Tumor necrosis factor-induced long myosin light chain kinase transcription is regulated by differentiation-dependent signaling events - Characterization of the human long myosin light chain kinase promoter
Tumor necrosis factor-induced long myosin light chain kinase transcription is regulated by differentiation-dependent signaling events - Characterization of the human long myosin light chain kinase promoter
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DOI：
10.1074/jbc.m602164200
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发表时间：
2006-09-08
影响因子：
4.8
通讯作者：
Turner, Jerrold R.
中科院分区：
文献类型：
--
作者：
Graham, W. Vallen;Wang, Fengjun;Turner, Jerrold R.
肌球蛋白轻链激酶（MLCK）在单个基因内的独特转录起始位点表达为长和短同种型。肿瘤坏死因子（TNF）增强肠上皮细胞长MLCK表达，这对细胞骨架调节至关重要。我们发现，TNF增加长MLCK mRNA的转录，无论是在体外人肠上皮细胞和小鼠肠上皮细胞在体内。5 '-RACE鉴定了两个新的外显子1A和1B，它们编码交替的长MLCK转录起始位点。染色质免疫沉淀（ChIP）和定点突变确定了两个必不可少的Sp1位点的外显子1A长MLCK转录起始位点上游。缺失和截短突变体的分析表明，一个102 bp的区域，包括这些Sp1位点是必要的基础转录。包括外显子1A上游4-kb的启动子构建体对TNF、AP-1或NF κ B有应答，但在较短的2-kb构建体中，除了NF κ B应答外，所有应答均不存在，在1-kb构建体中，所有应答均不存在。电泳迁移率变动分析、ChIP和定点突变通过鉴定外显子1A上游2- 4 kb之间的三个功能性AP-1位点和外显子1A上游1- 2 kb之间的两个NF κ B位点来解释这些数据。分化上皮细胞的分析表明，只有分化良好的肠上皮细胞激活的4 kb长的MLCK启动子响应TNF，和共识启动子报告表明，TNF诱导的NF κ B B激活减少分化过程中，而TNF诱导的AP-1激活增加。因此，AP-1或NF κ B可以上调长MLCK转录，但TNF上调肠上皮长MLCK转录外显子1A的机制是分化依赖性的。
Myosin light chain kinase (MLCK) is expressed as long and short isoforms from unique transcriptional start sites within a single gene. Tumor necrosis factor (TNF) augments intestinal epithelial long MLCK expression, which is critical to cytoskeletal regulation. We found that TNF increases long MLCK mRNA transcription, both in human enterocytes in vitro and murine enterocytes in vivo. 5'-RACE identified two novel exons, 1A and 1B, which encode alternative long MLCK transcriptional start sites. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis identified two essential Sp1 sites upstream of the exon 1A long MLCK transcriptional start site. Analysis of deletion and truncation mutants showed that a 102-bp region including these Sp1 sites was necessary for basal transcription. A promoter construct including 4-kb upstream of exon 1A was responsive to TNF, AP-1, or NF kappa B, but all except NF kappa B responses were absent in a shorter 2-kb construct, and all responses were absent in a 1-kb construct. Electrophoretic mobility shift assays, ChIP, and site-directed mutagenesis explained these data by identifying three functional AP-1 sites between 2- and 4-kb upstream of exon 1A and two NF kappa B sites between 1- and 2-kb upstream of exon 1A. Analysis of differentiating epithelia showed that only well differentiated enterocytes activated the 4-kb long MLCK promoter in response to TNF, and consensus promoter reporters demonstrated that TNF-induced NF kappa B activation decreased during differentiation while TNF-induced AP-1 activation increased. Thus either AP-1 or NF kappa B can up-regulate long MLCK transcription, but the mechanisms by which TNF up-regulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.