Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease of unknown etiology that results in significant morbidity and mortality. The pathogenesis of IPF is not completely understood. Because recent studies have implicated insulin-like growth factor-I (IGF-I) in the pathogenesis of fibrosis, we examined the expression and function of insulin-like growth factor binding proteins (IGFBP)-3 and -5 in IPF. IGFBP-3 and -5 levels were increased in vivo in IPF lung tissues and in vitro in fibroblasts cultured from IPF lung. The IGFBPs secreted by IPF fibroblasts are functionally active and can bind IGF-I, and IGFBPs secreted by primary fibroblasts bind extracellular matrix components. Our results also suggest that IGFBPs may be involved in the initiation and/or perpetuation of fibrosis by virtue of their ability to induce the production of extracellular matrix components such as collagen type I and fibronectin in normal primary adult lung fibroblasts. Although transforming growth factor-beta increased IGFBP-3 production by primary fibroblasts in a time-dependent manner, IGFBP-5 levels were not increased by transforming growth factor-beta. Taken together, our results suggest that IGFBPs play an important role in the development of fibrosis in IPF.

Figure 1

IGFBP-3 and -5 levels in IPF lung tissues. Serial sections of lung tissues from a healthy donor (A–D) and an IPF patient undergoing lung transplantation (E–H) were analyzed by immunohistochemistry using anti-fibronectin (B, F), anti-IGFBP-3 (C, G), or anti-IGFBP-5 (D, H) antibodies. A and E are representative images from tissue sections in which primary antibody was omitted. Fibronectin antibody labeled alveolar septae (arrowhead in B) and fibroblasts (arrows in F). IGBFP-3 and -5 antibodies labeled cells having the appearance of alveolar type II cells (arrows in C and D) but not normal alveolar septae (arrowheads in C and D). In IPF samples, IGFBP-3 and -5 strongly labeled fibroblasts in thickened alveolar septae (arrows in G and H). Data are representative of three NL and three IPF lung tissues. Original magnifications, ×400.

Figure 2

IGFBP-5 expression in IPF. IGFBP-5 antibody labeled interstitial fibroblasts (arrows) and epithelial cells in residual alveolar spaces (arrowheads in A) and in bronchiolar epithelial cells (arrowheads in B). Original magnifications, ×400.

Figure 3

Increased IGFBP-5 and -3 in IPF fibroblasts. Increased IGFBP-5 and -3 mRNA in fibroblasts from IPF lung tissue. A: IGFBP-5 mRNA levels: IGFBP-5 mRNA levels were analyzed by RPA in passage 3 lung fibroblasts from two healthy donors (NL) and five IPF patients (IPF). Yeast RNA was used as control (lane C). GAPDH mRNA levels were used to normalize the signal. B: IGFBP-3 mRNA levels: IGFBP-3 mRNA levels were analyzed by RPA as described in A. C: Graphical presentation of the data analyzed by scanning densitometry. Values represent mean ± SE.

Figure 4

A: Increased secretion of IGFBP-3 and -5 by IPF fibroblasts. Fibroblasts from control (NL) and IPF lung tissues were cultured and used in early passage. Cells were plated at equal numbers and conditioned media collected. Supernatant from 1 × 10⁵ fibroblasts was analyzed by Western blot using anti-IGFBP-5 and anti-IGFBP-3 antibodies. B: Graphical presentation of the data analyzed by scanning densitometry. Values represent mean ± SE.

Figure 5

Increased levels of collagen I, fibronectin, and IGFBP-3 and -5 in ECM from IPF fibroblasts: Western blot analysis of IGFBP-3 and -5 in ECM from control and IPF fibroblasts. ECM was prepared from NL and IPF fibroblasts and analyzed by Western blot using anti-IGFBP-3, IGFBP-5, collagen, and fibronectin antibodies. ECM fractions from 1 × 10⁵ fibroblasts were loaded in each lane. Comparisons are representative of fibroblasts from seven different IPF patients and four healthy controls.

Figure 6

Cell-associated IGFBP-3 and -5 expression is increased in cultured IPF fibroblasts compared to normal fibroblasts. Primary fibroblasts from normal and IPF lung tissues were cultured in four-chamber slides. Cells were fixed and stained with anti-IGFBP-3 and -5 antibodies as described in the Materials and Methods section. As a control (C), primary antibody was omitted from the incubation steps. Original magnifications, ×200.

Figure 7

Western ligand blot analysis of IGFBP. Functional IGFBPs in ECM were analyzed by Western ligand blot using biotinylated IGF-I. ECM extracted from 1 × 10⁵ cells was loaded in each lane. IGFBP-3 and -5 monomers correspond to the 30- to 35-kd and 40- to 48-kd bands indicated by asterisked arrows. Higher molecular weight bands represent dimers and multimers of IGFBP-3 and -5 based on molecular weight estimates. Comparisons are representative of two normal and two IPF lung fibroblast lines. Experiments with each pair were repeated three times.

Figure 8

Binding of IGFBP-3 and -5 to collagen type I. Supernatant (SUP) and ECM fractions of fibroblasts from control and IPF lung were incubated with protein A agarose-bound IGFBP-3 or IGFBP-5. Samples were analyzed by Western blot using a polyclonal anti-collagen I antibody. Note increased collagen I-bound IGFBP-3 and -5 in IPF relative to control.

Figure 9

Culture of normal lung fibroblasts on collagen or fibronectin increases IGFBP-3 and -5 levels. Tissue culture dishes were coated with collagen I or fibronectin. Primary normal lung fibroblasts were cultured on uncoated, collagen-coated, or fibronectin-coated dishes for 4 days. ECM from 1 × 10⁵ fibroblasts was collected and analyzed by Western blot analysis for IGFBP-3, IGFBP-5, and fibronectin.

Figure 10

IGFBP-3 and -5 induce ECM production. Primary adult lung fibroblasts were incubated with recombinant IGFBP-3 or -5 for 48 hours (A) or with IGFBP-5 for the indicated time points (B). ECM was extracted and analyzed by Western blot using anti-fibronectin antibody. Note induction of fibronectin by both IGFBP-3 and -5. C: Primary lung fibroblasts were infected with adenovirus expressing IGFBP-5. Increased expression of IGFBP-5 in adenovirus-IGFBP-5-infected fibroblasts was confirmed by Western blot analysis. D: ECM and conditioned media were collected from uninfected (lane C) and adenovirus-IGFBP-5-infected fibroblasts 4 or 5 days after infection, and analyzed by Western blot analysis using anti-fibronectin and anti-collagen antibodies, respectively. Lysates from cells used for the extraction of ECM were analyzed using anti-β actin antibody. E: As a control, fibroblasts were uninfected (No Ad) or infected with a control adenovirus (C-Ad). ECM was extracted and analyzed as described above.

Figure 11

TGF-β stimulates IGFBP-3, but not IGFBP-5 production by normal fibroblasts. Primary normal lung fibroblasts were treated with 1 ng/ml of TGF-β for the indicated times. Conditioned media from 4.5 × 10³ fibroblasts were analyzed by Western blot for IGFBP-3 and IGFBP-5. C denotes absence of TGF-β.