Fagerström S, Påhlman S, Gestblom C, Nånberg E
Department of Pathology, University Hospital, Uppsala, Sweden.
Cell Growth Differ. 1996 Jun;7(6):775-85.
A combination of basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I) or 16 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) and serum induces human SH-SY5Y neuroblastoma cells to undergo differentiation and acquire a neuronal phenotype. Nerve growth factor (NGF) added to SH-SY5Y cells stably transfected with the NGF-receptor TRK-A (SH-SY5Y/trk) induces a similar differentiated phenotype. SH-SY5Y cells express protein kinase C (PKC)-alpha, PKC-beta I, PKC-epsilon, and PKC-zeta protein, and phorbol ester- or growth factor-induced differentiation results in a sustained activation of PKC. The specific PKC inhibitor GF 109203X blocked TPA- and bFGF-IGF-I-induced neurite outgrowth in wild-type SH-SY5Y cells and NGF-induced neurite outgrowth in SH-SY5Y/trk cells. When added to differentiated cells, GF 109203X caused rapid retraction of growth cone filopodia. In TPA- and bFGF-IGF-I-treated cells, addition of GF 109203X also blocked induced expression of growth associated protein-43 and neuropeptide tyrosine while the increase in expression of these two genes was only slightly affected by the inhibitor in NGF-treated SH-SY5Y/trk cells. Thus, a portion of the NGF-induced phenotypic changes appears not to be mediated via PKC-dependent signaling. A high concentration of TPA (1.6 microM) down regulated PKC-alpha and PKC-beta I almost completely and PKC-epsilon partially in wild-type SH-SY5Y and SH-SY5Y/trk cells. Cells with down-regulated PKC-alpha and PKC-beta I after 1.6 microM TPA treatment still differentiated with growth factors. In these cells, the PKC-epsilon level was restored, and the PKC-epsilon protein was enriched in the growth cones. The 1.6 microM TPA-induced down-regulation of PKC-epsilon was counteracted by bFGF and NGF but not by platelet-derived growth factor or IGF-I. These data indicate that PKC activity is vital for neurite formation, and that the cells can differentiate under conditions when PKC-alpha and PKC-beta I are extensively down regulated. The close correlation between differentiation and presence of PKC-epsilon protein suggests an important function for this isoform during this process.
碱性成纤维细胞生长因子（bFGF）与胰岛素样生长因子-I（IGF-I）联合使用，或16 nM的12-O-十四烷酰佛波醇-13-乙酸酯（TPA）与血清共同作用，可诱导人SH-SY5Y神经母细胞瘤细胞发生分化并获得神经元表型。向稳定转染了神经生长因子（NGF）受体TRK-A的SH-SY5Y细胞（SH-SY5Y/trk）中添加NGF，可诱导出类似的分化表型。SH-SY5Y细胞表达蛋白激酶C（PKC）-α、PKC-βI、PKC-ε和PKC-ζ蛋白，佛波酯或生长因子诱导的分化会导致PKC的持续激活。特异性PKC抑制剂GF 109203X可阻断野生型SH-SY5Y细胞中TPA和bFGF-IGF-I诱导的神经突生长，以及SH-SY5Y/trk细胞中NGF诱导的神经突生长。当添加到分化细胞中时，GF 109203X会导致生长锥丝状伪足迅速回缩。在TPA和bFGF-IGF-I处理的细胞中，添加GF 109203X还可阻断生长相关蛋白-43和神经肽酪氨酸的诱导表达，而在NGF处理的SH-SY5Y/trk细胞中，这两种基因表达的增加仅受到该抑制剂的轻微影响。因此，NGF诱导的部分表型变化似乎不是通过PKC依赖性信号传导介导的。高浓度的TPA（1.6 microM）可使野生型SH-SY5Y和SH-SY5Y/trk细胞中的PKC-α和PKC-βI几乎完全下调，PKC-ε部分下调。经1.6 microM TPA处理后PKC-α和PKC-βI下调的细胞，在生长因子作用下仍可分化。在这些细胞中，PKC-ε水平得以恢复，并在生长锥中富集。1.6 microM TPA诱导的PKC-ε下调可被bFGF和NGF抵消，但不能被血小板衍生生长因子或IGF-I抵消。这些数据表明，PKC活性对于神经突形成至关重要，并且细胞在PKC-α和PKC-βI被广泛下调的条件下仍可分化。分化与PKC-ε蛋白的存在之间的密切相关性表明该同工型在此过程中具有重要功能。
