MAP Kinase Abnormalities in Hyperproliferative Cultured Fibroblasts from Psoriatic Skin Stéphanie Dimon-Gadal, Françoise Raynaud, Danièle Evain-Brion,

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MAP Kinase Abnormalities in Hyperproliferative Cultured Fibroblasts from Psoriatic Skin Stéphanie Dimon-Gadal, Françoise Raynaud, Danièle Evain-Brion, Guy Keryer Journal of Investigative Dermatology Volume 110, Issue 6, Pages 872-879 (June 1998) DOI: 10.1046/j.1523-1747.1998.00203.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Morphologic aspect of cultured fibroblasts from normal subjects (A, C) and psoriatic patients (B, D). (A, B) Optical microscopy; scale bar, 20 μm. (C, D) Confocal microscopy; cells were labeled with an antibody against a cell surface antigen (N-aminopeptidase); scale bar, 4 μm. Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Time course of stimulation by serum or by PDGF. Subconfluent normal and psoriatic fibroblasts were growth-arrested by 72h of serum depletion, and then stimulated with 10% fetal calf serum (A) or with 1 ng PDGF per ml (B) for the periods indicated. Cells were rinsed twice with cold PBS, and extracts were obtained as described in Materials and Methods. MAPK activity was measured using MBP in the presence of PKA, PKC, and Ca2+/calmodulin kinase inhibitors. MAPK activity is expressed in picomoles of inorganic phosphorus incorporated per min per mg protein. Immunoblots of serum-deprived normal and psoriatic cell extracts (C) and immunoblots of various cell extracts obtained during PDGF stimulation for the psoriatic fibroblasts (D) were prepared using anti-MAPK antibody (anti-rat MAPK R2). Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Characterization of Mono Q fractions by MBP phosphorylation activity and immunoblotting. Extracts from normal (Nor) and psoriatic (Pso) fibroblasts deprived of serum (– serum) or stimulated with serum (+ serum) were chromatographed on Mono Q as described in Materials and Methods, and fractions were assayed for MBP phosphorylation activity (A, D). Results are expressed as picomoles of inorganic phosphorus incorporated per 30min per 10 mg protein loaded onto the column. Mono Q fractions 37–48 were analyzed by western blotting with an anti-MAPK (B, E) or an anti-PKC antibody (C). Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of 8-bromo-cAMP on MAPK phosphorylation. Subconfluent normal (Nor) and psoriatic (Pso) fibroblasts were stimulated with 10% fetal calf serum for 10min with or without pretreatment with 100 μM 8-bromo-cAMP for 1h. (A) Cytosol extracts (10 μg protein) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with an anti-MAPK antibody (anti-rat MAPK R2). Scanning densitometry values (means ± SEM) of phosphorylated and unphosphorylated Erk1 and Erk2 bands are reported for normal (B) and psoriatic (C) fibroblast extracts (n = 3). Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunolocalization of phosphorylated Erk1 and Erk2 after serum stimulation. Normal (A, C, E) and psoriatic (B, D, F) fibroblasts were cultured on glass slides and then subject to serum depletion for 72h. Cells were not treated (A, B) or were stimulated with serum for 1h (C, D) or for 3h (E, F) and fixed in 60% methanol and 40% acetone at –20°C. They were incubated with the phospho-specific MAPK antibody, then with a biotinylated anti-rabbit IgG antibody and finally with Texas Red-streptavidin. The samples were examined with a fluorescence microscope under epifluorescent illumination with excitation-emission filters for rhodamine; scale bar, 10 μm. Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Immunolocalization of phosphorylated Erk1 and Erk2 after PDGF stimulation. After 72h of serum depletion, normal (A, C, E, G) and psoriatic (B, D, F, H) fibroblasts were not treated (A, B) or were stimulated with PDGF for 30min (C, D), for 1h (E, F), or for 3h (G, H). Immunocytochemistry was as described for Figure 3; scale bar, 10 μm. Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Immunohistochemical analysis of human skin. Thin sections (6 μm) of either normal skin (A) or nonlesional psoriatic skin (B) fixed at –20°C in 60% methanol and 40% acetone were examined by fluorescence microscopy following staining with the phospho-specific MAPK antibody. White arrows indicate positive staining in nucleus of epidermic and dermic cells; scale bar, 50 μm. Journal of Investigative Dermatology 1998 110, 872-879DOI: (10.1046/j.1523-1747.1998.00203.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions