Bladh Lars-Göran, Johansson-Haque Krishan, Rafter Ingalill, Nilsson Stefan, Okret Sam
Department of Biosciences and Nutrition, Karolinska Institutet, Novum, Huddinge, Sweden.
Biochim Biophys Acta. 2009 Mar;1793(3):439-46. doi: 10.1016/j.bbamcr.2008.11.013. Epub 2008 Dec 9.
Glucocorticoid (GC) effects are mediated via the GC-receptor (GR), which either stimulates or represses gene expression. Repression of target genes often involves negative cross-talk between the GR and other transcription factors e.g. NF-kappaB, important for gene activation. Using HEK293 cells we here describe that repression of NF-kappaB requires functions of the GR that are dependent on the signaling pathways employed to activate NF-kappaB. While a GR mutant was able to repress NF-kappaB activity following activation by TNFalpha, it did not so following activation by the phorbol ester TPA. In these cells, TPA stimulation but not TNFalpha, activated extracellular signal-regulated kinase (ERK). We demonstrated that the ability of the dexamethasone activated GR mutant to repress TPA-induced NF-kappaB activity was restored in conjunction with ERK1/2 inhibition. Previous reports have shown GC-mediated inhibition of ERK1/2 phosphorylation to involve GC induction of MAPK phosphatase-1 (MKP-1). Here, we demonstrated that the GRR488Q mutant was incapable of inducing gene expression of endogenous MKP-1 following dexamethasone treatment, in contrast to the GRwt. However, TPA treatment alone resulted in much stronger MKP-1 expression in both GRwt and GRR488Q containing cells than that of dexamethasone suggesting that the inability of GRR488Q to inhibit TPA-induced NF-kappaB activity did not involve a lack of MKP-1 expression. In line with this, RNAi targeted towards MKP-1 did not abolish or inhibit the ability of the GRwt to repress NF-kappaB activity. Importantly, we observed no difference in activated ERK1/2 (phospho-ERK1/2) expression over time between GRwt and GRR488Q containing cells following co-treatment with TPA and dexamethasone. Based on these results we suggest that GRwt does not directly regulate ERK1/2 but rather alters ERK1/2-mediated effects allowing it to repress NF-kappaB activity, a capacity lacked by the GRR488Q mutant.
糖皮质激素（GC）的作用是通过糖皮质激素受体（GR）介导的，GR可刺激或抑制基因表达。对靶基因的抑制通常涉及GR与其他转录因子（如对基因激活很重要的核因子κB（NF-κB））之间的负性相互作用。我们在此利用人胚肾293（HEK293）细胞描述，对NF-κB的抑制需要GR的功能，而这些功能取决于用于激活NF-κB的信号通路。虽然一个GR突变体在被肿瘤坏死因子α（TNFα）激活后能够抑制NF-κB活性，但在被佛波酯TPA激活后却不能。在这些细胞中，TPA刺激而非TNFα刺激激活了细胞外信号调节激酶（ERK）。我们证明，地塞米松激活的GR突变体抑制TPA诱导的NF-κB活性的能力在ERK1/2受到抑制时得以恢复。先前的报道显示，GC介导的对ERK1/2磷酸化的抑制涉及GC对丝裂原活化蛋白激酶磷酸酶-1（MKP-1）的诱导。在此，我们证明，与野生型GR（GRwt）相比，GRR488Q突变体在地塞米松处理后不能诱导内源性MKP-1的基因表达。然而，单独的TPA处理在含有GRwt和GRR488Q的细胞中导致的MKP-1表达比地塞米松处理更强，这表明GRR488Q不能抑制TPA诱导的NF-κB活性并不涉及MKP-1表达的缺乏。与此一致的是，针对MKP-1的RNA干扰并未消除或抑制GRwt抑制NF-κB活性的能力。重要的是，在用TPA和地塞米松共同处理后，我们观察到含有GRwt和GRR488Q的细胞之间，随着时间推移，活化的ERK1/2（磷酸化ERK1/2）表达没有差异。基于这些结果，我们认为GRwt并不直接调节ERK1/2，而是改变ERK1/2介导的效应，使其能够抑制NF-κB活性，而GRR488Q突变体缺乏这种能力。
