Calcium-sensing Receptor and Keratinocyte Differentiation

描述(申请人提供)：表皮由多层角质形成细胞组成，角质形成细胞分化并产生渗透性屏障，保护皮肤免受环境伤害。细胞外钙(Ca2o)是启动角质形成细胞分化和维持表皮功能所必需的。Ca~(2+)浓度升高触发细胞内游离Ca~(2+)水平升高，并诱导细胞间黏附，这是促进角质形成细胞分化的两个关键信号事件。Ca~(2+)i水平的升高是由于细胞内库的Ca~(2+)释放和质膜通道内的Ca~(2+)内流所致。钙离子浓度升高还通过激活Rho A GTP酶和Src/Fyn酪氨酸激酶信号通路，诱导E-钙粘附素介导的细胞-细胞黏附。E-钙粘附素介导的细胞黏附募集并激活PI3K，PI3K是细胞存活和分化的重要调节因子。角质形成细胞中钙信号转导细胞反应的机制尚未明确。钙敏感受体(CAR)是一种G蛋白偶联受体，在角质形成细胞中表达。CAR不仅定位在细胞膜上检测钙离子的变化，而且与钙离子储存和储存操作通道(SOC)的调节物形成蛋白质复合体，包括IP3R、PLC31和高尔基体中的钙-ATPase SPCA1，高尔基体是角质形成细胞中主要的钙离子储存库。体外抑制CAR的表达可通过减少钙离子池显著抑制钙离子对钙离子的反应，并阻断E-钙粘附素介导的细胞黏附，导致细胞分化受损。CAR很可能通过与其他信号效应因子如G1、Rho鸟嘌呤核苷酸交换因子和丝氨酸相互作用来传递Ca2o信号，从而激活下游的细胞反应。为了确定CAR是否负责在体内通过角质形成细胞感知钙离子，我们通过Cre-lox重组产生了角质形成细胞特异的CAR基因敲除小鼠EpidCaR-/-。这些小鼠的表皮表现出钙梯度的丧失，板层小体和角化包膜的产生减少，分化标志物的表达减少，通透性屏障功能受损。这只小鼠的角质形成细胞也表现出对钙离子的异常反应和细胞与细胞间的黏附缺陷。这些数据有力地支持了CAR在表皮发育中的作用。我们将使用这个模型和成熟的细胞培养系统来解决这一假说，即CAR通过与调节钙离子储存和SOC的分子直接相互作用来调节钙离子信号，并通过Rho依赖的Src/Fyn信号级联通路激活E-钙粘素/PI3K通路来促进细胞与细胞的黏附，从而介导了钙离子诱导的角质形成细胞分化。我们提出了以下具体目标：(1)确定CAR在介导钙离子诱导的分化和调节钙离子储存中的作用；(2)确定CAR在调节E-钙粘素介导的细胞间黏附和PI3K活化中的作用；(3)确定CAR偶联蛋白G1、RhoGF和细丝蛋白A在钙离子诱导的细胞内钙离子动员、E-钙粘附素介导的细胞-细胞黏附和角质形成细胞分化中的作用。我们的研究将极大地促进我们对促进表皮发育的钙信号机制的了解，以及对表现为角质形成细胞分化异常的皮肤病的发病机制的理解。 与公共健康相关：CA2通过作用于钙敏感受体(CAR)，在控制皮肤细胞的生长、分化和屏障功能方面发挥关键作用。这项建议将研究钙如何启动皮肤细胞分化，以及CAR如何调节这一过程。该项目的成功完成将极大地提高我们对皮肤细胞分化的认识，并有助于了解各种表现为分化异常的皮肤病的发病机制。

DESCRIPTION (provided by applicant): Epidermis consists of multiple layers of keratinocytes, which differentiate and produce a permeability barrier that provides protection against environmental insults. Extracellular calcium (Ca2+o) is essential for initiating keratinocyte differentiation and maintaining epidermal functions. Elevating Ca2+o concentration triggers an increase in the level of intracellular free Ca2+ (Ca2+i) and induces cell-cell adhesion, two key signaling events promoting keratinocyte differentiation. The increased Ca2+i level is due to Ca2+ release from internal stores and Ca2+ influx through channels in the plasma membrane. Raising Ca2+o also induces E-cadherin-mediated cell-cell adhesion by activating Rho A GTPase and Src/Fyn tyrosine kinase signaling pathways. The E-cadherin-mediated cell adhesion recruits and activates PI3K, an important regulator for cell survival and differentiation. The mechanisms transducing Ca2+o signals to cellular responses in keratinocytes have not been defined. The Ca2+-sensing receptor (CaR), a G-protein-coupled receptor, is expressed in keratinocytes. The CaR not only localizes on the cell membrane to detect changes in Ca2+o, but also forms a protein complex with modulators of Ca2+i stores and store-operated channels (SOC), including IP3R, PLC31 and a Ca2+-ATPase SPCA1 in the Golgi, which is a major Ca2+I reservoir in keratinocytes. Inhibition of CaR expression in vitro markedly suppresses Ca2+i responses to Ca2+o by reducing Ca2+i pools and blocks E-cadherin-mediated cell adhesion, leading to impaired cell differentiation. It is likely that the CaR conveys Ca2+o signals to activate downstream cellular responses by interacting with other signaling effectors such as G1, Rho guanine nucleotide exchange factor (RhoGEF) and filamin. To determine whether the CaR is responsible for sensing Ca2+o by keratinocytes in vivo, we generated keratinocyte-specific CaR knockout mice,EpidCaR-/-, by Cre-lox recombination. The epidermis of these mice manifest a loss of Ca2+ gradient, decreased production of lamellar bodies and cornified envelope, reduced expression of differentiation markers, and impaired permeability barrier functions. Keratinocytes from this mouse also display abnormal Ca2+I responses to Ca2+o and defective cell-cell adhesion. These data strongly support a role for the CaR in epidermal development. We will use this model and a well-established cell culture system to address the Hypothesis that the CaR mediates Ca2+o-induced keratinocyte differentiation by modulating Ca2+i signaling through direct interactions with molecules regulating Ca2+i stores and SOCs, and by promoting cell-cell adhesion via the activation of E-cadherin/PI3K pathway through Rho-dependent Src/Fyn signaling cascade. We propose the following Specific Aims: (1) to determine the role of CaR in mediating Ca2+o-induced differentiation and in regulating Ca2+i stores; (2) to determine the role of CaR in regulating E-cadherin-mediated cell-cell adhesion and activation of PI3K; (3) to determine the role of CaR- coupling proteins G1, RhoGEF and filamin A in Ca2+o-induced Ca2+i mobilization, E-cadherin-mediated cell-cell adhesion and keratinocyte differentiation. Our studies will greatly advance our knowledge of the Ca2+ signaling mechanisms that promote epidermal development and understanding of pathogenesis of skin disorders manifesting abnormal keratinocyte differentiation. PUBLIC HEALTH RELEVANCE: Ca2+ plays a critical role in controlling the growth, differentiation, and barrier function in skin cells by acting on a calcium-sensing receptor (CaR). This proposal will investigate how Ca2+ initiates skin cell differentiation and how the CaR mediates this process. Successful completion of this project will greatly advance our knowledge of skin cell differentiation and help understanding the pathogenesis of various skin disorders manifesting abnormal differentiation.