Bhandari B, Woodruff K, Abboud H E
Department of Medicine, University of Texas Health Science Center at San Antonio.
Mol Cell Biochem. 1994 Nov 9;140(1):31-6. doi: 10.1007/BF00928363.
We have investigated the effect of phorbol 12-myristate 13-acetate (PMA) on platelet-derived growth factor (PDGF) B-chain gene transcription as well as on mRNA stability in cultured human mesangial cells. Addition of actinomycin to cells stimulated with PMA decreases steady state levels of PDGF-B chain mRNA analysed by solution hybridization assay. PDGF-B chain gene transcription was also assayed directly by measuring elongation of transcripts in isolated nuclei followed by hybridization of labeled RNA transcripts to a cDNA encoding for PDGF-B chain. Our data show that PMA induces PDGF-B chain gene transcription by approximately 2-fold. alpha-Amanitin, an RNA polymerase II inhibitor, blocked transcription by more than 70%. In addition, we determined the effect of PMA on the halflife of PDGF-B chain mRNA directly by pulse chase method. In human mesangial cells, the PDGF-B chain mRNA exhibited halflife of approximately 105 min. In the presence of PMA, the halflife of PDGF-B chain mRNA was reduced to approximately 72 min. These studies indicate that regulation of PDGF-B chain gene by PMA in human mesangial cells involves a coordinate effort at the level of transcription and mRNA stability.
我们研究了佛波酯12 -肉豆蔻酸酯13 -乙酸酯（PMA）对培养的人系膜细胞中血小板衍生生长因子（PDGF）B链基因转录以及mRNA稳定性的影响。在用PMA刺激的细胞中加入放线菌素会降低通过溶液杂交分析测定的PDGF - B链mRNA的稳态水平。还通过测量分离细胞核中转录本的延伸，然后将标记的RNA转录本与编码PDGF - B链的cDNA杂交，直接测定了PDGF - B链基因的转录。我们的数据表明，PMA可使PDGF - B链基因转录增加约2倍。RNA聚合酶II抑制剂α-鹅膏蕈碱可使转录受阻超过70%。此外，我们通过脉冲追踪法直接测定了PMA对PDGF - B链mRNA半衰期的影响。在人系膜细胞中，PDGF - B链mRNA的半衰期约为105分钟。在PMA存在的情况下，PDGF - B链mRNA的半衰期缩短至约72分钟。这些研究表明，PMA对人系膜细胞中PDGF - B链基因的调控涉及转录水平和mRNA稳定性的协同作用。
