Coordinated Induction of Inducible Nitric Oxide Synthase and GTP-Cyclohydrolase I Is Dependent on Inflammatory Cytokines and Interferon-γ in HaCaT Keratinocytes: Implications for the Model of Cutaneous Wound Repair  Stefan Frank, Nicole Kolb, Josef Pfeilschifter  Journal of Investigative Dermatology  Volume 111, Issue 6, Pages 1065-1071 (December 1998) DOI: 10.1046/j.1523-1747.1998.00433.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Induction of GTP-CH I and iNOS mRNA expression by serum growth factors and inflammatory cytokines in cultured keratinocytes. (a) Cells were rendered quiescent by serum starvation and stimulated with 10% fetal calf serum, 10 ng EGF, PDGF, or TGF-β2 per ml for different time periods as indicated. Samples of 20 μg of total cellular RNA from these cells were analyzed for GTP-CH I mRNA expression using RNase protection analysis. The serum and serum growth factor-induced increase in GTP-CH I mRNA after stimulation as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gel is shown. (b, c) Serum-starved keratinocytes were stimulated for 2, 5, 8, or 24 h with 2 nM IL-1β or TNF-α, or 100 units IFN-γ per ml. Twenty micrograms of total cellular RNA from these cells were analyzed for the expression of GTP-CH I and iNOS mRNA. The degree of induction as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gel is shown schematically for GTP-CH I (b) and iNOS (c). The cytokine used for induction is indicated in the figure. Journal of Investigative Dermatology 1998 111, 1065-1071DOI: (10.1046/j.1523-1747.1998.00433.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Induction of GTP-CH I protein expression by EGF and IL-1β in keratinocytes. (a) RNase protection assay demonstrating the induction of GTP-CH I mRNA expression by EGF and IL-1β. Cells were rendered quiescent by serum starvation and stimulated with EGF (10 ng per ml) and 2 nM IL-1β for different time periods as indicated. Samples of 20 μg of total cellular RNA from these cells were analyzed for GTP-CH I mRNA expression. One thousand counts per min of the hybridization probe were used as a size marker. EGF- and IL-1β-stimulated expression of GTP-CH I protein is shown in (b). Serum-starved cells were harvested before and at different time points after treatment with EGF and IL-1β as indicated. Cytoplasmic fractions of these cells were analyzed by immunoblotting for the presence of GTP-CH I protein. GTP-CH I protein of 31 kDa, which was detected in EGF- and IL-1β-treated cells 2 h after stimulation, is indicated with anarrow. The protein was only hardly detectable in lysates from serum-starved cells (ctrl). Journal of Investigative Dermatology 1998 111, 1065-1071DOI: (10.1046/j.1523-1747.1998.00433.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 GTP-CH I and iNOS induction is independent ofde novo protein synthesis. Serum-starved keratinocytes were stimulated for different time periods with 10 ng EGF per ml, or 2 nM IL-1β and TNF-α in the presence of 20 μg per ml of the protein synthesis inhibitor cycloheximide as indicated. Because cycloheximide was dissolved in 5 μl of dimethyl sulfoxide (DMSO), medium containing 5 μl of the solvent was used as a control. Twenty micrograms of total cellular RNA from these cells were analyzed by RNase protection assay for the expression of GTP-CH I (a) and iNOS mRNA (b). The degree of GTP-CH I and iNOS mRNA induction as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gel is shown schematically in the right panels. The time points and cytokines used for GTP-CH I and iNOS induction is indicated in the figure. Journal of Investigative Dermatology 1998 111, 1065-1071DOI: (10.1046/j.1523-1747.1998.00433.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Co-regulated induction of GTP-CH I and iNOS expression by inflammatory cytokines IL-1β, TNF-α, and IFN-γ. Keratinocytes were rendered quiescent by serum starvation. They were simultaneously stimulated with IL-1β and TNF-α (2 nM), and IFN-γ (100 units per ml) for 1, 3, 5, 7, 9, 12, or 24 h as indicated (a, c, d). Twenty micrograms of total cellular RNA from these cells were analyzed by RNase protection assay for iNOS and GTP-CH I mRNA expression. Fifty micrograms of tRNA were used as a negative control. One thousand counts per ml of the hybridization probe were used as a size marker. An ethidium bromide stain of 2 μg of the same batch of RNA is shown in (b). The cytokine-induced increase in iNOS and GTP-CH I mRNA as assessed by PhosphoImager (Fuji) analysis of the radiolabeled gel is shown in (c). IL-1β, TNF-α, IFN-γ-stimulated expression of iNOS and GTP-CH I protein is shown in (d). Cells were treated with these cytokines for 5, 9, 12, or 24 h as indicated. Cytoplasmic fractions of these cells were analyzed by immunoblotting for the presence of iNOS and GTP-CH I proteins. GTP-CH I (left panel) and iNOS (right panel) protein with an expected size of 31 kDa and 130 kDa, respectively, was detected in stimulated cells and indicated with anarrow. Journal of Investigative Dermatology 1998 111, 1065-1071DOI: (10.1046/j.1523-1747.1998.00433.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Cytomix-stimulated GTP-CH I and iNOS expression is followed by nitrite accumulation. Keratinocytes were rendered quiescent by serum starvation and subsequently treated with a combination of inflammatory cytokines (2 nM IL-1β, 2 nM TNF-α, 100 U IFN-γ per ml) in the presence or absence of 2 mM L-NMMA for different time periods as indicated. Nitrite accumulation in the cell culture supernatants during the incubation period was measured as a readout for iNOS enzymatic activity.Frank S, Madlener M, Pfeilschifter J, Werner S: Induction of inducible nitric oxide synthase and its corresponding tetrahydrobiopterin-cofactor-synthesizing enzyme GTP-cyclohydrolase I during cutaneous wound repair.J Invest Dermatol 111:1058–1064 1998 Journal of Investigative Dermatology 1998 111, 1065-1071DOI: (10.1046/j.1523-1747.1998.00433.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions