Magócsi M, Apáti A, Gáti R, Kolonics A
Department of Cell Metabolism, National Institute of Haematology and Immunology, Budapest, Hungary.
Immunol Lett. 1999 May 3;68(1):187-95. doi: 10.1016/s0165-2478(99)00048-6.
Erythropoietin (Epo) is the principal regulator of the production of circulating erythrocytes by controlling the proliferation, the differentiation and the survival of the erythroid progenitor cells. Early down-regulation of c-myb expression in erythroleukemia cells is a common feature of the action of Epo and chemical inducers of differentiation such as DMSO. Previously we have shown that in our Epo-responsive murine erythroleukemia cell line ELM-I-1, [Ca2+]i increasing agents can mimic the effect of Epo on c-myb expression and activate nuclear signal transduction processes involved in the induction of hemoglobin synthesis. These results also indicated that the Ca2+-induced down-regulation of c-myb expression and hemoglobin synthesis are mediated by the Ca2+/calmodulin dependent serine/threonine-specific protein phosphatase PP2B, calcineurin, but the Epo induced processes are not mediated by PP2B. In spite of this, we demonstrated in this paper that in ELM-I-1 cells the Epo-induced down-regulation of c-myb expression and hemoglobin production can be effectively enhanced by the simultaneously added [Ca2+]i-increasing agent, cyclopiazonic acid (CPA). This observation further supports the existence of at least two independent signalling pathways in the mechanism of Epo and [Ca2+]i increasing agents and the strong correlation between c-myb expression and hemoglobin production in differentiating cells. Although the c-AMP-response element binding protein (CREB) could be the common target of both calcium-dependent and -independent dephosphorylation, our results do not support the involvement of CREB in the regulation of c-myb gene expression. In addition to the calcineurin mediated down-regulation of c-myb expression, we have found a negative regulatory effect in the Ca2+-mediated transcriptional activation of certain genes. In response to [Ca2+]i-increasing agents in ELM-I-1 cells, both, egr-1 and c-fos mRNA expression increased significantly after the inhibition of calcineurin by cyclosporine A. Cyclosporin A exerted stimulatory effects on the egr-1 and c-fos expression also at lower (150-400 nM) intracellular Ca2+ levels. This potential co-regulation of c-myb, egr-1 and c-fos expression by calcineurin suggests that the negative modulation of egr-1 and c-fos expression may also be important for the induction of erythroid differentiation by [Ca2+]i-increasing agents. This negative modulation may also contribute to the Epo-induced differentiation in the case of a moderate increase of [Ca2+]i.
促红细胞生成素（Epo）是循环红细胞生成的主要调节因子，它通过控制红系祖细胞的增殖、分化和存活来实现这一功能。红白血病细胞中c-myb表达的早期下调是Epo和化学分化诱导剂（如二甲基亚砜）作用的共同特征。此前我们已经表明，在我们的Epo反应性小鼠红白血病细胞系ELM-I-1中，[Ca2+]i升高剂可以模拟Epo对c-myb表达的影响，并激活参与血红蛋白合成诱导的核信号转导过程。这些结果还表明，Ca2+诱导的c-myb表达下调和血红蛋白合成是由Ca2+/钙调蛋白依赖性丝氨酸/苏氨酸特异性蛋白磷酸酶PP2B（钙调神经磷酸酶）介导的，但Epo诱导的过程不是由PP2B介导的。尽管如此，我们在本文中证明，在ELM-I-1细胞中，同时添加的[Ca2+]i升高剂环匹阿尼酸（CPA）可以有效增强Epo诱导的c-myb表达下调和血红蛋白生成。这一观察结果进一步支持了在Epo和[Ca2+]i升高剂的作用机制中至少存在两条独立的信号通路，以及在分化细胞中c-myb表达与血红蛋白生成之间的强相关性。虽然c-AMP反应元件结合蛋白（CREB）可能是钙依赖性和非依赖性去磷酸化的共同靶点，但我们的结果不支持CREB参与c-myb基因表达的调控。除了钙调神经磷酸酶介导的c-myb表达下调外，我们还发现了Ca2+介导的某些基因转录激活中的负调控作用。在ELM-I-1细胞中，响应[Ca2+]i升高剂，用环孢素A抑制钙调神经磷酸酶后，egr-1和c-fos mRNA表达均显著增加。环孢素A在较低（150 - 400 nM）的细胞内Ca2+水平时也对egr-1和c-fos表达产生刺激作用。钙调神经磷酸酶对c-myb、egr-1和c-fos表达的这种潜在共同调节表明，egr-1和c-fos表达的负调控对[Ca2+]i升高剂诱导的红系分化可能也很重要。在[Ca2+]i适度增加的情况下，这种负调控也可能有助于Epo诱导的分化。
