CRYPTDIN EFFECTS ON CRYPT EPITHELIA

描述（申请人摘要）：本提案的目标 目的是阐明crytpdin诱导的隐窝通道形成的生物学机制 上皮细胞Cryptdins是肠道（潘氏细胞）特异性α-防御素- 阳离子肽首先被认为是中性粒细胞的产物， 抗菌性能。十九种穴蛋白同种型已在文献中描述。 小鼠和两个，HD-5和HD-6，在人类。的抗微生物作用 α-防御素被认为是存在于它们的能力， 生物膜，并产生阴离子传导孔。还有HD-5和HD-6 已知从潘氏细胞释放到隐窝腔中， 隐窝细胞类型，主要的是分泌氯化物的"未分化" 隐窝细胞天然暴露于这些通道形成肽。pi和 他的合作者最近发现，cryptdins 2和3诱导了一种 人肠T84细胞的生理性氯分泌反应可能通过 在顶端（内腔）膜中形成阴离子传导通道。研究 提出的将测试假设潘氏细胞衍生的cryptdin的行为， 通过旁分泌插入调节肠隐窝的生理学 （自组装）氯离子传导孔进入顶端膜 邻近的隐窝上皮细胞和通过内吞作用下调， 根尖膜恢复体外实验将通过以下方式定义该机制： 其中小鼠穴蛋白2和3诱导氯化物分泌。PI和他的 合作者将使用matrilysin-/-小鼠和CR2-tox176小鼠来阐明 Cryptdins对离体和体内小肠功能的影响。的 Cryptdin 3、HD-5和HD-6的生物物理性质和结构功能 将在完整和半透化T84和肾HEK细胞模型中进行检查 系统和单通道膜片钳。最后，研究建议， 阐明cryptdin 3诱导的通道形成的细胞机制， 下调。这些研究的重要性是强调， 越来越多的证据表明肠隐窝在 肠上皮细胞个体发育，生理学（溶质和水运输）， 以及特异性和非特异性粘膜防御。Cryptdins以旁分泌方式发挥作用 调节隐窝细胞功能的时尚并且似乎有一种独特的机制 的行动。拟议的研究将阐明这些新的生物学 α-防御素，并可能为α-防御素的未来发展奠定基础 抑制剂或这些（或相关肽）在治疗疾病中的用途， 肠或呼吸道的粘膜表面。

DESCRIPTION ( applicant's abstract): The goal of this proposal is to elucidate the biology of crytpdin-induced channel formation in crypt epithelia. Cryptdins are intestinal (Paneth cell) specific alpha-defensins - cationic peptides first recognized as neutrophil products, which exhibit potent antimicrobial properties. Nineteen cryptdin isoforms have been described in the mouse and two, HD-5 and HD-6, in the human. The antimicrobial action of alpha-defensins is thought to reside in their ability to partition into biomembranes and produce anion conductive pores. Cryptdins, and HD-5 and HD-6 are known to be released from Paneth cells into the crypt lumen and thus other crypt cell types, the major one being the chloride secreting "undifferentiated" crypt cell, are naturally exposed to these channel forming peptides. The PI and his collaborators have recently shown the cryptdins 2 and 3 induce a physiologic chloride secretory response in human intestinal T84 cells likely by forming anion conductive channels in the apical (lumenal) membrane. Studies proposed will test the hypothesis that Paneth cell-derived cryptdins act to regulate the physiology of the intestinal crypt by paracrine insertion (self-assembly) of chloride conducting pores into apical membranes of neighboring crypt epithelial cells and down regulation by endocytosis and apical membrane restitution. In vitro experiments will define the mechanism by which mouse cryptdins 2 and 3 induce chloride secretion. The PI and his collaborators will use matrilysin -/- mouse and CR2-tox176 mouse to elucidate the effects of cryptdins on small intestinal functions ex vivo and in vivo. The biophysical properties and structure function of cryptdin 3 and HD-5 and HD-6 will be examined in intact and semi-permeabilized T84 and renal HEK cell model systems and by single channel patch clamp. Finally studies are proposed to elucidate the cellular mechanism(s) of cryptdin 3-induced channel formation and down regulation. The significance of these studies is emphasized by accumulating evidence that the intestinal crypt plays a central role in intestinal epithelial cell ontogeny, physiology (solute and water transport), and specific and non-specific mucosal defense. Cryptdins act in paracrine fashion to regulate crypt cell function and appear to have a unique mechanism of action. Proposed studies will elucidate the biology of these novel alpha-defensins and may set the stage for future development of alpha-defensin inhibitors or for use of these (or related peptides) in treatment of disease at the mucosal surfaces of the intestine or respiratory tract.