Anatomy, Department of Medicine, University of Fribourg, Route Albert-Gockel 1, CH-1700, Fribourg, Switzerland.
BMC Cancer. 2018 Apr 27;18(1):475. doi: 10.1186/s12885-018-4385-7.
The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood.
We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments.
Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7.
Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.
钙结合蛋白 calretinin（基因名称：CALB2）目前被认为是诊断恶性间皮瘤（MM）最敏感和最特异的标志物。MM 是一种非常侵袭性的肿瘤，与石棉暴露密切相关，迄今为止尚无治愈方法。calretinin 调节的机制及其在 MM 中的独特功能仍知之甚少。
我们寻找与 CALB2 启动子结合并调节 calretinin 表达的转录因子。为此，使用 DNA 结合测定法和肽 shotgun-质谱分析。通过双荧光素酶报告基因检测评估 CALB2 启动子活性。此外，我们通过慢病毒介导的 MM 细胞中鉴定的蛋白质的过表达或下调来分析 CALB2 启动子结合蛋白的作用。通过随后的 Western blot 分析确定了丁酸对这些蛋白质表达的调节。胚胎小鼠肺组织的免疫组织化学分析用于验证在早期发育过程中 calretinin 与与 CALB2 启动子相互作用的蛋白质的同时共表达。最后，通过共免疫沉淀实验证实了 calretinin 与靶蛋白的直接相互作用。
鉴定出 septin 7 是一种依赖于丁酸盐的转录因子，它与包含丁酸盐反应元件（BRE）的 CALB2 启动子区域结合，导致 calretinin 表达减少。因此，septin 7 的过表达降低了 MM 细胞中的 calretinin 表达水平。该调节被发现是双向的，即 calretinin 的过表达也降低了 septin 7 的水平。在小鼠胚胎发育过程中，calretinin 和 septin 7 被发现在胚胎间充质和未分化的间皮细胞中共同表达。在 MM 细胞中，calretinin 和 septin 7 在细胞分裂沟的不同区域和有丝分裂细胞的中部区域共定位。共免疫沉淀实验表明这种共定位是 calretinin 和 septin 7 之间直接相互作用的结果。
我们的研究结果表明，septin 7 不仅作为一种“细胞骨架”蛋白，而且作为一种转录因子抑制 calretinin 的表达。septin 7 对 calretinin 的负调节和反之亦然，为 MM 形成中可能涉及的机制提供了新的认识，并将这些蛋白质鉴定为转录调节剂和 MM 治疗的潜在靶点。
