HIGH GLUCOSE AND TGF-BETA(1), STIMULATE FIBRONECTIN GENE-EXPRESSION THROUGH A CAMP RESPONSE ELEMENT
HIGH GLUCOSE AND TGF-BETA(1), STIMULATE FIBRONECTIN GENE-EXPRESSION THROUGH A CAMP RESPONSE ELEMENT
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DOI：
10.1038/ki.1994.362
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发表时间：
1994-10-01
影响因子：
19.6
通讯作者：
AYO, SH
中科院分区：
文献类型：
--
作者：
KREISBERG, JI;GARONI, JA;AYO, SH
以前，我们报告说，肾小球系膜细胞增加纤维连接蛋白，层粘连蛋白和IV型胶原蛋白的合成时，在高葡萄糖（30 mM）的存在下培养。尽管所有三种细胞外基质（ECM）蛋白的mRNA水平在高糖条件下也增加，但这种增加的机制尚不清楚。为了确定所观察到的纤连蛋白mRNA水平的增加是否涉及转录的增加，用含有与氯霉素乙酰转移酶（CAT）基因的编码区[FN-CAT（-510）]融合的纤连蛋白（FN）基因的5 '侧翼区[位置+69至-510碱基对（bp）]的构建体转染肾小球系膜细胞。用该构建体瞬时且稳定地转染细胞。在无血清条件下，高糖仅在TGF β（1）（称为TGF β）存在的情况下增加CAT活性。由于FN-CAT（-510）含有血清响应元件，因此在无血清条件下进行实验。CAT的增加在瞬时转染的细胞中约为两倍，在稳定转染的细胞中约为三倍。TGF β单独增加CAT活性约30%。纤连蛋白基因表达的刺激似乎发生在位于FN基因的~ 170 bp的cAMP反应元件（CRE）水平，因为用含有编码该CRE的寡核苷酸的构建体转染的细胞融合到最小纤连蛋白启动子（~ 56 bp）和CAT报告基因[CRE（~ 170）FN-CAT]。在用高糖和TGF β处理后，CAT活性显示出类似的增加。CRE寡核苷酸的凝胶迁移率分析显示与系膜细胞核蛋白的多个复合物。在测试的不同条件下，没有观察到复合物的流动性或丰度的差异。
Previously, we reported that mesangial cells increased fibronectin, laminin and type IV collagen synthesis when cultured in the presence of high glucose (30 mM). Although mRNA levels for all three extracellular matrix (ECM) proteins were also increased in high glucose conditions, the mechanism for this increase was not known. In order to determine whether increased transcription was involved in the observed increase in fibronectin mRNA levels mesangial cells were transfected with a construct containing the 5'-flanking region of the fibronectin (FN) gene [position +69 to -510 base pairs (bp)] fused to the coding region of the chloramphenicol acetyltransferase (CAT) gene [FN-CAT (-510)]. Cells were transiently and stably transfected with this construct. Under serum-free conditions, high glucose increased CAT activity only in the presence of TGF beta(1) (referred to as TGF beta). The experiments were performed without serum because FN-CAT (-510) contains a serum responsive element. The increase in CAT was approximately twofold in transiently transfected cells and threefold in stably transfected cells. TGF beta alone increased CAT activity approximately 30%. Stimulation of fibronectin gene expression appeared to occur at the level of a cAMP response element (CRE) located -170 bp of the FN gene because cells transfected with a construct containing an oligonucleotide encoding for this CRE fused to a minimal fibronectin promoter (-56 bp) and a CAT reporter gene [CRE (-170) FN-CAT] displayed similar increments of CAT activity after treatment with high glucose and TGF beta. Gel shift mobility assays with a CRE oligonucleotide revealed multiple complexes with mesangial cell nuclear proteins. No differences in the mobility or abundance of complexes were observed in the different conditions tested.