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甲酸（13-顺式维甲酸（RA）），这是一种已知的致畸剂。异维甲酸是仅次于沙利度胺的第二种药物，由于公众对致畸性的关注而被列入FDA国家注册表。该登记处包括患者、药剂师、开处方者和批发商。确定13-顺式类风湿性关节炎的作用机制，是开发一种无严重副作用的有效替代药物的重要途径。该实验室通过对13顺式类风湿性关节炎患者皮肤的基因阵列表达分析和对皮脂细胞系的研究，参与确定对异维甲酸反应的关键因素。在13顺式RA治疗1周时表达增加最多的基因是脂钙蛋白2 (Icn2)，它编码中性粒细胞明胶酶相关脂钙蛋白（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接受异维甲酸治疗的患者皮肤上的NGAL分泌是否增加。皮肤上存在的NGAL量将通过对特定区域进行皮肤清洗，然后进行ELISA来评估。在异维甲酸治疗过程中，患者的皮肤NGAL水平与血浆NGAL水平相关。这些研究的结果应该揭示NGAL在13-顺式RA治疗的皮肤反应中是否有效以及反应的持续时间。实验室的初步数据表明，脂细胞表达最近发现的NGAL受体，并在NGAL的作用下发生凋亡。本研究的第二个目的是确定NGAL是否通过改变TSS-1脂质细胞内铁水平诱导细胞凋亡。这将首先通过含铁（holo-NGAL）和不含铁（载铁-NGAL）的重组人NGAL进行测试。TSS-1细胞将用13-顺式维甲酸、载脂蛋白ngal和全脂蛋白ngal处理，并使用western blotting检测铁蛋白和细胞上清比色法评估细胞的铁状态。这些实验将有助于阐明NGAL对脂质细胞系铁通量的影响。长期目标是确定NGAL是否是一种有希望的替代13-顺式RA治疗痤疮的方法。这项研究将有助于表征中性粒细胞明胶酶相关脂钙蛋白（NGAL）对皮肤的影响，并有望发现13-顺式类风湿性关节炎的替代疗法，已知13-顺式类风湿性关节炎会导致出生缺陷。

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.