The Suppressor of Cytokine Signaling-3 Is Upregulated in Impaired Skin Repair: Implications for Keratinocyte Proliferation  Itamar Goren, Andreas Linke, Elke Müller, Josef Pfeilschifter, Stefan Frank  Journal of Investigative Dermatology  Volume 126, Issue 2, Pages 477-485 (February 2006) DOI: 10.1038/sj.jid.5700063 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of SOCS-3 in normal skin repair. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in non-wounded back skin (ctrl skin) and wound tissue isolated from Balb/C mice. The time after injury is indicated for each lane. For the RNase protection assay (upper panel), every single experimental time point represents 16 wounds (n=16) isolated from four individual mice (n=4). A GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA (x-fold of control skin) is shown in the lower panel. **P<0.01; *P<0.05. All data points depict means±s.d. obtained from wounds (n=48) isolated from animals (n=12) from three independent animal experiments (n=3). (b) Immunoblot demonstrating the expression of SOCS-3 protein upon skin injury (upper panel). Every experimental time point represents wounds (n=8) isolated from four individual animals (n=4). A Ponceau S staining is given to demonstrate equal loading. Cytokine-stimulated murine primary keratinocytes (mKC-Cyt) and human embryonal kidney (HEK) cells transfected with a human SOCS-3 cDNA (HEKhSOCS-3) served to control the specificity of the antiserum. Immunohistochemical localization of SOCS-3 protein at the wound site on day 5 after injury (lower panel). e, epidermis; g, granulation tissue. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Induction of SOCS-3 expression in human HaCaT keratinocytes. Serum-starved HaCaT keratinocytes were stimulated for the indicated time periods with (a) EGF (10ng/ml) or TGF-α (10ng/ml) (b) DETA-NO (500μM), and (c) a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ). Total cellular RNA was analyzed by RNase protection assay for the presence of SOCS-3 mRNA (upper panels). The stimulation time is indicated for each lane. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA (x-fold of control) is shown in the middle panels. **P<0.01; *P<0.05. Data represent means±standard deviation (SD) obtained from four independent cell culture experiments (n=4). [35S]methionine-labeled SOCS-3 protein was immunoprecipitated from total cellular lysates after stimulation of cells for the indicated time points and analyzed by SDS gel electrophoresis (lower panels). Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Induction of SOCS-3 in human and murine primary keratinocytes. (a) Quiescent human primary keratinocytes were stimulated with EGF (10ng/ml) or a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) for the indicated time periods. Total cellular RNA was analyzed by RNase protection assay for the presence of SOCS-3 mRNA. The time after stimulation is indicated for each lane. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. (b) Quiescent murine primary keratinocytes were stimulated for the indicated time periods with TGF-α (10ng/ml) (c) with a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) in the presence or absence of the proteasomal inhibitor lactacystin (lac, 1μM). SOCS-3 protein was detected by immunoblot. Detection of actin was used to control for equal loading. One representative experiment out of three is shown. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Overexpression of SOCS-3 inhibits proliferation and promotes differentiation in cultured keratinocytes. (a) Immunoblot demonstrating different SOCS-3 expression levels in SOCS-3-overexpressing HaCaT cell lines OS-8 and OS-16. A mock-transfected HaCaT cell line and human embryonal kidney (HEK) cells transfected with a human SOCS-3 cDNA (HEK/SOCS-3) served as controls. (b) Serum-stimulated proliferation (percent of mock control) of SOCS-3-overexpressing cell lines (OS-8, OS-16) as assessed after 24hours of treatment. **P<0.01; *P<0.05. Data represent means±SD obtained from four independent cell culture experiments (n=4). (c) Total cellular RNA from different control (mock) and SOCS-3-overexpressing cell lines (OS) and Ca2+ (1.8mM)-differentiated HaCaT keratinocytes was analyzed by RNase protection assay for the presence of keratin 1 and keratin 10 mRNA (upper panels). GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. tRNA hybridization was used to control specificity of the antisense probe. A quantification of keratin 1 and 10 mRNA is shown in the lower panels. *P<0.05. Data represent means±standard deviation (SD) obtained from three independent SOCS-3-overexpressing cell lines (n=3). (d) Immunoblot showing expression of loricrin in unstimulated HaCaT (HaCaT), in mock cell lines (mock), in SOCS-3-overexpressing cell lines (OS) after passage 3 or 8 (p3, p8), or in Ca2+ (1.8mm)-differentiated HaCaT (high Ca2+) cells. Detection of actin and staining with Ponceau S were used to control for equal loading. (e) Immunoblot showing phosphorylated STAT-3 (Y-705) after a 20-minute stimulation of a control cell line (mock) and SOCS-3-overexpressing cell lines (OS) with a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) or IL-6 (10ng/ml) as indicated. Detection of actin was used to control for equal loading. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Localization of SOCS-3-expressing and proliferating wound margin keratinocytes during normal repair. Directly neighbored serial frozen sections from 3-, 5-, and 7-day wounds as indicated were immunohistologically analyzed for SOCS-3 (left panels) or the Ki67 marker of proliferation (right panels). e, epidermis; g, granulation tissue. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 SOCS-3 and Ki67 expression in NO-deficient wound healing. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in non-wounded back skin (ctrl skin) and wounds isolated from C57 wild-type (iNOS+/+) or C57 iNOS knockout (iNOS−/−) mice. The time after injury is indicated for each lane. Every single experimental time point represents 16 wounds (n=16) isolated from four individual mice. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification (x-fold of control skin) of SOCS-3 mRNA expression in non-wounded back skin (ctrl) and wound tissue isolated from wild-type (C57 iNOS+/+) and iNOS-deficient (C57 iNOS−/−) mice for the indicated time points is shown in the lower panel. **P<0.01; *P<0.05 as indicated by the brackets. All data points depict means±SD obtained from wounds (n=48) isolated from individual animals (n=12). (b) Directly neighbored serial frozen sections from 5-day wounds of iNOS-deficient mice (iNOS−/−) were immunohistologically analyzed for the Ki67 marker of proliferation (left panel) and SOCS-3 (right panel). Total keratinocyte numbers (lower left panel) and percentage of SOCS-3-expressing keratinocytes (lower right panel) in 5-day neo-epithelia of wild-type (iNOS+/+) and iNOS knockout (iNOS−/−) mice are shown. **P<0.01; *P<0.05. Data represent means±SD obtained from wounds (n=4) isolated from individual animals. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 SOCS-3 and Ki67 expression in diabetes-impaired wound healing. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in day-13 wound tissue isolated from PBS- or leptin-treated ob/ob mice. Every single experimental time point represents four wounds (n=4) isolated from individual PBS- or leptin-treated animals (PBS no. 1, 2, 3 or Lep no. 1, 2, 3). GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA expression in 13-day wounds of leptin- or PBS-treated ob/ob mice is shown in the right panel. **P<0.01. Data depict means±SD obtained from wounds (n=16) isolated from four individual animals (n=4). (b) Frozen serial sections from 13-day wounds of leptin- (left panels) or PBS- (right panels) treated ob/ob mice were analyzed for SOCS-3 protein and Ki67 expression as indicated. e, epidermis; g granulation tissue. Journal of Investigative Dermatology 2006 126, 477-485DOI: (10.1038/sj.jid.5700063) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions