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1.
Genes (Basel) ; 10(12)2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31810288

RESUMO

The EphB4 gene encodes for a transmembrane tyrosine kinase receptor involved in embryonic blood vessel differentiation and cancer development. Although EphB4 is known to be regulated at the post-translational level, little is known about its gene regulation. The present study describes the core promoter elements' identification and cloning, the cis-regulatory elements' mapping and the serum regulation of the human EphB4 gene promoter region. Using bioinformatic analysis, Sanger sequencing and recombinant DNA technology, we analyzed the EphB4 gene upstream region spanning +40/-1509 from the actual transcription start site (TSS) and proved it to be a TATA-less gene promoter with dispersed regulatory elements characterized by a novel motif-of-ten element (MTE) at positions +18/+28, and a DPE-like motif and a DPE-like-repeated motif (DRM) spanning nt +27/+30 and +32 +35, respectively. We also mapped both proximal (multiple Sp1) and distal (HoxA9) trans-activating/dispersed cis-acting transcription factor (TF)-binding elements on the region we studied and used a transient transfection reporter assay to characterize its regulation by serum and IGF-II using EphB4 promoter deletion constructs with or without the identified new DNA-binding elements. Altogether, these findings shed new light on the human EphB4 promoter structure and regulation, suggesting mechanistic features conserved among Pol-II TATA-less genes phylogenetically shared from Drosophila to Human genomes.


Assuntos
Regulação da Expressão Gênica , Motivos de Nucleotídeos , Receptor EphB4 , Elemento de Resposta Sérica , Transativadores , Transcrição Genética , Animais , Linhagem Celular , Clonagem Molecular , Drosophila , Humanos , Camundongos , Filogenia , Receptor EphB4/biossíntese , Receptor EphB4/genética , Transativadores/genética , Transativadores/metabolismo
2.
Chin J Physiol ; 62(2): 63-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31243176

RESUMO

One of the principal signaling pathway outcomes from brain-derived neurotrophic factor (BDNF) is the activation of antiapoptotic pathways. In addition to the role of extracellular signal-regulated kinase 1/2 and phosphatidylinositol-3 kinase, BDNF activates protein kinase CK2 to mediate its neuroprotective effect. The inhibition of CK2 activity has been shown to induce apoptosis. Although serum response element (SRE)-mediated transcription has been reported to be activated by BDNF and that the phosphorylation of serum response factor (SRF) by CK2 has been shown to enhance its DNA binding activity, the biological relevance of these interactions remains largely unclear. In the present study, we found that SRE-mediated transcription, CK2 activity, and SRF phosphorylation increased in PC12 cells under BDNF treatment. The transfection of CK2α siRNA blocked the enhancing effect of BDNF on SRE-mediated transcription, SRF phosphorylation, and Mcl-1 gene expression. Moreover, the blockade of CK2 diminished the antiapoptotic effects of BDNF on SRE-mediated transcription, Mcl-1 gene expression, and cell viability under rotenone-induced cytotoxicity. Our data may assist in the development of therapeutic strategies for inhibiting apoptosis during neurodegeneration.


Assuntos
Caseína Quinase II/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo , Proteína Quinase 3 Ativada por Mitógeno , Fosforilação , Ratos , Elemento de Resposta Sérica , Transdução de Sinais
3.
Small GTPases ; 10(5): 361-366, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-28489964

RESUMO

PLEKHG2 is a Gßγ- and Gαs-dependent guanine nucleotide exchange factor for Rac1 and Cdc42 small GTPases and has been shown to mediate signaling pathways such as those for actin cytoskeletal reorganization and serum response element (SRE)-dependent gene transcription. We have shown that the four-and-a-half LIM domains (FHL) 1 acts as a positive regulator of PLEKHG2. Here, we evaluated the other FHL family members and found that the FHL1A specifically regulate the PLEKHG2 activity. Moreover, FHL1A further enhanced Gßγ- and PLEKHG2-induced SRE-dependent gene transcription, whereas FHL1A partially restored the attenuated PLEKHG2-induced SRE-dependent gene transcription by Gαs. Our results suggest that FHL1A specifically interacts with PLEKHG2 to regulate a function of PLEKHG2 that is modified by the interaction of Gßγ and Gαs.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Elemento de Resposta Sérica , Transcrição Genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas Musculares/genética , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Front Immunol ; 9: 1906, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30197642

RESUMO

Arachidonate 15-lipoxygenase (ALOX15) and arachidonate 15-lipoxygenase, type B (ALOX15B) catalyze the dioxygenation of polyunsaturated fatty acids and are upregulated in human alternatively activated macrophages (AAMs) induced by Th2 cytokine interleukin-4 (IL-4) and/or interleukin-13. Known primarily for roles in bioactive lipid mediator synthesis, 15-lipoxygenases (15-LOXs) have been implicated in various macrophage functions including efferocytosis and ferroptosis. Using a combination of inhibitors and siRNAs to suppress 15-LOX isoforms, we studied the role of 15-LOXs in cellular cholesterol homeostasis and immune function in naïve and AAMs. Silencing or inhibiting the 15-LOX isoforms impaired sterol regulatory element binding protein (SREBP)-2 signaling by inhibiting SREBP-2 processing into mature transcription factor and reduced SREBP-2 binding to sterol regulatory elements and subsequent target gene expression. Silencing ALOX15B reduced cellular cholesterol and the cholesterol intermediates desmosterol, lanosterol, 24,25-dihydrolanosterol, and lathosterol as well as oxysterols in IL-4-stimulated macrophages. In addition, attenuating both 15-LOX isoforms did not generally affect IL-4 gene expression but rather uniquely impacted IL-4-induced CCL17 production in an SREBP-2-dependent manner resulting in reduced T cell migration to macrophage conditioned media. In conclusion, we identified a novel role for ALOX15B, and to a lesser extent ALOX15, in cholesterol homeostasis and CCL17 production in human macrophages.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Quimiocina CCL17/biossíntese , Colesterol/metabolismo , Homeostase , Macrófagos/imunologia , Macrófagos/metabolismo , Araquidonato 15-Lipoxigenase/genética , Movimento Celular/genética , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Humanos , Metabolismo dos Lipídeos , Ligação Proteica , RNA Interferente Pequeno/genética , Elemento de Resposta Sérica , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo
5.
Zoolog Sci ; 35(2): 109-114, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29623784

RESUMO

Human, mouse, and zebrafish ovarian cancer G protein-coupled receptors (OGR1s) are activated by both metals and extracellular protons. In the present study, we examined whether pig, rat, chicken, and Xenopus OGR1 homologs could sense and be activated by protons and metals. We found that all homologs stimulated serum response element (SRE)-driven promoter activities when they are stimulated by protons. On the other hand, metals differentially activated the homologs. The results using chimeric receptors of human and zebrafish OGR1s indicate that the specificity of the metal-induced activation lies in the extracellular region. These results suggest that protons are an evolutionally conserved agonist of OGR1. However, the types of metals that activated the receptor differed among the homologs.


Assuntos
Galinhas/genética , Metais/administração & dosagem , Prótons , Ratos/genética , Receptores Acoplados a Proteínas-G/genética , Sus scrofa/genética , Xenopus/genética , Animais , Galinhas/metabolismo , Feminino , Células HEK293 , Humanos , Neoplasias Ovarianas/genética , Ratos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Sus scrofa/metabolismo , Xenopus/metabolismo
6.
Mol Cell Endocrinol ; 472: 126-139, 2018 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-29225069

RESUMO

Stimulation of pancreatic ß-cells with glucose activates the protein kinases B-Raf and extracellular signal-regulated protein kinase that participate in glucose sensing. Inhibition of both kinases results in impairment of glucose-regulated gene transcription. To analyze the signaling pathway controlled by B-Raf, we expressed a conditionally active form of B-Raf in INS-1 insulinoma cells. Here, we show that stimulation of B-Raf strongly activated the transcription factor AP-1 which is accompanied by increased c-Jun and c-Fos promoter activities, an upregulation of c-Jun and c-Fos biosynthesis, and elevated transcriptional activation potentials of c-Jun and c-Fos. Mutational analysis identified the AP-1 sites within the c-Jun promoter and the serum response element (SRE) within the c-Fos promoter as the essential genetic elements connecting B-Raf stimulation with AP-1 activation. In line with this, the transcriptional activation potential of the SRE-binding protein Elk-1 was increased following B-Raf activation. The signal pathway from B-Raf to AP-1 required the activation of c-Jun. We identified the cyclin D1 gene as a delayed response gene for AP-1 following stimulation of B-Raf in insulinoma cells. Moreover, MAP kinase phosphatase-1 and the Ca2+/calmodulin-dependent protein phosphatase calcineurin were identified to function as shut-off-devices for the signaling cascade connecting B-Raf stimulation with the activation of AP-1. The fact that stimulation with glucose, activation of L-type voltage-gated Ca2+ channels, and stimulation of B-Raf all trigger an activation of AP-1 indicates that AP-1 is a point of convergence of signaling pathways in ß-cell.


Assuntos
Insulinoma/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fator de Transcrição AP-1/genética , Transcrição Genética , Regulação para Cima/genética , Animais , Calcineurina/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Ciclina D1/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Regulação Neoplásica da Expressão Gênica , Modelos Biológicos , Mutação/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Ratos , Elemento de Resposta Sérica/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/genética , Proteínas Elk-1 do Domínio ets/metabolismo
7.
J Cell Physiol ; 233(2): 1005-1016, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28407230

RESUMO

Sterol regulatory element binding protein 1 (SREBP-1) is well-known as the master regulator of lipogenesis in rodents. Acyl-CoA synthetase short-chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl-CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl-CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non-lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p < 0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p < 0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP-1. Furthermore, SREBP-1 interacted with an SRE (SREBP response element) spanning at -475 to -483 bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP-1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2.


Assuntos
Acetato-CoA Ligase/metabolismo , Células Epiteliais/enzimologia , Ácidos Graxos/biossíntese , Lactação , Glândulas Mamárias Animais/enzimologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Acetato-CoA Ligase/genética , Animais , Células Cultivadas , Feminino , Regulação Enzimológica da Expressão Gênica , Cabras , Gotículas Lipídicas/metabolismo , Lipogênese/genética , Glândulas Mamárias Animais/citologia , Mutagênese Sítio-Dirigida , Mutação , Regiões Promotoras Genéticas , Interferência de RNA , Elemento de Resposta Sérica , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Transfecção , Triglicerídeos/biossíntese
8.
Biol Reprod ; 96(5): 1043-1051, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28863434

RESUMO

We examined direct effect of kisspeptin on pituitary gonadotrophs. Kisspeptin-10 (KP10) significantly increased the promoter activities of the gonadotropin subunits, common alpha-glycoprotein (Cga), luteinizing hormone beta (Lhb), and follicle-stimulatinghormone beta (Fshb) in LbetaT2 cells overexpressing kisspeptin receptor (Kiss1r). KP10 and gonadotropin-releasing hormone (GnRH) increased gonadotropin subunit levels to similar degrees and combined treatment with GnRH and KP10 did not potentiate their individual effects. Adenylate cyclase-activating polypeptide 1 (ADCYAP1) also stimulates all three gonadotropin subunits. When cells were stimulated with both KP10 and ADCYAP1, expression of gonadotropin subunits was further increased compared to KP10 or ADCYAP1 alone. KP10 and GnRH dramatically increased serum response element (Sre) promoter levels but only slightly increased cAMP response element (Cre) promoter levels. Combined stimulation with KP10 and GnRH further increased Sre promoter levels. In contrast, ADCYAP1 slightly increased Sre promoter expression but did not modify the effect of KP10. However, ADCYAP1 increased Cre promoter to greater levels than KP10 alone, and combined treatment with KP10 and ADCYAP1 further increased Cre promoter expression. KP10 increased the expression of ADCYAP1 type I receptor (Adcyap1r) and the basal activity of the Cga promoter was increased at a higher Adcyap1r transfection level. The KP10-induced fold increase in all three gonadotropin subunit promoters was not altered by transfection with a higher amount of Adcyap1r vector. Our findings using model cells show that distinct signaling activation by ADCYAP1 potentiates the action of KP10. We also found that KP10 increases Adcyap1r expression.


Assuntos
Kisspeptinas/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Animais , Subunidade beta do Hormônio Folículoestimulante/metabolismo , Regulação da Expressão Gênica/genética , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Kisspeptinas/genética , Hormônio Luteinizante Subunidade beta/metabolismo , Camundongos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Plasmídeos , Regiões Promotoras Genéticas , Receptores de Kisspeptina-1/genética , Elemento de Resposta Sérica/genética
9.
Bioorg Med Chem Lett ; 27(8): 1744-1749, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28285914

RESUMO

We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.


Assuntos
Ácidos Nipecóticos/farmacocinética , Ácidos Nipecóticos/uso terapêutico , Fator Rho/antagonistas & inibidores , Escleroderma Sistêmico/tratamento farmacológico , Pele/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Administração Oral , Animais , Modelos Animais de Doenças , Fibrose , Células HEK293 , Humanos , Camundongos , Ácidos Nipecóticos/administração & dosagem , Ácidos Nipecóticos/química , Fator Rho/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Elemento de Resposta Sérica/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Transativadores/antagonistas & inibidores , Transativadores/metabolismo
10.
Can J Physiol Pharmacol ; 95(3): 275-280, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28157379

RESUMO

Lysophosphatidic acid (LPA), one component of oxidized low-density lipoprotein (ox-LDL), is a potent bioactive phospholipid. Our recent data reveal that LPA induces matricellular protein CCN1 (also known as Cyr61) expression in aortic smooth muscle cells (SMCs) and that CCN1 bridges LPA and integrin signaling pathways leading to SMC migration. Whether and how LPA regulates the transcriptional machinery of the CCN1 gene are unknown. In this study, we found that LPA markedly induces CCN1 mRNA expression in SMCs. Using deleting mutation and reporter gene strategies, we demonstrated regions from -2038 to -1787 and from -101 to +63 of the CCN1 promoter contain the essential regulatory elements. The serum response element (SRE) and cyclic AMP-response element (CRE) are located in these regions. LPA induced time-dependent phosphorylation of serum response factor (SRF) and CRE-binding protein (CREB) in mouse SMCs. Luciferase assays of a series of deleted, mutated CCN1 promoter-reporter gene constructs and dominant negative construct revealed the distal SRE and the proximal CRE in the CCN1 promoter are required for LPA-induced CCN1 gene expression. Our results imply that elevated LPA levels may trigger SMC migration and exacerbate restenosis and atherosclerotic lesions through the induced CCN1, which communicates with a set of plasma membrane proteins and intracellular kinases.


Assuntos
Proteína Rica em Cisteína 61/genética , Lisofosfolipídeos/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Elemento de Resposta Sérica/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Sítios de Ligação , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Genes Reporter , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosforilação , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima
11.
Cell Signal ; 32: 115-123, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28108261

RESUMO

PLEKHG2 is a Gßγ-dependent guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42, and has been shown to mediate signalling pathways such as actin cytoskeleton reorganization and serum response element (SRE)-dependent gene transcription. Here we show that the constitutively active mutant of the Gαs subunit significantly attenuated PLEKHG2-induced SRE-mediated gene transcription. Strikingly, we observed that the constitutive activation of endogenous Gαs by treatment with CTx caused a similar inhibitory effect on PLEKHG2-induced activation of SRE. However, both the enforced expression of the catalytic subunit ß of protein kinase A and the treatment with dibutyl-cyclic AMP failed to mimic the inhibitory effect of Gαs on PLEKHG2. Furthermore, the dominant negative mutant of protein kinase A had no effect on PLEKHG2-mediated SRE activation. Performing immunoprecipitation and an in vitro pulldown assay, we found that PLEKHG2 directly interacted with the active form of the Gαs subunit in cells. The interaction between PLEKHG2 and Gαs required the N-terminal region of PLEKHG2, which includes the DH domain, a functional domain of GEF, suggesting that Gαs directly masks the DH domain of PLEKHG2. In a previous study, we reported that Gßγ accelerates PLEKHG2-mediated SRE-dependent gene transcription. Interestingly, Gαs also inhibited the hyperactivation of SRE induced by the co-expression of Gßγ and PLEKHG2; however, Gαs and Gßγ bind to different regions of PLEKHG2. This is the first report to show that PLEKHG2 is a novel effector of Gαs, and is negatively regulated by the Gαs subunit through direct interaction.


Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Ligação Proteica , Elemento de Resposta Sérica/genética , Transcrição Genética
12.
Biochemistry ; 56(3): 473-486, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28005346

RESUMO

GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.


Assuntos
Lisofosfolipídeos/química , Piperazinas/química , Pirrolidinas/química , Receptores Acoplados a Proteínas-G/química , Proteínas Recombinantes de Fusão/química , Elemento de Resposta Sérica , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Expressão Gênica , Células HEK293 , Humanos , Cinética , Ligantes , Lisofosfolipídeos/farmacologia , Simulação de Acoplamento Molecular , Mutação , Piperazinas/farmacologia , Ligação Proteica , Pirrolidinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Opioides delta/química , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Resposta Sérica/química , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Soja , Homologia Estrutural de Proteína , Termodinâmica
13.
J Biol Chem ; 291(48): 25227-25238, 2016 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-27765816

RESUMO

PLEKHG2/FLJ00018 is a Gßγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gßγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gßγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteínas Musculares/metabolismo , Elemento de Resposta Sérica/fisiologia , Transcrição Genética/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM/genética , Proteínas Musculares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
14.
Cancer Lett ; 370(1): 91-9, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26515162

RESUMO

The polyphenolic flavone chrysin has been evaluated as a natural chemopreventive agent due to its anti-cancer effects in a variety of cancer cell lines. However, the mechanism of the chemopreventive effect has been not well established, especially in human colorectal cancer cells. We evaluated the chemopreventive effect of chrysin in three different human colorectal cancer cell lines. We found that chrysin treatment consequently reduced cell viability via induction of apoptosis. We identified that the involvement of up-regulation of pro-apoptotic cytokines tumor necrosis factor (Tnf) α and ß genes and consequent activation of the TNF-mediated transcriptional pathway in chrysin-induced apoptosis. Using our generated AHR siRNA expressing colorectal cancer cells, we demonstrated that the chrysin-induced up-regulation of Tnfα and ß gene expression was dependent on the aryl hydrocarbon receptor (AHR), which is a ligand-receptor for chrysin. Subsequently, we found that the AHR siRNA expressing colorectal cancer cells were resistant to chrysin-induced apoptosis. Therefore, we concluded that AHR is required for the chrysin-induced apoptosis and the up-regulation of Tnfα and ß gene expression in human colorectal cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Flavonoides/farmacologia , Receptores de Hidrocarboneto Arílico/fisiologia , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A2/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Linfotoxina-alfa/genética , Elemento de Resposta Sérica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia
15.
PLoS One ; 10(12): e0145484, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26691724

RESUMO

Serum stimulation of mammalian cells induces, via the MAPK pathway, the nuclear protein DUSP5 (dual-specificity phosphatase 5), which specifically interacts with and inactivates the ERK1/2 MAP kinases. However, molecular mechanisms underlying DUSP5 induction are not well known. Here, we found that the DUSP5 mRNA induction depends on a transcriptional regulation by the MAPK pathway, without any modification of the mRNA stability. Two contiguous CArG boxes that bind serum response factor (SRF) were found in a 1 Kb promoter region, as well as several E twenty-six transcription factor family binding sites (EBS). These sites potentially bind Elk-1, a transcription factor activated by ERK1/2. Using wild type or mutated DUSP5 promoter reporters, we demonstrated that SRF plays a crucial role in serum induction of DUSP5 promoter activity, the proximal CArG box being important for SRF binding in vitro and in living cells. Moreover, in vitro and in vivo binding data of Elk-1 to the same promoter region further demonstrate a role for Elk-1 in the transcriptional regulation of DUSP5. SRF and Elk-1 form a ternary complex (Elk-1-SRF-DNA) on DUSP5 promoter, consequently providing a link to an important negative feedback tightly regulating phosphorylated ERK levels.


Assuntos
Fosfatases de Especificidade Dupla/genética , Fosfatases de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Resposta Sérica/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Sítios de Ligação , Regulação Enzimológica da Expressão Gênica , Meia-Vida , Camundongos , Mutação , Células NIH 3T3 , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Elemento de Resposta Sérica , Fator de Resposta Sérica/genética , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/genética
16.
PLoS One ; 10(8): e0133751, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26241044

RESUMO

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.


Assuntos
Proteínas Musculares/genética , Miócitos de Músculo Liso/metabolismo , RNA Mensageiro/genética , Elemento de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Navegador , Animais , Sítios de Ligação , Proteínas de Transporte/genética , Colo/citologia , Simulação por Computador , Biblioteca Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Código das Histonas , Histonas/metabolismo , Canais Iônicos/genética , Jejuno/citologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Especificidade de Órgãos , Proteômica , Fator de Resposta Sérica/deficiência , Transcriptoma
17.
PLoS One ; 10(4): e0124444, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25923532

RESUMO

The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE.


Assuntos
DNA/química , Oligonucleotídeos/química , Fosfatos/química , Elemento de Resposta Sérica/genética , Fator de Resposta Sérica/química , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fator de Resposta Sérica/genética , Análise Espectral Raman , Eletricidade Estática , Termodinâmica , Transcrição Genética
18.
Arterioscler Thromb Vasc Biol ; 35(4): 918-29, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25722434

RESUMO

OBJECTIVE: In this study, we attempted to uncover the functional impact of microRNA-22 (miR-22) and its target gene in smooth muscle cell (SMC) differentiation and delineate the molecular mechanism involved. APPROACH AND RESULTS: miR-22 was found to be significantly upregulated during SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells. Enforced expression of miR-22 by its mimic, while knockdown of miR-22 by its antagomiR, promotes or inhibits SMC differentiation from embryonic stem cells and adventitia stem/progenitor cells, respectively. Expectedly, miR-22 overexpression in stem cells promoted SMC differentiation in vivo. Methyl CpG-binding protein 2 (MECP2) was predicted as one of the top targets of miR-22. Interestingly, the gene expression levels of MECP2 were significantly decreased during SMC differentiation, and MECP2 was dramatically decreased in miR-22 overexpressing cells but significantly increased when miR-22 was knockdown in the differentiating stem cells. Importantly, luciferase assay showed that miR-22 substantially inhibited wild-type, but not mutant MECP2-3' untranslated region-luciferase activity. In addition, modulation of MECP2 expression levels affects multiple SMC-specific gene expression in differentiated embryonic stem cells. Mechanistically, our data showed that MECP2 could transcriptionally repress SMC gene expression through modulating various SMC transcription factors, as well as several proven SMC differentiation regulators. Evidence also revealed that enrichment of H3K9 trimethylation around the promoter regions of the SMC differentiation regulators genes were significantly increased by MECP2 overexpression. Finally, miR-22 was upregulated by platelet-derived growth factor-BB and transforming growth factor-ß through a transcriptional mechanism during SMC differentiation. CONCLUSIONS: miR-22 plays an important role in SMC differentiation, and epigenetic regulation through MECP2 is required for miR-22 mediated SMC differentiation.


Assuntos
Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteína 2 de Ligação a Metil-CpG/metabolismo , MicroRNAs/metabolismo , Miócitos de Músculo Liso/metabolismo , Regiões 3' não Traduzidas , Animais , Becaplermina , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Epigênese Genética , Regulação da Expressão Gênica , Histonas/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Metilação , Camundongos , MicroRNAs/genética , Mutação , Miócitos de Músculo Liso/efeitos dos fármacos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleotídeos/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , Interferência de RNA , Elemento de Resposta Sérica , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Transdução de Sinais , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética , Transfecção , Fator de Crescimento Transformador beta/farmacologia
19.
Biochem J ; 467(3): 425-38, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25695333

RESUMO

Constitutive activation of the extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are central to regulating the proliferation and survival of many cancer cells. The current inhibitors of ERK1/2 target ATP binding or the catalytic site and are therefore limited in their utility for elucidating the complex biological roles of ERK1/2 through its phosphorylation and regulation of over 100 substrate proteins. To overcome this limitation, a combination of computational and experimental methods was used to identify low-molecular-mass inhibitors that are intended to target ERK1/2 substrate-docking domains and selectively interfere with ERK1/2 regulation of substrate proteins. In the present study, we report the identification and characterization of compounds with a thienyl benzenesulfonate scaffold that were designed to inhibit ERK1/2 substrates containing an F-site or DEF (docking site for ERK, FXF) motif. Experimental evidence shows the compounds inhibit the expression of F-site containing immediate early genes (IEGs) of the Fos family, including c-Fos and Fra1, and transcriptional regulation of the activator protein-1 (AP-1) complex. Moreover, this class of compounds selectively induces apoptosis in melanoma cells containing mutated BRaf and constitutively active ERK1/2 signalling, including melanoma cells that are inherently resistant to clinically relevant kinase inhibitors. These findings represent the identification and initial characterization of a novel class of compounds that inhibit ERK1/2 signalling functions and their potential utility for elucidating ERK1/2 and other signalling events that control the growth and survival of cancer cells containing elevated ERK1/2 activity.


Assuntos
Genes Precoces/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Simulação por Computador , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Células Jurkat , Ligantes , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Melanoma/patologia , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Fosforilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Elemento de Resposta Sérica , Fator de Transcrição AP-1/genética
20.
J Dent Res ; 94(3): 464-72, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25604255

RESUMO

Ephrin-A2-EphA2 and ephrin-B2-EphB4 interactions have been implicated in the regulation of bone remodeling. We previously demonstrated a potential role for members of the Eph-ephrin family of receptor tyrosine kinases for bone remodeling during orthodontic tooth movement: compression-dependent upregulation of ephrin-A2 in fibroblasts of the periodontal ligament (PDL) attenuated osteogenesis in osteoblasts of the alveolar bone. However, factors affecting the regulation of ephrin-A2 expression upon the application of compressive forces remained unclear. Here, we report a mechano-dependent pathway of ephrin-A2 induction in PDL fibroblasts (PDLFs) involving extracellular signal-regulated kinases (ERK) 1/2 and c-fos. PDLF subjected to compressive forces (30.3 g/cm(2)) upregulated c-fos and ephrin-A2 mRNA and protein expression and displayed increased ERK1/2 phosphorylation. Inhibition of the MAP kinase kinase (MEK)/ERK1/2 pathway using the specific MEK inhibitor U0126 significantly reduced ephrin-A2 messenger RNA upregulation upon compression. Silencing of c-fos using a small interfering RNA approach led to a significant inhibition of ephrin-A2 induction upon the application of compressive forces. Interestingly, ephrin-A2 stimulation of PDLF induced c-fos expression and led also to the induction of ephrin-A2 expression. Using a reporter gene construct in murine 3T3 cells, we found that ephrin-A2 was able to stimulate serum response element (SRE)-dependent luciferase activity. As the regulation of c-fos is SRE dependent, ephrin-A2 might induce c-fos via SRE activation. Taken together, we provide evidence for an ERK1/2- and c-fos-dependent regulation of ephrin-A2 in compressed PDLF and suggest a novel pathway for ephrin-A2 induction emanating from ephrin-A2 itself. We showed previously that ephrin-A2 at compression sites might contribute to tooth movement by inhibiting osteogenic differentiation. The regulatory pathway of ephrin-A2 induction during tooth movement identified in this study might be accessible for pharmacological interventions.


Assuntos
Efrina-A2/biossíntese , Fibroblastos/metabolismo , Ligamento Periodontal/citologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Células 3T3 , Adolescente , Animais , Fenômenos Biomecânicos , Butadienos/farmacologia , Técnicas de Cultura de Células , Células Cultivadas , Criança , Inativação Gênica , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Nitrilos/farmacologia , Pressão , RNA Interferente Pequeno/administração & dosagem , Elemento de Resposta Sérica/fisiologia , Estresse Mecânico , Ativação Transcricional , Regulação para Cima , Adulto Jovem
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