ABSTRACT
Hepatocyte nuclear factor 4alpha (HNF4alpha) is essential for the establishment and maintenance of liver-specific gene expression. The HNF4alpha gene codes for several isoforms whose developmental and physiological relevance has not yet been explored. HNF4alpha1 and HNF4alpha7 originate from different promoters, while alternative splicing in 3' leads to HNF4alpha2 and HNF4alpha8, respectively. HNF4alpha7/alpha8 were abundantly expressed in embryonic liver and fetal-like hepatoma cells. HNF4alpha1/alpha2 transcripts were up-regulated at birth and represented the only isoforms in adult-like hepatoma cells. In line with its expression profile, HNF4alpha7 activated more avidly than HNF4alpha1 reporter plasmids for genes that are expressed early. The expression patterns of both isoforms together with the differences observed in their transcriptional activities provide elements accounting for fine-tuning of the activity of HNF4alpha. The sequential expression of HNF4alpha7/alpha8 and HNF4alpha1/alpha2 during mouse liver development is the only modification in liver-enriched transcription factors thus far recorded, which parallels the transition from the fetal to the adult hepatic phenotype.
Subject(s)
DNA-Binding Proteins , Liver/embryology , Phosphoproteins/biosynthesis , Phosphoproteins/chemistry , RNA Splicing , Transcription Factors/biosynthesis , Transcription Factors/chemistry , 3T3 Cells , Alternative Splicing , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Blotting, Northern , Blotting, Western , Carcinoma, Hepatocellular/metabolism , Cell Line , Dimerization , Exons , Genes, Reporter , Hepatocyte Nuclear Factor 4 , Liver/metabolism , Mice , Models, Genetic , Phenotype , Plasmids/metabolism , Promoter Regions, Genetic , Protein Isoforms , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Time Factors , Transcription, Genetic , Transcriptional Activation , TransfectionABSTRACT
We have characterized a 700 bp enhancer element around -6 kb relative to the HNF4alpha1 transcription start. This element increases activity and confers glucocorticoid induction to a heterologous as well as the homologous promoters in differentiated hepatoma cells and is transactivated by HNF4alpha1, HNF4alpha7, HNF1alpha and HNF1beta in dedifferentiated hepatoma cells. A 240 bp sub-region conserves basal and hormone-induced enhancer activity. It contains HNF1, HNF4, HNF3 and C/EBP binding sites as shown by DNase I footprinting and electrophoretic mobility shift assays using nuclear extracts and/or recombinant HNF1alpha and HNF4alpha1. Mutation analyses showed that the HNF1 site is essential for HNF1alpha transactivation and is required for full basal enhancer activity, as is the C/EBP site. Glucocorticoid response element consensus sites which overlap the C/EBP, HNF4 and HNF3 sites are crucial for optimal hormonal induction. We present a model that accounts for weak expression of HNF4alpha1 in the embryonic liver and strong expression in the newborn/adult liver via the binding sites identified in the enhancer.
Subject(s)
Enhancer Elements, Genetic/genetics , Glucocorticoids/pharmacology , Phosphoproteins/genetics , Promoter Regions, Genetic/genetics , Transcription Factors/genetics , Transcription Factors/physiology , Animals , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Binding Sites/genetics , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , DNA-Binding Proteins/metabolism , Dexamethasone/pharmacology , Enhancer Elements, Genetic/physiology , Gene Expression Regulation/drug effects , Hepatocyte Nuclear Factor 1 , Hepatocyte Nuclear Factor 1-alpha , Hepatocyte Nuclear Factor 1-beta , Hepatocyte Nuclear Factor 3-alpha , Hepatocyte Nuclear Factor 4 , Liver/metabolism , Luciferases/genetics , Luciferases/metabolism , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Plasmids/genetics , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Regulatory Sequences, Nucleic Acid/physiology , Transcription Factors/metabolism , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
In rat-1 fibroblasts stably expressing alpha1d-adrenoceptors BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil decreased basal [Ca2+]i. WB 4101 induced a very small effect on this parameter but when added before the other antagonists it blocked their effect. All these agents inhibited the action of norepinephrine. Phorbol myristate acetate also blocked the effect of norepinephrine and decreased basal [Ca2+]i. Staurosporine inhibited these effects of the phorbol ester. Our results suggest that: (1) alpha1d-adrenoceptors exhibit spontaneous ligand-independent activity, (2) BMY 7378, phentolamine, chloroethylclonidine and 5-methyl urapidil act as inverse agonists and (3) protein kinase C activation blocks spontaneous and agonist-stimulated alpha1d-adrenoceptor activity.
Subject(s)
Adrenergic alpha-Agonists/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Calcium/metabolism , Receptors, Adrenergic, alpha-1/physiology , Tetradecanoylphorbol Acetate/pharmacology , Adrenergic alpha-1 Receptor Agonists , Adrenergic alpha-1 Receptor Antagonists , Animals , Cell Line , Clonidine/analogs & derivatives , Clonidine/pharmacology , Dioxanes/pharmacology , Dose-Response Relationship, Drug , Endothelin-1/pharmacology , Fibroblasts , Norepinephrine/pharmacology , Phentolamine/pharmacology , Piperazines/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/physiology , Rats , Staurosporine/pharmacology , TransfectionABSTRACT
The action of bradykinin was studied in rat-1 fibroblasts stably expressing alpha1b-adrenoceptors. It was observed that bradykinin and kallidin markedly increase cytosol calcium concentration, but that the B1 agonist, des-Arg9-bradykinin, only mimicked this effect to a minimal extent. Antagonists, selective for the B2 subtype, such as Hoe 140, blocked this effect of bradykinin and kallidin. Similarly, bradykinin and kallidin stimulated the production of inositol phosphates and B2 antagonists blocked their actions. The possibility that bradykinin could modulate alpha1b-adrenoceptors was studied. It was observed that bradykinin and kallidin increased alpha1b-adrenoceptor phosphorylation and that such effect was also blocked by Hoe 140. Interestingly, the ability of norepinephrine to increase intracellular calcium concentration was not altered by pretreatment of the cells with bradykinin, i.e. bradykinin induced alpha1b-adrenoceptor phosphorylation but this did not lead to receptor desensitization.
