SEMA3B inhibits TGFβ-induced extracellular matrix protein production and its reduced levels are associated with a decline in lung function in IPF

Abstract

Idiopathic pulmonary fibrosis (IPF) is marked by the activation of fibroblasts, leading to excessive production and deposition of extracellular matrix (ECM) within the lung parenchyma. Despite the pivotal role of ECM overexpression in IPF, potential negative regulators of ECM production in fibroblasts have yet to be identified. Semaphorin class 3B (SEMA3B), a secreted protein highly expressed in lung tissues, has established roles in axonal guidance and tumor suppression. However, the role of SEMA3B in ECM production by fibroblasts in the pathogenesis of IPF remains unexplored. Here, we show the downregulation of SEMA3B and its cognate binding receptor, neuropilin 1 (NRP1), in IPF lungs compared with healthy controls. Notably, the reduced expression of SEMA3B and NRP1 is associated with a decline in lung function in IPF. The downregulation of SEMA3B and NRP1 transcripts was validated in the lung tissues of patients with IPF, and two alternative mouse models of pulmonary fibrosis. In addition, we show that transforming growth factor-β (TGFβ) functions as a negative regulator of SEMA3B and NRP1 expression in lung fibroblasts. Furthermore, we demonstrate the antifibrotic effects of SEMA3B against TGFβ-induced ECM production in IPF lung fibroblasts. Overall, our findings uncovered a novel role of SEMA3B in the pathogenesis of pulmonary fibrosis and provided novel insights into modulating the SEMA3B-NRP1 axis to attenuate pulmonary fibrosis.NEW & NOTEWORTHY The excessive production and secretion of collagens and other extracellular matrix proteins by fibroblasts lead to the scarring of the lung in severe fibrotic lung diseases. This study unveils an antifibrotic role for semaphorin class 3B (SEMA3B) in the pathogenesis of idiopathic pulmonary fibrosis. SEMA3B functions as an inhibitor of transforming growth factor-β-driven fibroblast activation and reduced levels of SEMA3B and its receptor, neuropilin 1, are associated with decreased lung function in idiopathic pulmonary fibrosis.

Keywords: SEMA3B; TGFβ; extracellular matrix; fibroblasts; pulmonary fibrosis.

Graphical abstract

Figure 1.

Reduced semaphorin class 3B (SEMA3B) and neuropilin 1 (NRP1) levels associate with lung function loss in idiopathic pulmonary fibrosis (IPF). A: SEMA3B transcript levels were lower in patients with IPF (n = 160) compared to healthy subjects (n = 108) based on the RNA microarray dataset GSE47460. ****P < 0.0001. B and C: GSE47460 data show a positive correlation between SEMA3B transcript levels and both percent forced vital capacity (FVC) and diffusing capacity of the lungs for carbon monoxide (DL_CO) in both IPF (red) and healthy subjects (blue). The blue line represents the linear correlation. D: RNA-sequencing data from GSE150910 confirms decreased SEMA3B transcript levels in IPF lungs compared to healthy controls (CTRL; n = 40–60/group). ****P < 0.0001. E and F: similar to GSE47460, GSE150910 data show a positive correlation between SEMA3B transcript levels and percent FVC or DL_CO in IPF (red) and healthy subjects (blue). G: NRP1 transcript levels were also reduced in IPF lungs compared to healthy lungs according to the RNA-sequencing data from GSE150910. ****P < 0.0001. H and I: a positive correlation between NRP1 transcript levels and both percent FVC and DL_CO is observed in IPF (red) and healthy subjects (blue) from the GSE150910 dataset. The correlation coefficient (r) and P value are displayed within the scatter plots.

Figure 2.

Downregulation of semaphorin class 3B (SEMA3B) and neuropilin 1 (NRP1) in idiopathic pulmonary fibrosis (IPF) lungs. A: violin plot showing the expression levels of SEMA3B and NRP1 across various cell types in non-IPF and IPF lungs. Data are from a publicly available dataset (GSE136831) for non-IPF and IPF lung scRNA-seq. B and C: representative immunohistochemical images of distal lung biopsies from normal and IPF lungs stained with antibodies against SEMA3B (B) and NRP1 (C). Scale bar = 50 μm. Arrows highlight spindle-shaped mesenchymal cells positive for SEMA3B or NRP1 (n = 5 per group). D and E: transcript levels of SEMA3B and NRP1 were quantified in fibroblasts isolated from the lungs of IPF patients and healthy controls using RT-PCR. Student’s t test was performed with n = 10–14 per group. ***P < 0.001, **P < 0.01.

Figure 3.

Downregulation of semaphorin class 3B (SEMA3B) and neuropilin 1 (NRP1) in mouse models of pulmonary fibrosis. A and B: quantification of SEMA3B and Nrp1 transcripts in fibroblasts that were treated with profibrotic growth factors connective tissue growth factor (CTGF; 100 ng/mL), transforming growth factor-α (TGFα; 100 ng/mL), TGFβ1 (20 ng/mL), and IGF-1 (20 ng/mL) for 16 h using RT-PCR with n = 4/group, Data are shown as means ± SE. One-way ANOVA was used. *P < 0.05. C and D: SEMA3B and NRP1 transcripts were measured using RT-PCR in the lungs of mice treated with saline or bleomycin (BLM) for 4 weeks. Student’s t test was performed with n = 8/group. ****P < 0.0001. E and F: SEMA3B and NRP1 transcripts were measured using RT-PCR in the lungs of TGFα transgenic mice on doxycycline (DOX) food for 0, 4, and 8 wk. One-way ANOVA was used. Data are shown as means ± SE. **P < 0.01.

Figure 4.

Semaphorin class 3B (SEMA3B) inhibits TGFβ1-driven extracellular matrix (ECM) gene expression in fibroblasts. A: upregulation of the transcripts of ECM genes [collagen 1A1 (COL1A1), elastin (ELN), and α-smooth muscle actin (αSMA)] in normal lung fibroblasts treated with media, transforming growth factor-β (TGFβ), SEMA3B, or both TGFβ and SEMA3B for 16 h. B: upregulation of the transcripts of ECM genes (COL1A1, ELN, and αSMA) in IPF fibroblasts treated with media, TGFβ, SEMA3B, or both TGFβ and SEMA3B for 16 h. Data are shown as the means ± SE. Student’s t test was performed with n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5.

Semaphorin class 3B (SEMA3B) inhibits the transforming growth factor-β1 (TGFβ1)-driven extracellular matrix (ECM) protein production in Idiopathic pulmonary fibrosis (IPF) lung fibroblasts. A: IPF lung fibroblasts were treated with media, SEMA3B (400 ng/mL), and/or TGFβ1 (20 ng/mL) for 72 hours, and cell lysates were immunoblotted with antibodies against collagen 1 (COL1), fibronectin 1 (FN1), α-smooth muscle actin (αSMA), and GAPDH. B: IPF lung fibroblasts were treated with media, SEMA3B (400 ng/mL), and/or TGFβ1 (20 ng/mL) for 72 h, and cell lysates were immunoblotted with antibodies against elastin (ELN) and GAPDH. C: densitometric quantification of COL1, FN1, αSMA, and ELN protein levels normalized to GAPDH. Data are shown as the means ± SE. A one-way ANOVA test was performed with n = 3/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.