Pendaries Valérie, Verrecchia Franck, Michel Serge, Mauviel Alain
INSERM U532, Université Paris VII, Institut de Recherche sur la Peau, Pavillon Bazin, Hôpital Saint-Louis, 1, avenue Claude Vellefaux, F-75475 Paris Cedex 10, France.
Oncogene. 2003 Nov 6;22(50):8212-20. doi: 10.1038/sj.onc.1206913.
Transforming growth factor-beta (TGF-beta) and retinoic acid (RA) are important regulators of cell growth and differentiation. The TGF-beta receptors utilize Smad proteins to transduce signals intracellularly and regulate transcription of target genes, either directly or in combination with other sequence-specific transcription factors. Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors, are involved in mediating transcriptional responses to RA. Given the known interactions between the TGF-beta and RAR pathways, we have investigated the role played by RAR ligands in modulating functional interactions between Smad3 and RARs. Using transient cell transfection experiments with an artificial Smad3/Smad4-dependent reporter construct, we demonstrate that RAR overexpression enhances Smad-driven transactivation, an effect that requires both Smad3 and Smad4. We provide evidence that RAR effect on Smad3/Smad4-driven transcription is prevented by natural and synthetic RAR agonists, and potentiated by synthetic RAR antagonists. The activity of two TGF-beta-responsive human gene promoter constructs was regulated in a parallel fashion. Using both mammalian two-hybrid and immunoprecipitation/Western methods, we demonstrate a direct interaction between the region DEF of RARgamma and the MH2 domain of Smad3, inhibited by RAR agonists and enhanced by their antagonists. We propose that RARs may function as coactivators of the Smad pathway in the absence of RAR agonists or in the presence of their antagonists, a phenomenon that contrasts with their known role as agonist-activated transcriptional regulators of RA-dependent genes.
转化生长因子-β（TGF-β）和视黄酸（RA）是细胞生长和分化的重要调节因子。TGF-β受体利用Smad蛋白在细胞内转导信号，并直接或与其他序列特异性转录因子结合来调节靶基因的转录。两类核受体，即视黄酸受体（RARs）和视黄酸X受体，参与介导对RA的转录反应。鉴于TGF-β和RAR途径之间已知的相互作用，我们研究了RAR配体在调节Smad3和RARs之间功能相互作用中所起的作用。通过使用人工Smad3/Smad4依赖性报告构建体进行瞬时细胞转染实验，我们证明RAR的过表达增强了Smad驱动的反式激活，这一效应需要Smad3和Smad4两者。我们提供的证据表明，天然和合成的RAR激动剂可阻止RAR对Smad3/Smad4驱动转录的影响，而合成的RAR拮抗剂则可增强这种影响。两种TGF-β反应性人类基因启动子构建体的活性以平行方式受到调节。使用哺乳动物双杂交和免疫沉淀/蛋白质印迹方法，我们证明RARγ的DEF区域与Smad3的MH2结构域之间存在直接相互作用，RAR激动剂可抑制这种相互作用，而其拮抗剂则可增强这种相互作用。我们提出，在没有RAR激动剂或存在其拮抗剂的情况下，RARs可能作为Smad途径的共激活因子发挥作用，这一现象与其作为RA依赖性基因的激动剂激活转录调节因子的已知作用形成对比。
