PO.MCB09.02 · 分子与细胞生物学
雌激素相关受体γ通过谷氨酰胺来源的回补作用维持氧化代谢
Estrogen-related receptor gamma maintains oxidative metabolism via glutamine-derived anaplerosis
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
谷氨酰胺（Gln）传统上被认为是一种非必需氨基酸，但在癌细胞中由于Gln的消耗速率超过内源性生物合成速率而变得必需。这种代谢依赖性构成了一种独特的"谷氨酰胺成瘾"。尽管许多遗传和环境因素已被证明参与Gln代谢的调控，但癌症中Gln利用的精确调控在很大程度上仍不清楚。在此，我们确定雌激素相关受体γ（ERRγ）为谷氨酰胺分解酶类和细胞Gln利用的关键调控因子。对四种原发性视网膜母细胞瘤细胞系（有或无shRNA介导的ERRγ敲低）进行的RNA测序显示，相对于shGFP对照，shERRγ下调的基因富集于缺氧适应和糖酵解通路（FDR < 0.05），而氧化磷酸化未见改变，表明其向产乳酸的葡萄糖流转变。与此一致，乳酸发酵的关键基因（PDK1、LDH、MCT1）显著减少（p < 0.05）。shERRγ还降低了谷氨酰胺分解相关基因的表达（FDR < 0.05），提示在"类Warburg"状态下依赖Gln来源的碳来维持TCA活性。功能上，RB006（一种具有高基础ERRγ表达的模型癌细胞系）需要外源性Gln以维持活力，在Gln撤除时表现出明显的细胞死亡以及剂量依赖性的增殖增加，最大生长需要≥4mM。表达低ERRγ的间充质干细胞（MSCs）不依赖Gln；然而，ERRγ过表达（MSC[ERRγ]）诱导了Gln依赖性和剂量反应性增殖。为进一步评估Gln利用，向细胞中补充递增浓度的Gln，并定量TCA中间产物（谷氨酸、α-KG、柠檬酸、丙酮酸、ATP）。所有细胞系均以剂量依赖方式将Gln转化为谷氨酸，但仅RB006和MSC[ERRγ]表现出α-KG和柠檬酸的平行增加，与ERRγ介导的GLUD1/2和下游还原性羧化的调控一致。高ERRγ细胞在Gln水平与所有代谢物之间表现出强相关性（r > 0.9），每单位Gln产生的代谢物输出高于MSCs，并在Gln来源的通量上表现出更大的阶梯式增加，反映出增强的谷氨酰胺处理能力。我们的结果表明，ERRγ可能是通过谷氨酰胺来源的回补作用支持氧化磷酸化所必需的。我们提供了ERRγ是谷氨酰胺分解通路所必需的证据，提示ERRγ可能是高增殖细胞系代谢适应中的关键参与者。
查看英文原文 English abstract
Glutamine (Gln), traditionally considered a nonessential amino acid, becomes essential in cancer cells as the rate of Gln consumption exceeds that of endogenous biosynthesis. This metabolic dependency constitutes a unique “glutamine addiction”. Although many factors, both genetic and environmental, have been shown to contribute to the control of Gln metabolism, the precise regulation of Gln utilization in cancer still remains largely unclear. Here, we identify estrogen-related receptor gamma (ERRgamma) as a critical regulator of glutaminolytic enzymes and cellular Gln utilization. RNA-sequencing of four primary retinoblastoma cells lines with or without shRNA-mediated ERRgamma knockdown showed that, relative to shGFP controls, shERRgamma downregulated genes were enriched for hypoxic adaptation and glycolysis pathways ( FDR < 0.05 ), with no change in oxidative phosphorylation, indicating a shift toward lactate-producing glucose flux. Consistent with this, key genes in lactic acid fermentation (PDK1, LDH, MCT1) were significantly reduced ( p < 0.05 ). shERRgamma also decreased expression of glutaminolysis-related genes ( FDR < 0.05 ), suggesting reliance on Gln-derived carbon to sustain TCA activity in a “Warburg-like” state. Functionally , RB006, a model cancer cell line with high basal ERRgamma expression, requires exogenous Gln for viability, exhibiting marked cell death upon Gln withdrawal and a dose-dependent increase in proliferation, with ≥ 4mM required for maximal growth. Mesenchymal stem cells (MSCs), which express low ERRgamma, were Gln-independent; however, ERRgamma overexpression (MSC[ERRgamma]), induced both Gln dependence and dose-responsive proliferation. To further assess Gln utilization, cells were supplemented with increasing Gln, and TCA intermediates (glutamate, alpha-KG, citrate, pyruvate, ATP) were quantified. All lines converted Gln to glutamate in a dose-dependent manner, but only RB006 and MSC[ERRgamma] showed parallel increases in alpha-KG and citrate, consistent with ERRgamma-mediated control of GLUD1/2 and downstream reductive carboxylation. High- ERRgamma cells demonstrated strong correlations between Gln levels and all metabolites (r > 0.9), produced greater metabolite output per unit Gln than MSCs, and exhibited larger stepwise increases in Gln-derived flux, reflecting enhanced glutamine-handling capacity. Our results indicate ERRgamma may be necessary to support oxidative phosphorylation via glutamine-derived anaplerosis. We provide evidence that ERRgamma is required for glutaminolytic pathways, suggesting that ERRgamma may be a key player in the metabolic adaptation of highly proliferative cell lines.
利益披露 Disclosure
S. A. McLaughlin, None..
Z. M. Correa, None..
J. W. Harbour, None..
D. Pelaez, None.