Role of CRH in skin physiology and pathology

描述(申请人提供)：皮肤既是前阿片黑素皮质素(POMC)衍生神经肽的目标和来源。促肾上腺皮质激素释放激素(CRH)和CRH受体(CRH-R)在啮齿动物和人类皮肤中也有表达，并具有完全的功能，但其意义尚不清楚。CRH和CRH受体被认为是全身应激反应的主要中枢调节因子，CRH多肽在调节局部内环境平衡方面具有多效性作用。因此，我们将研究CRH是否能够模拟垂体生理调节POMC基因的局部表达，和/或阐明其在皮肤中的具体作用。在此背景下，CRH本身是一种强大的免疫调节因子，作用于血管功能，也是一种局部生长因子。这些作用表明，在皮肤中，CRH多肽在调节局部动态平衡方面可能具有多效性。我们最初的目标是研究哺乳动物皮肤中CRH-CRH受体系统的生化和功能特征。其具体目的如下：1.明确CRH在正常黑素细胞和角质形成细胞中表达的局部调控机制；2.通过a)确定CRH结合位点属性，b)测量第二信使包括cAMP、IP3和Ca的产生，c)检测受体后CRH信号转导通路是否包括核因子-kappaβ、Cre、AP1和STAT等激活转录调节因子；3.检测CRH对正常黑素细胞和角质形成细胞产生POMC mRNA和POMC衍生肽的影响；4.检测CRH对正常黑素细胞和角质形成细胞表型的影响；5.检测CRH对人头发生长和色素沉着的调节作用。这将在体外培养的人毛囊中进行评估，并在毛囊来源的毛囊黑素细胞、角质形成细胞和毛乳头细胞的孤立亚群中进行评估。为了实现这些目标，我们将使用我们实验室目前可用的标准分子、生化和细胞生物学工具。作为实验模型，我们将使用表皮和毛囊角质形成细胞、黑素细胞以及毛乳头细胞。上述目标的实现应该阐明CRH和CRH受体在外周(皮肤)应激反应中的作用。该项目的核心是实现皮肤和下丘脑CRH激活系统之间的功能性生化重叠。虽然CRH与CRH-Rs之间的中枢相互作用协调了对全身应激的行为、自主神经和内分泌反应，但目前的工作将测试这一作用是否延伸到外周器官。因此，我们将测试常见应激源对CRH和CRH-Rs表达的影响，CRH对POMC表达的影响。此外，我们将确定这些CRH受体的特征，并确定它们在调节表皮最重要成分(如黑素细胞和角质形成细胞)的表型中的作用，并测试CRH/CRH-R1轴在调节人类头发生长中的作用。

DESCRIPTION (provided by applicant): The skin is both target and source for proopiomelanocortin (POMC)-derived neuropeptides. Corticotropin releasing hormone (CRH) and CRH receptors (CRH-R) are also expressed and fully functional in rodent and human skin, but its significance is not known. CRH and CRH receptors are recognized as main central regulators in the response to systemic stress and CRH peptide has pleiotropic role in regulation of local homeostasis. Therefore, we will investigate whether CRH can regulate local expression of POMC gene, mimicking therefore pituitary physiology, and/or to clarify its specific actions in the skin. Within this context, CRH itself is a potent immunomodulatory factor, acts on vascular function, and is also a local growth factor. These actions suggest that in the skin the role of CRH peptide could be pleiotropic in the regulation of local homeostasis. Our initial objective is the biochemical and functional characterization of the CRH-CRH receptor system in mammalian skin. The specific aims are as follow: 1. To define local mechanisms regulating the expression of CRH in normal melanocytes and keratinocytes; 2. To fully characterize the CRH-R1 receptors in normal melanocytes and keratinocytes by a) defining CRH binding site properties, b) measuring production of second messengers including cAMP, IP3 and Ca, c) testing whether postreceptor CRH signal transduction includes activation transcriptional regulators such as NF-kappa beta, CRE, AP1 and STAT; 3. To test the effect of CRH on the production of the POMC mRNA and POMC-derived peptides in normal melanocytes and keratinocytes; 4. To test the phenotypic effect of CRH on normal epidermal melanocytes and keratinocytes; 5. To test CRH participation in regulation of human hair growth and pigmentation. This will be evaluated in human hair follicle grown ex vivo and in isolated sub-populations of hair follicle-derived follicular melanocytes and keratinocytes and dermal papilla cells. To accomplish these goals we will use standard molecular, biochemical, and cell biology tools, which are currently available in our laboratory. As experimental models we will use epidermal and follicular keratinocytes and melanocytes as well as dermal papilla cells. The realization of the above aims should clarify the role of CRH and CRH receptors in the peripheral (skin) response to stress. The core of the project is the realization of the functional biochemical overlap between the cutaneous and hypothalamic CRH-activating systems. Whereas the central interactions between CRH together with CRH-Rs coordinates the behavioral, autonomic and endocrine responses to systemic stress, the present work will test whether that role extends to peripheral organs. Thus, we will test the effects of common stressors on expression of CRH and CRH-Rs, CRH effect on POMC expression. Moreover, we will characterize these CRH receptors and define their role in the regulation of phenotype in the most important components of the epidermis, e.g., melanocytes and keratinocytes, and test the role of CRH/CRH-R1 axis in the regulation of human hair growth.