两种机制调节角质形成细胞中角蛋白 K15 的表达：PKC/AP-1 和 FOXM1 介导的信号通路的作用。
Two mechanisms regulate keratin K15 expression in keratinocytes: role of PKC/AP-1 and FOXM1 mediated signalling.
机构信息
Centre for Clinical and Diagnostic Oral Sciences, Institute of Dentistry, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.
出版信息
PLoS One. 2012;7(6):e38599. doi: 10.1371/journal.pone.0038599. Epub 2012 Jun 27.
BACKGROUND
Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes.
METHODOLOGY/PRINCIPAL FINDINGS: Human keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter.
CONCLUSIONS/SIGNIFICANCE: The data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving PKC/AP-1 pathway, and basal-specific mediated by FOXM1, and therefore the use of K15 expression as a marker of stem cells must be viewed with caution.
背景
角蛋白 15（K15）是一种 I 型角蛋白，可用作干细胞标志物。其表达仅限于复层上皮的基底层和毛囊的隆起处。然而，在某些临床情况下，包括口腔扁平苔藓，K15 在基底上层被诱导，这与干细胞标志物的作用不一致。本研究提供了角蛋白 15 在基底层和分化角蛋白细胞中表达的机制的见解。
方法/主要发现：通过三种不同的方法将人角质形成细胞分化；悬浮在甲基纤维素中、高密度和用佛波酯处理。通过定量 PCR 确定 mRNA 的表达，通过 Western blot 和免疫染色确定蛋白质的表达。悬浮培养的角质形成细胞抑制β1-整合素的表达，诱导分化特异性标志物和 K15，而 FOXM1（细胞周期调节蛋白）和 K14 下调。通过纤连蛋白或精氨酸-甘氨酸-天冬氨酸肽（Arg-Gly-Asp，RGD）来拯救β1-整合素，可抑制 K15，但诱导 K14 和 FOXM1 的表达。通过 siRNA 特异性抑制 PKCδ，以及通过 TAM67（显性负性 c-Jun）抑制 AP-1 转录因子，可抑制 K15 的表达，表明 PKC/AP-1 通路在 K15 的分化特异性表达中起作用。基底细胞特异性 K15 表达可能涉及 FOXM1，因为后者的异位表达已知可诱导 K15。通过染色质免疫沉淀，我们在 K15 启动子中鉴定出单个 FOXM1 结合基序。
结论/意义：数据表明，K15 是在由β1-整合素下调介导的终末分化过程中诱导的。然而，这不能成为分层上皮中基底/干细胞特异性 K15 表达的机制，因为基底角质形成细胞不会经历终末分化。我们提出，有两种机制调节分层上皮中的 K15 表达；涉及 PKC/AP-1 通路的分化特异性，以及由 FOXM1 介导的基底特异性，因此必须谨慎看待将 K15 表达用作干细胞标志物。
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