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1.
Endocrinology ; 155(12): 4762-73, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25203139

ABSTRACT

Several drugs currently used in the management of mood disorders, epilepsy (ie, valproic acid), or the control of inflammation (ie, corticosteroids) have been shown to promote visceral obesity in humans by increasing the number of newly formed adipocytes. Valproic acid is classified as a nonspecific histone deacetylase (HDAC) inhibitor, along with trichostatin A and butyric acid. In vitro experiments have demonstrated that such molecules greatly enhance the rate of preadipocyte differentiation, similarly to the effect of corticosteroids. The glucocorticoid receptor stimulates adipogenesis in part by enhancing the transcription of C/ebpa through the titration, and subsequent degradation, of HDAC1 from the C/ebpα promoter. There is, however, controversy in the literature as to the role of HDACs during adipogenesis. In this study, we sought to demonstrate, using 2 different strategies, the definite role of HDAC1 in adipogenesis. By using small interference RNA-mediated knockdown of HDAC1 and by generating an enzymatically inactive HDAC1D181A by site-directed mutagenesis, we were able to show that HDAC1, but not HDAC2, suppresses glucocorticoid receptor-potentiated preadipocyte differentiation by decreasing CCAAT/enhancer-binding protein (C/ebp)α and Pparγ expression levels at the onset of differentiation. Finally, we demonstrate that HDAC1D181A acts as a dominant negative mutant of HDAC1 during adipogenesis by modulating C/EBPß transcriptional activity on the C/ebpα promoter.


Subject(s)
Adipogenesis/drug effects , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Histone Deacetylase 1/metabolism , Histone Deacetylase Inhibitors/adverse effects , Obesity/chemically induced , 3T3-L1 Cells , Animals , COS Cells , Chlorocebus aethiops , Dexamethasone , Down-Regulation , Gene Knockdown Techniques , Histone Deacetylase 2/metabolism , Mice , Mutation , NIH 3T3 Cells , PPAR gamma/metabolism , RNA, Small Interfering
2.
Endocrinology ; 154(4): 1454-64, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23456364

ABSTRACT

Glucocorticoids promote adipogenesis and contribute to the metabolic syndrome through a number of mechanisms. One of the effectors of glucocorticoid action is the CCAAT/enhancer binding protein ß (C/EBPß). C/EBPß is a basic leucine-zipper transcription factor involved in diverse processes including differentiation, cellular proliferation, and inflammation. C/EBPß transcriptional activity is regulated, in part, by its acetylation profile resulting from its dynamic interaction with either acetylases general control nonrepressed protein 5/p300/CBP associated factor (GCN5/PCAF) or deacetylase complexes (mSin3A/histone deacetylase 1 [HDAC1]). Glucocorticoid treatment of preadipocytes promotes C/EBPß acetylation, leading to mSin3A/HDAC1 dissociation from C/EBPß and resulting in C/ebpα promoter activation at the onset of adipogenesis, thus increasing the differentiation rate. We recently showed that the regulatory domain 1 (RD1) of C/EBPß contains four residues (153-156) required for its interaction with HDAC1, therefore supporting RD1 proposed inhibitory role. In an attempt to further elucidate the intrinsic regulatory property of RD1, we sought to characterize the regulatory potential of the N terminus region of RD1 (residues 141-149). In this study, we show that C/EBPßΔ141-149 transcriptional activity was compromised on the C/ebpα, but not on the Pparγ, promoter. Additionally, the ability of C/EBPßΔ141-149 to induce adipogenesis in NIH 3T3 cells was compromised when compared with C/EBPßwt owing to a delayed expression of C/ebpα at the onset of differentiation. Furthermore, the data suggest that the reduced expression of C/ebpα in cells expressing C/EBPßΔ141-149 was due to a persistent recruitment of HDAC1 to the C/ebpα promoter after glucocorticoid treatment. Together, these results suggest that amino acids 141-149 of C/EBPß act as a positive regulatory domain required for maximum transcriptional activity.


