Jin Chang, Yin Furong, Lin Mimi, Li Hongxia, Wang Zhenlian, Weng Jinsheng, Liu Mingyao, Da Dong Xiang, Qu Jia, Tu LiLi
School of Ophthalmology and Optometry, Eye Hospital, Wenzhou Medical College, Wenzhou, Zhejiang, PR China.
Invest Ophthalmol Vis Sci. 2008 Oct;49(10):4245-53. doi: 10.1167/iovs.08-1860. Epub 2008 May 16.
Eyelid development is a dynamic process involving cell proliferation, differentiation, and migration regulated by a number of growth factors and cytokines. Mice deficient in the orphan G protein-coupled receptor 48 (GPR48) showed an eye open at birth (EOB) phenotype. In this study, the authors attempted to clarify the role of GPR48 in eyelid development and the molecular mechanisms leading to the EOB phenotype.
Phenotypic analysis of the eyelids of Gpr48(-/-) mice was carried out using histology and scanning electron microscopy. GPR48 expression pattern was determined using X-gal staining. In vitro scratch assay was used to determine cell motility defects in Gpr48(-)(/)(-) keratinocytes. The molecular mechanism underlying GPR48-mediated eyelid closure was explored using Western blot and immunostaining analyses. Expression levels of EGFR and its phosphorylated counterpart were examined in Gpr48(-/-) and wild-type keratinocytes and in eyelids.
GPR48 is highly expressed in the epithelium and apical mesenchymal cells of eyelids during embryonic development. Detailed analysis revealed that Gpr48(-/-) mice exhibited delayed leading-edge extension, reduced filopodia formation, and decreased rounded periderm cell formation around eyelid margins. Keratinocytes lacking GPR48 are defective in cell proliferation and migration with reduced F-actin staining. In addition, the phosphorylation of EGFR was dramatically decreased in cultured keratinocytes and developing eyelids in the absence of GPR48.
Inactivation of GPR48 induces the EOB phenotype by reducing epithelial cell proliferation and migration, indicating that GPR48 plays an essential role in eyelid development. Furthermore, GPR48 contributes to eyelid development through the regulation of the EGFR signaling pathway.
眼睑发育是一个动态过程，涉及细胞增殖、分化和迁移，受多种生长因子和细胞因子调控。缺乏孤儿G蛋白偶联受体48（GPR48）的小鼠表现出出生时睁眼（EOB）的表型。在本研究中，作者试图阐明GPR48在眼睑发育中的作用以及导致EOB表型的分子机制。
使用组织学和扫描电子显微镜对Gpr48(-/-)小鼠的眼睑进行表型分析。使用X-gal染色确定GPR48的表达模式。体外划痕试验用于确定Gpr48(-)(/)(-)角质形成细胞的细胞运动缺陷。使用蛋白质免疫印迹和免疫染色分析探索GPR48介导的眼睑闭合的分子机制。在Gpr48(-/-)和野生型角质形成细胞以及眼睑中检测表皮生长因子受体（EGFR）及其磷酸化对应物的表达水平。
在胚胎发育过程中，GPR48在眼睑的上皮细胞和顶端间充质细胞中高度表达。详细分析显示，Gpr48(-/-)小鼠表现出前沿延伸延迟、丝状伪足形成减少以及眼睑边缘周围圆形周皮细胞形成减少。缺乏GPR48的角质形成细胞在细胞增殖和迁移方面存在缺陷，F-肌动蛋白染色减少。此外，在缺乏GPR48的情况下，培养的角质形成细胞和发育中的眼睑中EGFR的磷酸化显著降低。
GPR48的失活通过减少上皮细胞增殖和迁移诱导EOB表型，表明GPR48在眼睑发育中起重要作用。此外，GPR48通过调节EGFR信号通路促进眼睑发育。
