RESUMO
Extracellular acidosis is a characteristic of solid tumours, resulting from hypoxia-induced glycolytic metabolism as well as from the "Warburg effect" (aerobic glycolysis). The acidic environment has shown to affect functional tumour properties (proliferation, migration, invasion) and thus the aim of the study was to identify signalling mechanisms, mediating these pH-dependent effects. Therefore, the serum response factor (Srf) and the activation of the serum response element (SRE) by acidosis were analysed in AT-1 prostate carcinoma cells. Furthermore, the expression of downstream targets of this cascade, namely the early growth response 1 (Egr1), which seems to be involved in tumour proliferation, and the cellular communication network factor 1 (Ccn1), which both contain SRE in their promotor region were examined in two tumour cell lines. Extracellular acidification led to an upregulation of Srf and a functional activation of the SRE. Egr1 expression was increased by acidosis in AT-1 cells whereas hypoxia had a suppressive effect. In experimental tumours, in vivo Egr1 and Ccn1 were also found to be acidosis-dependent. Also, it turned out that pH regulated expression of Egr1 was followed by comparable changes of p21, which is an important regulator of the cell cycle.This study identifies the Srf-SRE signalling cascade and downstream Egr1 and Ccn1 to be acidosis-regulated in vitro and in vivo, potentially affecting tumour progression. Especially linked expression changes of Egr1 and p21 may mediate acidosis-induced effects on cell proliferation.
Assuntos
Acidose , Hipóxia , Neoplasias da Próstata , Animais , Humanos , Masculino , Acidose/genética , Acidose/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/farmacologia , Hipóxia/genética , Hipóxia/metabolismo , Neoplasias Experimentais , Ativação Transcricional , Ratos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Elemento de Resposta Sérica/genética , Elemento de Resposta Sérica/fisiologiaRESUMO
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 Gênica/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/metabolismoRESUMO
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/fisiologiaRESUMO
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 , Nitrilas/farmacologia , Pressão , RNA Interferente Pequeno/administração & dosagem , Elemento de Resposta Sérica/fisiologia , Estresse Mecânico , Ativação Transcricional , Regulação para Cima , Adulto JovemRESUMO
Transcriptional regulation is essential for any gene expression including microRNA expression. MiR-1-1 and miR-133a-2 are essential microRNAs (miRs) involved in cardiac and skeletal muscle development and diseases. Early studies reveal two regulatory enhancers, an upstream and an intragenic, that direct the miR-1-1 and miR-133a-2 transcripts. In this study, we identify a unique serum response factor (SRF) binding motif within the enhancer through bioinformatic approaches. This motif is evolutionarily conserved and is present in a range of organisms from yeast, flies, to humans. We provide evidence to demonstrate that this regulatory motif is SRF-dependent in vitro by electrophoretic mobility shift assay, luciferase activity assay, and endogenous chromatin immunoprecipitation assay followed by DNA sequence confirmation, and in vivo by transgenic lacZ reporter mouse studies. Importantly, our transgenic mice indicate that this motif is indispensable for the expression of miR1-1/133a-2 in the heart, but not necessary in skeletal muscle, while the enhancer is sufficient for miR1-1/133a-2 gene expression in both tissues. The mutation of the motif alone completely abolishes miR-1-1/133a-2 gene expression in the animal heart, but not in the skeletal muscle. Our findings reveal an additional architecture of regulatory complex directing miR-1-1/133a-1 gene expression, and demonstrate how this intragenic enhancer differentially manages the expression of the two miRs in the heart and skeletal muscle, respectively.
Assuntos
Regulação da Expressão Gênica/fisiologia , MicroRNAs/biossíntese , Mioblastos Cardíacos/metabolismo , Miocárdio/metabolismo , Elemento de Resposta Sérica/fisiologia , Animais , Linhagem Celular , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , Mioblastos Cardíacos/citologia , Miocárdio/citologia , Especificidade de Órgãos/fisiologiaRESUMO
FLJ00018, a heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gßγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes.
Assuntos
Membrana Celular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Lipídeos de Membrana/metabolismo , Fracionamento Celular , DNA Complementar/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HEK293 , Humanos , Luciferases/genética , Plasmídeos , Ligação Proteica , Fatores de Troca de Nucleotídeo Guanina Rho , Elemento de Resposta Sérica/fisiologia , Transcrição GênicaRESUMO
We examined the effects of sex steroids on prolactin promoter activity in rat somatolactotrophic GH3 cells. Both estradiol (E2) and progesterone (P4) were found to inhibit basal prolactin promoter activity, but to potentiate Thyrotropin-releasing hormone (TRH)-induced prolactin promoter activity. P4 had a greater inhibitory effect on basal prolactin promoter activity than E2, and P4 also potentiated TRH-induced prolactin promoter more potently than E2. Combined treatment with E2 and P4 further increased TRH-induced prolactin promoter activity. E2 and P4 also both reduced basal serum response element (SRE) promoter activity, and increased TRH-induced SRE promoter activity. Combination treatment with E2 and P4 reduced basal activity of SRE promoter and increased TRH-induced SRE activity more potently than E2 or P4 alone. In contrast, basal cAMP response element (CRE) promoter activity was not influenced by either E2 or P4, although TRH-induced CRE promoter was potentiated by each of these steroids, and was further increased by E2 and P4 combination treatment. Both E2 and P4 increased TRH-induced extracellular signal-regulated kinase (ERK) phosphorylation; however, intracellular cAMP levels was not influenced by E2 or P4. TRH-induced CRE promoter was inhibited by mitogen-activated protein kinase/ERK kinase (MEK) inhibitor and was increased by overexpression of MEK kinase (MEKK). This study showed that ERK and SRE transcriptional pathways, but not the cAMP/CRE pathway, may be involved in the suppression of basal prolactin promoter activity, whereas both the ERK/SRE and MAP kinase-mediated CRE pathways appear to be involved in the increased transcriptional efficiency of the prolactin promoter induced by TRH stimulation.
Assuntos
Estradiol/farmacologia , Lactotrofos/metabolismo , Progesterona/farmacologia , Prolactina/biossíntese , Animais , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Ratos , Elemento de Resposta Sérica/genética , Elemento de Resposta Sérica/fisiologia , Hormônio Liberador de Tireotropina/farmacologiaRESUMO
Smooth muscle cell (SMC) differentiation is defined largely by a number of cell-restricted genes governed directly by the serum response factor (SRF)/myocardin (MYOCD) transcriptional switch. Here, we describe a new SRF/MYOCD-dependent, SMC-restricted gene known as Leiomodin 1 (Lmod1). Conventional and quantitative RT-PCRs indicate that Lmod1 mRNA expression is enriched in SMC-containing tissues of the mouse, whereas its two paralogs, Lmod2 and Lmod3, exhibit abundant expression in skeletal and cardiac muscle with very low levels in SMC-containing tissues. Western blotting and immunostaining of various adult and embryonic mouse tissues further confirm SMC-specific expression of the LMOD1 protein. Comparative genomic analysis of the human LMOD1 and LMOD2 genes with their respective mouse and rat orthologs shows high conservation between the three exons and several noncoding sequences, including the immediate 5' promoter region. Two conserved CArG boxes are present in both the LMOD1 and LMOD2 promoter regions, although LMOD1 displays much higher promoter activity and is more responsive to SRF/MYOCD stimulation. Gel shift assays demonstrate clear binding between SRF and the two CArG boxes in human LMOD1. Although the CArG boxes in LMOD1 and LMOD2 are similar, only LMOD1 displays SRF or MYOCD-dependent activation. Transgenic mouse studies reveal wild type LMOD1 promoter activity in cardiac and vascular SMC. Such activity is abolished upon mutation of both CArG boxes. Collectively, these data demonstrate that Lmod1 is a new SMC-restricted SRF/MYOCD target gene.
Assuntos
Autoantígenos/biossíntese , Diferenciação Celular/fisiologia , Proteínas do Citoesqueleto/biossíntese , Regulação da Expressão Gênica/fisiologia , Proteínas Musculares/biossíntese , Miócitos de Músculo Liso/metabolismo , Elemento de Resposta Sérica/fisiologia , Fator de Resposta Sérica/imunologia , Animais , Autoantígenos/genética , Células COS , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/biossíntese , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Miócitos de Músculo Liso/citologia , Células NIH 3T3 , Especificidade de Órgãos/fisiologia , Ratos , Fator de Resposta Sérica/genéticaRESUMO
G2A is a G protein-coupled receptor that can be induced by various stressors. G2A is reported to have proton-sensing activity that mediates intracellular inositol phosphate (IP) accumulation with decreasing pH. We previously showed that G2A is also activated by some oxidized free fatty acids such as 9-hydroxyoctadecadienoic acid (9-HODE). In this study, we identified a novel alternative splice variant of G2A (G2A-b) that has a partially different N terminus compared with the G2A originally reported (G2A-a). The two splice variants of G2A show similar tissue distributions, but G2A-b is expressed more abundantly. There was no difference between the two variants in 9-HODE-induced cellular responses, such as intracellular calcium mobilization and GDP/GTP exchange of Galpha protein, and in proton-sensitive IP accumulation. However, G2A-b showed a higher basal activity in terms of IP accumulation. Mutagenesis study revealed that the difference in the basal activity is attributable to the K7 residue that exists only in G2A-a. We further demonstrated that an R42A mutation largely impaired both the basal and proton-sensing activities, but did not affect the 9-HODE-induced intracellular calcium increase. Taken together, we found an additional novel G2A variant (G2A-b) that is the major transcript with functional response to ligand stimulation as well as G2A-a, and succeeded in discriminating proton-sensing and oxidized fatty acid-sensing activities of G2A.
Assuntos
Processamento Alternativo , Proteínas de Ciclo Celular/genética , Isoformas de Proteínas/genética , Receptores Acoplados a Proteínas G/genética , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Cálcio/metabolismo , Proteínas de Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteína A de Ligação a Elemento de Resposta do AMP Cíclico/metabolismo , Guanosina Trifosfato/metabolismo , Células HL-60 , Humanos , Fosfatos de Inositol/metabolismo , Leucócitos , Ácidos Linoleicos Conjugados/farmacologia , Dados de Sequência Molecular , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Elemento de Resposta Sérica/fisiologia , TransfecçãoRESUMO
Com o objetivo de estudar a influência do exercício físico prolongado na indução de lesão miocárdica e na desqualificação de eqüinos em provas de enduro, colheram-se 87 amostras de sangue de eqüinos adultos Árabes e mestiços para determinação da concentração sérica da isoenzima MB (CKMB), da atividade sérica de creatina quinase (CK) e do índice CKMB/CK. Dividiram-se as amostras da seguinte forma: C (controle, n=34): eqüinos em repouso, no dia anterior à competição; G1 (n=24): eqüinos que finalizaram as competições; G2 (n=14): eqüinos desqualificados durante as competições por causa metabólica e G3 (n=15): eqüinos desqualificados durante as competições por claudicação. Os valores medianos obtidos de CKMB, para os grupos C, G1, G2 e G3, foram respectivamente 2,25; 2,95; 1,95 e 2,40 ng/mL; para a atividade de CK, os resultados foram 164,2; 1228,0; 840,0 e 581,0 U/L a 30oC; e para o índice CKMB/CK, os resultados foram 1,41; 0,21; 0,23 e 0,42%. Houve média correlação positiva entre distância percorrida e CKMB e CK, e negativa entre distância e índice CKMB/CK. O exercício físico de resistência não leva a aumento significante dos valores da isoenzima CKMB, mas sim da atividade sérica da enzima CK, indicando dano à musculatura esquelética. Animais desqualificados por causa metabólica parecem não apresentar envolvimento de lesão miocárdica na causa de desqualificação. Não foi possível verificar a presença de lesão cardíaca induzida pelo exercício em eqüinos de enduro e sua relação com causas metabólicas de desqualificação a partir da utilização de CKMB como único marcador cardíaco.
With the purpose of studying the influence of prolonged phyisical exercise causing myocardial lesion and disqualifying horses in endurance competitions, 87 blood samples were collected from adult Arabian and crossbred horses, to determine the serum concentration of the isoenzyme MB (CKMB), the serum activity of creatine kinase (CK), and the CKMB/CK index. Samples were divided as follows: C (control, n=34): horses at rest, one day before competition; G1 (n=24): horses which finished competitions; G2 (n=14): horses disqualified during competitions by metabolic causes, and G3 (n=15): horses disqualified during competitions after lameness was diagnosed. Median values obtained for CKMB, for groups C, G1, G2, and G3, were, respectively, 2.25; 2.95; 1.95, and 2.40 ng/mL; for CK activity, the results were 164.2; 1228.0; 840.0, and 581.0 in U/L at 30oC; and for CKMB/CK index, the results were 1.41; 0.21; 0.23, and 0.42%. There was mean positive correlation between distance and CKMB and CK, and negative correlation between distance and CKMB/CK index. It was concluded that endurance exercise does not cause significant increases in CKMB values, but in CK serum activity, indicating skeletal muscle injury. Horses disqualified by metabolic causes seem not to present myocardial injury involved in the cause of disqualification. It was not possible to verify the presence of exercise-induced cardiac injury in endurance horses and its relationship with metabolic causes of disqualification only by the utilization of CKMB as the only cardiac marker.
Assuntos
Animais , Masculino , Feminino , Adulto , Condicionamento Físico Animal/efeitos adversos , Elemento de Resposta Sérica/fisiologia , Isoenzimas/sangue , Isoenzimas , Cavalos/lesões , Avaliação de Danos , MiocárdioRESUMO
The hypothalamic-pituitary-adrenal (HPA) axis is activated under various stressors. Corticotropin-releasing factor (CRF) plays a central role in controlling stress response, and regulating the HPA axis. CRF, produced in the hypothalamic paraventricular nucleus (PVN), stimulates adrenocorticotropic hormone (ACTH) production via CRF receptor type 1 (CRF(1) receptor) from the corticotrophs of the anterior pituitary (AP). Cyclic AMP (cAMP)-protein kinase A (PKA) pathway takes a main role in stimulating CRF gene transcription. Forskolin and pituitary adenylate cyclase-activating polypeptide (PACAP) stimulate adenylate cyclase, intracellular cAMP production, and then CRF and arginine vasopressin (AVP) gene expression in hypothalamic 4B cells. Interleukin (IL)-6, produced in the PVN, both directly and indirectly stimulates CRF and AVP gene expression. Estradiol may enhance the activation of CRF gene expression in response to stress. The HPA axis is regulated by a negative feedback mechanism, because glucocorticoids inhibit both CRF production in the hypothalamic PVN and ACTH production in the pituitary. Hypothalamic parvocellular neurons in the PVN are known to express glucocorticoid receptors, and glucocorticoids are able to regulate CRF gene transcription and expression levels directly in the PVN. Glucocorticoids-dependent repression of cAMP-stimulated CRF promoter activity is mainly localized to promoter sequences between -278 and -233 bp. Both negative glucocorticoid regulatory element (nGRE) and serum response element (SRE) are involved in the repression of the CRF gene in the hypothalamic cells.
Assuntos
Hormônio Liberador da Corticotropina/biossíntese , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Animais , Arginina Vasopressina/fisiologia , Colforsina/farmacologia , Hormônio Liberador da Corticotropina/genética , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dexametasona/farmacologia , Receptor alfa de Estrogênio/fisiologia , Receptor beta de Estrogênio/fisiologia , Estrogênios/fisiologia , Retroalimentação , Humanos , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/fisiologia , Interleucina-6/fisiologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Sistema Hipófise-Suprarrenal/fisiologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Elemento de Resposta Sérica/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
In the mammalian hippocampus, changes in the expression of immediate early genes (IEGs) is thought to contribute to long term plastic changes in neurons brought about by learning tasks and high frequency stimulation of synapses. The phosphatase calcineurin has emerged as an important negative regulator of hippocampus-dependent learning and long term potentiation. Here we investigated the possibility that the constraining action of calcineurin on hippocampal plasticity is mediated in part by regulation of gene expression through negative control of transcription factors, such as cAMP-response element (CRE)-binding protein (CREB). We assessed the effect of calcineurin inhibitors on CREB activation by neuronal activity and show that calcineurin activity is in fact required for CREB-mediated gene expression. However, inhibition of calcineurin had disparate effects on the transcriptional induction of CREB-dependent IEGs. We find that the IEG c-fos is unaffected by suppression of calcineurin activity, the plasticity-related genes Egr1/Zif268 and Egr2/Krox-20 are up-regulated, and genes encoding the orphan nuclear hormone receptors Nor1 and Nur77 are down-regulated. We further show that the up-regulation of particular IEGs is probably due to the presence of serum response elements (SREs) in their promoters, because SRE-mediated gene expression is enhanced by calcineurin blockers. Moreover, expression of the c-fos gene, which is unaffected by calcineurin inhibitors, could be down-regulated by mutating the SRE. Conversely, SRE-mediated c-fos induction in the absence of a functional CRE was enhanced by calcineurin inhibitors. Our experiments thus implicate calcineurin as a negative regulator of SRE-dependent neuronal genes.
Assuntos
Calcineurina/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Proteína de Ligação a CREB/metabolismo , Inibidores de Calcineurina , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , Proteína 2 de Resposta de Crescimento Precoce/biossíntese , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas Proto-Oncogênicas c-fyn/biossíntese , Ratos , Ratos Wistar , Receptores de Esteroides/biossíntese , Elemento de Resposta Sérica/fisiologia , Transcrição Gênica/fisiologiaAssuntos
Aorta/metabolismo , Proteínas do Citoesqueleto/biossíntese , Íntrons/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Elemento de Resposta Sérica/fisiologia , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Animais , Aorta/citologia , Movimento Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas com Domínio LIM , Masculino , Camundongos , Miócitos de Músculo Liso/citologia , Proteínas Nucleares/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Transcrição Gênica/fisiologiaRESUMO
Lipoma preferred partner (LPP) was recently recognized as a smooth muscle marker that plays a role in smooth muscle cell migration. In this report, we focus on the transcriptional regulation of the LPP gene. In particular, we investigate whether LPP is directly regulated by serum response factor (SRF). We show that the LPP gene contains 3 evolutionarily conserved CArG boxes and that 1 of these is part of an alternative promoter in intron 2. Quantitative RT-PCR shows that this alternative promoter directs transcription specifically to smooth muscle containing tissues in vivo. By using chromatin immunoprecipitation, we demonstrate that 2 of the CArG boxes, including the promoter-associated CArG box, bind to endogenous SRF in cultured aortic smooth muscle cells. Electrophoretic mobility-shift assays show that the conserved CArG boxes bind SRF in vitro. In reporter experiments, we show that the alternative promoter has transcriptional capacity that is dependent on SRF/myocardin and that the promoter associated CArG box is required for that activity. Finally, we show by quantitative RT-PCR that the alternative promoter is strongly downregulated in SRF-deficient embryonic stem cells and in smooth muscle tissues derived from conditional SRF knockout mice. Collectively, our data demonstrate that expression of LPP in smooth muscle is mediated by an alternative promoter that is regulated by SRF/myocardin.
Assuntos
Aorta/metabolismo , Proteínas do Citoesqueleto/biossíntese , Íntrons/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Elemento de Resposta Sérica/fisiologia , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Animais , Aorta/citologia , Movimento Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas com Domínio LIM , Masculino , Camundongos , Miócitos de Músculo Liso/citologia , Proteínas Nucleares/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Transcrição Gênica/fisiologiaRESUMO
Leupaxin is a LIM domain-containing adapter protein belonging to the paxillin family that has been previously reported to be preferentially expressed in hematopoietic cells. Herein, we identified leupaxin in a screen for focal adhesion kinase binding partners in aortic smooth muscle, and we show that leupaxin is enriched in human and mouse vascular smooth muscle and that leupaxin expression is dynamically regulated during development. In addition, our studies reveal that leupaxin can undergo cytoplasmic/nuclear shuttling and functions as an serum response factor cofactor in the nucleus. We found that leupaxin forms a complex with serum response factor and associates with CArG-containing regions of smooth muscle promoters and that ectopic expression of leupaxin induces smooth muscle marker gene expression in both 10T1/2 cells and rat aortic smooth muscle cells. Subsequent studies indicated that enhanced focal adhesion kinase activity (induced by fibronectin or expression of constitutively active focal adhesion kinase) attenuates the nuclear accumulation of leupaxin and limits the ability of leupaxin to enhance serum response factor-dependent gene transcription. Thus, these studies indicate that modulation of the subcellular localization of serum response factor cofactors is 1 mechanism by which extracellular matrix-dependent signals may regulate phenotypic switching of smooth muscle cells.
Assuntos
Moléculas de Adesão Celular/fisiologia , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/fisiologia , Elemento de Resposta Sérica/fisiologia , Animais , Aorta/citologia , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Transporte Biológico , Moléculas de Adesão Celular/farmacologia , Diferenciação Celular , Células Cultivadas/efeitos dos fármacos , Vasos Coronários/citologia , Feminino , Quinase 1 de Adesão Focal/fisiologia , Adesões Focais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miométrio/citologia , Especificidade de Órgãos , Fosfoproteínas/farmacologia , Mapeamento de Interação de Proteínas , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Elemento de Resposta Sérica/efeitos dos fármacos , Fator de Resposta Sérica/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição , Transcrição GênicaRESUMO
Dual-targeted therapy for antiangiogenesis and antilymphangiogenesis represents a potentially effective strategy for the treatment of various malignancies. Therefore, the goal of the present study was to identify genes that encode inhibitors of both angiogenesis and lymphangiogenesis. Using a cDNA library obtained from Lewis lung carcinoma (LL/2), a candidate gene was identified by the evaluation of growth inhibition in aortic and lymphatic endothelial cells (EC) as that coding for the mouse cold shock domain protein A (mCSDA). Overexpression of mCSDA significantly repressed cell proliferation and c-fos promoter activity in aortic, venous and lymphatic ECs. CSDA is a DNA-binding protein that binds to the hypoxia response element (HRE). Furthermore, of importance, we revealed that CSDA could directly bind to the serum response element (SRE) sequence, resulting in the inhibition of SRE activity, which may lead to growth inhibition in ECs. In an LL/2-inoculated mouse model, tumor growth was significantly repressed in an mCSDA-injected group. Histopathological analysis revealed that expression of blood and lymphatic EC markers was significantly decreased in mCSDA-injected groups. In conclusion, these data suggest that expression of CSDA can repress angiogenesis and lymphangiogenesis via direct binding to SRE in addition to HRE.
Assuntos
Carcinoma Pulmonar de Lewis/prevenção & controle , Proteínas de Ligação a DNA/fisiologia , Linfangiogênese/fisiologia , Neovascularização Patológica/prevenção & controle , Elemento de Resposta Sérica/fisiologia , Inibidores da Angiogênese/farmacologia , Animais , Aorta/citologia , Células COS , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Bovinos , Proliferação de Células , Células Cultivadas , Chlorocebus aethiops , Cães , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Biblioteca Gênica , Genes fos/fisiologia , Humanos , Hipóxia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Elementos de Resposta , Fatores de TranscriçãoRESUMO
Using a luciferase reporter assay we found that human serum transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) promoter three to fourfold. Confirming this effect, addition of human serum to both Caco-2 cells and fresh human ileal biopsies caused an approximate 2.0-fold increase in endogenous ASBT mRNA production. Alteration of non-esterified fatty acid (NEFA) content and cortisol content did not affect the transactivation potential of serum. Site-directed mutagenesis of response elements for corticosteroid, peroxisome proliferation-activated alpha (PPARalpha), hepatocyte nuclear factor 1alpha (HNF1alpha), and retinoic acid (RAR/RXR) did not affect transactivation potential of serum. Three putative serum response elements (SRE) were identified on the promoter, but all were determined inactive using site-directed mutagenesis and electrophoretic mobility shift assay. Promoter deletion analysis demonstrated that >80% of the response to serum was located within the last 273 bp of the 5'-UTR, an area containing one of two activate protein 1 (AP-1) response elements. Site-directed mutagenesis of this downstream AP-1 response element reduced the effect of serum on the promoter by about 50% while full deletion of the response element completely eliminated the effect of serum. These studies demonstrate that one or more constituents of human stimulate ASBT gene expression largely via the down-stream AP-1 response element.
Assuntos
Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Soro/fisiologia , Simportadores/genética , Ativação Transcricional , Animais , Células CACO-2 , Proteínas de Transporte/farmacologia , Galinhas , Corticosterona , Ácidos Graxos não Esterificados/farmacologia , Genes Reporter/efeitos dos fármacos , Humanos , Hidrocortisona/farmacologia , Íleo/metabolismo , Luciferases/genética , Mifepristona/farmacologia , Mutagênese Sítio-Dirigida , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/metabolismo , Elemento de Resposta Sérica/efeitos dos fármacos , Elemento de Resposta Sérica/fisiologia , Simportadores/metabolismo , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional/efeitos dos fármacos , Transfecção , Células Tumorais CultivadasRESUMO
We previously reported that Gbetagamma signaling regulates cell spreading or cell shape change through activation of a Rho family small GTPase, suggesting the existence of a Gbetagamma-regulated Rho guanine-nucleotide exchange factor (RhoGEF). In this study we examined various RhoGEF clones, found FLJ00018 to beaGbetagamma-activated RhoGEF, and investigated the molecular mechanism of Gbetagamma-induced activation of Rho family GTPases. Co-expression of the genes for FLJ00018 and Gbetagamma enhanced serum response element-mediated gene transcription in HEK-293 cells. Combined expression of Gbetagamma and FLJ00018 significantly induced activation of Rac and Cdc42 but not RhoA. FLJ00018 also enhanced gene transcription induced by carbachol-stimulated m2 muscarinic acetylcholine receptor, and this enhancement was blocked by pertussis toxin. Furthermore, we demonstrated Gbetagamma to interact directly with the N-terminal region of FLJ00018 and the N-terminal fragment of this molecule to inhibit serum response element-dependent transcription induced by Gbetagamma/FLJ00018 and carbachol. In NIH3T3 cells, FLJ00018 enhanced lysophosphatidic acid-induced cell spreading, which was also blocked by the N-terminal fragment of FLJ00018. These results provide evidence for a signaling pathway by which G(i)-coupled receptor specifically induces Rac and Cdc42 activation through direct interaction of Gbetagamma with FLJ00018.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Neuropeptídeos/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Carbacol/farmacologia , Forma Celular/fisiologia , Agonistas Colinérgicos/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Lisofosfolipídeos/metabolismo , Camundongos , Células NIH 3T3 , Neuropeptídeos/genética , Toxina Pertussis/farmacologia , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Elemento de Resposta Sérica/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/fisiologia , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Id2, a negative regulator of basic helix-loop-helix transcription factors, is involved in regulating cell differentiation and proliferation. To obtain insight into the role of Id2 in cell cycle control, we investigated the mechanisms underlying the immediate early response of Id2 expression to serum stimulation in NIH3T3 cells. Luciferase reporter analysis with deletion and point mutants demonstrated the serum response element of Id2 (Id2-SRE) to be a consensus binding site for RFX1 (regulatory factor for X-box 1) present 3.0 kb upstream of the transcription initiation site of Id2. Gel shift and chromatin immunoprecipitation assays confirmed the binding of RFX1 to Id2-SRE in vitro and in vivo, respectively. In both assays, RFX1 binding was observed not only in serum-stimulated cells, but also in serum-starved cells. Knockdown of RFX1 by RNA interference disturbed the immediate early response of Id2 expression in cells and abrogated the Id2-SRE-mediated induction of luciferase activity by serum. These alterations were rescued by the introduction of RNA interference-resistant RFX1 into cells. On the other hand, in the Id2-SRE-mediated reporter assay, RFX1 with an N-terminal deletion abrogated the serum response, whereas RFX1 with a C-terminal deletion enhanced the reporter activity in serum-starved cells. Furthermore, HDAC1 was recruited to Id2-SRE in serum-starved cells. These results demonstrate that RFX1 mediates the immediate early response of the Id2 gene by serum stimulation and suggest that the function of RFX1 is regulated intramolecularly in its suppression in growth-arrested cells. Our results unveil a novel transcriptional control of immediate early gene expression.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína 2 Inibidora de Diferenciação/biossíntese , Proteína 2 Inibidora de Diferenciação/metabolismo , Elemento de Resposta Sérica/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Sequência de Aminoácidos , Animais , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Células HeLa , Histona Desacetilase 1 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Proteína 2 Inibidora de Diferenciação/genética , Camundongos , Células NIH 3T3 , Mutação Puntual , Interferência de RNA , Fatores de Transcrição de Fator Regulador X , Fator Regulador X1 , Deleção de Sequência , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Adaptor proteins are important mediators in signal transduction. In the present study, we report the cloning and characterization of a novel adaptor protein, XB130. This gene is located on human chromosome 10q25.3 and encodes a protein of 818 amino acids. It contains several Src homology (SH)2- and SH3-binding motifs, two pleckstrin homology domains, a coiled-coil region, and a number of potential tyrosine or serine/threonine phosphorylation sites. Endogenous XB130 interacts with c-Src tyrosine kinase. Their co-expression in COS-7 cells resulted in activation of c-Src and elevated tyrosine phosphorylation of multiple proteins, including XB130 itself. XB130 expression in HEK293 cells enhanced serum response element- and AP-1-dependent transcriptional activation mediated by c-Src. XB130DeltaN, an N-terminal deletion mutant lacking a putative SH3-binding motif and several putative SH2-binding sites, reduced its ability to mediate Src signal transduction. Down-regulation of endogenous XB130 with siRNA reduced c-Src activity, IL-8 production, EGF-induced phosphorylation of Akt and GSK3beta, and altered cell cycles in human lung epithelial cells. These data suggest that XB130 as an adaptor may play an important role in the regulation of signal transduction and cellular functions.