Nitric Oxide Inhibits Cornified Envelope Formation in Human Keratinocytes by Inactivating Transglutaminases and Activating Protein 1  Antonello Rossi, Maria Valeria Catani, Eleonora Candi, Francesca Bernassola, Pietro Puddu, Gerry Melino  Journal of Investigative Dermatology  Volume 115, Issue 4, Pages 731-739 (October 2000) DOI: 10.1046/j.1523-1747.2000.00116.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CE formation is influenced by NO. Normal human epidermal keratinocyte cells were grown in 0.05 mM Ca2+ (black bar), 1.2 mM Ca2+ (gray bar), 1.2 mM Ca2+ plus 1 mM SNAP (white bar). The NO donor was replaced daily in the culture medium. After 1 wk, CEs were extracted by exhaustive boiling and sonication and quantified by spectrophotometric analysis. Results are shown as OD600 nm per 1 × 106 cells. The values are the means ± SD of three independent determinations. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Release of free NO by SNAP. Measurement of free NO concentrations in culture medium following addition of increasing concentrations of SNAP, by using a modified Clarke electrode (see Materials and Methods). NO meter readings were taken over 24 h after addition of the NO donor to 1 ml of culture medium supplemented with 10% FBS and incubated at 37°C: ○, 0.1 mM SNAP; •, 1 mM SNAP; ▪, 10 mM SNAP. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 In vitro inhibition of loricrin crosslinking by SNAP. Equivalent amount of enzyme activity of full-length recombinant TGase 1, TGase 2, and TGase 3 (A, B, and C, respectively) were reacted with recombinant human 35S-loricrin for 5 and 10 min in the presence of 5 mM Ca2+, as described in Materials and Methods. Aliquots were taken at specific time points and stopped by the addition of EDTA (7 mM final concentration). The crosslinked products were boiled in loading buffer, separated on a 4%-12% gradient sodium dodecyl sulfate gel and autoradiographed. In each experiment, zero represents the control in which reaction was carried out for 10 min without calcium and in the presence of 7 mM EDTA. Treatments with SNAP are indicated at the top of the three panels. SNAP was added at a final concentration of 5 mM (shown) or 10 mM (not shown), producing comparable results. SNAP was added in each reaction tube together with the enzymes. Protein size markers are shown on the left. One of three identical experiments is shown. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 NO inhibits activity of purified TGase 1 and 3 enzymes by interacting with their thiol groups. (A) Inhibition of purified cytosolic TGase 1 (•), membrane bound TGase 1 (○), and TGase 3 (▪) by SNAP. One hundred nanograms of purified TGases were incubated with increasing concentrations of SNAP for 20 min. The NO donor was added to the enzyme preparations immediately before the assay. Activity was evaluated as [3H]-putrescine incorporated into dimethylcasein. Results are shown as percent of enzyme activity inhibition. The values are the means ± SD of quadruplicate determinations of three separate experiments. (B) Titration of thiol groups, evaluated by alkylation with [14C]-iodoacetamide of purified cytosolic TGase 1. Purified enzyme was incubated with increasing concentrations of SPER-NO (•), GSNO (○), SNAP (▪), and SIN-1 (□) and titration was performed after 5 min. Data are the means ± SD of quadruplicate determinations carried out in two separate experiments. (C) Titration of thiol groups, evaluated by alkylation with [14C]-iodoacetamide of pure TGase 3 enzyme treated with SNAP. Results are shown as the number of titratable thiol groups. The values are the mean ± SD of three independent determinations. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 NO inhibits TGase activity in crude cell extracts. (A) Inhibition of TGase activity in HaCaT cell extracts. Proteins from cell extracts (0.1 mg) were exposed immediately before the assay to increasing concentrations of SIN-1 (□), GSNO (▪), SPER-NO (○), and SNAP (•), for 20 min. (B) Inhibition of enzyme activity in normal human epidermal keratinocyte cell extracts after incubation with SIN-1 (□), GSNO (▪), SPER-NO (○), and SNAP (•). (C) Inhibition of total TGase activity in cell extracts from SNAP-exposed normal human epidermal keratinocytes. Culture medium was supplemented daily with 1 (•) or 10 (○) mM NO donor. Activity was evaluated as [3H]-putrescine incorporated into dimethylcasein. Results are shown as percent of enzyme activity inhibition. Data are the means ± SD of quadruplicate determinations of three separate experiments. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 NO interferes with the transcription of differentiation-specific genes by modulating AP-1 activity. (A) EMSA of nuclear extracts of normal human epidermal keratinocyte cells. Keratinocytes were grown in 0.05 mM Ca2+ (lane 1), 1.2 mM Ca2+ (lane 2), 1.2 mM Ca2+ plus 1 mM SNAP (lane 3) as described in Materials and Methods. The sequences of the mutant and consensus AP-1 oligonucleotides are also shown in Materials and Methods. Lanes 4 and 5 represent specificity controls consisting of binding competition experiments with a 50-fold molar excess of unlabeled consensus AP-1 oligonucleotide (lane 4) and mutant AP-1 oligonucleotide (lane 5). (B) RT-PCR analysis of total RNA extracted by normal human epidermal keratinocyte cells. Keratinocytes were grown in 0.05 mM Ca2+ (lane 1), 1.2 mM Ca2+ (lane 2), 1.2 mM Ca2+ plus 1 mM SNAP (lane 3) as described in Materials and Methods. Purified total RNA was reverse transcribed and amplified either with TGase 1 and loricrin (upper and middle panels) specific primers, or primers specific for the 18S rRNA (lower panel) as a loading control. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Effect of NO donors on loricrin, involucrin, and TGase 1 promoter-regulated gene transcription induced by TPA. Cells were transfected with the TGase 1 (A), loricrin (B), and involucrin (C) promoter CAT reporter constructs and left untreated or stimulated with 10 ng per ml TPA for 48 h. Where indicated, cells were also incubated with increasing concentrations (each column corresponds to 0.01, 0.1, 1, 10 mM, respectively) of SNAP, GSNO, SIN-1, or SPER-NO before being harvested, as described in Materials and Methods. (D) Normal human epidermal keratinocytes were transiently transfected either with wild-type TGase 1, loricrin, and involucrin promoter CAT vectors, or with their counterparts containing AP-1 mutated sites, and treated with 10 ng per ml TPA in the absence or in presence of SNAP (0.01–10 mM). Results are means ± SD of duplicate determinations carried out in three different experiments. Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 8 Schematic model representing the interference of NO with the pathway of skin cornification. Activation of AP-1 transcription factor by extracellular signals (e.g., calcium, TPA) leads to transactivation of AP-1-responsive genes, including TGases, loricrin, and involucrin (dotted lines). NO might inhibit this mechanism through two main pathways. NO might act at post-translational level by chemically modifying TGases, probably by S-nitrosylation of their active cysteine residues (pathway a), or it might exert a transcriptional control by S-nitrosylating proteins of the AP-1 complex (pathway b). Journal of Investigative Dermatology 2000 115, 731-739DOI: (10.1046/j.1523-1747.2000.00116.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions