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BCAT1 Binds the RNA-Binding Protein ZNF423 to Activate Autophagy Via the IRE1-XBP-1-RIDD Axis in Hypoxic Pasmcs

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BCAT1 Binds the RNA-Binding Protein ZNF423 to Activate Autophagy Via the IRE1-XBP-1-RIDD Axis in Hypoxic Pasmcs Xin et al. Cell Death and Disease (2020) 11:764 https://doi.org/10.1038/s41419-020-02930-y Cell Death & Disease ARTICLE Open Access BCAT1 binds the RNA-binding protein ZNF423 to activate autophagy via the IRE1-XBP-1-RIDD axis in hypoxic PASMCs Wei Xin1,2,MinZhang1,2,3,YangYu1,2, Songlin Li4,CuiMa2,5, Junting Zhang1,2,YuanJiang1,2, Yiying Li1,2, Xiaodong Zheng6, Lixin Zhang2,5, Xijuan Zhao2,5, Xuzhong Pei1,2 and Daling Zhu1,2,7,8 Abstract Abnormal functional changes in pulmonary artery smooth muscle cells are the main causes of many lung diseases. Among, autophagy plays a crucial role. However, the specific molecular regulatory mechanism of autophagy in PASMCs remains unclear. Here, we first demonstrate that BCAT1 played a key role in the autophagy of hypoxic PASMCs and hypoxic model rats. BCAT1-induced activation and accumulation of the autophagy signaling proteins BECN1 and Atg5 by the endoplasmic reticulum (ER) stress pathway. Interestingly, we discovered that BCAT1 bound IRE1 on the ER to activate expression of its downstream pathway XBP-1-RIDD axis to activate autophagy. More importantly, we identified an RNA-binding protein, zinc finger protein 423, which promoted autophagy by binding adenylate/uridylate (AU)-rich elements in the BCAT1 mRNA 3′-untranslated region. Overall, our results identify BCAT1 as a potential therapeutic target for the clinical treatment of lung diseases and reveal a novel posttranscriptional regulatory mechanism and signaling pathway in hypoxia-induced PASMC autophagy. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction functions and regulation mechanisms of PASMCs, and Smooth muscle cells in the vessel wall are the main cell the need to find new therapeutic targets is urgent. type essential for the structural and functional integrity of The homeostasis of oxygen is an important factor in blood vessels. Abnormal functional changes in pulmonary maintaining normal lung structure and function7. In fact, artery smooth muscle cells (PASMCs) are the main cause hypoxia is a key cause of lung diseases such as pulmonary of many lung diseases. At present, lung diseases, including hypertension and pulmonary fibrosis. Increasing evidence pulmonary hypertension, pulmonary fibrosis, chronic shows that hypoxia has a significant effect on the behavior obstructive pulmonary disease, and even lung cancer, are of many kinds of tumors, including non-small cell lung – – a type of disease with extremely high mortality1 3. cancer (NSCLC)8 10. Hypoxia occurs in early NSCLC and Although different diseases have different characteristics is related to the increased expression of osteopontin and and pathogenesis, PASMC dysfunction plays an important carbonic anhydrase IX10. Importantly, hypoxia is an – role in the pathogenesis of various lung diseases4 6. important pathogenic factor for PASMC proliferation, Therefore, it is extremely important to further study the migration, apoptosis, and pyroptosis11. Hypoxia led to the upregulation of mitofusin 1, which maintains mitochon- drial homeostasis through mir-125a, and was shown to play an important role in promoting PASMCs12.We Correspondence: Daling Zhu ([email protected]) 1College of Pharmacy, Harbin Medical University, Harbin 150081, P.R. China reported that programmed death ligand 1 triggered pyr- 2Central Laboratory of Harbin Medical University (Daqing), Daqing 163319, P.R. optosis and pulmonary fibrosis in hypoxic PASMCs9. China Therefore, study of the molecular mechanism of hypoxia Full list of author information is available at the end of the article These authors contributed equally: Wei Xin, Min Zhang Edited by G. M. Fimia © 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 Xin et al. Cell Death and Disease (2020) 11:764 Page 2 of 16 in PASMCs may provide a new target for the diagnosis involved in increased BCAT1 expression through its and treatment of many lung diseases. binding of AREs in the 3′-UTR of BCAT1 mRNA for the Autophagy is a programmed intracellular homeostatic first time. Our results indicate a novel posttranscriptional process that plays an important role in maintaining the regulatory mechanism and signaling pathway in PASMC balance between protein synthesis and degradation in autophagy. cells13. Autophagy occurs in many lung diseases, such as chronic obstructive pulmonary disease, idiopathic pul- Results – monary fibrosis, and pulmonary hypertension14 16. Our Hypoxia upregulates the expression of BCAT1 previous studies confirmed that acetylated cyclophilin A is First, to confirm the expression of BCAT1 in PASMCs, an important mediator of hypoxia-induced autophagy and PASMCs were maintained under hypoxia for 24 h. pulmonary angiogenesis, indicating that hypoxia can BCAT1 expression was highly upregulated in the hypoxic induce autophagy in pulmonary artery endothelial cells group compared with the normal group (Fig. 1a). Coim- (PAECs)17. However, the role of hypoxia in PASMC munostaining for BCAT1 with α-SMA, a nonspecific autophagy remains unclear. marker of smooth muscle cells, showed that BCAT1 was Branched-chain amino transferases (BCAT) are substantially upregulated in hypoxic PASMCs (Fig. 1b). enzymes that carry out the reversible transamination of all Interestingly, we found that BCAT1 expression was three branched-chain amino acids (BCAAs) to branched- markedly increased in hypoxic PASMCs in a time- chain α-ketoacids and glutamate. There are two BCAT dependent manner and peaked at 24 h after hypoxia isoenzymes, cytosolic BCAT1 and mitochondrial exposure (Fig. 1e). We further explored BCAT1 expres- BCAT218. The differential expression of BCAT1 plays a sion in hypoxic model rats to confirm these results in vivo. key role in many diseases. Overexpression of BCAT1- We verified the hypoxia model rats successfully through induced proliferation, migration and invasion of cells in two types of in vitro hypoxia model experiments (Fig. S1) nasopharyngeal carcinoma19. BCAT1-promoted cell pro- and found that BCAT1 expression was upregulated in liferation through amino acid catabolism in human pulmonary arterial tissues of rats exposed to hypoxia for glioma carrying wild-type isocitrate dehydrogenase 120. 21 days (Fig. 1c, d) and a BCAT1 inhibitor, gabapentin, However, the relationship between BCAT1 and autophagy could significantly improve the relevant hypoxia indica- is not clear. For this reason, BCAT1 may serve as a reg- tors of hypoxia model rats (Fig. S1). This result is con- ulatory factor that participates in the regulation of sistent with the results obtained in vitro. PASMC autophagy. There are two BCAT isoenzymes18. To determine The posttranscriptional regulation of specifically tar- whether BCAT1 was the isoenzyme mainly affected by geted mRNA is crucial for maintaining proper cell phy- hypoxia, BCAT2 expression was also tested. Under siology and alters the cellular response to stress, hypoxia, there was no significant change in the expression autophagy, proliferation, apoptosis, and immune stimu- of BCAT2 (Fig. S2a). Finally, we also examined the – lation21 23. However, the posttranscriptional regulatory expression of BCAT1 in PAECs and found no significant mechanism of autophagy in PASMCs remains unknown. difference in expression between the hypoxia group and (Adenylate/uridylate) (AU)-rich elements (AREs) in 3′- the control group (Fig. S2b). These results indicated that untranslated regions (UTRs) can provide binding sites for the expression of BCAT1 was upregulated in hypoxia- transcriptionally active RNA-binding proteins (RBPs), induced PASMCs and hypoxic model rats. thereby regulating the stability and translation of tran- scripts24. Interestingly, certain types of zinc finger pro- Upregulation of BCAT1 expression by hypoxia leads to teins are RBPs that regulate RNA metabolism25. ZNF423, PASMCs autophagy – which is required for cerebellar development26 28,is Next, we explored the role of BCAT1 in the regulation involved in the development of B lymphoblastic leuke- of autophagy in hypoxic PASMCs. PASMCs were treated mia29,30. Silencing of ZNF423 has been reported to sig- with gabapentin and BCAT1-specific small interfering nificantly inhibit cell proliferation and invasion of the RNA (siRNA) to knock down the expression of BCAT1. cholangiocarcinoma cell line31. However, it is unclear The autophagy-related indicators beclin-1 (BECN1) and whether ZNF423 can be used as an RBP to activate Atg5 were detected by Western blot and immuno- autophagy in PASMCs through posttranscriptional reg- fluorescence analyses. As shown in (Figs. 2a, b, d and S5a), ulatory mechanisms. the autophagy-associated accumulation of BECN1 and In this study, we found for the first time that hypoxia Atg5 was
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