Fandrey J, Huwiler A, Frede S, Pfeilschifter J, Jelkmann W
Department of Physiology I, University of Bonn, Germany.
Eur J Biochem. 1994 Dec 1;226(2):335-40. doi: 10.1111/j.1432-1033.1994.tb20057.x.
Hypoxia-induced erythropoietin (Epo) production in vitro is suppressed by interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF) and phorbol esters. Herein, the Epo-synthesizing human hepatoma cell line HepG2 was used to investigate whether protein kinase C (PKC) is involved in the inhibitory action of the cytokines. Within 1 h after the onset of hypoxia, Epo mRNA levels were markedly increased in untreated HepG2 cells as quantitated by competitive reverse transcription PCR. The cytokines IL-1 beta and TNF prevented this hypoxia-induced increase in Epo mRNA levels. In phorbol-ester-treated cells first inhibitory effects on Epo mRNA levels were observed only after 3 h. Western blot analyses revealed the presence of four isoenzymes of PKC in HepG2 cells. None of these isoenzymes was translocated in response to TNF or IL-1 beta, suggesting that the cytokines do not activate PKC in HepG2 cells. In contrast, phorbol esters translocated and, upon prolonged exposure, down-regulated PKC isoenzymes alpha and epsilon. Activation of protein kinase A by dibutyryl-cAMP partially antagonized the cytokine-dependent inhibition of Epo production but did not influence the inhibitory effect of phorbol esters. Endogenous cAMP levels in HepG2 cells were unchanged by cytokine treatment. Obviously, at least two signaling pathways exist that can confer inhibition of Epo production in HepG2 cells. One of these may be mediated by down-regulation of the PKC alpha or epsilon isoenzyme. The other pathway, however, which is triggered by IL-1 beta and TNF, is independent of PKC.
白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF）和佛波酯可抑制体外缺氧诱导的促红细胞生成素（Epo）产生。在此，利用合成Epo的人肝癌细胞系HepG2来研究蛋白激酶C（PKC）是否参与细胞因子的抑制作用。通过竞争性逆转录PCR定量分析，在缺氧开始后1小时内，未处理的HepG2细胞中Epo mRNA水平显著升高。细胞因子IL-1β和TNF可阻止这种缺氧诱导的Epo mRNA水平升高。在佛波酯处理的细胞中，仅在3小时后才观察到对Epo mRNA水平的首次抑制作用。蛋白质印迹分析显示HepG2细胞中存在四种PKC同工酶。这些同工酶均未因TNF或IL-1β而发生转位，这表明细胞因子不会在HepG2细胞中激活PKC。相比之下，佛波酯可使PKC同工酶α和ε发生转位，并且在长时间暴露后使其下调。二丁酰环磷腺苷（dibutyryl-cAMP）激活蛋白激酶A可部分拮抗细胞因子依赖性的Epo产生抑制作用，但不影响佛波酯的抑制作用。细胞因子处理后，HepG2细胞内源性环磷腺苷（cAMP）水平未发生变化。显然，至少存在两条可导致HepG2细胞中Epo产生受到抑制的信号通路。其中一条可能由PKCα或ε同工酶的下调介导。然而，另一条由IL-1β和TNF触发的通路与PKC无关。
