RESUMO
BACKGROUND: Adrenogonadal cell growth and differentiation are controlled by nuclear receptor NR5A1 (Ad4BP/SF-1) that regulates the expression of adrenal and gonadal genes. In addition, SF-1 also resides in the centrosome and controls centrosome homeostasis by restricting the activity of centrosomal DNA-PK and CDK2/cyclin A. RESULTS: Here we show that SF-1 depletion resulted in centriole splitting and amplification due to aberrant activation of DNA-PK in the centrosome of mouse adrenocortical Y1 cells. In the absence of SF-1, GSK3ß was aberrantly phosphorylated during G1 phase and ß-catenin was accumulated in the centrosome, but not in the nucleus. DNA-PK inhibitor vanillin reversed these phenomena. SF-1 overexpression led to inhibition of centrosomal DNA-PK activation caused by SF-1 depletion. Both full-length SF-1 and truncated SF-1 devoid of its DNA-binding domain rescued the multiple centrosome phenotype caused by SF-1 depletion, indicating that the effect of SF-1 in the centrosome is not contributed by its DNA-binding domain. Furthermore, SF-1 interacted with cyclin A in the centrosome, but not in the nucleus. Depletion of SF-1 also resulted in centriole splitting, genomic instability and reduced growth of mouse testicular Leydig MA10 cells. CONCLUSION: Centrosomal DNA-PK signaling triggers the accumulation of ß-catenin, leading to centrosome over-duplication and centriole splitting. This cascade of centrosomal events results in genomic instability and reduced cell numbers.
Assuntos
Centrossomo/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Fator Esteroidogênico 1/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Células HEK293 , Humanos , Camundongos , Fator Esteroidogênico 1/genéticaRESUMO
Steroids are synthesized in adrenal glands and gonads under the control of pituitary peptides. These peptides bind to cell surface receptors to activate the cyclic AMP (cAMP) signaling pathway leading to an increase of steroidogenic gene expression. Exactly how cAMP activates steroidogenic gene expression is not clear, except for the knowledge that transcription factor SF-1 plays a key role. Investigating the factors participating in SF-1 action, we found that c-Jun and homeodomain-interacting protein kinase 3 (HIPK3) were required for basal and cAMP-stimulated expression of one major steroidogenic gene, CYP11A1. HIPK3 enhanced SF-1 activity, and c-Jun was required for the functional interaction of HIPK3 with SF-1. Furthermore, after cAMP stimulation, both c-Jun and Jun N-terminal kinase (JNK) were phosphorylated through HIPK3. These phosphorylations were important for SF-1 activity and CYP11A1 expression. Thus, we have defined HIPK3-mediated JNK activity and c-Jun phosphorylation as important events that increase SF-1 activity for CYP11A1 transcription in response to cAMP. This finding has linked three common factors, HIPK3, JNK, and c-Jun, to the cAMP signaling pathway leading to increased steroidogenic gene expression.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , AMP Cíclico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-jun/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Fator Esteroidogênico 1 , Fatores de Transcrição/genética , Transcrição Gênica/genéticaRESUMO
Steroids are synthesized mainly from the adrenal glands catalyzed by steroidogenic enzymes; the expression of these enzymes is controlled by transcription factor steroidogenic factor-1 (SF-1; NR5A1). To understand the physiological effect of genetic changes on steroid secretion, we used Cre-LoxP and gene targeting technology to mutate the binding sequence for SF-1 (SF-1 response element) on the promoter of the mouse Cyp11a1 gene, which encodes a critical enzyme for steroid biosynthesis. The resulting Cyp11a1 L/L mice expressed about 7-fold less cytochrome P450 side-chain cleavage enzyme (CYP11A1) in the adrenal and testis but expressed normal amounts of CYP11A1 in the placenta and ovary. This tissue-specific reduction of gene expression did not affect basal steroid secretion but attenuated the circadian rhythm of glucocorticoid secretion. These mice also failed to induce glucocorticoid secretion in response to stress, leading to retention of CD4+CD8+ double-positive thymocytes. Unlike complete Cyp11a1 disruption, which causes neonatal death, promoter mutation did not decrease life span and caused no defect in reproduction. Thus, CYP11A1 appears in normal mice to be expressed above the minimal required level, providing a large capacity for use in response to stress. Mutation of the SF-1 response element of Cyp11a1 results in reduced stress response due to decreased adrenal CYP11A1 expression and insufficient stress-induced glucocorticoids secretion.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Mutação , Ovário/metabolismo , Regiões Promotoras Genéticas/genética , Testículo/metabolismo , Glândulas Suprarrenais/metabolismo , Glândulas Suprarrenais/patologia , Glândulas Suprarrenais/ultraestrutura , Hiperplasia Suprarrenal Congênita/genética , Animais , Sítios de Ligação/genética , Western Blotting , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Ritmo Circadiano , Corticosterona/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Fertilidade/genética , Citometria de Fluxo , Expressão Gênica , Longevidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Placenta/metabolismo , Reprodução/genética , Fator Esteroidogênico 1/metabolismoRESUMO
Steroidogenic factor 1 (SF-1 or NR5A1) is a nuclear receptor that controls adrenogenital cell growth and differentiation. Adrenogenital primordial cells from SF-1 knockout mice die of apoptosis, but the mechanism by which SF-1 regulates cell survival is not entirely clear. Besides functioning in the nucleus, SF-1 also resides in the centrosome and controls centrosome homeostasis. Here, we show that SF-1 restricts centrosome overduplication by inhibiting aberrant activation of DNA-dependent protein kinase (DNA-PK) in the centrosome. SF-1 was found to be associated with Ku70/Ku80 only in the centrosome, sequestering them from the catalytic subunit of DNA-PK (DNA-PKcs). In the absence of SF-1, DNA-PKcs was recruited to the centrosome and activated, causing aberrant activation of centrosomal Akt and cyclin-dependent kinase 2 (CDK2)/cyclin A and leading to centrosome overduplication. Centrosome overduplication caused by SF-1 depletion was averted by the elimination of DNA-PKcs, Ku70/80, or cyclin A or by the inhibition of CDK2 or Akt. In the nucleus, SF-1 did not interact with Ku70/80, and SF-1 depletion did not activate a nuclear DNA damage response. Centriole biogenesis was also unaffected. Thus, centrosomal DNA-PK signaling triggers centrosome overduplication, and this centrosomal event, but not the nuclear DNA damage response, is controlled by SF-1.
Assuntos
Centrossomo/metabolismo , Proteína Quinase Ativada por DNA/metabolismo , Fator Esteroidogênico 1/metabolismo , Animais , Antígenos Nucleares/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Autoantígeno Ku , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator Esteroidogênico 1/genéticaRESUMO
Steroidogenic Factor 1 (SF-1, Ad4bp, NR5A1) is a nuclear receptor expressed mainly in the adrenals and gonads. It activates the transcription of genes in steroidogenesis, reproduction, and energy metabolism. In addition, it also regulates the growth and differentiation of adrenogonadal primodial cells. SF-1 resides in the nucleus and the centrosome. SF-1 moves dynamically in the nucleus, and SF-1 location and activity are dynamically regulated by post-translational modifications. In the centrosome, SF-1 maintains genomic integrity by controlling centrosome homeostasis. SF-1 prevents centrosome amplification by restricting aberrant activation of centrosomal DNA-PK. Upon SF-1 removal, DNA-PK is activated and centrosomes are amplified. This leads to genomic instability and cell growth defects. These data indicate that SF-1 at both the nucleus and the centrosome contributes to cell growth control, but the mechanisms of SF-1 action in different locations are different.