Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

Molecular species of oxidized phospholipids in brain differentiate between learning- and memory impaired and unimpaired aged rats.

Loss of cognitive function is a typical consequence of aging in humans and rodents. The extent of decline in spatial memory performance of rats, assessed by a hole-board test, reaches from unimpaired and comparable to young individuals to severely memory impaired. Recently, proteomics identified peroxiredoxin 6, an enzyme important for detoxification of oxidized phospholipids, as one of several synaptosomal proteins discriminating between aged impaired and aged unimpaired rats. In this study, we investigated several components of the epilipidome (modifications of phospholipids) of the prefrontal cortex of young, aged memory impaired (AI) and aged unimpaired (AU) rats. We observed an age-related increase in phospholipid hydroperoxides and products of phospholipid peroxidation, including reactive aldehydophospholipids. This increase went in hand with cortical lipofuscin autofluorescence. The memory impairment, however, was paralleled by additional specific changes in the aged rat brain epilipidome. There was a profound increase in phosphocholine hydroxides, and a significant decrease in phosphocholine-esterified azelaic acid. As phospholipid-esterified fatty acid hydroxides, and especially those deriving from arachidonic acid are both markers and effectors of inflammation, the findings suggest that in addition to age-related reactive oxygen species (ROS) accumulation, age-related impairment of spatial memory performance has an additional and distinct (neuro-) inflammatory component.

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.

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.

Bioinformatics approach for choosing the correct reference genes when studying gene expression in human keratinocytes.

Reverse transcription polymerase chain reaction (qRT-PCR) has become a mainstay in many areas of skin research. To enable quantitative analysis, it is necessary to analyse expression of reference genes (RGs) for normalization of target gene expression. The selection of reliable RGs therefore has an important impact on the experimental outcome. In this study, we aimed to identify and validate the best suited RGs for qRT-PCR in human primary keratinocytes (KCs) over a broad range of experimental conditions using the novel bioinformatics tool 'RefGenes', which is based on a manually curated database of published microarray data. Expression of 6 RGs identified by RefGenes software and 12 commonly used RGs were validated by qRT-PCR. We assessed whether these 18 markers fulfilled the requirements for a valid RG by the comprehensive ranking of four bioinformatics tools and the coefficient of variation (CV). In an overall ranking, we found GUSB to be the most stably expressed RG, whereas the expression values of the commonly used RGs, GAPDH and B2M were significantly affected by varying experimental conditions. Our results identify RefGenes as a powerful tool for the identification of valid RGs and suggest GUSB as the most reliable RG for KCs.

Deletion of NRF2 disturbs composition, morphology, and differentiation of the murine tail epidermis in chronological aging.

NRF2 is a master regulator of the cellular protection against oxidative damage in mammals and of multiple pathways relevant in the mammalian aging process. In the epidermis of the skin NRF2 contributes additionally to the formation of an antioxidant barrier to protect from environmental insults and is involved in the differentiation process of keratinocytes. In chronological aging of skin, the capacity for antioxidant responses and the ability to restore homeostasis after damage are impaired. Surprisingly, in absence of extrinsic stressors, NRF2 deficient mice do not show any obvious skin phenotype, not even at old age. We investigated the differences in chronological epidermal aging of wild type and NRF2-deficient mice to identify the changes in aged epidermis that may compensate for absence of this important transcriptional regulator. While both genotypes showed elevated epidermal senescence markers (increased Lysophospholipids, decreased LaminB1 expression), the aged NRF2 deficient mice displayed disturbed epidermal differentiation manifested in irregular keratin 10 and loricrin expression. The tail skin displayed less age-related epidermal thinning and a less pronounced decline in proliferating basal epidermal cells compared to the wildtype controls. The stratum corneum lipid composition also differed, as we observed elevated production of barrier protective linoleic acid (C18:2) and reduced abundance of longer chain saturated lignoceric acid (C24:0) among the stratum corneum fatty acids in the aged NRF2-deficient mice. Thus, despite epidermal differentiation being disturbed in aged NRF2-deficient animals in homeostasis, adaptations in keratinocyte proliferation and barrier lipid synthesis could explain the lack of a more severe phenotype.

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 ß-galactosidase activity (SA-ßgal) 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-ßgal 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.

Multi-Level Analysis of Adipose Tissue Reveals the Relevance of Perivascular Subpopulations and an Increased Endothelial Permeability in Early-Stage Lipedema.

