Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research, Clayton, Australia.
Cardiovascular Endocrinology Laboratory, Baker Heart and Diabetes Institute, Melbourne, Australia.
J Mol Endocrinol. 2022 Dec 7;70(1). doi: 10.1530/JME-21-0279. Print 2023 Jan 1.
The mineralocorticoid receptor (MR) is a ligand-activated transcription factor that regulates cardiorenal physiology and disease. Ligand-dependent MR transactivation involves a conformational change in the MR and recruitment of coregulatory proteins to form a unique DNA-binding complex at the hormone response element in target gene promoters. Differences in the recruitment of coregulatory proteins can promote tissue-, ligand- or gene-specific transcriptional outputs. The goal of this study was to evaluate the circadian protein TIMELESS as a selective regulator of MR transactivation. TIMELESS has an established role in cell cycle regulation and DNA repair. TIMELESS may not be central to mammalian clock function and does not bind DNA; however, RNA and protein levels oscillate over 24 h. Co-expression of TIMELESS down-regulated MR transactivation of an MR-responsive reporter in HEK293 cells, yet enhanced transactivation mediated by other steroid receptors. TIMELESS markedly inhibited MR transactivation of synthetic and native gene promoters and expression of MR target genes in H9c2 cardiac myoblasts. Immunofluorescence showed aldosterone induces colocalisation of TIMELESS and MR, although a direct interaction was not confirmed by coimmunoprecipitation. Potential regulation of circadian clock targets cryptochrome 1 and 2 by TIMELESS was not detected. However, our data suggest that these effects may involve TIMELESS coactivation of oestrogen receptor alpha (ERα). Taken together, these data suggest that TIMELESS may contribute to MR transcriptional outputs via enhancing ERα inhibitory actions on MR transactivation. Given the variable expression of TIMELESS in different cell types, these data offer new opportunities for the development of MR modulators with selective actions.
盐皮质激素受体 (MR) 是一种配体激活的转录因子，调节心脏肾生理学和疾病。配体依赖性 MR 反式激活涉及 MR 的构象变化和辅助调节蛋白的募集，以在靶基因启动子中的激素反应元件处形成独特的 DNA 结合复合物。辅助调节蛋白的募集差异可以促进组织、配体或基因特异性转录产物。本研究的目的是评估昼夜节律蛋白 TIMELSS 作为 MR 反式激活的选择性调节剂。TIMELSS 在细胞周期调控和 DNA 修复中具有既定作用。TIMELSS 可能不是哺乳动物生物钟功能的核心，也不与 DNA 结合；然而，RNA 和蛋白质水平在 24 小时内波动。在 HEK293 细胞中，TIMELSS 的共表达下调了 MR 对 MR 反应报告基因的反式激活，但增强了其他类固醇受体介导的反式激活。TIMELSS 显著抑制了 H9c2 心肌细胞中合成和天然基因启动子的 MR 反式激活和 MR 靶基因的表达。免疫荧光显示醛固酮诱导 TIMELSS 和 MR 的共定位，尽管通过共免疫沉淀未证实直接相互作用。未检测到 TIMELSS 对昼夜节律钟靶基因隐色体 1 和 2 的潜在调节作用。然而，我们的数据表明，这些影响可能涉及 TIMELSS 对雌激素受体 α (ERα) 的共激活。总之，这些数据表明，TIMELSS 可能通过增强 ERα 对 MR 反式激活的抑制作用来促进 MR 转录产物。鉴于 TIMELSS 在不同细胞类型中的表达不同，这些数据为开发具有选择性作用的 MR 调节剂提供了新的机会。
