Functional antagonism between CCAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma on the leptin promoter
Functional antagonism between CCAAT/enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma on the leptin promoter
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DOI：
10.1074/jbc.272.8.5283
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发表时间：
1997-02-21
影响因子：
4.8
通讯作者：
Lowell, BB
中科院分区：
文献类型：
--
作者：
Hollenberg, AN;Susulic, VS;Lowell, BB
肥胖基因产物Leptin是食欲和脂肪细胞质量的主要激素调节因子，最近的工作表明抗糖尿病药物TZ也是PPARγ的高亲和力配体，可以抑制啮齿动物Leptin的表达，为了研究这类药物对脂肪细胞Leptin基因的影响，我们对原代培养的大鼠脂肪细胞进行Northern分析，TZ使Leptin mRNA水平降低75%，以确定这种作用是否在转录水平介导。我们分离了6510个碱基对的瘦素启动子5‘侧翼序列，并研究了原代大鼠脂肪细胞和CV-1细胞中的报告结构，序列分析表明，在-3951和-3939之间存在一个共同的直接重复序列，在-55和-47之间存在一个共同的CCAAT/增强子结合蛋白(C/EBP)位点。我们在转基因的原代大鼠脂肪细胞中的功能分析表明，尽管存在一个带有1个碱基对缺口的规范的直接重复序列，但TZ单独降低了瘦素启动子结构的报告基因表达，范围从-6510到+9到-65到+9。在含有内源性PPARγ的CV-1细胞中，单独TZ处理对这些结构物几乎没有影响，然而，TZ处理确实抑制了C/EBPα介导的瘦素启动子的反式激活，这种下调瘦素报告结构的启动子被映射到在凝胶迁移率改变分析中与C/EBPα结合的-65到+9启动子片段，但不单独结合PPARγ2或作为异二聚体与9-顺式维甲酸受体结合，相反，另一种脂肪细胞特异性基因aP2的启动子(-5400至+24碱基对)被TZ诱导7.3倍，与C/EBPα共转染对aP2启动子的激活作用最小，但被TZ显著阻断。这些数据表明，PPAR-γ和C/EBPα至少可以在两个单独的启动子上相互拮抗，这一机制可能解释了噻唑烷二酮类药物下调瘦素表达的机制。
The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass, Recent work has suggested that the antidiabetic agents, the thiazolidinediones (TZ), which are also high affinity ligands of peroxisome proliferator-activated receptor-gamma (PPAR gamma), inhibit leptin expression in rodents, To examine the effects of this class of drug on the leptin gene in adipocytes we performed Northern analysis on primary rat adipocytes cultured in the presence or absence of TZ, TZ reduced leptin mRNA levels by 75%, To determine whether this effect was mediated at the transcriptional level, we isolated 6510 base pairs of 5'-flanking sequence of the leptin promoter and studied reporter constructs in primary rat adipocytes and CV-1 cells, Sequence analysis demonstrated the presence of a consensus direct repeat with a 1-base-pair gap site between -3951 and -3939 as well as a consensus CCAAT/enhancer binding protein (C/EBP) site between -55 and -47, Our functional analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promoter constructs ranging from -6510 to +9 to -65 to +9, In CV-1 cells, which contain endogenous PPAR gamma, TZ treatment alone had little effect on these constructs, However, TZ treatment did inhibit C/EBP alpha-mediated transactivation of the leptin promoter, This down-regulation of leptin reporter constructs mapped to a -65 to +9 promoter fragment which binds C/EBP alpha in gel mobility shift assays but does not bind PPAR gamma 2 alone or as a heterodimer with 9-cis-retinoic acid receptor, Conversely, the promoter (-5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ, Co-transfection with C/EBP alpha minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ, These data indicate that PPAR gamma and C/EBP alpha can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of leptin expression by thiazolidinediones.