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Isgylation and Inhibits TGF-Β1- Induced Fibronectin Expression

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Isgylation and Inhibits TGF-Β1- Induced Fibronectin Expression Original research J Investig Med: first published as 10.1136/jim-2019-001145 on 23 December 2019. Downloaded from FOXO3a is stabilized by USP18- mediated de- ISGylation and inhibits TGF-β1- induced fibronectin expression Ban Wang,1 Yanhui Li,2,3 Heather Wang,3 Jing Zhao,3 Yutong Zhao ,3 Zhonghui Liu,1 Haichun Ma2 1Department of Immunology, ABSTRact College of Basic Medical FOXO3a belongs to a family of transcription factors Significance of this study Sciences, Jilin University, Changchun, China characterized by a conserved forkhead box DNA- 2 What is already known about this subject? Department of Anesthesia, binding domain. It has been known to regulate Jilin University First Hospital, various cellular processes including cell proliferation, ► FOXO3a regulates various cellular Changchun, China and biological functions including cell 3 apoptosis and differentiation. Post- translational Department of Physiology modifications of FOXO3a and their roles in the proliferation and death. and Cell Biology, Ohio State ► FOXO3a can be post- translational modified University, Columbus, Ohio, regulation of FOXO3a activity have been well- USA documented. FOXO3a can be phosphorylated, by phosphorylation, ubiquitination and acetylated and ubiquitinated, however, the acetylation. Correspondence to ISGylation of FOXO3a has not been reported. ► Post- translational modification regulates Dr Zhonghui Liu; Protein overexpression, ISGylation and half-life FOXO3a’s stability and activity. liuzh@ jlu. edu. cn were measured to determine the post- translational Dr Haichun Ma; What are the new findings? modification of FOXO3a. Human fibroblast cells were mahc@ jlu. edu. cn ► FOXO3a can be ISGylated. treated with transforming growth factor (TGF)- 1 to β ► ISGylation of FOXO3a determines FOXO3a Accepted 26 November determine the role of FOXO3a ISGylation in TGF-β1 half- life. 2019 signaling. FOXO3a’s half-life is around 3.7 hours. Published Online First ► USP18 negatively regulates FOXO3a 23 December 2019 Inhibition of the proteasome, not lysosome, extends ISGylation. its half- life. ISGylation, but not ubiquitination ► FOXO3a overexpression attenuates of FOXO3a, is increased in the presence of the transforming growth factor (TGF)-β1 proteasome inhibitor. Overexpression of ISG15 signaling. increases FOXO3a degradation, while overexpression of USP18 stabilizes FOXO3a through de- ISGylation. How might these results change the focus These results suggest that FOXO3a is degraded in of research or clinical practice? http://jim.bmj.com/ the ISGylation and proteasome system, which can be ► This is first report to reveal a new reversed by USP18, an ISG15-specific deubiquitinase. modification of FOXO3a. This study reveals a new molecular mechanism by ► This study will open a new window to which ISGylation regulates FOXO3a degradation. understand molecular regulation of Furthermore, we show that the overexpression of FOXO3a. FOXO3a attenuated TGF-β1- induced fibronectin ► Understanding the mechanism lead to on September 27, 2021 by guest. Protected copyright. expression in human lung fibroblast cells without develop new therapeutic strategies to treat altering Smad2/3 expression and activation. FOXO3a TGF-β1- mediated fibrotic diseases. can be ISGylated, which can regulate FOXO3a stability. USP18/FOXO3a pathway is a potential target for treating TGF-β1-mediated fibrotic diseases suppressor.1–3 FOXO3a transcriptional activity such as idiopathic pulmonary fibrosis. upregulates the gene expression of cell cycle inhibitors (such as p27 and p21) and apoptosis- related proteins (such as Bim, FasL, TRAIL and INTRODUCTION PUMA).4–6 FOXO3a also plays an important Forkhead box (FOX) proteins contain a DNA- role in autophagy.7 8 Recently, the role of binding domain and function as transcription FOXO3a in transforming growth factor (TGF)- © American Federation for factors. In mammals, the FOX protein family β1- mediated signaling has been demonstrated. Medical Research 2020. consists of A to S subgroups based on their TGF-β1 treatment induced nuclear exclusion Re- use permitted under structural similarity. FOXO3a belongs to the of FOXO3a and inhibited its transcriptional CC BY- NC. No commercial class O subgroup and plays important roles in activity in mesangial cells,9 while TGF-β1 treat- re- use. Published by BMJ. regulating various gene expressions and diverse ment increased FOXO3a nuclear transloca- 10 To cite: Wang B, Li Y, cellular and biological processes. FOXO3a tion in leukemia- initiating cells. An increase Wang H, et al. J Investig Med is known to suppress cell proliferation and in FOXO3 mRNA by a PKC inhibitor (UCN- 2020;68:786–791. promote cell death and function as a tumor 01) significantly inhibited TGF-β1- induced 786 Wang B, et al. J Investig Med 2020;68:786–791. doi:10.1136/jim-2019-001145 Original research J Investig Med: first published as 10.1136/jim-2019-001145 on 23 December 2019. Downloaded from myofibroblast differentiation markers (Col1A1 and smooth In vivo ubiquitination and ISGylation assay muscle actin).11 This is a modified IP protocol under denaturing conditions. Post- translational modifications of FOXO3a has been Cells were washed and collected with cold phosphate- shown to determine FOXO3a activity and biological buffered saline (PBS). After centrifuging at 1000 rpm for functions. Phosphorylation of FOXO3a is primarily regu- 5 min, 50–80 µL of 2% sodium dodecyl sulfate (SDS) lysis lated by the PI3K/Akt signaling pathway.12–14 Activation buffer containing 1 µg/mL ubiquitin aldehyde and 5 mM of Akt triggers FOXO3a serine/threonine phosphoryla- N- ethylmaleimide (NEM) were added to cell pellets and tion, thereby leading to its nuclear exclusion.12–14 Other then sonicated on ice for 12 s followed by boiling at 100°C kinases such as JNK and MST1 have been identified for 10 min. The denatured samples were diluted with PBS. to phosphorylate FOXO3a, while the kinase-induced Equal amounts of cell lysates (1 mg) were incubated with phosphorylation promotes its nuclear translocation.15 16 a FOXO3a antibody overnight at 4°C, followed by the Acetylation of FOXO3a is mediated by CBP/p300, which addition of 40 µL of protein A/G agarose and incubation attenuates FOXO3a transcriptional activity,17 18 while the for an additional 2 hours at 4°C. The immunoprecipi- effect can be reversed by the deacetylation of FOXO3a tated complex was washed three times with cold PBS and by SIRT1.19 Both phosphorylation and acetylation of analyzed by immunoblotting with antibodies against ubiq- FOXO3a trigger FOXO3a degradation.20 Ubiquitination uitin and ISG15. of FOXO3a is believed to increase its degradation in the 21 22 proteasome. Immunoblotting Similar to ubiquitination, ISGylation is important for Cells were washed with cold PBS and collected in the cell protein localization and turn over. Interferon- stimulated lysis buffer containing 20 mM Tris·HCl (pH 7.4), 150 mM gene 15 (ISG15) is an ubiquitin-lik e protein and it can cova- NaCl, 2 mM EGTA (ethylene glycol-bis( β-aminoethyl 23 24 lently conjugate to target proteins to induce ISGylation. ether)- N,N,N′,N′-tetraacetic acid), 5 mM β-glycerophos- Even several ubiquitin E3 ligases have been identified to phate, 1 mM MgCl2, 1% Triton X-100, 1 mM sodium ortho- catalyze ISGylation, USP18 is the major enzyme known for vanadate, 10 µg/mL protease inhibitors, 1 µg/mL aprotinin, de- ISGylation.25 The role of ISGylation in protein stability 1 µg/mL leupeptin and 1 µg/mL pepstatin. An equal amount has been studied, while the effect of ISGylation on protein of cell lysates (20 µg) was subjected to SDS- PAGE without degradation in the proteasome is controversial. Huang and boiling, electrotransferred to membranes and immuno- Bulavin reported that ISGylation increased p53 protea- blotted following standard protocol. somal degradation,26 while others believed that ISGylation 24 negatively regulates the ubiquitin-proteasome system. Transfection of plasmids Here, we found that FOXO3a can be ISGylated and ISG15 Human FOXO3a and USPs cDNA were inserted into facilitates FOXO3a degradation, the effects are reversed by pcDNA3.1/V5- His TOPO expression vectors (Invit- the overexpression of USP18. Furthermore, we show that rogen). Human ISG15 cDNA with HA tag was inserted the stabilization of FOXO3a attenuates TGF-β1- induced into pcDNA3.1 TOPO expression vector. HBE2 or Mrc5 fibronectin expression in human lung fibroblast cells. This cells were grown on 6- well plates or D100 dishes to http://jim.bmj.com/ study indicates that the ISGylation of FOXO3a may play 60%–70% confluence. The cells were then transfected with an important role in TGF-β1- mediated extracellular matrix varying amounts of plasmid using the Polyjet In vitro DNA accumulation in fibroblast cells. Transfection Reagent (SignaGen Laboratories) system based on the manufacturer’s protocol. MatERIALS AND METHODS Cell culture and reagents Statistical analysis on September 27, 2021 by guest. Protected copyright. Human bronchial epithelial cell line (HBE2) was cultured All results were subjected to statistical analysis using with HITES medium containing 10% fetal bovine serum one- way analysis of variance. The data are presented as (FBS). HEK293 cells were cultured with Dulbecco’s mean±SD. Data were collected from at least three indepen- Modified Eagle Medium containing 10% FBS. Human dent experiments, and p<0.05 was considered to be statis- lung fibroblast cells (Mrc5) were cultured with Eagle’s tically significant (*p<0.05; **p<0.01). Minimum Essential Medium containing 10%
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