Insulin-Like Growth Factor-I Enhances Transforming Growth Factor-β-Induced Extracellular Matrix Protein Production Through the P38/Activating Transcription Factor- 2 Signaling Pathway in Keloid Fibroblasts  Takehiro Daian, Hiroshi Ishihara, Akiyoshi Hirano, Tohru Fujii  Journal of Investigative Dermatology  Volume 120, Issue 6, Pages 956-962 (June 2003) DOI: 10.1046/j.1523-1747.2003.12143.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Western blotting of ECM proteins in culture media from dermal fibroblasts. (A) Concentration-dependent effect of TGF-β1 stimulation on collagen type I secretion by cultured human fibroblasts. Lanes 1–3: normal fibroblasts (N-1–3); lanes 4–6: keloid fibroblasts (K-1–3). Equivalent protein load was controlled by Bio-Rad protein assay kit. (B) Collagen type I expression after treatment with TGF-β1 (1 or 10 ng per ml) and IGF-I (10 or 100 ng per ml) separately, or in combination in normal (N-1) and keloid fibroblasts (K-1, 2). (C) Fibronectin expression after treatment with TGF-β1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml), or both in combination in normal (N-3) and keloid fibroblasts (K-2, 3). (D) PAI-1 expression after treatment with TGF-β1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml), or both in combination in normal (N-1) and keloid fibroblasts (K-1, 3). Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of collagen type I mRNA after treatment with TGF-β1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml) separately, or in combination in cultured fibroblasts. RNA was extracted from cultures, reverse transcribed, and analyzed by quantitative reverse transcription–PCR using real-time PCR. Results are expressed as fold increases of collagen I mRNA after normalization to α-tubulin. *p<0.05. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 (A) 3TP-lux activity in BALB/C 3T3 mouse fibroblasts and HepG2 cells after treatment with TGF-β1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml), or with both in combination. 3T3 fibroblasts and HepG2 cells transfected with 3TP-Lux were incubated for 12 h with various stimulants. (B) ARE-lux activity in BALB/C 3T3 mouse fibroblasts and HepG2 cells after treatment with TGF-β1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml) separately, or both in combination. 3T3 fibroblasts and HepG2 cells were cotransfected with ARE-Lux in the presence or absence of FAST-1 before stimulation. Results are expressed as fold increases above the values for untreated controls (mean ± SE, n=6). *p<0.03, N.S., nonsignificant. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 3TP-lux activity in normal and keloid human fibroblasts treated with TGF-β1 (10 ng per ml), IGF-I (100 ng per ml) separately, or with both in combination. Normal and keloid fibroblasts transfected with 3TP-Lux were incubated 12 h with the stimulants. Results are expressed as fold increases above the values for untreated controls (mean ± SE, n=6) in normal fibroblasts. *p<0.01;**p<0.03. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 3TP-lux activity in normal and keloid fibroblasts. The effect on MAPK pathways was examined by pretreatment with either PD98059 or SB203580 before administration of IGF-I (100 ng per ml), TGF-β1 (10 ng per ml) separately, or both in combination. PD98059 is a specific inhibitor of MEK1, and SB203580 is a specific inhibitor of p38. *p<0.03 Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 3TP-lux activity in normal and keloid fibroblasts. The effect on PI3K pathways was examined by pretreatment with wortmannin (10 nM or 100 nM) before administration of IGF-I (100 ng per ml), TGF-β1 (10 ng per ml) or both in combination. Results are expressed as fold increases above the values for untreated controls (mean±SE, n=6). *p<0.03. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Western blotting showing phosphorylation of ATF-2, p38, and JNK after treatment with TGF-1 (1 or 10 ng per ml), IGF-I (10 or 100 ng per ml) separately, or both in combination in keloid fibroblasts. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 A possible molecular mechanism of keloid formation mediated by TGF-β1 and IGF-I cross-talk. Journal of Investigative Dermatology 2003 120, 956-962DOI: (10.1046/j.1523-1747.2003.12143.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions