Jung E M, Betancourt-Calle S, Mann-Blakeney R, Griner R D, Bollinger Bollag W
Program in Cell Signaling, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta 30912-2630, USA.
Carcinogenesis. 1999 Apr;20(4):569-76. doi: 10.1093/carcin/20.4.569.
Our previous results and data in the literature have suggested a potential role for phospholipase D (PLD) in the regulation of epidermal keratinocyte growth and differentiation. Therefore, we investigated the effect of agents reported to modulate keratinocyte growth and differentiation on PLD activation. The purported protein kinase C (PKC) 'inhibitor', staurosporine (Stsp), has been reported to activate PKC in keratinocytes, eliciting many of the same effects as active tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Stsp also induces a programmed pattern of differentiation similar to that seen in keratinocytes in vivo; TPA, on the other hand, appears to preferentially elicit markers consistent with late (granular) differentiation. In contrast, bradykinin is reported to stimulate keratinocyte proliferation. We found that these three agents had different effects on PLD activation in primary mouse epidermal keratinocytes. TPA increased PLD activity acutely and in a sustained fashion. In contrast, Stsp did not acutely activate PLD and inhibited acute TPA-induced activation of PLD. However, treatment of keratinocytes with Stsp for longer time periods (3-5 h) induced sustained PLD activation and this long-term effect was additive with that of TPA. Bradykinin activated PLD acutely but transiently. Both TPA and Stsp increased transglutaminase activity, a marker of late differentiation, whereas bradykinin had little or no effect on either cell proliferation or transglutaminase activity. These results suggest that a sustained activation of PLD is associated with the induction of keratinocyte differentiation. We hypothesize that PLD activity mediates late keratinocyte differentiation through generation of diacylglycerol and activation of specific PKC isoforms. Furthermore, we propose that the profound and immediate TPA-induced stimulation of PLD activity 'drives' the keratinocytes to late differentiation steps. However, the less efficacious (and more gradual) sustained activation of PLD by Stsp may allow a patterned differentiation more like that observed in skin.
我们之前的研究结果以及文献中的数据表明，磷脂酶D（PLD）在调节表皮角质形成细胞的生长和分化过程中可能发挥作用。因此，我们研究了据报道可调节角质形成细胞生长和分化的试剂对PLD激活的影响。所谓的蛋白激酶C（PKC）“抑制剂”星形孢菌素（Stsp），据报道可在角质形成细胞中激活PKC，引发许多与活性肿瘤启动子（如12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA））相同的效应。Stsp还能诱导出一种类似于体内角质形成细胞中所见的程序性分化模式；另一方面，TPA似乎优先引发与晚期（颗粒状）分化一致的标志物。相比之下，据报道缓激肽可刺激角质形成细胞增殖。我们发现这三种试剂对原代小鼠表皮角质形成细胞中PLD激活具有不同的影响。TPA能迅速且持续地增加PLD活性。相比之下，Stsp不会急性激活PLD，反而会抑制TPA诱导的PLD急性激活。然而，用Stsp处理角质形成细胞较长时间（3 - 5小时）会诱导PLD持续激活，并且这种长期效应与TPA的效应具有叠加性。缓激肽能急性但短暂地激活PLD。TPA和Stsp均能增加转谷氨酰胺酶活性，这是晚期分化的一个标志物，而缓激肽对细胞增殖或转谷氨酰胺酶活性几乎没有影响。这些结果表明，PLD的持续激活与角质形成细胞分化的诱导有关。我们推测PLD活性通过生成二酰基甘油和激活特定的PKC亚型来介导角质形成细胞的晚期分化。此外，我们提出TPA对PLD活性的强烈且即时的刺激“推动”角质形成细胞进入晚期分化阶段。然而，Stsp对PLD的激活效果较差（且较为缓慢），这种持续激活可能会使分化模式更类似于在皮肤中观察到的情况。
