RESUMO
Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1) (NR5A1), nerve growth factor-inducible protein B (nurr77), COUP-TF, and SET. The -447/-419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, nerve growth factor-inducible protein B, and cAMP. We identified a novel factor, steroidogenic factor-inducer of transcription-2, that binds to this -447/-419 region. We have now purified steroidogenic factor-inducer of transcription-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27-kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promoter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the -447/-419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double- and single-stranded DNA showed that translin binds to single-stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170-225 in SF-1 eliminates translin's ability to augment SF-1-dependent transcription. A translin-like protein, called translin-associated factor X, can substitute for a translin moiety; translin homomers and translin/translin-associated factor X heteromers activated SF-1-stimulated transcription equally. Thus, we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Proteínas de Homeodomínio/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Animais , Sítios de Ligação , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , Células HeLa , Proteínas de Homeodomínio/metabolismo , Humanos , Espectrometria de Massas , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Ligação Proteica , Proteínas de Ligação a RNA , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/química , Fator Esteroidogênico 1 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Rat P450c17 gene transcription is regulated by several nuclear factors, including steroidogenic factor-1 (SF-1), nerve growth factor-inducible protein B (NGF-IB, Nurr77), COUP-TF, SET, and Ku autoimmune antigen. A region of this gene, -447/-419, that mediates both basal and cAMP-stimulated transcription, contains two binding sites for orphan nuclear receptors. While SF-1 activates transcription through a single binding site, we show that both binding sites at -447/-419 are required for transcriptional activation by SF-1 and cAMP. Both SF-1 and a novel factor, Steroidogenic Factor-Inducer of Transcription-2 (StF-IT-2) bind to this region, suggesting that a DNA-dependent interaction between StF-IT-2 and SF-1 may be required for full transcriptional activity. Each of the two orphan nuclear receptor sites -429/-424 and at -444/-439 are sufficient for SF-1 binding but are insufficient for SF-1-mediated transcription. Increasing the distance between or changing the orientation of these two sites does not affect basal or SF-1-stimulated activity. Circular permutation analysis, which measures the degree of DNA bending caused by protein binding, indicates that SF-1 binding to -447/-419 induces a different degree of DNA bending than it does at another SF-1-responsive site. However, similar domains of the SF-1 protein are required for its actions at these two regions. Southwestern blots suggest that StF-IT-2 is a approximately 33 kDa protein, and gel shift assays suggest it is expressed primarily in the gonad and brain early in rodent development. These data suggest that the mechanism by which SF-1 stimulates transcription is DNA sequence dependent, and may require additional proteins, such as StF-IT-2, for activation at specific regions of DNA.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Gônadas/embriologia , Gônadas/metabolismo , Proteínas de Homeodomínio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Fator Esteroidogênico 1 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Cytochrome P4501B1 (CYP1B1), the major constitutively expressed CYP in the rat mammary gland, is induced by Ah-receptor (AhR) ligands, while CYP1A1 is predominantly expressed only after induction. These CYPs contribute to carcinogenic activation of polycyclic aromatic hydrocarbons (PAHs). AhR, ARNT, and CYP1B1 were only weakly expressed, even after 2,3,7,8-tetrachlorodibenzo-p-dioxin induction, when rat mammary epithelial cells (RMEC) were cultured on plastic. RMEC cultured on the extracellular matrix (ECM), Matrigel, or on a floating gel of collagen I demonstrated branching morphogenesis and substantially increased basal CYP1B1 and induced CYP1A1 expression, in parallel with large increases in AhR and ARNT expression. Branching was more pronounced in the Wistar Kyoto than in the Wistar Furth rat strain. Although EGF enhanced branching, neither strain nor growth factor treatment substantially impacted CYP expression. Increased AhR and ARNT expression is observed within 24 h of dispersal on Matrigel, substantially prior to branch formation. Culture on thin layers of collagen I, collagen IV, and laminin, respectively, failed to reproduce the branching morphogenesis or increases in AhR, ARNT, or CYP expression. However, adherent, gelled collagen I recapitulated the increased protein expression, without supporting branching. This increased protein expression was closely paralleled by enhanced expression of beta-catenin and E-cadherin, components of cell-cell adhesion complexes. A synthetic peptide that selectively antagonizes integrin-ECM interactions reduced branch formation, without diminishing AhR, ARNT, and CYP expression. These data demonstrate that early ECM surface adhesion interactions mediate AhR and ARNT expression, which enhances CYP expression, independent of branching morphogenesis.
Assuntos
Hidrocarboneto de Aril Hidroxilases/biossíntese , Citocromo P-450 CYP1A1/biossíntese , Proteínas de Ligação a DNA/biossíntese , Matriz Extracelular/metabolismo , Glândulas Mamárias Animais/metabolismo , Morfogênese/fisiologia , Receptores de Hidrocarboneto Arílico/biossíntese , Fatores de Transcrição/biossíntese , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto , Células Cultivadas , Colágeno/metabolismo , Citocromo P-450 CYP1B1 , Combinação de Medicamentos , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Laminina/metabolismo , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Proteoglicanas/metabolismo , Ratos , Ratos Endogâmicos WF , Ratos Endogâmicos WKY , Especificidade da EspécieRESUMO
CYP1B1 is unique among P450 cytochromes in exhibiting inductive responses mediated by both the Ah receptor (AhR) and cAMP. cAMP induction was mediated either by a 189bp far upstream enhancer region (FUER, -5110 to -5298) or by a 230bp AhR-responsive enhancer region (AhER) (-797 to -1026). CYP1B1 luciferase reporters respond selectively to cAMP and TCDD in adrenal Y-1 cells (only cAMP), testis MA10 cells (cAMP>TCDD), and C3H10T1/2 mouse embryo fibroblasts (only TCDD). In Y-1 cells, which lack AhR, cAMP induction is totally dependent on the FUER, including absolute requirements for upstream and downstream halves of this region, and for CREB activity at a CRE sequence located at the 3(')-end. cAMP stimulation of the FUER was remarkably high (27-fold) and equally effective when linked to an HSV-TK promoter, indicating direct cAMP activation of the FUER. Binding of CREB to the essential CRE was demonstrated along with dominant negative effects of functionally impaired mutants. cAMP induction in MA10 cells was partially mediated by the FUER mechanism but was regulated additionally by AhER through AhR activity. MA10 cells also exhibit cAMP-dependent AhR down-regulation and AhR/Arnt complex formation. Mutations in AhER including XRE5 were similarly inhibitory to cAMP stimulation in MA10 cells and to TCDD stimulation in C3H10T1/2 cells. Transfection of AhR into the AhR-deficient Y-1 cells did not introduce this second mechanism, which indicated a need for additional components that are present in MA10 cells.