Rogers M V, Buensuceso C, Montague F, Mahadevan L
Department of Cell Biology, Wellcome Research Laboratories, Beckenham, Kent, U.K.
Neuroscience. 1994 May;60(2):479-94. doi: 10.1016/0306-4522(94)90259-3.
We show here that a protein tyrosine phosphatase inhibitor, sodium orthovanadate, induces rat pheochromocytoma cells to express neurites, a prominent morphological marker of neuronal phenotype. Vanadate-induced differentiation and neurite outgrowth in pheochromocytoma cells was not as extensive as that induced by the positive control employed, nerve growth factor. However, neurite outgrowth responses were comparable between nerve growth factor-treated pheochromocytoma cells and cells primed and then restimulated with vanadate. In the human neuroblastoma cell line, SH-SY5Y, a single exposure to vanadate induced neurite extension in this cell line equal to that initiated by nerve growth factor. In both cell lines vanadate treatment resulted in tyrosine phosphorylation of several high-molecular-weight proteins and using anti-phosphotyrosine antibodies, intense fluorescence was observed in the cell body and neurites of pheochromocytoma cells exposed to vanadate. Vanadate mediated differentiation and neurite outgrowth in pheochromocytoma cells could be ablated by the tyrosine kinase inhibitor erbastatin, whereas nerve growth factor-induced neurite outgrowth was only partially inhibited. In SH-SY5Y cells, erbstatin mediated partial inhibition of both vanadate and nerve growth factor-induced neurite elongation with similar kinetics. In contrast, K252b, a trk tyrosine kinase inhibitor, exhibited only a 30% reduction of neurite outgrowth in vanadate treated pheochromocytoma cells but an 80% reduction in nerve growth factor-treated cells. In SH-SY5Y cells, K252a did not have a statistically significant effect on neurite elongation induced by vanadate in contrast to a 60% reduction in nerve growth factor-treated cells. The membrane impermeable analogue K252b, had no effect on neurite elongation induced with either vanadate or nerve growth factor in these cells. The effects of vanadate were not mimicked by ouabain (0.1-50 microM) indicating that vanadate does not induce differentiation and/or neurite extension by inhibiting ion channel Na,K-ATPase, which is one of its other well-characterised inhibitory activities. Evidence for the selective action of vanadate on some but not all neuronal cell lines comes from the fact that it did not induce neurite extension in the human neuroblastoma cell line SK-N-MC. These data imply that vanadate-induced neurite outgrowth responses in pheochromocytoma and SH-SY5Y cells can be induced by the inhibition of tyrosine phosphatases and appears not to simply mimic nerve growth factor signals. The target(s) of vanadate action in the two cell lines are currently being sought.
我们在此表明，一种蛋白酪氨酸磷酸酶抑制剂原钒酸钠可诱导大鼠嗜铬细胞瘤细胞表达神经突，这是神经元表型的一个显著形态学标志物。钒酸盐诱导的嗜铬细胞瘤细胞分化和神经突生长不如阳性对照神经生长因子诱导的那样广泛。然而，神经生长因子处理的嗜铬细胞瘤细胞与用钒酸盐预处理然后再刺激的细胞之间的神经突生长反应相当。在人神经母细胞瘤细胞系SH-SY5Y中，单次暴露于钒酸盐可诱导该细胞系中的神经突延伸，其程度与神经生长因子引发的相当。在这两种细胞系中，钒酸盐处理均导致几种高分子量蛋白的酪氨酸磷酸化，并且使用抗磷酸酪氨酸抗体，在暴露于钒酸盐的嗜铬细胞瘤细胞的细胞体和神经突中观察到强烈荧光。酪氨酸激酶抑制剂厄巴斯汀可消除钒酸盐介导的嗜铬细胞瘤细胞分化和神经突生长，而神经生长因子诱导的神经突生长仅被部分抑制。在SH-SY5Y细胞中，厄巴斯汀以相似的动力学介导对钒酸盐和神经生长因子诱导的神经突伸长的部分抑制。相比之下，trk酪氨酸激酶抑制剂K252b在钒酸盐处理的嗜铬细胞瘤细胞中仅使神经突生长减少30%，而在神经生长因子处理的细胞中减少80%。在SH-SY5Y细胞中，K252a对钒酸盐诱导的神经突伸长没有统计学上的显著影响，而在神经生长因子处理的细胞中减少60%。膜不可渗透类似物K252b对这些细胞中钒酸盐或神经生长因子诱导的神经突伸长均无影响。哇巴因（0.1 - 50 microM）不能模拟钒酸盐的作用，这表明钒酸盐不是通过抑制离子通道Na,K-ATP酶（这是其另一种已充分表征的抑制活性）来诱导分化和/或神经突延伸。钒酸盐对某些而非所有神经元细胞系具有选择性作用的证据来自于它未在人神经母细胞瘤细胞系SK-N-MC中诱导神经突延伸这一事实。这些数据表明，钒酸盐诱导的嗜铬细胞瘤和SH-SY5Y细胞中的神经突生长反应可能是通过抑制酪氨酸磷酸酶诱导的，并且似乎并非简单地模拟神经生长因子信号。目前正在寻找这两种细胞系中钒酸盐作用的靶点。
