Transcriptional Profiling of Insulin-like Growth Factor Signaling Components in Embryonic Lung Development and Idiopathic Pulmonary Fibrosis

Abstract

Insulin-like growth factor (IGF) signaling controls the development and growth of many organs, including the lung. Loss of function of Igf1 or its receptor Igf1r impairs lung development and leads to neonatal respiratory distress in mice. Although many components of the IGF signaling pathway have shown to be dysregulated in idiopathic pulmonary fibrosis (IPF), the expression pattern of such components in different cellular compartments of the developing and/or fibrotic lung has been elusive. In this study, we provide a comprehensive transcriptional profile for such signaling components during embryonic lung development in mice, bleomycin-induced pulmonary fibrosis in mice and in human IPF lung explants. During late gestation, we found that Igf1 is upregulated in parallel to Igf1r downregulation in the lung mesenchyme. Lung tissues derived from bleomycin-treated mice and explanted IPF lungs revealed upregulation of IGF1 in parallel to downregulation of IGF1R, in addition to upregulation of several IGF binding proteins (IGFBPs) in lung fibrosis. Finally, treatment of IPF lung fibroblasts with recombinant IGF1 led to myogenic differentiation. Our data serve as a resource for the transcriptional profile of IGF signaling components and warrant further research on the involvement of this pathway in both lung development and pulmonary disease.

Keywords: IGF1; IGF1R; bleomycin-induced pulmonary fibrosis; idiopathic pulmonary fibrosis; lung development.

Figure 1

Expression profile of IGF signaling components during lung development. (a–j) qPCR on lung homogenates at the indicated developmental stages; (k) Scheme for experimental design; (l–r) qPCR on primary mesenchymal cells at the indicated developmental stages. (s) Immunohistochemistry for TTF1 and IGF1 on E18.5 mouse lungs. (t) Immunohistochemistry for TTF1 and IGF1R on E18.5 mouse lungs. One-way ANOVA was used to compare the means. (a–j) E14.5: n = 5, E16.5: n = 5, E18.5: n = 6; (l–r) E14.5: n = 5–6, E16.5: n = 5–6, E18.5: n = 6. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Alteration of IGF signaling in the bleomycin model of lung fibrosis. (a,b) Hematoxylin/eosin and Masson Goldner staining showing clear fibrosis in bleomycin-treated mouse lungs compared with saline-treated controls. (c) Western blot for PAI-1, COL1A1 and ACTB. (d–m) qPCR on lung homogenates at the indicated timepoints. (n,o) Hematoxylin/eosin stain, Masson Goldner stain, IGF1 immunohistochemistry and TTF1 immunohistochemistry on saline- and bleomycin-treated mouse lungs. (p) IGF1R immunohistochemistry on saline- and bleomycin-treated mouse lungs. One-way ANOVA was used to compare the means. SAL: n = 6, BLM d7: n = 4, BLM d14: n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001. SAL: Saline; BLM: Bleomycin; IHC: Immunohistochemistry.

Figure 3

Alteration of IGF1 signaling in idiopathic pulmonary fibrosis. (a–k) qPCR on homogenates of lung explants derived from donor or IPF patients; (l,m) Immunohistochemistry for IGF1 and IGF1R on donor and IPF lung sections. t-test (b–e,g–j) or Mann–Whitney test (a,f,k) was performed to compare the groups. Donor: n = 8–10, IPF: n = 14–16. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. IHC: Immunohistochemistry.

Figure 4

Effect of recombinant IGF1 treatment on primary human lung fibroblasts. (a) Scheme for experimental design. (b–e) qPCR on primary IPF lung fibroblasts cultured on uncoated plates and treated with vehicle or recombinant human IGF1. (f–i) Similar analysis using coated plates. (j–m) Neutral lipid stain on primary IPF lung fibroblasts cultured on uncoated plates. Nuclei are stained with DAPI. (n) Quantification of LipidTOX staining. rhIGF1: Recombinant human IGF1; Veh: Vehicle. t-test was used to compare the means. (b–e) Veh: n = 8, rhIGF1: n = 7–8; (f–i) n = 3 per group; (n) n = 4 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.