CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia
CA IX Stabilizes Intracellular pH to Maintain Metabolic Reprogramming and Proliferation in Hypoxia
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DOI：
10.3389/fonc.2020.01462
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发表时间：
2020-09-02
影响因子：
4.7
通讯作者：
Pastorekova, Silvia
中科院分区：
文献类型：
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作者：
Benej, Martin;Svastova, Eliska;Pastorekova, Silvia
肿瘤缺氧是一种严重的微环境应激，通常与酸中毒有关。癌细胞通过改变基因表达来应对这些压力，至少部分通过pH调节和代谢重编程来促进生存。缺氧诱导的碳酸酐酶IX （CA IX）通过催化胞外CO(2)水合产生碳酸氢盐缓冲细胞内pH (pHi)，在缺氧和酸性环境中起关键的适应性作用。我们使用蛋白质组分析来研究缺氧条件下CA IX基因瞬间敲低的细胞反应，发现关键的糖酵解酶和乳酸脱氢酶a （LDHA）水平下降。有趣的是，通过天然凝胶内活性测定，LDH的活性也降低了。这些变化导致体外癌细胞糖酵解通量和细胞外乳酸水平显著降低，有助于减少增殖。有趣的是，添加替代LDH底物α -酮丁酸盐可以恢复LDHA活性、细胞外酸化、pHi和细胞增殖。这些结果表明，在缺乏CA IX的情况下，pHi的减少会破坏LDHA活性，阻碍细胞再生NAD（+）和向细胞外空间分泌质子的能力。因此，缺氧诱导的caix通过酶促细胞外CO(2)转化为碳酸氢盐直接介导对微环境缺氧和酸中毒的适应，通过维持允许糖酵解的细胞内环境间接介导。
Tumor hypoxia represents a severe microenvironmental stress that is frequently associated with acidosis. Cancer cells respond to these stresses with changes in gene expression that promote survival at least in part through pH regulation and metabolic reprogramming. Hypoxia-induced carbonic anhydrase IX (CA IX) plays a critical adaptive role in response to hypoxic and acidic environments by catalytically hydrating extracellular CO(2)to produce bicarbonate for buffering intracellular pH (pHi). We used proteome-wide profiling to study the cellular response to transient CA IX knockdown in hypoxia and found a decrease in the levels of key glycolytic enzymes and lactate dehydrogenase A (LDHA). Interestingly, the activity of LDH was also decreased as demonstrated by native in-gel activity assay. These changes led to a significant reduction in glycolytic flux and extracellular lactate levels in cancer cellsin vitro, contributing to a decrease in proliferation. Interestingly, addition of the alternative LDH substrate alpha-ketobutyrate restored LDHA activity, extracellular acidification, pHi, and cellular proliferation. These results indicate that in the absence of CA IX, reduction of pHi disrupts LDHA activity and hinders the cellular capacity to regenerate NAD(+)and secrete protons to the extracellular space. Hypoxia-induced CA IX therefore mediates adaptation to microenvironmental hypoxia and acidosis directly, by enzymatically converting extracellular CO(2)to bicarbonate, and indirectly, by maintaining glycolysis-permissive intracellular milieu.