RTN3 regulates collagen biosynthesis and profibrotic macrophage differentiation to promote pulmonary fibrosis via interacting with CRTH2

Abstract

Background: As an endoplasmic reticulum (ER) protein, Reticulum 3 (RTN3) has been reported to play a crucial role in neurodegenerative diseases, lipid metabolism, and chronic kidney disease. The involvement of RTN3 in idiopathic pulmonary fibrosis (IPF), a progressive and fatal interstitial lung disease, remains unexplored.

Methods: In this study, we explored the role of RTN3 in pulmonary fibrosis using public datasets, IPF patient samples, and animal models. We investigated its pathogenic mechanisms in lung fibroblasts and alveolar macrophages.

Results: We found decreased levels of RTN3 in IPF patients, bleomycin-induced mice, and TGFβ-treated cell lines. RTN3-null mice exhibited more severe pulmonary fibrosis phenotypes in old age or after bleomycin treatment. Collagen synthesis was significantly increased in RTN3-null mice lung tissues and lung fibroblasts. Mechanistic studies revealed that RTN3 deficiency reduced the ER-anchored CRTH2 in lung fibroblasts, which serves as an antifibrotic molecule via antagonizing collagen biosynthesis. Simultaneously, RTN3 deficiency reduced the autophagy degradation of CRTH2 which acts as an activator of profibrotic macrophage differentiation. Both effects of RTN3 and CRTH2 in lung fibroblasts and alveolar macrophages aggravated age-or bleomycin-induced pulmonary fibrosis. Additionally, we also identified a mutation of RTN3 in patients with ILD.

Conclusions: Our research demonstrated that RTN3 plays a significant role in the lung, and reduction of RTN3 levels may be a risk factor for IPF and related diseases.

Keywords: CRTH2; Collagen biosynthesis; IPF; Idiopathic pulmonary fibrosis; Profibrotic macrophage differentiation; RTN3.

Fig. 1

A strong linkage between low expression of RTN3 and IPF. The mRNA levels of RTN3 GSE150910 dataset (A). B HE staining, Masson staining, and IHC showed the RTN3 levels in lung tissue of mice treated by bleomycin on day 0, day14.and day 21. C Western blot analysis showed the RTN3 levels in the lung tissue of mice treated by bleomycin on day 0, day14.and day 21. D Western blot analysis exhibited the levels of COL1A1 and RTN3 in MRC5 cell lines treated with TGFβ on 0 h, 12 h and 24 h. E HE staining, Masson staining, and IHC showed the RTN3 levels in in lung tissues of healthy controls and IPF patients

Fig. 2

RTN3 deficiency aggravated pulmonary fibrosis mice model induced by bleomycin and age. A RTN3 protein levels in the lung tissues of Wild type mice and RTN3-null mice were detected by Western blot. B HE staining and Masson staining showed the pathological changes of lung tissues for Wild type mice and RTN3-null mice treated with PBS or bleomycin on day 21. ELISA detection showed the SP-A levels (C) and SP-B (D) levels in the BALF isolated from Wild type mice and RTN3-null mice treated with PBS or bleomycin on day 21. E Western blot analysis exhibited the protein levels of Fn1, p-SMAD3, SMAD3, α-SMA and RTN3 levels in the lung tissues from Wild type mice and RTN3-null mice treated with PBS or bleomycin on day 21. F HE staining and Masson staining showed the pathological changes of lung tissues for Wild type mice and RTN3-null mice at 16 months old. ELISA detection showed the SP-A levels (G) and SP-B (H) levels in the BALF isolated from Wild type mice and RTN3-null mice at 16 months old

Fig. 3

RTN3 reduction can promote the synthesis of collagen. A Significantly differentially expressed genes between Wild type and RTN3 KO mice lung tissues revealed by RNA-seq data. B Real-time PCR detected the RNA levels of collagen related genes in lung tissues of Wild type and RTN3 KO mice. Western blot analysis showed the COL1A1 levels in lung tissues of Wild type and RTN3 KO mice (C), primary cultured lung fibroblasts from Wild type and RTN3 KO mice (D) and MRC5 cell lines transfected with or without shRNA (E). ELISA detection exhibited the hydroxyproline levels in primary cultured lung fibroblasts from Wild type and RTN3 KO mice (F) and MRC5 cell lines transfected with or without shRNA (G)

Fig. 4

RTN3 deficiency reduced the antagonism of ER-anchored CRTH2 on collagen synthesis in lung fibroblasts. A, B Co-IP showed the interaction between RTN3 with CRTH2 in primary cultured lung fibroblasts. Primary cultured lung fibroblasts were transfected with MYC-RTN3 plasmids. C Immunofluorescence staining showed the colocalization between RTN3 and CRTH2 in primary lung fibroblasts and MRC5 cell lines. D Immunofluorescence staining showed subcellular localization of RTN3 and CRTH2 in Wild type and RTN3-null primary lung fibroblasts. E Western blot analysis showed the expression of CRTH2 in ER in in Wild type and RTN3-null primary lung fibroblasts. Real-time PCR showed the stability of COL1A1 mRNA and COL1A2 mRNA in Wild type and RTN3-null primary lung fibroblasts (F), as well as in RTN3-null primary lung fibroblasts transfected with pcDNA3.1( +)-CRTH2 (G). H Western blot analysis showed the levels of COL1A1 and LARP6 in RTN3-null primary lung fibroblasts transfected with or without si-LARP6

Fig. 5

Whole-exome sequencing identified a loss-of-function mutations in the RTN3 gene in the Chinese ILD population. A High resolution computed tomography of the patients who carried RTN3 mutations. B Sanger sequencing of RTN3 confirmed the c.548A > G/p.E183G mutation. C Immunofluorescence staining showed subcellular localization of Wild type and mutated RTN3. D Immunofluorescence staining showed subcellular localization of CRTH2 in RTN3-null lung fibroblasts transfected with Wild type and mutated RTN3 plasmids. E ELISA detection exhibited the hydroxyproline levels in RTN3-null lung fibroblasts transfected with Wild type and mutated RTN3 plasmids

Fig. 6

RTN3 deficiency promote the profibrotic macrophage differentiation via reducing CRTH2 levels. A IHC showed the CD206 levels in lung tissue of mice treated by bleomycin or PBS on day 21. B Real-time PCR detected the mRNA levels of CD206, Arf1 and Fizz1 in alveolar macrophages isolated from Wild type and RTN3-null mice. C Flow cytometry detection showed the expression of CD206 in alveolar macrophages isolated from Wild type and RTN3-null mice. D Western blot analysis showed the levels of CRTH2, DNM2, Beclin1 and LC3A/B in alveolar macrophages isolated from Wild type and RTN3-null mice. E Immunofluorescence staining showed the colocalization between CRTH2 (red) and autophagy marker DNM2 (greed) in alveolar macrophages isolated from Wild type and RTN3-null mice

Fig. 7

Potential mechanism of how RTN3 reduction induced pulmonary fibrosis. In fibroblasts, RTN3 deficiency reduced the localization of CRTH2 on the endoplasmic reticulum to protect collagen mRNA from degradation. In macrophages, RTN3 deficiency reduced the autophagy of CRTH2 in alveolar macrophages which acted as an activator for profibrotic macrophage differentiation. The Figure was drawn by Figdraw (ID: IRPTI14fc7)