Subject(s)
Adipogenesis/genetics , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Glucocorticoids/metabolism , Histone Deacetylase 1/metabolism , RNA, Messenger/metabolism , Adipogenesis/physiology , Animals , CCAAT-Enhancer-Binding Protein-alpha/metabolism , COS Cells , Chlorocebus aethiops , Glucocorticoids/genetics , Mice , NIH 3T3 Cells , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic/physiology
3.
J Biol Chem ; 286(24): 21488-99, 2011 Jun 17.
Article in English | MEDLINE | ID: mdl-21521687

ABSTRACT

The CCAAT/enhancer-binding protein ß (C/EBPß) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBPß to stimulate preadipocyte differentiation through activation of C/ebpα transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebpα promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebpα transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebpα promoter, mutations in C/ebpß that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBPα transcription and preadipocyte differentiation are discussed.


Subject(s)
Adipocytes/cytology , CCAAT-Enhancer-Binding Protein-beta/genetics , Gene Expression Regulation , Animals , COS Cells , Cell Differentiation , Chlorocebus aethiops , Dimerization , Gene Deletion , Mice , Mutation , NIH 3T3 Cells , Promoter Regions, Genetic , Protein Binding , Transcription, Genetic
4.
Mol Endocrinol ; 24(1): 104-13, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19887648

ABSTRACT

Glucocorticoids are synthesized locally in adipose tissue and contribute to metabolic disease through the facilitation of adipose tissue expansion. Here we report that exposure of human primary preadipocytes to glucocorticoids increases their sensitivity to insulin and enhances their subsequent response to stimuli that promote differentiation. This effect was observed in primary human preadipocytes but not in immortalized 3T3-L1 murine preadipocytes or in fully differentiated primary human adipocytes. Stimulation of insulin signaling was mediated through induction of insulin receptor (IR), IR substrate protein 1 (IRS1), IRS2, and the p85 regulatory subunit of phosphoinositide-3-3-kinase, which led to enhanced insulin-mediated activation of Akt. Although induction of IRS2 was direct, induction of IR and IRS1 by glucocorticoids occurred subsequent to primary induction of the forkhead family transcription factors FoxO1A and FoxO3A. These results reveal a new role for glucocorticoids in preparing preadipocytes for differentiation.


Subject(s)
Adipocytes/drug effects , Adipogenesis/drug effects , Forkhead Transcription Factors/metabolism , Gene Expression Regulation/drug effects , Glucocorticoids/pharmacology , Insulin/pharmacology , 3T3-L1 Cells , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Female , Forkhead Transcription Factors/genetics , Gene Expression Profiling , Glucocorticoids/physiology , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Mice , Oligonucleotide Array Sequence Analysis , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Time Factors
5.
Mol Oncol ; 4(1): 38-51, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19858003

ABSTRACT

Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.


Subject(s)
Antigens, Neoplasm/immunology , Antineoplastic Agents, Phytogenic/therapeutic use , DNA Damage/drug effects , DNA Repair/genetics , DNA Topoisomerases, Type II/pharmacology , DNA-Binding Proteins/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/pharmacology , Apoptosis/drug effects , Cell Cycle Proteins/drug effects , Cell Cycle Proteins/metabolism , Cell Survival/drug effects , Cell Survival/physiology , DNA/drug effects , DNA Breaks, Double-Stranded/drug effects , DNA Damage/genetics , DNA Repair/drug effects , DNA Replication/drug effects , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Etoposide , Flow Cytometry , G2 Phase/drug effects , G2 Phase/genetics , Humans , K562 Cells , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Tumor Suppressor Proteins
6.
Proc Natl Acad Sci U S A ; 104(8): 2703-8, 2007 Feb 20.
Article in English | MEDLINE | ID: mdl-17301242

ABSTRACT

Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98-102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.


