Malek R L, Halvorsen S W
Department of Biochemical Pharmacology, State University of New York at Buffalo 14260, USA.
J Neurobiol. 1997 Jan;32(1):81-94.
We have used SH-SY5Y neuroblastoma cells as a model for differentiating neurons to examine the mechanisms that regulate responses to the neuropoietic cytokine ciliary neurotrophic factor (CNTF). Retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) each induced differentiation of SH-SY5Y cells. Cells treated for 24 h with retinoic acid (10 microM) showed a threefold increase in 125I-CNTF binding sites and were up to five times more sensitive to CNTF than untreated cells in stimulating the tyrosine phosphorylation of the transcription factor STAT3. TPA (10 nM) induced a transient 42% decrease in 125I-CNTF binding sites after 4 h of treatment that recovered to near control levels after 7 h of continuous exposure. TPA-treated cells showed a decreased sensitivity to CNTF and a sevenfold decrease in levels of STAT3. The retinoic acid-induced increase in 125I-CNTF binding could be prevented by administration of either cycloheximide or actinomycin D, whereas neither agent altered the TPA-induced decrease in 125I-CNTF binding. In addition, levels of mRNA for both the CNTF receptor alpha and gp130 subunits increased twofold as measured by RNase protection after treatment with retinoic acid for 30 h. The increase in CNTF receptor alpha subunit mRNA was not due to a decrease in its turnover rate, and therefore, was likely due to an increase in gene expression. Thus, retinoic acid and TPA regulate CNTF receptors on neuroblastoma cells differently, and the results demonstrate the importance of transcriptional control of CNTF receptors and also implicate translational and post-translational mechanisms in the regulation of cytokine receptors and responses on neurons.
我们已将SH-SY5Y神经母细胞瘤细胞作为分化神经元的模型，以研究调节对神经营养细胞因子睫状神经营养因子（CNTF）反应的机制。视黄酸和12-O-十四酰佛波醇-13-乙酸酯（TPA）均可诱导SH-SY5Y细胞分化。用视黄酸（10 microM）处理24小时的细胞，其125I-CNTF结合位点增加了三倍，并且在刺激转录因子STAT3的酪氨酸磷酸化方面，对CNTF的敏感性比未处理的细胞高五倍。TPA（10 nM）处理4小时后，125I-CNTF结合位点短暂下降42%，持续暴露7小时后恢复到接近对照水平。TPA处理的细胞对CNTF的敏感性降低，STAT3水平降低七倍。视黄酸诱导的125I-CNTF结合增加可通过施用放线菌酮或放线菌素D来阻止，而这两种试剂均未改变TPA诱导的125I-CNTF结合减少。此外，用视黄酸处理30小时后，通过核糖核酸酶保护测定，CNTF受体α和gp130亚基的mRNA水平均增加了两倍。CNTF受体α亚基mRNA的增加不是由于其周转率降低，因此，可能是由于基因表达增加。因此，视黄酸和TPA对神经母细胞瘤细胞上的CNTF受体的调节方式不同，结果证明了CNTF受体转录控制的重要性，也暗示了翻译和翻译后机制在细胞因子受体调节以及神经元反应中的作用。
