Hartmut Rokos

Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair

Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT‐Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H2O2) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met‐S=O) repair in the entire gray hair follicle. Accordingly, Met‐S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT‐Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L‐ methionine. In summary, our data feed the long‐voiced, but insufficiently proven, concept of H2O2‐induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.—Wood, J. M., Decker, H., Hartmann, H., Chavan, B., Rokos, H., Spencer, J. D., Hasse, S., Thornton, M. J., Shalbaf, M., Paus, R., Schallreuter, K. U. Senile hair graying: H2O2‐mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair. FASEB J. 23, 2065–2075 (2009)

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT‐PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG‐CoA reductase and transport cholesterol via LDL/Apo‐B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time‐ and dose‐dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo‐B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.

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.

Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 1: epidermal H2O2/ONOO−-mediated stress abrogates tryptophan hydroxylase and dopa decarboxylase activities, leading to low serotonin and melatonin levels

Vitiligo is characterized by a progressive loss of inherited skin color. The cause of the disease is still unknown. To date, there is accumulating in vivo and in vitro evidence for massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO–) in the skin of affected individuals. Autoimmune etiology is the favored theory. Since depletion of the essential amino acid L‐tryptophan (Trp) affects immune response mechanisms, we here looked at epidermal Trp metabolism via tryptophan hydroxylase (TPH) with its downstream cascade, including serotonin and melatonin. Our in situ immunofluorescence and Western blot data reveal significantly lower TPH1 expression in patients with vitiligo. Expression is also low in melanocytes and keratinocytes under in vitro conditions. Although in vivo Fourier transform‐Raman spectroscopy proves the presence of 5‐hydroxytryptophan, epidermal TPH activity is completely absent. Regulation of TPH via microphthalmia‐associated transcription factor and L‐type calcium channels is severely affected. Moreover, dopa decarboxylase (DDC) expression is significantly lower, in association with decreased serotonin and melatonin levels. Computer simulation supports H2O2/ONOO–‐mediated oxidation/nitration of TPH1 and DDC, affecting, in turn, enzyme functionality. Taken together, our data point to depletion of epidermal Trp by Fenton chemistry and exclude melatonin as a relevant contributor to epidermal redox balance and immune response in vitiligo.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Martinez, A., Slominski, R., Lüdemann, J., Rokos, H. Blunted epidermal L‐tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 1: epidermal H2O2/ONOO–‐mediated stress abrogates tryptophan hydroxylase and dopa decarboxylase activities, leading to low serotonin and melatonin levels. FASEB J. 26, 2457‐2470 (2012). www.fasebj.org

Molecular evidence that halo in Sutton's naevus is not vitiligo.

Both halo naevus and vitiligo are acquired leucodermas of unknown aetiology. To date a significant contribution of oxidative stress through accumulation of hydrogen peroxide (H2O2) has been documented in the pathomechanism of vitiligo but not in halo naevus. Both epidermal pterin-4a-carbinolamine dehydratase (PCD) and catalase are sensitive markers to follow H2O2 concentration-dependent deactivation of these proteins. In situ protein expression of PCD and catalase was examined in full-skin biopsies from skin phototype-matched controls (n=5), untreated and treated vitiligo patients (n=5) and patients with untreated halo naevus in association with vitiligo (n=3). Vitiligo was treated with pseudocatalase (PC-KUS) only. Catalase levels were determined in epidermal suction blister extracts using fast protein liquid chromatography (FPLC). In addition, epidermal H2O2 levels were followed in vivo by Fourier-transform Raman spectroscopy. The results of this study ruled out a contribution of H2O2 in the millimolar range in the depigmentation process of halo naevus as previously documented in vitiligo. Therefore, it can be concluded that both leucodermas exercise distinct concentration-dependent H2O2 signalling in their pathomechanisms.

In vivo and in vitro evidence for autocrine DCoH/HNF‐1α transcription of albumin in the human epidermis

Abstract:  The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF‐1α in suprabasal keratinocytes in human full‐thickness skin sections and in keratinocytes cultured in serum‐free medium. Moreover, albumin and HNF‐1α protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse‐transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a λmax at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10−3 M, underlining the importance of this protein in epidermal homeostasis.

The redox – biochemistry of human hair pigmentation

The biochemistry of hair pigmentation is a complex field involving a plethora of protein and peptide mechanisms. The in loco factory for melanin formation is the hair follicle melanocyte, but it is common knowledge that melanogenesis results from a fine tuned concerted interaction between the cells of the entire dermal papilla in the anagen hair follicle. The key enzyme is tyrosinase to initiate the active pigmentation machinery. Hence, an intricate understanding from transcription of mRNA to enzyme activity, including enzyme kinetics, substrate supply, optimal pH, cAMP signaling, is a must. Moreover, the role of reactive oxygen species on enzyme regulation and functionality needs to be taken into account. So far our knowledge on the entire hair cycle relies on the murine model of the C57BL/6 mouse. Whether this data can be translated into humans still needs to be shown. This article aims to focus on the effect of H2O2‐redox homeostasis on hair follicle pigmentation via tyrosinase, its substrate supply and signal transduction as well as the role of methionine sulfoxide repair via methionine sulfoxide reductases A and B (MSRA and B).

Turmeric (curcumin): A widely used curry ingredient, can contribute to oxidative stress in asian patients with acute vitiligo

Restoration of the skin color can be achieved after reduction of epidermal H 2 O 2 levels using topical pseudocatalase cream (PC-KUS). Recently, we showed that epidermal catalase as well as other affected enzymes recover after treatment with this modality. However, to our surprise, part of our Asian patient population showed only moderate improvement or no response at all. Since Asian cuisine uses a variety of spices, we asked the question whether turmeric (haldi), which is widely used in curries, could possibly contribute to the poor outcome of this treatment.

Vitix® : a new treatment for vitiligo?

It has been demonstrated that a reduction in epidermal H 2 O 2 levels correlates with a cessation of the disease in 95% of affected individuals in association with extensive repigmentation. 4,7,8 Moreover, it has been shown that the reduction in epidermal H 2 O 2 levels induced by a low-dose, narrow-band, ultraviolet-B (UV-B)-activated pseudocatalase (PC-KUS) decreases systemic H 2 O 2 stress, underlining the importance of H 2 O 2 transfer from the epidermal compartment to the vascular system. 6,9

Human epidermal acetylcholinesterase (AchE) is regulated by hydrogen peroxide (H2O2)

Previously it has been demonstrated that the human epidermis synthesizes and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10−3M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)‐L‐erytho 5, 6, 7, 8 tetrahydrobiopterin via pterin‐4a‐carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunohistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetylcholinesterase (AchE) in association with accumulation of epidermal acetylcholine. This result was confirmed by Fluorescence excitation spectroscopy following the Trp fluorescence at λmax 280 nm. A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2(10−6M) activate this enzyme by increasing the Vmax > 2 fold, meanwhile high concentrations of H2O2(10−3M) deactivate the enzyme with a significant decrease in Vmax. Molecular modelling based on the established 3D structure of human AchE supported that H2O2‐mediated oxidation of Trp432, Trp435 and Met436 moves and disorients the active site His440 of the enzyme, thus explaining the deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2‐mediated oxidation of AchE contributes significantly to the well established oxidative stress in vitiligo.