Doyle G A, Saarialho-Kere U K, Parks W C
Department of Medicine (Dermatology), Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1997 Mar 4;36(9):2492-500. doi: 10.1021/bi962161e.
Upon exposure to 12-O-tetradecanoylphorbol 13-acetate (PMA), promonocyte-like U937 cells differentiate into macrophage-like cells and begin to express certain metalloproteinases and TIMP-1. We report here that distinct mechanisms regulate TIMP-1 production in PMA-treated U937 cells. TIMP-1 protein and steady-state mRNA levels increased about 10-fold in PMA-differentiated cells compared to undifferentiated cells. TIMP-1 transcription increased about 2.5-fold, but this stimulation was not detected until at least 48 h post-PMA. In contrast, the half-life for TIMP-1 mRNA increased about 3-fold and was detected at 8 h post-PMA. Using in vitro translation assays, we found that TIMP-1 mRNA from PMA-differentiated cells translated about 5-fold less efficiently than that from basal cells, suggesting structural differences in TIMP-1 mRNA in basal and differentiated U937 cells. Although primer extension and RNase protection analyses showed 5' heterogeneity of TIMP-1 transcripts, all forms were equally stimulated in response to PMA-mediated differentiation. The poly(A) tail length of TIMP-1 mRNA, however, was longer in PMA-treated cells. Our findings suggested that up-regulation of TIMP-1 expression in PMA treated U937 cells is mediated early by enhanced TIMP-1 mRNA stability, possibly related to increased poly(A) tail length, and later by an increase in transcription rate.
在暴露于12-O-十四烷酰佛波醇-13-乙酸酯（PMA）后，前单核细胞样U937细胞分化为巨噬细胞样细胞，并开始表达某些金属蛋白酶和TIMP-1。我们在此报告，不同的机制调节PMA处理的U937细胞中TIMP-1的产生。与未分化细胞相比，PMA分化细胞中TIMP-1蛋白和稳态mRNA水平增加了约10倍。TIMP-1转录增加了约2.5倍，但这种刺激直到PMA处理后至少48小时才被检测到。相反，TIMP-1 mRNA的半衰期增加了约3倍，并在PMA处理后8小时被检测到。使用体外翻译试验，我们发现PMA分化细胞中的TIMP-1 mRNA翻译效率比基础细胞中的低约5倍，这表明基础和分化的U937细胞中TIMP-1 mRNA存在结构差异。尽管引物延伸和核糖核酸酶保护分析显示TIMP-1转录本存在5'异质性，但所有形式在PMA介导的分化反应中均受到同等刺激。然而，PMA处理细胞中TIMP-1 mRNA的聚腺苷酸尾长度更长。我们的研究结果表明，PMA处理的U937细胞中TIMP-1表达的上调早期由TIMP-1 mRNA稳定性增强介导，可能与聚腺苷酸尾长度增加有关，后期由转录速率增加介导。
