Yavin E, Gabai A, Gil S
Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel.
J Neurochem. 1991 Jan;56(1):105-12. doi: 10.1111/j.1471-4159.1991.tb02568.x.
C6 rat glioma cells incubated in serum-free medium with D-[14C]glucosamine secrete, on stimulation with nerve growth factor (NGF) or monosialogangliosides (MSGs), several glycoproteins (Gps), the most prominent of which are a 270-, 220-, and 69-kDa Gp. Several growth factors, hormones, phorbol ester, and disialo- and trisialogangliosides did not stimulate secretion. Western blot analysis of the conditioned medium from C6 cells stimulated with NGF or MSG identified one distinct band of approximately 220 kDa for fibronectin and J1/tenascin, which comigrated. Antiserum to NGF prevented NGF-stimulated release and also blocked MSG-evoked release. The 220-kDa band was labeled after pulse labeling with [35S]methionine in the presence of NGF, and by a 15-min chase period radioactively labeled J1/tenascin could be immunoprecipitated. Tunicamycin drastically inhibited almost completely release of the 220-kDa Gp labeled by D-[14C]glucosamine or [35S]methionine. These results extend the range of neurotrophic properties attributed to NGF to cells of glial origin and suggest that NGF regulates secretion of extracellular matrix proteins. MSG stimulation of fibronectin and J1/tenascin secretion may be mediated by NGF or an NGF-like molecule also secreted by the C6 glioma cells.
在含有D-[14C]葡萄糖胺的无血清培养基中培养的C6大鼠胶质瘤细胞,在神经生长因子(NGF)或单唾液酸神经节苷脂(MSG)刺激下,会分泌几种糖蛋白(Gp),其中最显著的是270 kDa、220 kDa和69 kDa的Gp。几种生长因子、激素、佛波酯以及二唾液酸和三唾液酸神经节苷脂均未刺激分泌。对用NGF或MSG刺激的C6细胞的条件培养基进行蛋白质印迹分析,发现纤连蛋白和J1/腱生蛋白有一条约220 kDa的明显条带,二者迁移率相同。抗NGF血清可阻止NGF刺激的释放,也能阻断MSG引起的释放。在NGF存在的情况下用[35S]甲硫氨酸脉冲标记后,220 kDa的条带被标记,经过15分钟的追踪期,放射性标记的J1/腱生蛋白可被免疫沉淀。衣霉素几乎完全抑制了由D-[14C]葡萄糖胺或[35S]甲硫氨酸标记的220 kDa Gp的释放。这些结果将归因于NGF的神经营养特性范围扩展到了神经胶质来源的细胞,并表明NGF调节细胞外基质蛋白的分泌。MSG对纤连蛋白和J1/腱生蛋白分泌的刺激可能由NGF或C6胶质瘤细胞分泌的一种NGF样分子介导。