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RNF2 mediates pulmonary fibroblasts activation and proliferation by regulating mTOR and p16-CDK4-Rb1 signaling pathway

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Abstract

Background

Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease with unknown etiology, associated with increasing morbidity and pessimistic prognosis. Pulmonary fibroblasts (PFbs) are the key effector cells of PF, in which abnormal activation and proliferation is an important pathogenesis of PF. Ring finger protein 2 (RNF2), is identified as the catalytic subunit of poly-comb repressive complex 1, which is closely related to occurrence and development of lung cancer, but its function in PF has not been revealed. In this paper, we sought to identify the regulatory role of RNF2 in lung fibrogenesis and its underlying mechanisms.

Methods

The expression of RNF2 in lung fibrosis tissue (human and Bleomycin-induced mouse) and cell model (TGF-β1-induced HFL1 cells) was examined by immunoblotting analysis and immunofluorescence. Western blot, qRT-PCR were performed to evaluate the expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/ TIMPs) induced by TGF-β1 in HFL1 cells. Cell proliferation, cycle progression and apoptosis were examined by fow cytometric. Molecular interactions were tested by Co-IP assays.

Results

RNF2 expression was elevated in PF tissues compared to normal adjacent tissues and in PFbs (HFL1) induced by TGF-β1. Furthermore, knockdown of RNF2 could evidently inhibit the abnormal expression of pro-fibrogenic cytokines (including α-SMA, ECM and MMPs/TIMPs) induced by TGF-β1 in HFL1 cells. Functionally, RNF2 silencing could significantly suppress TGF-β1-induced anomalous proliferation, cell cycle progression, apoptosis and autophagy in HFL1 cells. Mechanistically, RNF2 deficiency could effectively inhibit the abnormal activation of mTOR signaling pathway in TGF-β1-induced HFL1 cells, and mTOR pathway had feedback regulation on the expression of RNF2. Further studies RNF2 could regulate the phosphorylation level of RB1 through interacting with p16 to destroy the binding of p16 and CDK4 competitively. Simultaneously, overexpression of RNF2 could show the opposite results.

Conclusions

These results indicated that RNF2 is a potent pro-fibrogenic molecule for PFbs activation and proliferation through mTOR and p16-CDK4-Rb signaling pathways, and RNF2 inhibition will be a potential therapeutic avenue for treating PF.

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Abbreviations

PF:

Pulmonary fibrosis

PFbs:

Pulmonary fibroblasts

RNF2:

Ring finger protein 2

ILD:

Interstitial lung disease

AECs:

Alveolar epithelial cells

EMT:

Epithelial-to-mesenchymal transition

MMT:

Mesothelial-to-mesenchymal transition

BM:

Basement membrane

ECM:

Secrete extracellular matrix

NSCLC:

Non-small-cell lung cancer

DMEM:

Dulbecco’s modified Eagle’s medium

TBLB:

Trans-bronchial wall lung biopsy

H&E:

Hematoxylin–eosin staining

MTS:

Masson's trichrome staining

IHC:

Immunohistochemistry

RT-PCR:

Real-time PCR

WB:

Western blot

MFbs:

Myofibroblasts

α-SMA:

Alpha-smooth muscle actin

FN:

Fibronectin

MMPs:

Matrix metalloproteinases

TIMPs:

Matrix metalloproteinases

PI:

Proliferation index

HFL1:

Human embryonic lung fibroblast 1

TGF-β:

Transforming growth factor-β

4E-BP1:

4E-binding protein 1

p70-S6K1:

Ribosomal protein S6 kinase beta-1

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Acknowledgements

Thanks for the support of Center for Scientific Research, Anhui Medical University.

Funding

This work was supported by the Natural Science Foundation of China (Grant Numbers: 81700522), Natural Science Foundation of Anhui Province (Grant Numbers: 2108085QH309, 2208085MH203), Anhui Medical University "Three Complete Education" Comprehensive Reform Pilot Project (2021xsqyr05) and Anhui University Postgraduate Scientific Research Project (Grant Numbers: YJS20210293).

Author information

Author notes

  1. Linxin Pan, Ying Hu, Cheng Qian and Wanrong Shi have equally contributed to this study.

Authors and Affiliations

  1. School of Life Science, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, People’s Republic of China

    Linxin Pan

  2. Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, School of Pharmacy, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, People’s Republic of China

    Ying Hu, Yan Yao, Shuxian Wang & Tao Xu

  3. Institute for Liver Diseases of Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, People’s Republic of China

    Ying Hu, Yan Yao, Shuxian Wang & Tao Xu

  4. Center for Scientific Research, Anhui Medical University, 81 Meishan Road, Hefei, Anhui, 230032, People’s Republic of China

    Cheng Qian

  5. General Medicine and Health Management Center, Fourth Affiliated Hospital, Anhui Medical University, Wuhu Road street, Hefei, Anhui, 230002, People’s Republic of China

    Wanrong Shi

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  1. Linxin Pan
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Contributions

TX and LXP: designed the experiments and took part in the critical revision of the manuscript. YH and CQ: carried out experiments and participated in drafting of the manuscript and provided a series of experimental instructions and help. YY, SXW: conducted the relevant experiments. WRS: analyzed and interpreted the data.

Corresponding authors

Correspondence to Wanrong Shi or Tao Xu.

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The authors declared that there is no conflict of interest.

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Pan, L., Hu, Y., Qian, C. et al. RNF2 mediates pulmonary fibroblasts activation and proliferation by regulating mTOR and p16-CDK4-Rb1 signaling pathway. Inflamm. Res. 71, 1283–1303 (2022). https://doi.org/10.1007/s00011-022-01617-8

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  • DOI: https://doi.org/10.1007/s00011-022-01617-8

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