Calcium-sensing Receptor and Keratinocyte Differentiation

钙敏感受体和角质形成细胞分化

• 批准号: 8230598
• 负责人: Chia-Ling Tu
• 金额: $ 32.01万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8230598
• 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; 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

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.

描述（由申请人提供）：表皮由多层角质形成细胞组成，可区分和产生渗透性屏障，从而保护环境损害。细胞外钙（Ca2+O）对于启动角质形成细胞分化和保持表皮功能至关重要。提高Ca2+O浓度会触发细胞内游离Ca2+（Ca2+I）水平的增加，并诱导细胞细胞粘附，这是两个关键的信号事件，促进了角质形成细胞分化。 Ca2+ I水平增加是由于内部存储中的Ca2+释放以及通过质膜中通道的Ca2+涌入所致。升高Ca2+O还通过激活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募集并激活PI3K，PI3K是细胞存活和分化的重要调节剂。尚未定义将Ca2+O信号转换为细胞反应的机制。 Ca2+传感受体（CAR）是一种G蛋白偶联受体，在角质形成细胞中表达。该汽车不仅将其定位在细胞膜上，以检测Ca2+O的变化，而且还形成具有CA2+I商店和商店操作通道的调节剂（包括IP3R，PLC31和GOLGI的CA2+-ATPase SPCA1）的蛋白质复合物，在Golgi中是主要的Ca2+I reservoir Inservoir In Keratinocilocystinocystinocystinocy。通过减少Ca2+I池并阻止E-钙粘着蛋白介导的细胞粘附，可以显着抑制体外CAR表达对Ca2+I对Ca2+O的反应，从而导致细胞分化受损。该汽车可能通过与其他信号效应子（例如G1，Rho Guanine核苷酸交换因子（RhoGEF）和Filamin）相互作用来传达CA2+O信号激活下游细胞反应。为了确定汽车是否负责通过体内角质形成细胞感测Ca2+O，我们通过Cre-lox重组产生了角质形成细胞特异性的汽车敲除小鼠Epidcar - / - 。这些小鼠的表皮表现出Ca2+梯度的损失，层状体的产生和晶状包膜的产生降低，分化标记的表达降低以及渗透性屏障功能受损。该小鼠的角质形成细胞还显示出异常的Ca2+I对Ca2+O和缺陷细胞细胞粘附的反应。这些数据强烈支持汽车在表皮开发中的作用。我们将使用该模型和一个完善的细胞培养系统来解决以下假设：CAR通过与调节CA2+I商店和SOC的分子进行直接相互作用来调节Ca2+O诱导的角质细胞分化，并通过通过E-Cadhery/PI3K的活性来促进细胞粘附来调节CA2+I商店和SOC的分子，从而介导CA2+I信号传导。级联。我们提出以下特定目的：（1）确定CAR在介导Ca2+O诱导的分化和调节Ca2+I商店中的作用； （2）确定CAR在调节E-钙粘蛋白介导的细胞粘附和PI3K激活中的作用； （3）确定CA2+O- O诱导的Ca2+I动员，E-钙粘蛋白介导的细胞细胞粘附和角质形成细胞分化中CA2+O诱导的Ca2+I在Ca2+O-诱导的Ca2+I中的作用。我们的研究将大大提高我们对CA2+信号传导机制的了解，这些机制促进表皮发育和对表现异常角质形成细胞分化的皮肤疾病发病机理的理解。 公共卫生相关性：CA2+通过作用于钙感密度受体（CAR）来控制皮肤细胞的生长，分化和屏障功能至关重要。该建议将研究CA2+如何启动皮肤细胞分化以及汽车如何介导该过程。成功完成该项目将大大提高我们对皮肤细胞分化的了解，并有助于理解表现出异常分化的各种皮肤疾病的发病机理。