Jutta Wenk

Photoaging of the skin from phenotype to mechanisms

The skin is increasingly exposed to ambient UV-irradiation thus increasing its risk for photooxidative damage with longterm detrimental effects like photoaging, which is characterized by wrinkles, loss of skin tone, and resilience. Photoaged skin displays prominent alterations in the cellular component and the extracellular matrix of the connective tissue with an accumulation of disorganized elastin and its microfibrillar component fibrillin in the deep dermis and a severe loss of interstitial collagens, the major structural proteins of the dermal connective tissue. The unifying pathogenic agents for these changes are UV-generated reactive oxygen species (ROS) that deplete and damage non-enzymatic and enzymatic antioxidant defense systems of the skin. As well as causing permanent genetic changes, ROS activate cytoplasmic signal transduction pathways in resident fibroblasts that are related to growth, differentiation, senescence, and connective tissue degradation. This review focuses on the role of UV-induced ROS in the photodamage of the skin resulting in biochemical and clinical characteristics of photoaging. In addition, the relationship of photoaging to intrinsic aging of the skin will be discussed. A decrease in the overall ROS load by efficient sunscreens or other protective agents may represent promising strategies to prevent or at least minimize ROS induced photoaging.

Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts.

Reactive oxygen species (ROS) have been shown to be important messenger molecules in the induction of several genes. In human dermal fibroblasts the herbicide paraquat (PQ2+) was used to induce intracellular oxidative stress that was modulated by the inhibition of copper, zinc superoxide dismutase (Cu,ZnSOD), glutathione peroxidase (GSHPx), catalase, and blocking of the Fenton reaction. Interstitial collagenase (MMP-1) mRNA increased time dependently for up to 72 h following paraquat treatment. A correlation with the translation of MMP-1 could, however, only be detected up to 24 h, indicating an uncoupling of transcription and translation. Interleukin-1 alpha and beta mRNA showed two peaks at 6 h and 72 h. The inhibition of catalase by aminotriazol (ATZ), inhibition of GSHPx by buthionine sulfoximine (BSO), and blocking the Fenton reaction by the iron chelator desferrioxamine (DFO) in concert led to an increase in steady-state MMP-1 mRNA levels, possibly dependent on intracellular H2O2 increase. This combined treatment potentiated MMP-1 mRNA induction up to 6.5-fold compared to paraquat treated controls. Furthermore, exogenously added H2O2 caused an increase in MMP-1 mRNA levels. In contrast, inhibition of Cu,ZnSOD by diethyldithiocarbamate (DDC), leading to diminished H2O2 production from O2.-, decreased MMP-1 mRNA induction. Collectively, our data provide evidence that H2O2 is an important intermediate in the downstream signalling pathway finally leading to the induction of increased steady state MMP-1 mRNA levels. The synthesis of MMPs may contribute to connective tissue damage in vivo related to photoaging, inflammatory diseases, and tumor invasion.

Singlet oxygen is an early intermediate in cytokine-dependent ultraviolet-A induction of interstitial collagenase in human dermal fibroblasts in vitro

Ultraviolet (UV) A irradiation of human dermal fibroblasts elicits an increase in specific mRNA amounts and bioactivities of the cytokines IL‐1α, IL‐1β, and IL‐6. These effects are enhanced in deuterium oxide‐based medium and are diminished in the presence of non‐toxic concentrations of sodium azide. Furthermore, generating singlet oxygen outside the cells by irradiation of rose bengal‐coated resin particles with visible light (λ>450 nm) results in the induction of interstitial collagenase, IL‐1 and IL‐6, similar to the response observed with UVA irradiation. These observations suggest that singlet oxygen is an early intermediate in the signaling pathway of IL‐1 and IL‐6 mediating UVA induction of interstitial collagenase (E.C. 3.4.24.7). Furthermore, singlet oxygen appears to initiate this complex UV response at the cell membrane.

Ultraviolet B Wavelength Dependence for the Regulation of Two Major Matrix-Metalloproteinases and Their Inhibitor TIMP-1 in Human Dermal Fibroblasts

Abstract— The wavelength dependence for the regulation of two major matrix‐metalloproteinases, interstitial collagenase (MMP‐1) and stromelysin‐1 (MMP‐3), and their major inhibitor, tissue inhibitor of metalloproteinases (TIMP‐1), was studied in human dermal fibroblasts in vitro. Monochromatic irradiation at 302, 307, 312 and 317 nm with intensities ranging from 20 to 300 J/m2 increased MMP‐1 and MMP‐3 mRNA steady‐state levels and the secretion of the corresponding proteins up to 4.4‐fold, whereas almost no increase was observed at wavelengths <290 nm. In contrast, the synthesis of TIMP‐1 increased only marginally. This unbalance may contribute to the severe connective tissue damage related to photoaging of the skin. The wavelengths responsible for MMP‐1 and MMP‐3 induction reported here are distinct from the absorption spectrum of DNA and are different from results previously reported in the literature. Importantly, they overlap with wavelengths whose intensity is predicted to increase on the earths surface upon ozone depletion. Intensities and particular wavelengths used in our studies in vitro can be absorbed readily by fibroblasts within the skin in vivo and, thus, are relevant for risk assessment and development of protective agents.

Adaptive antioxidant response protects dermal fibroblasts from UVA-induced phototoxicity

In response to the attack of reactive oxygen species (ROS) produced upon UV irradiation, the skin has developed a complex antioxidant defense system. Here we report that, in addition to the previously published induction of manganese superoxide dismutase (MnSOD) activity, single and, to a higher extent, repetitive low-dose UVA irradiation also leads to a substantial upregulation of glutathione peroxidase (GPx) activity. This concomitant adaptive response of two antioxidant enzymes acting in the same detoxification pathway coincided with the protection from high-UVA-dose-induced cytotoxicity conferred by low-dose UVA preirradiation. Whereas an interval of 24 h did not, an interval of 12 h did lead to the induction of MnSOD activity and, under selenium-supplemented conditions, of GPx activity as well, conferring definite cellular protection from UVA-induced phototoxicity. Moreover, under selenium-deficient conditions, which abrogate the UVA-mediated induction of GPx activity, adaptive protection against the cytotoxic effects of high UVA doses was significantly lower compared with selenium supplementation. Isolated 4.6-fold overexpression of MnSOD activity in stably transfected fibroblasts led to specific resistance from UVA-mediated phototoxicity under selenium-deficient conditions. Collectively, these data indicate that the concomitant induction of MnSOD and GPx activity is related to the optimal adaptive protection from photooxidative damage. This adaptive antioxidant protection clearly depends on the irradiation interval and a sufficient selenium concentration, findings that may have important implications for the improvement of photoprotective and phototherapeutic strategies in medicine.

Activation of p70 ribosomal protein S6 kinase is an essential step in the DNA damage-dependent signaling pathway responsible for the ultraviolet B-mediated increase in interstitial collagenase (MMP-1) and stromelysin-1 (MMP-3) protein levels in human dermal fibroblasts.

Ultraviolet B (UVB) irradiation has been shown to stimulate the expression of matrix-degrading metalloproteinases via generation of DNA damage and/or reactive oxygen species. Matrix-degrading metalloproteinases promote UVB-triggered detrimental long term effects like cancer formation and premature skin aging. Here, we were interested in identifying components of the signal transduction pathway that causally link UVB-mediated DNA damage and induction of matrix-degrading metalloproteinase (MMP)-1/interstitial collagenase and MMP-3/stromelysin-1 in human dermal fibroblasts in vitro. The activity of p70 ribosomal S6 kinase, a downstream target of the FK506-binding protein-12/rapamycin-associated protein kinase (FRAP) kinase (RAFT1, mTOR), was identified to be 4.8 ± 0.8-fold, and MMP-1 and MMP-3 protein levels 2.4- and 11.5-fold increased upon UVB irradiation compared with mock-irradiated controls. The FRAP kinase inhibitor rapamycin and the DNA repair inhibitor aphidicolin significantly suppressed the UVB-mediated increase in p70 ribosomal S6 kinase activity by 50–65% and MMP-1 and MMP-3 protein levels by 34–68% and 42–88% compared with UVB-irradiated fibroblasts. By contrast, the interleukin-1β-mediated increase in MMP-1 and MMP-3 protein levels could not be suppressed by rapamycin. Collectively, our data suggest that the FRAP-controlled p70 ribosomal S6 kinase is an essential component of a DNA damage-dependent, but not of the interleukin-1/cell membrane receptor-dependent signaling.

Ultraviolet-B induction of interstitial collagenase and stromelyin-1 occurs in human dermal fibroblasts via an autocrine interleukin-6-dependent loop.

Ultraviolet‐B irradiation of human dermal fibroblasts has earlier been shown to induce matrix‐degrading metalloproteinases, thus driving connective tissue degradation in photoaging and photocarcinogenesis. Herein, we report that Ultraviolet‐B irradiation led to a dramatic increase in specific mRNA and protein levels of interstitial collagenase, stromelysin and interleukin‐6. By contrast, the major tissue inhibitor of matrix‐degrading metalloproteinases, TIMP‐1, was unaffected. Monospecific neutralizing antibodies directed against human interleukin‐6 significantly reduced the interstitial collagenase and stromelysin‐1 protein levels. Taken together, our data provide the first evidence that Ultraviolet‐B induction of interstitial collagenase and stromelysin‐1 occurs via the synthesis and release of interleukin‐6. Hence, this newly identified autocrine mechanism may contribute to dermal photodamage.

PCR-based subtractive hybridization identifies repressed genes in growth-arrested human dermal fibroblasts following combined treatment with 8-methoxypsoralen and UVA irradiation (PUVA).

To identify genes which are repressed in growth-arrested human dermal fibroblasts upon a single treatment with 8-methoxypsoralen and UVA irradiation (PUVA) we have used a PCR-based subtractive hybridization protocol resulting in cloning of four PUVA-repressed genes. Sequence analysis and homology searches identified three known genes related to growth control, lipid and connective tissue metabolism. One cDNA clone represented a novel gene. Northern blot analyses confirmed a PUVA-dependent reduction in mRNA expression in fibroblasts in vitro. The identification of growth arrest related repressed genes in PUVA-treated fibroblasts may stimulate further research addressing the causal role of these genes in the control and regulation of the postmitotic phenotype of fibroblasts on a molecular and cellular level.

Free Radicals and Dermal Damage in Photoaging and Photocarcinogenesis

The increase in UV irradiation on earth due to the stratospheric ozone depletion represents a major environmental threat to the skin increasing its risk of photooxidative damage by reactive oxygen species (ROS). ROS have been implicated in several photo dermatological disorders including photoaging. The clinical manifestations of cutaneous photoaging are due to quantitative and qualitative alterations of the dermal connective tissue. Therefore, we were interested in a better understanding of the involvement of ROS in the up-regulation of matrix metalloproteinases (MMP) responsible for connective tissue degradation in photoaging, tumor invasion and metastasis. For this purpose fibroblast monolayer cultures have been subjected to various UV modalities. In parallel fibroblasts have been challenged by distinct ROS generating systems. Using scavengers, enhancers, and inhibitors for the activity of ROS detoxifying enzymes, using stably transfected fibroblast cell lines overexpressing ROS detoxifying enzymes, and using iron chelators blocking the Fenton reaction, distinct ROS have been increased peri-or intracellularly. A time and dose dependent increase in mRNA and protein levels for collagenase was observed after exposure of fibroblasts to UV-B, UV-A, O2, H2O2, O2 -,or OH generating systems. After UV-B irradiation inhibition of the Fenton reaction using iron chelators abrogated the MMP induction substantially. Scavengers for the hydroxyl radical reduced the MMP induction after UV-B, thus pointing to the importance of the Fenton reaction and the hydroxyl radical in the UV-B response. By modulating the lifetime of O2 by enhancers and quenchers, singlet oxygen could be identified as a crucial mediator of the UV-A response. In addition, the inhibition of glutathion peroxidase, catalase and the Fenton reaction enhanced MMP mRNA induction whereas inhibition of superoxide dismutase diminished the MMP mRNA increase. Furthermore, exposure to UV-A of a stably transfected fibroblast cell line overexpressing the manganese superoxide dismutase resulted in a substantial increase in MMP mRNA. These results indicate that following UV-A irradiation, beside O2, H2O2 is mainly responsible for the induction of MMP synthesis. In conclusion, we have identified distinct ROS involved in matrix-degrading enzyme induction resulting in tissue degradation follow-ing UV irradiation. This will allow a rational risk assessment for the dermal damage of different UV modalities in photoaging and photocarcinogenesis.

A newly adapted pulsed-field gel electrophoresis technique allows to detect distinct types of DNA damage at low frequencies in human dermal fibroblasts upon exposure to non-toxic H2O2 concentrations

Reactive oxygen species (ROS) comprise several oxygen containing compounds, among them hydrogen peroxide (H2O2), which are generated by internal and external sources and play pleiotropic roles in physiological and pathological states. Skin cells as well as cells from other tissues have developed antioxidant defense mechanisms to protect themselves from high concentrations of ROS. Although biological and pathological roles of ROS have previously been elucidated, so far only limited knowledge exists regarding ROS-mediated generation of DNA breaks and base lesions occurring at low frequency in intact skin cells. This study was therefore designed to probe a newly adapted pulsed-field gel electrophoresis technique for the adequate measurement of high molecular weight DNA fragments as well as to investigate the protective role of the antioxidant enzyme catalase against H2O2-mediated damage in human dermal fibroblasts. We stably transfected and overexpressed the full-length catalase cDNA in the human dermal fibroblast cell line 1306 in culture and found that these cells are significantly more protected from cytotoxicity, overall DNA strand breaks, and 8-oxodeoxyguanine base lesions resulting from H2O2-triggered oxidative stress compared to vector-transfected 1306 cells or secondary dermal fibroblasts. This work has outlined the importance of catalase in the protection from H2O2-mediated cytotoxicity and DNA damage which--if unbalanced--even when occurring at low frequency are known to lead to genomic instability, a hallmark in carcinogenesis and premature aging.