Wang H Q, Kim M P, Tiano H F, Langenbach R, Smart R C
Cell Signaling and Cancer Group, Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633, USA.
Mol Pharmacol. 2001 Apr;59(4):860-6. doi: 10.1124/mol.59.4.860.
Transgenic mice (K5-PKC alpha) in which the keratin 5 promoter directs the expression of protein kinase C-alpha (PKC alpha) to epidermal keratinocytes display a 10-fold increase in PKC alpha protein in their epidermis and alterations in phorbol ester-induced cutaneous inflammation [J Cell Science 1999;112:3497-3506]. In the current study, we have used these K5-PKC alpha mice to examine the role of PKC alpha in keratinocyte phospholipid metabolism/eicosanoid production and cutaneous inflammation. Primary keratinocytes from wild-type and transgenic mice were prelabeled in culture with [(3)H]arachidonic acid (AA) and subsequently treated with TPA. Compared with wild-type keratinocytes, K5-PKC alpha keratinocytes displayed a 2-fold increase in AA release. TPA treatment resulted in the phosphorylation of cPLA(2). PKC inhibitors GF-109203X or H7, but not mitogen-activated protein/extracellular signal-regulated protein kinase (MEK) inhibitor PD 98059, could inhibit phosphorylation and AA release. Topical 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of K5-PKC alpha mice resulted in a 5-fold increase in epidermal COX-2 induction and a 2- to 3-fold increase in prostaglandin (PG) E(2) levels above that observed in TPA-treated wild-type mice. PD 98059, GF-109203X, or H7 could block cyclooxygenase-2 (COX-2) induction by TPA. Because C/EBP beta, a basic leucine zipper transcription factor, can be activated via a PKC alpha/mitogen-activated protein kinase pathway and can influence COX-2 expression, we examined whether C/EBP beta is involved in TPA-induced epidermal COX-2 expression. TPA-induced COX-2 expression was similar in C/EBP beta nullizygous and wild-type mice. In summary, our results indicate that epidermal PKC alpha coordinately regulates cPLA(2) activity and COX-2 expression resulting in increased levels of AA and PGE(2). Furthermore, PKC alpha-induced AA release and cPLA(2) phosphorylation are independent of MEK, whereas PKC alpha-induced COX-2 expression and PGE(2) production are MEK-dependent and C/EBP beta-independent events.
在转基因小鼠（K5-PKCα）中，角蛋白5启动子将蛋白激酶C-α（PKCα）的表达导向表皮角质形成细胞，其表皮中PKCα蛋白增加了10倍，并且佛波酯诱导的皮肤炎症也发生了改变[《细胞科学杂志》1999年；112:3497 - 3506]。在当前研究中，我们利用这些K5-PKCα小鼠来研究PKCα在角质形成细胞磷脂代谢/类花生酸生成及皮肤炎症中的作用。将野生型和转基因小鼠的原代角质形成细胞在培养中用[³H]花生四烯酸（AA）预标记，随后用佛波酯（TPA）处理。与野生型角质形成细胞相比，K5-PKCα角质形成细胞的AA释放增加了2倍。TPA处理导致胞质型磷脂酶A2（cPLA₂）磷酸化。PKC抑制剂GF-109203X或H7能够抑制磷酸化和AA释放，但丝裂原活化蛋白/细胞外信号调节蛋白激酶（MEK）抑制剂PD 98059则不能。对K5-PKCα小鼠进行局部12-O-十四酰佛波醇-13-乙酸酯（TPA）处理后，其表皮环氧化酶-2（COX-2）的诱导增加了5倍，前列腺素（PG）E₂水平比TPA处理的野生型小鼠高出2至3倍。PD 98059、GF-109203X或H7能够阻断TPA诱导的COX-2表达。由于碱性亮氨酸拉链转录因子C/EBPβ可通过PKCα/丝裂原活化蛋白激酶途径被激活，并能影响COX-2表达，我们研究了C/EBPβ是否参与TPA诱导的表皮COX-2表达。在C/EBPβ纯合缺失小鼠和野生型小鼠中，TPA诱导的COX-2表达相似。总之，我们的结果表明，表皮PKCα协同调节cPLA₂活性和COX-2表达，导致AA和PGE₂水平升高。此外，PKCα诱导的AA释放和cPLA₂磷酸化不依赖于MEK，而PKCα诱导的COX-2表达和PGE₂生成是依赖于MEK且不依赖于C/EBPβ的事件。
