ABSTRACT
p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.
Subject(s)
Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , S Phase/physiology , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Animals , Cells, Cultured , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase Inhibitor p21/physiology , Enzyme Activation/physiology , Epidermal Growth Factor/antagonists & inhibitors , Epidermal Growth Factor/physiology , Male , Phosphatidylinositol 3-Kinases/physiology , Rats , Rats, Wistar , Tumor Suppressor Protein p53/antagonists & inhibitors , Tumor Suppressor Protein p53/physiologyABSTRACT
INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.
Subject(s)
Cell Cycle , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase 4/metabolism , Hepatocytes/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Active Transport, Cell Nucleus , Animals , Cell Nucleus/enzymology , Cell Proliferation , Cells, Cultured , Cyclin D1/metabolism , Cyclin E/biosynthesis , Cyclin-Dependent Kinase 4/chemistry , Epidermal Growth Factor/pharmacology , Hepatocytes/drug effects , Male , Rats , Rats, Wistar , Threonine/metabolismABSTRACT
We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.
Subject(s)
ErbB Receptors/metabolism , Hepatectomy , Liver/metabolism , Signal Transduction/physiology , Animals , Cell Cycle , Cell Fractionation , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Liver Regeneration , Male , Membrane Proteins/metabolism , Phosphorylation , Proliferating Cell Nuclear Antigen/metabolism , Rats , Rats, Inbred F344 , Tetradecanoylphorbol Acetate/metabolism , Vesicular Transport ProteinsABSTRACT
The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.
Subject(s)
Adaptor Proteins, Signal Transducing , Endosomes/metabolism , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Signal Transduction/physiology , Ubiquitin-Protein Ligases , Antigens, CD/analysis , Antigens, CD/metabolism , Antimalarials/pharmacology , Cell Compartmentation/physiology , Chloroquine/pharmacology , Endosomes/chemistry , Endosomes/ultrastructure , ErbB Receptors/analysis , GRB2 Adaptor Protein , HeLa Cells , Humans , Intramolecular Transferases/metabolism , Lysosomal Membrane Proteins , Lysosomes/drug effects , Lysosomes/metabolism , Membrane Glycoproteins/analysis , Membrane Glycoproteins/metabolism , Membrane Proteins/analysis , Microscopy, Electron , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Platelet Membrane Glycoproteins/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-cbl , Signal Transduction/drug effects , Tetraspanin 30 , Tyrosine/metabolism , Vesicular Transport ProteinsABSTRACT
The hepatic carcinogen 2-acetylaminofluorene (AAF) exerts its effect as a tumor promoter by mitoinhibition of normal hepatocytes. Initiated cells proliferate selectively and develop into preneoplastic foci and subsequently into carcinomas. To study whether some of the mitoinhibitory effects of AAF could be attributed to an influence on intracellular signal transduction, growth factor signaling was studied in cultured hepatocytes from rats fed AAF for 7 d. Activation through the epidermal growth factor receptor (EGFR) was used to probe possible changes in downstream mitogenic signaling mechanisms. The proliferative response to epidermal growth factor (EGF), measured as proliferating cell nuclear antigen expression and thymidine incorporation, was almost completely inhibited in hepatocytes exposed to AAF. Neither EGFR protein levels nor EGF binding was notably altered in AAF-exposed hepatocytes as opposed to normal hepatocytes. The initial tyrosine phosphorylation of EGFR and downstream activation of Sos, Raf-1, and extracellular signal-regulated protein kinase (ERK) were similar in AAF-treated and control hepatocytes. Even though ERK phosphorylation was unaffected, a remarkable (80%) reduction of ERK nuclear accumulation was observed in AAF-exposed hepatocytes immediately after mitogen stimulation. EGFR tyrosine phosphorylation and downstream signaling lasted 6 h in control cells versus 2 h in AAF-exposed hepatocytes. We previously demonstrated that AAF inhibits the growth factor-dependent induction of cyclin D1 and arrests hepatocyte cell-cycle progression before the p21/CIP1-controlled DNA-damage check point. The present data indicate that the DNA-damaging carcinogen AAF induces growth inhibition by a distinct inhibition of ERK nuclear accumulation after mitogen stimulation. Inhibition of intracellular signal transduction may represent a novel mechanism of growth arrest. Mol. Carcinog. 28:84-96, 2000.
Subject(s)
2-Acetylaminofluorene/pharmacology , Carcinogens/pharmacology , Cell Nucleus/enzymology , Epidermal Growth Factor/pharmacology , Liver/drug effects , Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Division/drug effects , Cells, Cultured , ErbB Receptors/metabolism , Liver/enzymology , Phosphorylation , Protein Binding , RatsABSTRACT
Growth arrest in G(1) is a common cellular response to DNA damage. In the present study, liver regeneration was combined with continuous exposure for 2-acetylaminofluorene (AAF) to study mechanisms of carcinogen-induced growth arrest in vivo. Growth arrest of uninitiated hepatocytes is central for AAF-induced promotion of premalignant lesions in rat liver. To characterize this growth arrest, we examined the activity of cyclin-dependent kinase (Cdk) 2 in unexposed liver and in AAF-exposed liver after growth induction by partial hepatectomy (PH). Rats were fed either a control diet or an AAF-supplemented diet. After 7 d, a two-third PH was performed and the animals were killed after 0, 12, 18, 24, and 36 h. Kinase assays showed that cyclin E- and Cdk2-associated activities were lower in AAF-exposed liver than in unexposed liver after PH. Although the total cellular levels of cyclin E and Cdk2 were similar, cyclin E-Cdk2 assembly was markedly reduced. In unexposed hepatocytes, Cdk2 translocated to the nuclei after PH. Much of the nuclear Cdk2 was in a rapidly migrating form, presumably representing the Thr160-phosphorylated form of Cdk2. In contrast, in AAF-exposed liver both nuclear Cdk2 accumulation and Thr160-phosphorylation of Cdk2 were reduced. Although p53 and p21(waf1/cip1) were induced by AAF, the binding of p21 to cyclin E and Cdk2 was not increased in growth arrested liver. In conclusion, hepatocyte growth arrest caused by AAF exposure was characterized by a lowered Cdk2 activity that was accompanied by a reduced assembly of cyclin E-Cdk2 complexes but not by binding of p21.
Subject(s)
2-Acetylaminofluorene/pharmacology , CDC2-CDC28 Kinases , Carcinogens/pharmacology , Cell Nucleus/metabolism , Cyclin-Dependent Kinases/metabolism , Liver/drug effects , Protein Serine-Threonine Kinases/metabolism , Animals , Cell Division , Cyclin E/metabolism , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , Enzyme Activation , Liver/enzymology , Liver/metabolism , Liver Regeneration , Male , Phosphorylation , Rats , Rats, Inbred F344 , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.
Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Endosomes/ultrastructure , Epidermal Growth Factor/pharmacology , ErbB Receptors/isolation & purification , Phosphoproteins/isolation & purification , Proteins/isolation & purification , Binding, Competitive , Biological Transport , Cell Compartmentation , Cell Membrane/metabolism , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , HeLa Cells , Humans , Intracellular Membranes/metabolism , Microscopy, Immunoelectron , Phosphorylation , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1ABSTRACT
The scandal in Belgium last spring has drawn attention to the environmental hazards of dioxins. Previous production of pesticides and widespread combustion of organic material in the presence of chloride have lead to environmental accumulation of these toxicants, which more precisely are termed polychlorinated dibenzo-p-dioxins and dibenzofurans. Their very long biological half-lives in combination with detectable biological effects at very low concentrations have caused health concerns. Chloracne is the only well documented health effect in man, but there are experimental evidence for carcinogenic, teratogenic, reproductive and immunosuppressive effects. In this presentation we review current knowledge about the cellular effects of dioxins. Dioxins bind to and exert their effects through the cytoplasmic aryl hydrocarbon receptor, which acts as a transcription factor and regulates a number of cytokines and microsomal enzymes. Furthermore, dioxins interfere with hormonal signalling, and anti-oestrogenic effects, vitamin A inhibition and thyroxin mimicry have been reported. Recently, effects on intracellular growth factor signalling have been demonstrated. Dioxins inhibit epidermal growth factor receptor, activate protein kinase C and other intracellular signal transducers, and activate transcription factors. As overall understanding of their cellular mechanisms of toxicity is lacking, we do not possess a complete basis for estimating the adverse health effects of this group of environmental toxicants.
Subject(s)
Dioxins/adverse effects , Environmental Pollutants/adverse effects , Polychlorinated Dibenzodioxins/adverse effects , Animals , Apoptosis/drug effects , Benzofurans/adverse effects , Benzofurans/chemistry , Benzofurans/poisoning , Benzofurans/toxicity , Carcinogens , Dioxins/chemistry , Dioxins/poisoning , Dioxins/toxicity , Environmental Pollutants/poisoning , Environmental Pollutants/toxicity , Hormones/metabolism , Humans , Immunosuppressive Agents/adverse effects , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/poisoning , Immunosuppressive Agents/toxicity , Polychlorinated Dibenzodioxins/chemistry , Polychlorinated Dibenzodioxins/poisoning , Polychlorinated Dibenzodioxins/toxicity , Receptors, Cell Surface/drug effects , Receptors, Cell Surface/metabolism , Reproduction/drug effects , Risk Factors , TeratogensABSTRACT
Minutes comprise > 50 phenotypically similar mutations scattered throughout the genome of Drosophila, many of which are identified as mutations in ribosomal protein (rp) genes. Common traits of the Minute phenotype are short and thin bristles, slow development, and recessive lethality. By mobilizing a P element inserted in the 5' UTR of M(3)95A, the gene encoding ribosomal protein S3 (RPS3), we have generated two homozygous viable heteroalleles that are partial revertants with respect to the Minute phenotype. Molecular characterization revealed both alleles to be imprecise excisions, leaving 40 and 110 bp, respectively, at the P-element insertion site. The weaker allele (40 bp insert) is associated with a approximately 15% decrease in RPS3 mRNA abundance and displays a moderate Minute phenotype. In the stronger allele (110 bp insert) RPS3 mRNA levels are reduced by approximately 60%, resulting in an extreme Minute phenotype that includes many morphological abnormalities as well as sterility in both males and females due to disruption of early gametogenesis. The results show that there is a correlation between reduced RPS3 mRNA levels and the severity of the Minute phenotype, in which faulty differentiation of somatic tissues and arrest of gametogenesis represent the extreme case. That heteroalleles in M(3)95A can mimic the phenotypic variations that exist between different Minute/rp-gene mutations strongly suggests that all phenotypes primarily are caused by reductions in maximum protein synthesis rates, but that the sensitivity for reduced levels of the individual rp-gene products is different.