Calcium-sensing Receptor and Keratinocyte Differentiation

描述（由申请人提供）：表皮由多层角质形成细胞组成，这些细胞分化并产生渗透性屏障，提供对环境损害的保护。细胞外钙（Ca2+o）是启动角质细胞分化和维持表皮功能所必需的。升高Ca2+o浓度触发细胞内游离Ca2+ (Ca2+i)水平的增加并诱导细胞-细胞粘附，这是促进角质形成细胞分化的两个关键信号事件。Ca2+i水平的增加是由于Ca2+从内部储存释放和Ca2+通过质膜通道流入。升高Ca2+o也通过激活Rho A GTPase和Src/Fyn酪氨酸激酶信号通路诱导e -cadherin介导的细胞-细胞粘附。e -钙粘蛋白介导的细胞粘附招募并激活PI3K， PI3K是细胞存活和分化的重要调节因子。在角质形成细胞中，Ca2+o信号传导到细胞反应的机制尚未明确。Ca2+感应受体（CaR）是一种g蛋白偶联受体，在角质形成细胞中表达。CaR不仅定位于细胞膜上以检测Ca2+o的变化，而且还与Ca2+i储存和储存操作通道（SOC）的调节剂形成蛋白质复合物，包括IP3R， PLC31和高尔基体中的Ca2+- atp酶SPCA1，这是角化细胞中主要的Ca2+i储存库。体外抑制CaR表达，通过减少Ca2+i池，显著抑制Ca2+i对Ca2+o的反应，阻断e -cadherin介导的细胞粘附，导致细胞分化受损。CaR可能通过与其他信号效应物如G1、Rho鸟嘌呤核苷酸交换因子（RhoGEF）和丝蛋白相互作用，传递Ca2+o信号来激活下游细胞反应。为了确定CaR是否负责体内角质形成细胞感知Ca2+o，我们通过Cre-lox重组产生了角质形成细胞特异性CaR敲除小鼠EpidCaR-/-。这些小鼠的表皮表现为Ca2+梯度的丧失，板层体和锥形包膜的产生减少，分化标记物的表达减少，通透性屏障功能受损。来自该小鼠的角质形成细胞也表现出对Ca2+o的异常Ca2+I反应和细胞-细胞粘附缺陷。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一个完善的细胞培养系统来解决CaR介导Ca2+o诱导的角质细胞分化的假设，即通过与调节Ca2+i储存和soc的分子直接相互作用来调节Ca2+i信号，并通过rho依赖的Src/Fyn信号级联激活E-cadherin/PI3K途径来促进细胞间的粘附。我们提出以下具体目标：(1)确定CaR在介导Ca2+o诱导的分化和调节Ca2+i储存中的作用；(2)确定CaR在调节e -cadherin介导的细胞-细胞粘附和PI3K活化中的作用；(3)确定CaR偶联蛋白G1、RhoGEF和丝蛋白A在Ca2+o诱导的Ca2+i动员、e -cadherin介导的细胞-细胞粘附和角化细胞分化中的作用。我们的研究将极大地推进我们对促进表皮发育的Ca2+信号机制的认识，并了解皮肤疾病的发病机制，表现为角质细胞异常分化。

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.