Mechanisms for transcriptional activation of Reg (Regenerating gene)

1984年，我们发现给90%的去胰岛大鼠应用聚(ADP-核糖)合成酶抑制剂如烟酰胺(NA)可诱导胰岛再生，从而改善外科糖尿病(糖尿病33,401)。我们从再生的胰岛来源的cDNA文库(J.Biol.化学。263、2111、1988)，证明了Reg蛋白诱导β细胞再生以改善实验性糖尿病(Proc.娜塔莉。阿卡德。SCI。美国91,3589,1994；内分泌学139,2369,1998)。REG基因在β-细胞的再生过程中表达，但在正常胰岛中不表达。因此，REG基因的激活被认为是β-细胞再生的关键步骤。在本研究中，我们发现在RINm5F-β细胞中，IL-6、地塞米松(Dx)和去甲肾上腺素(NA)可以激活REG基因。RT-PCR法和免疫印迹分析表明，IL-6(5000u/ml)和Dx(100nM)联合作用可诱导RINm5F细胞REG基因的表达，并诱导…添加5-10 mM的NA显著提高了更多的On。将一系列5‘端缺失的大鼠荧光素酶基因导入RINm5F细胞。从-2303到-81的序列缺失没有引起IL-6/Dx/NA诱导的显著变化。进一步缺失到-70会导致诱导作用的显著丧失。使用一系列突变的竞争对手进行的凝胶漂移分析表明，该区域的GC盒样序列参与了诱导。事实上，在GC盒样序列中用于荧光素酶检测的双碱基突变结构没有显示出IL-6/Dx/NA的诱导作用。经Southwest分析，发现一个120 kDa的蛋白与GC盒结合，但抗GC盒结合蛋白的抗体如Sp1-4在活性转录复合体中没有表现出任何“超移位”。这些结果表明，IL-6和再生过程中产生的糖皮质激素通过将120 kDa的因子与GC盒样序列结合来激活REG基因，NA通过增强REG基因的激活来实现β细胞的再生。较少

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