Tsukada J, Saito K, Waterman W R, Webb A C, Auron P E
Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115.
Mol Cell Biol. 1994 Nov;14(11):7285-97. doi: 10.1128/mcb.14.11.7285-7297.1994.
A site located between -2782 and -2729 of the human prointerleukin-1 beta (IL1B) gene functions as a strong lipopolysaccharide (LPS)-responsive enhancer independent of the previously identified enhancer located between -2896 and -2846 (F. Shirakawa, K. Saito, C.A. Bonagura, D.L. Galson, M. J. Fenton, A. C. Webb, and P. E. Auron, Mol. Cell. Biol. 13:1332-1344, 1993). Although these two enhancers appear to function cooperatively in the native sequence context, they function independently as LPS-responsive elements upon removal of an interposed silencer sequence. The new enhancer is not induced by dibutyryl cyclic AMP (dbcAMP) alone but is superinduced by costimulation with LPS-dbcAMP. This pattern of induction depends upon the nature of the sequence, a composite NF-IL6-cAMP response element (CRE) binding site. This pseudosymmetrical sequence is shown to contrast with a classical symmetric CRE which responds to dbcAMP but not LPS. DNA binding studies using in vivo nuclear extract, recombinant proteins, and specific antibodies show that LPS induces the formation of two different complexes at the enhancer: (i) an NF-IL6-CREB heterodimer and (ii) a heterodimer consisting of NF-IL6 and a non-CREB, CRE-binding protein. Cotransfection studies using NF-IL6 and CREB expression vectors show that NF-IL6 transactivates the enhancer in the presence of LPS, whereas CREB acts either positively or negatively, depending upon its cAMP-regulated phosphorylation state. Our data demonstrate that the newly identified enhancer is a specialized LPS-responsive sequence which can be modulated by cAMP as a result of the involvement of NF-IL6-CRE-binding protein heterodimers.
人白细胞介素-1β（IL1B）基因-2782至-2729之间的一个位点作为一个强大的脂多糖（LPS）反应增强子发挥作用，该增强子独立于先前鉴定的位于-2896至-2846之间的增强子（F. Shirakawa、K. Saito、C.A. Bonagura、D.L. Galson、M.J. Fenton、A.C. Webb和P.E. Auron, Mol. Cell. Biol. 13:1332 - 1344, 1993）。尽管这两个增强子在天然序列背景下似乎协同发挥作用，但在去除插入的沉默子序列后，它们作为LPS反应元件独立发挥作用。新的增强子单独不受二丁酰环磷酸腺苷（dbcAMP）诱导，但与LPS - dbcAMP共刺激时会被超诱导。这种诱导模式取决于序列的性质，即一个复合的NF - IL6 - 环磷酸腺苷反应元件（CRE）结合位点。该假对称序列与经典对称CRE形成对比，经典对称CRE对dbcAMP有反应，但对LPS无反应。使用体内核提取物、重组蛋白和特异性抗体进行的DNA结合研究表明，LPS在增强子处诱导形成两种不同的复合物：（i）一种NF - IL6 - CREB异二聚体和（ii）一种由NF - IL6和一种非CREB的CRE结合蛋白组成的异二聚体。使用NF - IL6和CREB表达载体进行的共转染研究表明，在LPS存在的情况下，NF - IL6可反式激活增强子，而CREB的作用则取决于其cAMP调节的磷酸化状态，可能为正向或负向。我们的数据表明，新鉴定的增强子是一个专门的LPS反应序列，由于NF - IL6 - CRE结合蛋白异二聚体的参与，其可被cAMP调节。
