Epidermal Growth Factor Induces Fibronectin Expression in Human Dermal Fibroblasts via Protein Kinase C δ Signaling Pathway  Yoshihiro Mimura, Hironobu Ihn, Masatoshi Jinnin, Yoshihide Asano, Kenichi Yamane, Kunihiko Tamaki  Journal of Investigative Dermatology  Volume 122, Issue 6, Pages 1390-1398 (June 2004) DOI: 10.1111/j.0022-202X.2004.22618.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Concentration-dependent effects of epidermal growth factor (EGF) on the fibronectin level. Human dermal fibroblasts were serum starved for 24 h and incubated in the absence or presence of indicated doses of EGF or transformation growth factor (TGF)α for an additional 24 h. (A) Medium (adjusted by the protein concentration of the cell lysates) was subjected to immunoblotting with anti-fibronectin antibodies. Levels of β-actin are shown as a loading control. One representative result of three independent experiments is shown. (B) Northern blot analysis was performed to evaluate fibronectin mRNA expression. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown as a loading control. (C) Fibronectin mRNA levels quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples are shown relative to the level in untreated cells (1.0). Data are expressed as the mean±SD of three independent experiments. *p<0.05, as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Time course of fibronectin expression levels in dermal fibroblasts stimulated by epidermal growth factor (EGF). Human dermal fibroblasts were serum starved for 24 h and incubated in the presence of 20 ng per mL EGF for the indicated times. (A) Medium samples (adjusted by the protein concentration of the cell lysates) were subjected to immunoblotting using anti-fibronectin antibodies. Levels of β-actin are shown as a loading control. One representative result of three independent experiments is shown. (B) Northern blot analysis was performed to evaluate fibronectin mRNA expression. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown as a loading control. One representative result of three independent experiments is shown. (C) Fibronectin mRNA levels quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples are shown relative to the level at 0 h (1.0). Data are expressed as the mean±SD of three independent experiments. *p<0.05, as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effects of actinomycin D or cycloheximide on epidermal growth factor (EGF)-mediated fibronectin upregulation. Human dermal fibroblasts were serum starved for 24 h and incubated in the absence or presence of 400 ng per mL actinomycin D or 10 μg per mL cycloheximide, with or without 20 ng per mL EGF, for 12 h. (A) Northern blot analysis was performed to evaluate fibronectin mRNA expression. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown as a loading control. One representative result of three independent experiments is shown. (B) Fibronectin mRNA levels quantitated by scanning densitometry and corrected for the levels of GAPDH in the same samples are shown relative to the level in untreated cells (1.0). Data are expressed as the mean±SD of three independent experiments. *p<0.05, as compared with the value in untreated cells. #p<0.05, as compared with the value in the cells treated only with actinomycin D. †p<0.05, as compared with the value in the cells treated only with cycloheximide. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Epidermal growth factor (EGF)-mediated induction of the fibronectin promoter activity in human dermal fibroblasts. Human dermal fibroblasts were transiently transfected with the fibronectin promoter-pGL3LUC (-508/+18). After incubation for 24 h, the cells were stimulated with EGF or transformation growth factor (TGF)β at the indicated doses for 18 h. EGF or TGFβ induction was determined as the fold increase in the luciferase activity relative to that in untreated cells (1.0). Data are expressed as the mean±SD of three independent experiments. *p<0.05, as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Effects of Calphostin C, Rottlerin, Gö6976, PD98059, and SB203580 on the epidermal growth factor (EGF)-induced fibronectin. Human dermal fibroblasts were serum starved for 24 h and pre-treated with 50 μM Calphostin C, 3 μM Rottlerin, 50nM Gö6976, 30 μM PD98059, 10 μM SB203580 for 1 h prior to the addition of 20 ng per mL of EGF for 24 h. (A) Northern blot analysis was performed to evaluate fibronectin mRNA expression. Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown as a loading control. One representative result of three independent experiments is shown. (B) Fibronectin mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to the level in untreated cells (1.0). (C) Calphostin C and Rottlerin blocked the EGF-mediated upregulation of fibronectin mRNA expression in a concentration-dependent manner. Human dermal fibroblasts were serum starved for 24 h and treated with 20 or 100 μM Calphostin C, 1 or 3 μM Rottlerin for 1 h prior to the addition of 20 ng per mL of EGF for 24 h. Northern blot analysis was performed to evaluate the fibronectin mRNA expression. Levels of GAPDH mRNA are shown as a loading control. One representative result of three independent experiments is shown. (D) Fibronectin mRNA levels quantitated by scanning densitometry and corrected for the level of GAPDH in the same samples are shown relative to the level in untreated cells (1.0). *p<0.05, as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effect of protein kinase C (PKC) inhibitors or dominant-negative PKCδ on epidermal growth factor (EGF)-induced fibronectin protein expression. (A) Calphostin C and Rottlerin blocked the EGF-mediated upregulation of fibronectin protein expression in a concentration-dependent manner. Medium samples (adjusted by counting cell numbers) were subjected to immunoblotting with anti-fibronectin antibodies. One representative result of three independent experiments is shown. (B) Transfection with dominant-negative mutant form of PKCδ blocked the EGF-mediated induction of fibronectin expression. Human dermal fibroblasts were transiently transfected with either empty vector or with dominant-negative mutant constructs for PKCδ. After incubation overnight, the transfected cells were either treated or untreated with 20 ng per mL EGF for 48 h. Medium samples (adjusted by counting cell numbers) were subjected to immunoblotting with anti-fibronectin antibodies. One representative result of three independent experiments is shown. Data are expressed as the mean±SD of three independent experiments. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Upregulation of protein kinase C (PKC)δ signaling pathway by epidermal growth factor (EGF) in human dermal fibroblasts. Human dermal fibroblasts were serum starved for 24 h and treated with 20 ng per mL EGF for the indicated times. (A) Cell lysates (30 μg of protein per sample) were subjected to immunoblotting with anti-PKCα, PKCδ, or PKCε antibodies. That the total amounts of proteins were unchanged was confirmed by immunoblotting using anti-β-actin antibodies. One representative result of three independent experiments is shown. (B) The levels of PKCα, PKCδ, or PKCε quantitated by scanning densitometry and corrected for the levels of β-actin in the same samples are shown relative to the level at 0 h (1.0). Data are expressed as the mean±SD of three independent experiments. *p<0.05, as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Effects of epidermal growth factor (EGF) on localization of protein kinase C (PKC)δ in human dermal fibroblasts. The localization of PKCδ was investigated with immunofluorescence analysis using human dermal fibroblasts as described in Materials and Methods. PKCδ was located mainly in the cytoplasm of fibroblasts without stimulation (A). PKCδ was translocated to the membrane 60 min after exposure to 20 ng per mL EGF (B). Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 9 Effect of Rottlerin on the epidermal growth factor (EGF)-induced upregulation of fibronectin mRNA stability. Human dermal fibroblasts were serum starved for 24 h and incubated in the absence (a) or presence of 20 ng per mL EGF (b) or EGF plus 3 μM Rottlerin (c) for 12 h before the addition of 400 ng per mL actinomycin D. (A) Northern blot analysis was performed to evaluate fibronectin mRNA expression in the cells with no treatment (a), only with 20 ng per mL EGF (b), or with EGF plus 3 μM Rottlerin (c). Levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA are shown as a loading control. One representative result of three independent experiments is shown. (B) Time-dependent degradation of fibronectin mRNA. The corrected density was expressed as a percent of the value at time 0. The squares with dashed line indicate the EGF-treated cells, and the filled circles with continuous line indicate control (untreated) cells and the open circles with irregularly dashed line indicate the cells treated with EGF and Rottlerin-treated levels. Data are expressed as the mean±SD of three independent experiments. *p<0.05 as compared with the value in untreated cells. Journal of Investigative Dermatology 2004 122, 1390-1398DOI: (10.1111/j.0022-202X.2004.22618.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions