RESUMO
ATF1 (activating transcription factor 1), a stimulus-induced CREB family transcription factor, plays important roles in cell survival and proliferation. Phosphorylation of ATF1 at Ser63 by PKA (cAMP-dependent protein kinase) and related kinases was the only known post-translational regulatory mechanism of ATF1. Here, we found that HIPK2 (homeodomain-interacting protein kinase 2), a DNA-damage-responsive nuclear kinase, is a new ATF1 kinase that phosphorylates Ser198 but not Ser63. ATF1 phosphorylation by HIPK2 activated ATF1 transcription function in the GAL4-reporter system. ATF1 is a transcriptional repressor of ferritin H, the major intracellular iron storage gene, through an ARE (antioxidant-responsive element). HIPK2 overrode the ATF1-mediated ARE repression in a kinase-activity-dependent manner in HepG2 cells. Furthermore, DNA-damage-inducing agents doxorubicin, etoposide and sodium arsenite induced ferritin H mRNA expression in HIPK2(+/+) MEF cells, whereas it was significantly impaired in HIPK2(-/-) MEF cells. Induction of other ARE-regulated detoxification genes such as NQO1 (NADPH quinone oxidoreductase 1), GST (glutathione S-transferase) and HO1 (heme oxygenase 1) by genotoxic stress was also decreased in HIPK2-deficient cells. Taken together, these results suggest that HIPK2 is a new ATF1 kinase involved in the regulation of ferritin H and other antioxidant detoxification genes in genotoxic stress conditions.
Assuntos
Fator 1 Ativador da Transcrição/genética , Proteínas de Transporte/genética , Dano ao DNA , Ferritinas/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Fator 1 Ativador da Transcrição/metabolismo , Proteínas de Transporte/metabolismo , Processos de Crescimento Celular/genética , Sobrevivência Celular/genética , Ferritinas/metabolismo , Regulação da Expressão Gênica , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismoRESUMO
An effective utilization of intracellular iron is a prerequisite for erythroid differentiation and hemoglobinization. Ferritin, consisting of 24 subunits of H and L, plays a crucial role in iron homeostasis. Here, we have found that the H subunit of the ferritin gene is activated at the transcriptional level during hemin-induced differentiation of K562 human erythroleukemic cells. Transfection of various 5' regions of the human ferritin H gene fused to a luciferase reporter into K562 cells demonstrated that hemin activates ferritin H transcription through an antioxidant-responsive element (ARE) that is responsible for induction of a battery of phase II detoxification genes by oxidative stress. Gel retardation and chromatin immunoprecipitation assays demonstrated that hemin induced binding of cJun, JunD, FosB, and Nrf2 b-zip transcription factors to AP1 motifs of the ferritin H ARE, despite no significant change in expression levels or nuclear localization of these transcription factors. A Gal4-luciferase reporter assay did not show activation of these b-zip transcription factors after hemin treatment; however, redox factor 1 (Ref-1), which increases DNA binding of Jun/Fos family members via reduction of a conserved cysteine in their DNA binding domains, showed induced nuclear translocation after hemin treatment in K562 cells. Consistently, Ref-1 enhanced Nrf2 binding to the ARE and ferritin H transcription. Hemin also activated ARE sequences of other phase II genes, such as GSTpi and NQO1. Collectively, these results suggest that hemin activates the transcription of the ferritin H gene during K562 erythroid differentiation by Ref-1-mediated activation of these b-zip transcription factors to the ARE.
Assuntos
Antioxidantes/farmacologia , Diferenciação Celular , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Células Eritroides/citologia , Ferritinas/genética , Hemina/farmacologia , Elementos de Resposta/genética , Antioxidantes/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Glutationa S-Transferase pi/metabolismo , Humanos , Células K562 , NAD(P)H Desidrogenase (Quinona)/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/genéticaRESUMO
CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2-/- MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2-/- cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress.
Assuntos
Proteínas de Transporte/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dano ao DNA , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Células K562 , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Serina/genética , Transcrição Gênica/efeitos dos fármacos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Gene transcription is coordinately regulated by the balance between activation and repression mechanisms in response to various external stimuli. Ferritin, composed of H and L subunits, is the major intracellular iron storage protein involved in iron homeostasis. We previously identified an enhancer, termed antioxidant-responsive element (ARE), in the human ferritin H gene and its respective transcriptional activators including Nrf2 and JunD. Here we found that ATF1 (activating transcription factor 1) is a transcriptional repressor of the ferritin H ARE. Subsequent yeast two-hybrid screening identified PIAS3 (protein inhibitor of activated STAT3) as an ATF1-binding protein. Further investigation of the human ferritin H ARE regulation showed that 1) PIAS3 reversed ATF1-mediated repression of the ferritin H ARE; 2) ATF1 was sumoylated, but PIAS3, a SUMO E3 ligase, did not appear to play a major role in SUMO1-mediated ATF1 sumoylation or ATF1 transcription activating function; 3) PIAS3 decreased ATF1 binding to the ARE; and 4) ATF1 knockdown with siRNA increased ferritin H expression, whereas PIAS3 knockdown decreased basal expression and oxidative stress-mediated induction of ferritin H. These results suggest that PIAS3 antagonizes the repressor function of ATF1, at least in part by blocking its DNA binding, and ultimately activates the ARE. Collectively our results suggest that PIAS3 is a new regulator of ATF1 that regulates the ARE-mediated transcription of the ferritin H gene.