Lipedema is a chronic, progressive disease of adipose tissue with unknown etiology. Based on the relevance of the stromal vascular fraction (SVF) cell population in lipedema, we performed a thorough characterization of subcutaneous adipose tissue, SVF isolated thereof and the sorted populations of endothelial cells (EC), pericytes and cultured adipose-derived stromal/stem cells (ASC) of early-stage lipedema patients. We employed histological and gene expression analysis and investigated the endothelial barrier by immunofluorescence and analysis of endothelial permeability in vitro. Although there were no significant differences in histological stainings, we found altered gene expression of factors relevant for local estrogen metabolism (aromatase), preadipocyte commitment (ZNF423) and immune cell infiltration (CD11c) in lipedema on the tissue level, as well as in distinct cellular subpopulations. Machine learning analysis of immunofluorescence images of CD31 and ZO-1 revealed a morphological difference in the cellular junctions of EC cultures derived from healthy and lipedema individuals. Furthermore, the secretome of lipedema-derived SVF cells was sufficient to significantly increase leakiness of healthy human primary EC, which was also reflected by decreased mRNA expression of VE-cadherin. Here, we showed for the first time that the secretome of SVF cells creates an environment that triggers endothelial barrier dysfunction in early-stage lipedema. Moreover, since alterations in gene expression were detected on the cellular and/or tissue level, the choice of sample material is of high importance in elucidating this complex disease.

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

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

The epidermis is a multi-layered epithelium that consists mainly of keratinocytes which proliferate in its basal layer and then differentiate to form the stratum corneum, the skin's ultimate barrier to the environment. During differentiation keratinocyte function, chemical composition, physical properties, metabolism and secretion are profoundly changed. Extrinsic or intrinsic stressors, like ultraviolet (UV) radiation thus may differently affect the epidermal keratinocytes, depending on differentiation stage. Exposure to UV elicits the DNA damage responses, activation of pathways which detoxify or repair damage or induction of programmed cell death when the damage was irreparable. Recently, rapid diversion of glucose flux into the pentose phosphate pathway (PPP) was discovered as additional mechanism by which cells rapidly generate reduction equivalents and precursors for nucleotides - both being in demand after UV damage. There is however little known about the correlation of such metabolic activity with differentiation state, cell damage and tissue localization of epidermal cells. We developed a method to correlate the activity of G6PD, the first and rate-limiting enzyme of this metabolic UV response, at cellular resolution to cell type, differentiation state, and cell damage in human skin and in organotypic reconstructed epidermis. We thereby could verify rapid activation of G6PD as an immediate UVB response not only in basal but also in differentiating epidermal keratinocytes and found increased activity in cells which initiated DNA damage responses. When keratinocytes had been UVB irradiated before organotypic culture, their distribution within the skin equivalent was abnormal and the G6PD activity was reduced compared to neighboring cells. Finally, we found that the anti-diabetic and potential anti-aging drug metformin strongly induced G6PD activity throughout reconstructed epidermis. Activation of the protective pentose phosphate pathway may be useful to enhance the skin's antioxidant defense systems and DNA damage repair capacity on demand.

The impact of recent advances in lipidomics and redox lipidomics on dermatological research.

Dermatological research is a major beneficiary of the rapidly developing advances in lipid analytic technology and of bioinformatic tools which help to decipher and interpret the accumulating big lipid data. At its interface with the environment, the epidermis develops a blend of lipids that constitutes the epidermal lipid barrier, essential for the protection from water loss and entry of dangerous noxae. Apart from their structural role in the barrier, novel intra- and inter-cellular signaling functions of lipids and their oxidation products have been uncovered in most cutaneous cell types over the last decades, and the discovery rate has been boosted by the advent of high resolution and -throughput mass spectrometric techniques. Our understanding of epidermal development has benefited from studies on fetal surface lipids, which appear to signal for adaptation to desiccation post partum, and from studies on the dynamics of epidermal lipids during adjustment to the atmosphere in the first months of life. At birth, external insults begin to challenge the skin and its lipids, and recent years have yielded ample insights into the dynamics of lipid synthesis and -oxdiation after UV exposure, and upon contact with sensitizers and irritants. Psoriasis and atopic dermatitis are the most common chronic inflammatory skin diseases, affecting at least 3% and 7% of the global population, respectively. Consequently, novel (redox-) lipidomic techniques have been applied to study systemic and topical lipid abnormalities in patient cohorts. These studies have refined the knowledge on eicosanoid signaling in both diseases, and have identified novel biomarkers and potential disease mediators, such as lipid antigens recognized by psoriatic T cells, as well as ceramide species, which specifically correlate with atopic dermatitis severity. Both biomarkers have yielded novel mechanistic insights. Finally, the technological progress has enabled studies to be performed that have monitored the consequences of diet, lifestyle, therapy and cosmetic intervention on the skin lipidome, highlighting the translational potential of (redox-) lipidomics in dermatology.

A novel role for NUPR1 in the keratinocyte stress response to UV oxidized phospholipids.

Ultraviolet light is the dominant environmental oxidative skin stressor and a major skin aging factor. We studied which oxidized phospholipid (OxPL) mediators would be generated in primary human keratinocytes (KC) upon exposure to ultraviolet A light (UVA) and investigated the contribution of OxPL to UVA responses. Mass spectrometric analysis immediately or 24 h post UV stress revealed significant changes in abundance of 173 and 84 lipid species, respectively. We identified known and novel lipid species including known bioactive and also potentially reactive carbonyl containing species. We found indication for selective metabolism and degradation of selected reactive lipids. Exposure to both UVA and to in vitro UVA - oxidized phospholipids activated, on transcriptome and proteome level, NRF2/antioxidant response signaling, lipid metabolizing enzyme expression and unfolded protein response (UPR) signaling. We identified NUPR1 as an upstream regulator of UVA/OxPL transcriptional stress responses and found this protein to be expressed in the epidermis. Silencing of NUPR1 resulted in augmented expression of antioxidant and lipid detoxification genes and disturbed the cell cycle, making it a potential key factor in skin reactive oxygen species (ROS) responses intimately involved in aging and pathology.

OLR1 scavenger receptor knockdown affects mitotic gene expression but is dispensable for oxidized phospholipid- mediated stress signaling in SZ 95 sebocytes.

Phospholipid oxidation products (OxPL) are versatile stress signaling mediators in the skin. These lipid signaling molecules can be generated non-enzymatically or enzymatically by ultraviolet light, the major extrinsic skin aging factor. OxPL regulate cytoprotective, immunological and metabolic adaptation of the skin to oxidant stress. We here investigated whether the scavenger receptor Oxidized Low Density Lipoprotein Receptor 1 (OLR1, LOX-1) would have a function in cutaneous oxPL signaling. We found, that OLR1 is expressed in several cutaneous cell types, most prominently in cells of the sebaceous gland and in keratinocytes. We repressed OLR1 expression with siRNA in SZ95 sebocytes, exposed cells to oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and performed transcriptomic profiling. Bioinformatic analysis revealed that OxPL exposure induced the Nrf2 antioxidant stress response and aldosterone signaling. The analysis also revealed that OLR1 is not required for the transcriptional regulation induced by oxidized PAPC but interestingly, OLR1 knockdown affected expression of CNN2, HMRR, ITGB6 and KIF20A, all genes governing cell proliferation and motility. We identify sebocytes as cutaneous cells responsive to lipid mediated redox stress which is not dependent on the scavenger receptor OLR1.

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7.

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7f/f Tyr-Cre mice relative to Atg7f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.

Different pro-angiogenic potential of γ-irradiated PBMC-derived secretome and its subfractions.

Secretomes from various cell sources exert strong regenerative activities on numerous organs, including the skin. Although secretomes consist of many diverse components, a growing body of evidence suggests that small extracellular vesicles (EVs) account for their regenerative capacity. We previously demonstrated that the secretome of γ-irradiated peripheral blood mononuclear cells (PBMCs) exhibits wound healing capacity. Therefore, we sought to dissect the molecular composition of EVs present in the secretome and compared wound healing-related activities of these EVs to other subfractions of the secretome and the fully supplemented secretome (MNCaposec). Compared to EVs derived from non-irradiated PBMCs, γ-irradiation significantly increased the size and number and changed the composition of released EVs. Detailed characterization of the molecular components of EVs, i.e. miRNA, proteins, and lipids, derived from irradiated PBMCs revealed a strong association with regenerative processes. Reporter gene assays and aortic ring sprouting assays revealed diminished activity of the subfractions compared to MNCaposec. In addition, we showed that MNCaposec accelerated wound closure in a diabetic mouse model. Taken together, our results suggest that secretome-based wound healing represents a promising new therapeutic avenue, and strongly recommend using the complete secretome instead of purified subfractions, such as EVs, to exploit its full regenerative capacity.

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 (γH2AX) 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 γH2AX 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.

Autophagy deficient melanocytes display a senescence associated secretory phenotype that includes oxidized lipid mediators.

Autophagy is a recycling program which allows cells to adapt to metabolic needs and to stress. Defects in autophagy can affect metabolism, aging, proteostasis and inflammation. Autophagy pathway genes, including autophagy related 7 (Atg7), have been associated with the regulation of skin pigmentation, and autophagy defects disturb the biogenesis and transport of melanosomes in melanocytes as well as transfer and processing of melanin into keratinocytes. We have previously shown that mice whose melanocytes or keratinocytes lack Atg7 (and thus autophagy) as a result of specific gene knockout still retained functioning melanosome synthesis and transfer, and displayed only moderate reduction of pigmentation. In cell culture the Atg7 deficient melanocytes were prone to premature senescence and dysregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling. To elucidate the biochemical basis of this phenotype, we performed a study on global gene expression, protein secretion and phospholipid composition in Atg7 deficient versus Atg7 expressing melanocytes. In cell culture Atg7 deficient melanocytes showed a pro-inflammatory gene expression signature and secreted higher levels of C-X-C motif chemokine ligand -1,-2,-10 and -12 (Cxcl1, Cxcl2, Cxcl10, Cxcl12), which are implicated in the pathogenesis of pigmentary disorders and expressed higher amounts of matrix metalloproteinases -3 and -13 (Mmp3, Mmp13). The analysis of membrane phospholipid composition identified an increase in the arachidonic- to linoleic acid ratio in the autophagy deficient cells, as well as an increase in oxidized phospholipid species that act as danger associated molecular patterns (DAMPs). The secretion of inflammation related factors suggests that autophagy deficient melanocytes display a senescence associated secretory phenotype (SASP), and we propose oxidized lipid mediators as novel components of this SASP.

Nrf2 deficiency causes lipid oxidation, inflammation, and matrix-protease expression in DHA-supplemented and UVA-irradiated skin fibroblasts.

Fish oil rich in docosahexaenoic acid (DHA) has beneficial effects on human health. Omega-3 polyunsaturated fatty acids are precursors of eicosanoids and docosanoids, signaling molecules that control inflammation and immunity, and their dietary uptake improves a range of disorders including cardiovascular diseases, ulcerative colitis, rheumatoid arthritis, and psoriasis. The unsaturated nature of these fatty acids, however, makes them prone to oxidation, especially when they are incorporated into (membrane) phospholipids. The skin is an organ strongly exposed to oxidative stress, mainly due to solar ultraviolet radiation. Thus, increased levels of PUFA in combination with oxidative stress could cause increased local generation of oxidized lipids, whose action spectrum reaches from signaling molecules to reactive carbonyl compounds that can crosslink biomolecules. Here, we investigated whether PUFA supplements to fibroblasts are incorporated into membrane phospholipids and whether an increase of PUFA within phospholipids affects the responses of the cells to UV exposure. The redox-sensitive transcription factor Nrf2 is the major regulator of the fibroblast stress response to ultraviolet radiation or exposure to oxidized lipids. Here we addressed how Nrf2 signaling would be affected in PUFA-supplemented human dermal fibroblasts and mouse dermal fibroblasts from Nrf2-deficient and wild type mice. We found, using HPLC-tandem MS, that DHA supplements to culture media of human and murine fibroblasts were readily incorporated into phospholipids and that subsequent irradiation of the supplemented cells with UVA resulted in an increase in 1-palmitoyl-2-(epoxyisoprostane-E2)-sn-glycero-3-phosphorylcholine and Oxo-DHA esterified to phospholipid, both of which are Nrf2 agonists. Also, induction of Nrf2 target genes was enhanced in the DHA-supplemented fibroblasts after UVA irradiation. In Nrf2-deficient murine fibroblasts, the expression of the target genes was, as expected, decreased, but surprisingly, expression of TNFα and MMP13 was strongly induced in DHA-supplemented, UVA-irradiated cells. Also, Nrf2-deficient cells had increased levels of oxidized phospholipids relative to the unoxidized precursors after UVA irradiation. Our data suggest that under ultraviolet stress a functioning Nrf2 system is required to prevent DHA-induced inflammation and matrix degradation in dermal fibroblasts.

Suppression of autophagy dysregulates the antioxidant response and causes premature senescence of melanocytes.

Autophagy 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.