Insulin-like growth factor-II is increased in systemic sclerosis-associated pulmonary fibrosis and contributes to the fibrotic process via Jun N-terminal kinase- and phosphatidylinositol-3 kinase-dependent pathways

Abstract

Systemic sclerosis (SSc)-related pulmonary fibrosis, for which there are few effective therapies, is the most common cause of SSc-related mortality. We examined insulin-like growth factor (IGF)-II expression in explanted lung tissues from control and SSc patients to determine its role in the pathogenesis of fibrosis. IGF-II levels in vivo were detected using immunohistochemistry. Primary lung fibroblasts were cultured from lung tissues, and IGF-II mRNA was measured using reverse transcriptase-polymerase chain reaction. Western blot analysis measured extracellular matrix (ECM) production and phosphorylated signaling molecules. Immunostaining revealed increased IGF-II expression in fibroblastic foci of SSc lungs. Furthermore, primary SSc lung fibroblasts had a fourfold increase in IGF-II mRNA and a twofold increase in IGF-II protein compared with normal lung fibroblasts. IGF-II mRNA in SSc lung fibroblasts was expressed primarily from the P3 promoter of the IGF-II gene, and IGF-II induced both a dose- and time-dependent increase in collagen type I and fibronectin production. IGF-II triggered the activation of both phosphatidylinositol-3 kinase and Jun N-terminal kinase signaling cascades, the inhibition of which diminished IGF-II-induced ECM production. Our study demonstrates increased local IGF-II expression in SSc-associated pulmonary fibrosis both in vitro and in vivo as well as IGF-II-induced ECM production through both phosphatidylinositol-3 kinase- and Jun N-terminal kinase-dependent pathways. Our results provide novel insights into the role of IGF-II in the pathogenesis of SSc-associated pulmonary fibrosis.

Figure 1

IGF-II levels are increased in vivo in SSc lung tissues. A: Immunohistochemical staining of SSc was performed using anti-IGF-II antibody (a–d). Goat IgG was used as an antibody control (e, f). Normal lung was also stained with anti-IGF-II antibody (g, h). SSc lungs have increased expression of IGF-II in fibroblastic foci (arrowheads) and epithelial cells (arrows). B: Immunofluorescent staining of SSc lung tissue using IGF-II (red) or α-SMA antibodies (green) (a–c), SSc lung with control antibodies (d–f), and normal lung with IGF-II and α-SMA antibodies (g–i). IGF-II co-localizes with α-SMA in some interstitial cells in SSc lungs (yellow arrowheads). IGF-II is also expressed in non-α-SMA-producing cells (white arrowheads). Pulmonary vessels stain for α-SMA, but not IGF-II (white arrows). Original magnifications: ×200 [A (a, e, g), B); ×400 [A (b–d, f, h)].

Figure 2

Steady-state IGF-II mRNA (A) and protein levels (B) are increased in SSc lung fibroblasts. A: RT-PCR analysis of total IGF-II mRNA levels in primary lung fibroblasts shows significantly increased expression in SSc versus normal fibroblasts (n = 6, 4.5 ± 2.3 versus 1.0 ± 0.5, respectively; P = 0.013). Relative expression was determined by scanning densitometry of PCR products normalized to β-actin mRNA levels. B: IGF-II protein was detected by immunofluorescence using polyclonal anti-IGF-II antibody and compared to goat isotype control antibody. Signal intensity was quantified with Metamorph imaging software (Molecular Devices) and shown in arbitrary units. SSc lung fibroblasts show a twofold increase in IGF-II protein compared to normal lung fibroblasts (n = 4, P = 1 × 10⁻⁷). Values and bars represent mean and SD, respectively. Original magnifications, ×400.

Figure 3

IGF-II gene expression is derived from P3 in SSc lung fibroblasts. A: RT-PCR of total RNA was performed using primer pairs specific for mRNA species generated from each promoter. As previously described, expected PCR product sizes were 363 bp for P1, 249 and 409 bp for P2, 151 bp for P3, and 76 bp for P4. B: IGF-II expression from one representative SSc lung fibroblast shows abundant expression from P3. There is no detectable mRNA from P1, P2, or P4. IGF-II expression from a representative normal lung fibroblast shows no IGF-II expression from any of the four promoters. Adult liver tissue and HepG2 cells served as positive controls for each primer pair. C: Comparison of mRNA from six SSc lung fibroblasts show increased IGF-II expression from P3 compared to six normal lung samples.

Figure 4

IGF-II induces collagen type I and fibronectin production in SSc primary lung fibroblasts in a dose-dependent manner (A) compared to normal fibroblasts (B). Equal numbers of fibroblasts were cultured with increasing concentrations of recombinant IGF-II for 48 hours. Collagen (C) and fibronectin (D) expression was quantified using scanning densitometry, and values were normalized to baseline SSc expression levels. SSc fibroblasts show a statistically significant increase in collagen (at 200 ng/ml IGF-II, 3.09 versus 1.0, ±1.8, in arbitrary units; P = 0.016) and fibronectin production (2.39 versus 1.0 ± 1.3; P = 0.019). Normal fibroblasts show peak collagen expression at 50 ng/ml of IGF-II (1.48 versus 0.64 ± 0.64; P = 0.020). Data shown represent experiments using five different SSc and five different normal fibroblasts. Asterisks indicate P < 0.05 compared with no IGF-II stimulation in SSc (one asterisk) and normal fibroblasts (two asterisks).

Figure 5

IGF-II induces collagen type I and fibronectin production in a time-dependent manner in primary lung fibroblasts from SSc (A) and normal lungs (B). Equal numbers of SSc and normal fibroblasts were cultured in media with or without IGF-II (200 ng/ml) for 24 to 72 hours. C: Both SSc and normal fibroblasts secreted significantly increased amounts of collagen throughout time compared to media alone. D: Only SSc fibroblasts showed significant fibronectin production at 48 hours. Data represent four different SSc and four different normal fibroblasts. Asterisk denotes P < 0.05.

Figure 6

IGF-II does not induce fibroblast proliferation. SSc and normal lung fibroblasts were treated with IGF-II for a total of 48 hours and proliferation measured using ³H-thymidine incorporation. SSc and normal fibroblasts do not show a statistically significant increase in proliferation using different IGF-II concentrations. Data are shown as a ratio of IGF-II stimulated to unstimulated fibroblast proliferation.

Figure 7

Activation of Akt (A) and JNK (B) signaling cascades in SSc and normal lung fibroblasts. Fibroblasts were treated with recombinant IGF-II (200 ng/ml) for 1 to 60 minutes or media alone (0 minutes). A: Both SSc and normal fibroblasts show peak activation of Akt and GSK-3β within 5 minutes of IGF-II stimulation. B: SAPK/JNK and c-Jun also show activation within 5 to 10 minutes after IGF-II stimulation.

Figure 8

IGF-II-induced ECM is inhibited by PI-3 kinase and JNK inhibitors. Primary lung fibroblasts from SSc (lanes 1 to 6) and normal lungs (lanes 7 to 12) were incubated with 10 μmol/L LY294002 (LY), U0126 (U), JNK inhibitor II (J), or vehicle control (veh) for 1 hour before the addition of IGF-II (200 ng/ml). Fibroblasts were stimulated with IGF-II for 48 hours before collection of lysates and supernatants. IGF-II induced collagen and fibronectin production (lanes 2, 3, 8, 9). This effect is blocked with the addition of a PI-3 kinase inhibitor (lanes 4 and 10) and JNK inhibitor (lanes 6 and 12). MEK inhibition did not have any effect on IGF-II-induced ECM production (lanes 5 and 11).