Production of Lysophosphatidic Acid in Blister Fluid: Involvement of a Lysophospholipase D Activity Juliette Mazereeuw-Hautier, Sandra Gres, Madie Fanguin,

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Production of Lysophosphatidic Acid in Blister Fluid: Involvement of a Lysophospholipase D Activity Juliette Mazereeuw-Hautier, Sandra Gres, Madie Fanguin, Clotilde Cariven, Josette Fauvel, Bertrand Perret, Hugues Chap, Jean-Pierre Salles, Jean-Sébastien Saulnier-Blache Journal of Investigative Dermatology Volume 125, Issue 3, Pages 421-427 (September 2005) DOI: 10.1111/j.0022-202X.2005.23855.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Soluble extracellular lysophosphatidic acid (LPA) synthesis activity in blister fluid and influence of quinacrine and phenanthrolin. LPA was quantified in blister fluids from five patients, before (t0) and after 6 h (t6 h) incubation at 37°C without (cont) or with quinacrine (quin) at 100 μM or phenanthrolin (phen) at 100 μM. Values are means±SEM from four independent repetitive assays realized for each patient. Statistical significance was considered when p<0.05 (*), ns, not statistically significant. Journal of Investigative Dermatology 2005 125, 421-427DOI: (10.1111/j.0022-202X.2005.23855.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of lysophosphatidic acid (LPA) receptor subtypes and autotaxin (ATX) in skin biopsies using real-time PCR. Normal skin (filled bars) and blister skin (unfilled bars) were both obtained from the same individuals suffering from bullous dermatoses (four patients). Total RNA was isolated and mRNA encoding LPA1-receptor (LPA1-R), LPA2-receptor (LPA2-R) and LPA3-receptor (LPA3-R), autotaxin (ATX), and hypoxanthine phosphoribosyl transferase (HPRT) was quantified using real-time PCR as described in Materials and Methods. Values are means±SEM from four independent repetitive assays realized for each patient. mRNA in normal skin was compared to RNA in blister skin (*p<0.05). Journal of Investigative Dermatology 2005 125, 421-427DOI: (10.1111/j.0022-202X.2005.23855.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of lysophosphatidic acid (LPA) on keratinocyte migration. LPA was solubilized in phosphate-buffered saline using 1% bovine serum albumin prior to addition of RPMI-1% bovine serum albumin. HaCaT cells were treated or not (control (cont): RPMI containing 1% bovine serum albumin) to increased concentrations of LPA. Cell migration was assessed using modified Boyden chambers as described in Materials and Methods. The migration was expressed as a migration index, and represented the percentage of migrated cells with LPA compared to the percentage of migrated cells with the control. Values are means±SEM from three independent repetitive experiments realized with three independent pools of cells. The migration index obtained after treatment by various concentrations of LPA was compared with migration index of the control (*p<0.05). Journal of Investigative Dermatology 2005 125, 421-427DOI: (10.1111/j.0022-202X.2005.23855.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions