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1.
Mol Biol Cell ; 26(11): 2139-50, 2015 Jun 01.
Article in English | MEDLINE | ID: mdl-25833708

ABSTRACT

Glycogen synthase kinase-3 (Gsk-3) is a key regulator of multiple signal transduction pathways. Recently we described a novel role for Gsk-3 in the regulation of DNA methylation at imprinted loci in mouse embryonic stem cells (ESCs), suggesting that epigenetic changes regulated by Gsk-3 are likely an unrecognized facet of Gsk-3 signaling. Here we extend our initial observation to the entire mouse genome by enriching for methylated DNA with the MethylMiner kit and performing next-generation sequencing (MBD-Seq) in wild-type and Gsk-3α(-/-);Gsk-3ß(-/-) ESCs. Consistent with our previous data, we found that 77% of known imprinted loci have reduced DNA methylation in Gsk-3-deficient ESCs. More specifically, we unambiguously identified changes in DNA methylation within regions that have been confirmed to function as imprinting control regions. In many cases, the reduced DNA methylation at imprinted loci in Gsk-3α(-/-);Gsk-3ß(-/-) ESCs was accompanied by changes in gene expression as well. Furthermore, many of the Gsk-3-dependent, differentially methylated regions (DMRs) are identical to the DMRs recently identified in uniparental ESCs. Our data demonstrate the importance of Gsk-3 activity in the maintenance of DNA methylation at a majority of the imprinted loci in ESCs and emphasize the importance of Gsk-3-mediated signal transduction in the epigenome.


Subject(s)
DNA Methylation , Embryonic Stem Cells/metabolism , Genetic Loci , Genomic Imprinting , Glycogen Synthase Kinase 3/metabolism , Animals , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , High-Throughput Nucleotide Sequencing , Mice , Mice, Knockout , Signal Transduction
2.
Biol Reprod ; 92(3): 65, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25568307

ABSTRACT

The signaling enzyme glycogen synthase kinase 3 (GSK3) exists as two isoforms-GSK3A and GSK3B. Protein phosphorylation by GSK3 has important signaling roles in several cells. In our past work, we found that both isoforms of GSK3 are present in mouse sperm and that catalytic GSK3 activity correlates with motility of sperm from several species. Here, we examined the role of Gsk3a in male fertility using a targeted gene knockout (KO) approach. The mutant mice are viable, but have a male infertility phenotype, while female fertility is unaffected. Testis weights of Gsk3a(-/-) mice are normal and sperm are produced in normal numbers. Although spermatogenesis is apparently unimpaired, sperm motility parameters in vitro are impaired. In addition, the flagellar waveform appears abnormal, characterized by low amplitude of flagellar beat. Sperm ATP levels were lower in Gsk3a(-/-) mice compared to wild-type animals. Protein phosphatase PP1 gamma2 protein levels were unaltered, but its catalytic activity was elevated in KO sperm. Remarkably, tyrosine phosphorylation of hexokinase and capacitation-associated changes in tyrosine phosphorylation of proteins are absent or significantly lower in Gsk3a(-/-) sperm. The GSK3B isoform was present and unaltered in testis and sperm of Gsk3a(-/-) mice, showing the inability of GSK3B to substitute for GSK3A in this context. Our studies show that sperm GSK3A is essential for male fertility. In addition, the GSK3A isoform, with its highly conserved glycine-rich N terminus in mammals, may have an isoform-specific role in its requirement for normal sperm motility and fertility.


Subject(s)
Glycogen Synthase Kinase 3/deficiency , Glycogen Synthase Kinase 3/physiology , Infertility, Male/etiology , Infertility, Male/physiopathology , Sperm Motility/physiology , Spermatozoa/physiology , Animals , Disease Models, Animal , Genotype , Glycogen Synthase Kinase 3/genetics , Infertility, Male/genetics , Isoenzymes , Male , Mice , Mice, Knockout , Mutation/genetics , Phenotype , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Signal Transduction/genetics , Signal Transduction/physiology , Sperm Motility/genetics , Spermatogenesis/genetics , Spermatogenesis/physiology
3.
J Biol Chem ; 285(53): 41337-47, 2010 Dec 31.
Article in English | MEDLINE | ID: mdl-21047779

ABSTRACT

Glycogen synthase kinase-3 (Gsk-3) isoforms, Gsk-3α and Gsk-3ß, are constitutively active, largely inhibitory kinases involved in signal transduction. Underscoring their biological significance, altered Gsk-3 activity has been implicated in diabetes, Alzheimer disease, schizophrenia, and bipolar disorder. Here, we demonstrate that deletion of both Gsk-3α and Gsk-3ß in mouse embryonic stem cells results in reduced expression of the de novo DNA methyltransferase Dnmt3a2, causing misexpression of the imprinted genes Igf2, H19, and Igf2r and hypomethylation of their corresponding imprinted control regions. Treatment of wild-type embryonic stem cells and neural stem cells with the Gsk-3 inhibitor, lithium, phenocopies the DNA hypomethylation at these imprinted loci. We show that inhibition of Gsk-3 by phosphatidylinositol 3-kinase (PI3K)-mediated activation of Akt also results in reduced DNA methylation at these imprinted loci. Finally, we find that N-Myc is a potent Gsk-3-dependent regulator of Dnmt3a2 expression. In summary, we have identified a signal transduction pathway that is capable of altering the DNA methylation of imprinted loci.


Subject(s)
DNA Methylation , Embryonic Stem Cells/metabolism , Gene Expression Regulation, Enzymologic , Genomic Imprinting , Glycogen Synthase Kinase 3/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Insulin-Like Growth Factor II/metabolism , Mice , Mice, Transgenic , Models, Biological , Oligonucleotide Array Sequence Analysis , RNA, Long Noncoding , RNA, Untranslated/metabolism , Receptor, IGF Type 2/metabolism , Signal Transduction
4.
Dev Dyn ; 239(12): 3235-46, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20981831

ABSTRACT

In Wnt/ß-catenin signaling pathway, Gsk3ß functions to facilitate ß-catenin degradation. Inactivation of Gsk3ß in mice causes a cleft palate formation, suggesting an involvement of Wnt/ß-catenin signaling during palatogenesis. In this study, we have investigated the expression pattern, tissue-specific requirement and function of Gsk3ß during mouse palatogenesis. We showed that Gsk3ß is primarily expressed in the palatal epithelium, particularly in the medial edge epithelium overlapping with ß-catenin. Tissue-specific gene inactivation studies demonstrated an essential role for Gsk3ß in the epithelium for palate elevation, and disruption of which contributes to cleft palate phenotype in Gsk3ß mutant. We observed that expression of Aixn2, a direct target gene of Wnt/ß-catenin signaling, is ectopically activated in the mutant tongue, but not in the palate. Our results indicate that Gsk3ß is an intrinsic regulator required in the epithelium for palate elevation, and could act through a pathway independent of Wnt/ß-catenin signaling to regulate palate development.


Subject(s)
Epithelium/embryology , Glycogen Synthase Kinase 3/metabolism , Palate/embryology , Animals , Cell Proliferation , Cleft Palate/embryology , Cleft Palate/genetics , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , In Situ Hybridization , In Situ Nick-End Labeling , Male , Mice , Mice, Knockout , Organ Culture Techniques , Signal Transduction/genetics , Signal Transduction/physiology , Wnt Proteins/metabolism , beta Catenin/metabolism
5.
Oncogene ; 23(20): 3521-9, 2004 Apr 29.
Article in English | MEDLINE | ID: mdl-15116090

ABSTRACT

Bone morphogenetic protein 3B (BMP3B) is a member of the TGF-beta superfamily. The BMP3B promoter sequence was previously identified as a target for aberrant DNA methylation in non-small-cell lung cancer (NSCLC). Aberrant DNA hypermethylation in the BMP3B promoter is associated with downregulation of BMP3B transcription in both primary human lung cancers as well as lung cancer cell lines. In order to understand the mechanisms of BMP3B silencing in lung cancer, a sample set of 91 primary NSCLCs was used to detect aberrant BMP3B promoter methylation, mutations in the coding sequence of BMP3B, and loss of heterozygosity (LOH). Our results showed that 45 of 91 (or 49.5%) tested primary NSCLCs exhibited increased promoter methylation, and 40% demonstrated LOH in at least one of the flanking microsatellite markers sJRH and D10S196 (63 kb upstream or 3.338 Mbp downstream of BMP3B). The lung cancer cell line A549, a type II alveolar epithelial human lung cancer cell line, is characterized by aberrant DNA promoter methylation. We used retroviral vector constructs containing the BMP3B cDNA to re-express the gene in A549 cells and to investigate the effects on cell growth. No change in the cell growth rate was observed after BMP3B re-expression, as compared to the vector controls. Although the number of colonies formed in anchorage-dependent assays was only slightly decreased, the colony-forming ability of A549 cells after BMP3B expression in anchorage-independent assays in soft agar was significantly reduced to 10% (P<0.005, t-test). Moreover, the in vivo tumorigenicity assay in nude mice indicated that cells re-expressing BMP3B grew significantly slower than cells not expressing BMP3B (P<0.05, t-test). In conclusion, this study provides evidence that BMP3B expression is repressed by different mechanisms in lung cancer, and that the silencing of BMP3B promotes lung tumor development.


