The Neuronal Nitric Oxide Synthase Is Upregulated in Mouse Skin Repair and in Response to Epidermal Growth Factor in Human HaCaT Keratinocytes Jean-Paul.

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The Neuronal Nitric Oxide Synthase Is Upregulated in Mouse Skin Repair and in Response to Epidermal Growth Factor in Human HaCaT Keratinocytes Jean-Paul Boissel, Dorothea Ohly, Matthias Bros, Ute Gödtel-Armbrust, Ulrich Förstermann, Stefan Frank Journal of Investigative Dermatology Volume 123, Issue 1, Pages 132-139 (July 2004) DOI: 10.1111/j.0022-202X.2004.22731.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 nNOS mRNA expression in human tissues and cells. (a) nNOS exonic organization (exons 13–15). Arrows show the localization of the sense and antisense primers used for PCR amplification. Exonic localization of primers indicate that PCR amplicons (222 bp) must represent expressed nNOS mRNA. (b) Amplifications of nNOS-specific sequences were performed using reverse-transcribed mRNA from human tissues and cells as indicated. nNOS, neuronal nitric oxide synthase. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of nNOS upon skin wounding. Regulation of nNOS mRNA (a) and protein (b) expression in BALB/c mice as assessed by real-time PCR or immunoblot. The time after injury is indicated. Ctrl skin represents back skin biopsies of non-wounded mice. A quantification of nNOS mRNA (x-fold induction compared with control skin) is shown in (a). **, p<0.01 (ANOVA) compared with control skin. Bars represent mean±SD obtained from wounds (n=48) isolated from animals (n=12) from three independent animal experiments. RNAs have been equilibrated by analysis of constitutive porphobilinogen deaminase. (b) Total protein (50 μg) from non-wounded control skin and wound tissue was analyzed for nNOS-specific protein. A lysate from murine brain tissue was used as to control the specificity of the used anti-nNOS antiserum. A shorter exposure time of the immunoblot is shown in the right panel. nNOS, neuronal nitric oxide synthase. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Localization of nNOS protein in regenerating skin. Frozen sections from mouse wounds (day 5 post-wounding) were incubated with a polyclonal antiserum directed against nNOS. Sections were subsequently stained with the avidin–biotin–peroxidase complex system using 3,3-diaminobenzidine tetrahydrochloride as a chromogenic substrate. Nuclei were counterstained with hematoxylin. Immunopositive signals within the sections are indicated with arrows. d, dermis; e, epidermis. nNOS, neuronal nitric oxide synthase. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Human HaCaT keratinocytes increase nNOS expression upon EGF treatment. (a) Quiescent human HaCaT keratinocytes were stimulated with EGF (100 ng per mL) for 24 h. RNase protection assay to detect nNOS-, eNOS-, or iNOS-specific mRNA was performed as indicated. Ctrl, non-stimulated cells. One representative experiment is shown. β-actin expression is shown as a loading control within the same gel. A quantification of nNOS mRNA (x-fold induction compared with control) over a time period of 24 h as indicated is shown in (b). *, p<0.05; **, p<0.01 (ANOVA) compared with control. Bars indicate the mean±SD obtained from four independent cell culture experiments. (c) Quiescent HaCaT keratinocytes were treated with increasing amounts of EGF for 18 h as indicated. A quantification of nNOS mRNA (x-fold induction compared with control) is shown. **, p<0.01 (ANOVA) compared with control. Bars indicate the mean±SD obtained from four independent cell culture experiments. (d) Total protein (100 μg) from quiescent and EGF-treated (100 ng per mL) HaCaT cells was analyzed for nNOS-specific protein by immunoblot as indicated. Lysate from porcine cerebellum was used to control specificity of the anti-nNOS antiserum. β-tubulin was used as a loading control. One representative experiment is shown. nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS, inducible nitric oxide synthase; EGF, epidermal growth factor. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Upregulation of nNOS mRNA by ErbB receptor ligands in HaCaT cells. Quiescent human HaCaT keratinocytes were stimulated with EGF, heparin-binding EGF (HB-EGF), heregulin (H)-α, H-β1, or transforming growth factor-α (TGF-α) (100 ng per mL) for 18 h as indicated. RNase protection assay to detect nNOS-specific mRNA was performed. Ctrl, non-stimulated cells. A quantification of nNOS mRNA (x-fold induction compared with control) is shown. **, p<0.01 (ANOVA) compared with control. Bars indicate the mean±SD obtained from four independent cell culture experiments. nNOS, neuronal nitric oxide synthase. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Expression of activated epidermal growth factor receptor (EGFR) at the wound site. (a) Total protein (50 μg) from non-wounded (ctrl skin) and wounded skin was analyzed for the presence of phosphorylated (Y1173) EGFR-specific protein by immunoblot as indicated. One representative experiment is shown. Frozen sections from mouse wounds isolated at day 5 (b) and day 7 (c) post-wounding were incubated with a polyclonal antiserum directed against phosphorylated (Y1173) EGFR. Sections were subsequently stained with the avidin–biotin–peroxidase complex system using 3,3-diaminobenzidine tetrahydrochloride as a chromogenic substrate. Nuclei were counterstained with hematoxylin. Immunopositive signals within the sections are indicated with arrows. d, dermis; e, epidermis. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 EGFR antagonists inhibit nNOS mRNA expression in HaCaT cells. Quiescent HaCaT keratinocytes were treated with EGF (100 ng per mL) in the presence or absence of increasing amounts of the EGFR antagonist PD153035 (a) or the EGFR/JAK2/3 inhibitor AG490 (b) for 24 h as indicated. RNase protection assay to detect nNOS-specific mRNA was performed. A quantification of nNOS mRNA (x-fold induction compared with control) is shown. **, p<0.01 (ANOVA) compared with control. Bars indicate the mean±SD obtained from four independent cell culture experiments (n=4). EGFR, epidermal growth factor receptor; nNOS, neuronal nitric oxide synthase. Journal of Investigative Dermatology 2004 123, 132-139DOI: (10.1111/j.0022-202X.2004.22731.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions