The repair enzyme peptide methionine-S-sulfoxide reductase is expressed in human epidermis and upregulated by UVA radiation.

Recently, we reported that photoaging correlates well with the amount of oxidized protein accumulated in the upper dermis, while protein oxidation levels in the viable epidermis are very low. We hypothesized that this might be due to epidermal expression of the repair enzymes methionine sulfoxide reductases (MSRs). The expression of human methionine sulfoxide reductase A (MSRA) was investigated in HaCaT cells, primary human keratinocytes, and in human skin. High MSRA mRNA and protein levels as well as MSR activity were found in cultured human keratinocytes. MSRA was expressed in human epidermis, as shown by immunohistochemistry in healthy human skin. Repetitive in vivo exposure of human skin to solar-simulated light on 10 consecutive days (n=10 subjects) significantly increased epidermal MSRA expression. To further assess the functional relevance of the enzyme, its expression in response to UVB, UVA, and H(2)O(2) was investigated in HaCaT cells. While UVB lowered protein expression of MSRA, an upregulation was observed in response to low doses of UVA and H(2)O(2). In summary, MSRA represents the only enzyme so far identified in human skin that is capable of repairing oxidative protein damage. In addition to melanogenesis and DNA repair systems, a wavelength-specific activation of epidermal MSRA may be involved in epidermal photoprotection.

Necrobiotic xanthogranuloma-rapid progression under treatment with melphalan.

Necrobiotic xanthogranuloma is a systemic disease associated in most cases with monoclonal paraproteinaemia. Although the causative role of the paraproteinaemia is supposed, the pathogenesis of this disease remains unknown. We report a 65-year-old woman with a 28-year history of disseminated indurated plaques involving her face, trunk and extremities. Since 1994 an IgG kappa paraproteinaemia was present. Current biopsies showed typical histological features of necrobiotic xanthogranuloma. We treated this extraordinary widespread condition with melphalan and prednisolone. Although the serum levels of IgG kappa light chains and gamma globulins decreased, the cutaneous plaques extended rapidly in size and number, which casts doubt on the causative significance of the paraproteinaemia in the pathogenesis of necrobiotic xanthogranuloma. The paraproteinaemia in patients with necrobiotic xanthogranuloma may rather reflect a secondary phenomenon than the originating cause. Pathogenetic and therapeutical concepts based thereon that have been proposed so far should be critically reconsidered.

Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis.

Melanoma and nonmelanoma skin cancers are among the most prevalent cancers in the human population. Solar ultraviolet radiation is considered a major etiological factor but the relationship between dose, timing, and nature of exposure to tumor development is still unclear. Free radicals are generated by normal physiologic processes, including aerobic metabolism and inflammatory response, but may inflict cellular damage when generation is increased and antioxidant defense mechanisms are overwhelmed. Important findings supporting the free radical hypothesis in skin carcinogenesis are: (1) Reactive oxygen species (ROS) are generated in UVA- and UVB-irradiated skin in excessive doses, (2) the natural cutaneous antioxidant defense is impaired upon UV-exposure, (3) free radicals are involved in all steps of carcinogenesis, (4) supplementation with antioxidants can inhibit skin carcinogenesis, and (5) conditions that increase ROS generation enhance photocarcinogenesis. These findings provide a promising rationale for the development of powerful new antioxidant strategies in the prevention and therapy of skin cancer.

Detection of poly(ADP-ribose) by immunocytochemistry: a sensitive new method for the early identification of UVB- and H2O2-induced apoptosis in keratinocytes.

Apoptosis is an active form of cell death that is initiated by a variety of stimuli, including reactive oxygen species (ROS) and ultraviolet (UV) radiation. Poly (ADP-ribose) (PAR) is formed upon activation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), and therefore was suggested as a new marker of apoptosis. Since DNA of epidermal cells represents a well-known chromophore for UVB irradiation, and UVB is known to generate H2O2 in keratinocytes, we hypothesized that PAR is a very sensitive marker of UVB- and H2O2-induced apoptosis in keratinocytes. In order to test this hypothesis, human immortalized keratinocytes (HaCaT) were UVB-irradiated or treated with H2O2, and subsequently apoptosis was identified by comparing conventional parameters such as morphological analysis, DNA laddering, and TUNEL assay, with PAR formation. Both, UVB and H2O2 treatment induced PAR formation in HaCaT cells in a dose-dependent manner, and its formation was detected as early as 4 h after irradiation, and at lower UVB doses (10 mJ/cm2) than observed by DNA laddering and the TUNEL assay. In conclusion, the detection of PAR formation is a very sensitive and early method for the identification of apoptotic cells in UVB-induced apoptosis of human keratinocytes.

Photoaging is associated with protein oxidation in human skin in vivo.

There is increasing evidence for the generation of reactive oxygen species in skin upon ultraviolet exposure, but little is known about their pathophysiologic relevance in human skin in vivo. We hypothesized that chronic and acute photodamage is mediated by depleted antioxidant enzyme expression and increased oxidative protein modifications. Biopsies from patients with histologically confirmed solar elastosis, from non-ultraviolet-exposed sites of age-matched controls, and from young subjects were analyzed. To evaluate the influence of acute ultraviolet exposures, buttock skin of 12 healthy subjects was irradiated repetitively on 10 d with a solar simulator and compared intraindividually to non-ultraviolet-treated contralateral sites. The antioxidant enzymes catalase, copper-zinc superoxide dismutase, and manganese superoxide dismutase were investigated by immunohistochemistry. Protein carbonyls were analyzed by immunohistochemical and immunoblotting techniques in human skin and in cell models. Whereas overall expression of antioxidant enzymes was very high in the epidermis, low baseline levels were found in the dermis. In photoaged skin, a significant depletion of antioxidant enzyme expression was observed within the stratum corneum and in the epidermis. Importantly, an accumulation of oxidatively modified proteins was found specifically within the upper dermis of photoaged skin. Upon acute ultraviolet exposure of healthy subjects, depleted catalase expression and increased protein oxidation were detected. Exposures of keratinocytes and fibroblasts to ultraviolet B, ultraviolet A, and H2O2 led to dose-dependent protein oxidation and thus confirmed in vivo results. In conclusion, the correlation between photodamage and protein oxidation was demonstrated for the first time, which hence may be a relevant pathophysiologic factor in photoaging.