Raghow R, Gossage D, Seyer J M, Kang A H
J Biol Chem. 1984 Oct 25;259(20):12718-23.
Recently Hatahara and Seyer (Hatahara, T., and Seyer, J.M. Biochim. Biophys. Acta (1982) 716, 377-382) isolated a factor from fibrotic rat liver which stimulates collagen synthesis in cultured fibroblasts without affecting their rate of proliferation. To investigate the mechanism of fibrogenic factor-mediated enhancement of type I collagen synthesis, we quantitated the levels of mRNAs coding for pro-alpha 1(I) and pro-alpha 2(I) chains in rat dermal fibroblasts. Cell-free translation experiments revealed that the fibrogenic factor caused greater than 5-fold increase in the translatable levels of type I mRNAs. We also quantitated collagen mRNAs by techniques of Northern blotting of glyoxylated poly(A+) RNA followed by hybridization to nick-translated human cDNA clones containing the coding sequence of pro-alpha 1(I) and pro-alpha 2(I) chains. Furthermore, we investigated the relative rates of collagen mRNA transcription in the isolated nuclei of treated and control fibroblasts. Similar quantitation of beta-actin mRNA transcription, which remains unaffected by the treatment with fibrogenic factor, was used as an internal control. We demonstrate that the fibrogenic factor causes a 4-6-fold increase in the rate of transcription of pro-alpha 1(I) and pro-alpha 2(I) genes. Finally, we also show that the rate of intracellular degradation of collagen is not significantly altered in cells treated with fibrogenic factor. These results combined with data on cell-free translation strongly suggest that the increased accumulation of type I collagen mRNA in fibrogenic factor-treated fibroblasts is a consequence of enhanced rates of collagen mRNA transcription.
最近，幡原和塞耶（幡原，T.，以及塞耶，J.M.《生物化学与生物物理学学报》（1982年）716卷，377 - 382页）从纤维化大鼠肝脏中分离出一种因子，该因子可刺激培养的成纤维细胞中的胶原蛋白合成，而不影响其增殖速率。为了研究纤维化因子介导的I型胶原蛋白合成增强的机制，我们对大鼠真皮成纤维细胞中编码前α1（I）链和前α2（I）链的mRNA水平进行了定量。无细胞翻译实验表明，纤维化因子使I型mRNA的可翻译水平增加了5倍以上。我们还通过对经乙醛酸处理的聚（A +）RNA进行Northern印迹分析，然后与含有前α1（I）链和前α2（I）链编码序列的切口平移人cDNA克隆杂交的技术，对胶原蛋白mRNA进行了定量。此外，我们研究了经处理的成纤维细胞和对照成纤维细胞分离细胞核中胶原蛋白mRNA的相对转录速率。对β-肌动蛋白mRNA转录进行类似的定量分析，其不受纤维化因子处理的影响，用作内部对照。我们证明，纤维化因子使前α1（I）和前α2（I）基因的转录速率增加了4 - 6倍。最后，我们还表明，在用纤维化因子处理的细胞中，胶原蛋白的细胞内降解速率没有显著改变。这些结果与无细胞翻译数据相结合，有力地表明，在经纤维化因子处理的成纤维细胞中I型胶原蛋白mRNA积累的增加是胶原蛋白mRNA转录速率增强的结果。
