Epidermal Aquaporin-3 in Psoriasis

 描述(由申请人提供)： 牛皮癣是一种常见的人类皮肤病，影响大约2%的人口，包括估计40万退伍军人。除了免疫系统的激活和炎症，银屑病的特征是皮肤的表皮角质形成细胞过度增殖和异常分化。本实验室前期研究表明，在表皮角质形成细胞中，脂质代谢酶磷脂酶D2(PLD2)和甘油转运体水通道蛋白-3(AQP3)在物理和功能上相互作用。AQP3将甘油运输到细胞内，使其可供相关的PLD2使用，然后PLD2可以使用该甘油来合成磷脂酰甘油(PG)。我们的结果进一步证明PG作为脂质第二信使抑制角质形成细胞的增殖，刺激分化和抑制炎症。缺乏AQP3基因的小鼠表现出以表皮甘油含量减少和屏障修复延迟为部分特征的表皮表型，这一事实强调了AQP3在皮肤中的重要性。这种表型可以通过药物剂量的甘油来纠正，但不能通过其他保湿剂来纠正，这与甘油只有在没有AQP3等水甘油疏松蛋白的情况下才会无效进入细胞的事实一致。此外，AQP3已被发现在人类皮肤病中调节失调。因此，例如，我们之前已经报道，AQP3蛋白水平在银屑病中降低并弥漫；然而，其他研究人员发现AQP3 mRNA在银屑病皮损中表达上调。我们首次提出通过对银屑病患者和健康人的表皮进行Western和qRT-PCR分析来确定AQP3蛋白水平(与mRNA水平相比)在银屑病中是增加还是减少。此外，我们将测量这些样本中PLD2和甘油的水平，认为这两个参数中的一个或两个的减少也可能导致抗增殖和促进分化的PG水平下降。在这些患者中，我们还将研究甘油治疗银屑病的效果，以期该药能改善银屑病的皮损。尽管AQP3对皮肤功能很重要，但关于AQP3在健康或疾病中表达的调节机制，现有的信息很少。在初步结果中，我们发现广谱组蛋白脱乙酰酶(HDAC)抑制剂处理后AQP3的mRNA和蛋白水平增加，这表明基本上一个或多个HDAC通常抑制AQP3的表达。对一组HDAC抑制剂和HDAC3(相对于HDAC1)的过度表达和敲除的进一步研究表明，这种抑制性HDAC就是HDAC3。在拟议的研究中，我们将探索HDAC3调节AQP3表达的机制，验证HDAC3通常通过降低P53家族一个或多个成员的乙酰化和转录活性来抑制AQP3表达的假设，以及它对组蛋白和染色质组织的影响。我们将在体外和体内的表皮特异性条件HDAC3基因敲除小鼠模型中检验这一想法，监测AQP3的mRNA和蛋白水平，并与开花的HDAC3对照窝产仔进行比较。我们还将在咪喹莫特银屑病小鼠模型中检测角质形成细胞特异性HDAC3缺失对表皮结构和功能以及银屑病样皮损发展的影响，无论是否使用甘油共同治疗。这项拟议研究的完成将有助于理解HDAC3在调节AQP3表达中的作用，并可能证明HDAC3抑制作用与甘油一起或不与甘油一起用于治疗牛皮癣。

 DESCRIPTION (provided by applicant): Psoriasis is a common human skin disease that affects approximately 2% of the population including an estimated 400,000 veterans. In addition to activation of the immune system and inflammation, psoriasis is characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes of the skin. Previous data from our laboratory indicate that in epidermal keratinocytes the lipid-metabolizing enzyme phospholipase D2 (PLD2) and the glycerol transporter aquaporin-3 (AQP3) physically and functionally interact. AQP3 transports glycerol into the cell, making it available to the associated PLD2, which can then use this glycerol to synthesize phosphatidylglycerol (PG). Our results further demonstrate that PG acts as a lipid second messenger to inhibit keratinocyte proliferation, stimulate differentiation and inhibit inflammation. The importance of AQP3 in skin is emphasized by the fact that mice lacking the gene for AQP3 exhibit an epidermal phenotype characterized in part by decreased epidermal glycerol content and delayed barrier repair. The phenotype can be corrected by pharmacologic doses of glycerol but not other humectants, consistent with the fact that glycerol only inefficienty enters cells in the absence of aquaglyceroporins like AQP3. In addition, AQP3 has been found to be dysregulated in human skin diseases. Thus, for example, we have previously reported that AQP3 protein levels are decreased and diffuse in psoriasis; however, other investigators have found an up-regulation of AQP3 mRNA expression in psoriatic lesions. We first propose to resolve whether AQP3 protein levels (compared with mRNA levels) are increased or decreased in psoriasis using western and qRT-PCR analysis of psoriatic and healthy epidermis. In addition, we will measure PLD2 and glycerol levels in these samples, with the idea that reductions in one or both of these parameters could also lead to decreased levels of anti-proliferative, pro-differentiative PG. In these patients we will also investigate the effect of glycerol administratio on psoriasis with the expectation that this agent will improve psoriatic lesions. Despite the importance of AQP3 to skin function, there is little information available concerning the mechanisms regulating AQP3 expression in health or disease. In initial results we have found that AQP3 mRNA and protein levels are increased by treatment with a broad-spectrum histone deacetylase (HDAC) inhibitor, suggesting that basally one or more HDACs normally represses AQP3 expression. Additional studies with a panel of HDAC inhibitors and overexpression and knockdown of HDAC3 (versus HDAC1) indicate that this repressive HDAC is HDAC3. In the proposed studies we will explore the mechanism by which HDAC3 regulates AQP3 expression, testing the hypothesis that HDAC3 normally suppresses AQP3 expression by decreasing the acetylation and transcriptional activity of one or more members of the p53 family of transcription factors, in addition to its effects on histones and chromatin organization. We will examine this idea in vitro and in an epidermal-specific conditional HDAC3 knockout mouse model in vivo, monitoring the mRNA and protein levels of AQP3 in comparison with floxed HDAC3 control littermates. We will also examine the effect of keratinocyte-specific HDAC3 deletion on epidermal structure and function and the development of psoriasiform lesions in the imiquimod mouse model of psoriasis, with and without co-treatment with glycerol. Completion of the proposed research should result in an understanding of the role of HDAC3 in regulating AQP3 expression as well as a potential proof-of-principle for HDAC3 inhibition, with or without concomitant glycerol administration, as a therapy for psoriasis.