sPLA2-X Stimulates Cutaneous Melanocyte Dendricity and Pigmentation Through a Lysophosphatidylcholine-Dependent Mechanism  Glynis A. Scott, Stacey E. Jacobs, Alice P. Pentland  Journal of Investigative Dermatology  Volume 126, Issue 4, Pages 855-861 (April 2006) DOI: 10.1038/sj.jid.5700180 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 (A and B) Expression of HA-tagged sPLA2-X from COS1 cell lysates. (a) Elutions from Ni2+ chromatography of COS1 cell lysates expressing recombinant (HA-tagged) sPLA2-X resolved on 15% SDS-PAGE and blotted with anti-HA antibodies. Elutions 1 and 2 demonstrated detectable HA-tagged sPLA2-X whereas elutions 3 and 4 did not show detectable HA-tagged sPLA2-X. (b) An aliquot from elution fraction 2 (5μg) shows a single band of approximately 17kDa when resolved on 15% SDS-PAGE and stained with Coomassie. Journal of Investigative Dermatology 2006 126, 855-861DOI: (10.1038/sj.jid.5700180) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 sPLA2-X and LPC, but not AA, activate tyrosinase in human melanocytes. Order of magnitude change in tyrosinase activity in melanocytes treated with sPLA2-X, LPC, or AA for 5 days. For cells treated with LPC or AA, controls consisted of cells treated with diluent alone. For cells treated with sPLA2-X, the control consisted of elution buffer. For each experiment, samples were assessed in duplicate and the results averaged. Presented are the averaged results of five separate experiments on individual Caucasian melanocyte cultures±SEM. *Statistically significant change (P<0.05). Journal of Investigative Dermatology 2006 126, 855-861DOI: (10.1038/sj.jid.5700180) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 (a) Human melanocytes become highly dendritic following treatment with sPLA2-X and LPC. Representative photographs of human melanocytes treated with sPLA2-X (0.6μg/ml), LPC (50nM), or AA (50nM). Nontreated cells exhibit a bipolar morphology whereas cells treated with sPLA2-X or LPC extend multiple dendrites. In contrast, cells treated with AA did not show increased dendricity. Bar=10μm. (b) Quantitative analysis of dendricity in human melanocytes following treatment with sPLA2-X, LPC, or AA for 24hours. Results shown are percent of cells with more than two dendrites. For sPLA2-X, controls consisted of cells treated with elution buffer; for LPC and AA, controls consisted of cells treated with diluent. Both sPLA2-X and LPC induced a dose-dependent increase in the number of dendrites, which was statistically significant. In contrast, AA had no statistically significant effect on dendricity. The results represent the averaged results of four individual cultures±SEM. Journal of Investigative Dermatology 2006 126, 855-861DOI: (10.1038/sj.jid.5700180) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 (a) Melanocytes express message for PLA2R and the LPC receptors GPR119 and G2A. Reverse transcription-PCR of human melanocyte RNA for the sPLA2-receptor (PLA2R) and the receptors for LPC G2A and GPR119 were performed and showed bands of the expected size that were confirmed by sequencing. β-Actin served as a positive control. NT=no template. Shown is a 1% agarose gel. Results shown are representative of at least two experiments using pooled cultures from three or more human melanocyte cultures. (b) Melanocytes express G2A protein. Western blot of human melanocyte crude cell lysate (60μg) and positive control lysates (WEHI-231 cell lysates; 5 and 10μg) blotted for G2A. Two bands were detected in melanocyte cell lysates, one that co-migrated with the positive control at ∼42kDa on 12% SDS-PAGE, and a prominent band of ∼30kDa. Membranes blotted with antibodies preabsorbed with blocking peptide to the G2A antibody show loss of the ∼42kDa band in melanocyte lysates and WEHI-231 cell lysates, as well as the lower band in melanocyte lysates, suggesting that the lower band represents degraded G2A protein. Results are representative of three separate experiments. Journal of Investigative Dermatology 2006 126, 855-861DOI: (10.1038/sj.jid.5700180) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions