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Calcium-sensing Receptor and Keratinocyte Differentiation

钙敏感受体和角质形成细胞分化

基本信息

批准号：
8619584
负责人：
Chia-Ling Tu
金额：
$ 31.37万
依托单位：
NORTHERN CALIFORNIA INSTITUTE/RES/EDU
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8619584
关键词：
1-Phosphatidylinositol 3-Kinase Abnormal Keratinocyte Acute Address Adherens Junction Adhesions Agonist Apoptosis Binding Ca(2+)-Transporting ATPase Cadherins Calcium Calcium-Sensing Receptors Cell Adhesion Cell Culture System Cell Differentiation process Cell Survival Cell membrane Cell-Cell Adhesion Cells Complex Cyclic AMP Cytoskeletal Proteins Cytosol Data Development Differentiation Antigens Differentiation and Growth E-Cadherin Endoplasmic Reticulum Epidermis Event Family member G-Protein-Coupled Receptors GTP-Binding Proteins Gene Expression Genetic Recombination Golgi Apparatus Guanine Nucleotide Exchange Factors Guanosine Triphosphate Phosphohydrolases ITPR1 gene In Vitro Inositol Intercellular Junctions Knock-out Knockout Mice Knowledge Length Mediating Mediator of activation protein Membrane Microfilaments Modeling Mus Organelles Pathogenesis Pathway interactions Permeability Phosphatidylinositol 4,5-Diphosphate Phosphatidylinositols Phospholipase C Play Process Production Protein Subunits Protein Tyrosine Kinase Proteins Proto-Oncogene Proteins c-fyn Recruitment Activity Regulation Rho-associated kinase Role Scaffolding Protein Signal Pathway Signal Transduction Skin Stress System Tyrosine Phosphorylation Work alveolar lamellar body base cell type extracellular filamin in vivo keratinocyte keratinocyte differentiation phosphatidylinositol 3,4,5-triphosphate protein complex public health relevance receptor receptor coupling receptor expression response rho rho GTP-Binding Proteins rho guanine nucleotide exchange factor p115 skin disorder src-Family Kinases uptake

项目摘要

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.

描述（由申请人提供）：表皮由多层角质形成细胞组成，其分化并产生渗透性屏障，提供对环境损害的保护。细胞外钙（Ca 2 + 0）对于启动角质形成细胞分化和维持表皮功能是必不可少的。升高的Ca 2 + 0浓度触发细胞内游离Ca 2+（Ca 2 + 1）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传导事件。Ca ~（2+）水平的增加是由于Ca ~（2+）从内部储存释放和Ca ~（2+）通过质膜通道内流。升高Ca 2 + 0还通过激活Rho A GT3和Src/Fyn酪氨酸激酶信号传导途径诱导E-钙粘蛋白介导的细胞-细胞粘附。E-钙粘蛋白介导的细胞粘附招募并激活PI 3 K，PI 3 K是细胞生存和分化的重要调节因子。在角质形成细胞中将Ca 2 + 0信号转导至细胞应答的机制尚未确定。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不仅定位于细胞膜上以检测Ca 2 + i的变化，而且还与Ca 2 +i储存和储存操纵通道（SOC）的调节剂形成蛋白复合物，包括高尔基体中的IP 3R、PLC 31和Ca 2 +-ATP酶SPCA 1，高尔基体是角质形成细胞中的主要Ca 2 +I储存库。在体外抑制CaR表达通过减少Ca 2 +i池显著抑制Ca 2 +i对Ca 2 +o的反应，并阻断E-钙粘蛋白介导的细胞粘附，导致受损的细胞分化。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）和细丝蛋白相互作用来传递Ca 2 +o信号以激活下游细胞反应。为了确定CaR是否负责在体内通过角质形成细胞感测Ca 2 + 0，我们通过Cre-lox重组产生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Ca 2+梯度的损失，板层体和皮质包膜的产生减少，分化标志物的表达减少，以及渗透屏障功能受损。来自该小鼠的角质形成细胞也显示出对Ca 2 + 0的异常Ca 2 +I反应和有缺陷的细胞-细胞粘附。这些数据有力地支持了钙调素受体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的假设，即CaR介导的钙诱导角质形成细胞分化通过调节Ca 2 +i信号通过直接相互作用的分子调节Ca 2 +i商店和SOC，并通过激活E-cadherin/PI 3 K途径通过Rho依赖的Src/Fyn信号级联促进细胞-细胞粘附。本研究的主要目的是：（1）研究CaR在介导Ca ~（2+）诱导的分化和调节Ca ~（2+）储备中的作用，（2）研究CaR在调节E-cadherin介导的细胞-细胞粘附和PI 3 K激活中的作用，（3）研究CaR在细胞内CaR的表达和调节细胞内CaR的表达和调节细胞内CaR的变化。（3）探讨CaR偶联蛋白G1、RhoGEF和细丝蛋白A在Ca ~（2+）诱导的Ca ~（2+）动员中的作用。钙黏蛋白介导的细胞间黏附与角质形成细胞分化。我们的研究将大大推进我们的知识的钙离子信号机制，促进表皮发育和理解的发病机制的皮肤疾病表现出异常角质形成细胞分化。