Subject(s)
Bone Morphogenetic Proteins/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Gene Silencing/physiology , Lung Neoplasms/metabolism , Bone Morphogenetic Protein 3 , Bone Morphogenetic Proteins/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic/physiology , Growth Differentiation Factor 10 , Humans , Promoter Regions, Genetic , Tumor Cells, Cultured
6.
Neoplasia ; 5(4): 362-6, 2003.
Article in English | MEDLINE | ID: mdl-14511407

ABSTRACT

In the present studies, we investigated the correlation between RASSF1A promoter methylation status and Kras2 mutations in 65 primary non small cell lung cancer (NSCLC) including 33 adenocarcinomas, 12 large cell carcinomas, and 20 squamous cell carcinomas. Mutational analysis of Kras2 showed: 30% (10 of 33) of adenocarcinomas, 25% (3 of 12) of large cell carcinomas, and only 5% (1 of 20) of squamous cell carcinomas contained activated Kras2 mutation at codon 12 or 13. RASSF1A promoter region CpG island methylation was detected in adenocarcinomas (55%), large cell carcinomas (25%), and squamous cell carcinomas (25%). Interestingly, combined RASSF1A methylation and Kras2 mutation data show that only - 7% adenocarcinomas/large cell carcinomas exhibited both KRASSF1A promoter methylation and Kras2 mutation, whereas 24% adenocarcinomas, 50% large cell carcinomas, and 70% squamous cell carcinomas showed neither Kras2 mutation nor RASSF1A promoter methylation. These results showed that the majority of the primary NSCLCs with Kras2 mutations lack RASSF1A inactivation, and both RASSF1A inactivation and Kras2 mutation events occur frequently in adenocarcinomas and large cell carcinomas. Our results indicate a trend of inverse relationship between Kras2 activation and RASSF1A promoter methylation in the majority of human lung adenocarcinomas and large cell carcinomas.


Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Methylation , Lung Neoplasms/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins/genetics , Tumor Suppressor Proteins/genetics , Adenocarcinoma/genetics , Carcinoma, Large Cell/genetics , Carcinoma, Squamous Cell/genetics , Cell Line, Tumor , Colonic Neoplasms/genetics , DNA/metabolism , Humans , Mutation , Polymerase Chain Reaction , Proto-Oncogene Proteins p21(ras) , Sequence Analysis, DNA , Signal Transduction , ras Proteins
7.
Cancer Res ; 63(11): 2864-71, 2003 Jun 01.
Article in English | MEDLINE | ID: mdl-12782592

ABSTRACT

Thyroid cancer is common, occurring in 1% of the general population. The relative frequencies of two of the most common subtypes of thyroid carcinoma, follicular (FTC) and papillary (PTC), vary depending on the regional prevalence of iodine deficiency. Although PTC has been more extensively studied, the etiology of sporadic FTC is poorly understood. To further elucidate this, we conducted microarray expression comparison of FTC tumors and normal thyroid tissue. Three commonly down-regulated genes, caveolin-1, caveolin-2, and GDF10/BMP3b, were chosen for further study on the basis of their localization to two chromosomal regions, 7q31.1 and 10q11.1, that commonly show loss of heterozygosity in FTC. Two additional genes, glypican-3 and a novel chordin-like gene, were also analyzed in view of their involvement in bone morphogenetic protein signaling and possible interaction with GDF10. Each of these five genes was down-regulated in >or=15 of 19 FTC tumors (79%) by semiquantitative reverse transcription-PCR. Caveolin-1 showed preferential down-regulation of its beta-isoform at both the mRNA and protein level, suggesting a distinct function for this isoform. Caveolin-1 is of particular functional interest because it has been shown to interact with PTEN, the tumor suppressor gene mutated in Cowden syndrome, an inherited multiple hamartoma syndrome that includes predisposition to FTC. Immunohistochemical analysis of 141 thyroid tumors of various histological types showed significantly fewer caveolin-1-positive tumors in FTCs, including insular type tumors, and Hurthle cell carcinomas in comparison with normal thyroid. PTC and anaplastic thyroid carcinomas did not show significant down-regulation, and thus, caveolin-1 may become a useful molecular marker to differentiate the various histologies of thyroid malignancies.


Subject(s)
Adenocarcinoma, Follicular/genetics , Bone Morphogenetic Proteins/genetics , Caveolins/genetics , Genes, Tumor Suppressor , Thyroid Neoplasms/genetics , Adenocarcinoma, Follicular/metabolism , Bone Morphogenetic Protein 3 , Bone Morphogenetic Proteins/biosynthesis , Caveolin 1 , Caveolin 2 , Caveolins/biosynthesis , DNA Methylation , Down-Regulation , Female , Growth Differentiation Factor 10 , Humans , Loss of Heterozygosity , Male , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Thyroid Neoplasms/metabolism
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