IGFBP-5 Promotes Fibrosis via Increasing Its Own Expression and That of Other Pro-fibrotic Mediators

Abstract

Pulmonary fibrosis is a hallmark of diseases such as systemic sclerosis (SSc, scleroderma) and idiopathic pulmonary fibrosis (IPF). To date, the therapeutic options for patients with pulmonary fibrosis are limited, and organ transplantation remains the most effective option. Insulin-like growth factor-binding protein 5 (IGFBP-5) is a conserved member of the IGFBP family of proteins that is overexpressed in SSc and IPF. In this study, we demonstrate that both exogenous and adenovirally expressed IGFBP-5 promote fibrosis by increasing the production of extracellular matrix (ECM) genes and the expression of pro-fibrotic genes in primary human lung fibroblasts. IGFBP-5 increased expression of the pro-fibrotic growth factor CTGF and levels of the matrix crosslinking enzyme lysyl oxidase (LOX). Silencing of IGFBP-5 had different effects in lung fibroblasts from normal donors and patients with SSc or IPF. Moreover, we show that IGFBP-5 increases expression of ECM genes, CTGF, and LOX in human lung tissues maintained in organ culture. Together, our data extend our previous findings and demonstrate that IGFBP-5 exerts its pro-fibrotic activity by directly inducing expression of ECM and pro-fibrotic genes. Further, IGFBP-5 promotes its own expression, generating a positive feedback loop. This suggests that IGFBP-5 likely acts in concert with other growth factors to drive fibrosis and tissue remodeling.

Keywords: extracellular matrix (ECM); fibrosis; idiopathic pulmonary fibrosis (IPF); insulin-like growth factor binding protein-5 (IGFBP-5); systemic sclerosis (SSc).

Figure 1

IGFBP-5 mRNA levels are increased in lung tissues and primary fibroblasts of SSc patients. (A) Expression of IGFBP-5 was evaluated in lung tissues from 7 normal controls (NL) and 8 patients with SSc pulmonary fibrosis using real-time PCR. (B) Expression of IGFBP-5 was evaluated in human primary lung fibroblasts from 8 normal controls (NL) and 8 patients with SSc pulmonary fibrosis using real-time PCR. Each dot represents one individual sample. Graphical presentation of the data analyzed by Mann-Whitney U-test. Values represent mean ± standard deviation. ^*P < 0.05, ^***P < 0.001.

Figure 2

IGFBP-5 exogenously promotes extracellular matrix (ECM) and pro-fibrotic factor production in vitro. Primary human lung fibroblasts were treated with vehicle control or recombinant protein IGFBP-5 (rBP5). Samples were harvested after 1 and 3 h of stimulation. Gene expression levels were quantified using qPCR, and fold-change estimates were calculated to compare rBP5 to vehicle. The following genes were analyzed: (A) Collagen 1A1. (B) FN. (C) CTGF. (D) IGFBP-5. (E) LOX. The data were obtained from 9 different experiments using fibroblasts from lung tissues of 9 different individual normal donors. Graphical presentation of the data analyzed by one-sided Wilcoxon signed rank tests. A dotted line at a fold-change of 1.0 (i.e., which would represent no change) is provided in each graph for reference. (F) Immunoblotting results of extracellular matrix fractions of primary human lung fibroblasts from three different donors treated with vehicle or rBP5 for 1 h. Upper images: Collagen1α1 (COL) and Fibronectin (FN) in ECM fractions from an equivalent number of fibroblasts were detected by immunoblotting and signals were normalized to vitronectin (VN). Lower graphs: Graphical presentation of the data analyzed by one-sided paired t-test. Values represent mean ± standard deviation. ^*P < 0.05, ^**P < 0.01.

Figure 3

IGFBP-5 expression promotes extracellular matrix (ECM) and pro-fibrotic factor expression in vitro. Primary human lung fibroblasts were infected with control adenovirus AdCN-Flag or adenovirus expressing IGFBP-5-Flag (AdBP-5-Flag). Samples were harvested after 22 and 28 h of infection. Levels of expression were quantified using qPCR at 22 and 28 h, and post-infection fold-change estimates were calculated to compare AdBP-5-Flag to control. The following genes were analyzed: (A) IGFBP-5. (B) Collagen 1A1. (C) FN. (D) CTGF. (E) LOX. The data are obtained from 7 different experiments using fibroblasts from lung tissues of 7 different normal donors. Graphical presentation of the data analyzed by one-sided Wilcoxon signed rank tests. Values represent mean ± standard deviation. A dotted line at a fold-change of 1.0 (i.e., which would represent no change) is provided in each graph for reference. (F) Representative protein levels of Collagen 1A1 (Col) and Fibronectin (FN) in the ECM fraction from an equivalent number of fibroblasts infected with AdCN-Flag or adenovirus expressing IGFBP-5-Flag (AdBP-5-Flag) for 24 hr and analyzed by immunoblotting. GAPDH was detected in the corresponding lysates using immunoblotting. ^*P < 0.05, ^**P < 0.01.

Figure 4

Knockdown of IGFBP-5 shows variable effects in primary pulmonary fibroblasts from different donors. IGFBP-5 was silenced in primary human lung fibroblasts from normal lung (NL, circles) and lung tissues of patients with IPF (squares) and SSc (triangles) using small interfering IGFBP-5 (siBP-5) or scramble siRNA as a negative control (siCN) for 48 h. Levels of expression were quantified using qPCR, and fold-change estimates were calculated to compare siBP-5 to siCN. The following genes were analyzed: (A) IGFBP-5. (B) Collagen 1A1. (C) FN. (D) CTGF. (E) LOX. The data shown are obtained using fibroblasts from 4 different normal donors (NL), 5 patients with IPF and 4 patients with SSc. Graphical presentation of the data analyzed by one-sided Wilcoxon signed rank tests. A dotted line at a fold-change of 1.0 (i.e., which would represent no change) is provided in each graph for reference. Values represent mean ± standard deviation. ^*P < 0.05.

Figure 5

IGFBP-5 promotes ECM and pro-fibrotic factor production ex vivo in human lung tissues. Human lung tissue cores were treated with vehicle control (VC, 10 mM HCl) or recombinant IGFBP-5 (rBP5, 500 ng/ml) for 16 and 30 h. Gene expression levels were measured using qPCR at the indicated time points, and fold-change estimates were calculated to compare IGFBP-5 to VC. The following genes were analyzed: (A) Collagen 1A1 mRNA levels at 16 h. (B) FN mRNA levels at 16 h. (C) CTGF mRNA levels at 16 h. (D). LOX mRNA levels at 16 h. (E) LOX mRNA levels at 30 h. The data shown are obtained from lung tissues of 7–9 different donors. Graphical presentation of the data analyzed by one-sided Wilcoxon signed rank tests. A dotted line at a fold-change of 1.0 (i.e., which would represent no change) is provided in each graph for reference. Values represent mean ± standard deviation. ^*P < 0.05; ^**P < 0.01.