Subject(s)
Adipocytes/cytology , Adipocytes/drug effects , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Differentiation/drug effects , Glucocorticoids/pharmacology , Histone Acetyltransferases/metabolism , 3T3-L1 Cells , Acetylation/drug effects , Animals , Cell Cycle Proteins/metabolism , Dexamethasone/pharmacology , Green Fluorescent Proteins/metabolism , Humans , Lysine/metabolism , Mice , NIH 3T3 Cells , Protein Binding , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Transcription Factors/metabolism , p300-CBP Transcription Factors
7.
J Biol Chem ; 282(15): 10963-71, 2007 Apr 13.
Article in English | MEDLINE | ID: mdl-17314103

ABSTRACT

The glucocorticoid receptor (GR) cycles between a naive chaperone-complexed form in the cytoplasm and a transcriptionally active steroid-bound nuclear form. Nuclear import of GR occurs rapidly and is mediated through the importin alpha/beta karyopherin import pathway. By contrast, nuclear export of GR occurs only slowly under most conditions, despite a dependence on active signaling. In this study we have defined a nuclear retention signal (NRS) in the hinge region of GR that actively opposes the nuclear export of GR as well as the nuclear export mediated through an ectopic CRM1-dependent nuclear export signal (NES). The GR NRS overlaps closely with the basic NL1 nuclear localization signal (NLS) but can be distinguished from NL1 by targeted mutagenesis. Substitution of the classical NLS from SV40 T antigen for the GR NL1 results in a receptor in which nuclear export is accelerated. Remarkably, although the SV40-modified GR remains predominantly nuclear in the presence of steroid and is recruited to transcriptional regulatory regions indistinguishably from wild-type GR, the substitution dramatically weakens the ability of GR to activate transcription of a mouse mammary tumor virus reporter gene. These results suggest that active nuclear retention of GR plays an integral role in glucocorticoid signaling.


Subject(s)
Receptors, Glucocorticoid/metabolism , Signal Transduction , Transcriptional Activation/genetics , Active Transport, Cell Nucleus , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/metabolism , Animals , COS Cells , Chlorocebus aethiops , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Rats , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics
8.
Endocrinology ; 147(11): 5284-93, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16873539

ABSTRACT

Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing's syndrome. Glucocorticoid treatment also strongly potentiates the differentiation of human preadipocytes in culture. However, the molecular basis of these stimulatory effects remains to be defined. In this study, we provide a detailed analysis of the specific contribution of glucocorticoid treatment to the differentiation of primary human preadipocytes cultured in chemically defined medium. Contrary to previous descriptions of glucocorticoids being required throughout the course of differentiation, our results show that glucocorticoid treatment is stimulatory only during the first 48 h of differentiation. Furthermore, stimulation by glucocorticoids and the peroxisome proliferator activator receptor-gamma agonist troglitazone is mediated sequentially. Several details of the early events in the differentiation of human preadipocytes and the contribution of steroid to these events differ from the responses observed previously in murine preadipocyte models. First, glucocorticoid treatment stimulated the early accumulation of CCAAT enhancer binding protein-beta (C/EBPbeta) in primary human preadipocytes. Second, induction of C/EBPalpha in primary human preadipocytes was noted within 4 h of adipogenic stimulus, whereas C/EBPalpha induction is not detected until 24-48 h in the murine 3T3 L1 preadipocyte model. Remarkably, by contrast to human primary preadipocytes, which do not undergo postconfluent mitosis, 3T3 L1 murine preadipocytes stimulated to differentiate under chemically defined conditions required glucocorticoids to survive the clonal expansion that precedes terminal differentiation, revealing a novel signal imparted by glucocorticoids in this immortalized murine cell system.


Subject(s)
Adipocytes/drug effects , Cell Differentiation/drug effects , Dexamethasone/pharmacology , Stem Cells/drug effects , 3T3-L1 Cells , Adipocytes/cytology , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , CCAAT-Enhancer-Binding Protein-beta/genetics , CCAAT-Enhancer-Binding Protein-beta/metabolism , Chromans/pharmacology , DNA Replication , Humans , Mice , Receptors, Glucocorticoid/physiology , Stem Cells/cytology , Thiazolidinediones/pharmacology , Transcription, Genetic , Troglitazone
9.
Am J Respir Cell Mol Biol ; 35(6): 668-80, 2006 Dec.
Article in English | MEDLINE | ID: mdl-16809634

ABSTRACT

Antiinflammatory effects of glucocorticoids are critical to treatment of airway inflammation in such common disorders as asthma. There is considerable variation in responsiveness to glucocorticoid, and prolonged exposure can result in glucocorticoid resistance. We cloned LGL2, a glucocorticoid-inducible gene in fetal rat lung. We described the characterization of lgl2 as a nuclear transport protein, classified as importin 13 (IPO13), and demonstrated developmental regulation of IPO13 nucleocytoplasmic shuttling. We now report on the identification of the glucocorticoid receptor (GR) as a cargo substrate for IPO13. Binding of GR and IPO13 was demonstrated by GR-GST pulldown and coimmunoprecipitation. To investigate the role of IPO13 in modulating GR signaling in the lung, we studied IPO13-regulated GR transport in airway epithelial cells. Small interfering RNAs that inhibited IPO13 synthesis prevented nuclear translocation of GR. Silencing of IPO13 also abrogated the ability of cortisol to inhibit synthesis of the inflammatory cytokine IL-8 after stimulation with TNF-alpha. Our findings support a role for IPO13 in promoting nuclear occupancy of GR in a way that strongly potentiates the antiinflammatory effects of glucocorticoids. We speculate that variation in cellular levels of IPO13 and intracellular IPO13 shuttling rates may contribute to glucocorticoid resistance.


Subject(s)
Cell Nucleus/metabolism , Receptors, Glucocorticoid/metabolism , Respiratory Mucosa/metabolism , beta Karyopherins/metabolism , Active Transport, Cell Nucleus/drug effects , Animals , Anti-Inflammatory Agents/pharmacology , Cell Nucleus/drug effects , Drug Resistance , HeLa Cells , Humans , Hydrocortisone/pharmacology , Immunoprecipitation , Interleukin-8/biosynthesis , Protein Binding , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Rats, Wistar , Receptors, Glucocorticoid/drug effects , Receptors, Glucocorticoid/genetics , Recombinant Fusion Proteins/metabolism , Respiratory Mucosa/cytology , Respiratory Mucosa/drug effects , Transfection , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects , beta Karyopherins/genetics
10.
J Mol Biol ; 358(5): 1200-11, 2006 May 19.
Article in English | MEDLINE | ID: mdl-16600297

ABSTRACT

Artemis is a nuclear phosphoprotein required for genomic integrity whose phosphorylation is increased subsequent to DNA damage. Artemis phosphorylation by the DNA-dependent protein kinase (DNA-PK) and the association of Artemis with DNA-PK catalytic subunit (DNA-PKcs) have been proposed to be crucial for the variable, diversity, joining (V(D)J) reaction, genomic stability and cell survival in response to double-stranded DNA breaks. The exact nature of the effectors of Artemis phosphorylation is presently being debated. Here, we have delimited the interface on Artemis required for its association with DNA-PKcs and present the characterization of six DNA-PK phosphorylation sites on Artemis whose phosphorylation shows dependence on its association with DNA-PKcs and is induced by double-stranded DNA damage. Surprisingly, DNA-PKcs Artemis association appeared to be dispensable in a V(D)J recombination assay with stably integrated DNA substrates. Phosphorylation at two of the sites on Artemis, S516 and S645, was verified in vivo using phosphospecific antibodies. Basal Artemis S516 and S645 phosphorylation in vivo showed a significant dependence on DNA-PKcs association. However, regardless of its association with DNA-PKcs, phosphorylation of Artemis at both S516 and S645 was stimulated in response to the double-stranded DNA-damaging agent bleomycin, albeit to a lesser extent. This suggests that additional factors contribute to promote DNA damage-induced Artemis phosphorylation. Intriguingly, pS516/pS645 Artemis was concentrated in chromatin-associated nuclear foci in naïve cells. These foci were maintained upon DNA damage but failed to overlap with the damage-induced gammaH2AX. These results provide the expectation of a specific role for DNA-PK-phosphorylated Artemis in both naïve and damaged cells.


Subject(s)
Chromatin/metabolism , DNA-Activated Protein Kinase/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Binding Sites , Cell Line , DNA Damage , DNA-Binding Proteins , Endonucleases , Humans , In Vitro Techniques , Nuclear Proteins/genetics , Phosphorylation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombination, Genetic , Transfection
11.
Cancer Res ; 65(23): 10750-8, 2005 Dec 01.
Article in English | MEDLINE | ID: mdl-16322220

ABSTRACT

The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to gamma radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5' regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress.


Subject(s)
Gene Expression Regulation/physiology , Octamer Transcription Factor-1/physiology , 3T3 Cells , Animals , Doxorubicin , Fibroblasts/drug effects , Fibroblasts/radiation effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Hydrogen Peroxide , Mice , Octamer Transcription Factor-1/deficiency , Octamer Transcription Factor-1/genetics , Oxidative Stress , Reverse Transcriptase Polymerase Chain Reaction
12.
J Biol Chem ; 280(17): 17549-61, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15737989

ABSTRACT

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.


Subject(s)
Cell Nucleus/metabolism , Nuclear Localization Signals , Receptors, Mineralocorticoid/metabolism , Serine/chemistry , Threonine/chemistry , Active Transport, Cell Nucleus , Amino Acid Motifs , Amino Acid Sequence , Animals , Biological Transport , Blotting, Western , COS Cells , Cytoplasm/metabolism , Fluorescence Recovery After Photobleaching , Fluorescent Antibody Technique, Indirect , Glutathione Transferase/metabolism , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Ligands , Molecular Sequence Data , Nuclear Matrix/metabolism , Phosphorylation , Plasmids/metabolism , Protein Structure, Tertiary , Receptors, Glucocorticoid/metabolism , Steroids/metabolism , Time Factors , Transfection
13.
Eur J Immunol ; 34(11): 3146-55, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15468306

ABSTRACT

Artemis is a DNA repair factor required for V(D)J recombination, repair of DNA damage induced by ionizing radiation (IR) or radiomimetic drugs, and the maintenance of genome integrity. During V(D)J recombination, Artemis participates in the resolution of hairpin-sealed coding ends, a step crucial to the constitution of the gene encoding for the antigen receptor of lymphocytes. The precise role of Artemis in the repair of IR-induced DNA damage remains to be elucidated. Here we show that Artemis is constitutively phosphorylated in cultured cells and undergoes additional phosphorylation events after irradiation. The IR-induced phosphorylation is mainly, although not solely, dependent on Ataxia-telangiectasia-mutated kinase (ATM). The physiological role of these phosphorylation events remains unknown, as in vitro-generated Artemis mutants, which present impaired IR-induced phosphorylation, still display an activity sufficient to complement the V(D)J recombination defect and the increased radiosensibility of Artemis-deficient cells. Thus, Artemis is an effector of DNA repair that can be phosphorylated by ATM, and possibly by DNA-PKcs and ATR depending upon the type of DNA damage.


Subject(s)
DNA Damage/physiology , DNA Repair/physiology , DNA/radiation effects , Nuclear Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Blotting, Western , CHO Cells , Cell Cycle Proteins/immunology , Cricetinae , DNA-Binding Proteins , Endonucleases , Fibroblasts/immunology , Humans , Kinetics , Molecular Sequence Data , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Phosphorylation , Point Mutation , Protein Serine-Threonine Kinases/immunology , Radiation, Ionizing , Sequence Alignment , Tumor Suppressor Proteins
14.
Eur J Biochem ; 271(18): 3776-84, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15355354

ABSTRACT

Phosphorylation at multiple sites within the N-terminus of p53 promotes its dissociation from hdm2/mdm2 and stimulates its transcriptional regulatory potential. The large phosphoinositide 3-kinase-like kinases ataxia telangiectasia mutated gene product and the ataxia telangectasia and RAD-3-related kinase promote phosphorylation of human p53 at Ser15 and Ser20, and are required for the activation of p53 following DNA damage. DNA-dependent protein kinase (DNA-PK) is another large phosphoinositide 3-kinase-like kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhance phosphorylation of these sites in vivo. Moreover, recent studies support a role for DNA-PK in the regulation of p53-mediated apoptosis. We have shown previously that colocalization of p53 and DNA-PK to structured single-stranded DNA dramatically enhances the potential for p53 phosphorylation by DNA-PK. We report here the identification of p53 phosphorylation at two novel sites for DNA-PK, Thr18 and Ser9. Colocalization of p53 and DNA-PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared to be dependent upon additional determinants of p53 beyond the N-terminal 65 amino acids. Our results suggest a role for DNA-PK in the modulation of p53 activity resultant from the convergence of p53 and DNA-PK on structured DNA.


Subject(s)
DNA-Binding Proteins , DNA/metabolism , Protein Serine-Threonine Kinases/metabolism , Serine/metabolism , Threonine/metabolism , Tumor Suppressor Protein p53/metabolism , Amino Acid Sequence , DNA, Single-Stranded/metabolism , DNA-Activated Protein Kinase , Electrophoresis, Polyacrylamide Gel , Humans , Nuclear Proteins , Phosphorylation , Recombinant Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity
15.
Cancer Res ; 63(21): 7197-205, 2003 Nov 01.
Article in English | MEDLINE | ID: mdl-14612514

ABSTRACT

Cells respond to double-stranded DNA breaks (DSBs) by pausing cell cycle progression to allow the repair machinery to restore genomic integrity. DNA-dependent protein kinase (DNA-PK), comprising a large catalytic subunit (DNA-PK(cs)) and the Ku antigen regulatory subunit (Ku70/Ku80), is activated in response to DSBs and is required for DNA repair through the nonhomologous end-joining pathway. Here we provide evidence that DNA-PK participates in altering specific gene expression in response to DNA damage by modulating the stability and transcriptional regulatory potential of the essential transcription factor octamer transcription factor 1 (Oct-1). Histone H2B and U2 RNA, whose expression are highly dependent on Oct-1, were strongly decreased in response to ionizing radiation in a DNA-PK-dependent manner, and Oct-1-dependent reporter gene transcription was repressed. Furthermore, Oct-1 phosphorylation in response to ionizing radiation increased in a DNA-PK-dependent manner. Paradoxically, down-regulation of transactivation correlated with the rapid DNA-PK-dependent stabilization of Oct-1. Stabilization of Oct-1 was dependent on the NH(2)-terminal region of Oct-1, which contains a transcriptional activation domain and which was phosphorylated by DNA-PK in vitro. These results suggest a mechanism for the regulation of Oct-1 in response to DNA damage through specific phosphorylation within the NH(2)-terminal transcriptional regulatory domain.


Subject(s)
DNA Damage/physiology , DNA-Binding Proteins/physiology , Histones/biosynthesis , Protein Serine-Threonine Kinases/physiology , Transcription Factors/physiology , Animals , Cricetinae , Cricetulus , DNA-Activated Protein Kinase , DNA-Binding Proteins/metabolism , Down-Regulation , HeLa Cells , Histones/antagonists & inhibitors , Histones/genetics , Host Cell Factor C1 , Humans , Nuclear Proteins , Octamer Transcription Factor-1 , Phosphorylation , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , RNA, Small Nuclear/biosynthesis , RNA, Small Nuclear/genetics , Transcription Factors/metabolism , Transcriptional Activation
16.
J Biol Chem ; 278(39): 37858-64, 2003 Sep 26.
Article in English | MEDLINE | ID: mdl-12869547

ABSTRACT

Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.


Subject(s)
Active Transport, Cell Nucleus , Calreticulin/physiology , Cell Fusion , Receptors, Glucocorticoid/metabolism , Animals , COS Cells , Cytoplasm/metabolism
17.
EMBO J ; 22(9): 2135-45, 2003 May 01.
Article in English | MEDLINE | ID: mdl-12727880

ABSTRACT

Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non-transcriptional mechanism mediated through the ligand-binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPalpha) expression by potentiating its initial transcriptional activation by C/EBPbeta. In the absence of steroid, C/EBPbeta associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPalpha promoter with C/EBPbeta and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPalpha expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand-binding domain suggests a conserved mechanism relevant to many aspects of steroid-mediated differentiation.


Subject(s)
Adipocytes/drug effects , Cell Differentiation/drug effects , Glucocorticoids/pharmacology , Histone Deacetylases/metabolism , 3T3 Cells , Adipocytes/cytology , Animals , CCAAT-Enhancer-Binding Protein-alpha/genetics , COS Cells , HeLa Cells , Histone Deacetylase 1 , Humans , Mice , Repressor Proteins/metabolism , Sin3 Histone Deacetylase and Corepressor Complex
18.
DNA Cell Biol ; 22(2): 65-78, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12713733

ABSTRACT

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.


Subject(s)
Antigens, Nuclear/metabolism , Antigens, Nuclear/physiology , DNA Helicases , DNA Replication , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Base Sequence , Binding Sites , Cell Nucleus/enzymology , Cell Nucleus/genetics , DNA Footprinting , DNA-Activated Protein Kinase , Electrophoretic Mobility Shift Assay , HeLa Cells , Humans , Ku Autoantigen , Molecular Sequence Data , Nuclear Proteins , Protein Serine-Threonine Kinases/physiology , Replication Origin , Response Elements , Ultraviolet Rays
19.
Cancer Res ; 63(6): 1198-201, 2003 Mar 15.
Article in English | MEDLINE | ID: mdl-12649176

ABSTRACT

DNA-dependent protein kinase (DNA-PK) is required for the repair of double-stranded DNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH(2) terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.


Subject(s)
DNA-Binding Proteins , Protein Serine-Threonine Kinases/metabolism , Radiation Tolerance/physiology , Threonine/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , DNA Damage , DNA Repair , DNA-Activated Protein Kinase , Enzyme Activation , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Threonine/genetics
20.
Gene ; 302(1-2): 53-64, 2003 Jan 02.
Article in English | MEDLINE | ID: mdl-12527196

ABSTRACT

Ku antigen is an abundant nuclear protein with multiple functions that depend mainly on Ku's prolific and highly verstatile interactions with DNA. We have shown previously that the direct binding of Ku in vitro to negative regulatory element 1 (NRE1), a transcriptional regulatory element in the long terminal repeat of mouse mammary tumour virus, correlates with the regulation of viral transcription by Ku. In this study, we have sought to explore the interaction of Ku with NRE1 in vivo in yeast one-hybrid experiments. Unexpectedly, we observed that human Ku70 carrying a transcriptional activation domain from the yeast Gal4 protein induced transcription of yeast reporter genes pleiotrophically, independent of NRE1, promoter, reporter gene and chromosomal location. Ku80 with the same activation domain had no effect on transcription when expressed alone, but reconstituted activation when co-expressed with native human Ku70. The requirements for transcriptional activation by Ku-Gal4 activation domain proteins correlated with previous descriptions of the requirements for DNA sequence-independent DNA binding by Ku, but were distinct from determinants for DNA-end binding by a truncated Ku heterodimer determined recently by crystallography. These results suggest a preferential targeting of Ku to transcriptionally active chromatin that indicate a possible function for Ku within the RNA polymerase II holoenzyme.


Subject(s)
Antigens, Nuclear/metabolism , DNA Helicases , DNA-Binding Proteins/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic/genetics , Animals , Antigens, Nuclear/genetics , Binding Sites/genetics , Cells, Cultured , DNA-Binding Proteins/genetics , Gene Expression Regulation , Genes, Reporter/genetics , Humans , Ku Autoantigen , Lac Operon/genetics , Mutation , Promoter Regions, Genetic/genetics , Protein Binding , Saccharomyces cerevisiae/genetics
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