Functional analysis of rat cerebellum-derived neural differentiation factor

寻找未知的神经分化因子对于更好地理解复杂的中枢神经系统具有重要意义。为了筛选新的神经分化因子，我们利用pCD-2载体构建了大鼠小脑cDNA表达文库。将文库中的每个cDNA克隆在COS-1/7细胞中瞬时表达，并测定所得条件培养基对PC 12细胞的神经分化诱导活性，所述PC 12细胞在NGF、cAMP和IL-6存在下分化为交感神经元样细胞。结果，我们分离了cDNA克隆398（grp 75 R3），它编码67个氨基酸残基，与大鼠grp 75（热休克蛋白之一）的C-末端区域具有高度同源性。为了进一步研究grp 75 R3的功能，我们用GST融合的grp 75 R3蛋白制备了抗grp 75 R3抗体，并建立了稳定表达grp 75 R3蛋白的COS 75 R3细胞。在PC 12细胞中检测，COS 75 R3细胞的条件培养液中含有高活性的神经分化因子，但用抗grp 75 R3抗体进行Western blot检测，未检测到grp 75 R3蛋白，表明grp 75 R3不能直接诱导PC 12细胞分化。通过抗NGF、BDNF和NT-3等神经营养因子的抗体实验和RT-PCR分析，证明grp 75 R3和grp 75都能选择性地增强NGF基因的表达。将grp 75 R3 cDNA表达载体导入大鼠胶质瘤C6细胞，也得到了同样的结果。考虑到这些数据和饥饿24小时的大鼠脑中grp 75表达的显著诱导，神经胶质细胞中的grp 75 R3/grp 75很可能是由应激诱导的，并且维持了NGF应答性胆碱能神经元的存活。

It is important to find out unknown neural differentiation factors for better understanding of complex central nervous system. We constructed the rat cerebellar cDNA expression library using pCD-2 vector to isolate novel neural differentiation factors. Each cDNA clone in the library was transiently expressed in COS-1/7 cells and the resulting conditioned medium was assayed for the neural differentiation inducing activity against PC12 cells, which are differentiated into sympathetic neuron-like cells in the presence of NGF, cAMP and IL-6. As a result, we isolated cDNA clone 398 (grp75R3), which encodes 67 amino acid residues having high homology to the C-terminal region of rat grp75, one of the heat shock proteins. For the detailed functional analysis of grp75R3, we prepared anti-grp75R3 antibody using GST-fused grp75R3 protein, and also established COS75R3 cells stably expressing grp75R3 protein. The conditioned medium from COS75R3 cells contained a highly active neural differentiation factor when assayed in PC12 cells, however grp75R3 protein was not detected by Western blot with anti-grp75R3 antibody, showing that grp75R3 did not directly differentiate PCl2 cells. By the expriment using antibodies against NGF family neurotrophins such as NGF, BDNF and NT-3 and RT-PCR analysis, it has been shown that the gene expression of NGF was selectively enhnced by grp 75R3 as well as grp75. The same result was obtained when grp75R3 cDNA expression vector was introduced into rat glioma C6 cells. Taking into these data and the significant induction of grp75 expression in the brain of rat starved for 24 hr, it is highly possible that grp75R3/grp75 in glial cells is induced by stress and maintains the survival of NGF-responsive cholinergic neurons.