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1.
Int J Biochem Cell Biol ; 78: 315-326, 2016 09.
Article in English | MEDLINE | ID: mdl-27477312

ABSTRACT

Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC.


Subject(s)
Carcinoma, Hepatocellular/pathology , Hepacivirus/physiology , Hepatocyte Nuclear Factor 4/genetics , Hepatocyte Nuclear Factor 4/metabolism , Liver Neoplasms/pathology , Carrier Proteins/genetics , Cell Line, Tumor , Down-Regulation , Gene Expression Regulation, Neoplastic , Hepatocyte Nuclear Factor 1-alpha/genetics , Humans , MicroRNAs/genetics , Phosphorylation , Promoter Regions, Genetic/genetics , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism , Transcription, Genetic
2.
PLoS One ; 11(1): e0147117, 2016.
Article in English | MEDLINE | ID: mdl-26784701

ABSTRACT

The control of hyperlipidemia plays a central role in cardiovascular disease. Previously, we have shown that camphene, a constituent of mastic gum oil, lowers cholesterol and triglycerides (TG) in the plasma of hyperlipidemic rats without affecting HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. In the present study, we examine the mechanism by which camphene exerts its hypolipidemic action. We evaluated the effect of camphene on the de novo synthesis of cholesterol and TG from [14C]-acetate in HepG2 cells, along with the statin mevinolin. Camphene inhibited the biosynthesis of cholesterol in a concentration-dependent manner, and a maximal inhibition of 39% was observed at 100 µM while mevinolin nearly abolished cholesterol biosynthesis. Moreover, treatment with camphene reduced TG by 34% and increased apolipoprotein AI expression. In contrast, mevinolin increased TG by 26% and had a modest effect on apolipoprotein AI expression. To evaluate the mode of action of camphene, we examined its effects on the expression of SREBP-1, which affects TG biosynthesis and SREBP-2, which mostly affects sterol synthesis. Interestingly, camphene increased the nuclear translocation of the mature form of SREBP-1 while mevinolin was found to increase the amount of the mature form of SREBP-2. The effect of camphene is most likely regulated through SREBP-1 by affecting MTP levels in response to a decrease in the intracellular cholesterol. We propose that camphene upregulates SREBP-1 expression and MTP inhibition is likely to be a probable mechanism whereby camphene exerts its hypolipidemic effect.


Subject(s)
Cholesterol/metabolism , Gene Expression Regulation/drug effects , Hypolipidemic Agents/pharmacology , Membrane Transport Proteins/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Terpenes/pharmacology , Triglycerides/metabolism , Animals , Bicyclic Monoterpenes , Blotting, Western , Hep G2 Cells , Humans , Membrane Transport Proteins/genetics , RNA, Messenger/genetics , Rats , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/genetics
3.
PLoS One ; 8(9): e73616, 2013.
Article in English | MEDLINE | ID: mdl-24040001

ABSTRACT

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future.


Subject(s)
Colorectal Neoplasms/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Interferon-gamma/genetics , Interleukin-17/genetics , Interleukin-23/genetics , Interleukins/genetics , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Adult , Aged , Aged, 80 and over , Apoptosis/drug effects , Apoptosis/genetics , Caco-2 Cells , Cell Survival/drug effects , Cell Survival/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Disease Progression , Dose-Response Relationship, Drug , Female , Gene Expression Profiling , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Interferon-gamma/metabolism , Interleukin-17/metabolism , Interleukin-23/metabolism , Interleukins/metabolism , Male , Middle Aged , Mutation , Neoplasm Staging , Polyenes/pharmacology , Polyunsaturated Alkamides/pharmacology , Proto-Oncogene Proteins p21(ras) , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Interleukin-22
4.
Cancer Lett ; 335(2): 387-96, 2013 Jul 28.
Article in English | MEDLINE | ID: mdl-23474496

ABSTRACT

Vanadium is known for its antitumorigenicity. Poised to investigate the impact of well-defined forms of vanadium on processes and specific biomolecules (oncogenes-proteins) involved in cancer cell physiology, a novel ternary V(V)-peroxido-betaine compound was employed in experiments targeting cell viability, apoptosis, reactive oxygen species (ROS) production, H-ras signaling, and matrix metalloproteinase-2 (MMP-2) expression in human breast cancer epithelial and lung adenocarcinoma cells. The results reveal that vanadium imparts a significant decrease in cancer cell viability, reducing H-ras and MMP-2 expression by increasing ROS-mediated apoptosis, distinctly emphasizing the nature, structure and properties of ternary ligands on vanadium anti-tumor activity and its future potential as a metallodrug.


Subject(s)
Adenocarcinoma/drug therapy , Betaine/pharmacology , Breast Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Peroxides/pharmacology , Vanadium/pharmacology , Adenocarcinoma of Lung , Apoptosis/drug effects , Betaine/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Damage/drug effects , Female , Humans , MCF-7 Cells , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 2/drug effects , Matrix Metalloproteinase 2/metabolism , Peroxides/chemistry , Proto-Oncogene Proteins p21(ras)/biosynthesis , Proto-Oncogene Proteins p21(ras)/drug effects , Proto-Oncogene Proteins p21(ras)/metabolism , Reactive Oxygen Species/metabolism , Vanadium/chemistry
5.
J Inorg Biochem ; 121: 100-7, 2013 Apr.
Article in English | MEDLINE | ID: mdl-23376331

ABSTRACT

Cadmium (Cd) is a well-known metal carcinogen associated with tumor formation and carcinogenesis. It has been shown to induce cancer through various cellular mechanisms involving inhibition of DNA repair, abnormal gene expression, induction of oxidative stress, and triggering apoptosis. It is well-established that the H-ras oncogene is involved in the process of carcinogenesis with direct effects on cellular proliferation and tumorigenesis. Given the biotoxicity of cadmium and its association with carcinogenesis, the effect of that metal ion (Cd(II)) was investigated, in a concentration-dependent fashion, on cell viability, cell proliferation, caspase-3 mediated apoptosis and H-ras gene expression in human breast cancer epithelial MCF-7 cells transfected with the H-ras oncogene (wild type and G12V mutation). The findings show a significant modulation effect of cadmium on H-ras gene expression accompanied by up-regulation of caspase-3-related apoptosis in the concentration range of 100-1000 nΜ cadmium. Concurrently, there is a decrease in MCF-7 proliferation. Collectively, the results a) indicate an interplay of cadmium with H-ras(wt and G12V), with cadmium exhibiting a significant concentration-dependent effect on the modulation of H-ras expression, cell viability and proliferation, and b) project distinctly interwoven roles for both cadmium and H-ras in aberrant physiologies in cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Cadmium Chloride/pharmacology , Caspase 3/genetics , Gene Expression Regulation, Neoplastic/drug effects , Oncogene Protein p21(ras)/genetics , Apoptosis/drug effects , Breast Neoplasms/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Female , Humans , Mutation , Oncogene Protein p21(ras)/metabolism , Signal Transduction/drug effects
6.
Eur J Nutr ; 51(2): 181-90, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21598030

ABSTRACT

BACKGROUND: Flavonoids have physiological activity and a variety of pharmacological properties, including anticancer activity in vitro, but structure-anticancer activity relationships are unclear. AIM: The objectives of this work were to investigate the activity of dietary flavonol congeners against cell lines derived from human solid tumours and to examine whether the in vitro activity was associated with specific structural feature(s) of the molecules. METHODS: Antiproliferative activity of the flavonol congeners was investigated against eight different human cancer cell lines representing different types of human solid tumour, using the sulforhodamine B (SRB) assay in accordance with the instructions published by the NCI. Cell cycle perturbations caused by the congeners were monitored by flow-cytometric analysis of DNA stained with propidium iodide. RESULTS: Most of the flavonols examined had weak antiproliferative and cytotoxic activity. Of all the flavonol congeners tested peracetylated tiliroside found to be the most powerful, with significant antiproliferative and cytotoxic activity. Most flavonols induced similar cell cycle perturbations, whereas induction of apoptosis was significant only for cells treated with peracetylated tiliroside. CONCLUSIONS: These findings indicated that the -OH groups of aromatic ring B were not linked to the cytotoxic and antiproliferative activity of the tested flavonols whereas peracetylation of the glycosides resulted in moderate improvement. In contrast, acetylation of tiliroside esterified with coumaric acid at position 5 of the sugar moiety greatly improved the activity of this congener. Overall, the results of this study suggest a critical role of sugar moiety substituents in the anticancer activity of the flavonols.


Subject(s)
Cell Division/drug effects , Cell Proliferation/drug effects , Diet , Flavonols/pharmacology , Apoptosis/drug effects , Cell Line, Tumor/drug effects , Coumaric Acids/metabolism , Flavonoids/pharmacology , Glycosides/metabolism , Humans , Neoplasms/pathology , Structure-Activity Relationship
7.
Cell ; 147(6): 1233-47, 2011 Dec 09.
Article in English | MEDLINE | ID: mdl-22153071

ABSTRACT

Hepatocyte nuclear factor 4α (HNF4α) is essential for liver development and hepatocyte function. Here, we show that transient inhibition of HNF4α initiates hepatocellular transformation through a microRNA-inflammatory feedback loop circuit consisting of miR-124, IL6R, STAT3, miR-24, and miR-629. Moreover, we show that, once this circuit is activated, it maintains suppression of HNF4α and sustains oncogenesis. Systemic administration of miR-124, which modulates inflammatory signaling, prevents and suppresses hepatocellular carcinogenesis by inducing tumor-specific apoptosis without toxic side effects. As we also show that this HNF4α circuit is perturbed in human hepatocellular carcinomas, our data raise the possibility that manipulation of this microRNA feedback-inflammatory loop has therapeutic potential for treating liver cancer.


Subject(s)
Carcinoma, Hepatocellular/metabolism , Cell Transformation, Neoplastic , Hepatocyte Nuclear Factor 4/metabolism , Inflammation/metabolism , Liver Neoplasms/metabolism , MicroRNAs/metabolism , Animals , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Receptors, Interleukin-6/metabolism , STAT3 Transcription Factor/metabolism
8.
PLoS One ; 6(11): e20516, 2011.
Article in English | MEDLINE | ID: mdl-22073134

ABSTRACT

BACKGROUND: Central to the pathology of coronary heart disease is the accumulation of lipids, cholesterol and triglycerides, within the intima of arterial blood vessels. The search for drugs to treat dislipidemia, remains a major pharmaceutical focus. In this study, we evaluated the hypolipidemic properties of the essential oil from Chios mastic gum (MGO). METHODOLOGY/PRINCIPAL FINDINGS: The hypolipidemic effect of MGO was investigated in naïve as well as in rats susceptible to detergent-induced hyperlipidemia. Serum cholesterol and triglycerides were determined using commercial kits. HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase activity was measured in HepG2 cell extracts using a radioactive assay; cellular cholesterol and cholesterol esters were assessed using gas chromatography. MGO administration into naïve rats resulted in a dose-dependent reduction in the constitutive synthesis of serum cholesterol and triglycerides. In hyperlipidemic rats, MGO treatment had also a strong hypolipidemic effect. By testing various components of MGO, we show for the first time that the hypolipidemic action is associated with camphene. Administration of camphene at a dose of 30 µg/gr of body weight in hyperlipidemic rats resulted in a 54.5% reduction of total cholesterol (p<0.001), 54% of Low Density Lipoprotein (LDL)-cholesterol (p<0.001) and 34.5% of triglycerides (p<0.001). Treatment of HepG2 cells with camphene led to a decrease in cellular cholesterol content to the same extend as mevinolin, a known HMG-CoA reductase inhibitor. The hypolipidemic action of camphene is independent of HMG-CoA reductase activity, suggesting that its hypocholesterolemic and hypotriglyceridemic effects are associated with a mechanism of action different than that of statins. CONCLUSIONS: Given the critical role that the control of hyperlipidemia plays in cardiovascular disease, the results of our study provide insights into the use of camphene as an alternative lipid lowering agent and merits further evaluation.


Subject(s)
Acyl Coenzyme A/metabolism , Cholesterol/blood , Hypolipidemic Agents/therapeutic use , Terpenes/therapeutic use , Triglycerides/blood , Animals , Bicyclic Monoterpenes , Cell Line , Chromatography, Gas , Dose-Response Relationship, Drug , Female , Humans , Rats , Rats, Inbred F344
9.
FEBS Lett ; 585(1): 78-84, 2011 Jan 03.
Article in English | MEDLINE | ID: mdl-21130767

ABSTRACT

A significant amount of nuclear p53 is found associated with the nuclear matrix in cells that were exposed to genotoxic stress. In this study we identified Scaffold attachment factor B1 (SAFB1), a nuclear matrix-associated protein that binds the scaffold or matrix attachment regions (S/MARs) of genomic DNA, as a novel p53-interacting protein. SAFB1 was able to associate with p53 through its C-terminal domain, while significant co-localization of the two proteins was observed in cells treated with 5-fluorouracil or mithramycin. Binding of p53 to SAFB1 had a significant functional outcome, since SAFB1 was shown to suppress p53-mediated reporter gene expression. These data suggest that nuclear matrix-associated proteins may play a critical role in regulating p53 localization and activity.


Subject(s)
Matrix Attachment Region Binding Proteins/metabolism , Nuclear Matrix-Associated Proteins/metabolism , Receptors, Estrogen/metabolism , Transcriptional Activation , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/pharmacology , Cell Nucleus/metabolism , Fluorouracil/pharmacology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Hep G2 Cells , Humans , Immunoblotting , K562 Cells , Matrix Attachment Region Binding Proteins/genetics , Microscopy, Fluorescence , Nuclear Matrix-Associated Proteins/genetics , Plicamycin/pharmacology , Protein Binding , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Transport/drug effects , RNA Interference , Receptors, Estrogen/genetics , Transfection , Tumor Suppressor Protein p53/genetics , Two-Hybrid System Techniques
10.
Mol Cell Biochem ; 340(1-2): 291-300, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20232113

ABSTRACT

Besides the well-characterized genomic action of thyroid hormone (TH), mediated by thyroid hormone receptors (TRs), accumulating data support the so-called non-genomic action of TH, which is often related to activation of signalling pathways. In this study, we sought to determine whether TH activates intracellular signalling pathways in the adult cardiac myocytes and whether such activation modulates cell growth and the expression of target proteins important in cardiac function. We demonstrate that TH promotes a rapid increase in the phosphorylation of several kinases, ERK1/2, PKCdelta, p38-MAPK and Akt. This activation is inhibited by triiodothyroacetic acid (triac), which is a TH analogue known to displace the hormone from membrane bound receptors, indicating that this TH effect is mediated through a cell membrane-initiated mechanism. Furthermore, using specific inhibitors of the TH-activated kinases, we show that the long-term effects of TH on the expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), alpha- and beta-myosin heavy chain (MHC) and cell growth are reverted, implying that what is initiated as a non-genomic action of the hormone interfaces with genomic effects. These data provide further insights into the underlying mechanisms of TH action in the heart with potentially important implications in the management of cardiac pathology.


Subject(s)
Cell Enlargement , Myocytes, Cardiac/metabolism , Signal Transduction , Triiodothyronine/metabolism , Age Factors , Animals , Cell Enlargement/drug effects , Cells, Cultured , Enzyme Activation , Gene Expression Regulation , Male , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Myocytes, Cardiac/drug effects , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Phosphorylation , Protein Kinase C-delta/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Rats , Rats, Wistar , Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Signal Transduction/drug effects , Time Factors , Triiodothyronine/analogs & derivatives , Triiodothyronine/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
11.
FEBS J ; 273(9): 1948-58, 2006 May.
Article in English | MEDLINE | ID: mdl-16640558

ABSTRACT

Hepatocyte nuclear factor-4 (HNF-4alpha), a member of the nuclear receptor superfamily, binds DNA exclusively as a homodimer. Dimerization controls important aspects of receptor function, such as DNA binding, protein stability, ligand binding and interaction with coactivators. Crystallographic data of the HNF-4alpha ligand-binding domain (LBD) demonstrated that the homodimer interface is composed of residues in helices 7, 9 and 10 with intermolecular salt bridges, hydrogen bonds and hydrophobic interactions contributing to the stability of the interface. To investigate the importance of the proposed ionic interactions for HNF-4alpha dimerization, interactions critical for formation of the LBD homodimer interface were disrupted by introducing point mutations in residues D261N (H7), E269Q (H7), Q307L (H9), D312N (H9) and Q336L (H10). Mutants were analysed for transactivation, coactivator interaction, DNA binding and dimerization. EMSA analysis showed that the mutants are able to bind DNA as dimers and coimmunoprecipitation assays confirmed dimerization in solution. Furthermore, the mutations do not compromise HNF-4alpha activity and are responsive to PPAR-gamma coactivator-1 (PGC-1). Finally, residue R324, located in the H9/H10 loop, which was suspected to be involved in dimer stabilization via an ionic interaction with residue E276, was studied. In contrast to the conservative substitution R324H the mutation R324L abolishes HNF-4alpha transcriptional activity and coactivator recruitment, revealing that the nature of substitution may play an important role in HNF-4alpha function.


Subject(s)
Hepatocyte Nuclear Factor 4/chemistry , Hepatocyte Nuclear Factor 4/genetics , Mutation , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , COS Cells , Chlorocebus aethiops , DNA/metabolism , Dimerization , Hepatocyte Nuclear Factor 4/metabolism , Hepatocyte Nuclear Factor 4/physiology , Humans , Molecular Sequence Data , Protein Binding/genetics , Rats
12.
J Biol Chem ; 280(23): 21810-9, 2005 Jun 10.
Article in English | MEDLINE | ID: mdl-15826954

ABSTRACT

Hepatocyte nuclear factor-4 (HNF-4) is a transcription factor of the nuclear hormone receptor superfamily that is constitutively active without the addition of exogenous ligand. Crystallographic analysis of the HNF-4alpha and HNF-4gamma ligand binding domains (LBDs) demonstrated the presence of endogenous ligands that may act as structural cofactors for HNF-4. It was also proposed by crystallographic studies that a combination of ligand and coactivator might be required to lock the receptor in its active state. We previously showed that mutations in amino acid residues Ser-181 and Met-182 in H3, Leu-219 and Leu-220 and Arg-226 in H5, Ileu-338 in H10, and Ileu-346 in H11, which line the LBD pocket in HNF-4alpha and come in contact with the ligand, impair its transactivation potential. In the present study, physical and functional interaction assays were utilized with two different coactivators, PGC-1 and SRC-3, to address the role of coactivators in HNF-4 function. We show that the integrity of the hinge (D) domain of HNF-4alpha and the activation function (AF)-2 activation domain region are critical for coactivation. Surprisingly, a different mode of coactivation is observed among the LBD point mutants that lack transcriptional activity. In particular, coactivation is maintained in mutants Ser-181, Arg-226, and Ile-346 but is abolished in mutants Met-182, Leu-219, and Ile-338. Physical interactions confirm this pattern of activation, implying that distinct amino acid residues may be involved in coactivator and ligand interactions, although some residues may be critical for both functions. Our results provide evidence and expand predictions based on the crystallographic data as to the role of coactivators in HNF-4alpha constitutive transcriptional activity.


