Sybille Hasse

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)

Towards the development of a simplified long‐term organ culture method for human scalp skin and its appendages under serum‐free conditions

Abstract:u2002 Organ culture of human scalp skin is usually performed with serum‐containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum‐free Williams E medium for more than 2u2003weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM)u2003+u200310% fetal bovine serum (FBS). Moreover, Williams E medium protected better against cell death than MEMu2003+u200310% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki‐67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum‐free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13u2003days in culture. The percentage of surviving hair follicles (mostly with catagen‐ or telogen‐like morphology) gradually increased over time displaying mostly catagen hair follicles after 17u2003days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long‐term‐surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum‐free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2u2003weeks under well‐defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:u2002 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 30u2003min and later after 6 and 24u2003h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72u2003h. 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.

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.

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:u2002 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 295u2003nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10−3u2003M, underlining the importance of this protein in epidermal homeostasis.

Basic research confirms coexistence of acquired Blaschkolinear Vitiligo and acrofacial Vitiligo

We report about a female patient with bilateral and unilateral blaschkolinear depigmentation on the extremities and coexistence of acrofacial vitiligo, who initially presented her first signs of depigmentation at the age of 32xa0years. The patient was otherwise healthy. The correct diagnosis was based on the latest up to date technology utilizing in vivo FT-Raman and Fluorescence spectroscopy, Wood’s light examination of the depigmented skin and immunoreactivity of epidermal catalase expression in 3xa0mm punch biopsies from the linear depigmented area. The results yielded decreased catalase protein expression compared to healthy controls as well as complete absence of melanocytes. FT-Raman spectroscopy identified the presence of hydrogen peroxide (H2O2) in the mM range and Fluorescence spectroscopy demonstrated H2O2-mediated oxidation of tryptophan residues in the depigmented area. The results were in agreement with vitiligo. Repigmentation of the linear lesion was initiated after reduction/removal of epidermal H2O2 with pseudocatalase PC-KUS further supporting the correct diagnosis. To the best of our knowledge this is the first case documented with vitiligo following Blaschko lines in coexistence with classical acrofacial vitiligo. This observation raises the question whether besides H2O2-mediated stress in association with genomic mosaicism could play a role in some cases with vitiligo.

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).

Significant Immediate and Long-term Improvement in Quality of Life and Disease Coping in Patients with Vitiligo after Group Climatotherapy at the Dead Sea

Quality of life in patients with vitiligo is impaired. This study explored the immediate effect of 20 days of climatotherapy at the Dead Sea on quality of life, coping with the disease, general well-being and individual stress levels in a group of 71 patients with vitiligo and 42 matched controls. The long-term effect was assessed after 12 months in 33/71 patients and 12/42 controls. Study instruments were Dermatology Life Quality Index, Beck Depression Inventory and the Adjustment to Chronic Skin Disorders Questionnaire. Stress measurements were based on cortisol and β-endorphin concentrations in saliva samples. Quality of life was significantly improved at day 20 at the Dead Sea compared with day 1, and this was still significant after 12 months. Moreover, social anxiety/avoidance, anxious-depressive mood and helplessness as measured by the Adjustment to Chronic Skin Disorders Questionnaire were significantly reduced. There was no difference in levels of cortisol and β-endorphin between patients and controls, indicating that stress per se is not a significant contributor in vitiligo. In conclusion, therapy in patient groups offers an effective tool for long-lasting improvement in quality of life and patients well-being.