Bonkobara M, Das A, Takao J, Cruz P D, Ariizumi K
Department of Dermatology, The University of Texas South-western Medical Center and Dermatology Section (Medical Service), Dallas Veterans Affairs Medical Center, Dallas, TX, USA.
Br J Dermatol. 2003 Apr;148(4):654-64. doi: 10.1046/j.1365-2133.2003.05244.x.
Both intercellular and intracellular signals are transduced primarily by interactions of secreted and/or membrane-anchored polypeptides, and they play a pivotal role in regulating proliferation, differentiation and apoptosis of keratinocytes within the epidermis. Despite recent identification of these polypeptides, it is likely that several important molecules remain undisclosed.
To identify novel genes encoding secreted or membrane-anchored polypeptides expressed by human keratinocytes.
We employed a signal sequence (SS) trap of a 5'-end-enriched cDNA library prepared from primary cultured human keratinocytes. Gene expression analysis was performed using Northern blotting. R Screening of 4018 cDNA clones yielded 82 positive clones (57 independent genes), most of which encoded SSs in their N-termini. Most of the positive clones were known genes registered in the GenBank database. Seven genes were identified in the EST database, four of which encoded novel membrane-anchored polypeptides with features of type I transmembrane proteins; the other three genes encoded novel non-type I transmembrane polypeptides. These EST genes were expressed differentially by keratinocytes subjected to low vs. high calcium concentrations and by basal vs. squamous cell carcinomas.
Using the SS trap, we isolated many genes known to be involved in constituting epidermal structures and others that had not previously been associated with keratinocytes. In addition, we identified novel genes (EST genes) that differ in kinetics of gene expression in keratinocyte differentiation. Our results validate the effective use of this SS trap method for identifying secreted and membrane-anchored polypeptides expressed by human keratinocytes. The identification will better illuminate the molecular mechanisms responsible for co-ordinated regulation of epidermal homeostasis.
细胞间和细胞内信号主要通过分泌型和/或膜锚定多肽的相互作用进行转导，它们在调节表皮内角质形成细胞的增殖、分化和凋亡中起关键作用。尽管最近已鉴定出这些多肽，但仍可能有一些重要分子未被发现。
鉴定编码人角质形成细胞表达的分泌型或膜锚定多肽的新基因。
我们采用了从原代培养的人角质形成细胞制备的5'端富集cDNA文库的信号序列（SS）捕获法。使用Northern印迹进行基因表达分析。对4018个cDNA克隆进行筛选，得到82个阳性克隆（57个独立基因），其中大多数在其N端编码信号序列。大多数阳性克隆是GenBank数据库中登记的已知基因。在EST数据库中鉴定出7个基因，其中4个编码具有I型跨膜蛋白特征的新型膜锚定多肽；另外3个基因编码新型非I型跨膜多肽。这些EST基因在低钙与高钙浓度处理的角质形成细胞以及基底细胞癌与鳞状细胞癌中差异表达。
利用SS捕获法，我们分离出许多已知参与构成表皮结构的基因以及其他先前与角质形成细胞无关的基因。此外，我们鉴定出在角质形成细胞分化过程中基因表达动力学不同的新基因（EST基因）。我们的结果证实了这种SS捕获法可有效用于鉴定人角质形成细胞表达的分泌型和膜锚定多肽。这些鉴定将更好地阐明负责协调调节表皮稳态的分子机制。
