Signaling Events During Induction of Plasminogen Activator Inhibitor-1 Expression by Sphingosylphosphorylcholine in Cultured Human Dermal Fibroblasts

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Signaling Events During Induction of Plasminogen Activator Inhibitor-1 Expression by Sphingosylphosphorylcholine in Cultured Human Dermal Fibroblasts Kyung-Chae Kye, Eun-Kyung Chae, Yong-Jun Piao, Seonyang Park, Jang-Kyu Park, Chang Deok Kim, Jeung-Hoon Lee, Ki-Beom Suhr Journal of Investigative Dermatology Volume 122, Issue 6, Pages 1365-1371 (June 2004) DOI: 10.1111/j.0022-202X.2004.22615.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 PAI-1 induction by SPC in healing wounds. (A) The inner ears of rabbit were wounded using a biopsy punch at size of about 6 mm. Wounded areas were covered with a plastic film, then injected daily with 5 μM SPC solution for 4 d. (B) Sections were prepared with paraffin, and stained with Hematoxylin–Eosin (H&E stain). (C) Immunohistochemical staining (IHC stain) was carried out with monoclonal anti-PAI-1 antibody. After washing in PBS, sections were incubated sequentially with biotinylated rabbit anti-mouse IgG and with peroxidase-conjugated avidin. Black arrows indicate the wound edges. Red arrows represent the PAI-1 staining in the dermis. Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 SPC-induced PAI-1 expression in cultured human dermal fibroblasts. (A) Cells were treated with 5 μM SPC at the indicated time points. Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in conditioned media were quantified using ELISA kits. Values represent the means and SEM from three independent measurements (*p<0.01 vs control). (B) PAI-1 content of cellular extracts was analyzed by western blotting (upper panel), and enzyme activity was determined by reverse fibrin autography (lower panel). Cells were treated with 5 μM SPC at the indicated time points, and extracted in lysis buffer. Proteins (100 μg per lane) were separated on 10% polyacrylamide gels. PAI-1 denotes the purified PAI-1 positive control, which was obtained commercially. All bands migrated to a location corresponding to a molecular mass of 50 kDa. (C) Concentration-dependence of the effect of SPC on PAI-1 expression. Cells were treated with SPC at the indicated concentrations and cultured for 12 h. The released PAI-1 and uPA were measured as in (A). (D) Cells were treated with SPC at the indicated concentrations for 12 h. PAI-1 content and enzyme activity were assayed as in (B). Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Northern blot analysis of PAI-1 expression in cultured human dermal fibroblasts. Cells were treated with 5 μM SPC at the indicated time points, and total RNA was isolated. Aliquots of 10 μg of total RNA were loaded in each lane, and the ethidium bromide-stained gel was photographed as a loading control. Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effects of pertussis toxin (PTx) and tyrphostin A47 (A47) on SPC-induced PAI-1 expression. Cells were preincubated for 4 h with the G protein inhibitor PTx (A), or the receptor tyrosine kinase inhibitor A47 (B) at the indicated concentrations. After the addition of 5 μM SPC, incubation was continued for a further 12 h. Cellular content of PAI-1 was assessed by western blot analysis. Each lane contained 100 μg of protein extracts. Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Measurements of intracellular Ca2+ level. Cells were loaded with 4 μM Fluo-3, AM for 40 min, and then subjected to confocal laser scanning microscopy. SPC (5 μM) was added alone (A) or following pre-treatment with 100 ng per mL pertussis toxin (PTx) (B). Results are presented as relative fluorescence intensity (RFI). Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Effects of Ca2+ on SPC-induced PAI-1 expression. (A) Fibroblasts were pre-treated with the intracellular Ca2+ chelator thapsigargin (Thap) at 30 nM or with the extracellular chelator EGTA (1 mM) for 30 min. SPC (5 μM) was then added and incubation was continued for 12 h. (B) The calcium ionophore A23187 (1 μM) was added to cultures, and cellular extracts were prepared at the indicated time points (upper panel). In cotreatment experiments, SPC (5 μM) was added 12 h before the treatment of A23187 (1 μM) (lower panel). Western blot analyses were performed with 100 μg of cellular protein per lane. Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effects of protein kinase C (PKC) activation on SPC-induced PAI-1 expression. (A) Fibroblasts were pre-treated with 100 nM phorbol-12-myristate-13-acetate (PMA) for 0.5 or 4 h, followed by the addition of 5 μM SPC. Cells were further incubated for 12 h, and extracts were subjected to western blotting for PAI-1. (B) Cells were pre-treated with the PKC inhibitor Ro 31-8220 (Ro) at the indicated concentrations for 30 min, followed by SPC treatment as described above. Each lane contained 100 μg of protein. Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Involvement of a mitogen-activated protein kinase (MAPK) pathway in SPC-induced PAI-1 expression. (A) Cells were incubated with the specific MAPK kinase 1/2 inhibitor PD98059 at the indicated concentrations for 30 min, and then stimulated with 5 μM SPC for 12 h. Cellular PAI-1 was assayed by western blot analysis. (B) Cells were treated with 5 μM SPC at the indicated time points. Cellular extracts were separated on 8% polyacrylamide gels and subjected to western blot analyses for phospho-p42/44 ERK (p-ERK) or p42/44 ERK (total-ERK). Journal of Investigative Dermatology 2004 122, 1365-1371DOI: (10.1111/j.0022-202X.2004.22615.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions