Overexpressed ski efficiently promotes neurorestoration, increases neuronal regeneration, and reduces astrogliosis after traumatic brain injury.

Traumatic brain injury (TBI) survivors suffer from long-term disability and neuropsychiatric sequelae due to irreparable brain tissue destruction. However, there are still few efficient therapies to promote neurorestoration in damaged brain tissue. This study aimed to investigate whether the pro-oncogenic gene ski can promote neurorestoration after TBI. We established a ski-overexpressing experimental TBI mouse model using adenovirus-mediated overexpression through immediate injection after injury. Hematoxylin-eosin staining, MRI-based 3D lesion volume reconstruction, neurobehavioral tests, and analyses of neuronal regeneration and astrogliosis were used to assess neurorestorative efficiency. The effects of ski overexpression on the proliferation of cultured immature neurons and astrocytes were evaluated using imaging flow cytometry. The Ski protein level increased in the perilesional region at 3 days post injury. ski overexpression further elevated Ski protein levels up to 14 days post injury. Lesion volume was attenuated by approximately 36-55% after ski overexpression, with better neurobehavioral recovery, more newborn immature and mature neurons, and less astrogliosis in the perilesional region. Imaging flow cytometry results showed that ski overexpression elevated the proliferation rate of immature neurons and reduced the proliferation rate of astrocytes. These results show that ski can be considered a novel neurorestoration-related gene that effectively promotes neurorestoration, facilitates neuronal regeneration, and reduces astrogliosis after TBI.

Understanding Early Stage Myelodysplastic Syndrome Pathobiology

Understanding myelodysplastic-syndrome (MDS) pathobiology is critical to derive and evaluate new therapeutic interventions. Transforming growth factor-beta (TGFβ) signaling is abnormally active in early-stage MDS patient hematopoietic stem and progenitors, and the level of TGFβ-signal-induced transcriptional changes may have prognostic value. However, the mechanism of TGFβ signaling resulting in ineffective hematopoiesis in MDS is not known. TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7) which block promiscuous activity. Upon TGFβ receptor signaling, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by downstream signaling and subsequently terminate the TGFβ signal (a negative feedback loop). The Verma lab demonstrated that human MDS marrow cells have elevated levels of microRNA-21 (miR-21) that target SMAD7. Their work identifies miR-21 as one factor in MDS that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal. We used transient transcription assays to reveal that miR-21 targets the SKI corepressor, a TGFβ-signaling antagonist that binds to R-SMADS to block downstream SMAD-target gene activation. Immunofluorescence staining of primary bone-marrow samples from early-stage MDS patients (with elevated miR-21 and chronic TGFβ) shows significant reduction of not only SMAD7 but also SKI protein. TGFβ signaling induces ubiquitin mediated degradation of SKI, so low SKI expression in MDS is the sum of miR-21 and chronic TGFβ effects. To determine the functional impact of low SKI we examined Ski-/- mice. We show that SKI loss of function is itself sufficient to impair hematopoietic stem cell (HSC) fitness. While flow cytometric evaluation of Ski-/- hematopoiesis indicates little phenotypic change in HSC fractions, and Ski-/- HSC engraft lethally irradiated recipients, competitive HSC transplantation reveals profound Ski-/- HSC fitness defects. Ski-like (Sno/Skil) encodes a SKI-related compressor that is not a miR-21 target. Ski-/- defects are not phenocopied in Skil-/- HSC; underscoring the specificity of the HSC defects to the hypothesized miR-21-SKI axis. In agreement with this, retroviral or lentiviral vector forced expression of miR-21 in HSC is counter-selected within 60 days after transplantation. The Growth factor independent-1 (GFI1) transcriptional repressor is required for HSC fitness and is an independent prognostic indicator in AML/MDS. We previously demonstrated GFI1 transcriptional repression of miR21 . Subsequently, SKI protein levels are significantly decreased in Gfi1-/- bone marrow cells (which express elevated miR-21). SKI expression is rescued in Gfi1-/-miR-21-/- knockout mice but Gfi1-/- HSC fitness is not restored. Thus, it is unlikely that loss of SKI downstream of Gfi1 loss of function explains Gfi1-/- HSC fitness defects. Instead, we show that the SKI corepressor binds the GFI1 transcriptional repressor. Native GFI1 and SKI proteins are in the same complex by immunoprecipitation, and synthetic-protein pull-down assays validated direct interaction and mapped the interacting domains. ChIP Seq reveals that Ski is coincident with Gfi1 on a subset of common peaks. We conclude that: 1) in normal cells GFI1 repression of miR-21 protects expression of SKI, an HSC-relevant GFI1 corepressor, and 2) chronic TGFβ signaling in early stage MDS directly affects HSC fitness by targeting SKI. Disclosures No relevant conflicts of interest to declare.

Ski promotes proliferation and inhibits apoptosis in fibroblasts under high-glucose conditions via the FoxO1 pathway.

ObjectivesThe present study clarified the role and signalling pathway of Ski in regulating proliferation and apoptosis in fibroblasts under high‐glucose (HG) conditions.Materials and MethodsThe proliferation and apoptosis of rat primary fibroblasts were assessed using EdU incorporation and TUNEL assays. The protein and phosphorylation levels of the corresponding factors were measured using immunofluorescence staining and Western blotting. Immunoprecipitation was used to determine the interactions between Ski and FoxO1 or Ski and HDAC1. The Ski protein was overexpressed via recombinant adenovirus transfection, and FoxO1 and HDAC1 were knocked down using targeted small‐interfering RNA.ResultsThe present study found that HG inhibited fibroblast proliferation, increased apoptosis and reduced Ski levels in rat primary fibroblasts. Conversely, increasing Ski protein levels alleviated HG‐induced proliferation inhibition and apoptosis promotion. Increasing Ski protein levels also increased Ski binding to FoxO1 to decrease FoxO1 acetylation, and interfering with FoxO1 caused loss of the regulatory effect of Ski in fibroblasts under HG. Increasing Ski protein levels decreased FoxO1 acetylation via HDAC1‐mediated deacetylation.ConclusionsTherefore, these findings confirmed for the first time that Ski regulated fibroblast proliferation and apoptosis under HG conditions via the FoxO1 pathway.

The Oncoprotein SKI Acts as A Suppressor of NK Cell-Mediated Immunosurveillance in PDAC.

Simple SummaryPancreatic ductal adeno carcinoma is one of the most lethal solid tumors and the survival rate has not improved significantly over the past decades. The disease is characterized by an immune-suppressive tumor microenvironment, which promotes the limited response to novel immunotherapies. The aim of our study was to contribute to a better understanding of the diminished Natural Killer (NK) cell-activity in pancreatic cancer. We showed that oncoprotein SKI, which is involved in CBP/p300-mediated acetylation, diminished the expression of activating ligands for the cytotoxicity receptor NKG2D on tumor cells, thereby counteracting NK cell-dependent cytotoxicity. Treatment of tumor cells with histone deacetylase inhibitors (HDACi) induced the expression of these ligands and improved NK cell-dependent killing. Thus, we unraveled a so far unknown role of SKI in NK cell-mediated immunosurveillance. Our results suggest that the combination of HDACi with NK cell-based immunotherapies may be beneficial for pancreatic cancer patients.Drugs targeting epigenetic mechanisms such as histone deacetylase inhibitors (HDACi) suppress tumor growth. HDACi also induce the expression of ligands for the cytotoxicity receptor NKG2D rendering tumors more susceptible to natural killer (NK) cell-dependent killing. The major acetylases responsible for the expression of NKG2D ligands (NKG2D-L) are CBP and p300. The role of the oncogene and transcriptional repressor SKI, an essential part of an HDAC-recruiting co-repressor complex, which competes with CBP/p300 for binding to SMAD3 in TGFβ signaling, is unknown. Here we show that the siRNA-mediated downregulation of SKI in the pancreatic cancer cell lines Panc-1 and Patu8988t leads to an increased target cell killing by primary NK cells. However, the higher cytotoxicity of NK cells did not correlate with the induction of NKG2D-L. Of note, the expression of NKG2D-L and consequently NK cell-dependent killing could be induced upon LBH589 (LBH, panobinostat) or valproic acid (VPA) treatment irrespective of the SKI expression level but was significantly higher in pancreatic cancer cells upon genetic ablation of SKI. These data suggest that SKI represses the inducible expression of NKG2D-L. The combination of HDACi with NK cell-based immunotherapy is an attractive treatment option for pancreatic tumors, specifically for patients with high SKI protein levels.

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.

Inside the USCAP Journals

Inside the USCAP Journals

SKI Controls MDS-Associated Chronic TGFb Signaling, Aberrant Splicing, and Stem Cell Fitness

The transforming growth factor-beta (TGFβ) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGFb signaling becomes chronic in early-stage myelodysplasia (MDS) where it may select MDS-genic HSC clones. In MDS, the level of TGFb signaling may have prognostic value while TGFβ-receptor inhibitors improve hematopoiesis in MDS samples. Thus, to better understand MDS pathobiology, it is vital to understand the mechanisms underlying chronic TGFb signaling. Normally, TGFβ signaling is tightly controlled by antagonists (e.g. SMAD7, SKI) which block promiscuous activity. Upon ligand-receptor engagement, these antagonists are transiently eliminated to amplify the signal; however, they are re-induced by TGFβ signaling and subsequently terminate the signal (a negative feedback loop). Early-stage MDS marrow cells have significantly diminished expression of SMAD7 and elevated levels of microRNA-21 (miR-21), which targets SMAD7. Thus, miR-21 is one factor that interferes with the TGFβ negative feedback loop to generate a chronic TGFβ signal in MDS.We bioinformatically reanalyzed a recently published RNA-Seq dataset of MDS patient samples and find that a TGFb-signal-correlated gene signature is sufficient to identify a population of MDS patients with abnormal RNA splicing (e.g. CSF3R) independent of splicing factor mutations, and coincident with low HNRNPK activity. Elevated levels of SKI mRNA, encoding a transcriptional corepressor and TGFb-antagonist, are sufficient to identify these patients. We questioned why elevated SKI mRNA (encoding a TGFβ-antagonist) would be associated with chronic activation of TGFβ. We show that miR-21 targets SKI to block translation, and that event is associated with slight elevation in SKI mRNA abundance. Moreover, SKI protein is reduced in primary bone marrow samples from early-stage MDS patients with elevated miR-21 and chronic TGFβ signaling. To determine the impact of SKI loss, we examined murine Ski-/- HSC. Ski-/- HSC are initially specified and rescue transplant recipients in the absence of competitors, where they participate in multilineage hematopoiesis. However, when challenged with wild-type competitors, Ski-/- HSC display a profound defect in HSC fitness that can be rescued by increasing the number of Ski-/- HSC transplanted. Ski wild type and null embryonic stem cell - blastocyst complementation assays confirm an intrinsic Ski-/- HSC defect in the absence of transplantation. Using single-cell RNA-Seq, Ski-/- HSC exhibited striking upregulation of TGFb signaling, including Tgfb1 itself. Novel bioinformatics single-cell-splicing analyses revealed splicing alterations in Ski-/- HSC concordant with low HNRNPK activity. Strikingly, a large fraction of the differentially expressed genes and alternative splicing events in Ski-/- HSC are found in SKI-high MDS patients. We conclude that miR-21-mediated loss of SKI contributes to early stage MDS pathogenesis by activating TGFβ signaling and alternative splicing while hindering HSC fitness. DisclosuresNo relevant conflicts of interest to declare.

SKI controls MDS-associated chronic TGF-β signaling, aberrant splicing, and stem cell fitness

AbstractThe transforming growth factor beta (TGF-β) signaling pathway controls hematopoietic stem cell (HSC) behavior in the marrow niche; however, TGF-β signaling becomes chronic in early-stage myelodysplastic syndrome (MDS). Although TGF-β signaling normally induces negative feedback, in early-stage MDS, high levels of microRNA-21 (miR-21) contribute to chronic TGF-β signaling. We found that a TGF-β signal–correlated gene signature is sufficient to identify an MDS patient population with abnormal RNA splicing (eg, CSF3R) independent of splicing factor mutations and coincident with low HNRNPK activity. Levels of SKI messenger RNA (mRNA) encoding a TGF-β antagonist are sufficient to identify these patients. However, MDS patients with high SKI mRNA and chronic TGF-β signaling lack SKI protein because of miR-21 activity. To determine the impact of SKI loss, we examined murine Ski−/− HSC function. First, competitive HSC transplants revealed a profound defect in stem cell fitness (competitive disadvantage) but not specification, homing, or multilineage production. Aged recipients of Ski−/− HSCs exhibited mild phenotypes similar to phenotypes in those with macrocytic anemia. Second, blastocyst complementation revealed a dramatic block in Ski−/− hematopoiesis in the absence of transplantation. Similar to SKI-high MDS patient samples, Ski−/− HSCs strikingly upregulated TGF-β signaling and deregulated expression of spliceosome genes (including Hnrnpk). Moreover, novel single-cell splicing analyses demonstrated that Ski−/− HSCs and high levels of SKI expression in MDS patient samples share abnormal alternative splicing of common genes (including those that encode splicing factors). We conclude that miR-21–mediated loss of SKI activates TGF-β signaling and alternative splicing to impair the competitive advantage of normal HSCs (fitness), which could contribute to selection of early-stage MDS-genic clones.

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. DisclosuresNo relevant conflicts of interest to declare.

Novel Regulation of the Stability of Transcriptional Cofactors Ski and SnoN by GPCR Signals and Actin‐Cytoskeleton Dynamics

TGF‐β signaling controls several cellular processes from growth arrest to apoptosis in epithelial cells. The transcriptional cofactors Ski and SnoN are main regulators of TGF‐β signaling by sequestering SMAD proteins or recruiting transcriptional repressor complexes at TGF‐β/SMADs target genes. TGF‐β signaling regulates the stability of Ski and SnoN proteins through the ubiquitin‐proteasome system. Even though the important role that Ski and SnoN proteins play in early development and homeostasis, cell cycle was the unique signal besides TGF‐β pathway that regulated their stability. Nevertheless, the mechanisms that regulate Ski and SnoN protein stability in diverse cellular processes remain unknown. In this study, we found out that the actin cytoskeleton dynamics is a novel regulator of Ski and SnoN protein stability in hepatocytes. Inhibitors of actin cytoskeleton dynamics and cell confluence status regulate Ski and SnoN protein levels. Actin polymerization is a signal for Ski protein degradation via proteasome. We also demonstrated that G protein‐coupled receptors (GPCR) signaling control Ski and SnoN protein levels through promoting and decreasing its stability by cAMP or Rho signals, respectively. In conclusion, we identified a new regulation for Ski and SnoN protein stability by changes in actin cytoskeleton rearrangements. These findings suggest that changes in Ski and SnoN protein stability could be controlled by this novel regulation in physiological and pathological events as a result of changes in actin cytoskeleton dynamics induced by cell contact, extracellular matrix remodeling and soluble ligands; thus, it will be important to evaluate TGF‐β/SMADs signaling in all these cellular contexts.Support or Funding InformationThis work is supported by grants from DGAPA/PAPIIT/UNAM (IN208115) and CONACYT (No. 240224).

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

The Ski Protein is Involved in the Transformation Pathway of Aurora Kinase A.

Oncogenic kinase Aurora A (AURKA) has been found to be overexpresed in several tumors including colorectal, breast, and hematological cancers. Overexpression of AURKA induces centrosome amplification and aneuploidy and it is related with cancer progression and poor prognosis. Here we show that AURKA phosphorylates in vitro the transcripcional co-repressor Ski on aminoacids Ser326 and Ser383. Phosphorylations on these aminoacids decreased Ski protein half-life. Reduced levels of Ski resulted in centrosomes amplification and multipolar spindles formation, same as AURKA overexpressing cells. Importantly, overexpression of Ski wild type, but not S326D and S383D mutants inhibited centrosome amplification and cellular transformation induced by AURKA. Altogether, these results suggest that the Ski protein is a target in the transformation pathway mediated by the AURKA oncogene.

