Reddy S P, Chuu Y J, Lao P N, Donn J, Ann D K, Wu R
California Regional Primate Research Center, School of Medicine, University of California at Davis 95616, USA.
J Biol Chem. 1995 Nov 3;270(44):26451-9. doi: 10.1074/jbc.270.44.26451.
Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by co-transfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5'-flanking region contains the basal promoter activity, while the region between -162 and -96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: -141 TRE, -131 GT, -123 ETS-like, and -111 TRE-like motifs and in the enhanced protections in -141 TRE and -111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both -141 TRE and -111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the -111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.
通常不表达鳞状细胞分化标志物基因SPR1的气管支气管上皮（TBE）细胞，可被十四酰佛波醇乙酯（TPA）诱导产生该基因。TPA对SPR1基因表达的调控部分发生在原代人及猴TBE细胞的转录水平。通过瞬时转染实验，我们观察到TPA可使转染的TBE细胞中报告基因氯霉素乙酰转移酶的活性提高2至4倍。然而，这种氯霉素乙酰转移酶活性具有细胞类型特异性，在转化的上皮细胞系中活性显著降低，在非上皮细胞类型中则无活性。与过表达JUN蛋白家族（尤其是c-JUN）的质粒DNA共转染，也可证明TPA依赖性刺激。c-JUN的过表达与TPA处理协同刺激SPR1启动子活性超过40倍。启动子区域的缺失分析表明，5'侧翼区域前98个碱基对的DNA片段包含基础启动子活性，而-162至-96之间的区域包含基础及TPA/c-JUN刺激启动子活性的顺式增强子元件。体内基因组足迹研究支持了这一观察结果，该研究揭示了该区域以下基序中存在持续的保护：-141 TRE、-131 GT、-123 ETS样和-111 TRE样基序，以及TPA处理后细胞中-141 TRE和-111 TRE样基序的增强保护。该区域的定点诱变表明，-141 TRE和-111 TRE样基序均参与TPA/c-JUN依赖性刺激以及增强的基础转录活性。然而，主要是-111 TRE样基序参与介导人SPR1基因增强的基础启动子活性。凝胶迁移率变动分析进一步支持了这些结果，该分析表明c-JUN和这些TRE基序参与了DNA-蛋白质复合物的形成。
