Department of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany.
DZHK Standort Hamburg, Kiel, Lübeck, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 2023 Jun;396(6):1223-1233. doi: 10.1007/s00210-023-02401-z. Epub 2023 Jan 26.
The dual leucine zipper kinase (DLK) and the ubiquitously expressed transcription factor c-FOS have important roles in beta-cell proliferation and function. Some studies in neuronal cells suggest that DLK can influence c-FOS expression. Given that c-FOS is mainly regulated at the transcriptional level, the effect of DLK on c-FOS promoter activity was investigated in the beta-cell line HIT. The methods used in this study are the following: Luciferase reporter gene assays, immunoblot analysis, CRISPR-Cas9-mediated genome editing, and real-time quantitative PCR. In the beta-cell line HIT, overexpressed DLK increased c-FOS promoter activity twofold. Using 5'-,3'-promoter deletions, the promoter regions from - 348 to - 339 base pairs (bp) and from a - 284 to - 53 bp conferred basal activity, whereas the promoter region from - 711 to - 348 bp and from - 53 to + 48 bp mediated DLK responsiveness. Mutation of the cAMP response element within the promoter prevented the stimulatory effect of DLK. Treatment of HIT cells with KCl and the adenylate cyclase activator forskolin increased c-FOS promoter transcriptional activity ninefold. Since the transcriptional activity of those promoter fragments activated by KCl and forskolin was decreased by DLK, DLK might interfere with KCl/forskolin-induced signaling. In a newly generated, genome-edited HIT cell line lacking catalytically active DLK, c-Fos mRNA levels were reduced by 80% compared to the wild-type cell line. DLK increased c-FOS promoter activity but decreased stimulated transcriptional activity, suggesting that DLK fine-tunes c-FOS promoter-dependent gene transcription. Moreover, at least in HIT cells, DLK is required for FOS mRNA expression.
双亮氨酸拉链激酶（DLK）和普遍表达的转录因子 c-FOS 在β细胞增殖和功能中具有重要作用。一些神经元细胞的研究表明，DLK 可以影响 c-FOS 的表达。鉴于 c-FOS 主要在转录水平上受到调控，因此在β细胞系 HIT 中研究了 DLK 对 c-FOS 启动子活性的影响。本研究采用的方法如下：荧光素酶报告基因检测、免疫印迹分析、CRISPR-Cas9 介导的基因组编辑和实时定量 PCR。在β细胞系 HIT 中，过表达的 DLK 使 c-FOS 启动子活性增加了两倍。通过 5'、3'启动子缺失，从-348 到-339 个碱基对（bp）和从-284 到-53bp 的启动子区域赋予基础活性，而从-711 到-348bp 和从-53 到+48bp 的启动子区域介导了 DLK 的反应性。启动子内 cAMP 反应元件的突变阻止了 DLK 的刺激作用。用 KCl 和腺苷酸环化酶激活剂 forskolin 处理 HIT 细胞可使 c-FOS 启动子转录活性增加九倍。由于 KCl 和 forskolin 激活的那些启动子片段的转录活性被 DLK 降低，因此 DLK 可能干扰 KCl/forskolin 诱导的信号。在一个新生成的、基因组编辑的缺乏催化活性的 DLK 的 HIT 细胞系中，与野生型细胞系相比，c-Fos mRNA 水平降低了 80%。DLK 增加了 c-FOS 启动子活性，但降低了受刺激的转录活性，表明 DLK 微调了 c-FOS 启动子依赖性基因转录。此外，至少在 HIT 细胞中，DLK 是 FOS mRNA 表达所必需的。
