Monaghan T K, Pou C, MacKenzie C J, Plevin R, Lutz E M
Strathclyde Institute of Pharmacy and Biomedical Sciences, Royal College, 204 George Street, Glasgow, G1 1XW, UK.
J Mol Neurosci. 2008 Nov;36(1-3):45-56. doi: 10.1007/s12031-008-9082-6. Epub 2008 May 28.
The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.
在人神经母细胞瘤SH-SY5Y细胞中研究了垂体腺苷酸环化酶激活多肽（PACAP）-38和白血病抑制因子（LIF）的神经营养作用。通过监测形态变化和对神经元标志物蛋白表达进行蛋白质印迹分析来评估对分化的影响。与诱导有神经突细胞数量增加5.5倍的PACAP-38不同，在4天的时间进程中，与对照细胞相比，LIF对细胞形态没有显著影响。与单独用PACAP-38处理的细胞相比，用PACAP-38+LIF共同处理的细胞中有神经突细胞数量有类似增加。用PACAP-38处理的细胞和用PACAP-38+LIF共同处理的细胞形态相似，形成具有长而细的神经突的双极神经元样细胞，顶部有生长锥样结构和膨体。SH-SY5Y细胞表达酪氨酸羟化酶（TH），但仅表达低水平的神经元标志物蛋白：Bcl-2、GAP-43和胆碱乙酰转移酶（ChAT）。用PACAP-38处理细胞可诱导Bcl-2、GAP-43和ChAT的表达，但似乎不会改变TH的表达。LIF未能诱导GAP-43的表达，对TH的表达影响很小，但可诱导Bcl-2的表达并上调ChAT的表达。与LIF共同处理对PACAP-3诱导的Bcl-2、GAP-43和ChAT的表达没有影响。用PACAP-38分化4天或用LIF处理的细胞对低氧条件以及H2O2和TNFα处理也表现出增加的抗性。PACAP分化细胞对低氧条件增加的抗性被p38丝裂原活化蛋白激酶抑制剂SB203580阻断，但未被MEK1抑制剂PD98059阻断。此外，细胞增殖试验表明，LIF而非PACAP-38刺激SH-SY5Y细胞的增殖，并且观察到的LIF引起的增殖增加不会因与PACAP共同处理而减弱。对介导PACAP对SH-SY5Y细胞神经营养作用的细胞内信号通路的进一步研究表明，磷脂酶C激活和Ca2+/钙调蛋白依赖性激酶II（CAMKII）均未参与。
