Neutrophil gelatinase-associated lipocalin mediated retinoid effects in the skin

描述（由申请人提供）：痤疮是一种常见疾病，尤其是在青春期。严重痤疮最有效的治疗方法之一是异维甲酸（13-顺式维甲酸（RA）），这是一种已知的致畸剂。异维甲酸是第二个药物（后沙利度胺）被放置在FDA的国家注册，由于公共卫生问题有关致畸性。该登记研究包括患者、药剂师、处方医生和批发商。确定13-cis RA的作用机制，以开发有效的替代药物而没有严重的副作用是重要的。该实验室一直参与确定关键球员的反应，异维甲酸通过基因阵列表达分析的皮肤与13顺式RA治疗的患者，并通过与皮脂细胞系的工作。在用13-顺式RA处理1周时具有最大表达增加的基因是脂质运载蛋白2（Icn 2），其编码亲脂明胶酶相关脂质运载蛋白（NGAL）蛋白。本研究的总体目标是确定13-顺式RA诱导皮脂腺细胞凋亡的作用是否由NGAL介导。这项工作的第一个目的是确定异维甲酸治疗的患者皮肤上NGAL分泌是否增加。将通过对特定区域进行皮肤洗涤，然后进行ELISA来评估皮肤上存在的NGAL的量。在用异维甲酸治疗的过程中，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NGAL是否在皮肤上对13-顺式RA治疗有反应以及反应的持续时间。实验室的初步数据表明，皮脂腺细胞表达最近鉴定的NGAL受体，并响应于NGAL而发生凋亡。该建议的第二个目的是确定NGAL是否通过改变TSS-1皮脂腺细胞中的细胞内铁水平来诱导细胞凋亡。这将首先通过使用负载铁的重组人NGAL（holo-NGAL）和不负载铁的重组人NGAL（apo-NGAL）进行测试。将用13-顺式视黄酸、apo-NGAL和holo-NGAL处理TSS-1细胞，并使用铁蛋白的蛋白质印迹和细胞上清液的比色测定来评估细胞的铁状态。这些实验将有助于阐明NGAL对皮脂腺细胞系铁通量的影响。长期目标是确定NGAL是否是13-顺式RA作为痤疮治疗的有希望的替代品。这项研究将有助于表征亲脂明胶酶相关脂质运载蛋白（NGAL）对皮肤的影响，并有望发现13-cis RA的替代疗法，已知13-cis RA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.