Adrenocorticotropic Hormone Receptors In Adrenal Tumors,

Objectives:Adrenalcorticotropin,depending of its receptor,plays the important role in the differentiation,steroidgenesis and proliferation of adrenal cortex cells.In according to CT imaging improvement,the detection rate of adrenal incidental adenoma obviously increases.It is important to elucidate the pathogenesy of adrenalcortical adenoma.It was reported that the frequency of loss of heterozygosity(LOH) in ACTH gene increased in adrenalcortical carcinoma,with reduced expression of ACTH receptor mRNA in the tumors.It is suggested that ACTH receptor gene may act as a tumor-suppressor gene in the development of adrenalcortical tumors.The aim of this study is to analyze adrenocoticotropin receptor mRNA expression of in human adrenalcortical tumors,pheochromocytoma,normal adrenal cortex and medulla and gain an insight into the action of ACTH receptor on the development of adrenal tumors.Methods:Total RNA was exracted from 6 normal human adrenal cortex,6 normal human adrenal medulla,2 adrenal tissues of idiopathatic hyperaldosteronism patients,20 pheochromacytomas and 24 adrenalcortical tumors(14 aldoterone-producing adenomas and 10 cortisol-producing adenomas).RT-PCR was used to analyze the ACTH receptor and GAPDH mRNA expression in different tissues.The mRNA expression of ACTH receptor was semi-quantified by dividing the grayness value of ACTH-R/GAPDH in the same sample and compared among the different tissues.It was studied whether the mRNA expression of ACTH receptor correlated with the tumor diameter and basal plasma aldosterone level and so on.Results:1.The ratio ACTH-R mRNA to GAPDH was 0.67±0.22 in normal adrenal cortex,and was 1.76±0.46 in aldosterone-producing adenoma.Compared with normal adrenal cortex,the difference was significant(p＜0.01).The ratio ACTH-R mRNA to GAPDH was 0.27±0.09 in cortisol-pruducing adenoma,and it was much lower than normal adrenal cortex(p=0.01). 2.There was a significant difference in ACTH-R mRNA expression between APA and cortisol-producing adenoma(p＜0.01),3.In APA,the ACTH receptor mRNA expression had a negative correlation with the increase of plasma aldosterone level to upright stimulation(r=-0.712,p = 0.002), but had no correlation with tumor diameter and basal plasma aldosterone level (p＞0.05).4.The mRNA expression of ACTH receptor in normal adrenal medulla was lower than in the normal adrenal cortex(p=0.006 );The mRNA expression of ACTH receptor was not detected in pheochromocytoma tissues.Conclusions:1.The mRNA expression of ACTH receptor in aldosterone-producing adenoma tissues could determine the responsive of APA patients to angiotensinⅡ,it is suggested that ACTH could contribute to the development of a part of APA.2.It is indicated that ACTH have nothing to do with the development of pheochromocytoma. Objectives:The aim of this study is to analyze the protein expression of adrenocoticotropin receptor in human adrenalcortical tumors,pheochromocytoma,normal adrenal cortex and medulla and gain an insight into ACTH receptor action on the development of adrenocortical tumors.Methods:Total protein was exracted from 6 normal human adrenal cortex,6 normal human adrenal medulla,2 adrenal tissues of idiopathatic hyperaldosteronism patients,10 pheochromacytomas and 24 adrenalcortical tumors(14 aldoterone-producing adenomas and 10 cortisol-producing adenomas).The concentration of protein was evaluated by the means of Bradford.Using western bolt we analyzed the protein expression of ACTH receptor andβ-actin.The protein expression of ACTH receptor was semi-quantified by dividing the grayness value of ACTH-R/β- actin in the same sample and compared among the different tissues.Results:1.The ratio ACTH-R protein toβ-actin was 0.80±0.37 in normal adrenal cortex, and was 1.09±0.09 in aldosterone-producing adenoma.Compared with normal adrenal cortex,the difference was significant(p＜0.001).The ratio ACTH-R protein toβ-actin in cortisol-producing adenoma was 0.47±0.09,and it was much lower than in normal adrenal cortex(p=0.002).2.There was a significant difference in ACTH-R protein expression between APA and cortisol-producing adenoma(p＜0.001).3.In APA,the ACTH receptor protein expression had a negative correlation with the increase of plasma aldosterone level to upright stimulation(r=- 0.733,p= 0.003),but had no correlation with tumor diameter and basal plasma aldosterone level(p＞0.05).4.The expression of ACTH receptor protein was not detected in normal adrenal medulla and pheochromocytoma tissues. Conclusions:1.In accord with the mRNA,the protein expression of ACTH-R in APA tissues determines the responsive of APA patients to angiotensinⅡ.It is confirmed that ACTH could contribute to the development of a part of APA at the aspect of the protein expression.2.It is suggested that ACTH could not participate in the physiologica and pathological function of adrenal medulla. Objective:Although its pathogenesis is unclear,genetic factors are assumed to play a determining role in the development of adrenocortical adenoma.Adrenalcorticotropin, the principal hormone regulator of adrenal steroidogenesis,plays the important role in the differentiation and proliferation of adrenal cortex cells.It is indicated that the polymorphism of ACTH receptor gene promoter region might impact on either corticosteoid or androgen production.It is not known whether ACTH-R gene influence the expression of ACTH-R in adrenocortical adenoma tissues.The aims of this study are to search gene mutations and single nucleotide porlymorphisms in ACTH-R gene promoter and coding region in sporadic and familial patients with adrenocortical adenoma and investigate the association of ACTH receptor genetic variation with adrenocortical adenoma.Methods:1.We recruited 78 patients with primary aldosteronism(including 32 IHA and 46 APA) and 16 patients with cortisol-producing adenoma and 52 normal controls,and isolated genomic DNA from full blood samples.Tissue samples from 29 patients undergoing surgery for APA were studied.All subjects were obtained from PUMCH.2.ACTH receptor gene mutations were detected in 16 subjects from 2 pedigrees with adrenal adenoma family history and a familial Addison kindred.3.The coding and promoter region of ACTH recepor gene were amplifiacted.PCR products directly sequence to detect gene mutation and polymorphic sites.4.Visual inspection and alignment were performed using BioEdit version 5.0.9. Hardy-Weinberg test,linkage disequilibrium and haplotype analysis were carried out by SHEsis software.Results:1.Two IHA(2/32) and 4 APA patients(4/46) have germline mutations in ACTH receptor gene non-coding region,and the frequency of mutations in the promoter -2bp and -347bp in APA patients was 8.7%(4/46).2.In non-coding region of ACTH receptor gene seven SNPs sites were detected.We describe a new polymorthism in the promoter -58bp site altering the allele from C to T.In 52 health adults the frequency of T allele was 8%,and the frequency of CC and CT genotype was respectively 85%and 15%;no TT genotype was detected.3.The promoter -759bp A and -2bp C alleles of ACTH receptor gene were significantly more common among patients with cortisol-producing adenoma than in patients with APA(p≤0.05).4.We analyzed the distribution of haplotype in three SNPs site with linkage disequilibrium(rs4797824,rs1893219,and -2bp in promoter).The frequency of ATT(15%vs 1%,p＜0.01) and GCT(9%vs 1%,p=0.01) haplotypes was significantly more common among adenoma tissues from APA patients than blood samples from health controls.5.In APA patients some polymorphic sites(rs4797825 and rs4797824) correlated with plasma aldosterone.Patients with AA genotype(rs4797825) and AG/AA genotypes(rs4797824) had a higher plasma aldosterone level(p≤0.05).Conclusion:1.In pimary aldoteronism,particularly in aldosterone-producing adenoma the frequency of ACTH-R gene mutation in promoter region increased.2.The common polymorphyism of adrenalcoticotropin receptor gene promoter could predispose to the development of aldosterone-producing adenoma.