ABSTRACT
BACKGROUND: CCAAT/enhancer binding protein-delta (C/EBP-delta) is a member of the highly conserved C/EBP family of basic region leucine zipper transcription factors. C/EBP family members regulate cell growth and differentiation and "loss of function" alterations in C/EBPs have been reported in a variety of human cancers. C/EBP-delta gene expression is upregulated by G0 growth arrest, IL-6 family cytokines and endotoxin treatments. C/EBP-delta exhibits properties of a tumor suppressor gene, including reduced expression and promoter methylation-induced silencing in transformed cell lines and primary tumors. In addition, C/EBP-delta gene expression is repressed by c-Myc, an oncogene that is over-expressed in a wide range of human cancers. "ChIP-chip" studies demonstrated that C/EBP-delta functions as a transcriptional activator of target genes that function in intracellular signal transduction, transcription, DNA binding/repair, cell cycle control, cell adhesion, and apoptosis. Despite progress in determining the biochemical functions of C/EBP-delta, the specific cellular defects that are induced by C/EBP-delta "loss of function" alterations are poorly understood. This study investigated the impact of C/EBP-delta "loss of function" alterations on growth arrest, migration/invasion and differentiation in nontransformed mouse mammary epithelial cells (MECs) and primary mouse embryo fibroblasts (MEFs). RESULTS: C/EBP-delta siRNA transfected MECs exhibited ~90% reduction in C/EBP-delta mRNA and protein levels. C/EBP-delta siRNA treatment resulted in defective growth arrest as demonstrated by persistently elevated BrdU labeling, 3H-thymidine incorporation and cyclin D1 levels in response to growth arrest treatments. C/EBP-delta siRNA treatment also resulted in increased migration/invasion and defective differentiation. C/EBP-delta knockout MEFs exhibited defective growth arrest and increased proliferation/migration. Re-introduction of C/EBP-delta expression restored the growth arrest response of C/EBP-delta knockout MEFs. Finally, deletion of the C/EBP-delta DNA binding domain or the C/EBP-delta bZIP domain resulted in the loss of C/EBP-delta growth inhibition in clonogenic assays. CONCLUSIONS: This study demonstrates that C/EBP-delta functions in the regulation of critical cell fate determining programs such as growth arrest, migration, and differentiation. These results support the tumor suppressor function of C/EBP-delta and identify potential mechanisms in which "loss of function" alterations in C/EBP-delta could promote cell transformation and tumorigenesis.
ABSTRACT
BACKGROUND: "Loss of function" alterations in CCAAT/Enhancer Binding Proteindelta (C/EBPdelta) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPdelta gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPdelta exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing. We previously reported that C/EBPdelta expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPalpha, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPdelta expression. RESULTS: Myc represses C/EBPdelta promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPdelta promoter in proliferating cells, when C/EBPdelta expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPdelta promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPdelta promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPdelta promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPdelta promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPdelta expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPdelta promoter activity. CONCLUSION: Myc represses C/EBPdelta expression by associating with the C/EBPdelta proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPdelta promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPdelta, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , CCAAT-Enhancer-Binding Protein-delta/biosynthesis , Gene Expression Regulation/genetics , Kruppel-Like Transcription Factors/metabolism , Mammary Glands, Animal/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Repressor Proteins/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Blotting, Western , CCAAT-Enhancer-Binding Protein-delta/genetics , Chromatin Immunoprecipitation , Electrophoretic Mobility Shift Assay , Epithelial Cells , Female , Gene Expression , Immunoprecipitation , Kruppel-Like Transcription Factors/genetics , Mice , Promoter Regions, Genetic , Proto-Oncogene Proteins c-myc/genetics , RNA, Small Interfering , Repressor Proteins/genetics , TransfectionABSTRACT
