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1.  Beneficial Effects of Canagliflozin in Combination with Pioglitazone on Insulin Sensitivity in Rodent Models of Obese Type 2 Diabetes 
PLoS ONE  2015;10(1):e0116851.
Background
Despite its insulin sensitizing effects, pioglitazone may induce weight gain leading to an increased risk of development of insulin resistance. A novel sodium glucose co-transporter 2 (SGLT2) inhibitor, canagliflozin, provides not only glycemic control but also body weight reduction through an insulin-independent mechanism. The aim of this study was to investigate the combined effects of these agents on body weight control and insulin sensitivity.
Methods
Effects of combination therapy with canagliflozin and pioglitazone were evaluated in established diabetic KK-Ay mice and prediabetic Zucker diabetic fatty (ZDF) rats.
Results
In the KK-Ay mice, the combination therapy further improved glycemic control compared with canagliflozin or pioglitazone monotherapy. Furthermore, the combination significantly attenuated body weight and fat gain induced by pioglitazone and improved hyperinsulinemia. In the ZDF rats, early intervention with pioglitazone monotherapy almost completely prevented the progressive development of hyperglycemia, and no further improvement was observed by add-on treatment with canagliflozin. However, the combination significantly reduced pioglitazone-induced weight gain and adiposity and improved the Matsuda index, suggesting improved whole-body insulin sensitivity.
Conclusions
Our study indicates that combination therapy with canagliflozin and pioglitazone improves insulin sensitivity partly by preventing glucotoxicity and, at least partly, by attenuating pioglitazone-induced body weight gain in two different obese diabetic animal models. This combination therapy may prove to be a valuable option for the treatment and prevention of obese type 2 diabetes.
doi:10.1371/journal.pone.0116851
PMCID: PMC4304810  PMID: 25615826
2.  Novel Indole-N-glucoside, TA-1887 As a Sodium Glucose Cotransporter 2 Inhibitor for Treatment of Type 2 Diabetes 
Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.
doi:10.1021/ml400339b
PMCID: PMC4027614  PMID: 24900773
Type 2 diabetes; sodium glucose cotransporter 2 inhibitor; urinary glucose excretion; indole-N-glucoside; TA-1887; antihyperglycemic effect
3.  Skp2 deletion unmasks a p27 safeguard that blocks tumorigenesis in the absence of pRb and p53 tumor suppressors 
Cancer cell  2013;24(5):10.1016/j.ccr.2013.09.021.
SUMMARY
pRb and p53 are two major tumor suppressors. Here, we found that p53 activates expression of Pirh2 and KPC1, two of the three ubiquitin ligases for p27. Loss of p53 in the absence of Skp2, the third ubiquitin ligase for p27, shrinks the cellular pool of p27 ubiquitin ligases to accumulate p27 protein. In the absence of pRb and p53, p27 was unable to inhibit DNA synthesis in spite of its abundance, but could inhibit division of cells that maintain DNA replication with re-replication. This mechanism blocked pRb and p53 doubly deficient pituitary and prostate tumorigenesis lastingly coexistent with BrdU-labeling neoplastic lesions, revealing an unconventional cancer cell vulnerability when pRb and p53 are inactivated.
doi:10.1016/j.ccr.2013.09.021
PMCID: PMC3880806  PMID: 24229711
4.  Skp2 suppresses apoptosis in Rb1 deficient tumors by limiting E2F1 activity 
Nature communications  2014;5:3463.
One mechanism of tumor suppression by pRb is repressing E2F1. Hence, E2f1 deletion diminishes tumorigenesis following Rb1 loss. However, E2F1 promotes both proliferation and apoptosis. It therefore remains unclear how de-repressed E2F1 promotes tumorigenesis. Another mechanism of pRb function is repressing Skp2 to elevate p27 to arrest proliferation. However, Skp2 deletion induced apoptosis, not proliferation arrest, in Rb1 deficient pituitary tumorigenesis. Here, we show that Rb1 deletion induces higher expression of E2F1 target genes in the absence of Skp2. E2F1 binds less cyclin A but more target promoters when Rb1 is deleted with Skp2 knockout or p27T187A knockin, suggesting that stabilized p27 prevents cyclin A from binding and inhibiting E2F1. In Rb1 deficient pituitary tumorigenesis, Skp2 deletion or p27T187A mutation converts E2F1’s role from proliferative to apoptotic. These findings delineate a pRb-Skp2-p27-cyclin A-E2F1 pathway that determines whether E2F1 is proliferative or apoptotic in Rb1 deficient tumorigenesis.
doi:10.1038/ncomms4463
PMCID: PMC3982150  PMID: 24632684
Skp2 ubiquitin ligase; Rb1 tumor suppressor; E2F1 transcription factor; cyclin A; p27
5.  HCV Infection Enhances Th17 Commitment, Which Could Affect the Pathogenesis of Autoimmune Diseases 
PLoS ONE  2014;9(6):e98521.
Background
Various kinds of autoimmune diseases have been reported to have a significant relationship with persistent hepatitis c virus (HCV) infection and Th17 cells. Previously, our group reported that the existence of HCV in T lymphocytes could affect the development of CD4+ helper T cells and their proliferation, in addition to the induction of immunoglobulin hyper-mutation.
Methods
Therefore, we analyzed the relationship between persistent infection of HCV and the mechanism of Th17 cell induction ex vivo and in vitro.
Results
The prevalence of autoimmune-related diseases in chronic hepatitis c patients (CH-C) was significantly higher than in other types of chronic hepatitis (hepatitis B and NASH). A significantly higher frequency of IL6 and TGF-β double-high patients was detected in CH-C than in other liver diseases. Moreover, these double-high patients had significantly higher positivity of anti-nuclear antibody, cryoglobulinemia, and lymphotropic HCV and higher amounts of IL1-β, IL21, IL23. In addition to the previously reported lymphotropic SB-HCV strain, we found a novel, genotype 1b lymphotropic HCV (Ly-HCV), by deep sequencing analysis. Lymphotropic-HCV replication could be detected in the lymphoid cells with various kinds of cytokine-conditions including IL1β, IL23, IL6 and TGF-β in vitro. Infection by HCV could significantly enhance the development of Th17 cells. The HCV protein responsible for inducing the Th17 cells was HCV-Core protein, which could enhance the STAT-3 signaling and up-regulate the expression of RORγt as a Th17 master gene.
Conclusion
Infection by lymphotropic HCV might enhance the Th17 development and contribute to understanding the pathogenesis of autoimmune-related diseases.
doi:10.1371/journal.pone.0098521
PMCID: PMC4048196  PMID: 24905921
6.  Identification of acquired mutations by whole-genome sequencing in GATA-2 deficiency evolving into myelodysplasia and acute leukemia 
Annals of Hematology  2014;93(9):1515-1522.
Heterozygous GATA-2 germline mutations are associated with overlapping clinical manifestations termed GATA-2 deficiency, characterized by immunodeficiency and predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, there is considerable clinical heterogeneity among patients, and the molecular basis for the evolution of immunodeficiency into MDS/AML remains unknown. Thus, we conducted whole-genome sequencing on a patient with a germline GATA-2 heterozygous mutation (c. 988 C > T; p. R330X), who had a history suggestive of immunodeficiency and evolved into MDS/AML. Analysis was conducted with DNA samples from leukocytes for immunodeficiency, bone marrow mononuclear cells for MDS and bone marrow-derived mesenchymal stem cells. Whereas we did not identify a candidate genomic deletion that may contribute to the evolution into MDS, a total of 280 MDS-specific nonsynonymous single nucleotide variants were identified. By narrowing down with the single nucleotide polymorphism database, the functional missense database, and NCBI information, we finally identified three candidate mutations for EZH2, HECW2 and GATA-1, which may contribute to the evolution of the disease.
Electronic supplementary material
The online version of this article (doi:10.1007/s00277-014-2090-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00277-014-2090-4
PMCID: PMC4119934  PMID: 24782121
GATA-2; GATA-2 deficiency; MonoMAC; Myelodysplastic syndrome; Whole-genome sequencing; EZH2; GATA-1
7.  TBX1 Mutation Identified by Exome Sequencing in a Japanese Family with 22q11.2 Deletion Syndrome-Like Craniofacial Features and Hypocalcemia 
PLoS ONE  2014;9(3):e91598.
Background
Although TBX1 mutations have been identified in patients with 22q11.2 deletion syndrome (22q11.2DS)-like phenotypes including characteristic craniofacial features, cardiovascular anomalies, hypoparathyroidism, and thymic hypoplasia, the frequency of TBX1 mutations remains rare in deletion-negative patients. Thus, it would be reasonable to perform a comprehensive genetic analysis in deletion-negative patients with 22q11.2DS-like phenotypes.
Methodology/Principal Findings
We studied three subjects with craniofacial features and hypocalcemia (group 1), two subjects with craniofacial features alone (group 2), and three subjects with normal phenotype within a single Japanese family. Fluorescence in situ hybridization analysis excluded chromosome 22q11.2 deletion, and genomewide array comparative genomic hybridization analysis revealed no copy number change specific to group 1 or groups 1+2. However, exome sequencing identified a heterozygous TBX1 frameshift mutation (c.1253delA, p.Y418fsX459) specific to groups 1+2, as well as six missense variants and two in-frame microdeletions specific to groups 1+2 and two missense variants specific to group 1. The TBX1 mutation resided at exon 9C and was predicted to produce a non-functional truncated protein missing the nuclear localization signal and most of the transactivation domain.
Conclusions/Significance
Clinical features in groups 1+2 are well explained by the TBX1 mutation, while the clinical effects of the remaining variants are largely unknown. Thus, the results exemplify the usefulness of exome sequencing in the identification of disease-causing mutations in familial disorders. Furthermore, the results, in conjunction with the previous data, imply that TBX1 isoform C is the biologically essential variant and that TBX1 mutations are associated with a wide phenotypic spectrum, including most of 22q11.2DS phenotypes.
doi:10.1371/journal.pone.0091598
PMCID: PMC3956758  PMID: 24637876
8.  PKCδ Deficiency Perturbs Bone Homeostasis by Selective Uncoupling of Cathepsin K Secretion and Ruffled Border Formation in Osteoclasts 
Bone homeostasis requires stringent regulation of osteoclasts, which secrete proteolytic enzymes to degrade the bone matrix. Despite recent progress in understanding how bone resorption occurs, the mechanisms regulating osteoclast secretion, and in particular the trafficking route of cathepsin K vesicles, remain elusive. Using a genetic approach, we describe the requirement for PKCδ in regulating bone resorption by affecting cathepsin K exocytosis. Importantly, PKCδ deficiency does not perturb formation of the ruffled border or trafficking of lysosomal vesicles containing the v-ATPase. Mechanistically, we find that cathepsin K exocytosis is controlled by PKCδ through modulation of the actin bundling protein MARCKS. The relevance of our finding is emphasized in vivo as PKCδ−/− mice exhibit increased bone mass and are protected from pathological bone loss in a model of experimental post-menopausal osteoporosis. Collectively, our data provide novel mechanistic insights into the pathways that selectively promote secretion of cathepsin K lysosomes independently of ruffled border formation, providing evidence for the presence of multiple mechanisms that regulate lysosomal exocytosis in osteoclasts.
doi:10.1002/jbmr.1701
PMCID: PMC3498518  PMID: 22806935
9.  Ras-Induced Changes in H3K27me3 Occur after Those in Transcriptional Activity 
PLoS Genetics  2013;9(8):e1003698.
Oncogenic signaling pathways regulate gene expression in part through epigenetic modification of chromatin including DNA methylation and histone modification. Trimethylation of histone H3 at lysine-27 (H3K27), which correlates with transcriptional repression, is regulated by an oncogenic form of the small GTPase Ras. Although accumulation of trimethylated H3K27 (H3K27me3) has been implicated in transcriptional regulation, it remains unclear whether Ras-induced changes in H3K27me3 are a trigger for or a consequence of changes in transcriptional activity. We have now examined the relation between H3K27 trimethylation and transcriptional regulation by Ras. Genome-wide analysis of H3K27me3 distribution and transcription at various times after expression of oncogenic Ras in mouse NIH 3T3 cells identified 115 genes for which H3K27me3 level at the gene body and transcription were both regulated by Ras. Similarly, 196 genes showed Ras-induced changes in transcription and H3K27me3 level in the region around the transcription start site. The Ras-induced changes in transcription occurred before those in H3K27me3 at the genome-wide level, a finding that was validated by analysis of individual genes. Depletion of H3K27me3 either before or after activation of Ras signaling did not affect the transcriptional regulation of these genes. Furthermore, given that H3K27me3 enrichment was dependent on Ras signaling, neither it nor transcriptional repression was maintained after inactivation of such signaling. Unexpectedly, we detected unannotated transcripts derived from intergenic regions at which the H3K27me3 level is regulated by Ras, with the changes in transcript abundance again preceding those in H3K27me3. Our results thus indicate that changes in H3K27me3 level in the gene body or in the region around the transcription start site are not a trigger for, but rather a consequence of, changes in transcriptional activity.
Author Summary
Trimethylation of histone H3 at lysine-27 (H3K27) has been associated with silencing of gene expression. Abnormalities of this modification are thought to contribute to the epigenetic silencing of tumor suppressor genes and are regarded as a hallmark of cancer. It has remained unclear, however, whether the production of trimethylated H3K27 (H3K27me3) is the cause or the consequence of gene silencing. To address this issue, we examined the time courses of changes in H3K27me3 level and those in gene transcription induced by an oncogenic form of the Ras protein, the gene for which is one of the most frequently mutated in human cancer. We found that the amount of H3K27me3 was inversely related to transcriptional activity both at the genome-wide level and at the level of individual genes. However, we also found that the Ras-induced changes in H3K27me3 level occurred after those in transcriptional activity. Our results thus demonstrate that changes in H3K27me3 abundance are a consequence rather than a cause of transcriptional regulation, and they suggest that oncoprotein-driven changes in gene transcription can alter the pattern of histone modification in cancer cells.
doi:10.1371/journal.pgen.1003698
PMCID: PMC3757056  PMID: 24009517
10.  Loss of Protein Kinase C-δ Protects against LPS-Induced Osteolysis Owing to an Intrinsic Defect in Osteoclastic Bone Resorption 
PLoS ONE  2013;8(8):e70815.
Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (−α, −β and −γ), novel PKCs (−δ, −ε, −η and −θ) and atypical PKCs (−ι/λ and −ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC −α, −γ and −ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.
doi:10.1371/journal.pone.0070815
PMCID: PMC3738588  PMID: 23951014
11.  Acetylation-Dependent Regulation of Skp2 Function 
Cell  2012;150(1):179-193.
SUMMARY
Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.
doi:10.1016/j.cell.2012.05.038
PMCID: PMC3595190  PMID: 22770219
12.  Distinct MicroRNAs Expression Profile in Primary Biliary Cirrhosis and Evaluation of miR 505-3p and miR197-3p as Novel Biomarkers 
PLoS ONE  2013;8(6):e66086.
Background and Aims
MicroRNAs are small endogenous RNA molecules with specific expression patterns that can serve as biomarkers for numerous diseases. However, little is known about the expression profile of serum miRNAs in PBC.
Methods
First, we employed Illumina deep sequencing for the initial screening to indicate the read numbers of miRNA expression in 10 PBC, 5 CH-C, 5 CH-B patients and 5 healthy controls. Comparing the differentially expressed miRNAs in the 4 groups, analysis of variance was performed on the number of sequence reads to evaluate the statistical significance. Hierarchical clustering was performed using an R platform and we have found candidates for specific miRNAs in the PBC patients. Second, a quantitative reverse transcription PCR validation study was conducted in 10 samples in each group. The expression levels of the selected miRNAs were presented as fold-changes (2−ΔΔCt). Finally, computer analysis was conducted to predict target genes and biological functions with MiRror 2.0 and DAVID v6.7.
Results
We obtained about 12 million 32-mer short RNA reads on average per sample and the mapping rates to miRBase were 16.60% and 81.66% to hg19. In the statistical significance testing, the expression levels of 81 miRNAs were found to be differentially expressed in the 4 groups. The heat map and hierarchical clustering demonstrated that the miRNA profiles from PBC clustered with those of CH-B, CH-C and healthy controls. Additionally, the circulating levels of hsa-miR-505-3p, 197-3p, and 500a-3p were significantly decreased in PBC compared with healthy controls and the expression levels of hsa-miR-505-3p, 139-5p and 197-3p were significantly reduced compared with the viral hepatitis group.
Conclusions
Our results indicate that sera from patients with PBC have a unique miRNA expression profile and that the down-regulated expression of hsa-miR-505-3p and miR-197-3p can serve as clinical biomarkers of PBC.
doi:10.1371/journal.pone.0066086
PMCID: PMC3680413  PMID: 23776611
13.  A diacylglycerol-dependent signaling pathway contributes to regulation of anti-bacterial autophagy 
Cell host & microbe  2010;8(2):137-146.
Summary
Autophagy mediates the degradation of cytoplasmic contents in the lysosome and plays a significant role in innate and adaptive immune responses. Lipid second messengers are implicated in the regulation of autophagy but the nature of the lipids involved and their mechanisms of action have yet to be characterized. Here we demonstrate a novel signaling role for diacylglycerol (DAG) in antibacterial autophagy. DAG production was necessary for efficient autophagy of Salmonella and its localization to bacteria-containing phagosomes preceded autophagy. Previous studies have revealed a role for the ubiquitin binding adaptor molecules p62 and NDP52 in autophagy of S. Typhimurium. We observed bacteria-containing autophagosomes colocalizing individually with either DAG or ubiquitinated proteins, indicating that both signals can act independently to promote anti-bacterial autophagy. We determined that the actions of phospholipase D (PLD) and phosphatidic acid phosphatase (PAP) were required for DAG generation and autophagy. The DAG-responsive δ isoform of protein kinase C was required for anti-bacterial autophagy, as were its downstream targets JNK and NADPH oxidase. Pkc1, the single PKC isoform in yeast, was essential for starvation-induced autophagy in Saccharomyces cerevisiae. These findings reveal an important role for DAG-mediated PKC function in mammalian anti-bacterial autophagy, and suggest a conserved role for PKC in autophagy regulation in eukaryotes.
doi:10.1016/j.chom.2010.07.002
PMCID: PMC3668700  PMID: 20674539
14.  Nrf2–MafG heterodimers contribute globally to antioxidant and metabolic networks 
Nucleic Acids Research  2012;40(20):10228-10239.
NF-E2-related factor 2 (Nrf2) is a key transcription factor that is critical for cellular defense against oxidative and xenobiotic insults. Nrf2 heterodimerizes with small Maf (sMaf) proteins and binds to antioxidant response elements (AREs) to activate a battery of cytoprotective genes. However, it remains unclear to what extent the Nrf2–sMaf heterodimers contribute to ARE-dependent gene regulation on a genome-wide scale. We performed chromatin immunoprecipitation coupled with high-throughput sequencing and identified the binding sites of Nrf2 and MafG throughout the genome. Compared to sites occupied by Nrf2 alone, many sites co-occupied by Nrf2 and MafG exhibit high enrichment and are located in species-conserved genomic regions. The ARE motifs were significantly enriched among the recovered Nrf2–MafG-binding sites but not among the Nrf2-binding sites that did not display MafG binding. The majority of the Nrf2-regulated cytoprotective genes were found in the vicinity of Nrf2–MafG-binding sites. Additionally, sequences that regulate glucose metabolism and several amino acid transporters were identified as Nrf2–MafG target genes, suggesting diverse roles for the Nrf2–MafG heterodimer in stress response. These data clearly support the notion that Nrf2–sMaf heterodimers are complexes that regulate batteries of genes involved in various aspects of cytoprotective and metabolic functions through associated AREs.
doi:10.1093/nar/gks827
PMCID: PMC3488259  PMID: 22965115
15.  Use of Illumina Deep Sequencing Technology To Differentiate Hepatitis C Virus Variants 
Journal of Clinical Microbiology  2012;50(3):857-866.
Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus that shows diverse viral populations even in one individual. Though Sanger sequencing has been used to determine viral sequences, deep sequencing technologies are much faster and can perform large-scale sequencing. We demonstrate the successful use of Illumina deep sequencing technology and subsequent analyses to determine the genetic variants and amino acid substitutions in both treatment-naïve (patient 1) and treatment-experienced (patient 7) isolates from HCV-infected patients. As a result, almost the full nucleotide sequence of HCV was detectable for patients 1 and 7. The reads were mapped to the HCV reference sequence. The coverage was 99.8% and the average depth was 69.5× for patient 7, with values of 99.4% (coverage) and 51.1× (average depth) for patient 1. In patient 7, amino acid (aa) 70 in the core region showed arginine, with methionine at aa 91, by Sanger sequencing. Major variants showed the same amino acid sequence, but minor variants were detectable in 18% (6/34 sequences) of sequences, with replacement of methionine by leucine at aa 91. In NS3, 8 amino acid positions showed mixed variants (T72T/I, K213K/R, G237G/S, P264P/S/A, S297S/A, A358A/T, S457S/C, and I615I/M) in patient 7. In patient 1, 3 amino acid positions showed mixed variants (L14L/F/V, S61S/A, and I586T/I). In conclusion, deep sequencing technologies are powerful tools for obtaining more profound insight into the dynamics of variants in the HCV quasispecies in human serum.
doi:10.1128/JCM.05715-11
PMCID: PMC3295113  PMID: 22205816
16.  SCFFbw7 Modulates the NFκB Signaling Pathway by Targeting NFκB2 for Ubiquitination and Destruction 
Cell reports  2012;1(5):434-443.
SUMMARY
The NFκB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFκB2/p100 precursor has been characterized as the fourth IκB type of suppressor for NFκB. However, the molecular mechanism(s) underlying regulated destruction of NFκB2 remains largely unknown. Here, we report that, unlike other IκBs, ubiquitination and destruction of NFκB2 are governed by SCFFbw7 in a GSK3-dependent manner. In Fbw7−/− cells, elevated expression of NFκB2/p100 leads to a subsequent reduction in NFκB signaling pathways and elevated sensitivity to TNFα-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFκBactivity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFκB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFκB2′s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFκB activity.
doi:10.1016/j.celrep.2012.04.002
PMCID: PMC3375724  PMID: 22708077
17.  The Amelioration of Renal Damage in Skp2-Deficient Mice Canceled by p27 Kip1 Deficiency in Skp2−/− p27−/− Mice 
PLoS ONE  2012;7(4):e36249.
SCF-Skp2 E3 ubiquitin ligase (Skp2 hereafter) targets several cell cycle regulatory proteins for degradation via the ubiquitin-dependent pathway. However, the target-specific physiological functions of Skp2 have not been fully elucidated in kidney diseases. We previously reported an increase in Skp2 in progressive nephropathy and amelioration of unilateral ureteral obstruction (UUO) renal injury associated with renal accumulation of p27 in Skp2−/− mice. However, it remains unclear whether the amelioration of renal injury in Skp2−/− mice is solely caused by p27 accumulation, since Skp2 targets several other proteins. Using Skp2−/−p27−/− mice, we investigated whether Skp2 specifically targets p27 in the progressive nephropathy mediated by UUO. In contrast to the marked suppression of UUO renal injury in Skp2−/− mice, progression of tubular dilatation associated with tubular epithelial cell proliferation and tubulointerstitial fibrosis with increased expression of collagen and α-smooth muscle actin were observed in the obstructed kidneys in Skp2−/−p27−/− mice. No significant increases in other Skp2 target proteins including p57, p130, TOB1, cyclin A and cyclin D1 were noted in the UUO kidney in Skp2−/− mice, while p21, c-Myc, b-Myb and cyclin E were slightly increased. Contrary to the ameliorated UUO renal injure by Skp2-deficiency, the amelioration was canceled by the additional p27-deficiency in Skp2−/−p27−/− mice. These findings suggest a pathogenic role of the reduction in p27 targeted by Skp2 in the progression of nephropathy in UUO mice.
doi:10.1371/journal.pone.0036249
PMCID: PMC3338689  PMID: 22558406
18.  Deregulation of the p57-E2F1-p53 Axis Results in Nonobstructive Hydrocephalus and Cerebellar Malformation in Mice ▿ 
Molecular and Cellular Biology  2011;31(20):4176-4192.
The cyclin-dependent kinase inhibitor (CKI) p57Kip2 plays a pivotal role in cell cycle arrest during development, in particular, in the regulation of the entry of proliferating progenitors into quiescence. The gene encoding p57 undergoes genomic imprinting, and impairment of the regulation of p57 expression results in various developmental anomalies in humans and mice. We now show that p57 is expressed predominantly in the subcommissural organ and cerebellar interneurons in the mouse brain and that mice with brain-specific deletion of the p57 gene (Kip2) manifest prominent nonobstructive hydrocephalus as well as cerebellar malformation associated with the loss of Pax2-positive interneuron precursors and their descendants, including Golgi cells and γ-aminobutyric acid-containing neurons of the deep cerebellar nuclei. These abnormalities were found to be attributable to massive apoptosis of precursor cells in the developing brain. The morphological defects of the p57-deficient mice were corrected by knock-in of the gene for the related CKI p27Kip1 at the Kip2 locus. The abnormalities were also prevented by additional genetic ablation of p53 or E2F1. Our results thus implicate p57 in cell cycle arrest in the subcommissural organ and Pax2-positive interneuron precursors, with the lack of p57 resulting in induction of p53-dependent apoptosis due to hyperactivation of E2F1.
doi:10.1128/MCB.05370-11
PMCID: PMC3187288  PMID: 21844226
19.  Fbxw7 regulates lipid metabolism and cell fate decisions in the mouse liver 
E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box– and WD repeat domain–containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7–/– embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver.
doi:10.1172/JCI40725
PMCID: PMC3007132  PMID: 21123947
20.  Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1+/- mice 
Nature genetics  2009;42(1):83-88.
Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied pRb targets partially inhibited tumorigenesis in Rb1+/- mice 1,2,3,4,5,6. Here, we report that inactivation of pRb target Skp2 7,8 completely prevents spontaneous tumorigenesis in Rb1+/- mice. Targeted Rb1 deletion in melanotrophs ablates the entire pituitary intermediate lobe when Skp2 is inactivated. Skp2 inactivation does not inhibit aberrant proliferation of Rb1-deleted melanotrophs, but induces their apoptotic death. Eliminating p27 phosphorylation on T187 in p27T187A knockin mice reproduces the effects of Skp2 knockout, identifying p27 ubiquitination by SCFSkp2 ubiquitin ligase as the underlying mechanism for Skp2’s essential tumorigenic role in this setting. RB1-deficient human retinoblastoma cells also undergo apoptosis after Skp2 knockdown; and ectopic expression of p27, especially the p27T187A mutant, induces apoptosis. These results reveal that Skp2 becomes an essential survival gene when susceptible cells incur Rb1 deficiency.
doi:10.1038/ng.498
PMCID: PMC2990528  PMID: 19966802
21.  Inhibition of FOXO3 Tumor Suppressor Function by βTrCP1 through Ubiquitin-Mediated Degradation in a Tumor Mouse Model 
PLoS ONE  2010;5(7):e11171.
Background
The ubiquitin-proteasome system is the primary proteolysis machine for controlling protein stability of the majority of regulatory proteins including those that are critical for cancer development. The forkhead box transcription factor FOXO3 plays a key role in regulating tumor suppression; however, the control of FOXO3 protein stability remains to be established. It is crucial to elucidate the molecular mechanisms underlying the ubiquitin-mediated degradation of FOXO3 tumor suppressor.
Methodology and Principal Findings
Here we show that βTrCP1 oncogenic ubiquitin E3-ligase interacts with FOXO3 and induces its ubiquitin-dependent degradation in an IκB kinase-β phosphorylation dependent manner. Silencing βTrCP1 augments FOXO3 protein level, resulting in promoting cellular apoptosis in cancer cells. In animal models, increasing FOXO3 protein level by silencing βTrCP1 suppresses tumorigenesis, whereas decreasing FOXO3 by over-expressing βTrCP1 promotes tumorigenesis and tumor growth in vivo.
Conclusions/Significance
This is a unique demonstration that the βTrCP1-mediated FOXO3 degradation plays a crucial role in tumorigenesis. These findings significantly contribute to understanding of the control of FOXO3 stability in cancer cells and may provide opportunities for developing innovative anticancer therapeutic modalities.
doi:10.1371/journal.pone.0011171
PMCID: PMC2896402  PMID: 20625400
22.  Protein Kinase Cδ differentially regulates platelet functional responses 
Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors.
Objective:
To investigate the role of PKCδ in platelets.
Methods and Results:
We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3–induced injury in vivo thrombosis model, PKCδ −/− mice occluded similar to their wild type littermates.
Conclusions:
Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo.
doi:10.1161/ATVBAHA.109.184010
PMCID: PMC2742914  PMID: 19213940
23.  Mechanoregulation of Proliferation▿  
Molecular and Cellular Biology  2009;29(18):5104-5114.
The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.
doi:10.1128/MCB.00465-09
PMCID: PMC2738302  PMID: 19596792
24.  S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo 
Journal of Vascular Surgery  2009;50(5-4):1135-1142.
Objective
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
Conclusion
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
Clinical Relevance
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
doi:10.1016/j.jvs.2009.07.066
PMCID: PMC2774860  PMID: 19878790
25.  Conditional inactivation of Fbxw7 impairs cell-cycle exit during T cell differentiation and results in lymphomatogenesis 
The Journal of Experimental Medicine  2007;204(12):2875-2888.
Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4+CD8+ stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4+CD8+ stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.
doi:10.1084/jem.20062299
PMCID: PMC2118521  PMID: 17984302

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