cis-Urocanic Acid Enhances Prostaglandin E2 Release and Apoptotic Cell Death via Reactive Oxygen Species in Human Keratinocytes  Kazuyo Kaneko, Susan L. Walker, Joey Lai-Cheong, Mary S. Matsui, Mary Norval, Antony R. Young  Journal of Investigative Dermatology  Volume 131, Issue 6, Pages 1262-1271 (June 2011) DOI: 10.1038/jid.2011.37 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 cis-Urocanic acid (UCA) increases cyclooxygenase-2 (COX-2) protein expression and prostaglandin E2 (PGE2) release, and the COX inhibitor indomethacin abrogates PGE2 synthesis induced by cis-UCA in nTERT keratinocytes. (a) COX-2 protein expression 6hours after treatment, (b) kinetics of COX-2 protein expression, and (c) optical density values for COX-2 relative to actin in response to cis-UCA. (d–f) PGE2 release into the culture medium; (d) 24hours after treatment, (e) kinetics in response to cis-UCA, and (f) pretreatment with 0.5μM indomethacin for 1hour before treatment with phosphate-buffered saline (PBS) or cis-UCA for 24hours. Results are expressed as mean±SE of three independent experiments. *P<0.05, **P<0.01 versus PBS control (d, f) or before treatment (c, e). The blots are representative of three independent experiments. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 cis-Urocanic acid (UCA) increases apoptotic cells in primary and nTERT keratinocytes. (a) Mono- and oligonucleosome release 24hours after treatment, (b, c) light microscope images 24hours after treatment (scale bars=100μm in b and 50μm in c), (d) MTT cell proliferation/viability 24hours after treatment, and (e) flow cytometric analysis of apoptotic cells. The values indicate the percentage of living (lower left quadrant), early apoptotic (lower right quadrant), late apoptotic (upper right quadrant), and dead (upper left quadrant) cells. Similar results were obtained in at least three independent experiments. (f) Electron microscope images 24hours after treatment (scale bars=2 or 10μm as indicated). (a–c) Primary human keratinocytes and (c–f) nTERT keratinocytes. (a, d) Results are expressed as mean±SE of three independent experiments. *P<0.05, ***P<0.001 versus phosphate-buffered saline (PBS) control. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 cis-Urocanic acid (UCA) generates reactive oxygen species (ROS) mediated through nicotinamide adenine dinucleotide phosphateoxidase (NADPH), and the natural antioxidant α-tocopherol reduces prostaglandin E2 (PGE2) increase and apoptotic cell death induced by cis-UCA in nTERT keratinocytes. (a–c) ROS generation at 2hours after treatment. Cells were pretreated with 10μM diphenyleneiodonium chloride (DPI; b) or 10–100μM α-tocopherol (c) before cis-UCA treatment (scale bar=200μm). (d) PGE2 release into the culture medium for 24hours, (e) MTT cell proliferation/viability, and (f) flow cytometric analysis of apoptotic cells after 24hours. Cells were pretreated with 10 (d) or 100μM (e, f) α-tocopherol before cis-UCA treatment. (d, e) Results are expressed as mean±SE of three independent experiments. *P<0.05, **P<0.01. Similar results were obtained in at least three independent experiments. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 cis-Urocanic acid (UCA) activates EGFR and mitogen-activated protein kinase (MAPK), and inhibition of EGFR or MAPK blocks prostaglandin E2 (PGE2) increase induced by cis-UCA in nTERT keratinocytes. (a) Expression of phosphorylated and total EGFR after 2hours, (b) kinetics of EGFR expression in response to cis-UCA, (d) expression of phosphorylated and total extracellular signal-regulated kinase (ERK; after 2hours) and p38 (after 6hours), and (e) kinetics of ERK, p38, and c-Jun N-terminal kinase (JNK) expression in response to cis-UCA. Similar results were obtained in at least three independent experiments. (c, f) PGE2 release into the culture medium for 24hours. Cells were pretreated with 0.5μM EGFR or MAPK inhibitors for 1hour before cis-UCA treatment. Results are expressed as mean±SE of three independent experiments. *P<0.05, **P<0.01. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 cis-Urocanic acid (UCA) induces activation of caspase-3 and downregulation of EGFR in nTERT keratinocytes. (a) Kinetics of cleaved caspase-3 expression in response to cis-UCA, (b) expression of cleaved caspase-3 and EGFR 24hours after treatment, and (c) expression of cleaved caspase-3 and EGFR 24hours after phosphate-buffered saline (PBS) or cis-UCA treatment with or without pretreatment with 100μM α-tocopherol (α-Toc) for 24hours. The blots are representative of four independent experiments. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 A model of signaling pathways involved in cis-urocanic acid (UCA)-induced prostaglandin E2 (PGE2) synthesis and apoptosis in human keratinocytes. cis-UCA induces intracellular reactive oxygen species (ROS) generation that transiently activates EGFR. Subsequent activation of the downstream extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways leads to increased transcription of cyclooxygenase-2 (COX-2) that then stimulates PGE2 release. At higher concentrations, cis-UCA activates caspase-3 via intracellular ROS generation that downregulates EGFR, resulting in apoptosis. Journal of Investigative Dermatology 2011 131, 1262-1271DOI: (10.1038/jid.2011.37) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions