Short-Chain Dehydrogenases in Retinol/Sterol Metabolism

全反式维甲酸（RA）是维生素A和β-胡萝卜素的生物活性衍生物，对于胚胎组织的分化和发育以及成人器官和组织的维持和稳健性能至关重要。在细胞核中，RA通过与RXR/RAR异二聚体转录因子结合来调节超过530个基因的表达。还已知RA在细胞质中具有调节功能。在胚胎发生过程中，RA水平的变化在一个严格定义的时空模式。类似地，在成年期，各种组织和细胞中的RA浓度维持在对每种类型的细胞最佳的狭窄范围内。RA稳态的破坏导致胚胎畸形，而在成人组织中，RA稳态的畸变可导致导致疾病的病理生理变化。因此，关键是要了解：（1）细胞维持RA稳态的分子机制;（2）细胞如何调节RA水平以响应不同的生理需求;以及（3）为什么这些机制在疾病中失败。由于视黄醇氧化为视黄醇醛是反应中的限速步骤， 由于RA生物合成是RA生物合成的主要途径，控制RA生物合成的总体速率，因此重要的是鉴定和表征催化这一步骤的酶，并了解每种酶对总体RA稳态的贡献。在上一个资助周期中，已经确定细胞中RA的基线水平是由视黄醇脱氢酶10（RDH 10）和脱氢酶/还原酶3（DHRS 3）形成的异源寡聚类视黄醇氧化还原酶复合物（ROC）维持的。来自该实验室和其他实验室的数据表明，RDH 10也是负责在胚胎发生的早期阶段将视黄醇氧化为视黄醇醛的主要酶。然而，其他尚未确定的视黄醇脱氢酶似乎在成人组织中更重要。来自该实验室的初步数据表明，具有编码视黄醇脱氢酶表皮2（RDHE 2）和RDHE 2类似物（RDHE 2S）的基因的双敲除的小鼠显示出与皮肤毛囊皮脂腺单位和眼睑的睑板腺中的RA信号传导减少一致的表型。这些数据还表明，RDHE 2和RDHE 2S的表达在毛囊再生的不同阶段以昼夜模式振荡。我们假设，这些诱导型和RA敏感性酶负责毛囊再生周期中RA的波动，并负责ROC建立的基线RA水平的微调，以响应不同的生理需求或 病理生理条件。为了验证这一假设，我们将使用新的小鼠模型和在前一周期中产生的定制抗体来确定RDHE 2/E2 S对眼睑皮肤和睑板腺中RA生物合成的贡献。拟议的研究将提供一个全面的背景，以更好地了解维持和/或破坏RA稳态的分子机制，并将为未来的策略提供信息，以开发有针对性的和更有效的治疗干预措施。

All-trans-retinoic acid (RA) is the bioactive derivative of vitamin A and β-carotene that is essential for differentiation and development of embryonic tissues as well as the maintenance and robust performance of adult organs and tissues. In the nucleus, RA acts by binding to RXR/RAR heterodimeric transcription factors to regulate the expression of over 530 genes. RA is also known to have regulatory functions in the cytoplasm. During embryogenesis the levels of RA change in a strictly defined spatiotemporal pattern. Similarly, during adulthood the concentration of RA in various tissues and cells is maintained within narrow margins that are optimal for each type of cell. Disruption of RA homeostasis results in embryonic malformations, whereas in adult tissues aberrations in RA homeostasis can lead to pathophysiological changes that result in disease. Thus, it is critical to understand: (1) the molecular mechanisms whereby the cells maintain RA homeostasis; (2) how the cells adjust RA levels in response to varied physiological requirements; and (3) why these mechanisms fail in disease. Since the oxidation of retinol to retinaldehyde is the rate-limiting step in the pathway of RA biosynthesis that controls the overall rate of RA biosynthesis, it is important to identify and characterize the enzymes that catalyze this step and to understand the contribution of each enzyme to overall RA homeostasis. During the previous funding cycle, it was established that the baseline levels of RA in cells are maintained by a heterooligomeric retinoid oxidoreductase complex (ROC) formed by retinol dehydrogenase 10 (RDH10) and dehydrogenase/reductase 3 (DHRS3). Data from this and other laboratories indicate that RDH10 is also the primary enzyme responsible for the oxidation of retinol to retinaldehyde during early stages of embryogenesis. However, other yet unidentified retinol dehydrogenases appear to be more important in adult tissues. Preliminary data from this laboratory indicate that mice with a double knockout of genes encoding retinol dehydrogenase epidermal 2 (RDHE2) and RDHE2-similar (RDHE2S), display a phenotype consistent with reduced RA signaling in skin pilosebaceous unit and meibomian glands of eyelids. The data also suggest that the expression of RDHE2 and RDHE2S oscillates in a diurnal pattern and during various stages of hair follicle regeneration. We hypothesize that these inducible and RA-sensitive enzymes are responsible for the fluctuations of RA during the cycle of hair follicle regeneration, and for fine-tuning of the baseline RA levels, established by the ROC, in response to varied physiological requirements or pathophysiological conditions. To test this hypothesis, we will use the novel mouse models and custom-made antibodies generated during the previous cycle to determine the contribution of RDHE2/E2S to RA biosynthesis in skin and meibomian glands of the eyelid. The proposed studies will provide a comprehensive background to better understand the molecular mechanisms that maintain and/or disrupt RA homeostasis and will inform future strategies to develop targeted and more effective therapeutic interventions.