Melanocytes with loss of function mutations in the MC1 receptor were characterized for the response to the MC1R ligand -MSH as described (24). melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1C4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes. prostanoid receptor(s) that mediates PGE2 effects on melanocyte pigmentation. Our data show that UVR stimulates PGE2 synthesis and activates cPLA2 in melanocytes, indicating that PGE2 is an autocrine factor for melanocytes. Treatment of melanocytes with PGE2 stimulated the cAMP/PKA pathway, and increased tyrosinase activity, and modestly increased melanocyte proliferation. Through the use of selective agonists and antagonists of EP receptors, we show that EP4 receptor stimulates, and EP3 receptor inhibits, cAMP production in melanocytes. These data are the first to demonstrate that PGE2 is an UV-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation, and that signaling by EP3 and EP4 receptors modulate the cAMP/PKA signaling pathway, a critical regulatory pathway of melanocyte function. Materials and Methods Reagents Rabbit polyclonal antibodies to -actin and to the EP4 receptor (H-160) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibodies against EP2were purchased from Cayman Chemicals (Ann Arbor, MI); rabbit polyclonal antibodies to cPLA2 phosphorylated on Ser 505 and rabbit polyclonal antibodies to cPLA2 were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies were purchased from Sigma Co (St. Louis, MO). Full range rainbow molecular weight markers were purchased from Amersham Life Sciences (Arlington Heights, Il). PGE2, butaprost (EP2 receptor agonist), sulprostone (EP3 receptor agonist) and ELISA Assay kits for analysis of PGE2 were purchased from Cayman Chemicals. PAH6809 (EP2 receptor antagonist) and L 161982 (EP4 receptor antagonist) were purchased from Tocris Biosciences (Ellisville, MO). Cells and cell culture Human melanocyte cultures were derived from neonatal foreskins as previously described (18). Melanocytes with loss of function mutations in the MC1 receptor were characterized for the response to the MC1R ligand -MSH as described (24). For analysis of effects of UVR on PGE2 synthesis and on PGE2 synthetic enzymes, melanocytes were maintained in of MCDB 153, supplemented with 4% fetal bovine serum (FBS), 14 g/ml bovine pituitary extract (BPE), 5 g/ml insulin, 10 ng/ml -tocopherol acetate, 8 nM 12-melanocytes were established in medium as described above, and were changed into medium without TPA and BPE (?/? media), 4 days prior to the experiment. Removal of these two factors is important for determining the effects of factors that activate PKC, such as PGE2, since TPA down-regulates protein kinase C, and cAMP activity, since BPE contains high concentrations of melanocortins that stimulate cAMP formation (24C26). This medium does not support proliferation, however, melanocyte survival is maintained by the presence of bFGF and fetal bovine serum (personal observations). Irradiation Dihydrofolic acid of melanocytes with UVR Melanocytes were maintained in complete media and irradiation was carried out in phosphate buffered saline (PBS) using a bank of 6 FS20 sun lamps (Westinghouse) that have more than 75% emission in the UVR range (280C320 nm), with a peak emission of 313 nm, and less than 25% UVA rays ( 320 nm). Kodacel filter was used to remove UVC rays. For controls, cells were placed in PBS but not irradiated (sham irradiated). Western Blotting for EP receptors Cells were lysed in RIPA buffer (150 mM NaCl, 1%NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl) with protease inhibitors (Boehringer Mannheim, Gmbt, Germany) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Set II, Calbiochem). Total cell lysates were resolved on 10% SDS-PAGE and blotted using standard procedures. Visualization of the immunoreactive proteins was accomplished with an enhanced chemiluminescence reaction (Pierce Chemical, Rockford, IL). Tyrosinase activity assay Melanocytes were in triplicate dishes in complete medium, then switched to ?/? media 4 days prior to treatment with PGE2. Melanocytes were treated with PGE2 (1.5 nM, 3 nM or 30 nM) every other day, for 6 days, in the continuous presence of indomethacin (4 g/ml) to block endogenous PG production. Twenty-four.Two hours prior to the addition of PGE2 or prostanoid receptor agonists and antagonists, isobutylmethylxanthine (IBMX, 2 mM) was added to inhibit phosphodiesterase activity. melanocytes with UVR stimulates tyrosinase activity, independent of expression of a functional melanocortin 1 receptor, suggesting the presence of a autocrine factor. Irradiation of melanocytes activated cPLA2, the rate-limiting step in eicosanoid synthesis, and stimulated PGE2 secretion. PGE2 increased cAMP production, tyrosinase activity and proliferation in melanocytes. PGE2 binds to four distinct G-protein coupled receptors (EP1C4). We show that EP4 receptor signaling stimulates cAMP production in melanocytes. Conversely, stimulation of the EP3 receptor lowered basal cAMP levels. controls effects of PGE2 on cAMP in melanocytes. The data are the first to identify PGE2 as an UVR-inducible autocrine factor for melanocytes that stimulates tyrosinase activity and proliferation, and to show that EP3 and EP4 receptor signaling have opposing effects on cAMP production, a critical signaling pathway that regulates proliferation and melanogenesis in melanocytes. prostanoid receptor(s) that mediates PGE2 effects on melanocyte pigmentation. Our data show that UVR stimulates PGE2 synthesis and activates cPLA2 in melanocytes, indicating that PGE2 is an autocrine factor for melanocytes. Treatment of melanocytes with PGE2 stimulated the cAMP/PKA pathway, and increased tyrosinase activity, and modestly increased melanocyte proliferation. Through the use of selective agonists and antagonists of EP receptors, we show that EP4 receptor stimulates, and EP3 receptor inhibits, cAMP production in melanocytes. These data are the first to demonstrate that PGE2 is an UV-inducible autocrine factor for human melanocytes that stimulates tyrosinase activation, and that signaling by EP3 and EP4 receptors modulate the cAMP/PKA signaling pathway, a critical regulatory pathway of melanocyte DCHS1 function. Materials and Methods Reagents Rabbit polyclonal antibodies to -actin and to the EP4 receptor (H-160) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit polyclonal antibodies against EP2were purchased from Cayman Chemicals (Ann Arbor, MI); rabbit polyclonal antibodies to cPLA2 phosphorylated on Ser 505 and rabbit polyclonal antibodies to cPLA2 were purchased from Cell Signaling Technology (Danvers, MA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibodies were purchased from Sigma Co (St. Louis, MO). Full range rainbow molecular weight markers were purchased from Amersham Life Sciences (Arlington Heights, Il). PGE2, butaprost (EP2 receptor agonist), sulprostone (EP3 receptor agonist) and ELISA Assay kits for analysis of PGE2 were purchased from Cayman Chemicals. PAH6809 (EP2 receptor antagonist) and L 161982 (EP4 receptor antagonist) were purchased from Tocris Biosciences (Ellisville, MO). Cells and cell culture Human melanocyte cultures were derived from neonatal foreskins as previously described (18). Melanocytes with loss of function mutations in the MC1 receptor were characterized for the response to the MC1R ligand -MSH as described (24). For analysis of effects of UVR on PGE2 synthesis and on PGE2 synthetic enzymes, melanocytes were maintained in of MCDB 153, supplemented with 4% fetal bovine serum (FBS), 14 g/ml bovine pituitary extract (BPE), 5 g/ml insulin, 10 ng/ml -tocopherol acetate, 8 nM 12-melanocytes were established in medium as described above, and were changed into medium without TPA and BPE (?/? media), 4 days prior to the experiment. Removal of these two factors is important for determining the effects of factors that activate PKC, such as PGE2, since TPA down-regulates protein kinase C, and cAMP activity, since BPE contains high concentrations of melanocortins that stimulate cAMP formation (24C26). This medium does not support proliferation, however, melanocyte survival is maintained by the presence of bFGF and fetal bovine serum (personal observations). Irradiation of melanocytes with UVR Melanocytes were maintained in complete media and irradiation was carried out in phosphate buffered saline (PBS) using a standard bank of 6 Dihydrofolic acid FS20 sun lamps (Westinghouse) that have more than 75% emission in the UVR range (280C320 nm), having a maximum emission of 313 nm, and less than 25% UVA rays ( 320 nm). Kodacel filter was used to remove UVC rays. For settings, cells were placed in PBS but not irradiated (sham irradiated). European Blotting for EP receptors Cells were lysed in RIPA buffer (150 mM NaCl, 1%NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl) with protease inhibitors (Boehringer Mannheim, Gmbt, Germany) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail Collection II, Calbiochem). Total cell lysates were resolved on 10% SDS-PAGE and blotted using standard procedures. Visualization of the immunoreactive proteins was accomplished with an enhanced chemiluminescence reaction (Pierce Chemical, Rockford, IL). Tyrosinase activity assay Melanocytes were in triplicate dishes in complete medium, then switched to ?/? press 4 days prior to treatment with PGE2. Melanocytes were treated with PGE2 (1.5 nM, 3 nM or 30 nM) every other day, for 6 days, in the continuous presence of indomethacin (4 Dihydrofolic acid g/ml).