Transcriptional Control of HSC Fitness

To sustain lifetime hematopoiesis, adult hematopoietic stem cells (HSCs) are required to remain quiescent. Proper control over cell cycle status underlies HSC fitness. Although the identity of cell cycle mediators which control HSC cycling are basically understood, the gene regulatory networks controlling them are largely unknown. Gfi1 encodes a transcriptional repressor protein that is required to maintain HSC quiescence. Over-cycling of stem-progenitors causes Gfi1-/- HSC exhaustion and prevents long-term engraftment in lethally-ablated recipients. Gfi1 controls myeloid progenitor maintenance through the direct regulation of HoxA9, but the transcriptional programming necessary to control HSC function is not known. Gfi1-mediated repression is critically dependent upon the recruitment of known corepressors; including the Lsd1/CoREST complex, and Eto proteins. Moreover, Gfi1 directly represses a suite of microRNA genes, including miR-21, miR-196b and miR-302b. We were intrigued by a series of papers in which miR-302b was shown to target the Lsd1 corepressor, which was subsequently shown to be required for HSC fitness. Lsd1 serves as a transcriptional co-repressor for Gfi1, Gfi1b, Scl1/Tal1, Bcl11a, Sall4, and Runx1. Knockdown and genetic ablation studies demonstrated that loss of Lsd1 results in a derepression of stem/progenitor genes, and leads to defects in HSC self-renewal and marrow failure. Given this published example, we next sought to determine whether miR-196b and miR-21 might also affect Gfi1 cofactors. First, we find that miR-196b targets CBFA2T3/Mtg16, encoding Eto2 (in human and mouse, respectively). Eto2 is the most abundant Eto family protein in hematopoietic stem and progenitors, and functions as a transcriptional co-repressor with Gfi1, Gfi1b, Scl1/Tal1, Gata-1, Lmo2, and Ldb1. In luciferase-sensor assays for the CBFA2T3-3'UTR, cotransfection with a miR-196b mimic reduces luciferase activity, dependent upon a canonical miR-196b binding site. In agreement with published data, we find that Mtg16-/- HSC are reduced in number and are impaired in competitive transplant assays, but this defect can be partially alleviated by increasing Mtg16-/- HSC numbers. We also find that the initial serial transplant of Mtg16-/- HSC leads to immediate exhaustion. Second, we find that miR-21 targets Ski which functions as a transcriptional corepressor for Smad, Gli, MAD, and thyroid hormone receptor transcription factors. In luciferase-sensor assays for the Ski 3'UTR, cotransfection with a miR-21 mimic reduces luciferase activity, dependent upon a canonical miR-21 binding site. We find that both synthetic and endogenous Ski and Gfi1 proteins directly and specifically interact with one another, implicating Ski as a new Gfi1-corepressor. Recently, overexpression of Ski was shown to enhance HSC repopulation in transplant recipients and caused myeloproliferative disease by promoting HSC gene signatures independent of Tgf-β signaling. Ski-/- is perinatal lethal due to cleft palate, so we analyzed Ski-/- hematopoiesis by transplanting E14.5 fetal liver cells. Similar to Mtg16-/- HSC, Ski-/- HSCs display impaired competitive engraftment that can be rescued by increasing Ski-/- HSC numbers. We also find that the initial serial transplant of Ski-/- HSC leads to immediate exhaustion. Finally, in Gfi1-/- lineage negative bone marrow (which expresses elevated levels of miR-21 and miR-196b) the level of Ski and Eto2 protein are significantly decreased. Our findings suggest that Gfi1 repression of individual Gfi1-target miR genes is required for the proper control of Gfi1 co-factors and subsequent HSC biology. To test this concept we generated mice with tetracycline-inducible miR-196b expression (in mice with intact Gfi1 expression). After only one month of transgene induction we find significant HSC exhaustion. Our data suggests that repression of Gfi1 target genes miR-21, miR-196b and miR-302b is critical for normal HSC function, because these miR target essential transcriptional cofactors. DisclosuresNo relevant conflicts of interest to declare.

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

Sloan-Kettering virus gene, a product of a cellular proto-oncogene c-Ski is a unique nuclear pro-oncoprotein and belongs to the Ski/Sno proto-oncogene family. The aim of the present study was to locate Ski protein in rat ovaries in order to find insights into the possible involvement of Ski in follicular development. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). RT-PCR and in situ hybridization revealed that c-Ski mRNA was expressed in the ovaries of the adult rat on the day of estrous and localized mainly in the granulose cells. Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in rats having atretic follicles with double staining. These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles. Based on the present findings, Ski may play a role in the apoptosis of granulosa cells during follicular atresia.

Actin-cytoskeleton polymerization differentially controls the stability of Ski and SnoN co-repressors in normal but not in transformed hepatocytes

BackgroundSki and SnoN proteins function as transcriptional co-repressors in the TGF-β pathway. They regulate cell proliferation and differentiation, and their aberrant expression results in altered TGF-β signalling, malignant transformation, and alterations in cell proliferation. MethodsWe carried out a comparative characterization of the endogenous Ski and SnoN protein regulation by TGF-β, cell adhesion disruption and actin-cytoskeleton rearrangements between normal and transformed hepatocytes; we also analyzed Ski and SnoN protein stability, subcellular localization, and how their protein levels impact the TGF-β/Smad-driven gene transcription. ResultsSki and SnoN protein levels are lower in normal hepatocytes than in hepatoma cells. They exhibit a very short half-life and a nuclear/cytoplasmic distribution in normal hepatocytes opposed to a high stability and restricted nuclear localization in hepatoma cells. Interestingly, while normal cells exhibit a transient TGF-β-induced gene expression, the hepatoma cells are characterized by a strong and sustained TGF-β-induced gene expression. A novel finding is that Ski and SnoN stability is differentially regulated by cell adhesion and cytoskeleton rearrangements in the normal hepatocytes. The inhibition of protein turnover down-regulated both Ski and SnoN co-repressors impacting the kinetic of expression of TGF-β-target genes. ConclusionNormal regulatory mechanisms controlling Ski and SnoN stability, subcellular localization and expression are altered in hepatocarcinoma cells. General significanceThis work provides evidence that Ski and SnoN protein regulation is far more complex in normal than in transformed cells, since many of the normal regulatory mechanisms are lost in transformed cells.

Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes

TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

Protective Effect of Triptolide against Glomerular Mesangial Cell Proliferation and Glomerular Fibrosis in Rats Involves the TGF- β 1/Smad Signaling Pathway.

Triptolide as a main active ingredient of Tripterygium wilfordii is 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.

Downregulation of SnoN oncoprotein induced by antibiotics anisomycin and puromycin positively regulates transforming growth factor-β signals

BackgroundSnoN and Ski proteins function as Smad transcriptional corepressors and are implicated in the regulation of diverse cellular processes such as proliferation, differentiation and transformation. Transforming growth factor-β (TGF-β) signaling causes SnoN and Ski protein degradation via proteasome with the participation of phosphorylated R-Smad proteins. Intriguingly, the antibiotics anisomycin (ANS) and puromycin (PURO) are also able to downregulate Ski and SnoN proteins via proteasome. MethodsWe explored the effects of ANS and PURO on SnoN protein downregulation when the activity of TGF-β signaling was inhibited by using different pharmacological and non-pharmacological approaches, either by using specific TβRI inhibitors, overexpressing the inhibitory Smad7 protein, or knocking-down TβRI receptor or Smad2 by specific shRNAs. The outcome of SnoN and Ski downregulation induced by ANS or PURO on TGF-β signaling was also studied. ResultsSnoN protein downregulation induced by ANS and PURO did not involve the induction of R-Smad phosphorylation but it was abrogated after TGF-β signaling inhibition; this effect occurred in a cell type-specific manner and independently of protein synthesis inhibition or any other ribotoxic effect. Intriguingly, antibiotics seem to require components of the TGF-β/Smad pathway to downregulate SnoN. In addition, SnoN protein downregulation induced by antibiotics favored gene transcription induced by TGF-β signaling. ConclusionsANS and PURO require TGF-β/Smad pathway to induce SnoN and Ski protein downregulation independently of inducing R-Smad2 phosphorylation, which facilitates TGF-β signaling. General significanceAntibiotic analogs lacking ribotoxic effects are useful as pharmacological tools to study TGF-β signaling by controlling Ski and SnoN protein levels.

Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.