Regulation of Mineralocorticoid Receptor Action

盐皮质激素受体作用的调节

• 批准号: 10293532
• 负责人: Celso Enrique Gomez-Sanchez
• 金额: --
• 依托单位: G V SONNY MONTGOMERY VA MEDCIAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10293532
• 关键词: Address; Affect; Affinity; Alcohol Oxidoreductases; Aldosterone; Amino Acids; Angiotensins; Antibodies; Area; Binding; Blood Pressure; Brain; CXCR3 gene; Cardiovascular system; Cell Line; Cells; Complications of Diabetes Mellitus; Congestive Heart Failure; Corticosterone; Cortisone; Cultured Cells; Dahl Hypertensive Rats; Data; Development; Diet; Distal; Dominant-Negative Mutation; Enzymes; Epithelial; Essential Hypertension; Gene Expression; Genomics; Glucocorticoid Receptor; Heart; Histocytochemistry; Hormones; Human; Hydrocortisone; Hydroxysteroid Dehydrogenases; Hyperaldosteronism; Hypertension; Immunofluorescence Immunologic; Intercalated Cell; Kidney; Ligand Binding Domain; Ligands; Luciferases; Measures; Mediating; Mineralocorticoid Receptor; Mouse Mammary Tumor Virus; Movement; Nephrons; Normal Range; Organ; Pathogenicity; Pathologic; Patients; Phosphorylation; Phosphotransferases; Plasma; Positioning Attribute; Post-Translational Protein Processing; Potassium; Protein Tyrosine Kinase; Rattus; Receptor Activation; Regulation; Reporter Genes; Reporting; Role; Serine; Site; Sleep Apnea Syndromes; Sodium; Sodium Chloride; Specificity; Steroids; System; TBK1 gene; Threonine; Time; Tissues; Transactivation; Water; cell type; inorganic phosphate; kidney cell; non-genomic; normotensive; overexpression; receptor; receptor binding; response; serine receptor; side effect; steroid hormone; transcription factor; vector

The mineralocorticoid receptor (MR) is a transcription factor expressed in many cells that regulates the expression of genes in involved myriad cell type-specific functions. Inappropriate activation of the MR causes hypertension and pathological tissue remodeling even in conditions of normal aldosterone levels. Activity of the MR is regulated by post-translational modifications including phosphorylation of serines and threonines. Phosphorylation activates or suppresses MR activity depending upon the amino acid involved. The kinase ULK1 was recently reported to phosphorylate serine 843 of the MR, thereby suppressing its activity, and to be expressed with the MR only in intercalated cells of the distal nephron. Our own specific antibodies reveal more extensive co-expression of ULK1 and MR in the kidney. ULK1 expression is widespread in the body and in multiple cell lines. Other kinases also have the potential to phosphorylate the MR at S843, thus inhibit MR activity. The MR has similar affinity for aldo, cortisol or corticosterone. Specificity for aldo is conferred by conversion of cortisol and corticosterone to the inactive cortisone and 11-dehydrocorticosterone by the 11β-hydroxysteroid dehydrogenase 2 (11-HSD2), however this enzyme is not present in most non-epithelial aldo target tissues. Cortisol and corticosterone are also converted to 20β-dihydro-cortisol and -corticosterone by carbonyl reductase 1 (Cbr1). We that found the 20β-dihydro metabolites do not activate the MR, thus providing an alternative mechanism for MR specificity for aldo where 11-HSD2 is not expressed. Hypotheses: 1) Phosphorylation of the MR at serine 843 is an important negative regulator of MR action that occurs in multiple tissues. 2) Reduction of the 20-keto of corticosterone and cortisol by carbonyl reductase 1 regulates ligand selectivity for the MR by converting corticosterone and cortisol into the inactive metabolites 20β-dihydrocorticosterone (20β-DHC) and 20β-dihydrocortisol. Both potentially modulate MR activity. Specific Aim 1: Study the role of phosphorylation of the MR at serine 843 (human) on genomic and non- genomic activity of the MR. Study the effects of over-expression and suppression of several kinases, ULK1, ULK2, TBK1, NEK2 and PAK1, with putative ability to phosphorylate the MR at S843 in cells expressing an MR reporter gene system. Study the distribution and co-localization of the MR with ULK1, ULK2 and other kinases that catalyze S843 phosphorylation in the kidney, heart, vessels and brain. Specific Aim 2: Study the role of the 20-keto reduction of cortisol and corticosterone on MR ligand selectivity. Determine the dynamics of the 20-keto reduction of corticosterone and cortisol by the human and rat Cbr1 in cells. Measure the conversion of corticosterone to 20β-dihydrocorticosterone in various tissues including in micropunches from specific areas of the rat brain where we demonstrate that Cbr1 and MR are co-expressed by immunofluorescent histochemistry. Measure plasma levels of corticosterone and 20β-DHC in plasma and tissues of rats fed different amounts of salt. The mechanisms for the cell-specific modulation of MR activity will never be known if not explored and are crucial for the development of rational therapy with minimal side effects for inappropriate MR activation.

