RESUMO
Drug options for the life-threatening Cushing's disease are limited, and surgical resection or radiation therapy is not invariably effective. Testicular receptor 4 (TR4) has been identified as a novel drug target to treat Cushing's disease. We built the structure model of TR4 and searched the TR4 antagonist candidate via in silico virtual screening. Bexarotene was identified as an antagonist of TR4 that can directly interact with TR4 ligand binding domain (TR4-LBD) and induces a conformational change in the secondary structure of TR4-LBD. Bexarotene suppressed AtT-20 cell growth, proopiomelanocortin (POMC) expression and adrenocorticotropin (ACTH) secretion. Mechanism dissection revealed that bexarotene could suppress TR4-increased POMC expression via promoting the TR4 translocation from the nucleus to the cytoplasm. This TR4 translocation might then result in reducing the TR4 binding to the TR4 response element (TR4RE) on the 5' promoter region of POMC. Results from in vivo mouse model also revealed that oral bexarotene administration markedly suppressed ACTH-secreting tumour growth, adrenal enlargement and the secretion of ACTH and corticosterone in mice with already established tumours. Together, these results suggest that bexarotene may be developed as a potential novel therapeutic drug to better suppress Cushing's disease.
Assuntos
Bexaroteno/farmacologia , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/antagonistas & inibidores , Pró-Opiomelanocortina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenoma Hipofisário Secretor de ACT , Hormônio Adrenocorticotrópico/biossíntese , Animais , Bexaroteno/química , Sítios de Ligação , Linhagem Celular Tumoral , Modelos Animais de Doenças , Descoberta de Drogas , Expressão Gênica , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/química , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/metabolismo , Hipersecreção Hipofisária de ACTH , Pró-Opiomelanocortina/genética , Ligação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Transcrição Gênica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Alternative lengthening of telomeres (ALT) is known to use homologous recombination (HR) to replicate telomeric DNA in a telomerase-independent manner. However, the detailed process remains largely undefined. It was reported that nuclear receptors COUP-TFII and TR4 are recruited to the enriched GGGTCA variant repeats embedded within ALT telomeres, implicating nuclear receptors in regulating ALT activity. Here, we identified a function of nuclear receptors in ALT telomere maintenance that involves a direct interaction between COUP-TFII/TR4 and FANCD2, the key protein in the Fanconi anemia (FA) DNA repair pathway. The COUP-TFII/TR4-FANCD2 complex actively induces the DNA damage response by recruiting endonuclease MUS81 and promoting the loading of the PCNA-POLD3 replication complex in ALT telomeres. Furthermore, the COUP-TFII/TR4-mediated ALT telomere pathway does not require the FA core complex or the monoubiquitylation of FANCD2, key steps in the canonical FA pathway. Thus, our findings reveal that COUP-TFII/TR4 regulates ALT telomere maintenance through a novel noncanonical FANCD2 pathway.
Assuntos
Fator II de Transcrição COUP/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/metabolismo , Telômero/metabolismo , Motivos de Aminoácidos , Fator II de Transcrição COUP/antagonistas & inibidores , Fator II de Transcrição COUP/genética , Linhagem Celular Tumoral , DNA Polimerase III/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/antagonistas & inibidores , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Fase G2 , Humanos , Mutagênese Sítio-Dirigida , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/antagonistas & inibidores , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/genética , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Homeostase do TelômeroRESUMO
The testicular receptor 4 (TR4) is a member of the nuclear receptor superfamily that controls various biological activities. A protective role of TR4 against oxidative stress has recently been discovered. We here examined the protective role of TR4 against ionizing radiation (IR) and found that small hairpin RNA mediated TR4 knockdown cells were highly sensitive to IR-induced cell death. IR exposure increased the expression of TR4 in scramble control small hairpin RNA expressing cells but not in TR4 knockdown cells. Examination of IR-responsive molecules found that the expression of Gadd45a, the growth arrest and DNA damage response gene, was dramatically decreased in Tr4 deficient (TR4KO) mice tissues and could not respond to IR stimulation in TR4KO mouse embryonic fibroblast cells. This TR4 regulation of GADD45A was at the transcriptional level. Promoter analysis identified four potential TR4 response elements located in intron 3 and exon 4 of the GADD45A gene. Reporter and chromatin immunoprecipitation (ChIP) assays provided evidence indicating that TR4 regulated the GADD45A expression through TR4 response elements located in intron 3 of the GADD45A gene. Together, we find that TR4 is essential in protecting cells from IR stress. Upon IR challenges, TR4 expression is increased, thereafter inducing GADD45A through transcriptional regulation. As GADD45A is directly involved in the DNA repair pathway, this suggests that TR4 senses genotoxic stress and up-regulates GADD45A expression to protect cells from IR-induced genotoxicity.