Imaging of metabolic activity adaptations to UV stress, drugs and differentiation at cellular resolution in skin and skin equivalents – Implications for oxidative UV damage

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Suppression of Autophagy Dysregulates the Antioxidant Response and Causes Premature Senescence of Melanocytes

YesAutophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10–15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2–related factor 2 (Nrf2)–dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs

Age-promoting and lipid-oxidizing stress in the skin

Die Haut ist aufgrund der Grenzflächenfunktion zwischen Körper und Umwelt nicht nur endogenem sondern auch umweltbedingtem oxidativen Stress ausgesetzt, der von ultravioletter (UV) Strahlung, Ozon und Umweltverschmutzung verursacht wird. Der Beitrag dieser Stressfaktoren zur Hautalterung steht im Interesse der Grundlagenforschung und der angewandten Hautforschung. Membranphospholipide können leicht durch UV oxidiert werden und haben in den letzten Jahren als zelluläre Signaltransduktoren stark an Bedeutung gewonnen. Sie spielen eine wichtige Rolle in Thrombose, Angiogenese, endothelialer Barrierefunktion, Immunregulation und Entzündung und wurden als Biomarker für altersassoziierte Krankheiten wie Atherosklerose und Alzheimer‘s angeführt. Über die Funktion und die zugrundeliegende Biologie oxidierter Phospholipide in Stress und Alterung der Haut ist jedoch wenig bekannt. Spezifische oxidierte Phospholipide können Proteine und andere Zellbestandteile chemisch modifizieren, und solche Modifikationen wurden in UV-geschädigter Haut nachgewiesen und sind Bestandteil von Alters-Pigment und Lipofuscin. Es ist daher für unser biologisches Verständnis wichtig, den Einfluss von spezifischen oxidierten Phospholipidmarkern in definierten Krankheitszuständen, Alterung, oder bei maligner Transformation eindeutig zu identifizieren, und hat daher großes diagnostisches Potenzial. Diese PhD-Arbeit untersuchte die Entstehung, Funktion und relative Konzentrationskinetik von bioaktiven oxidierten Phospholipiden in der Haut und in verschiedenen primären Hautzelltypen unter UVA-Stressbedingungen. Wir bestrahlten primäre Keratinozyten entweder mit UVA-1, dem wichtigsten oxidativen Hautstressor, oder behandelten sie mit extern UVA-oxidierten Phospholipiden (UVPAPC) um den Beitrag der Lipide zur UVA-induzierten Signaltransduktion und den Verbleib von extern zugegebenen oxidierten Lipiden in Keratinozyten zu untersuchen. Es wurde gezeigt, dass sowohl UVA als auch UVPAPC zur Induktion der Autophagie und der Nrf2-gesteuerten antioxidativen Stressantwort führen, zwei Mechanismen, die mit zunehmendem Alter stagnieren. Aus diesem Grund wurden zwei weitere Modellsysteme angewandt, um die Effekte der beiden zellulären Stressantworten auf die relative Menge und die biologische Aktivität von oxidierten Lipiden hin zu erforschen: Wir haben Atg7 in Maus-Melanocyten deletiert, um die Autophagie, einen zellulären Mechanismus zum Abbau geschädigter und modifizierter Moleküle stillzulegen. Des Weiteren deletierten wir den redox-sensitiven Transkriptionsfaktor Nrf2 in Maus-Fibroblasten, um die antioxidative Stressantwort, welche zelluläre Antioxidantien und entgiftende Enzyme bereitstellt, zu minimieren. Anschließend untersuchten wir, wie sich der Genverlust auf die relativen Mengen oxidierter Lipide und die zugrunde liegende Signaltransduktion auswirkte, sowohl in Zellhomöostase als auch unter Stress.Due to its interface function between the body and the environment, the skin is strongly challenged not only by internally generated- but also by environmental oxidative stress arising from solar ultraviolet radiation, ozone and pollution. These factors are all well recognized stress agents that have an impact on skin aging and are extensively studied regarding this aspect. Membrane phospholipids can be easily oxidized by environmental stressors, like UVA and have gained over the past years increasing biological importance in cellular signal transduction and as indicators of the cellular redox status. A role for oxPLs has been proposed in thrombosis, angiogenesis, endothelial barrier function, immune tolerance and inflammation and they have gained importance as biomarkers of age related diseases, like atherosclerosis and Alzheimers. Still, little is known about the function and the underlying biology of oxidized phospholipids in skin stress and -aging. Selected oxidized lipid species can easily modify other molecules like proteins, and the results of such modifications can be detected in photodamaged skin where they contribute to the age-related pigment and lipofuscin. To find specific oxidized phospholipid markers that would clearly identify a defined disease state, aging, tissue damage or cell configurations like malignant transformation would thus be of high value, both to biologically understand the contribution of oxidized lipids to these states and from a diagnostic point of view. This thesis explored the generation, the function and the relative concentration kinetics of bioactive oxidized phospholipids in the skin and in various primary skin cell types under UVA stress conditions. In one experimental system we aimed to study these aspects in human keratinocytes. We either irradiated primary skin cells with UVA-1, the major skin oxidative stressor, or treated them with externally UVA oxidized phospholipids (UVPAPC), in an effort to single out the contribution of oxidized lipids to UVA induced signaling events in keratinocytes and to study the fate of supplemented oxidized lipids on keratinocytes. UVA and UVPAPC were both shown to be inducers of autophagy and the Nrf2-driven antioxidant response, two mechanisms that are impaired with increased age. Therefore we used two additional model systems to study the putative effects of the identified cellular responses on abundance and biological activity of oxidized lipids: We either knocked out Atg7 to impair autophagy, a cellular mechanism to degrade damaged and modified molecules in mouse melanocytes or deleted the redox-sensitive transcription factor Nrf2 to dysregulate the antioxidant response that provides cellular antioxidants and detoxifying enzymes in mouse fibroblasts. With these approaches we further investigated how the gene deficiencies would affect oxidized lipid levels, cell signaling and downstream biological consequences in homeostasis and under age promoting stress.submitted by Marie-Sophie NarztAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2018OeBB(VLID)268647

Involvement of cutaneous SR-B1 in skin lipid homeostasis

Background: The main functions of the skin are to protect against environmental insults and prevent water loss, which are performed by the complex lipid- and protein matrix present in the outermost layers of the epithelium. The lipidome of these outer layers is mainly composed of ceramides, fatty acids, and cholesterol, which regulates keratinocyte differentiation and skin barrier function. SR-B1 is a multifunctional scavenger receptor that is best known for facilitating uptake of cholesterol from HDL particles in the liver, but it is also expressed in the skin. Objective: To determine the role of SR-B1 in keratinocyte differentiation. Methods: We investigated the relationship between SR-B1 and keratinocyte differentiation using a physiologically relevant model, organotypic skin equivalents (SEs), wherein SR-B1 was knocked down via siRNA transfection. To assess effects of SR-B1 knockdown on keratinocyte differentiation, we performed hematoxylin/eosin staining, RT-PCR, western blotting, and immunohistochemistry. We also examined the effect of SR-B1 knockdown on lipid production by performing Oil Red O staining and thin layer chromatography. Results: SR-B1 knockdown resulted in decreased lipid levels in SEs, specifically ceramides, and in decreased transcript levels of LDLR, PPAR-α and PPAR-γ which are factors involved in regulating ceramide synthesis. In addition, filaggrin levels increased in SR-B1 KD tissues, but neither keratin 14 nor keratin 10 were affected. Conclusion: We conclude that one of the main functions of SR-B1 in the skin is to regulate ceramide levels and thereby maintain the barrier function of the skin, resulting in the protection of cutaneous tissues from outdoor insults

Redox Biology / Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo

Autophagy allows cells fundamental adaptations to metabolic needs and to stress. Using autophagic bulk degradation cells can clear crosslinked macromolecules and damaged organelles that arise under redox stress. Accumulation of such debris results in cellular dysfunction and is observed in aged tissue and senescent cells. Conversely, promising anti-aging strategies aim at inhibiting the mTOR pathway and thereby activating autophagy, to counteract aging associated damage. We have inactivated autophagy related 7 (Atg7), an essential autophagy gene, in murine keratinocytes (KC) and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by paraquat (PQ), an oxidant drug commonly used to induce cellular senescence. Atg7 deficient KC displayed increased prostanoid signaling and a pro- mitotic gene expression signature as compared to the WT. After exposure to PQ, both WT and KO cells showed an inflammatory and stress-related transcriptomic response. However, the Atg7 deficient cells additionally showed drastic DNA damage- and cell cycle arrest signaling. Indeed, DNA fragmentation and oxidation were strongly increased in the stressed Atg7 deficient cells upon PQ stress but also after oxidizing ultraviolet A irradiation. Damage associated phosphorylated histone H2AX (H2AX) foci were increased in the nuclei, whereas expression of the nuclear lamina protein lamin B1 was strongly decreased. Similarly, in both, PQ treated mouse tail skin explants and in UVA irradiated mouse tail skin, we found a strong increase in H2AX positive nuclei within the basal layer of Atg7 deficient epidermis. Atg7 deficiency significantly affected expression of lipid metabolic genes. Therefore we performed lipid profiling of keratinocytes which demonstrated a major dysregulation of cellular lipid metabolism. We found accumulation of autophagy agonisitic free fatty acids, whereas triglyceride levels were strongly decreased. Together, our data show that in absence of Atg7/autophagy the resistance of keratinocytes to intrinsic and environmental oxidative stress was severely impaired and resulted in DNA damage, cell cycle arrest and a disturbed lipid phenotype, all typical for premature cell aging.(VLID)485638

Establishment of In Vitro Models by Stress-Induced Premature Senescence for Characterizing the Stromal Vascular Niche in Human Adipose Tissue

Acting as the largest energy reservoir in the body, adipose tissue is involved in longevity and progression of age-related metabolic dysfunction. Here, cellular senescence plays a central role in the generation of a pro-inflammatory environment and in the evolution of chronic diseases. Within the complexity of a tissue, identification and targeting of senescent cells is hampered by their heterogeneity. In this study, we generated stress-induced premature senescence 2D and 3D in vitro models for the stromal vascular niche of human adipose tissue. We established treatment conditions for senescence induction using Doxorubicin (Dox), starting from adipose-derived stromal/stem cells (ASCs), which we adapted to freshly isolated microtissue-stromal vascular fraction (MT-SVF), where cells are embedded within their native extracellular matrix. Senescence hallmarks for the established in vitro models were verified on different cellular levels, including morphology, cell cycle arrest, senescence-associated β-galactosidase activity (SA-βgal) and gene expression. Two subsequent exposures with 200 nM Dox for six days were suitable to induce senescence in our in vitro models. We demonstrated induction of senescence in the 2D in vitro models through SA-βgal activity, at the mRNA level (LMNB1, CDK1, p21) and additionally by G2/M phase cell cycle arrest in ASCs. Significant differences in Lamin B1 and p21 protein expression confirmed senescence in our MT-SVF 3D model. MT-SVF 3D cultures were composed of multiple cell types, including CD31, CD34 and CD68 positive cells, while cell death remained unaltered upon senescence induction. As heterogeneity and complexity of adipose tissue senescence is given by multiple cell types, our established senescence models that represent the perivascular niche embedded within its native extracellular matrix are highly relevant for future clinical studies

Consequences of Autophagy Deletion on the Age-Related Changes in the Epidermal Lipidome of Mice

Autophagy is a controlled mechanism of intracellular self-digestion with functions in metabolic adaptation to stress, in development, in proteostasis and in maintaining cellular homeostasis in ageing. Deletion of autophagy in epidermal keratinocytes does not prevent the formation of a functional epidermis and the permeability barrier but causes increased susceptibility to damage stress and metabolic alterations and accelerated ageing phenotypes. We here investigated how epidermal autophagy deficiency using Keratin 14 driven Atg7 deletion would affect the lipid composition of the epidermis of young and old mice. Using mass spectrometric lipidomics we found a reduction of age-related accumulation of storage lipids in the epidermis of autophagy-deficient mice, and specific changes in chain length and saturation of fatty acids in several lipid classes. Transcriptomics and immunostaining suggest that these changes are accompanied by changes in expression and localisation of lipid and fatty acid transporter proteins, most notably fatty acid binding protein 5 (FABP5) in autophagy knockouts. Thus, maintaining autophagic activity at an advanced age may be necessary to maintain epidermal lipid homeostasis in mammals

Cornification of nail keratinocytes requires autophagy for bulk degradation of intracellular proteins while sparing components of the cytoskeleton.

Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes

Cornification of nail keratinocytes requires autophagy for bulk degradation of intracellular proteins while sparing components of the cytoskeleton

Epidermal keratinocytes undergo cornification to form the cellular building blocks of hard skin appendages such as nails and the protective layer on the surface of the skin. Cornification requires the cross-linking of structural proteins and the removal of other cellular components to form mechanically rigid and inert corneocytes. Autophagy has been proposed to contribute to this intracellular remodelling process, but its molecular targets in keratinocytes, if any, have remained elusive. Here, we deleted the essential autophagy factor Atg7 in K14-positive epithelia of mice and determined by proteomics the impact of this deletion on the abundance of individual proteins in cornified nails. The genetic suppression of autophagy in keratinocytes resulted in a significant increase in the number of proteins that survived cornification and in alterations of their abundance in the nail proteome. A broad range of enzymes and other non-structural proteins were elevated whereas the amounts of cytoskeletal proteins of the keratin and keratin-associated protein families, cytolinker proteins and desmosomal proteins were either unaltered or decreased in nails of mice lacking epithelial autophagy. Among the various types of non-cytoskeletal proteins, the subunits of the proteasome and of the TRiC/CCT chaperonin were most strongly elevated in mutant nails, indicating a particularly important role of autophagy in removing these large protein complexes during normal cornification. Taken together, the results of this study suggest that autophagy is active during nail keratinocyte cornification and its substrate specificity depends on the accessibility of proteins outside of the cytoskeleton and their presence in large complexes.(VLID)365860