Concomitant DNA methylation and transcriptome signatures define epidermal responses to acute solar UV radiation.

The simultaneous analysis of different regulatory levels of biological phenomena by means of multi-omics data integration has proven an invaluable tool in modern precision medicine, yet many processes ultimately paving the way towards disease manifestation remain elusive and have not been studied in this regard. Here we investigated the early molecular events following repetitive UV irradiation of in vivo healthy human skin in depth on transcriptomic and epigenetic level. Our results provide first hints towards an immediate acquisition of epigenetic memories related to aging and cancer and demonstrate significantly correlated epigenetic and transcriptomic responses to irradiation stress. The data allowed the precise prediction of inter-individual UV sensitivity, and molecular subtyping on the integrated post-irradiation multi-omics data established the existence of three latent molecular phototypes. Importantly, further analysis suggested a form of melanin-independent DNA damage protection in subjects with higher innate UV resilience. This work establishes a high-resolution molecular landscape of the acute epidermal UV response and demonstrates the potential of integrative analyses to untangle complex and heterogeneous biological responses.

An integrative metabolomics and transcriptomics study to identify metabolic alterations in aged skin of humans in vivo.

BACKGROUND: Aging human skin undergoes significant morphological and functional changes such as wrinkle formation, reduced wound healing capacity, and altered epidermal barrier function. Besides known age-related alterations like DNA-methylation changes, metabolic adaptations have been recently linked to impaired skin function in elder humans. Understanding of these metabolic adaptations in aged skin is of special interest to devise topical treatments that potentially reverse or alleviate age-dependent skin deterioration and the occurrence of skin disorders. RESULTS: We investigated the global metabolic adaptions in human skin during aging with a combined transcriptomic and metabolomic approach applied to epidermal tissue samples of young and old human volunteers. Our analysis confirmed known age-dependent metabolic alterations, e.g. reduction of coenzyme Q10 levels, and also revealed novel age effects that are seemingly important for skin maintenance. Integration of donor-matched transcriptome and metabolome data highlighted transcriptionally-driven alterations of metabolism during aging such as altered activity in upper glycolysis and glycerolipid biosynthesis or decreased protein and polyamine biosynthesis. Together, we identified several age-dependent metabolic alterations that might affect cellular signaling, epidermal barrier function, and skin structure and morphology. CONCLUSIONS: Our study provides a global resource on the metabolic adaptations and its transcriptional regulation during aging of human skin. Thus, it represents a first step towards an understanding of the impact of metabolism on impaired skin function in aged humans and therefore will potentially lead to improved treatments of age related skin disorders.

Impaired glyoxalase activity is associated with reduced expression of neurotrophic factors and pro-inflammatory processes in diabetic skin cells.

Patients suffering from type II diabetes develop several skin manifestations including cutaneous infections, diabetic dermopathy, diabetic bullae and acanthosis nigricans. Diabetic micro- and macroangiopathy as well as diabetic neuropathy are believed to play a crucial role in the development of diabetic skin disorders. A reduced cutaneous nerve fibre density was reported in diabetic subjects, which subsequently leads to impaired sensory nerve functions. Using an innervated skin model, we investigated the impact of human diabetic dermal fibroblasts and keratinocytes on porcine sensory neurons. Diabetic skin cells showed a reduced capacity to induce neurite outgrowth due to a decreased support with neurotrophic factors, such as NGF. Furthermore, diabetic keratinocytes displayed insulin resistance and increased expression of pro-inflammatory cytokines demonstrating the persistent effect of diabetes mellitus on human skin cells. Dysregulations were related to a significantly reduced glyoxalase enzyme activity in diabetic keratinocytes as experimentally reduced glyoxalase activity mimicked the increase in pro-inflammatory cytokine expression and reduction in NGF. Our results demonstrate an impaired crosstalk of diabetic skin cells and sensory neurons favouring hypo-innervation. We suggest that reduced methylglyoxal detoxification contributes to an impaired neurocutaneous interaction in diabetic skin.

Reduced DNA methylation patterning and transcriptional connectivity define human skin aging.

Epigenetic changes represent an attractive mechanism for understanding the phenotypic changes associated with human aging. Age-related changes in DNA methylation at the genome scale have been termed 'epigenetic drift', but the defining features of this phenomenon remain to be established. Human epidermis represents an excellent model for understanding age-related epigenetic changes because of its substantial cell-type homogeneity and its well-known age-related phenotype. We have now generated and analyzed the currently largest set of human epidermis methylomes (N = 108) using array-based profiling of 450 000 methylation marks in various age groups. Data analysis confirmed that age-related methylation differences are locally restricted and characterized by relatively small effect sizes. Nevertheless, methylation data could be used to predict the chronological age of sample donors with high accuracy. We also identified discontinuous methylation changes as a novel feature of the aging methylome. Finally, our analysis uncovered an age-related erosion of DNA methylation patterns that is characterized by a reduced dynamic range and increased heterogeneity of global methylation patterns. These changes in methylation variability were accompanied by a reduced connectivity of transcriptional networks. Our findings thus define the loss of epigenetic regulatory fidelity as a key feature of the aging epigenome.

Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells.

Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i.e., of GAPDH or PKM2). Through ultra-short (13)C labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.

UV radiation induces CXCL5 expression in human skin.

CXCL5 has recently been identified as a mediator of UVB-induced pain in rodents. To compare and to extend previous knowledge of cutaneous CXCL5 regulation, we performed a comprehensive study on the effects of UV radiation on CXCL5 regulation in human skin. Our results show a dose-dependent increase in CXCL5 protein in human skin after UV radiation. CXCL5 can be released by different cell types in the skin. We presumed that, in addition to immune cells, non-immune skin cells also contribute to UV-induced increase in CXCL5 protein. Analysis of monocultured dermal fibroblasts and keratinocytes revealed that only fibroblasts but not keratinocytes displayed up regulated CXCL5 levels after UV stimulation. Whereas UV treatment of human skin equivalents, induced epidermal CXCL5 mRNA and protein expression. Up regulation of epidermal CXCL5 was independent of keratinocyte differentiation and keratinocyte-keratinocyte interactions in epidermal layers. Our findings provide first evidence on the release of CXCL5 in UV-radiated human skin and the essential role of fibroblast-keratinocyte interaction in the regulation of epidermal CXCL5.

Glutathione-conjugated sulfanylalkanols are substrates for ABCC11 and γ-glutamyl transferase 1: a potential new pathway for the formation of odorant precursors in the apocrine sweat gland.

We have previously shown that precursors of odorous components characteristic of axillary sweat are hardly detectable or undetectable in individuals carrying the 538G > A SNP in the ABCC11 transporter gene. However, it is unclear, whether ABCC11 is directly involved in the transport of these compounds. To approach this question, transport of peptide-conjugated potential precursors of 3-methyl-3-sulfanylhexanol (3M3SH), a key determinant of axillary malodour, was measured using membrane vesicles of Sf9 insect cells overexpressing human ABCC11. Whilst no ABCC11-mediated transport was detected for the dipeptide precursor Cys-Gly-3M3SH, the glutathione conjugate of 3M3SH (SG-3M3SH) was robustly taken up by ABCC11 at a transport rate of 0.47 pmol/mg/min. Collectively, these results illuminate SG-3M3SH as a putative precursor of 3M3SH, which then may undergo intra-vesicular maturation to generate Cys-Gly-3M3SH. Critically, the apocrine sweat gland was demonstrated to express γ-glutamyl transferase 1 (GGT1) protein, which is known to catalyse the deglutamylation of glutathionyl conjugates. Additionally, we provide evidence that recombinant and isolated hepatic human GGT1 is capable of transforming SG-3M3SH to Cys-Gly-3M3SH in vitro. To sum up, we demonstrate that the functionality of ABCC11 is likely to play an important role in the generation of axillary malodour. Furthermore, we identify GGT1 as a key enzyme involved in the biosynthesis of Cys-Gly-3M3SH.

A functional ABCC11 allele is essential in the biochemical formation of human axillary odor.

The characteristic human axillary odor is formed by bacterial action on odor precursors that originate from apocrine sweat glands. Caucasians and Africans possess a strong axillary odor ,whereas many Asians have only a faint acidic odor. In this study, we provide evidence that the gene ABCC11 (MRP8), which encodes an apical efflux pump, is crucial for the formation of the characteristic axillary odor and that a single-nucleotide polymorphism (SNP) 538G --> A, which is prominent among Asian people, leads to a nearly complete loss of the typical odor components in axillary sweat. The secretion of amino-acid conjugates of human-specific odorants is abolished in homozygotic carriers of the SNP, and steroidal odorants and their putative precursors are significantly reduced. Moreover, we show that ABCC11 is expressed and localized in apocrine sweat glands. These data point to a key function of ABCC11 in the secretion of odorants and their precursors from apocrine sweat glands. SNP 538G --> A, which also determines human earwax type, is present on an extended haplotype, which has reached >95% frequency in certain populations in recent human evolution. A strong positive selection in mate choice for low-odorant partners with a dysfunctional ABCC11 gene seems a plausible explanation for this striking frequency of a loss-of-function allele.

Noninvasive measurement of cell volume changes by negative staining.

To maintain the intracellular concentration of ions and small molecules on osmotic challenges, nature has developed highly sophisticated transport systems for regulating water and ion content. An ideal measurement technique for volume changes of cells during osmotic challenges has to fulfil two requirements: it has to be osmotically inert, and it should allow online monitoring of cell volume changes. Here, a simple fluorescence microscopy-based approach is presented. Using fluorescein as a negative stain, it is possible to monitor cell volume changes without affecting the functionality of cell membranes and cell osmolarity. Measurement of Madine-Darby canine kidney (MDCK) cells after hypo- and hyperosmotic challenges reveals the main advantages of this approach: besides providing precise and reproducible quantitative data on reversible cell volume changes, the viability of the cells can be assessed directly by the appearance of stain in the cytoplasm. This becomes evident especially after hypo-osmotic challenge of glutaraldehyde-treated cells, which become leaky after fixation, followed by a massive volume change. This new approach represents a very sensitive measurement technique for cell volume changes resulting from water or ion flux, and thus seems to be an ideal tool for studying cell volume regulatory processes.