N6-methyladenosine modification of CBLB affects LPS-induced endoplasmic reticulum stress in alveolar epithelial cells
1
Department of Respiratory and Critical Care Medicine, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou 412007, Hunan Province, P.R. China
2
Department of Trauma Center, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou 412007, Hunan Province, P.R. China
3
Intensive Care Medicine Department, Zhuzhou Hospital Affiliated to Xiangya School of Medicine, Central South University, Zhuzhou 412007, Hunan Province, P.R. China
Submission date: 2025-02-08
Final revision date: 2025-04-16
Acceptance date: 2025-05-20
Online publication date: 2025-12-18
Corresponding author
KEYWORDS
ABSTRACT
Introduction:
Acute lung injury (ALI) is defined as an acute hypoxic respiratory insufficiency that arises from injury to alveolar epithelial cells. The suppression of endoplasmic reticulum stress (ERS) was reported to protect against ALI. This study investigated the mechanism of N6-methyladenosine (m6A)-modified Casitas B lymphoma-b (CBLB) on ERS in lipopolysaccharide (LPS)-induced type II alveolar epithelial cells (AECIIs).
Material and Methods:
AECII viability was evaluated via Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was examined via an ROS kit. TUNEL staining was applied to assay apoptosis. The levels of CBLB, LIM domain kinase 1 (LIMK1), methyltransferase-like 14 (METTL14), and ERS-related proteins were assessed by western blot and immunofluorescence. The ubiquitination level of LIMK1 was analyzed using immunoprecipitation. The interrelationship between CBLB and LIMK1 was identified via Co-IP. The m6A modification of CBLB was analyzed through MeRIP. The binding of METTL14 to the CBLB mRNA was verified by RIP.
Results:
CBLB facilitated the ubiquitination and degradation of LIMK1. The suppressive action of upregulated CBLB on apoptosis and ERS in LPS-induced AECIIs was reversed by LIMK1 overexpression. METTL14 reduced the stability of CBLB mRNA in a manner dependent on m6A methylation. CBLB knockdown reversed the inhibitory effects of METTL14 silencing on apoptosis and ERS in LPS-stimulated AECIIs.
Conclusions:
METTL14-mediated m6A modification of CBLB impaired the mRNA stability of CBLB to block the degradation of LIMK1, which promoted ERS in LPS-induced AECIIs.
Acute lung injury (ALI) is defined as an acute hypoxic respiratory insufficiency that arises from injury to alveolar epithelial cells. The suppression of endoplasmic reticulum stress (ERS) was reported to protect against ALI. This study investigated the mechanism of N6-methyladenosine (m6A)-modified Casitas B lymphoma-b (CBLB) on ERS in lipopolysaccharide (LPS)-induced type II alveolar epithelial cells (AECIIs).
Material and Methods:
AECII viability was evaluated via Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was examined via an ROS kit. TUNEL staining was applied to assay apoptosis. The levels of CBLB, LIM domain kinase 1 (LIMK1), methyltransferase-like 14 (METTL14), and ERS-related proteins were assessed by western blot and immunofluorescence. The ubiquitination level of LIMK1 was analyzed using immunoprecipitation. The interrelationship between CBLB and LIMK1 was identified via Co-IP. The m6A modification of CBLB was analyzed through MeRIP. The binding of METTL14 to the CBLB mRNA was verified by RIP.
Results:
CBLB facilitated the ubiquitination and degradation of LIMK1. The suppressive action of upregulated CBLB on apoptosis and ERS in LPS-induced AECIIs was reversed by LIMK1 overexpression. METTL14 reduced the stability of CBLB mRNA in a manner dependent on m6A methylation. CBLB knockdown reversed the inhibitory effects of METTL14 silencing on apoptosis and ERS in LPS-stimulated AECIIs.
Conclusions:
METTL14-mediated m6A modification of CBLB impaired the mRNA stability of CBLB to block the degradation of LIMK1, which promoted ERS in LPS-induced AECIIs.
REFERENCES (35)
1.
Long ME, Mallampalli RK, Horowitz JC (2022): Pathogenesis of pneumonia and acute lung injury. Clin Sci (Lond) 136: 747-769.
2.
Meyer NJ, Gattinoni L, Calfee CS (2021): Acute respiratory distress syndrome. Lancet 398: 622-637.
3.
Huang CY, Deng JS, Huang WC, et al. (2020): Attenuation of lipopolysaccharide-induced acute lung injury by hispolon in mice, through regulating the TLR4/PI3K/Akt/mTOR and Keap1/Nrf2/HO-1 pathways, and suppressing oxidative stress-mediated ER stress-induced apoptosis and autophagy. Nutrients 12: 1742.
4.
Du Y, Zhu P, Wang X, et al. (2020): Pirfenidone alleviates lipopolysaccharide-induced lung injury by accentuating BAP31 regulation of ER stress and mitochondrial injury. J Autoimmun 112: 102464.
5.
Li A, Chen S, Wu J, et al. (2023): Ischemic postconditioning attenuates myocardial ischemia-reperfusion-induced acute lung injury by regulating endoplasmic reticulum stress-mediated apoptosis. Braz J Cardiovasc Surg 38: 79-87.
6.
Li P, Wang X, Liu Z, et al. (2016): Single nucleotide polymorphisms in CBLB, a regulator of T-cell response, predict radiation pneumonitis and outcomes after definitive radiotherapy for non-small-cell lung cancer. Clin Lung Cancer 17: 253-262.e5.
7.
Tang R, Langdon WY, Zhang J (2022): Negative regulation of receptor tyrosine kinases by ubiquitination: Key roles of the Cbl family of E3 ubiquitin ligases. Front Endocrinol (Lausanne) 13: 971162.
8.
Pu ZQ, Liu D, Lobo Mouguegue HPP, et al. (2020): NR4A1 counteracts JNK activation incurred by ER stress or ROS in pancreatic beta-cells for protection. J Cell Mol Med 24: 14171-14183.
9.
Bachmaier K, Toya S, Gao X, et al. (2007): E3 ubiquitin ligase Cblb regulates the acute inflammatory response underlying lung injury. Nat Med 13: 920-926.
10.
Liu Y, Yang D, Liu T, et al. (2023): N6-methyladenosine-mediated gene regulation and therapeutic implications. Trends Mol Med 29: 454-467.
11.
Jiang X, Liu B, Nie Z, et al. (2021): The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther 6: 74.
12.
Shi Q, Li Z, Dong Y, et al. (2023): LncRNA THRIL, transcriptionally activated by AP-1 and stabilized by METTL14- mediated m6A modification, accelerates LPS-evoked acute injury in alveolar epithelial cells. Int Immunopharmacol 123: 110740.
13.
Zhang H, Wu D, Wang Y, et al. (2023): METTL3-mediated N6-methyladenosine exacerbates ferroptosis via m6A- IGF2BP2-dependent mitochondrial metabolic reprogramming in sepsis-induced acute lung injury. Clin Transl Med 13: e1389.
14.
Chatterjee D, Preuss F, Dederer V, et al. (2022): Structural aspects of LIMK regulation and pharmacology. Cells 11: 142.
15.
Rossi E, Kauskot A, Saller F, et al. (2021): Endoglin is an endothelial housekeeper against inflammation: Insight in ECFC-related permeability through LIMK/cofilin pathway. Int J Mol Sci 22: 8837.
16.
Ni J, Li G, Dai N, et al. (2023): Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration. Int Immunopharmacol 119: 110177.
17.
Chen L, Sun K, Qin W, et al. (2023): LIMK1 m(6)A-RNA methylation recognized by YTHDC2 induces 5-FU chemoresistance in colorectal cancer via endoplasmic reticulum stress and stress granule formation. Cancer Lett 576: 216420.
18.
Li S, Li S, Gao Z, Liu Y (2024): LncRNA HOTTIP promotes LPS-induced lung epithelial cell injury by recruiting DNMT1 to epigenetically regulate SP-C. J Cell Commun Signal 18: e12020.
19.
Tao H, Xu Y, Zhang S (2023): The role of macrophages and alveolar epithelial cells in the development of ARDS. Inflammation 46: 47-55.
20.
Marciniak SJ, Chambers JE, Ron D (2022): Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov 21: 115-140.
21.
Gong J, Wang XZ, Wang T, et al. (2017): Molecular signal networks and regulating mechanisms of the unfolded protein response. J Zhejiang Univ Sci B 18: 1-14.
22.
Wang M, Yin H, Xia Y, et al. (2021): Huganbuzure granule attenuates concanavalin-A-induced immune liver injury in mice via regulating the balance of Th1/Th2/Th17/Treg cells and inhibiting apoptosis. Evid Based Complement Alternat Med 2021: 5578021.
23.
Qi X, Luo Y, Xiao M, et al. (2023): Mechanisms of alveolar type 2 epithelial cell death during acute lung injury. Stem Cells 41: 1113-1132.
24.
Zhang CY, Zhong WJ, Liu YB, et al. (2023): EETs alleviate alveolar epithelial cell senescence by inhibiting endoplasmic reticulum stress through the Trim25/Keap1/Nrf2 axis. Redox Biol 63: 102765.
25.
Baek AR, Hong J, Song KS, et al. (2020): Spermidine attenuates bleomycin-induced lung fibrosis by inducing autophagy and inhibiting endoplasmic reticulum stress (ERS)-induced cell death in mice. Exp Mol Med 52: 2034-2045.
26.
Li X, Sun W, Huang M, et al. (2024): Deficiency of CBL and CBLB ubiquitin ligases leads to hyper T follicular helper cell responses and lupus by reducing BCL6 degradation. Immunity 57: 1603-1617.e7.
27.
Hu X, Li E, Zhou Y, et al. (2024): Casitas b cell lymphoma‑ B (Cbl-b): A new therapeutic avenue for small-molecule immunotherapy. Bioorg Med Chem 102: 117677.
28.
You H, Chang F, Chen H, et al. (2024): Exploring the role of CBLB in acute myocardial infarction: transcriptomic, microbiomic, and metabolomic analyses. J Transl Med 22: 654.
29.
Seijkens TTP, Poels K, Meiler S, et al. (2019): Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death. Eur Heart J 40: 372-382.
30.
Su Q, Zhang P, Yu D, et al. (2019): Upregulation of miR-93 and inhibition of LIMK1 improve ventricular remodeling and alleviate cardiac dysfunction in rats with chronic heart failure by inhibiting RhoA/ROCK signaling pathway activation. Aging (Albany NY) 11: 7570-7586.
31.
Villalonga E, Mosrin C, Normand T, et al. (2023): LIM kinases, LIMK1 and LIMK2, are crucial node actors of the cell fate: Molecular to pathological features. Cells 12: 805.
32.
Cao F, Chen G, Xu Y, et al. (2024): METTL14 contributes to acute lung injury by stabilizing NLRP3 expression in an IGF2BP2-dependent manner. Cell Death Dis 15: 43.
33.
Zheng Y, Zhang Z, Zheng D, et al. (2023): METTL14 promotes the development of diabetic kidney disease by regulating m(6)A modification of TUG1. Acta Diabetol 60: 1567-1580.
34.
Zhang L, Wan Y, Zhang Z, et al. (2021): FTO demethylates m6A modifications in HOXB13 mRNA and promotes endometrial cancer metastasis by activating the WNT signalling pathway. RNA Biol 18: 1265-1278.
35.
He M, Lei H, He X, et al. (2022): METTL14 regulates osteogenesis of bone marrow mesenchymal stem cells via inducing autophagy through m6A/IGF2BPs/Beclin-1 signal axis. Stem Cells Transl Med 11: 987-1001.
Share
RELATED ARTICLE
| eISSN: | 1644-4124 |
| ISSN: | 1426-3912 |
We process personal data collected when visiting the website. The function of obtaining information about users and their behavior is carried out by voluntarily entered information in forms and saving cookies in end devices. Data, including cookies, are used to provide services, improve the user experience and to analyze the traffic in accordance with the Privacy policy. Data are also collected and processed by Google Analytics tool (more).
You can change cookies settings in your browser. Restricted use of cookies in the browser configuration may affect some functionalities of the website.
You can change cookies settings in your browser. Restricted use of cookies in the browser configuration may affect some functionalities of the website.
