Regulation of Mineralocorticoid Receptor Action

盐皮质激素受体作用的调节

• 批准号: 10436926
• 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/10436926
• 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，其调节细胞的增殖和分化。 基因的表达涉及无数细胞类型特异性功能。MR激活不当导致 高血压和病理性组织重塑，即使在正常的醛固酮水平的条件下。活性 MR受翻译后修饰调节，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。激酶 最近报道ULK 1磷酸化MR的丝氨酸843，从而抑制其活性，并被认为是一种新的蛋白质。 MR仅在远端肾单位的闰细胞中表达。我们自己的特异性抗体 肾脏中ULK 1和MR的广泛共表达。ULK 1表达在体内广泛存在， 多个细胞系。其他激酶也有可能在S843磷酸化MR，从而抑制MR 活动 MR对醛、皮质醇或皮质酮具有类似的亲和力。对Aldo的特异性是通过以下物质的转化而赋予的： 皮质醇和皮质酮通过11β-羟基类固醇转化为无活性的可的松和11-脱氢皮质酮 脱氢酶2（11-HSD 2），然而，这种酶不存在于大多数非上皮aldo靶组织中。 皮质醇和皮质酮也可通过羰基转化为20β-二氢-皮质醇和-皮质酮 还原酶1（Cbr 1）。我们发现20个β-二氢代谢物不激活MR，从而提供了一种新的方法。 对于不表达11-HSD 2的aldo的MR特异性的替代机制。 假设：1）MR在丝氨酸843处的磷酸化是MR作用的重要负调节剂， 发生在多种组织中。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无活性代谢物来调节MR的配体选择性 20β-二氢皮质酮（20β-DHC）和20β-二氢皮质醇。两者都可能调节MR活性。 具体目的1：研究MR丝氨酸843（人）磷酸化在基因组和非基因组中的作用。 研究过表达和抑制几种激酶，ULK 1， ULK 2、TBK 1、NEK 2和PAK 1，具有在表达MR的细胞中使S843处的MR磷酸化的推定能力 报告基因系统研究MR与ULK 1、ULK 2等激酶的分布及共定位 在肾脏、心脏、血管和大脑中催化S843磷酸化。 具体目标2：研究皮质醇和皮质酮的20-酮还原对MR配体选择性的作用。 测定人和大鼠Cbr 1对皮质酮和皮质醇的20-酮还原动力学， 细胞测量各种组织中皮质酮向20β-二氢皮质酮的转化，包括 从大鼠大脑的特定区域的微穿孔，我们证明Cbr 1和MR是共同表达的 通过免疫荧光组织化学。测量血浆中皮质酮和20β-DHC的血浆水平， 组织的老鼠喂食不同数量的盐。 细胞特异性调节MR活性的机制如果不探索就永远不会知道， 这对于开发具有最小副作用的合理治疗方法至关重要。