Endocytosis of Peptidase Inhibitor SerpinE2 promotes Myocardial Fibrosis through activating ERK1/2 and β-catenin Signaling Pathways

Figures

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  Figure 1. SerpinE2 promotes collagen expression in cardiac fibroblasts. A-B. Quantification of collagen content in cardiac fibroblasts and its supernatants after exogenous serpinE2 administration (n = 6-10, 20 ng/mL serpinE2 for 48 h); C. Immunofluorescence assay showed the distribution of exogenous serpinE2 in cardiac fibroblasts (n = 4, Scale bars = 20 µm). D. Relative level of serpinE2 in supernatants of fibroblast treated with heparin and serpinE2-antibody (serpinE2 to anti-serpinE2 at a ratio of 1:1 low dose or 1:2 high dose; heparin low dose 125 U, high dose 250 U; n = 6); E-F. Relative level of collagen in cardiac fibroblasts and supernatants of fibroblasts (n = 4 - 6); G-I. mRNA level of collagen I, collagen III and α-SMA after treated with serpinE2-antibody or heparin (n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined by student's paired t test (A-B), one-way ANOVA, Bonferroni's multiple comparisons test (D-I).

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  Figure 2. SerpinE2 can be internalized into cardiac fibroblasts. A. Immunofluorescence assay demonstrated that nystatin and β-cyclodextrin inhibited the endocytosis of SerpinE2 into cardiac fibroblasts (n = 4, Scale bars = 20 µm). B-C. SerpinE2 was marked by His tag at the C-terminus. Immunofluorescence assay was used to detect the level of His-SerpinE2 and the statistical graph. (n = 9, Scale bars = 10 µm). D-F. mRNA level of collagen I, collagen III and α-SMA. (n = 6); Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (C-F).

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  Figure 3. The effect of LRP1 and uPAR on endocytosis of serpinE2 in cardiac fibroblasts. A. Protein expression level of collagen I and serpinE2 in cardiac fibroblasts after silencing uPAR (n = 4); B. Protein expression level of collagen I and serpinE2 in cardiac fibroblasts after silencing LRP1 (n = 4); C. Immunofluorescence assay demonstrated that knockdown of LRP1 and uPAR reduced endocytosis of serpinE2 into cardiac fibroblasts (n = 4, Scale bars = 20 µm); D. The level of serpinE2 in supernatant of fibroblast after knockdown of LRP1 and uPAR (n = 8); E. The content of collagen in supernatant of fibroblast after knockdown of LRP1 or uPAR (n = 6); F. It was showed the direct interaction between serpinE2 and uPAR, serpinE2 and LRP1 in cardiac fibroblasts by Co-IP. The panel shows the presence of uPAR and LRP1 in the sample pulled down by anti-serpinE2. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined by one-way ANOVA, Bonferroni's multiple comparisons test (A, B) and Tukey's multiple comparisons test (D, E).

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  Figure 4. SerpinE2 activates the ERK1/2 signaling pathway in cardiac fibroblasts. A. Co-staining of p-ERK1/2 and serpinE2 in cardiac fibroblasts (n = 4, Scale bars = 20 µm). B. Relative expression level of phosphate-ERK1/2 in cardiac fibroblasts after administration of serpinE2 antibody, heparin, nystatin or β-cyclodextrin (n=4); C. Expression level of ERK1 was detected by ELISA assay after administration of serpinE2 antibody, heparin, nystatin or β-cyclodextrin (n = 6); D. Collagen expression level in CFs after treated with U0126 (n = 5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (B-D).

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  Figure 5. SerpinE2 activates β-catenin signaling pathway in cardiac fibroblasts. A. Co-staining of β-catenin and serpinE2 in cardiac fibroblasts (n = 4, Scale bars = 20 µm). B. Expression level of β-catenin was detected by ELISA assay after administration of serpinE2 antibody, heparin, nystatin, β-cyclodextrin (n = 6); C. mRNA level of β-catenin in cardiac fibroblasts after administration of serpinE2 antibody, heparin, nystatin, β-cyclodextrin (n = 6); D. Collagen expression level in CFs after treated with DKK1 (n = 5). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (B-D).

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  Figure 6. Knockdown of serpinE2 attenuated cardiac fibrosis induced by TAC. A. Masson staining showed the area of fibrosis (Scale bars = 100μm) and transmission electron microscope analysis of heart sections ( Scale bars = 2 µm); B. Fibrotic score was quantified by histological score from Masson staining (n = 4); C. Quantitative analysis of ejection fraction (EF) and fractional shortening (FS) of the heart (n = 5); D. Protein expression level of serpinE2, collagen, p-ERK44/42 and t-ERK44/42 (n = 4); E-G. Relative expression level of serpinE2, collagen and β-catenin detected by Elisa assay (n = 5-8); Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, determined one-way ANOVA, Bonferroni's multiple comparisons test (B-G).

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  Figure 7. Schematic model describing the effect and molecular mechanism of serpinE2 regulates collagen secretion in cardiac fibrosis. SerpinE2 was endocytosed by cardiac fibroblasts through the membrane protein LRP and uPAR through lipid-raft dependent endocytosis, this process then activated the intracellular signaling ERK1/2 and β-catenin signaling pathway to promote synthesis of collagen in cardiac fibroblasts.