眼睫状体上皮中的类视黄醇处理蛋白。

Retinoid processing proteins in the ocular ciliary epithelium.

作者信息

Salvador-Silva Mercedes, Ghosh Sikha, Bertazolli-Filho Rubens, Boatright Jeffrey H, Nickerson John M, Garwin Gregory G, Saari John C, Coca-Prados Miguel

机构信息

Department of Ophthalmology and Visual Science, Yale University, New Haven, CT 06510, USA.

出版信息

Mol Vis. 2005 May 18;11:356-65.

PMID:15928609

Abstract

PURPOSE

To identify retinoids and retinoid processing proteins in the ocular ciliary epithelium (CE), and to compare in cultured ciliary epithelial cell lines promoter activities of the cellular retinaldehyde binding protein (CRALBP) and interphotoreceptor retinoid binding protein (IRBP).

METHODS

Retinoid processing proteins were detected by RT-PCR, western analysis and immunocytochemistry. PCR products were verified by DNA sequence analysis. Retinoids were measured by normal phase HPLC and UV visible spectroscopy. Reporter product from CRALBP and IRBP promoter fragments was measured following transient transfection in bovine pigmented and nonpigmented CE cells.

RESULTS

CRALBP, IRBP, cellular retinol binding protein (CRBP), 11-cis-retinol dehydrogenase (11-cis-RDH), lecithin:retinol acyltransferase (LRAT), and ATP binding cassette receptor (ABCR) were detected in human CE tissue by RT-PCR. Retinal pigment epithelium specific protein 65 kDa (RPE65) mRNA and protein were also detected. CRALBP and IRBP were detected by western analysis in tissue extracts from bovine CE and were localized to the PE and NPE cell layers, respectively, by immunocytochemistry. IRBP immunoreactivity was also detected in aqueous humor. Retinoids identified in the bovine CE include retinyl esters (7.4+/-3.5 pMol/mg of protein) and all-trans-retinol (14.9+/-1.1 pMol/mg of protein). Betacarotene was also tentatively identified. 11-cis-Retinoids were not detected. In CE cell cultures, the CRALBP p2.1-kb promoter construct exhibited reporter activity 15-30 fold above basal level, with 2 fold more activity in pigmented than nonpigmented CE cells. IRBP promoter constructs exhibited low level reporter activities in vitro in both CE cell layers.

CONCLUSIONS

The ocular CE expresses genes encoding components of the rod visual cycle. The differential localization of CRALBP and IRBP along the bilayer of the CE suggests a potential role in retinoid transport and/or retinoid metabolism. However, the absence of 11-cis-retinoids suggests that the function of retinoid processing proteins in the CE differs from that of the retina.

摘要

目的

鉴定眼睫状体上皮（CE）中的类视黄醇及类视黄醇加工蛋白，并比较培养的睫状体上皮细胞系中细胞视黄醛结合蛋白（CRALBP）和光感受器间类视黄醇结合蛋白（IRBP）的启动子活性。

方法

通过逆转录聚合酶链反应（RT-PCR）、蛋白质印迹分析和免疫细胞化学检测类视黄醇加工蛋白。PCR产物经DNA序列分析验证。通过正相高效液相色谱法和紫外可见光谱法测定类视黄醇。在牛色素性和非色素性CE细胞中进行瞬时转染后，测量CRALBP和IRBP启动子片段的报告基因产物。

结果

通过RT-PCR在人CE组织中检测到CRALBP、IRBP、细胞视黄醇结合蛋白（CRBP）、11-顺式视黄醇脱氢酶（11-cis-RDH）、卵磷脂：视黄醇酰基转移酶（LRAT）和ATP结合盒受体（ABCR）。还检测到视网膜色素上皮特异性65 kDa蛋白（RPE65）的mRNA和蛋白。通过蛋白质印迹分析在牛CE组织提取物中检测到CRALBP和IRBP，通过免疫细胞化学分别将其定位到色素上皮（PE）和非色素上皮（NPE）细胞层。在房水中也检测到IRBP免疫反应性。在牛CE中鉴定出的类视黄醇包括视黄酯（7.4±3.5 pMol/mg蛋白）和全反式视黄醇（14.9±1.1 pMol/mg蛋白）。还初步鉴定出β-胡萝卜素。未检测到11-顺式类视黄醇。在CE细胞培养物中，CRALBP的2.1 kb启动子构建体表现出比基础水平高15 - 30倍的报告基因活性，在色素性CE细胞中的活性比非色素性CE细胞高2倍。IRBP启动子构建体在体外两种CE细胞层中均表现出低水平的报告基因活性。