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，这些角质形成细胞分化并产生渗透性屏障，提供针对环境侵害的保护。细胞外钙（Ca2O）对于启动角质形成细胞分化和维持表皮功能至关重要。提高 Ca2O 浓度会引发细胞内游离 Ca2 (Ca2i) 水平的增加并诱导细胞间粘附，这是促进角质形成细胞分化的两个关键信号传导事件。 Ca2i 水平增加是由于内部储存的 Ca2 释放和 Ca2 通过质膜通道流入。提高 Ca20 还可以通过激活 Rho A GTPase 和 Src/Fyn 酪氨酸激酶信号通路来诱导 E-钙粘蛋白介导的细胞粘附。 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。将 Ca2O 信号转导至角质形成细胞中的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 不仅定位在细胞膜上以检测 Ca2O 的变化，而且还与 Ca2i 储存和储存操纵通道 (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，高尔基体是角质形成细胞中主要的 Ca2I 储存库。体外抑制 CaR 表达可通过减少 Ca2i 池并阻断 E-钙粘蛋白介导的细胞粘附来显着抑制 Ca2i 对 Ca2o 的反应，从而导致细胞分化受损。 CaR很可能通过与G1、Rho鸟嘌呤核苷酸交换因子(RhoGEF)和细丝蛋白等其他信号传导效应物相互作用来传递Ca2O信号以激活下游细胞反应。为了确定CaR是否负责体内角质形成细胞感知Ca2O，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出Ca2梯度丧失、层状体和角化包膜的产生减少、分化标记物的表达减少以及通透性屏障功能受损。该小鼠的角质形成细胞还表现出对 Ca2O 的异常 Ca2I 反应和细胞间粘附缺陷。这些数据有力地支持了 CaR 在表皮发育中的作用。我们将使用该模型和完善的细胞培养系统来解决以下假设：CaR 通过与调节 Ca2i 储存和 SOC 的分子直接相互作用来调节 Ca2i 信号传导，并通过 Rho 依赖性 Src/Fyn 信号级联激活 E-c​​adherin/PI3K 途径来促进细胞与细胞粘附，从而介导 Ca2o 诱导的角质形成细胞分化。我们提出以下具体目标：（1）确定CaR在介导Ca2O诱导分化和调节Ca2i储存中的作用； (2)确定CaR在调节E-cadherin介导的细胞间粘附和PI3K激活中的作用； (3)确定CaR-偶联蛋白G1、RhoGEF和Filamin A在Ca2O诱导的Ca2i动员、E-钙粘蛋白介导的细胞间粘附和角质形成细胞分化中的作用。我们的研究将极大地增进我们对促进表皮发育的 Ca2 信号传导机制的了解，以及对表现为角质形成细胞分化异常的皮肤疾病发病机制的了解。

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.