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1.
Endocrinology ; 142(10): 4212-22, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11564677

ABSTRACT

We investigated the transcriptional regulation of the Na(+)/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na(+)/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na(+)/taurocholate-cotransporting polypeptide mRNA. In HepG2 cells transiently cotransfected with either the long form of the rat PRL receptor or rat GH receptor, signal transducer and activator of transcription-5a and a -5-responsive luciferase expression vector containing the Na(+)/taurocholate-cotransporting polypeptide promoter, mouse placental lactogen I, like ovine PRL, activated -5a via the long form of the rat PRL receptor; whereas rat GH activated -5a via rat GH receptor, leading to transactivation of the Na(+)/taurocholate-cotransporting polypeptide promoter. These data establish that PRL and placental lactogen I induce Na(+)/taurocholate-cotransporting polypeptide gene expression via signal transducer and activator of transcription-5 proteins in liver, and indicate that these hormones play an important role in regulating liver metabolic function.


Subject(s)
Carrier Proteins/physiology , DNA-Binding Proteins/physiology , Liver/physiology , Membrane Transport Proteins , Milk Proteins , Trans-Activators/physiology , Animals , Cells, Cultured , Female , Growth Hormone/pharmacology , Organic Anion Transporters, Sodium-Dependent , Placental Lactogen/pharmacology , Prolactin/pharmacology , Rats , Rats, Sprague-Dawley , STAT5 Transcription Factor , Symporters , Transcriptional Activation/drug effects
2.
Mol Carcinog ; 30(4): 199-208, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11346882

ABSTRACT

The promoting effects of polychlorinated biphenyls (PCBs) have been studied extensively in a variety of two-stage carcinogenesis models. However, the molecular mechanisms responsible for the promotion effects of PCBs have not been elucidated. We measured the effect of PCBs on DNA-binding proteins involved in cell proliferation and transformation. Male Sprague-Dawley rats were injected intraperitoneally with mono-, di-, tri-, tetra-, or hexachlorobiphenyls (300 micromol/kg/d) each day for 4 d and killed 4 h after the last injection. To detect alterations in nuclear proteins that could explain the tumor-promoter activity of PCBs, liver nuclear extracts were analyzed by electrophoretic mobility shift assays. Electrophoretic mobility shift assay analysis of signal transducers and activators of transcription (STAT)-binding activity to a consensus gamma-interferon-activated sequence (GAS) element was compared in liver nuclear extracts from treated rats. STAT-binding activity was eightfold to tenfold higher in nuclear extracts from animals treated with 2,4,4'-trichloro- (PCB 28) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153). Analysis of the protein complex binding to the GAS element, with antibodies specific for STAT3, STAT5, and STAT6, indicated that the protein complex was made up of STAT5 and STAT6 proteins. HepG2 cells transiently transfected with a luciferase reporter gene construct containing many STAT5 binding sites were treated with PCB 28 and PCB 153. PCB 28 stimulated a greater than 25-fold increase in luciferase activity at the highest concentration tested, 1.0 microg/mL. However, enhanced luciferase activity did not occur with PCB 153 treatment. 4-Chlorobiphenyl (PCB 3), PCB 28, and PCB 153 treatment of Sprague-Dawley rats resulted in a large increase in protein binding to a consensus activated protein-1 (AP-1) element. However, 3,4-dichlorobiphenyl (PCB 12) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) treatments did not increase AP-1 transcription activity. Further analysis of the proteins binding to the AP-1 consensus sequence with antibodies specific for c-fos, junD, and junB indicated that the protein composition consists of junD proteins. These data showed functional differences between noncoplanar and coplanar PCBs with respect to STAT activation and AP-1-DNA binding.


Subject(s)
DNA-Binding Proteins/genetics , Milk Proteins , Polychlorinated Biphenyls/pharmacology , Trans-Activators/genetics , Animals , Cytochrome P-450 Enzyme System/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , Electrophoresis, Agar Gel , Liver/metabolism , Luciferases/metabolism , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , STAT5 Transcription Factor , Trans-Activators/metabolism , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism
3.
J Biol Chem ; 276(13): 10485-91, 2001 Mar 30.
Article in English | MEDLINE | ID: mdl-11133982

ABSTRACT

The growth hormone family of cytokines transduces intracellular signals through the Jak2-Stat5 pathway to activate the transcription of target genes. Amino acids within the C termini of Stats constitute the transactivation domain but also regulate the time course of tyrosine phosphorylation and extent of DNA binding. We mutated Thr(757) in the C-terminal of Stat5a (Thr-Stat5) to Val (Val-Stat5) and Asp (Asp-Stat5) and examined the effect on nuclear translocation, DNA binding, and prolactin-induced transcriptional activation of a Stat5-responsive luciferase reporter gene. Val-Stat5 produced a 5-fold higher increase in transcriptional activity relative to Thr-Stat5; Asp-Stat5 produced a similar response to Thr-Stat5. The increased transactivation was ligand induced and was not due to differences in basal expression of Val-Stat5 or to a constitutively activated Stat5 protein. Similar rates of loss of DNA binding ability and phosphorylation of Val- and Thr-Stat5 were observed following a single pulse of prolactin, indicating that the dephosphorylation pathways were unaltered. The serine-threonine kinase inhibitor H7 inhibited the transactivation potential of Thr-, Val-, and Asp-Stat5 to a similar extent, eliminating phosphorylation of Thr(757) as a regulatory mechanism. The results suggest that Thr(757) modulates the transactivation potential of Stat5 by a mechanism(s) that is dependent on the formation of Stat5 dimers and/or their nuclear translocation.


Subject(s)
DNA-Binding Proteins/physiology , Milk Proteins , Threonine/chemistry , Trans-Activators/physiology , Valine/chemistry , Aspartic Acid/chemistry , Blotting, Western , Cell Line , Cell Nucleus/metabolism , DNA/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Humans , Ligands , Luciferases/metabolism , Phosphorylation , Plasmids/metabolism , Prolactin/pharmacology , Protein Structure, Tertiary , STAT5 Transcription Factor , Signal Transduction , Time Factors , Trans-Activators/chemistry , Trans-Activators/metabolism , Transcriptional Activation , Transfection , Tumor Suppressor Proteins , beta-Galactosidase/metabolism
4.
J Clin Invest ; 99(12): 2906-14, 1997 Jun 15.
Article in English | MEDLINE | ID: mdl-9185514

ABSTRACT

The intracellular mechanism(s) underlying the upregulation of the hepatic Na+/taurocholate cotransporting polypeptide (ntcp) by prolactin (PRL) are unknown. In this report, we demonstrate a time-dependent increase in nuclear translocation of phosphorylated liver Stat5 (a member of the ignal ransducers and ctivators of ranscription family) that correlated with suckling-induced increases in serum PRL levels. In electrophoretic mobility gel shift assays, nuclear Stat5 exhibited specific DNA-binding ability towards IFN-gamma-activated sequence (GAS)-like elements (GLEs; 5'TTC/A-PyNPu-G/TAA-3') located in the -937 to -904 bp region of the ntcp promoter. Transient cotransfections in HepG2 cells revealed that PRL inducibility (2.5-3-fold) required coexpression of the long form of the PRL receptor (PRLRL) and Stat5. Deletion analysis mapped the PRLinducible region to -1237 to -758 bp of the ntcp promoter. Linking this 0.5-kb region to a heterologous thymidine kinase (tk) promoter, or linking multimerized ntcp GLEs either upstream of the ntcp minimal promoter (-158 to +47 bp) or the heterologous promoter conferred dose-dependent PRL responsiveness. The short form of the PRL receptor failed to transactivate ntcp GLEs. These results indicate that PRL acts via the PRLRL to facilitate Stat5 binding to ntcp-GLEs and to transcriptionally regulate ntcp.


Subject(s)
Carrier Proteins/genetics , Gene Expression Regulation/drug effects , Liver/metabolism , Milk Proteins , Organic Anion Transporters, Sodium-Dependent , Prolactin/pharmacology , Sodium/pharmacology , Symporters , Animals , Animals, Suckling , Binding Sites , Cell Nucleus/metabolism , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , Interferon-gamma/pharmacology , Kinetics , Phosphorylation , Phosphotyrosine/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Prolactin/genetics , STAT5 Transcription Factor , Trans-Activators/genetics , Trans-Activators/metabolism , Transfection
6.
Drug Metab Dispos ; 23(9): 945-50, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8565785

ABSTRACT

The cDNA for the human monoamine-sulfating form of phenol sulfotransferase (hM-PST) was isolated from a T47D human breast carcinoma lambda gt10 cDNA library, and the active enzyme was expressed in Escherichia coli. Expressed hM-PST was very similar to the brain, intestinal, and platelet forms of the enzyme in its physical, immunological, and kinetic properties. The ability of hM-PST to sulfate a number of xenobiotics was examined and compared with the bacterially expressed human phenol-sulfating form of PST (hP-PST). The translation product of the T47D hM-PST cDNA was 92% identical to that of liver hP-PST. Monoamine neurotransmittors, such as epinephrine and dopamine, were maximally conjugated at lower concentrations by expressed hM-PST (2 and 20 microM, respectively) than by hP-PST (1 and 1 mM, respectively). In contrast, simple phenols--such as p-nitrophenol, acetaminophen, and alpha-naphthol--were maximally conjugated at lower concentrations (4 microM, 20 microM, and 0.5 microM, respectively) by hP-PST than by hM-PST (1 mM, 1.5 mM, and 50 microM, respectively). Minoxidil was sulfated at similar rates and concentrations (7 mM) by both forms of PST. None of the estrogens or related compounds, such as beta-estradiol, 17 alpha-ethinylestradiol, diethylstilbestrol, equilenin, or genistein tested as substrates were sulfated by hM-PST; however, all of these compounds were substrates for hP-PST. As with hP-PST, the hydroxysteroids dehydroepiandrosterone and cortisol were not sulfated by hM-PST.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Arylsulfotransferase/biosynthesis , Biogenic Monoamines/metabolism , Escherichia coli/enzymology , Amino Acid Sequence , Arylsulfotransferase/metabolism , Base Sequence , DNA, Neoplasm/biosynthesis , Escherichia coli/drug effects , Humans , Immunoblotting , Kinetics , Liver/enzymology , Molecular Sequence Data , Oligonucleotide Probes
7.
Biochem J ; 303 ( Pt 1): 33-6, 1994 Oct 01.
Article in English | MEDLINE | ID: mdl-7945260

ABSTRACT

We have shown that Na+/taurocholate co-transport activity is decreased in pregnancy, but rebounds post partum relative to non-pregnant controls, and that activity can be increased by treatment with ovine prolactin [Ganguly, Hyde and Vore (1993) J. Pharmacol. Exp. Ther. 267, 82-87]. To determine the basis for these effects, Na+/taurocholate co-transport was determined in purified basolateral liver plasma-membrane (bLPM) vesicles and compared with steady-state mRNA levels encoding the Na+/taurocholate-co-transporting polypeptide (Ntcp) in non-pregnant controls, pregnant rats (19-20 days pregnant), rats post partum (48 h post partum) and rats post partum treated with bromocriptine to inhibit prolactin secretion. Na+/taurocholate co-transport activity (nmol/5 s per mg of protein) in bLPM was decreased from 10.4 +/- 1.8 in non-pregnant controls to 7.9 +/- 0.6 in bLPM in pregnant rats, but rebounded to 17.5 +/- 1.3 post partum; treatment of rats post partum with bromocriptine to inhibit prolactin secretion decreased activity to 14.1 +/- 0.9. Northern and slot-blot analyses revealed similar changes in mRNA for Ntcp, so that a positive correlation was observed between Na+/taurocholate co-transport activity and Ntcp mRNA. Furthermore, treatment of ovariectomized rats with ovine prolactin increased Ntcp mRNA 10-fold compared with solvent-treated controls, consistent with the 2-fold increase in Vmax, for Na+/taurocholate co-transport in isolated hepatocytes. These data are the first to demonstrate endogenous physiological regulation by prolactin of Ntcp mRNA in parallel with Na+/taurocholate co-transport activity.


Subject(s)
Bromocriptine/pharmacology , Carrier Proteins/metabolism , Liver/metabolism , Organic Anion Transporters, Sodium-Dependent , Postpartum Period/metabolism , Pregnancy, Animal/metabolism , Prolactin/physiology , RNA, Messenger/biosynthesis , Sodium/metabolism , Symporters , Taurocholic Acid/metabolism , Animals , Biological Transport , Carrier Proteins/biosynthesis , Cell Membrane/metabolism , Female , Gene Expression/drug effects , Kinetics , Liver/drug effects , Pregnancy , Rats , Rats, Sprague-Dawley , Reference Values
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