Armendariz-Borunda J, Simkevich C P, Roy N, Raghow R, Kang A H, Seyer J M
VA Medical Center Research Service, Memphis, TN 28104.
Biochem J. 1994 Dec 15;304 ( Pt 3)(Pt 3):817-24. doi: 10.1042/bj3040817.
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.
肝星状细胞（Ito细胞）的激活表现为增殖增加、纤维化形成、细胞内视黄醇丧失以及细胞形态改变。在此，我们描述了新鲜分离的Ito细胞（FIC）与长期培养的Ito细胞（LTIC）之间的根本差异。这种激活过程与FIC中Proα1（I）基因表达缺失相关。LTIC表达丰富的Proα1（I）基因转录本。核转录实验表明，FIC无法支持Proα1（I）RNA转录，而与FIC相比，LTIC转录该基因的水平高出5倍以上。与对照LTIC相比，转化生长因子β（TGFβ）处理的LTIC中Proα1（I）基因转录速率优先增加。一种人I型胶原启动子-增强子构建体（pCOL-KT）[汤普森、西姆凯维奇、霍尔尼斯、康和拉格霍（1991年）《生物化学杂志》266卷，2549 - 2556页]在LTIC中易于表达，但在FIC中未能表达。此外，TGFβ处理LTIC导致pCOL-KT表达增加。pCOL-KT（S360）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报告基因表达降低，这表明这个真正的AP-1位点至少部分可以介导TGFβ的反式激活作用。通过DNA酶I保护实验，我们在S360片段中证明了一个位于+590至+625的单一位点；从TGFβ处理的LTIC制备的核提取物中这些AP-1结合蛋白表现出更高的活性。凝胶迁移实验证实并扩展了足迹观察结果。在FIC的核提取物中未发现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基序寡核苷酸则不能。用抗c-jun和c-fos抗体对对照和TGFβ处理的LTIC的核提取物进行预孵育，使AP-1蛋白与靶S360片段的结合减少。
