Liu S, Cieslinski D A, Funke A J, Humes H D
Veterans Administration Medical Center, Ann Arbor, Mich., USA.
Exp Nephrol. 1997 Jul-Aug;5(4):295-300.
BACKGROUND/AIMS: The pattern-forming event of kidney tubulogenesis is initiated by the inductive transition of mesenchymal cells to epithelial phenotype; a transition that is critically dependent on the regulated expression of the developmental control gene, Pax-2. Because of a defined role in in vitro renal tubulogenesis, the effects of epidermal growth factor (EGF), transforming growth factor (TGF)-beta 1 and retinoic acid on Pax-2 gene expression in proximal tubule cells (PTC) were evaluated.
Rabbit PTC were isolated and grown in tissue culture. Under confluent quiescent conditions, the effect of various factors on Pax-2 gene expression was assessed by Northern blot analysis. To assess whether the effect of TGF-beta 1 to alter Pax-2 gene expression was due to transcriptional or posttranscriptional events, nuclear run-on assays were also undertaken.
Under control, confluent growth conditions, PTC expressed high levels of Pax-2. A 24-hour exposure to EGF (10 nM), a potent mitogen of PTC, increased this level of expression. In contrast, Pax-2 gene expression was suppressed by treating PTC with retinoic acid (10 mM), a well-described differentiating factor, and with TGF-beta 1 (10 ng/ml), a recognized antiproliferative agent for these cells, which suggests that Pax-2 has a role in renal cell proliferation. The mechanism of the effect of TGF-beta 1 on Pax-2 mRNA levels was further detailed. TGF-beta 1 did not affect Pax-2 transcription rates, as assessed by nuclear run-on assays; however, in a dose-dependent manner, it diminished the stability of Pax-2 mRNA. At a concentration of 10 ng/ml, TGF-beta 1 reduced Pax-2 mRNA stability from a control half-life of 120 min to a half-life of less than 60 min.
This study demonstrates that various soluble inductive factors affect Pax-2 gene expression in renal tubule cells. Also, TGF-beta 1 downregulates Pax-2 gene expression through a posttranscriptional process, an acknowledged mechanism for modulating important growth regulatory gene products.
背景/目的：肾小管形成的模式形成事件由间充质细胞向上皮表型的诱导转变引发；这种转变严重依赖于发育控制基因Pax-2的调控表达。由于表皮生长因子（EGF）、转化生长因子（TGF）-β1和视黄酸在体外肾小管形成中具有明确作用，因此评估了它们对近端小管细胞（PTC）中Pax-2基因表达的影响。
分离兔PTC并在组织培养中生长。在汇合静止条件下，通过Northern印迹分析评估各种因素对Pax-2基因表达的影响。为了评估TGF-β1改变Pax-2基因表达的作用是由于转录事件还是转录后事件，还进行了核转录分析。
在对照汇合生长条件下，PTC表达高水平的Pax-2。用PTC的一种有效促有丝分裂原EGF（10 nM）处理24小时可增加这种表达水平。相反，用视黄酸（10 mM，一种众所周知的分化因子）和TGF-β1（10 ng/ml，这些细胞公认的抗增殖剂）处理PTC可抑制Pax-2基因表达，这表明Pax-2在肾细胞增殖中起作用。进一步详细研究了TGF-β1对Pax-2 mRNA水平影响的机制。通过核转录分析评估，TGF-β1不影响Pax-2转录速率；然而，它以剂量依赖方式降低了Pax-2 mRNA的稳定性。在10 ng/ml浓度下，TGF-β1将Pax-2 mRNA稳定性从对照半衰期120分钟降低至小于60分钟的半衰期。
本研究表明各种可溶性诱导因子影响肾小管细胞中Pax-2基因表达。此外，TGF-β1通过转录后过程下调Pax-2基因表达，这是调节重要生长调节基因产物的公认机制。
