Colangelo A M, Fink D W, Rabin S J, Mocchetti I
Department of Cell Biology, Georgetown University, School of Medicine, Washington D.C. 20007.
Glia. 1994 Oct;12(2):117-27. doi: 10.1002/glia.440120205.
Cells that lack the high affinity receptor component (trkA) for nerve growth factor (NGF) are unresponsive to NGF. We investigated whether C6-2B cells, a rat glioma derived cell line, express trkA and, as a consequence, are responsive to NGF. In these cells, NGF (100 ng/ml) failed to induce the mRNA encoding for c-fos protooncogene and the low affinity NGF receptor p75NGFR, two NGF-responsive genes. In contrast, both mRNAs were induced in PC12 cells by NGF. Using a RNase protection assay with a cRNA probe for rat trkA, the expected trkA RNA protected fragment was detected in PC12 but not in C6-2B glioma cells, indicating that C6-2B cells either do not express the gene or express it only in low amounts. Cross-linking of 125I-labeled NGF to PC12 cells identified two major bands with an apparent molecular weight of 158 kDa and 100 kDa corresponding to trkA and p75NGFR, respectively. In contrast, only the 100 kDa band could be detected in C6-2B cells by cross-linking analysis. In C6-2B cells stably transfected with the rat trkA cDNA, NGF increased c-fos mRNA, induced tyrosine phosphorylation of gp140trk, and SNT (suc-associated neurotrophic factor-induced tyrosine-phosphorylated target), and caused morphological changes within 72 h. All of these effects of NGF were blocked by the protein kinase inhibitor K-252a suggesting that NGF signal transduction was restored by trkA expression. Most important, in C6trk+ cells, NGF was a weaker (2-fold) inducer of [3H]thymidine incorporation when compared to bFGF (5-fold), suggesting that expression of trkA fails to confer to NGF a strong mitogenic effect. Our findings indicate that C6-2B glioma cells do not possess high affinity NGF receptor and thus are unresponsive to NGF and that expression of trkA in neuroectoderm derived cells elicits some of the NGF responses characteristic of neuronal cells.
缺乏神经生长因子（NGF）高亲和力受体成分（trkA）的细胞对NGF无反应。我们研究了大鼠胶质瘤衍生细胞系C6-2B细胞是否表达trkA，以及因此是否对NGF有反应。在这些细胞中，NGF（100 ng/ml）未能诱导编码原癌基因c-fos和低亲和力NGF受体p75NGFR的mRNA，这两个都是NGF反应性基因。相比之下，NGF可在PC12细胞中诱导这两种mRNA。使用针对大鼠trkA的cRNA探针进行核糖核酸酶保护试验，在PC12细胞中检测到预期的trkA RNA保护片段，但在C6-2B胶质瘤细胞中未检测到，这表明C6-2B细胞要么不表达该基因，要么仅少量表达。将125I标记的NGF与PC12细胞交联，鉴定出两条主要条带，表观分子量分别为158 kDa和100 kDa，分别对应trkA和p75NGFR。相比之下，通过交联分析在C6-2B细胞中只能检测到100 kDa的条带。在稳定转染大鼠trkA cDNA的C6-2B细胞中，NGF增加了c-fos mRNA，诱导了gp140trk和SNT（蔗糖相关神经营养因子诱导的酪氨酸磷酸化靶点）的酪氨酸磷酸化，并在72小时内引起形态变化。NGF的所有这些作用都被蛋白激酶抑制剂K-252a阻断，这表明trkA的表达恢复了NGF信号转导。最重要的是，与碱性成纤维细胞生长因子（bFGF）（5倍）相比，在C6trk+细胞中，NGF是较弱的（2倍）[3H]胸苷掺入诱导剂，这表明trkA的表达未能赋予NGF强大的促有丝分裂作用。我们的研究结果表明，C6-2B胶质瘤细胞不具有高亲和力NGF受体，因此对NGF无反应，并且神经外胚层来源细胞中trkA的表达引发了一些神经元细胞特有的NGF反应。
