Dlugosz A A, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland 20892.
J Cell Biol. 1993 Jan;120(1):217-25. doi: 10.1083/jcb.120.1.217.
The protective function of skin depends on successful completion of a tightly regulated multi-step differentiation program, during which the induction of markers for a specific stage in epidermal differentiation is coupled to repression of markers expressed at the preceding stage. We have explored the role of protein kinase C (PKC) in this process using an in vitro model system, in which cultures of primary mouse epidermal keratinocytes are induced to terminally differentiate by raising the Ca2+ concentration in the medium from 0.05 to 0.12 mM. At doses which activate PKC, 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1-oleoyl-2-acetylglycerol block Ca(2+)-mediated induction of the spinous cell markers keratins K1 and K10 at both the protein and mRNA level. TPA and 1-oleoyl-2-acetylglycerol also rapidly repress K1 and K10 mRNA expression when added to differentiating keratinocyte cultures already expressing these markers. The inhibition of K1 mRNA expression by TPA is blocked in cells where PKC has been inactivated with bryostatin. TPA-mediated loss of K1 mRNA is also blocked in cells exposed to cycloheximide or actinomycin D implicating a PKC-induced protein factor in this process. The loss of K1 mRNA in TPA-treated cultures is the result of both a selective destabilization of K1 transcripts and a rapid inhibition of K1 gene transcription. In contrast to the dramatic repression of mRNAs typical for spinous cell differentiation, activation of PKC concurrently enhances expression of mRNAs and proteins for the granular cell markers loricrin and filaggrin. This response does not occur in cells pre-treated with bryostatin to inactivate PKC. Our results suggest that PKC is a fundamental regulator of the coordinate changes in keratinocyte gene expression that occur during the spinous to granular cell transition in epidermis.
皮肤的保护功能依赖于一个严格调控的多步骤分化程序的成功完成，在此过程中，表皮分化特定阶段标志物的诱导与前一阶段表达的标志物的抑制相耦合。我们使用体外模型系统探索了蛋白激酶C（PKC）在此过程中的作用，在该系统中，通过将培养基中的Ca2+浓度从0.05 mM提高到0.12 mM，诱导原代小鼠表皮角质形成细胞培养物终末分化。在激活PKC的剂量下，12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）和1 - 油酰 - 2 - 乙酰甘油在蛋白质和mRNA水平上均阻断Ca(2+)介导的棘状细胞标志物角蛋白K1和K10的诱导。当将TPA和1 - 油酰 - 2 - 乙酰甘油添加到已经表达这些标志物的分化角质形成细胞培养物中时，它们也会迅速抑制K1和K10 mRNA的表达。在已用苔藓抑素使PKC失活的细胞中，TPA对K1 mRNA表达的抑制作用被阻断。TPA介导的K1 mRNA缺失在暴露于放线菌酮或放线菌素D的细胞中也被阻断，这表明在此过程中有一个PKC诱导的蛋白质因子参与。TPA处理的培养物中K1 mRNA的缺失是K1转录本选择性不稳定和K1基因转录迅速抑制的结果。与棘状细胞分化典型的mRNA的显著抑制相反，PKC的激活同时增强了颗粒细胞标志物兜甲蛋白和细丝聚集蛋白的mRNA和蛋白质表达。在用苔藓抑素预处理使PKC失活的细胞中不会出现这种反应。我们的结果表明，PKC是表皮中棘状细胞向颗粒细胞转变过程中角质形成细胞基因表达协同变化的基本调节因子。
