Mechanisms for transcriptional activation of Reg (Regenerating gene)

在1984年，我们发现给予90%去胰腺大鼠聚（ADP-核糖）合成酶抑制剂如烟酰胺（NA）诱导胰岛再生，从而改善手术糖尿病（Diabetes 33，401）。我们从再生胰岛衍生的cDNA文库（J.Biol.Chem.263，2111，1988）中分离出基因Reg（再生基因），并证明Reg蛋白诱导β细胞再生以改善实验性糖尿病（Proc.Natl.Chem.263，2111，1988）。Acad. Sci. USA 91，3589，1994 ; Endocrinology 139，2369，1998）。Reg基因在胰岛β细胞再生过程中表达，而在正常胰岛中不表达。本研究在RINm 5 F β细胞中发现了白细胞介素（IL）-6、地塞米松（Dx）和去甲肾上腺素（NA）对Reg基因的激活。RT-PCR和免疫印迹分析显示，IL-6（5，000 u/ml）和Dx（100 nM）联合作用可诱导RINm 5 F细胞中Reg基因表达，并且诱导的Reg基因表达水平与IL-6（5，000 u/ml）的表达水平呈负相关。 ...更多信息 通过加入5 - 10 mM NA显著增强。将用于荧光素酶测定的大鼠Reg基因的一系列5 '-缺失转染到RINm 5 F细胞中。-2303 ~-81序列的缺失对IL-6/Dx/NA的诱导作用无明显影响。进一步删除至-70导致诱导的显著丧失。使用一系列突变的竞争者进行的凝胶迁移试验表明，该区域中的GC盒样序列参与诱导。事实上，在GC盒样序列中用于荧光素酶测定的两个碱基突变构建体没有显示出IL-6/Dx/NA的诱导。通过Southwestern分析揭示了一种120 kDa的蛋白质结合GC盒样序列，但针对GC盒结合蛋白如Sp1-4的抗体未能在活性转录复合物中显示任何“超移位”。这些结果表明，再生过程（例如胰岛炎）中产生的IL-6和糖皮质激素通过120 kDa因子与GC盒样序列的结合来激活Reg基因，并且NA的β细胞再生是通过增强Reg基因来实现的。激活。少

In 1984, we found that the administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide (NA) to 90% depancreatized rats induced islet regeneration, thereby ameliorating the surgical diabetes (Diabetes 33, 401). We isolated a gene, Reg (regenerating gene) from the regenerating islet-derived cDNA library (J. Biol. Chem. 263, 2111, 1988) and demonstrated that Reg protein induced β-cell regeneration to ameliorate experimental diabetes (Proc. Natl. Acad. Sci. USA 91, 3589, 1994 ; Endocrinology 139, 2369, 1998). Reg gene is expressed in the regenerative processes of β-cell but not in normal islets. Therefore, the activation of Reg gene is considered to be a key step for β-cell regeneration.In the present study, we found the activation of Reg gene by interleukin (IL)-6, dexamethasone (Dx) and NA in RINm5F β-cells. RT-PCR and immunoblot analyses revealed that the combined addition of IL-6 (5,000 u/ml) and Dx (100 nM) induced Reg gene expression in RINm5F cells, and the inducti … More on was significantly enhanced by the addition of 5 - 10 mM NA. A series of 5'-deletions of rat Reg gene for luciferase assay was transfected into RINm5F cells. Deletion of the sequence from -2303 to -81 caused no significant changes of the induction by IL-6/Dx/NA. Further deletion to -70 caused a marked loss of the induction. Gel shift assay using a series of mutated competitors suggested a GC box-like sequence in the region is involved in the induction. In fact, a two-base mutation construct for luciferase assay in the GC box-like sequence did not show the induction by IL-6/Dx/NA. A 120 kDa protein was revealed to bind the GC box-like sequence by Southwestern analysis, but antibodies against GC box binding proteins such as Sp1-4 failed to show any "super-shift" in the active transcription complex. These results suggest that IL-6 and glucocorticoids produced in regenerative processes such as insulitis activate Reg gene via the binding of the 120 kDa factor to the GC box-like sequence and that β-cell regeneration by NA is achieved by the enhancement of the Reg gene activation. Less