Myocyte Enhancer Factor 2A Contributes to the TGF-β1-Mediated Cholangiocyte Epithelial to Mesenchymal Transition and Senescence in Cholestatic Liver Fibrosis

Guangxi Zhou^1,2, Fei Hou³, Heng He¹, Yuan Xue¹, Yibo Wang¹, Xueying Chen⁴, Fengqin Zhu^1,2,*

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¹ Department of Gastroenterology, Affiliated Hospital of Jining Medical University, Jining Medical University, 272000 Jining, Shandong, China

² Shandong University of Traditional Chinese Medicine, 250355 Jinan, Shandong, China

³ Department of Critical Liver Diseases, Liver Research Center, Beijing Friendship Hospital, Capital Medical University, 100015 Beijing, China

⁴ Department of Cardiology, Affiliated Hospital of Jining Medical University, Jining Medical University, 272000 Jining, Shandong, China

^*Correspondence: zhufqin@126.com (Fengqin Zhu)
Academic Editor: Vesna Jacevic

Front. Biosci. (Landmark Ed) 2022, 27(12), 324; https://doi.org/10.31083/j.fbl2712324

Submitted: 2 October 2022 | Revised: 1 November 2022 | Accepted: 21 November 2022 | Published: 20 December 2022

This is an open access article under the CC BY 4.0 license.

Abstract

Background: Cholangiocytes are primary targets in chronic cholestatic liver diseases. Myocyte enhancer factor 2A (MEF2A) is a transcription factor with a crucial role in some fibrogenic diseases. However, whether it contributes to cholestatic liver fibrosis is still obscure. Methods: A bile duct–ligated (BDL) mouse model was established to detect MEF2A expression during cholestatic liver fibrosis. In addition, human intrahepatic biliary epithelial cells (HIBECs) were transfected with lentivirus-expressing shMEF2A (LV-shMEF2A) to regulate the expression of MEF2A in vitro. Biomarkers of epithelial to mesenchymal transition (EMT), senescence, and fibrogenesis were evaluated using various assays: Quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, senescence-associated $\beta$ -galactosidase (SA- $\beta$ -gal), and immunofluorescence. Furthermore, MEF2A expression and cytoplasm translocation induced by transforming growth factor $\beta$ 1 (TGF- $\beta$ 1) in HIBECs were determined by qRT-PCR, western blotting, and immunofluorescence. The expression of TGF- $\beta$ 1-induced MEF2A, EMT, senescence, and fibrosis markers inhibited by p38 MAPK signaling were evaluated by western blotting. Finally, the peripheral blood from primary biliary cholangitis (PBC) patients and healthy controls (HCs) was collected to analyze expression of MEF2A using Enzyme-linked immunosorbent assay (ELISA). Results: We found that MEF2A expression increased in liver tissues of BDL mice, and positively related to the extent of fibrosis. Silencing MEF2A in HIBECs restrained TGF- $\beta$ 1-induced EMT, senescence, and fibrotic reaction. Moreover, TGF- $\beta$ 1 enhanced the expression of MEF2A and induced its cytoplasm translocation in a concentration- and time-dependent manner, partially through interacting with p38 MAPK. The expression of MEF2A was also higher in the serum of PBC patients than in HCs, and positively correlated with fibrosis degree. Conclusions: Our study demonstrates that MEF2A is a central mediator linking TGF- $\beta$ 1-induced EMT and senescence in HIBECs. We propose it as a novel biomarker of fibrogenesis in cholestatic liver fibrosis. We also suggest inhibiting MEF2A as a potential strategy in treating cholestatic liver fibrosis.

Keywords

MEF2A

EMT

senescence

fibrosis

cholangiocytes

Funding

tsqn202103190/Tai Shan Young Scholar Foundation of Shandong Province

Q-2022134/TCM Science and Technology Project of Shandong Province

2021YXNS045/Key research and development plan of Jining City

2021YXNS144/Key research and development plan of Jining City

JYFY303574/Postdoctoral Fund of of the Affiliated Hospital of Jining Medical University

Figures

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Fig. 1.

MEF2A is overexpressed in livers of BDL mice and positively correlates with degree of fibrosis. Bile duct-ligated model was established using C57BL/6 mice to induce liver fibrosis. Sham mice were used as control. Mice were sacrificed 7 or 28 days after BDL surgery, and liver tissues were harvested. (A) Histological examination of liver sections with H&E staining. Scale bar represents 50 $\mu{}$ m. (B) Expression of MEF2A in liver tissues of BDL and sham mice detected with immunohistochemical staining using second antibodies labeled with fluorochromes. Original magnification $\times{}$ 200. Scale bar represents 50 $\mu{}$ m. (C) Percentages of MEF2A ${}^{+}$ cells in mice livers shown in (B). **p $<$ 0.01, ***p $<$ 0.001. (D) MEF2A and TGF- $\beta$ 1 protein expression in liver tissues of BDL and sham mice identified by western blotting. Reference protein was GAPDH. (E) Relative MEF2A mRNA expression in liver tissues of BDL and sham mice examined by qRT-PCR. Gene expression was normalized to GAPDH in each group. **p $<$ 0.01, ***p $<$ 0.001.

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