Kuo Ya-Huei, Chen Thomas T
Department of Molecular and Cell Biology and Biotechnology Center, University of Conneticut, Storrs 06269, USA.
Exp Cell Res. 2002 Oct 15;280(1):75-89. doi: 10.1006/excr.2002.5628.
Insulin-like growth factor-I (IGF-I) is translated as a pre-pro-peptide that is posttranslationally processed to its mature form by proteolytic removal of the signal peptide and the E-domain peptide. Contrary to the mature human (h) IGF-I, the recombinant rtEa4 -peptide significantly reduced the anchorage-independent cell growth in human neuroblastoma cells (SK-N-F1), shown by colony formation assay in vitro. Significant inhibition of colony formation is also observed in SK-N-F1 cells stably transfected with a bicistronic expression construct encoding a secretory form of the rtEa4 peptide. Furthermore, treatment with the recombinant rtEa4 peptide, but not the mature hIGF-I, resulted in morphological differentiation of SK-N-F1 cells characterized by long neurite outgrowth. Similar morphological differentiation is also observed in SK-N-F1 cell clones stably transfected with the rtEa4 peptide expression construct. A spectrum of biological activities similar to those of rtEa4 peptide is also observed in the synthetic hEb peptide, but not-the hEa peptide. Results of further characterization reveal that neurites induced by rtEa4 or hEb peptide contain neuronal-specific MAP-2, Tau, and neurofilament (NF-160), accompanied by an increased expression of the neuronal marker gene neuropeptide tyrosine (NPY). Furthermore, effects of signal transduction inhibitors are indicative of the involvement of MAP-kinase PI-3-kinase cascades. The activation of ERK-1/-2 is markedly increased in response to rtEa-4 or hEb peptide stimulation, further indicating the involvement of MAPK signaling cascade. These unique biological activities exhibited by the rtEa4 or hEb peptide suggest that E peptide of the pro-IGF-I may play distinct roles in regulating cell growth and differentiation in neuroblastoma cells.
胰岛素样生长因子-I（IGF-I）最初被翻译为前原肽，经翻译后加工，通过蛋白水解去除信号肽和E结构域肽而形成成熟形式。与成熟的人（h）IGF-I相反，重组rtEa4肽在体外集落形成试验中显示，可显著降低人神经母细胞瘤细胞（SK-N-F1）的非锚定依赖性细胞生长。在稳定转染了编码分泌形式rtEa4肽的双顺反子表达构建体的SK-N-F1细胞中，也观察到集落形成受到显著抑制。此外，用重组rtEa4肽而非成熟的hIGF-I处理，导致SK-N-F1细胞发生形态分化，其特征为长出长长的神经突。在稳定转染了rtEa4肽表达构建体的SK-N-F1细胞克隆中，也观察到类似的形态分化。在合成的hEb肽中也观察到了一系列与rtEa4肽相似的生物学活性，但hEa肽没有。进一步的特性分析结果表明，rtEa4或hEb肽诱导的神经突含有神经元特异性微管相关蛋白2（MAP-2）、微管蛋白（Tau）和神经丝（NF-160），同时神经肽酪氨酸（NPY）这一神经元标记基因的表达增加。此外，信号转导抑制剂的作用表明丝裂原活化蛋白激酶（MAP）-激酶磷脂酰肌醇-3-激酶（PI-3-激酶）级联反应参与其中。响应rtEa-4或hEb肽刺激，细胞外信号调节激酶1/2（ERK-1/-2）的激活显著增加，进一步表明MAPK信号级联反应参与其中。rtEa4或hEb肽所展现的这些独特生物学活性表明，前胰岛素样生长因子-I的E肽在调节神经母细胞瘤细胞的生长和分化中可能发挥着独特作用。