Subject(s)
DNA-Binding Proteins/physiology , Phosphoproteins/physiology , Transcription Factors/physiology , Amino Acids/chemistry , Animals , Arginine/chemistry , Biotin/chemistry , COS Cells , Cell Line , Chloramphenicol O-Acetyltransferase/metabolism , DNA-Binding Proteins/chemistry , Detergents/pharmacology , Dimerization , Gene Deletion , Genetic Vectors , Glutathione Transferase/metabolism , Hepatocyte Nuclear Factor 4 , Humans , Isoleucine/chemistry , Leucine/chemistry , Ligands , Methionine/chemistry , Models, Biological , Mutation , Phosphoproteins/chemistry , Plasmids/metabolism , Point Mutation , Protein Binding , Protein Structure, Tertiary , Serine/chemistry , Transcription Factors/chemistry , Transcription, Genetic , Transfection
13.
J Mol Cell Cardiol ; 37(5): 1001-11, 2004 Nov.
Article in English | MEDLINE | ID: mdl-15522277

ABSTRACT

cAMP responsive element binding protein (CREB) is a stimulus induced transcription factor with possible relevance for the pathophysiology of the heart. In the present study, we provide evidence that the hypertrophic agonist, phenylephrine (PE), promotes phosphorylation of CREB in adult rat cardiac myocytes through alpha(1)- and beta-adrenergic receptors. PE-induced phosphorylation of CREB was partially inhibited by Ro318220 and H89, which were shown to be potent inhibitors of mitogen- and stress-activated protein kinase-1 (MSK1) activation, implicating the involvement of this kinase in the response. Similar results were obtained when cardiac myocytes were treated with the inhibitors of ERK1/2 and p38 MAPK pathways. In addition, inhibition of protein kinase A by RpcAMP reduced phosphorylation of CREB, suggesting that this pathway is also involved. Furthermore, PE stimulation was accompanied by an increase in CRE-binding activity, which was reduced by drugs that prevented phosphorylation of CREB. An enhanced CBP/phospho-CREB complex formation was also observed, suggesting recruitment of CBP to phosphorylated CREB. These results suggest that PE stimulates phosphorylation and DNA binding activity of CREB in adult rat ventricular myocytes through multiple signaling pathways involving ERK1/2, p38 MAPK, MSK1 and PKA. The same pathways seem to regulate atrial natriuretic peptide (ANF) mRNA expression, a highly conserved marker gene of cardiac hypertrophy, suggesting that the PE-stimulated activation of CREB is likely to play an important role in the hypertrophic response.


Subject(s)
Adrenergic Agonists/pharmacology , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic AMP-Dependent Protein Kinases/physiology , Myocytes, Cardiac/enzymology , Phenylephrine/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/physiology , Adrenergic alpha-1 Receptor Agonists , Animals , Atrial Natriuretic Factor/genetics , CREB-Binding Protein , Cardiomegaly/genetics , Cyclic AMP Response Element-Binding Protein/immunology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Gene Expression Regulation/drug effects , Immunoprecipitation , Indoles/pharmacology , Isoquinolines/pharmacology , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Mitogen-Activated Protein Kinase 3/physiology , Myocytes, Cardiac/drug effects , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Phosphorylation/drug effects , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Receptors, Adrenergic, alpha-1/physiology , Receptors, Adrenergic, beta/physiology , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Signal Transduction/drug effects , Signal Transduction/physiology , Sulfonamides/pharmacology , Trans-Activators/immunology , Trans-Activators/metabolism , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/physiology
14.
J Biol Chem ; 279(29): 30680-8, 2004 Jul 16.
Article in English | MEDLINE | ID: mdl-15123688

ABSTRACT

Hepatocyte nuclear factor-4alpha (HNF-4alpha), a member of the nuclear receptor superfamily, is a crucial regulator of a large number of genes involved in glucose, cholesterol, and fatty acid metabolism. Unlike other members of the superfamily, HNF-4alpha activates transcription in the absence of exogenously added ligand. Recently published crystallographic data show that fatty acids are endogenous ligands for HNF-4. Transcriptional analysis of point mutations of the residues that are located in helices H3, H5, H10, and H11, which have been shown to come in contact with the ligand, resulted in a dramatic decrease in activity, without affecting DNA binding and dimerization. Our results show the importance of residues Ser-181, Met-182 in H3, Leu-219, Leu-220 and Arg-226 in H5, Ile-338 in H10, and Ile-346 in H11 that line the ligand-binding domain pocket in HNF-4alpha and impair its transactivation potential. Structural modeling reveals that the mutations do not cause any large scale structural alterations, and the observed loss in transactivation can be attributed to local changes, demonstrating that these residues play a significant role in maintaining the structural integrity of the HNF-4alpha ligand binding pocket.


Subject(s)
DNA-Binding Proteins/chemistry , Phosphoproteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Cell Line , Chloramphenicol O-Acetyltransferase/metabolism , Crystallography, X-Ray , DNA/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dimerization , Hepatocyte Nuclear Factor 4 , Humans , Ligands , Models, Molecular , Molecular Sequence Data , Mutation , Phosphoproteins/genetics , Phosphoproteins/metabolism , Plasmids/metabolism , Point Mutation , Protein Binding , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Transcriptional Activation , Transfection
15.
Mol Biol Cell ; 14(3): 1279-94, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12631740

ABSTRACT

We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads.


Subject(s)
DNA-Binding Proteins/metabolism , Phosphoproteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Animals , Apolipoprotein C-III , Apolipoproteins C/genetics , Apolipoproteins C/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , COUP Transcription Factor I , Cell Line , Gene Expression Regulation , Hepatocyte Nuclear Factor 4 , Humans , Promoter Regions, Genetic , Protein Binding , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Steroid/genetics , Receptors, Steroid/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Smad3 Protein , Smad4 Protein , Transcription Factors/genetics , Transcriptional Activation
16.
Biochemistry ; 41(4): 1217-28, 2002 Jan 29.
Article in English | MEDLINE | ID: mdl-11802721

ABSTRACT

We have investigated the mechanism of functional cooperativity between specificity protein 1 (Sp1) and hepatocyte nuclear factor-4 (HNF-4) on the human apolipoprotein CIII (apoCIII) promoter. Cotransfections in Drosophila SL2 cells that lack endogenous Sp1 or Sp1-related activities showed that HNF-4 and Sp1 synergistically transactivate the -890/+24 apoCIII promoter up to 150-fold. Synergistic transactivation required the HNF-4 binding site of the apoCIII enhancer. Deletion of part of the Ser/Thr-rich and Gln-rich domain or the C-terminal domain of Sp1 decreased, and deletion of residues 501-610 of Sp1 increased, the functional cooperativity between Sp1 and HNF-4. Physical interactions between the two factors were demonstrated by glutathione S-transferase pull-down and co-immunoprecipitation assays. The amino terminal domain of both factors and the carboxy terminal domain of Sp1 contribute to these interactions. Antagonism between HNF-4 and Sp1 was demonstrated on homopolymeric promoters containing multiple binding sites for either factor, suggesting that the synergism between the two factors occurs only when both factors are bound simultaneously to the DNA. The observed physical interactions between Sp1 and HNF-4 in the context of the apoCIII promoter may explain in part their in vitro and in vivo synergism in the transcriptional activation of the apolipoprotein A-I/apoCIII/apolipoprotein A-IV gene cluster.


Subject(s)
Apolipoproteins C/genetics , DNA-Binding Proteins , Phosphoproteins/metabolism , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Animals , Apolipoprotein C-III , Apolipoproteins C/chemistry , Base Sequence , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Cells, Cultured , DNA Primers , Down-Regulation , Hepatocyte Nuclear Factor 4 , Humans , Protein Binding , Sequence Deletion
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