Pape M E, Kim K H
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Mol Cell Biol. 1989 Mar;9(3):974-82. doi: 10.1128/mcb.9.3.974-982.1989.
Acetyl coenzyme A (acetyl-CoA) carboxylase activity, amount, and mRNA levels increase during the differentiation of 30A-5 preadipocytes to adipocytes. Tumor necrosis factor (TNF) completely prevents this differentiation, with concomitant inhibition of acetyl-CoA carboxylase mRNA accumulation. To investigate the mechanisms by which TNF prevents acetyl-CoA carboxylase mRNA accumulation, we determined the effect of TNF on the transcription rate of the carboxylase gene and the half-life of carboxylase mRNA. Nuclear runoff transcription assays revealed no differences in the number of RNA polymerase molecules actively engaged in transcription of the acetyl-CoA carboxylase gene in preadipocytes, adipocytes, TNF-treated preadipocytes, or at any time during the course of differentiation. However, changes in adipsin, glycerophosphate dehydrogenase, and actin mRNAs, whose levels are also differentiation dependent, can be accounted for in part by changes in the number of polymerase complexes on their respective genes. To determine whether TNF caused a decrease in the stability of carboxylase RNA transcripts, we measured the rate of decay of prelabeled acetyl-CoA carboxylase mRNA. Control and TNF-treated cells showed no difference between the apparent half-lives of acetyl-CoA carboxylase mRNAs (9 h). However, the rate of acetyl-CoA carboxylase mRNA synthesis in vivo was decreased three- to fourfold in the presence of TNF. These data demonstrate that TNF prevents accumulation of acetyl-CoA carboxylase mRNA during preadipocyte differentiation by decreasing the rate of acetyl-CoA carboxylase gene transcription. However, transcriptional control is not due to a change in the number of RNA polymerase complexes actively engaged in carboxylase transcript elongation which could be measured by a number runoff assay. Instead, transcriptional control may be related to the rate at which RNA polymerase traverses the acetyl-CoA carboxylase gene.
在30A - 5前脂肪细胞向脂肪细胞分化过程中，乙酰辅酶A（乙酰 - CoA）羧化酶的活性、含量及mRNA水平均会升高。肿瘤坏死因子（TNF）可完全抑制这种分化，并同时抑制乙酰 - CoA羧化酶mRNA的积累。为研究TNF抑制乙酰 - CoA羧化酶mRNA积累的机制，我们测定了TNF对羧化酶基因转录速率及羧化酶mRNA半衰期的影响。核转录延伸分析显示，在前脂肪细胞、脂肪细胞、TNF处理的前脂肪细胞或分化过程中的任何时间，积极参与乙酰 - CoA羧化酶基因转录的RNA聚合酶分子数量均无差异。然而，脂肪分化蛋白、甘油磷酸脱氢酶和肌动蛋白mRNA水平的变化同样依赖于分化，其部分变化可归因于各自基因上聚合酶复合物数量的改变。为确定TNF是否导致羧化酶RNA转录本稳定性下降，我们测量了预先标记的乙酰 - CoA羧化酶mRNA的衰减速率。对照细胞和TNF处理的细胞中，乙酰 - CoA羧化酶mRNA的表观半衰期（9小时）并无差异。然而，在TNF存在的情况下，体内乙酰 - CoA羧化酶mRNA的合成速率降低了三到四倍。这些数据表明，TNF通过降低乙酰 - CoA羧化酶基因的转录速率，阻止前脂肪细胞分化过程中乙酰 - CoA羧化酶mRNA的积累。然而，转录控制并非由于积极参与羧化酶转录延伸的RNA聚合酶复合物数量的改变（这可通过核转录延伸分析来测量）。相反，转录控制可能与RNA聚合酶穿越乙酰 - CoA羧化酶基因的速率有关。