CCAAT/enhancer binding proteindelta (C/EBPdelta) plays a key role in mammary epithelial cell G(0) growth arrest, and "loss of function" alterations in C/EBPdelta have been reported in breast cancer and acute myeloid leukemia. C/EBPdelta is regulated at the transcriptional, post-transcriptional, and post-translational levels, suggesting tight control of C/EBPdelta content and function. Protein inhibitors of activated STATs (PIASs) regulate a growing number of transcription factors, including C/EBPs. HC11 nontransformed mammary epithelial cells express PIAS3, PIASxbeta, and PIASy, and all three PIAS family members repress C/EBPdelta transcriptional activity. PIASy is the most potent, however, repressing C/EBPdelta transcriptional activity by >80%. PIASy repression of C/EBPdelta transcriptional activity is dependent upon interaction between the highly conserved PIASy N-terminal nuclear matrix binding domain (SAPD) and the C/EBPdelta transactivation domain (TAD). PIASy repression of C/EBPdelta transcriptional activity is independent of histone deacetylase activity, PIASy E3 SUMO ligase activity, and C/EBPdelta sumoylation status. PIASy expression is associated with C/EBPdelta translocation from nuclear foci, where C/EBPdelta co-localizes with p300, to the nuclear periphery. PIASy-mediated translocation of C/EBPdelta is dependent upon the PIASy SAPD and C/EBPdelta TAD. PIASy reduces the expression of C/EBPdelta adhesion-related target genes and enhances repopulation of open areas within a cell monolayer in the in vitro "scratch" assay. These results demonstrate that PIASy represses C/EBPdelta by a mechanism that requires interaction between the PIASy SAPD and C/EBPdelta TAD and does not require PIASy SUMO ligase activity or C/EBPdelta sumoylation. PIASy alters C/EBPdelta nuclear localization, reduces C/EBPdelta transcriptional activity, and enhances cell proliferation/migration.
Subject(s)
CCAAT-Enhancer-Binding Protein-delta/metabolism , Cell Nucleus/metabolism , Protein Inhibitors of Activated STAT/metabolism , Trans-Activators/genetics , Animals , Cell Adhesion , Cell Line , Cell Movement , Cell Proliferation , Gene Expression Regulation , Mice , Protein Binding , Protein Inhibitors of Activated STAT/classification , Protein Processing, Post-Translational , SUMO-1 Protein/metabolismABSTRACT
The G(0) growth arrest (quiescent) state is highly conserved in evolution to promote survival under adverse environmental conditions. To maintain viability, G(0) growth arrested cells limit gene expression to essential growth control and pro-survival genes. CCAAT enhancer binding protein delta (C/EBPdelta), a member of the C/EBP family of nuclear proteins, is highly expressed in G(0) growth arrested mammary epithelial cells (MECs). Although C/EBPdelta gene transcription is elevated during G(0) growth arrest, C/EBPdelta mRNA and protein are relatively short lived, suggesting tight control of the cellular C/EBPdelta content in unstressed, quiescent cells. Treatment of G(0) growth arrested MECs with ultraviolet radiation (UVR) dramatically increases the C/EBPdelta mRNA half-life (approximately 4-fold) and protein content (approximately 3-fold). The mRNA stabilizing effects of UVR treatment are mediated by the C/EBPdelta mRNA 3'untranslated region, which contains an AU rich element. UVR increased p38 MAP kinase (MAPK) activation and SB203580, a p38 MAPK inhibitor, blocked UVR-induced C/EBPdelta mRNA stabilization. UVR increased the nuclear to cytoplasmic translocation of HuR, an ARE-binding protein that functions in mRNA stabilization. Finally, HuR siRNA treatment blocked UVR-induced stabilization of the C/EBPdelta and C/EBPbeta mRNAs but had no effect on C/EBPzeta (CHOP) mRNA stability. In summary, G(0) growth arrested MECs respond to UVR treatment by activating p38 MAPK, increasing HuR translocation and HuR/C/EBPdelta mRNA binding and stabilizing the C/EBPdelta mRNA. These results identify post-transcriptional stabilization of the C/EBPdelta mRNA as a mechanism to increase C/EBPdelta levels in the stress response of quiescent cells to UVR.
