Nitric oxide inhibits cornified envelope formation in human keratinocytes by inactivating transglutaminases and activating protein 1

Epidermal keratinocytes undergo terminal differentiation to form the stratu m corneum, which consists of many layers of flat dead cells. These cells as semble an insoluble cornified envelope composed of specific proteins deposi ted on the intracellular surface of the cell membrane. The proteins are cro sslinked by the action of transglutaminases, which catalyze the formation o f isodipeptide bonds between the epsilon-NH2 side chain of a lysine residue and the gamma-amide side chain of a glutamine residue. Transglutaminases s hare a conserved, highly reactive cysteine in their active site. In this st udy, we found that nitric-oxide-releasing compounds inhibited cornified env elope formation in cultured keratinocytes and the in vitro crosslinking of loricrin, a natural substrate of transglutaminases. The NO donors inhibited transglutaminase catalytic activity in a dose-dependent manner, in both pu rified enzymes and keratinocyte extracts. Titration of thiol groups of tran sglutaminases indicated that NO regulates their enzymatic activity by chemi cally modifying a cysteine residue, possibly by S-nitrosylation. NO was als o found to inhibit DNA-binding activity of activating protein 1 in keratino cyte nuclear extracts, and to interfere with the transactivation of activat ing protein 1 responsive genes such as transglutaminase 1, involucrin, and loricrin, whose expression is regulated during epidermal differentiation. I n conclusion, we propose that NO may inhibit keratinocyte differentiation, acting both at transcriptional level (inactivation of activating protein 1) and at post-translational level (inhibition of transglutaminase activity).