Differentially expressed genes in the lens of mimecan-null mice.
Differentially expressed genes in the lens of mimecan-null mice.
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发表时间：
2004-06
期刊：
影响因子：
2.2
通讯作者：
E. Tasheva;An Ke;Youping Deng;C. Jun;L. Takemoto;A. Koester;G. Conrad
中科院分区：
文献类型：
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作者：
E. Tasheva;An Ke;Youping Deng;C. Jun;L. Takemoto;A. Koester;G. Conrad
目的富含亮氨酸的小蛋白聚糖 (SLRP) 基因家族的成员对于各种结缔组织中正常胶原纤维生成至关重要，也是细胞生长、分化和组织修复的重要调节因子。 Mimecan 是该基因家族的成员，在许多结缔组织中表达。我们之前曾报道过，小鼠 mimecan 基因的敲除会导致胶原纤维生成异常，主要发生在角膜和皮肤中。在我们研究 mimecan 在眼睛中的生物学作用的过程中，我们发现该基因在小鼠晶状体中表达。在这里，我们试图确定晶状体中与 mimecan 缺失相关的基因表达变化。方法 采用逆转录聚合酶链式反应扩增 (RT-PCR)、原位杂交 (ISH) 和免疫组织化学 (IHC) 测定人和小鼠眼中 mimecan 的表达。微阵列杂交用于确定从mimecan缺失小鼠和野生型小鼠中分离的晶状体之间的基因表达差异。使用相对定量 RT-PCR 来验证已识别基因的子集的表达水平。结果通过ISH和IHC，在胚胎第16.5天（E16.5）和出生后第10天（P10）小鼠眼的角膜和晶状体中检测到mimecan mRNA。通过 RT-PCR，在人角膜、晶状体、虹膜和视网膜中检测到 mimecan mRNA。在没有 mimecan 的小鼠晶状体中，对 5,002 个小鼠基因的微阵列分析表明，65 个基因的表达增加了两倍以上，76 个基因的表达减少了两倍以上。表达增加的基因包括细胞粘附分子、G蛋白偶联受体、细胞内信号分子、参与蛋白质生物合成和降解的基因以及参与免疫功能的基因。细胞外基质分子、钙结合和转运蛋白以及已知其在调节细胞运动中的作用的基因的表达减少。有趣的是，观察到两个 SLRP 家族成员（双糖链蛋白聚糖和软骨粘蛋白）以及几种应激反应蛋白（包括 γA-晶状体蛋白、血红蛋白 α 1 和金属硫蛋白 1）的基因表达降低。定量 RT-PCR 证实了从阵列中选择的 12 个基因的表达变化。结论 在本报告中，我们首次证明 mimecan 在脊椎动物晶状体中组成型表达。基因表达谱的结果揭示了 mimecan 影响双糖链蛋白聚糖和软骨粘附素表达的能力，从而表明这些 SLRP 家族成员之间可能存在新的调控相互作用。与 mimecan 一样，软骨素在脊椎动物晶状体中的表达此前尚未有报道。我们的结果提供了对 mimecan 在晶状体中的功能的深入了解，并能够进一步表征该蛋白质发挥其生物学作用的分子机制。
PURPOSE Members of the small leucine-rich proteoglycans (SLRP) gene family are essential for normal collagen fibrillogenesis in various connective tissues and important regulators of cellular growth, differentiation, and tissue repair. Mimecan is a member of this gene family and is expressed in many connective tissues. We have previously reported that knockout of the mouse mimecan gene results in abnormal collagen fibrillogenesis, mainly in the cornea and skin. During the course of our studies on biological roles of mimecan in the eye, we found that this gene is expressed in the mouse lens. Here, we sought to identify gene expression changes in the lens that are associated with the absence of mimecan. METHODS Reverse transcription-polymerase chain reaction amplification (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC) were used to determine mimecan expression in human and mouse eyes. Microarray hybridization was used to determine gene expression differences between lenses isolated from mimecan-null and wild type mice. Relative quantitative RT-PCR was used to verify the expression levels of a subset of the identified genes. RESULTS By ISH and IHC, mimecan mRNA was detected in cornea and lens at embryonic day 16.5 (E16.5) and postnatal day 10 (P10) mouse eyes. By RT-PCR, mimecan mRNA was detected in human cornea, lens, iris, and retina. In mimecan-null mice lenses, microarray analysis of 5,002 mouse genes demonstrated a more than two fold increase in expression of 65 genes and a more than two fold decrease in expression of 76 genes. Among genes with increased expression were cell adhesion molecules, G-protein coupled receptors, intracellular signaling molecules, genes involved in protein biosynthesis and degradation, and genes involved in immune function. Decreased expression was found in extracellular matrix molecules, calcium binding and transporting proteins, and genes known for their roles in regulating cellular motility. Intriguingly, decreased gene expression was observed with two SLRP family members, biglycan and condroadherin, as well as with several stress-response proteins, including gammaA-crystallin, hemoglobin alpha 1, and metallothionein 1. Quantitative RT-PCR confirmed changes in expression of 12 genes selected from the arrays. CONCLUSIONS In this report we present the first demonstration that mimecan is constitutively expressed in the vertebrate lens. The results from gene expression profiling reveal the ability of mimecan to influence expression of biglycan and chondroadherin, thereby indicating possible novel regulatory interactions between these SLRP family members. As with mimecan, the expression of chondroadrein in vertebrate lens has not been reported previously. Our results provide insight into the function of mimecan in the lens and enable further characterization of molecular mechanisms by which this protein exerts its biological roles.