Subject(s)
CCAAT-Enhancer-Binding Protein-delta/biosynthesis , Epithelial Cells/metabolism , Mammary Glands, Animal/metabolism , RNA Stability/radiation effects , Resting Phase, Cell Cycle/radiation effects , Ultraviolet Rays , p38 Mitogen-Activated Protein Kinases/metabolism , 3' Untranslated Regions/metabolism , Animals , Cell Line , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/radiation effects , Half-Life , Imidazoles/pharmacology , Mice , Pyridines/pharmacology , RNA Stability/drug effects , RNA-Binding Proteins/metabolism , Resting Phase, Cell Cycle/drug effects , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
C/EBPdelta (CCAAT/enhancer-binding protein delta) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPdelta gene transcription is highly induced in G(0) growth-arrested mammary epithelial cells but the C/EBPdelta protein exhibits a t(1/2) of only approximately 120 min. The goal of the present study was to investigate the role of C/EBPdelta modification by ubiquitin and C/EBPdelta proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPdelta ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPdelta ubiquitination is not required for proteasome-mediated C/EBPdelta degradation and the presence of ubiquitin does not increase C/EBPdelta degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPdelta protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPdelta ubiquitination we treated G(0) growth-arrested mammary epithelial cells with DNA-damage- and oxidative-stress-inducing agents and found that C/EBPdelta ubiquitination is induced in response to H2O2. However, C/EBPdelta protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPdelta is ubiquitin-independent.
Subject(s)
CCAAT-Enhancer-Binding Protein-delta/metabolism , Proteasome Endopeptidase Complex/metabolism , Ubiquitin/physiology , Animals , Cells, Cultured , Epithelial Cells , HeLa Cells , Humans , Hydrogen Peroxide/pharmacology , Lysine/genetics , Mice , Protein Structure, TertiaryABSTRACT
BACKGROUND: Although a number of reports have investigated the effects of IL-6 family cytokines on prostate cell growth, there is limited information available identifying IL-6 inducible downstream effector genes and their function in growth control. Previous studies have demonstrated that IL-6 treatment results in the activation of signal transducer and activator of transcription3 (STAT3) in prostate cancer cells. The goal of this study was to investigate the influence of IL-6 treatment and activation of the Jak/STAT signal transduction pathway on C/EBPdelta gene expression and growth inhibition of human prostate cancer cells. METHODS: Expression of C/EBPdelta and STAT3 activation were assayed using Northern and Western blotting techniques. Proliferation was assessed by [(3)H] thymidine incorporation, flow cytometry, and colony formation analyses. The analysis of the transcriptional regulation of C/EBPdelta was performed using luciferase-reporter constructs. RESULTS: In this report, we demonstrate that IL-6 treatment induces STAT3 activation (pSTAT3), pSTAT3 binds to the human C/EBPdelta gene promoter and induces its expression. We also demonstrate that C/EBPdelta over-expression is capable of suppressing prostate cancer cell growth. CONCLUSIONS: These results demonstrate that C/EBPdelta gene expression is increased in IL-6 treated LNCaP cells. Increased C/EBPdelta gene expression plays an important role in IL-6/STAT3 mediated growth arrest of LNCaP prostate cancer cells. Ongoing studies are investigating the mechanism by which C/EBPdelta controls prostate cancer cell growth and the potential role of C/EBPdelta in the survival and chemo resistance of prostate cancer metastasis. (c) 2004 Wiley-Liss, Inc.
Subject(s)
CCAAT-Enhancer-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Interleukin-6/pharmacology , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/pathology , Trans-Activators/metabolism , Transcription Factors/biosynthesis , Binding Sites , Blotting, Northern , Blotting, Western , CCAAT-Enhancer-Binding Protein-delta , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Interleukin-6/biosynthesis , Interleukin-6/genetics , Interleukin-6/metabolism , Janus Kinase 2 , Male , Mutagenesis, Insertional , Promoter Regions, Genetic/physiology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , STAT3 Transcription Factor , Signal Transduction/drug effects , Thymidine/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , TransfectionABSTRACT
OBJECTIVE: To determine the effect of a single bout of exercise and increased substrate availability after exercise on gene expression and content of the glucose transporter-4 (GLUT-4) protein in equine skeletal muscle. ANIMALS: 6 healthy adult Thoroughbreds. PROCEDURES: The study was designed in a balanced, randomized, 3-way crossover fashion. During 2 trials, horses were exercised at 45% of their maximal rate of oxygen consumption for 60 minutes after which 1 group received water (10 mL/kg), and the other group received glucose (2 g/kg, 20% solution) by nasogastric intubation. During 1 trial, horses stood on the treadmill (sham exercise) and then received water (10 mL/kg) by nasogastric intubation. Muscle glycogen concentration and muscle GLUT-4 protein and mRNA content were determined before exercise and at 5 minutes and 4, 8, and 24 hours after exercise. RESULTS: Although exercise resulted in a 30% reduction in muscle glycogen concentration, no significant difference was detected in muscle GLUT-4 protein or mRNA content before and after exercise. Glycogen replenishment was similar in both exercised groups and was not complete at 24 hours after exercise. Horses that received glucose had significantly higher plasma glucose and insulin concentrations for 3 hours after exercise, but no effect of hyperglycemia was detected on muscle GLUT-4 protein or mRNA content. CONCLUSION: Under the conditions of this study, neither exercise nor the combination of exercise followed by hyperglycemia induced translation or transcription of the GLUT-4 protein in horses.
Subject(s)
Glucose/pharmacology , Hyperglycemia/physiopathology , Monosaccharide Transport Proteins/genetics , Muscle Proteins , Muscle, Skeletal/physiology , Physical Conditioning, Animal , Animals , Blood Glucose/metabolism , Exercise Test/veterinary , Female , Glucose/administration & dosage , Glucose Transporter Type 4 , Glycogen/metabolism , Horses , Intubation, Gastrointestinal , Lactates/blood , Male , Monosaccharide Transport Proteins/metabolism , Oxygen ConsumptionABSTRACT
CCAAT/Enhancer binding proteins (C/EBPs) are a family of nuclear proteins that function in the control of cell growth, death, and differentiation. We previously reported that C/EBPdelta plays a key role in mammary epithelial cell G(0) growth arrest. In this report, we investigated the role of C/EBPdelta in mammary gland development and function using female mice homozygous for a targeted deletion of C/EBPdelta (C/EBPdelta -/-). C/EBPdelta -/- females develop normally and exhibit normal reproductive and lactational performance. Adult nulliparous C/EBPdelta -/- females, however, exhibit mammary epithelial cell growth control defects. The mean number of mammary ductal branches is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ (wild-type control) females (66.8 +/- 5.2 vs 42.9 +/- 6.3 branch points/field, P < 0.01). In addition, the mean total mammary gland cellular volume occupied by epithelium is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls (29.0 +/- 1.4 vs 20.4 +/- 1.3, P < 0.001). Our results showed that the BrdU labeling index was significantly higher in mammary epithelial cells from nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls during the proestrus/estrus (4.55 +/- 0.70 vs 2.14 +/- 0.43, P < 0.01) and metestrus/diestrus (6.92 +/- 0.75 vs 3.98 +/- 0.43 P < 0.01) phases of the estrus cycle. In contrast, the percentage of mammary epithelial cells undergoing apoptosis during both phases of the estrus cycle did not differ between C/EBPdelta -/- and C/EBPdelta +/+ females. The increased epithelial cell content and proliferative capacity was restricted to the nulliparous C/EBPdelta -/- females as no differences in mammary gland morphology, ductal branching or total epithelial content were observed between multiparous C/EBPdelta -/- and C/EBPdelta +/+ females. These results demonstrate that C/EBPdelta plays a novel role in mammary epithelial cell growth control that appears to be restricted to the nulliparous mammary gland.
Subject(s)
CCAAT-Enhancer-Binding Proteins/physiology , Fibrocystic Breast Disease/genetics , Transcription Factors , Animals , Apoptosis , Blotting, Northern , CCAAT-Enhancer-Binding Protein-delta , CCAAT-Enhancer-Binding Proteins/genetics , Cell Division , Estrus , Female , Gene Expression , Mice , Mice, KnockoutABSTRACT
Oncostatin M (OSM), an interleukin 6-type cytokine, induces sustained up-regulation of CCAAT/enhancer-binding protein (C/EBP) delta mRNA and protein in nonneoplastic HC11 mouse mammary epithelial cells. This up-regulation is dependent on signaling by phospho-Stat3 (signal transducers and activators of transcription). The same signaling pathway is activated in two human breast cancer cell lines, a neoplastic mouse mammary epithelial cell line and a second nonneoplastic mouse mammary epithelial cell line. [3H]Thymidine incorporation and flow cytometry demonstrate that OSM inhibits the growth of HC11 cells by reducing the number of S-phase cells. These phenotypic changes are accompanied by reduced expression of S-phase genes with a corresponding increased expression of G0 genes in HC11 cells. Reduction of C/EBPdelta protein in HC11 cells by expression of a C/EBPdelta antisense construct inhibits OSM-mediated growth arrest. These data demonstrate that OSM induces up-regulation of C/EBPdelta via a Stat3-dependent pathway in mammary epithelial cells and that the growth inhibition induced by OSM depends on the presence of C/EBPdelta.
