Lindquist Jeffrey N, Parsons Christopher J, Stefanovic Branko, Brenner David A
Biochemistry and Biophysics and Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2004 May 28;279(22):23822-9. doi: 10.1074/jbc.M314060200. Epub 2004 Feb 17.
Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs). Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein alphaCP binds to the alpha1(I) collagen 3'-untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of alphaCP in the decay of transcripts containing the collagen 3'-UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, alphaCP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of alphaCP binding activity increased the rate of decay of wild type transcripts containing the alphaCP 3'-UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no alphaCP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for alphaCP. The addition of recombinant alphaCP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain alphaCP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting alphaCP showed a reduction of the binding activity of alphaCP and a concomitant reduction in intracellular levels of alpha1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of alphaCP in the stabilization of alpha1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs.
肝纤维化的特征是活化的肝星状细胞（HSCs）使包括I型胶原蛋白在内的细胞外基质蛋白沉积增加。先前的研究表明，这种增加主要由转录后机制介导。特别是，RNA结合蛋白αCP与α1（I）胶原蛋白3'-非翻译区（UTR）结合，并在活化而非静止的HSCs中使该RNA稳定。本研究探讨了αCP在从NIH成纤维细胞以及静止和活化的HSCs中提取的提取物中对含胶原蛋白3'-UTR转录本衰变的作用。使用体外衰变系统，通过添加野生型寡核苷酸可竞争出αCP结合活性，而添加突变型寡核苷酸则不能。αCP结合活性的竞争增加了含αCP 3'-UTR结合位点的野生型转录本的衰变率，但含突变结合位点的转录本的衰变率没有增加。静止的HSC提取物不具有αCP结合活性，并且在具有野生型和突变型αCP结合位点的转录本的衰变率上没有差异。添加重组αCP足以增加野生型转录本的半衰期，而突变型转录本的半衰期变化很小。用含有αCP结合活性活化的HSC提取物进行的体外衰变试验表明，与突变型转录本相比，野生型转录本的衰变率明显降低。针对αCP的体内小干扰RNA实验表明，αCP的结合活性降低，同时α1（I）胶原蛋白信使RNA的细胞内水平也降低。总之，本研究证明了αCP通过在活化的HSCs中阻断RNA降解，在稳定α1（I）胶原蛋白信使RNA方面的直接作用。