盐皮质激素受体(MR)是一种在许多细胞中表达的转录因子，它调节 涉及无数细胞类型特异性功能的基因表达。MR原因的不适当激活 高血压和病理组织重塑，即使在正常的醛固酮水平的条件下。该组织的活动 MR受翻译后修饰的调节，包括丝氨酸和苏氨酸的磷酸化。 磷酸化激活或抑制MR活性取决于所涉及的氨基酸。一种激活剂 最近报道ULK1使MR的丝氨酸843磷酸化，从而抑制其活性，并被认为是 MR仅在远端肾单位的间质细胞中表达。我们自己的特异性抗体揭示了更多 肾组织中ULK1和MR的广泛共表达。ULK1在体内广泛表达，在 多个细胞系。其他的激酶也有可能在S843使MR磷酸化，从而抑制MR 活动。 MR对Aldo、皮质醇或皮质酮也有类似的亲和力。ALDO的专一性是通过转换 皮质醇和皮质酮对非活性皮质酮和11-脱氢皮质酮的作用(11β-羟基类固醇) 脱氢酶2(11-HSD2)，然而该酶在大多数非上皮性Aldo靶组织中不存在。 皮质醇和皮质酮也通过羰基转化为20β-二氢皮质醇和-皮质酮。 还原酶1(Cbr1)。我们发现20种β-二氢代谢物不能激活MR，从而提供了一种 在11-HSD2不表达的情况下，ALDO的MR特异性的替代机制。 假设：1)MR丝氨酸843位的磷酸化是MR作用的一个重要的负调节因子， 出现在多个组织中。2)羰基还原酶1还原皮质酮和皮质醇的20-酮 通过将皮质酮和皮质醇转化为非活性代谢物来调节MR的配体选择性 20β-二氢皮质酮(20β-DHC)和20β-二氢皮质醇。两者都有可能调节MR的活动。 具体目标1：研究丝氨酸843位MR的磷酸化对基因组和非基因组的作用 MR的基因组活性研究几种激酶ULK1，ULK1， ULK2、TBK1、NEK2和PAK1，推测有能力在表达MR的细胞中磷酸化S843处的MR 报道基因系统。MR与ULK1、ULK2等激酶的分布及共定位研究 在肾脏、心脏、血管和大脑中催化S843的磷酸化。 具体目标2：研究皮质醇和皮质酮的20-酮还原对MR配体选择性的影响。 测定人和大鼠体内Cbr1对皮质酮和皮质醇的20-酮还原动力学 细胞。测定不同组织中皮质酮向20β-二氢皮质酮的转化 我们证明Cbr1和MR共表达的大鼠大脑特定区域的微针 采用免疫荧光组织化学方法。测定血浆皮质酮和血浆中20β-DHC的水平 喂食不同量盐的大鼠的组织。 如果不探索，就永远不会知道细胞特异性调节MR活动的机制，而且 对于开发合理的治疗方法至关重要，同时将不适当的MR激活的副作用降至最低。