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
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.071021
ISSN1525-2191
AutoresEileen Hsu, Carol Feghali‐Bostwick,
Tópico(s)Fibroblast Growth Factor Research
ResumoSystemic 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. 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. Systemic sclerosis (SSc)-related pulmonary fibrosis is the most common cause of SSc-related mortality.1Steen VD Medsger TA Changes in causes of death in systemic sclerosis, 1972–2002.Ann Rheum Dis. 2007; 66: 940-944Crossref PubMed Scopus (1152) Google Scholar Fibrosis in SSc is believed to result from the interaction of immune mediators and other growth factors with fibroblasts, which respond by increasing matrix production in the skin and internal organs. Insulin-like growth factors and their binding proteins (IGFBP) have been implicated in the pathogenesis of pulmonary fibrosis. Increased IGF-I has been reported in bronchoalveolar lavage fluid in patients with SSc-related pulmonary fibrosis2Harrison NK Cambrey AD Myers AR Southcott AM Black CM du Bois RM Laurent GJ McAnulty RJ Insulin-like growth factor-I is partially responsible for fibroblast proliferation induced by bronchoalveolar lavage fluid from patients with systemic sclerosis.Clin Sci (Lond). 1994; 86: 141-148PubMed Google Scholar as well as other forms of pulmonary fibrosis such as idiopathic pulmonary fibrosis, sarcoidosis, and coal miner's pneumoconiosis.3Homma S Nagaoka I Abe H Takahashi K Seyama K Nukiwa T Kira S Localization of platelet-derived growth factor and insulin-like growth factor I in the fibrotic lung.Am J Respir Crit Care Med. 1995; 152: 2084-2089Crossref PubMed Scopus (124) Google Scholar, 4Vanhee D Gosset P Wallaert B Voisin C Tonnel AB Mechanisms of fibrosis in coal workers' pneumoconiosis. Increased production of platelet-derived growth factor, insulin-like growth factor type I, and transforming growth factor beta and relationship to disease severity.Am J Respir Crit Care Med. 1994; 150: 1049-1055Crossref PubMed Scopus (87) Google Scholar IGF-II levels, on the other hand, have not been examined. We have found IGFBP-3 and -5 to be elevated in lung tissues of patients with pulmonary fibrosis.5Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar We have also shown that IGFBP-5 can induce fibrosis in vitro and in vivo.6Yasuoka H Jukic DM Zhou Z Choi AM Feghali-Bostwick CA Insulin-like growth factor binding protein 5 induces skin fibrosis: a novel murine model for dermal fibrosis.Arthritis Rheum. 2006; 54: 3001-3010Crossref PubMed Scopus (46) Google Scholar, 7Yasuoka H Zhou Z Pilewski JM Oury TD Choi AM Feghali-Bostwick CA Insulin-like growth factor-binding protein-5 induces pulmonary fibrosis and triggers mononuclear cellular infiltration.Am J Pathol. 2006; 169: 1633-1642Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Our findings suggest that the IGF/IGFBP system plays a role in the development and perpetuation of pulmonary fibrosis. IGF-II is a mitogenic peptide that is required for fetal growth and lung development.8Silva D Venihaki M Guo WH Lopez MF Igf2 deficiency results in delayed lung development at the end of gestation.Endocrinology. 2006; 147: 5584-5591Crossref PubMed Scopus (34) Google Scholar IGF-II is normally highly expressed in the fetus. Shortly after birth, IGF-II levels dramatically decrease in the systemic circulation, but IGF-II remains locally expressed in a variety of tissues. Elevated IGF-II levels in malignancy have classically been studied in Wilms' tumor9Scott J Cowell J Robertson ME Priestley LM Wadey R Hopkins B Pritchard J Bell GI Rall LB Graham CF Knott TJ Insulin-like growth factor-II gene expression in Wilms' tumour and embryonic tissues.Nature. 1985; 317: 260-262Crossref PubMed Scopus (329) Google Scholar and subsequently implicated in other malignancies such as colon adenocarcinoma and breast cancer.10Ghahary A Tredget EE Shen Q Kilani RT Scott PG Houle Y Mannose-6-phosphate/IGF-II receptors mediate the effects of IGF-1-induced latent transforming growth factor beta 1 on expression of type I collagen and collagenase in dermal fibroblasts.Growth Factors. 2000; 17: 167-176Crossref PubMed Scopus (37) Google Scholar, 11Giani C Pinchera A Rasmussen A Fierabracci P Bonacci R Campini D Bevilacqua G Trock B Lippman ME Cullen KJ Stromal IGF-II messenger RNA in breast cancer: relationship with progesterone receptor expressed by malignant epithelial cells.J Endocrinol Invest. 1998; 21: 160-165PubMed Google Scholar Elevated IGF-II expression has also been implicated in Beckwith-Wiedemann syndrome,12Morison IM Becroft DM Taniguchi T Woods CG Reeve AE Somatic overgrowth associated with overexpression of insulin-like growth factor II.Nat Med. 1996; 2: 311-316Crossref PubMed Scopus (121) Google Scholar which is characterized by somatic overgrowth, glossomegaly, and organomegaly. IGF-II has been shown to stimulate human lung fibroblast proliferation.13Hetzel M Bachem M Anders D Trischler G Faehling M Different effects of growth factors on proliferation and matrix production of normal and fibrotic human lung fibroblasts.Lung. 2005; 183: 225-237Crossref PubMed Scopus (165) Google Scholar, 14Scarpa RC Carraway RE Cochrane DE Insulin-like growth factor (IGF) induced proliferation of human lung fibroblasts is enhanced by neurotensin.Peptides. 2005; 26: 2201-2210Crossref PubMed Scopus (11) Google Scholar However, the role of IGF-II in fibrosis and wound healing has not been delineated. No published studies to date have examined the role of IGF-II in human pulmonary fibrosis or in SSc. IGF-II gene transcription can be initiated from one of four promoter sites (P1 to P4), producing unique mRNA species, which generate the same IGF-II peptide.15de Pagter-Holthuizen P Jansen M van der Kammen RA van Schaik FM Sussenbach JS Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and an mRNA encoding a putative IGF-II-associated protein.Biochim Biophys Acta. 1988; 950: 282-295Crossref PubMed Scopus (135) Google Scholar, 16Holthuizen P van der Lee FM Ikejiri K Yamamoto M Sussenbach JS Identification and initial characterization of a fourth leader exon and promoter of the human IGF-II gene.Biochim Biophys Acta. 1990; 1087: 341-343Crossref PubMed Scopus (78) Google Scholar Gene expression from each promoter is tissue- and development-specific.15de Pagter-Holthuizen P Jansen M van der Kammen RA van Schaik FM Sussenbach JS Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and an mRNA encoding a putative IGF-II-associated protein.Biochim Biophys Acta. 1988; 950: 282-295Crossref PubMed Scopus (135) Google Scholar, 17de Pagter-Holthuizen P Jansen M van Schaik FM van der Kammen R Oosterwijk C Van den Brande JL Sussenbach JS The human insulin-like growth factor II gene contains two development-specific promoters.FEBS Lett. 1987; 214: 259-264Crossref PubMed Scopus (135) Google Scholar Fetal tissues normally express IGF-II from P2, P3, and P4, whereas the adult liver expresses IGF-II from P1. A variety of malignancies have been found to express IGF-II from P3 and P4.18Vu TH Hoffman A Alterations in the promoter-specific imprinting of the insulin-like growth factor-II gene in Wilms' tumor.J Biol Chem. 1996; 271: 9014-9023Crossref PubMed Scopus (47) Google Scholar, 19Lu L Katsaros D Wiley A Rigault de la Longrais IA Puopolo M Schwartz P Yu H Promoter-specific transcription of insulin-like growth factor-II in epithelial ovarian cancer.Gynecol Oncol. 2006; 103: 990-995Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 20Singh P Dai B Given RL Lu X Holthuizen PE Differential activation of IGF-II promoters P3 and P4 in Caco-2 cells during growth and differentiation.Gastroenterology. 1998; 114: 1221-1229Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 21Tang SH Yang DH Huang W Zhou M Zhou HK Lu XH Ye G Differential promoter usage for insulin-like growth factor-II gene in Chinese hepatocellular carcinoma with hepatitis B virus infection.Cancer Detect Prev. 2006; 30: 192-203Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar We have identified IGF-II as a novel factor involved in the pathogenesis of pulmonary fibrosis in SSc. Using tissues derived from explanted lungs of patients with scleroderma-associated pulmonary fibrosis, we show that IGF-II is aberrantly expressed in lung fibroblasts. We also demonstrate that the IGF-II gene in SSc lung fibroblasts is transcribed primarily from the P3 promoter. Additionally, we show that IGF-II induces extracellular matrix (ECM) deposition in primary lung fibroblasts. We thus conclude that IGF-II local expression in SSc lung is increased and propose that IGF-II contributes to the pathogenesis of pulmonary fibrosis in SSc by inducing ECM deposition in an autocrine or paracrine manner. Lung explants were obtained from SSc patients undergoing lung transplantation at the University of Pittsburgh Medical Center under a protocol approved by the University of Pittsburgh Institutional Review Board. Normal lung tissues were obtained from organ donors whose lungs were not used for transplant surgery. Fibroblasts were cultured from minced tissue in Dulbecco's modified Eagle's medium (Invitrogen Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), penicillin, streptomycin, and antimycotic agent (Invitrogen). Cells were used in passages two to six. Fibroblasts were stimulated with recombinant human IGF-II (R&D Systems, Minneapolis, MN). Kinase inhibitors LY294002 (Cell Signaling Technology, Danvers, MA), U0126 (Cell Signaling Technology), and Jnk II inhibitor (Calbiochem, San Diego, CA) were added to media of cells 1 hour before IGF-II stimulation. Six-μm sections of paraffin-embedded lung tissues were deparaffinized, and endogenous peroxidases were quenched with 3% H2O2. Sections were blocked with 5% serum and incubated with polyclonal anti-IGF-II antibody or IgG isotype controls (R&D Systems). Sections were washed and incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA). Bound secondary antibody was detected using the Vectastain ABC kit (Vector Laboratories) and the AEC Red kit (Zymed, San Francisco, CA). A light hematoxylin counterstain was used to identify nuclei. Images were taken on an Eclipse 800 microscope (Nikon Instruments, Huntley, IL) using identical camera settings. Four separate SSc and normal lung tissues were analyzed and representative images were shown. Double-immunofluorescent staining was performed using polyclonal anti-IGF-II (R&D Systems) and monoclonal anti-α-smooth muscle actin (Sigma-Aldrich) antibodies or goat (R&D Systems) and mouse (Lab Vision Corporation, Fremont, CA) isotype controls. Sections were incubated with biotinylated secondary antibodies and visualized with Texas Red- and AMCA-avidin-conjugated stains (Vector Laboratories). Images were taken on an Olympus Provis microscope (Olympus America, Melville, NY) using identical camera settings. Primary lung fibroblasts were fixed in acetone and blocked with 5% serum. Cells were incubated with anti-IGF-II or goat IgG antibodies, washed, and incubated with biotinylated secondary anti-goat antibody. Bound secondary antibody was detected with Texas Red Avidin D stain (Vector Laboratories). Nuclei were stained with 4,6-diamidino-2-phenylindole (Vector Laboratories). Images were taken on an Olympus Fluoview 1000 (Olympus America) under identical settings. Signal intensity was measured using Metamorph imaging software (Molecular Devices, Sunnyvale, CA). Primary lung fibroblasts were grown in media containing 0.5% fetal bovine serum, and total mRNA was isolated using Trizol reagent (Invitrogen). Normal liver tissue was obtained through the Liver Tissue Procurement and Distribution System, Pittsburgh, Pennsylvania, which is supported by the National Institutes of Health contract no. N01-DK-9-2310. Total RNA from homogenized whole liver tissue was isolated using cesium chloride density-gradient ultracentrifugation as previously described.22Feghali CA Wright TM Identification of multiple, differentially expressed messenger RNAs in dermal fibroblasts from patients with systemic sclerosis.Arthritis Rheum. 1999; 42: 1451-1457Crossref PubMed Scopus (58) Google Scholar Total RNA from HepG2 hepatocarcinoma cell lines was isolated using Trizol. Complementary DNA was prepared using Superscript II reverse transcriptase and random hexamer primers (Invitrogen). PCR was performed using primers designed specifically for total IGF-II mRNA amplification: forward primer 5′-TCCTCCCTGGACAATCAGAC-3′ and reverse 5′-AGAAGCACCAGCATCGACTT-3′. Primers for promoter-specific IGF-II mRNA species were previously described.18Vu TH Hoffman A Alterations in the promoter-specific imprinting of the insulin-like growth factor-II gene in Wilms' tumor.J Biol Chem. 1996; 271: 9014-9023Crossref PubMed Scopus (47) Google Scholar Promoter-specific forward primers were 5′-CAGTGACTCCCCGGTCCTCTTTAT-3′ (P1, 362 bp), 5′-ACCGGGCATTGCCCCCAGTCTCC-3′ (P2, 249 and 409 bp), 5′-CAGAGCGGCGCTGGCAGAGGAGT-3′ (P3, 151 bp), and 5′-CAGCGAGCCTTCTGCTGAGCTGTA-3′ (P4, 76 bp). Reverse primer p11 used for promoter-specific mRNA was 5′-GAAGCTTAGAAGCACCAGCATCGACTTC-3′. Primers used for β-actin were forward primer 5′-ATGTTTGAGACCTTCAACAC-3′ and reverse primer 5′-CACGTCACACTTCATGATGG-3′. PCR products were separated by electrophoresis on an agarose or polyacrylamide gel and visualized after ethidium bromide staining. Expression levels were measured by scanning densitometry. Culture supernatants and cell lysates were obtained as previously reported.5Pilewski JM Liu L Henry AC Knauer AV Feghali-Bostwick CA Insulin-like growth factor binding proteins 3 and 5 are overexpressed in idiopathic pulmonary fibrosis and contribute to extracellular matrix deposition.Am J Pathol. 2005; 166: 399-407Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 7Yasuoka H Zhou Z Pilewski JM Oury TD Choi AM Feghali-Bostwick CA Insulin-like growth factor-binding protein-5 induces pulmonary fibrosis and triggers mononuclear cellular infiltration.Am J Pathol. 2006; 169: 1633-1642Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Samples were analyzed by Western blotting with polyclonal anti-type I collagen, monoclonal anti-fibronectin, monoclonal anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-GSK-3β, anti-phospho-SAPK/JNK, anti-JNK, and anti-phospho-c-Jun antibodies (Cell Signaling Technology). Signals were detected after incubation with horseradish peroxidase-conjugated secondary antibody and chemiluminescence (Perkin Elmer, Boston, MA). Equal numbers of fibroblasts were plated in triplicate in 96-well plates and cultured in serum-deprived media for 24 hours. Fibroblasts were then stimulated with IGF-II for a total of 48 hours. 3H-thymidine was added (1 μCi/well) for the last 24 hours of incubation. 3H-thymidine counts were measured in a β-emission counter in triplicate for each condition and results were averaged and normalized to counts obtained using unstimulated fibroblasts. All comparisons were performed using paired t-test. Lung sections were obtained from SSc patients undergoing lung transplantation at the University of Pittsburgh Medical Center between 1999 and 2006. All SSc lung sections were examined by a pulmonary pathologist and were characterized as usual interstitial pneumonia pattern (Table 1). Normal lung sections were obtained from a pool of potential organ donors through the Center for Organ Recovery and Education (Table 2). IGF-II protein expression was analyzed by immunohistochemistry in paraffin-embedded lung sections from patients and compared with lung sections from healthy donors (Figure 1A). Normal lungs did not show any significant IGF-II staining. IGF-II expression was increased in SSc lungs compared to normal lung tissues and localized to spindle-shaped cells in the interstitial space that were suggestive of fibroblastic foci. IGF-II also localized to epithelial cells of alveoli and airway. Immunohistochemical staining of four SSc and four normal lungs showed similar results.Table 1Clinical Characteristics of Patients with SScAge (years)/sexLung histologySmoking (pack years)Disease typeAutoantibodySSc-548/FUIP0DiffuseTopoisomeraseSSc-826/FUIP0SineU11/U12 RNPSSc-2444/MUIP0n/an/aSSc-2657/MUIP0Diffusen/aSSc-2741/FUIP5DiffuseU11/U12 RNPSSc-2858/MUIP30DiffuseRNA polymeraseSSc-3058/FUIP0n/an/aDiffuse, diffuse cutaneous skin involvement; sine, systemic sclerosis sine scleroderma; n/a, not available. Open table in a new tab Table 2Clinical Characteristics of Normal Lung DonorsAge (years)/sexCause of deathSmokingNL-1443/MCerebrovascular accidentYNL-1826/FAspirin overdosen/aNL-2257/FIntracranial hemorrhageNNL-2618/FTraumaNNL-3227/n/aMotor vehicle accidentNNL-4153/FSubarachnoid hemorrhageYNL-4222/Fn/an/an/a: not available. Open table in a new tab Diffuse, diffuse cutaneous skin involvement; sine, systemic sclerosis sine scleroderma; n/a, not available. n/a: not available. To determine whether myofibroblasts are a source of IGF-II, IGF-II and α-smooth muscle actin (α-SMA) were detected in SSc and normal lung tissues using double-immunofluorescence staining (Figure 1B). In SSc lung tissues, IGF-II was detected in both α-SMA-positive and -negative interstitial cells. This suggests that IGF-II is expressed by both activated and nonactivated lung fibroblasts in SSc-associated pulmonary fibrosis. In normal lungs, α-SMA was detected in pulmonary vessels whereas no significant IGF-II was detected, confirming our immunohistochemical findings. In vivo analysis of IGF-II expression in SSc lungs suggested that fibroblasts are a source of IGF-II expression in SSc lung fibrosis. To examine IGF-II production by pulmonary fibroblasts, we cultured primary lung fibroblasts from the same lung tissues. Equal numbers of fibroblasts from SSc and normal lungs were cultured, and equal amounts of RNA were used in RT-PCR for the detection of IGF-II mRNA levels. Steady-state mRNA levels were 4.5-fold higher in SSc lung fibroblasts compared to normal controls (4.5 ± 2.3 versus 1.0 ± 0.5, respectively; P = 0.013) (Figure 2A). Additionally, immunofluorescent analysis of fibroblasts showed a significant increase in IGF-II protein expression in primary lung fibroblasts from SSc lungs compared to normal fibroblasts (41.3 ± 8.3 versus 19.3 ± 5.4, respectively, in arbitrary units; P = 1 × 10−7) (Figure 2B). The increased IGF-II mRNA and protein levels in SSc lung fibroblasts suggest that IGF-II is aberrantly expressed from lung fibroblasts in SSc-related pulmonary fibrosis. Expression of the IGF-II gene is regulated by four promoters (P1 to P4) and generates a variety of mRNA species that ultimately encode the same IGF-II peptide. Promoter usage has been shown to be tissue- and development-specific.15de Pagter-Holthuizen P Jansen M van der Kammen RA van Schaik FM Sussenbach JS Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and an mRNA encoding a putative IGF-II-associated protein.Biochim Biophys Acta. 1988; 950: 282-295Crossref PubMed Scopus (135) Google Scholar, 17de Pagter-Holthuizen P Jansen M van Schaik FM van der Kammen R Oosterwijk C Van den Brande JL Sussenbach JS The human insulin-like growth factor II gene contains two development-specific promoters.FEBS Lett. 1987; 214: 259-264Crossref PubMed Scopus (135) Google Scholar We performed a semiquantitative analysis of IGF-II promoter usage by using previously described primer pairs designed to amplify specific IGF-II transcripts derived from P1 to P4 (Figure 3A).18Vu TH Hoffman A Alterations in the promoter-specific imprinting of the insulin-like growth factor-II gene in Wilms' tumor.J Biol Chem. 1996; 271: 9014-9023Crossref PubMed Scopus (47) Google Scholar Primary lung fibroblasts from patients with SSc showed an increased expression of IGF-II derived from P3, but no mRNA from P1, P2, and P4 was detected (Figure 3B). Additionally, primary lung fibroblasts derived from six patients with SSc all show IGF-II gene expression from P3 (Figure 3C). Only one of six primary fibroblasts from normal lungs showed any detectable levels of IGF-II mRNA from P3. Overall, normal fibroblasts did not express IGF-II P3 mRNA. Fibroblasts are the major source of ECM production and deposition in pulmonary fibrosis. To determine whether IGF-II contributes to ECM deposition, we measured expression of fibronectin and type I collagen, the major components of ECM in pulmonary fibrosis. Equal numbers of fibroblasts were cultured with varying concentrations of recombinant IGF-II for 48 hours. IGF-II increased ECM production and secretion in lung fibroblasts from SSc patients in a dose-dependent manner (Figure 4). SSc fibroblasts showed increased collagen and fibronectin production with the addition of 100 and 200 ng/ml of IGF-II compared to vehicle control. Normal fibroblasts showed significantly increased collagen production and a trend toward increased fibronectin production with 50 ng/ml of IGF-II, but these effects were more modest with increasing amounts of IGF-II. To determine whether prolonged exposure of fibroblasts affects ECM production, fibroblasts were incubated in media with and without IGF-II for 24 to 72 hours. SSc and normal lung fibroblasts secreted increasing amounts of collagen and fibronectin with prolonged incubation in growth media alone (Figure 5). Both SSc and normal lung fibroblasts showed significantly increased collagen production at 24, 48, and 72 hours with IGF-II compared to control treated cells. A significant increase in fibronectin production was seen in SSc fibroblasts stimulated with IGF-II for 48 hours. Although normal lung fibroblasts showed a trend toward a time-dependent increase in fibronectin production, differences in fibronectin levels did not reach statistical significance. The dose- and time-dependent effects of IGF-II on fibronectin and collagen secretion in SSc lung fibroblasts suggest that IGF-II directly contributes to ECM production and that SSc fibroblasts may be more susceptible to the ECM-inducing effects of IGF-II compared to normal fibroblasts. IGF-II is known to exert proliferative effects on a variety of cell types. We treated SSc and normal fibroblasts with different concentrations of IGF-II for 48 hours. IGF-II did not induce a significant difference in 3H-thymidine incorporation in SSc fibroblasts (P = 0.17) (Figure 6). Similarly IGF-II had no significant effect on the proliferation of normal fibroblasts (P = 0.14). IGF-II stimulates cell activity via tyrosine kinase receptors IGF-IR and insulin receptor isoform A (IR-A), which activate a variety of signaling cascades. The PI3 kinase pathway of intracellular signaling was examined by Western blot analysis of activated signaling molecules. Phosphorylation of Akt and GSK-3β increased within 5 minutes of IGF-II stimulation (Figure 7A). Similarly, the SAPK/JNK cascade was examined by detection of phosphorylated kinases. JNK and c-Jun displayed peak activation between 5 to 10 minutes of IGF-II stimulation (Figure 7B). These findings suggest that IGF-II causes activation of PI3 kinase and JNK signaling cascades in primary fibroblasts. To confirm the role of these signaling cascades in the response of primary fibroblasts to IGF-II, we pretreated SSc and normal lung fibroblasts with specific inhibitors of PI3K (LY294002), MEK (U0126), and JNK. As shown in Figure 8, IGF-II-induced fibronectin and collagen secretion was abrogated by PI3K and JNK inhibition. On the other hand, IGF-II-induced ECM secretion was not blocked by MEK inhibition. These findings further confirm that IGF-II induces ECM production in primary fibroblasts via PI3K- and JNK-dependent pathways. Pulmonary fibrosis is the leading cause of death in individuals with SSc, but few therapies are effective. Although the IGF system has been implicated in the pathogenesis of pulmonary fibrosis, no studies to date have examined the role of IGF-II in pulmonary fibrosis. We demonstrate IGF-II expression is increased in vivo in lung tissues of patients with SSc-related pulmonary fibrosis. Additionally, our studies show increased IGF-II expression in vitro in SSc lung fibroblasts compared to normal fibroblasts. Retrospective studies suggest that a nonspecific interstitial pneumonia pattern is more common than a usual interstitial pneumonia pattern in SSc patients who underwent lung biopsy.23Bouros D Wells AU Nicholson AG Colby TV Polychronopoulos V Pantelidis P Haslam PL Vassilakis DA Black CM du Bois RM Histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome.Am J Respir Crit Care Med. 2002; 165: 1581-1586Crossref PubMed Scopus (651) Google Scholar, 24Kim DS Yoo B Lee JS Kim EK Lim CM Lee SD Koh Y Kim WS Kim WD Colby TV Kitiaichi M The major histopathologic pattern of pulmonary fibrosis in scleroderma is nonspecific interstitial pneumonia.Sarcoidosis Vasc Diffuse Lung Dis. 2002; 19: 121-127PubMed Google Scholar These studies also show that nonspecific interstitial pneumonia carries a better prognosis compared to usual interstitial pneumonia. All of our patients underwent lung transplantation for end-stage pulmonary fibrosis. Not surprisingly, these lungs all had a usual interstitial pneumonia pattern. Normal lung samples were limited to a pool of potential lung donors, which generally are younger than lung transplant recipients. Therefore, one weakness of our study is the age difference between SSc patients and normal donors. Although no studies have examined the relationship of age and local IGF-II expression in the lung, serum IGF-II levels are known to decrease with age in adults.25Pfeilschifter J Scheidt-Nave C Leidig-Bruckner G Woitge HW Blum WF Wuster C Haack D Ziegler R Relationship between circulating insulin-like growth factor components and sex hormones in a population-based sample of 50- to 80-year-old men and women.J Clin Endocrinol Metab. 1996; 81: 2534-2540Crossref PubMed Scopus (143) Google Scholar, 26Ceda GP Dall'Aglio E Magnacavallo A Vargas N Fontana V Maggio M Valenti G Lee PD Hintz RL Hoffman AR The insulin-like growth factor axis and plasma lipid levels in the elderly.J Clin Endocrinol Metab. 1998; 83: 499-502Crossref PubMed Scopus (62) Google Scholar This suggests that our findings are significant despite the age di
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