Significance of metabolic activation of lumisterol in the skin

皮肤中光甾醇代谢激活的意义

• 批准号: 9756150
• 负责人: ANDRZEJ T SLOMINSKI
• 金额: $ 32.85万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9756150
• 关键词: 7-dehydrocholesterol; Adrenal Glands; Binding; Biochemical Pathway; Biochemistry; Bioinformatics; Biological; Blood Circulation; C10; CYP11A1 gene; Cells; Chemistry; Chinese Hamster Ovary Cell; Cholecalciferol; Cholesterol; Cleaved cell; Computer Simulation; Coupled; Crystallization; Cutaneous; DNA Damage; Data; Docking; Dose; Enzymes; Epidermis; Evaluation; Exposure to; Follow-Up Studies; Gene Expression; Gene Silencing; Genes; Genetic Complementation Test; Genetic Transcription; Genomics; Gonadal structure; Homeostasis; Human; Human body; Intoxication; Investigation; Ligand Binding Domain; Ligands; Measures; Mediating; Metabolic; Metabolic Activation; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Mus; Organ; Organ Culture Techniques; Outcome; Oxidative Stress; Pathology; Pathway interactions; Pharmacology; Phenotype; Physiological; Placenta; Play; Pregnenolone; Process; Production; Recombinants; Regulation; Reporter; Role; Sampling; Scheme; Serum; Side; Signal Transduction; Skin; Skin Physiology; Stereoisomer; Steroid biosynthesis; Steroids; Structure; System; Techniques; Testing; Tetanus Helper Peptide; Transactivation; UVB induced; Ultraviolet B Radiation; absorption; base; design; genetically modified cells; in vivo; interest; keratinocyte; keratinocyte differentiation; molecular modeling; multidisciplinary; novel; orphan nuclear receptor ROR-gamma; photoprotection; predictive modeling; prevent; programs; receptor; simulation; skin barrier; stressor; transcriptome sequencing; whole genome

UVB breaks the B-ring of 7-dehydrocholesterol leading to the formation of pre-vitamin D3 that either undergoes thermal isomerization to D3 or photoisomerization to lumisterol (L3) in a 9β,10α-configuration. L3 is the major photoisomer formed after prolonged exposure of the skin to UVB energy, in a process that is irreversible. L3 has been considered to be metabolically and biologically inactive with its formation explaining why UVB induced cutaneous production of pre-D3 does not lead to systemic intoxication by D3. Recently we found that L3 is metabolized by CYP11A1 to 22(OH)L3, 24(OH)L3, 20,22(OH)2D3 and pregnalumisterol (pL) [collectively called (OH)nL3]. Uning LC/qTOF-MS we demonstrated the presence of L3 in human serum at levels higher than D3, and detectable levels of (OH)nL3 in the epidermis and serum. Furthermore, we found that (OH)nL3 inhibits the proliferation of human epidermal keratinocytes and that 20(OH)L3 stimulates the expression of genes associated with keratinocyte differentiation and protection against oxidative stress. Our preliminary molecular modeling predicts that the major (OH)nL3 products can act on RORα and RORγ showing favorable docking scores in the ligand binding domain (LBD) of the receptors, similar to known natural ligands. Supporting these predictions we found that 20(OH)L3 inhibits RORγ and RORα transactivation activities in a Tet-on CHO cell reporter system, as well as reducing (RORE)-LUC reporter activity in skin cells. Thus, we have discovered a new lumisterogenic biochemical pathway that is biologically relevant. To investigate its role in skin physiology and pathology we formulate the hypothesis that skin-derived L3 is enzymatically activated generating (OH)nL3 compounds which regulate epidermal barrier and photoprotective functions through interaction with RORα and RORγ. This hypothesis will be tested via three aims. 1. Defining RORα and RORγ as functional receptors for novel hydroxylumisterol derivatives ((OH)nL3); 2. Defining the phenotypic activities of (OH)nL3 in cultured epidermal keratinocytes and in human skin histocultured ex vivo; 3. Defining the relative roles of RORα and RORγ in (OH)nL3-mediated regulation of the differentiation program and protective mechanisms against UVB radiation in the epidermis. Our highly mechanistic strategy combines techniques of primary epidermal and organ cultures, in silico methods, cell-based transcriptional studies coupled to the LBD of RORα/γ, the use of genetically modified cells, whole genome RNA sequencing with bioinformatics, and the techniques of biochemistry, chemistry and molecular biology. This proposal is a comprehensive, multi- disciplinary and state-of-the-art investigation. Its significance encompasses defining a role for a previously unrecognized pathway of activation of an important UV photoproduct, L3, in skin physiology and pathology. The practical outcome of the realization of aims 1-3 would be the use of the most potent (OH)nL3 as agent(s) that either enhance the epidermal barrier or prevent or reverse damage inflicted by noxious stressors and UVB. Establishing roles for RORs in these processes will define them as druggable pharmacological targets.

UVB破坏7-脱氢胆固醇的B环，导致形成前维生素D3， 热异构化为D3或光异构化为9β，10 α-构型的光甾醇（L3）。L3是主要的 在皮肤长时间暴露于UVB能量后，在一个不可逆的过程中形成的光异构体。L3 被认为是代谢和生物活性与它的形成解释了为什么UVB 前D3的诱导皮肤产生不会导致D3的全身中毒。最近我们发现， L3通过CYP 11 A1代谢为22（OH）L3、24（OH）L3、20，22（OH）2D 3和阿铝甾醇（pL）[统称 （OH）nL3）。使用LC/qTOF-MS，我们证明了L3在人血清中的存在水平高于 在表皮和血清中检测到（OH）nL 3水平。此外，我们发现，（OH）nL 3 抑制人表皮角质形成细胞的增殖，并且20（OH）L3刺激 与角质形成细胞分化和抗氧化应激相关的基因。我们的初步 分子模拟预测，主要的（OH）nL 3产物可以作用于RORα和RORγ，表现出有利的 受体的配体结合域（LBD）中的对接得分，类似于已知的天然配体。 支持这些预测，我们发现20（OH）L3抑制RORγ和RORα的反式激活活性， Tet-on CHO细胞报告基因系统，以及降低皮肤细胞中的（RORE）-LUC报告基因活性。因此我们 已经发现了一种新的生物学相关的发光生物化学途径。来研究它的作用 在皮肤生理学和病理学中，我们提出了皮肤来源的L3被酶激活的假设 产生（OH）nL 3化合物，其通过以下方式调节表皮屏障和光保护功能： 与RORα和RORγ相互作用。这一假设将通过三个目标进行检验。1.定义RORα和RORγ 作为新型羟基光甾醇衍生物（（OH）nL 3）的功能性受体; 2.定义表型活性 （OH）nL 3在培养的表皮角质形成细胞和离体培养的人皮肤组织中的表达; 3.定义relative RORα和RORγ在（OH）nL 3介导的分化程序调节和保护性细胞凋亡中的作用 在表皮中抵抗UVB辐射的机制。我们高度机械化的策略结合了 原代表皮和器官培养、计算机模拟方法、与LBD结合的基于细胞的转录研究 RORα/γ，转基因细胞的使用，全基因组RNA测序与生物信息学， 生物化学、化学和分子生物学技术。这是一个全面的、多方面的... 纪律和最先进的调查它的重要性包括为以前的 在皮肤生理学和病理学中，一种重要的UV光产物L3的未被认识到的活化途径。 实现目标1-3的实际结果将是使用最有效的（OH）nL 3作为试剂。 增强表皮屏障或预防或逆转有害应激源造成的损伤， UVB。确定ROR在这些过程中的作用将把它们定义为可药用的药理学靶点。