Neutrophil gelatinase-associated lipocalin mediated retinoid effects in the skin

描述(申请人提供)：粉刺是一种常见的疾病，尤其是在青春期。治疗严重痤疮最有效的方法之一是异维A酸(13-顺式维甲酸(RA))，它是一种已知的致畸物质。由于公众对致畸作用的担忧，异维A酸是继沙利度胺之后第二种被列入FDA国家登记的药物。这一登记包括患者、药剂师、处方者和批发商。为了开发一种有效的、无严重副作用的替代药物，确定13-顺式维甲酸的作用机制是非常重要的。该实验室通过对接受13-顺式维甲酸治疗的患者皮肤的基因阵列表达分析，以及通过皮脂细胞系的工作，参与了识别异维A酸反应的关键因素。在13-顺式维甲酸治疗1周后，表达增加最多的基因是Lipocalin 2(ICN2)，它编码中性粒细胞-明胶酶相关的Lipocalin(NGAL)蛋白。本研究的总体目标是确定13-顺式维甲酸诱导皮脂腺细胞凋亡的作用是否由NGAL介导。这项拟议工作的第一个目标是确定接受异维A酸治疗的患者皮肤上的NGAL分泌是否增加。皮肤上存在的NGAL的数量将通过对特定区域进行洗皮然后进行ELISA来评估。在异维A酸治疗过程中，皮肤NGAL水平将与患者的血浆NGAL水平相关。这些研究的结果应该揭示皮肤上是否有NGAL对13-顺式维甲酸治疗的反应以及反应的持续时间。实验室的初步数据表明，皮脂细胞表达最近发现的NGAL受体，并在NGAL反应中发生凋亡。这项建议的第二个目的是确定NGAL是否通过改变TSS-1皮脂细胞内的铁水平来诱导细胞凋亡。这将首先通过使用载铁的重组人NGAL(HALO-NGAL)和不含铁的重组人NGAL(APO-NGAL)进行测试。TSS-1细胞将用13-顺式维甲酸、apo-NGAL和全反式维甲酸处理，并用蛋白质印迹铁蛋白和细胞上清液比色法评估细胞的全铁和铁状态。这些实验将有助于阐明NGAL对皮脂细胞铁通量的影响。长期目标是确定NGAL是否是治疗痤疮的13-顺式RA的有前景的替代品。这项研究将有助于表征中性粒细胞-明胶酶相关脂蛋白(NGAL)对皮肤的影响，并有望发现替代13-顺式RA的治疗方法，后者已被认为会导致出生缺陷。

DESCRIPTION (provided by applicant): Acne is a common disorder, especially during adolescence. One of the most effective treatments for severe acne is isotretinoin (13-cis Retinoic Acid (RA)), which is a known teratogen. Isotretinoin is only the second drug (after thalidomide) to be placed on a FDA national registry due to public health concerns regarding teratogenicity. This registry includes patients, pharmacists, prescribers and wholesalers. It is important to determine the mechanism of action of 13-cis RA, in order to develop an effective alternative drug without serious side effects. The laboratory has been involved in identifying key players in the response to isotretinoin through gene array expression analysis of the skin of patients treated with 13-cis RA and through work with a sebocyte cell-line. The gene with the greatest increase in expression at 1 week of treatment with 13-cis RA is lipocalin 2 (Icn2), which encodes the neutrophil-gelatinase associated lipocalin (NGAL) protein. The overall goal of this investigation is to determine if the effects of 13-cis RA in inducing apoptosis in sebaceous glands is mediated by NGAL. The first aim of the proposed work is to determine if there is an increase in NGAL secretion on the skin of patients treated with isotretinoin. The amount of NGAL present on the skin will be assessed by performing skin washings of specific areas followed by an ELISA. Skin NGAL levels will be correlated with plasma NGAL levels of patients during the course of treatment with isotretinoin. The results of these studies should reveal whether or not NGAL is available on the skin in response to 13-cis RA treatment and the duration of the response. Preliminary data in the laboratory suggests that sebocytes express the recently identified NGAL receptor and undergo apoptosis in response to NGAL. The second aim of this proposal is to determine if NGAL induces apoptosis by changing intracellular iron levels in TSS-1 sebocytes. This will be tested first by using recombinant human NGAL loaded with iron (holo-NGAL) and without iron (apo-NGAL). TSS-1 cells will be treated with 13-cis retinoic acid, apo-NGAL, and holo-NGAL and iron status of the cells will be assessed using western blotting for ferritin and colormetric assay of cell supernatants. These experiments will help to elucidate the effect NGAL has on iron flux in sebocyte cell line. The long-term goal is to determine whether NGAL is a promising alternative to 13-cis RA as a treatment for acne. This research will help to characterize neutrophil-gelatinase associated lipocalin's (NGAL) effect on the skin and will hopefully lead to the discovery of therapies alternative to 13-cis RA, which is known to cause birth defects.