Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression.
Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression.
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Ito 细胞的激活涉及 AP-1 结合蛋白的调节和 I 型胶原蛋白基因表达的诱导。
DOI：
10.1042/bj3040817
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Seyer,JM
中科院分区：
文献类型：
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作者：
Armendariz-Borunda,J;Simkevich,CP;Roy,N;Raghow,R;Kang,AH;Seyer,JM
肝Ito细胞活化的特征在于增殖增加、纤维化、细胞类维生素A的损失和细胞形状的改变。在这里，我们已经描述了新鲜分离的Ito细胞（FIC）和长期培养的Ito细胞（LTIC）之间的根本差异。该活化过程与FIC中Pro α 1（I）基因表达的缺失相关。LTIC表达丰富的Pro α 1（I）基因转录本。核径流实验表明，FIC不能支持Pro α 1（I）RNA转录，而LTIC转录它大于5倍，与FIC相比。与对照LTIC相比，转化生长因子β（TGF β）处理的LTIC在Pro α 1（I）基因转录速率方面优先增加。人I型胶原启动子-增强子构建体（pCOL-KT）[Thompson，Simkevich，Holness，Kang和Raghow（1991）J.Biol.Chem.266，2549-2556]容易在LTIC中表达，但不能在FIC中表达。此外，LTIC的TGF β处理导致pCOL-KT的表达增加。pCOL-KT（S360）的360 bp增强子区域中激活蛋白-1（AP-1）结合位点（+598至+604）的缺失导致CAT报告基因的表达降低，这表明该真正的AP-1位点可以至少部分地介导TGF β的反式激活作用。使用DNA酶I保护，我们证明了位于S360片段+590至+625处的单一足迹;从TGF β处理的LTIC制备的核提取物显示出这些AP-1结合蛋白的更大活性。凝胶迁移率测定证实并扩展了足迹观察。在FIC的核提取物中没有发现AP-1结合活性。含有共有AP-1基序的双链寡核苷酸能够竞争结合;共有NF-1基序寡核苷酸未能这样做。来自对照和TGF β处理的LTIC的核提取物与针对c-jun和c-fos的抗体的预孵育使得AP-1蛋白与靶S360片段的结合减少。
Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.