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PDK4 Promotes Vascular Calcification by Interfering with Autophagic

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PDK4 Promotes Vascular Calcification by Interfering with Autophagic Ma et al. Cell Death and Disease (2020) 11:991 https://doi.org/10.1038/s41419-020-03162-w Cell Death & Disease ARTICLE Open Access PDK4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming Wen-Qi Ma 1,Xue-JiaoSun1,YiZhu1 and Nai-Feng Liu1 Abstract Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular energy regulation. Previous studies suggested that PDK4 is increased in the calcified vessels of patients with atherosclerosis and is closely associated with mitochondrial function, but the precise regulatory mechanisms remain largely unknown. This study aims to investigate the role of PDK4 in vascular calcification and the molecular mechanisms involved. Using a variety of complementary techniques, we found impaired autophagic activity in the process of vascular smooth muscle cells (VSMCs) calcification, whereas knocking down PDK4 had the opposite effect. PDK4 drives the metabolic reprogramming of VSMCs towards a Warburg effect, and the inhibition of PDK4 abrogates VSMCs calcification. Mechanistically, PDK4 disturbs the integrity of the mitochondria-associated endoplasmic reticulum membrane, concomitantly impairing mitochondrial respiratory capacity, which contributes to a decrease in lysosomal degradation by inhibiting the V-ATPase and lactate dehydrogenase B interaction. PDK4 also inhibits the nuclear translocation of the transcription factor EB, thus inhibiting lysosomal function. These changes result in the interruption of autophagic flux, which accelerates calcium deposition in VSMCs. In addition, glycolysis serves as a metabolic adaptation to improve VSMCs oxidative stress resistance, whereas inhibition of glycolysis by 2-deoxy-D-glucose induces the apoptosis of 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; VSMCs and increases the calcium deposition in VSMCs. Our results suggest that PDK4 plays a key role in vascular calcification through autophagy inhibition and metabolic reprogramming. Introduction are involved in vascular calcification, and among these, Vascular calcification is a prevalent complication that VSMCs are the predominant cell type involved, having occurs more often in patients with atherosclerosis, biosynthetic, proliferative and contractile roles in the chronic kidney disease, hypertension, or diabetes mellitus vessel wall3,4. Continuous intracellular or environmental or who are ageing or smoking1,2. At pathogenic and stimuli, including oxidative stress, hypoxia, abnormal clinical levels, vascular calcification shares many simila- mineral metabolism, apoptosis and chronic inflammation, rities with bone formation, and its severity is commonly contribute to a phenotypic change of VSMCs to accompanied by a heightened risk of cardiovascular osteoblast-like cells, in which master transcription factors events and all-cause mortality; however, no therapeutics of osteogenesis and chondrogenesis are upregulated are currently available. Multiple cardiovascular-related concomitant with the generation of mineralized extra- cells, including endothelial cells, pericytes, vascular cellular matrix; parallel with this process, VSMCs lose a smooth muscle cells (VSMCs) and valve interstitial cells, range of contractile proteins, eventually causing vascular calcification5. Under basal conditions, VSMCs display higher rates of glucose metabolism and lactate production Correspondence: Nai-Feng Liu ([email protected]) than do other normal cells6, despite oxidative phosphor- 1Department of Cardiology, Zhongda Hospital, School of Medicine, Southeast University, 87 Dingjiaqiao, Nanjing 210009, P.R. China ylation is the main site for the production of ATP in these Edited by J.-E. Ricci © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Ma et al. Cell Death and Disease (2020) 11:991 Page 2 of 23 mammalian cells. Furthermore, VSMCs phenotypic mammary gland, liver and vascular tissue19,22. Clinical data changes are tightly integrated with the overall energetic indicated that PDK4 is upregulated in the calcified vessels of state of the cells, as evidenced during VSMCs prolifera- patients with atherosclerosis22. Mice deficient for PDK4 in tion in atherosclerosis7 and pulmonary arterial hyper- VSMCs exhibit a significant reduction in aortic calcium tension8. Recently, our laboratory found that accumulation after vitamin D3 treatment compared with − − mitochondrial dysfunction, especially abnormal mito- this accumulation in the control mice22.InApoE / mice, chondrial biogenesis, is associated with the phenotypic inhibition of PDK4 by dichloroacetate (DCA), a well- switch of VSMCs9. These observations indicate that cel- established PDK inhibitor, attenuates atherosclerosis23. lular energy metabolic regulation is at the centre of vas- Recent studies from our laboratory and others have found cular calcification, but the precise regulatory mechanisms that PDK4 activity is closely related to mitochondrial remain largely uncertain. function in VSMCs9,22; however, the exact mechanisms by Autophagy is an evolutionarily conserved pathway for which it affects pathological processes are poorly under- the degradation of cellular components through lyso- stood. New evidence suggests that PDK4 is a novel mod- somes, and it is also required for cells that do not have ulator of the integrity of the mitochondria-associated ER sufficient energy to survive10. Autophagy is commonly membrane (MAM), a structural link between the endo- regarded as a protective mechanism against vascular cal- plasmic reticulum (ER) surface and the outer mitochondrial cification11, but its activity in this role is controversial. membrane that acts as crucial signalling hub in cellular Several studies have suggested an increase in autophagic energy homoeostasis, ER stress, mitochondrial injury, flux and the formation of autophagic vesicles during the autophagy, apoptosis, inflammation and calcium homo- – calcification process11, whereas other investigators have eostasis24 26. This evidence prompted us to investigate – identified that autophagic flux is defective4,12 14. These whether the regulation of PDK4 functions in mitochondria discrepancies may be caused by differences in cell types, are associated with alterations in ER-mitochondrial inter- assay conditions, or methods to measure autophagy. actions in the context of vascular calcification. Therefore, it is necessary to perform a comprehensive In light of previous observations, the goal of the current evaluation of autophagy. Transcription factor EB (TFEB) work was to determine how PDK4 affects mitochondrial was recently discovered as a master regulator of lysosomal metabolism in VSMCs and to identify whether such biogenesis and autophagy15. Once activated, TFEB is changes regulate the VSMCs phenotypic switch and vas- translocated into the nucleus to induce the transcription cular calcification. In addition, we also investigated the of target genes16. In response to the enhanced cellular correlation between PDK4 and autophagy. We observed clearance capability via autophagy, TFEB coordinates an that PDK4 could lead to mitochondrial fragmentation and efficient transcription programme to upregulate genes disruption of the integrity of the MAM, which might associated with either the early (autophagosome forma- result in a decrease in mitochondrial respiratory capacity. tion) or late (lysosome biogenesis) phases of autophagy17. Moreover, PDK4 inhibits the nuclear translocation of Recent evidence suggests that activation of TFEB alle- TFEB, thus inhibiting lysosomal function. These changes viates atherosclerosis development in mice by promoting result in decreased autophagic activity, which has been lysosomal biogenesis and autophagy induction18. implicated in the pathogenesis of vascular calcification. In Although atherosclerosis is a chronic inflammatory and addition, we found that PDK4 drives the metabolic age-related disease that shares several common patho- reprogramming of VSMCs towards a Warburg effect and genic mechanisms with vascular calcification, there has that lactate, a by-product of glycolysis, plays a protective been no direct evidence or precise regulatory mechanisms role to improve VSMCs resistance to oxidative stress discovered to determine whether TFEB and autophagy under a calcification microenvironment. cooperate in the regulation of vascular calcification. Pyruvate dehydrogenase kinases (PDKs) are key enzymes Results in mitochondria that negatively regulate the activity of PDK4 expression is upregulated during both vitamin D3 the pyruvate dehydrogenase (PDH) complex through plus nicotine-induced aortic calcification in rats and β-GP- the phosphorylation of its subunit,
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