Xue G Z, Zheng Z S, Chen R Z, Lloyd M B, Prystowsky J H
Department of Dermatology, Columbia University, New York, NY, USA.
Biochem J. 1996 Oct 15;319 ( Pt 2)(Pt 2):641-8. doi: 10.1042/bj3190641.
Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125-132]. In this report, 10(-10) M-10(-7) M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interfered with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41-57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cisretinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.
多项研究表明，小鼠和人类角质形成细胞对佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）的反应不同。通过体外试验，我们发现，与PMA对小鼠皮肤的作用相反，PMA在人类皮肤活检组织中并未诱导鸟氨酸脱羧酶（ODC）活性。为了探究PMA诱导的信号传导，并确定体外培养系统是否可用于预测类视黄醇在人类角质形成细胞中的生物学活性，我们研究了一种猿猴病毒40（SV40）转化的人类角质形成细胞系。表皮生长因子（EGF）以剂量和时间依赖性方式刺激这些细胞中的ODC活性，并增加ODC mRNA的稳态水平[普里斯托夫斯基、克莱文杰和郑（1993年）《实验皮肤病学》2，125 - 132页]。在本报告中，10(-10)M - 10(-7)M的PMA在7小时时诱导ODC mRNA和酶合成，但未显著诱导ODC活性，并抑制了EGF对ODC活性的诱导。为了探究PMA干扰EGF信号传导的机制，研究了PMA对EGF与其细胞表面受体结合的影响；用PMA急性处理（7小时内）使EGF结合减少至基线水平的41% - 57%。相反，用PMA慢性处理（24小时）使EGF结合增加至基线水平的156%，并与EGF受体蛋白数量增加相关。蛋白激酶C（PKC）激活与PMA处理后EGF结合的急性减少相关。总之，PMA诱导ODC mRNA和ODC酶合成，而可免疫沉淀的ODC酶蛋白和ODC活性的稳态水平并未增加，这表明ODC酶蛋白的周转可能增加。此外，PMA通过减少EGF结合抑制EGF对ODC的诱导，这可能是由PKC激活介导的。最后，用包括依曲替酯、Ro13 - 7410、阿维A、Ro40 - 8757、13 - 顺维甲酸和阿维A酸在内的类视黄醇处理角质形成细胞，阻断了PMA对ODC mRNA的诱导，表明该体外模型可能是预测人类生物学活性的有价值的筛选试验。
