Department of Pharmacology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan; Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
Life Sci. 2024 Aug 15;351:122807. doi: 10.1016/j.lfs.2024.122807. Epub 2024 Jun 7.
Differentiation-inducing factor-1 (DIF-1) is a polyketide produced by Dictyostelium discoideum that inhibits growth and migration, while promoting the differentiation of Dictyostelium stalk cells through unknown mechanisms. DIF-1 localizes in stalk mitochondria. In addition to its effect on Dictyostelium, DIF-1 also inhibits growth and migration, and induces mitochondrial fission followed by mitophagy in mammalian cells, at least in part by activating AMP-activated protein kinase (AMPK). In a previous study, we found that DIF-1 binds to mitochondrial malate dehydrogenase (MDH2) and inhibits its activity in HeLa cells. In the present study, we investigated whether MDH2 serves as a pharmacological target of DIF-1 in mammalian cells.
To examine the enzymatic activity of MDH, mitochondrial morphology, and molecular mechanisms of DIF-1 action, we conducted an MDH reverse reaction assay, immunofluorescence staining, western blotting, and RNA interference using mammalian cells such as human umbilical vein endothelial cells, human cervical cancer cells, mouse endothelial cells, and mouse breast cancer cells.
DIF-1 inhibited mitochondrial but not cytoplasmic MDH activity. Similar to DIF-1, LW6, an authentic MDH2 inhibitor, induced phosphorylation of AMPK, resulting in the phosphorylation of acetyl-CoA carboxylase (ACC) and the dephosphorylation of p70 S6 kinase with approximately the same potency. DIF-1 and LW6 induced mitochondrial fission. Furthermore, MDH2 knockdown using siRNA reproduced the DIF-1 action on the AMPK signaling and mitochondrial morphology. Conversely, an AMPK inhibitor prevented DIF-1-induced mitochondrial fission.
We propose that MDH2 is a mammalian target of DIF-1 for the activation of AMPK and induction of mitochondrial fission.
分化诱导因子-1（DIF-1）是一种由盘基网柄菌产生的聚酮化合物，通过未知机制抑制生长和迁移，同时促进盘基网柄菌柄细胞的分化。DIF-1 定位于柄细胞的线粒体中。除了对盘基网柄菌的作用外，DIF-1 还能抑制哺乳动物细胞的生长和迁移，并诱导线粒体裂变，随后引发线粒体自噬，至少部分是通过激活 AMP 激活的蛋白激酶（AMPK）。在之前的研究中，我们发现 DIF-1 与线粒体苹果酸脱氢酶（MDH2）结合并抑制其在 HeLa 细胞中的活性。在本研究中，我们研究了 MDH2 是否作为 DIF-1 在哺乳动物细胞中的药理学靶标。
为了研究 MDH 的酶活性、线粒体形态以及 DIF-1 作用的分子机制，我们使用哺乳动物细胞（如人脐静脉内皮细胞、人宫颈癌细胞、小鼠内皮细胞和小鼠乳腺癌细胞）进行 MDH 反转反应测定、免疫荧光染色、western blot 和 RNA 干扰。
DIF-1 抑制线粒体而不是细胞质 MDH 活性。与 DIF-1 相似，真实的 MDH2 抑制剂 LW6 诱导 AMPK 磷酸化，导致乙酰辅酶 A 羧化酶（ACC）磷酸化和 p70 S6 激酶去磷酸化，其效力大致相同。DIF-1 和 LW6 诱导线粒体裂变。此外，使用 siRNA 敲低 MDH2 可再现 DIF-1 对 AMPK 信号和线粒体形态的作用。相反，AMPK 抑制剂可阻止 DIF-1 诱导的线粒体裂变。
我们提出 MDH2 是 DIF-1 激活 AMPK 和诱导线粒体裂变的哺乳动物靶标。
