Desai D S, Hirai S, Karnes W E, Niles R M, Ohno S
Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa Ku, Yokohama, 236, Japan.
Biochem Biophys Res Commun. 1999 Sep 16;263(1):28-34. doi: 10.1006/bbrc.1999.1307.
Protein kinase C (PKC) is a family which consists of multiple isoforms whose distinct physiological roles within the cell are unknown. We have previously demonstrated that levels of PKC alpha mRNA, protein, and enzyme activity in B16 melanoma cells can be modulated by retinoic acid. We investigated this regulation by cloning and characterizing the promoter region of the murine PKC alpha gene. A 13 kb mouse genomic fragment containing the 5' flanking region, first exon, and first intron was isolated and sequenced. Two transcription initiation sites were identified at 919 and 925 bp upstream from the translation start site. The promoter region contained a TATA-like box at -93 bp upstream of the transcription start site, but no CAAT box. Promoter activity differed between cell lines and correlated with the levels of PKC alpha expressed in these cell lines. Reporter gene assays showed that the region between -179 and -452 bp likely contains a silencer element(s). The promoter activity of a -179 bp fragment in B16 cells was stimulated twofold by retinoic acid. Within this region (-93 to -65 bp) there is a retinoic acid response element. An oligonucleotide spanning this region specifically bound exogenous RAR-RXR heterodimers and endogenous RAR from B16 nuclear extracts. These results suggest that retinoic acid increases PKC alpha gene expression in B16 cells, at least in part, through direct transcriptional stimulation of its promoter.
蛋白激酶C（PKC）是一个由多种亚型组成的家族，其在细胞内不同的生理作用尚不清楚。我们之前已经证明，视黄酸可以调节B16黑色素瘤细胞中PKCα mRNA、蛋白质和酶活性的水平。我们通过克隆和鉴定小鼠PKCα基因的启动子区域来研究这种调控。分离并测序了一个包含5'侧翼区域、第一个外显子和第一个内含子的13 kb小鼠基因组片段。在翻译起始位点上游919和925 bp处鉴定出两个转录起始位点。启动子区域在转录起始位点上游-93 bp处含有一个类似TATA盒的结构，但没有CAAT盒。不同细胞系之间的启动子活性不同，并且与这些细胞系中表达的PKCα水平相关。报告基因分析表明，-179至-452 bp之间的区域可能含有一个沉默元件。视黄酸可使B16细胞中-179 bp片段的启动子活性提高两倍。在该区域（-93至-65 bp）内有一个视黄酸反应元件。跨越该区域的寡核苷酸能特异性结合外源性RAR-RXR异二聚体和来自B16核提取物的内源性RAR。这些结果表明，视黄酸至少部分通过直接转录刺激其启动子来增加B16细胞中PKCα基因的表达。
