Function Study Of NR3C1 Gene Mutation And Its Role In The Pathogenesis Of Pituitary ACTH-secreting Adenoma

ObjectiveCushing's disease is a disorder of metabolic disturbance caused by hypercortisolism due to ACTH-secreting pituitary adenomas,with severe complications and a high recurrence rate.Cortisol acts through glucocorticoid receptor(GR),which is coded by gene Nuclear Receptor Subfamily 3 Group C Member 1(NR3C1).As a hormone-regulated nuclear receptor and transcriptional factor,GR plays a significant role in the negative feedback regulation of its target gene Proopiomelanocortin(POMC)expression.We previously studied the whole genome sequence of ACTH-secreting pituitary adenomas and identified six somatic NR3C1 gene point mutations,c.1405C>T(p.Arg469TGA),c.2077T>G(p.Tyr693Asp),c.2173A>G(p_Met725Va1),c.2098G>C(p.Ala700Pro),c.1769A>G(p.Asp590Gly),and c.1750T>G(p.Phe584Val).NR3C1 mutations had been widely reported in previous studies,but for the first time it has been shown related to pituitary ACTH-secreting adenomas.Our study aims to discover the function changes of mutant GR molecule and its possible role in the pathogenesis of ACTH-secreting pituitary adenoma.MethodsWe retrospectively studied the clinicopathologic characteristics of patients with ACTH-secreting pituitary adenomas who were treated with transnasal transsphenoidal adenomectomy in Peking Union Medical College Hospital(n=58),and collected the pituitary adenoma specimens.The expression of GR in each sample was examined and quantified by immunohistochemistry.We built wild-type and three mutant NR3C1(c.1405C>T(p.R469*),c.1769A>G(p.D590G)and c.2077T>G(p.Y693D))expressing plasmid vectors and transfected into murine corticotropic adenoma cell line(AtT-20).We analyzed the protein expression differences through Western Blot,and compared the protein location difference using immunofluorescence technology.We then analyzed Pomc mRNA and protein levels in AtT-20 cells transfected with different mutation types of NR3C1 expressing plasmids.Aiming to discover the transcription factor activity of the mutant protein,we co-transfected the expression vectors with a reporter vector harboring human POMC gene promoter and a reference luciferase expressing vector,then performed dual-luciferase assays to evaluate the function of mutant NR3C1 gene.We also analyzed the cell cycle related gene Cdk5 and Abl enzyme substrate 1(Cables1)expression difference between wild-type and mutant NR3C1 expressing AtT-20 cells.ResultsACTH-secreting pituitary adenomas had higher GR protein level than other subtypes of pituitary adenomas.Recurrent ACTH-secreting pituitary adenomas showed significant higher GR immunohistochemistry score than non-recurrent pituitary adenoma.Recurrent-free interval is positively related with GR immunohistochemistry score.Sequencing of pcDNA3.1(+)-mut-NR3C1 confirmed the mutation.Compared with wild-type GR,the expression levels of mutant GR p.D590G and Y693D were significantly reduced in AtT-20 cells,and mutant GR p.R469*was truncated but with no expression decrease.GR subcellular distribution revealed a marked decrease in dexamethasone(DEX)-induced nuclear translocation of GR mutants.Dual-luciferase assays disclosed the lack of transcriptional activity of GR mutants with the presence of DEX,whereas POMC mRNA and protein levels in AtT-20 cells transfected with wild-type or mutant NR3C1 expressing plasmids showed no difference with the presence of DEX.POMC expression was increased in p.R469*and p.D590G mutant NR3C1 expressing AtT-20 cells.GR over-expression reduced the Cablesl mRNA level in AtT-20 cells,which was significantly increased treating with DEX.Wild-type and mutant GR showed identical results.ConclusionsACTH-secreting pituitary adenomas had higher GR protein level than other subtypes of pituitary adenomas,especially recurrent ACTH-secreting pituitary adenomas.In AtT-20 cells,the mutant NR3C1 p.D590G and Y693D were significantly reduced and p.R469*GR was truncated.DEX-induced nuclear translocation of GR mutants were impaired.The DEX-induced transcriptional activity of GR mutants to inhibit POMC expression was reduced.The ability of mutant GR to up-regulate the cell proliferation related gene Cablesl expression was not impaired.