Presence and distribution of 14-3-3 proteins in human ocular surface tissues
Presence and distribution of 14-3-3 proteins in human ocular surface tissues
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发表时间：
2008-12
期刊：
影响因子：
2.2
通讯作者：
J. Shankardas;M. Senchyna;S. Dimitrijevich
中科院分区：
文献类型：
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作者：
J. Shankardas;M. Senchyna;S. Dimitrijevich
目的14-3-3是一个高度保守、广泛表达的蛋白质家族。已知至少七种哺乳动物同种型（β、ε、γ、η、θ、σ和η）。这些蛋白质与超过200种不同的靶分子结合，并激活参与代谢、细胞周期、凋亡、蛋白质运输、转录、应激反应和恶性转化调节的几个下游信号级联。我们感兴趣的是这些蛋白质在调节人体眼表稳态和病理机制中的作用。因此，我们的目的是确定14-3-3蛋白在人角膜、结膜和包括这些组织的原代细胞中的表达。方法采用免疫荧光法检测14-3-3 β、ε、γ、η、θ、σ和γ在人角膜和结膜石蜡切片中的表达。使用间接免疫荧光和蛋白质印迹分析，我们还确定了这些异构体在原代角膜上皮细胞，角膜细胞，内皮细胞和原代结膜上皮细胞的表达。这些亚型在原代上皮细胞和内皮细胞中的表达与在几种角膜细胞系中的相同表达进行了比较。Western印迹分析用于确定来自角膜上皮细胞、细胞系和泪液的培养基中14-3-3同种型的存在。结果14-3-3亚型在角膜、结膜上皮、原代上皮细胞和细胞系中均有表达。14-3-3 σ的表达局限于上皮细胞，并分泌到原代细胞和细胞系的培养基中。我们还首次报告了两种分泌亚型14-3-3 γ和ζ也存在于人类泪液中。结论我们已经确定所有哺乳动物14-3-3异构体在人角膜、结膜和组成细胞中表达，并且发现14-3-3 σ异构体是上皮细胞特异性的。我们认为14-3-3 σ在细胞内和细胞外的存在表明它参与了上皮特异性信号通路。
Purpose 14–3-3 is a highly conserved, ubiquitously expressed family of proteins. At least seven mammalian isoforms (β, ε, γ, η, θ, σ, and ζ) are known. These proteins associate with over 200 different target molecules and activate several downstream signaling cascades involved in the regulation of metabolism, cell cycle, apoptosis, protein trafficking, transcription, stress responses, and malignant transformations. We are interested in the role of these proteins in the mechanisms regulating homeostasis and the pathologies of the human ocular surface. Therefore, our purpose is to determine the expression of the 14–3-3 proteins in the human cornea, the conjunctiva, and the primary cells comprising these tissues. Methods Using immunofluorescence, we determined the expression of 14–3-3 β, ε, γ, η, θ, σ, and ζ in paraffin sections of the human cornea and conjunctiva. Using indirect immunofluorescence and western blot analysis, we also determined the expression of these isoforms in primary corneal epithelial cells, keratocytes, endothelial cells, and primary conjunctival epithelial cells. The expressions of these isoforms in primary epithelial and endothelial cells were compared with the same expressions in several corneal cell lines. Western blot analysis was used to determine the presence of 14–3-3 isoforms in the culture medium from corneal epithelial cells, cell lines, and the tear fluid. Results All the 14–3-3 isoforms were expressed in the corneal and conjunctival epithelia as well as primary epithelial cells and cell lines. Expression of 14–3-3 σ was confined to epithelial cells and was secreted into the culture medium of primary cells and cell lines. We also report for the first time that two of the secreted isoforms, 14–3-3 γ and ζ, are also present in the human tear fluid. Conclusions We have determined that all the mammalian 14–3-3 isoforms are expressed in the human cornea, conjunctiva, and the component cells and that the 14–3-3 σ isoform was found to be epithelial cell specific. We propose that the intracellular and extracellular presence of 14–3-3 σ suggest its involvement in the epithelia specific signaling pathways.