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1.
J Biol Chem ; 276(1): 355-63, 2001 Jan 05.
Article in English | MEDLINE | ID: mdl-11035028

ABSTRACT

Infection of host cells by viruses leads to the activation of multiple signaling pathways, resulting in the expression of host genes involved in the establishment of the antiviral state. Among the transcription factors mediating the immediate response to virus is interferon regulatory factor-3 (IRF-3) which is post-translationally modified as a result of virus infection. Phosphorylation of latent cytoplasmic IRF-3 on serine and threonine residues in the C-terminal region leads to dimerization, cytoplasmic to nuclear translocation, association with the p300/CBP coactivator, and stimulation of DNA binding and transcriptional activities. We now demonstrate that IRF-3 is a phosphoprotein that is uniquely activated via virus-dependent C-terminal phosphorylation. Paramyxoviridae including measles virus and rhabdoviridae, vesicular stomatitis virus, are potent inducers of a unique virus-activated kinase activity. In contrast, stress inducers, growth factors, DNA-damaging agents, and cytokines do not induce C-terminal IRF-3 phosphorylation, translocation or transactivation, but rather activate a MAPKKK-related signaling pathway that results in N-terminal IRF-3 phosphorylation. The failure of numerous well characterized pharmacological inhibitors to abrogate virus-induced IRF-3 phosphorylation suggests the involvement of a novel kinase activity in IRF-3 regulation by viruses.


Subject(s)
DNA-Binding Proteins/metabolism , Protein Kinases/metabolism , Signal Transduction , Transcription Factors/metabolism , Virus Physiological Phenomena , Active Transport, Cell Nucleus/drug effects , DNA Damage/drug effects , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Enzyme Inhibitors/pharmacology , Genes, Reporter , Growth Substances/pharmacology , Humans , Interferon Regulatory Factor-3 , Jurkat Cells , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System/drug effects , Models, Biological , Mutagenesis , NF-kappa B/metabolism , Oxidative Stress , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors , Protein Structure, Tertiary , Respirovirus/physiology , Signal Transduction/drug effects , Transcription Factors/chemistry , Transcription Factors/genetics , Transcriptional Activation/drug effects
2.
Mol Cell Biochem ; 212(1-2): 99-109, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11108141

ABSTRACT

Angiotensin II (Ang II), the primary effector of the renin-angiotensin system, is a multifunctional hormone that plays an important role in vascular function. In addition to its classical vasoconstrictor action, more recent studies demonstrated that Ang II stimulates the growth of a number of cell types, including vascular smooth muscle cells (SMC) (reviewed in [1-3]). In vivo studies have shown that chronic infusion of Ang II leads to the development of vascular hypertrophy in rats, whereas administration of angiotensin-converting enzyme (ACE) inhibitors or Ang II receptor antagonists prevents or regresses vascular hypertrophy in models of genetic and experimental hypertension [4]. Consistent with in vivo data, several laboratories have shown that Ang II stimulates protein synthesis and induces cellular hypertrophy, but not cell proliferation, in cultured aortic SMC [5-9]. Ang II also induces directed migration (chemotaxis) of vascular SMC [10, 11], although its effect is less prominent than that of platelet-derived growth factor (PDGF). The cellular mechanisms underlying these diverse actions of Ang II are not clearly understood but are likely to involve the activation of distinct signaling pathways.


Subject(s)
Cyclic AMP/physiology , DNA-Binding Proteins/metabolism , Muscle, Smooth, Vascular/physiology , Protein-Tyrosine Kinases/metabolism , Receptors, Angiotensin/physiology , Signal Transduction/physiology , Angiotensin II/physiology , Animals , Humans , Muscle, Smooth, Vascular/cytology , Rats , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2
3.
J Virol ; 74(8): 3781-92, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10729153

ABSTRACT

Virus infection of target cells can result in different biological outcomes: lytic infection, cellular transformation, or cell death by apoptosis. Cells respond to virus infection by the activation of specific transcription factors involved in cytokine gene regulation and cell growth control. The ubiquitously expressed interferon regulatory factor 3 (IRF-3) transcription factor is directly activated following virus infection through posttranslational modification. Phosphorylation of specific C-terminal serine residues results in IRF-3 dimerization, nuclear translocation, and activation of DNA-binding and transactivation potential. Once activated, IRF-3 transcriptionally up regulates alpha/beta interferon genes, the chemokine RANTES, and potentially other genes that inhibit viral infection. We previously generated constitutively active [IRF-3(5D)] and dominant negative (IRF-3 DeltaN) forms of IRF-3 that control target gene expression. In an effort to characterize the growth regulatory properties of IRF-3, we observed that IRF-3 is a mediator of paramyxovirus-induced apoptosis. Expression of the constitutively active form of IRF-3 is toxic, preventing the establishment of stably transfected cells. By using a tetracycline-inducible system, we show that induction of IRF-3(5D) alone is sufficient to induce apoptosis in human embryonic kidney 293 and human Jurkat T cells as measured by DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay, and analysis of DNA content by flow cytometry. Wild-type IRF-3 expression augments paramyxovirus-induced apoptosis, while expression of IRF-3 DeltaN blocks virus-induced apoptosis. In addition, we demonstrate an important role of caspases 8, 9, and 3 in IRF-3-induced apoptosis. These results suggest that IRF-3, in addition to potently activating cytokine genes, regulates apoptotic signalling following virus infection.


Subject(s)
Apoptosis , DNA-Binding Proteins/metabolism , Gene Expression Regulation, Viral , Respirovirus/physiology , Transcription Factors/metabolism , Caspases/metabolism , Cell Line , DNA-Binding Proteins/genetics , Enzyme Activation , Humans , Interferon Regulatory Factor-3 , Interferons/biosynthesis , Jurkat Cells , Respirovirus/pathogenicity , Transcription Factors/genetics , Transgenes
4.
J Cell Biol ; 148(3): 543-56, 2000 Feb 07.
Article in English | MEDLINE | ID: mdl-10662779

ABSTRACT

Platelet-derived growth factor-BB (PDGF-BB) acts as a full mitogen for cultured aortic smooth muscle cells (SMC), promoting DNA synthesis and cell proliferation. In contrast, angiotensin II (Ang II) induces cellular hypertrophy as a result of increased protein synthesis, but is unable to drive cells into S phase. In an effort to understand the molecular basis for this differential growth response, we have examined the downstream effects of PDGF-BB and Ang II on regulators of the cell cycle machinery in rat aortic SMC. Both PDGF-BB and Ang II were found to stimulate the accumulation of G(1) cyclins with similar kinetics. In addition, little difference was observed in the expression level of their catalytic partners, Cdk4 and Cdk2. However, while both factors increased the enzymatic activity of Cdk4, only PDGF-BB stimulated Cdk2 activity in late G(1) phase. The lack of activation of Cdk2 in Ang II-treated cells was causally related to the failure of Ang II to stimulate phosphorylation of the enzyme on threonine and to downregulate p27(Kip1) expression. By contrast, exposure to PDGF-BB resulted in a progressive and dramatic reduction in the level of p27(Kip1) protein. The time course of p27(Kip1) decline was correlated with a reduced rate of synthesis and an increased rate of degradation of the protein. Importantly, the repression of p27(Kip1) synthesis by PDGF-BB was associated with a marked attenuation of Kip1 gene transcription and a corresponding decrease in Kip1 mRNA accumulation. We also show that the failure of Ang II to promote S phase entry is not related to the autocrine production of transforming growth factor-beta1 by aortic SMC. These results identify p27(Kip1) as an important regulator of the phenotypic response of vascular SMC to mitogenic and hypertrophic stimuli.


Subject(s)
Angiotensin II/pharmacology , CDC2-CDC28 Kinases , Cell Cycle Proteins , Gene Expression Regulation/drug effects , Microtubule-Associated Proteins/genetics , Mitogens/pharmacology , Platelet-Derived Growth Factor/pharmacology , Protein Processing, Post-Translational/drug effects , Tumor Suppressor Proteins , Animals , Aorta/cytology , Becaplermin , Cells, Cultured , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/metabolism , DNA/biosynthesis , Microtubule-Associated Proteins/biosynthesis , Muscle, Smooth, Vascular/cytology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-sis , RNA, Messenger , Rats , S Phase , Transcription, Genetic/drug effects , Transforming Growth Factor beta/metabolism
5.
Gene ; 237(1): 1-14, 1999 Sep 03.
Article in English | MEDLINE | ID: mdl-10524230

ABSTRACT

Interferons are a large family of multifunctional secreted proteins involved in antiviral defense, cell growth regulation and immune activation. Viral infection induces transcription of multiple IFN genes, a response that is in part mediated by the interferon regulatory factors (IRFs). The initially characterized members IRF-1 and IRF-2 are now part of a growing family of transcriptional regulators that has expanded to nine members. The functions of the IRFs have also expanded to include distinct roles in biological processes such as pathogen response, cytokine signaling, cell growth regulation and hematopoietic development. The aim of this review is to provide an update on the novel discoveries in the area of IRF transcription factors and the important roles of the new generation of IRFs--particularly IRF-3, IRF-4 and IRF-7.


Subject(s)
DNA-Binding Proteins/physiology , Interferons/genetics , Interferons/metabolism , Phosphoproteins/physiology , Transcription Factors/physiology , Amino Acid Sequence , Animals , Apoptosis/physiology , Cell Division/physiology , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Gene Expression Regulation , Humans , Immune System/metabolism , Interferon Regulatory Factor-1 , Interferon Regulatory Factor-2 , Interferon Regulatory Factor-3 , Interferon Regulatory Factor-7 , Interferon Regulatory Factors , Interferon-Stimulated Gene Factor 3 , Interferon-Stimulated Gene Factor 3, gamma Subunit , Leukemia, T-Cell/metabolism , Molecular Sequence Data , Phosphoproteins/chemistry , Repressor Proteins/metabolism , T-Lymphocytes/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism
6.
J Cardiovasc Pharmacol ; 34(3): 402-6, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10470999

ABSTRACT

We monitored cardiac angiotensin II concentration and AT1-receptor density after long-term blockade of the renin-angiotensin system in inbred control hamsters treated with placebo or losartan (100 mg/kg/day) and cardiomyopathic hamsters treated with placebo, low-(30 mg/kg/day), or high-dose (100 mg/kg/day) losartan or quinapril (100 mg/kg/day). All treatments were started at age 50 days. Angiotensin II-receptor density and affinity were measured by radioligand-binding assays, and ventricular angiotensin II concentration was determined by radioimmunoassay. After 125 and 275 days of treatment, both doses of losartan significantly reduced AT1-receptor density, whereas quinapril had no effect. The administration of both drugs resulted in significant reductions in ventricular angiotensin II concentration. The prolonged administration of losartan was associated with an increase in cardiac hypertrophy, suggesting that angiotensin II signaling is not directly involved or at least does not play a major role in the remodeling process observed in cardiomyopathic hamsters.


Subject(s)
Angiotensin II/metabolism , Cardiomegaly/metabolism , Receptors, Angiotensin/biosynthesis , Renin-Angiotensin System/physiology , Angiotensin Receptor Antagonists , Animals , Binding, Competitive , Cricetinae , Down-Regulation , Heart Ventricles/metabolism , Losartan/pharmacology , Male , Mesocricetus , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Receptors, Angiotensin/metabolism
7.
J Biol Chem ; 272(43): 26879-86, 1997 Oct 24.
Article in English | MEDLINE | ID: mdl-9341120

ABSTRACT

In the present study, we have examined the effect of increased cyclic AMP (cAMP) levels on the stimulatory action of angiotensin II (Ang II) on protein synthesis. Treatment with cAMP-elevating agents potently inhibited Ang II-induced protein synthesis in rat aortic smooth muscle cells and in rat fibroblasts expressing the human AT1 receptor. The inhibition was dose-dependent and was observed at all concentrations of the peptide. To explore the mechanism of cAMP action, we have analyzed the effects of forskolin and 3-isobutyl-1-methylxanthine on various receptor-mediated responses. Elevation of cAMP did not alter the binding properties of the AT1 receptor and did not interfere with the activation of phospholipase C or the induction of early growth response genes by Ang II. Likewise, Ang II-dependent activation of the mitogen-activated protein kinases ERK1/ERK2 and p70 S6 kinase was unaffected by cAMP. In contrast, we found that increased concentration of cAMP strongly inhibited the stimulatory effect of Ang II on protein tyrosine phosphorylation. Specifically, cAMP abolished Ang II-induced tyrosine phosphorylation of the focal adhesion-associated protein paxillin and of the tyrosine kinase Tyk2. These results identify a novel mechanism by which the cAMP signaling system may exert growth-inhibitory effects in specific cell types.


Subject(s)
Angiotensin II/pharmacology , Cyclic AMP/metabolism , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular/metabolism , Phosphotyrosine , Receptors, Angiotensin/physiology , 1-Methyl-3-isobutylxanthine/pharmacology , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Aorta , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Cholera Toxin/pharmacology , Colforsin/pharmacology , Fibroblasts , Gene Expression Regulation/drug effects , Humans , Isoproterenol/pharmacology , Kinetics , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Muscle, Smooth, Vascular/drug effects , Phosphorylation , Rats , Receptor, Angiotensin, Type 1 , Receptor, Angiotensin, Type 2 , Receptors, Angiotensin/biosynthesis , Recombinant Proteins/metabolism , Ribosomal Protein S6 Kinases/metabolism , Signal Transduction/drug effects , Transfection , Type C Phospholipases/metabolism
8.
Oncogene ; 15(6): 717-25, 1997 Aug 07.
Article in English | MEDLINE | ID: mdl-9264412

ABSTRACT

Mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) is a dual-specificity protein phosphatase encoded by an immediate-early gene responsive to growth factors and stress. The MKP-1 protein selectively inactivates MAP kinases in vitro by dephosphorylation of the regulatory Thr and Tyr residues. Little is known on the mechanisms that regulate MKP-1 gene expression. Here, we demonstrate that Ca2+ is both necessary and sufficient for the induction of MKP-1 gene expression. Treatment of Rat1 fibroblasts with the Ca2+ chelating agent BAPTA completely suppressed serum-induced MKP-1 expression in a dose- and time-dependent manner. The inhibitory effect of BAPTA was observed at the level of the protein and the mRNA. Importantly, Ca2+ chelation blocked the induction of MKP-1 expression in response to all stimuli tested and in different cell types. Increasing the intracellular concentration of Ca2+ with the ionophore A23187 was sufficient to induce MKP-1 mRNA and protein expression in rat fibroblasts. We also provide evidence that activation of MAP kinases is not an absolute requirement for induction of the MKP-1 gene. Exposure of rat fibroblasts to A23187 induced MKP-1 expression without activating the JNK and p38 MAP kinase pathways. Also, inhibition of the ERK pathway with the selective MEK inhibitor PD98059 did not interfere with serum-stimulated MKP-1 mRNA expression. These results will help define the regulatory mechanisms that govern MKP-1 gene transcription in target cells.


Subject(s)
Calcium/physiology , Cell Cycle Proteins , Gene Expression Regulation , Immediate-Early Proteins/genetics , Immediate-Early Proteins/metabolism , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases , Phosphoprotein Phosphatases , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Calcimycin/pharmacology , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Dual Specificity Phosphatase 1 , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Flavonoids/pharmacology , Ionophores/pharmacology , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Protein Kinases/metabolism , Protein Phosphatase 1 , Proteins/metabolism , RNA, Messenger/metabolism , Transcription, Genetic , p38 Mitogen-Activated Protein Kinases
9.
J Biol Chem ; 271(27): 16047-52, 1996 Jul 05.
Article in English | MEDLINE | ID: mdl-8663242

ABSTRACT

A common response of cells to mitogenic and hypertrophic factors is the activation of high rates of protein synthesis. To investigate the molecular basis of this action, we have used the recently developed MAP kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor PD 98059 to examine the involvement of the ERK pathway in the regulation of global protein synthesis by growth factors in rat aortic smooth muscle cells (SMC). Incubation with PD 98059 blocked angiotensin II (AII)-dependent phosphorylation and enzymatic activity of both MEK1 and MEK2 isoforms, leading to inhibition of the phosphorylation and activation of p44(mapk) and p42(mapk). The compound was found to selectively inhibit activation of the ERK pathway by AII, but not the stimulation of p70 S6 kinase, phospholipase C, or tyrosine phosphorylation. Most importantly, treatment of aortic SMC with PD 98059 potently inhibited AII-stimulated protein synthesis with a half-maximal inhibitory concentration of 4.3 microM. The effect of PD 98059 was not restricted to AII, since the compound also blocked to various extent the induction of protein synthesis by growth factors acting through tyrosine kinase receptors, G protein-coupled receptors, or protein kinase C. These results provide strong evidence that activation of ERK isoforms is an obligatory step for growth factor-induced protein synthesis in aortic SMC.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Growth Substances/pharmacology , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Muscle, Smooth, Vascular/metabolism , Protein Biosynthesis/drug effects , Amino Acid Sequence , Angiotensin II/antagonists & inhibitors , Angiotensin II/pharmacology , Animals , Aorta , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cells, Cultured , Fibroblast Growth Factor 2/pharmacology , Insulin/pharmacology , Kinetics , MAP Kinase Kinase 1 , MAP Kinase Kinase 2 , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Molecular Sequence Data , Muscle, Smooth, Vascular/drug effects , Phosphorylation , Polyenes/pharmacology , Protein Serine-Threonine Kinases/metabolism , Protein Synthesis Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/metabolism , Rats , Sirolimus , Substrate Specificity , Tetradecanoylphorbol Acetate/pharmacology , Thrombin/pharmacology
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