Jaeger C, Schaefer B M, Wallich R, Kramer M D
Department of Dermatology, University of Heidelberg, Heidelberg, Germany.
J Invest Dermatol. 2000 Sep;115(3):375-80. doi: 10.1046/j.1523-1747.2000.00071.x.
In order to isolate genes that are upregulated in human keratinocytes upon loss of cell/matrix contact, a subtractive cDNA library was constructed from dispase-treated versus untreated keratinocytes. Among the cloned cDNAs one was pKe#192 having an open reading frame of 411 bp. By database analysis pKe#192 was found to be identical with the gene "MAP17" previously isolated from human kidney. Kyte-Doolittle hydrophobicity analyzes showed a hydrophobic amino terminus of 13 amino acids, a transmembrane region and a 61 amino acid hydrophilic carboxy-terminus and two potential phosphorylation sites. In order to study regulation of pKe#192/MAP17 expression, RNA was extracted from resting human keratinocytes and from keratinocytes stimulated by dispase-induced detachment from the growth substratum. Reverse transcription polymerase chain reaction did not reveal specific mRNA in resting keratinocytes, whereas mRNA was detectable after detachment. For further characterization poly- and monoclonal antibodies were generated against a recombinant fusion protein. Immunohistologic studies using the mono- and polyclonal antibodies showed staining of the upper layers of the stratum granulosum in normal human epidermis. The staining was colocalized with involucrin. Immunhistologic staining of frozen sections derived from lesional skin of bullous pemphigoid und pemphigus vulgaris indicated that pKe#192/MAP17 was upregulated in the epidermis adjacent to the blister. Taken together, the data demonstrate that pKe#192/MAP17 is expressed in keratinocytes and may be involved in epidermal physiology and pathology.
bullous diseases/differentiation.
为了分离在人角质形成细胞失去细胞/基质接触后上调的基因，构建了一个消减cDNA文库，该文库来自经dispase处理和未经处理的角质形成细胞。在克隆的cDNA中，有一个是pKe#192，其开放阅读框为411 bp。通过数据库分析发现，pKe#192与先前从人肾中分离出的“MAP17”基因相同。Kyte-Doolittle疏水性分析显示有一个13个氨基酸的疏水氨基末端、一个跨膜区域和一个61个氨基酸的亲水羧基末端以及两个潜在的磷酸化位点。为了研究pKe#192/MAP17表达的调控，从静止的人角质形成细胞以及因dispase诱导脱离生长基质而受到刺激的角质形成细胞中提取RNA。逆转录聚合酶链反应未在静止的角质形成细胞中检测到特异性mRNA，而在脱离后可检测到mRNA。为了进一步表征，针对重组融合蛋白产生了多克隆和单克隆抗体。使用单克隆和多克隆抗体进行的免疫组织学研究显示，正常人表皮颗粒层上层有染色。该染色与内披蛋白共定位。对大疱性类天疱疮和寻常型天疱疮皮损皮肤的冰冻切片进行免疫组织学染色表明，pKe#192/MAP17在水疱相邻的表皮中上调。综上所述，数据表明pKe#192/MAP17在角质形成细胞中表达，可能参与表皮生理和病理过程。
大疱性疾病/分化
