Nicholas C.J. Gibbons

Presence of epidermal allantoin further supports oxidative stress in vitiligo

Abstract:  Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the hydroxylation of hypoxanthine to xanthine and finally to uric acid in purine degradation. These reactions generate H2O2 yielding allantoin from uric acid when reactive oxygen species accumulates. The presence of XO in the human epidermis has not been shown so far. As patients with vitiligo accumulate H2O2 up to mm levels in their epidermis, it was tempting to examine whether this enzyme and consequently allantoin contribute to the oxidative stress theory in this disease. To address this question, reverse transcription‐polymerase chain reaction, immunoreactivity, western blot, enzyme kinetics, computer modelling and high performance liquid chromatography/mass spectrometry analysis were carried out. Our results identified the presence of XDH/XO in epidermal keratinocytes and melanocytes. The enzyme is regulated by H2O2 in a concentration‐dependent manner, where concentrations of 10−6 m upregulates the activity. Moreover, we demonstrate the presence of epidermal allantoin in acute vitiligo, while this metabolite is absent in healthy controls. H2O2‐mediated oxidation of Trp and Met in XO yields only subtle alterations in the enzyme active site, which is in agreement with the enzyme kinetics in the presence of 10−3 m H2O2. Systemic XO activities are not affected. Taken together, our results provide evidence that epidermal XO contributes to H2O2‐mediated oxidative stress in vitiligo via H2O2‐production and allantoin formation in the epidermal compartment.

KdPT, a Tripeptide Derivative of α-Melanocyte–Stimulating Hormone, Suppresses IL-1β–Mediated Cytokine Expression and Signaling in Human Sebocytes

Acne is the most common inflammatory skin disease in which IL-1 plays a central role. Although α-melanocyte–stimulating hormone has immunomodulatory effects, its usefulness as an anti-inflammatory agent in acne is hampered owing to its lipid- and pigment-inducing effects via activation of melanocortin receptors (MC-Rs). We used the immortalized human sebocyte line SZ95 as an in vitro model to investigate the anti-inflammatory potential of KdPT, a tripeptide derivative of the C-terminal end of α-melanocyte–stimulating hormone. KdPT potently suppressed IL-1β–induced IL-6 and IL-8 expression. Mechanistically, KdPT decreased IL-1β–mediated IκBα degradation, reduced nuclear accumulation of p65, and attenuated DNA binding of NF-κB. Moreover, KdPT reduced IL-1β–mediated generation of intracellular reactive oxygen species, which contributed to IL-1β–mediated cytokine induction. KdPT also reduced cell surface binding of fluorochrome-labeled IL-1β in SZ95 sebocytes. Analysis of the crystal structure of the complex between IL-1β/IL-1R type I (IL-1RI), followed by computer modeling of KdPT and subsequent modeling of the peptide receptor complex with the crystal structure of IL-1RI via manual docking, further predicted that the tripeptide, through several H-bonds and one hydrophobic bond, interacts with the IL-1RI. Importantly, KdPT did not bind to MC-1Rs, as demonstrated by blocking experiments with a peptide analog of Agouti signaling protein and by binding assays using MC-1R–expressing B16 melanoma cells. Accordingly, KdPT failed to induce melanogenesis. Our data demonstrate a promising anti-inflammatory potential of KdPT and point toward novel future directions in the treatment of acne—as well as of various other IL-1–mediated inflammatory diseases—with this small molecule.

Computer simulation of heterogeneous single nucleotide polymorphisms in the catalase gene indicates structural changes in the enzyme active site, NADPH-binding and tetramerization domains : a genetic predisposition for an altered catalase in patients with vitiligo?

Abstract:  Patients with vitiligo have low levels/activities of catalase in their lesional and non‐lesional epidermis as well as in their epidermal melanocytes under in vitro conditions while the levels of catalase mRNA are unaltered. This defect leads to a build‐up of hydrogen peroxide (H2O2) in the 10−3 m range in the epidermis of these patients. In this context, it was realized that 10−3 m H2O2 deactivates catalase. Along this line, it was also suspected that catalase in patients with vitiligo possesses a special sensitivity to this reactive oxygen species (ROS), and indeed several heterozygous single nucleotide polymorphisms (SNPs) have been documented in the cat gene of these patients. Based on the 3D structure of human catalase monomer, we have modelled the influence of three selected SNPs on the enzyme active site, on the NADPH‐ as well as the tetramerization‐binding domains. Our results show that these SNPs severely alter catalase structurally, which in turn should make the enzyme more susceptible to ROS compared with wild‐type enzyme. Taken together, the work presented herein together with the earlier results on SNPs in the cat gene suggests a genetic predisposition for an altered catalase in patients with vitiligo.

In situ and in vitro evidence for DCoH/HNF-1α transcription of tyrosinase in human skin melanocytes

Human epidermal melanocytes hold the full capacity for autocrine de novo synthesis/regulation/recycling of the essential cofactor 6-tetrahydrobiopterin (6BH(4)) for conversion of L-phenylalanine via phenylalanine hydroxylase to L-tyrosine and for production of L-Dopa via tyrosine hydroxylase to initiate both pigmentation and catecholamine synthesis in these neural crest-derived cells. Earlier we have demonstrated pterin-4a-carbinolamine dehydratase (PCD) mRNA and enzyme activities in epidermal melanocytes and keratinocytes. This protein dimerises also the transcription factor hepatocyte nuclear factor 1 (HNF-1), leading to activation of multiple genes. This study demonstrates for the first time DCoH/HNF-1 alpha expression and transcriptional activity in human epidermal melanocytes in vitro and in situ and identified tyrosinase, the key enzyme for pigmentation, as a new transcriptional target. Specific binding of DCoH/HNF-1 complex to the human tyrosinase promoter was confirmed by gel shift analysis. These results provide a novel mechanism in the regulation of skin pigmentation.

Structural and functional alterations in the β2-adrenoceptor are caused by a point mutation in patients with atopic eczema

Abstract:  The density of β2‐adrenoceptors is significantly decreased in both keratinocytes and peripheral blood lymphocytes from patients with atopic eczema. Furthermore both cell types showed a sixfold increase in the KD for the specific binding of the non‐specific antagonists (–)‐[3H]CGP 12177 and [125 I]CYP to keratinocytes and lymphocytes respectively compared with healthy controls. Based on these results polymorphism in the β2‐adrenoceptor gene was suspected. Consequently the entire intronless β2‐adrenoceptor gene was isolated from whole blood and by RT‐PCR from keratinocyte extracts of nine patients with atopic eczema and four healthy controls. DNA sequence analysis of nine atopic eczema patients confirmed a substitution in codon (1618) GCC (Ala119) to GAC (Asp119). This point mutation is expressed on the third transmembrane helix only 13Å away from the established agonist/antagonist binding site at Asp113. Computer modelling of this third transmembrane helix revealed substantial structural changes in the mutant compared with the wild type. Epidermal keratinocytes were established from one patient with atopic eczema (homozygote), the mother (heterozygote) and one age‐matched healthy control. Cells were grown in media containing different concentrations of l‐phenylalanine and receptor densities were determined. The results showed that cells with atopic eczema showed an increased sensitivity to l‐phenylalanine concentrations with a narrow homeostasis compared with healthy controls. The heterozygous mother was only 50% as sensitive as the child. In summary, the results indicate that atopic eczema is associated with a single point mutation in the β2‐adrenoceptor gene leading to an impaired adrenergic response in the epidermis of these patients.