Długosz A A, Yuspa S H
Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, NIH, Bethesda, MD 20892.
J Invest Dermatol. 1994 Apr;102(4):409-14. doi: 10.1111/1523-1747.ep12372171.
During the final stage of epidermal differentiation, activation of keratinocyte transglutaminase results in covalent crosslinking of a variety of proteins to form highly protective cornified cell envelopes. We have studied the regulation of keratinocyte transglutaminase (TGK) gene expression in murine epidermal keratinocytes induced to terminally differentiate in vitro by increasing the level of extracellular Ca++ or treatment with the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Raising extracellular Ca++ induces squamous differentiation of cultured keratinocytes and elicits a concentration-dependent increase in expression of TGK mRNA; keratinocytes grown for 24 h in 0.12 mM Ca++ medium express approximately 12 times as much TGK mRNA as basal cells (grown in 0.05 mM Ca++ medium), whereas cultures exposed to 1.4 mM Ca++ express approximately 17 times as much. TPA induces squamous differentiation and TGK mRNA even in basal keratinocyte cultures grown in 0.05 mM Ca++ medium, suggesting that expression of this differentiation marker is regulated by the PKC signaling pathway. Induction of TGK mRNA in response to TPA treatment is transient, reaching a peak at 6-8 h and returning to baseline by 24 h. In contrast, elevation of TGK mRNA levels in response to Ca++ persists for at least 24 h. The increased abundance of TGK mRNA reflects increased transcription of the TGK gene, based on nuclear run-on analysis of Ca(++)- and TPA-treated keratinocytes. Induction of TGK mRNA by either TPA or Ca++ is blocked in the presence of cycloheximide, suggesting that a PKC-dependent protein factor is required for TGK gene expression in response to both stimuli. Furthermore, the accumulation of TGK mRNA in keratinocytes treated with TPA or Ca++ is blocked in cells treated with the PKC inhibitor GF 109203X or bryostatin. These results suggest that the induction of TGK gene expression by Ca++ is dependent on PKC, providing further support for the hypothesis that PKC plays a central role in regulating the late stages of epidermal differentiation.
在表皮分化的最后阶段，角质形成细胞转谷氨酰胺酶的激活导致多种蛋白质发生共价交联，形成高度保护性的角质化细胞包膜。我们研究了在体外通过提高细胞外Ca++水平或用蛋白激酶C（PKC）激活剂12-O-十四酰佛波醇-13-乙酸酯（TPA）处理诱导终末分化的小鼠表皮角质形成细胞中角质形成细胞转谷氨酰胺酶（TGK）基因表达的调控。提高细胞外Ca++可诱导培养的角质形成细胞发生鳞状分化，并引起TGK mRNA表达的浓度依赖性增加；在0.12 mM Ca++培养基中培养24小时的角质形成细胞表达的TGK mRNA约为基底细胞（在0.05 mM Ca++培养基中生长）的12倍，而暴露于1.4 mM Ca++的培养物表达的TGK mRNA约为17倍。TPA即使在0.05 mM Ca++培养基中生长的基底角质形成细胞培养物中也能诱导鳞状分化和TGK mRNA，这表明该分化标志物的表达受PKC信号通路调控。TPA处理后TGK mRNA的诱导是短暂的，在6-8小时达到峰值，到24小时恢复到基线水平。相比之下，Ca++诱导的TGK mRNA水平升高至少持续24小时。基于对Ca(++)和TPA处理的角质形成细胞的核转录分析，TGK mRNA丰度的增加反映了TGK基因转录的增加。在放线菌酮存在的情况下，TPA或Ca++诱导的TGK mRNA被阻断，这表明PKC依赖性蛋白因子是两种刺激下TGK基因表达所必需的。此外，用PKC抑制剂GF 109203X或苔藓抑素处理的细胞中，TPA或Ca++处理的角质形成细胞中TGK mRNA的积累被阻断。这些结果表明Ca++诱导的TGK基因表达依赖于PKC，为PKC在调节表皮分化后期起核心作用的假说提供了进一步支持。
