The Eumelanin Intermediate 5,6-Dihydroxyindole-2-Carboxylic Acid Is a Messenger in the Cross-Talk among Epidermal Cells  Daniela Kovacs, Enrica Flori, Vittoria Maresca, Monica Ottaviani, Nicaela Aspite, Maria Lucia Dell'Anna, Lucia Panzella, Alessandra Napolitano, Mauro Picardo, Marco d'Ischia  Journal of Investigative Dermatology  Volume 132, Issue 4, Pages 1196-1205 (April 2012) DOI: 10.1038/jid.2011.457 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Normal human keratinocyte (NHK) growth in response to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). (a) Cell growth evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Results are expressed as fold change relative to the untreated cell value at 24 hours, which was set as 1 by definition. (b) Percentage of Ki67-positive cells after 24, 48, and 72 hours of DHICA. (c) Immunofluorescence with anti-Ki67 antibody of NHKs following DHICA treatment. Arrows point at cells detected by the nuclear 4′-6-diamidino-2-phenylindole (DAPI) staining, which are negative for Ki67. Bar=20μm. (d, e) Count of total and viable cell number evaluated by trypan blue exclusion assay. Results are expressed as % of total cells relative to control (d) and % of viable cells/total cells for each condition (e). (f) Phase contrast microscopic analysis of cells after DHICA treatment for 72 hours. Bar=20μm. O.D., optical density; rel., relative. Journal of Investigative Dermatology 2012 132, 1196-1205DOI: (10.1038/jid.2011.457) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) stimulates normal human keratinocyte (NHK) differentiation. (a) Messenger RNA transcript levels of Involucrin, Loricrin, and Filaggrin evaluated by quantitative real-time reverse transcriptase–PCR after 24 hours of treatment with DHICA 50μM or with Ca++ 1.2mM. Values are normalized against the expression of GAPDH and are expressed relative to untreated control cells. (b, c) Immunofluorescence analysis and corresponding quantitative analysis of the percentage of positive cells for K1 (b, arrows) and K10 (c, arrows) on NHKs treated with DHICA for 48 hours. Cells maintained in high calcium condition were used as positive control. Nuclei are stained with 4′-6-diamidino-2-phenylindole (DAPI). Bar=20μm. (d) Immunocytochemical staining for filaggrin and corresponding quantitative analysis of the percentage of positive cells (d, arrows). Journal of Investigative Dermatology 2012 132, 1196-1205DOI: (10.1038/jid.2011.457) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) increases the activity and protein expression of intracellular enzymatic antioxidants. Superoxide dismutase (SOD) (a) and catalase (b) enzymatic activities on normal human keratinocytes treated with increasing concentration of DHICA for 24 and 48 hours. (c) Western blot analysis of Cu++Zn++SOD and catalase protein expression on cell lysate of keratinocytes treated with DHICA 50μM for 24 and 48 hours. β-tubulin was used as equal loading control. A representative experiment is shown. Journal of Investigative Dermatology 2012 132, 1196-1205DOI: (10.1038/jid.2011.457) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) counteracts reactive oxygen species-induced cell damage and death following UVA irradiation. (a) Viability of primary keratinocytes irradiated with UVA 10Jcm-2 after DHICA pretreatment (5 and 50μM) evaluated by trypan blue exclusion assay. (b) Phase contrast morphological analysis of irradiated keratinocytes in the absence or presence of DHICA pretreatment. Bar=20μm. (c) Analysis of polyunsaturated fatty acids (PUFA) content in UVA-irradiated keratinocytes upon pre-stimulation with DHICA evaluated immediately after the irradiation. (d) Effect of DHICA on UVA-induced keratinocyte apoptosis evaluated by FACS analysis using annexin V labeling. Rel., relative. Journal of Investigative Dermatology 2012 132, 1196-1205DOI: (10.1038/jid.2011.457) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 5,6-Dihydroxyindole-2-carboxylic acid (DHICA) induces peroxisome proliferator-activated receptor (PPAR)α messenger RNA (mRNA) expression and partially acts through its activation. (a) Expression of PPARα, PPARβ/δ, and PPARγ mRNA evaluated by quantitative real-time reverse transcriptase–PCR (qRT-PCR) after 6 hours of treatment with DHICA. Values are normalized against the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and are expressed relative to untreated cells. (b) PPARα mRNA level evaluated by qRT-PCR on normal human keratinocytes (NHKs) transfected with siRNA specific for PPARα (siPPARα) or nonspecific siRNA (siCtr). (c) Involucrin and Filaggrin mRNA expression evaluated by qRT-PCR in NHKs transfected with siPPARα or siCtr, and treated for 24 hours with DHICA. Values are normalized against the expression of GAPDH and are expressed relative to untreated control cells. (d, e) Immunofluorescence with anti-K1 and anti-K10 antibodies of NHKs transfected with siPPARα or siCtr following treatment with DHICA for 48 hours and corresponding quantitative analysis of the percentage of positive cells. Bar=20μm. DAPI, 4′-6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2012 132, 1196-1205DOI: (10.1038/jid.2011.457) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions