Gougat Claire, Jaffuel Dany, Gagliardo Rosalia, Henriquet Corinne, Bousquet Jean, Demoly Pascal, Mathieu Marc
Institut National de la Santé et de la Recherche Médicale U454-IFR3 and Service des Maladies Respiratoires, CHU de Montpellier, 34295 Montpellier Cedex 5, France.
J Mol Med (Berl). 2002 May;80(5):309-18. doi: 10.1007/s00109-001-0302-6. Epub 2001 Dec 7.
The human glucocorticoid receptor isoforms GRalpha and GRbeta are generated by alternative splicing. Upon hormone binding, GRalpha regulates positively or negatively transcription. In particular, it represses numerous genes encoding pro-inflammatory mediators by inhibiting the transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB. The observation that GRbeta, which does not bind the hormone, may act as a dominant negative receptor is subject to controversy. Because GRbeta must be more abundant than GRalpha to act as such, we evaluated the relative amounts of GRalpha and GRbeta in COS-1, A549 and HeLa cells using a monoclonal antibody that recognises the two isoforms equally well on western blots. Messenger RNA levels of GRalpha and GRbeta were compared by reverse transcriptase polymerase chain reaction analysis. To gain insight into the possible function of GRbeta, we examined the ability of overexpressed GRbeta to alter transcription of glucocorticoid, AP-1 and NF-kappaB inducible reporter genes using transient transfection in COS-1 and A549 cells. Subcellular localisation of GRbeta was determined in A549 cells by immunofluoresence microscopy. Data indicate that GRalpha is the predominant endogenous isoform in A549 and HeLa cells. GRbeta became the major form after transfection with the corresponding expression vector and translocated into cell nuclei even in the absence of hormone. Overexpression of GRbeta inhibited glucocorticoid-induced transcription markedly in COS-1 cells but weakly in A549 cells. We found that GRbeta did not act as a dominant negative modulator of GRalpha for repression of AP-1 and NF-kappaB activities. In fact, both GRbeta and GRalpha inhibited hormone-independently these activities by 25-60%. This property was not shared by the closely related mineralocorticoid receptor. Our results suggest that overexpression of either GRalpha or GRbeta may have an anti-inflammatory effect.
人类糖皮质激素受体亚型GRα和GRβ是通过可变剪接产生的。激素结合后，GRα对转录起正向或负向调节作用。特别是，它通过抑制转录因子激活蛋白（AP）-1和核因子（NF）-κB来抑制众多编码促炎介质的基因。不与激素结合的GRβ可能作为显性负性受体起作用这一观点存在争议。由于GRβ必须比GRα更丰富才能如此发挥作用，我们使用一种在蛋白质免疫印迹上能同等良好识别这两种亚型的单克隆抗体，评估了COS-1、A549和HeLa细胞中GRα和GRβ的相对含量。通过逆转录聚合酶链反应分析比较了GRα和GRβ的信使核糖核酸水平。为深入了解GRβ的可能功能，我们在COS-1和A549细胞中通过瞬时转染，检测了过表达的GRβ改变糖皮质激素、AP-1和NF-κB诱导型报告基因转录的能力。通过免疫荧光显微镜在A549细胞中确定了GRβ的亚细胞定位。数据表明，GRα是A549和HeLa细胞中的主要内源性亚型。用相应表达载体转染后，GRβ成为主要形式，即使在无激素的情况下也会转位到细胞核中。GRβ的过表达在COS-1细胞中显著抑制糖皮质激素诱导的转录，但在A549细胞中抑制作用较弱。我们发现，GRβ在抑制AP-1和NF-κB活性方面并非作为GRα的显性负性调节因子起作用。事实上，GRβ和GRα均能不依赖激素抑制这些活性达25% - 60%。密切相关的盐皮质激素受体不具有此特性。我们的结果表明，GRα或GRβ的过表达可能具有抗炎作用。
