Identification and functional validation of SRC and RAPGEF1 as new direct targets of miR-203, involved in regulation of epidermal homeostasis.

The epidermis is mostly composed of keratinocytes and forms a protecting barrier against external aggressions and dehydration. Epidermal homeostasis is maintained by a fine-tuned balance between keratinocyte proliferation and differentiation. In the regulation of this process, the keratinocyte-specific miR-203 microRNA is of the outmost importance as it promotes differentiation, notably by directly targeting and down-regulating mRNA expression of genes involved in keratinocyte proliferation, such as ΔNp63, Skp2 and Msi2. We aimed at identifying new miR-203 targets involved in the regulation of keratinocyte proliferation/differentiation balance. To this end, a transcriptome analysis of human primary keratinocytes overexpressing miR-203 was performed and revealed that miR-203 overexpression inhibited functions like proliferation, mitosis and cell cycling, and activated differentiation, apoptosis and cell death. Among the down-regulated genes, 24 putative target mRNAs were identified and 8 of them were related to proliferation. We demonstrated that SRC and RAPGEF1 were direct targets of miR-203. Moreover, both were down-regulated during epidermal morphogenesis in a 3D reconstructed skin model, while miR-203 was up-regulated. Finally silencing experiments showed that SRC or RAPGEF1 contributed to keratinocyte proliferation and regulated their differentiation. Preliminary results suggest their involvement in skin carcinoma hyperproliferation. Altogether this data indicates that RAPGEF1 and SRC could be new mediators of miR-203 in epidermal homeostasis regulation.

A precise analysis of the relative contribution of UVA1 and visible light colour domains in solar light-induced skin pigmentation.

BACKGROUND: Solar light induces or aggravates hyperpigmentation issues. The contribution of UVA1, as well as visible light (VL), especially high-energy blue-violet visible (HEV) light, is now clearly established. OBJECTIVES: This work aimed at determining the relative contribution of UVA1, HEV and VL wavelength bands and their sub-domains in pigmentation induction. METHODS: Two clinical studies using solar simulators equipped with specific bandpass physical filters were carried out. Volunteers (FSPT III-IV) were exposed on the back to UVA1 + HEV (350-450 nm), UVA1 (350-400 nm), HEV (400-450 nm) or part of UVA1 + HEV (370-450 nm) in Study 1 (n = 27) and to VL (400-700 nm), HEV (400-450 nm), Blue (400-500 nm), Green (500-600 nm) and Green+Red (500-700 nm) domains in Study 2 (n = 25). Pigmentation level was assessed by visual scoring and colorimetry at different time points postexposure, up to Day 43. RESULTS: Induced pigmentation was detected in all exposed conditions, peaking at 2 h and thereafter progressively decreasing but remaining persistent up to Day 43. In Study 1, UVA1 showed an additive effect with HEV, with a significant contribution coming from the Longest UVA1 rays (370-400 nm). Study 2 demonstrated that 24 h postexposure, the Blue domain accounted for 71% of VL-induced pigmentation, the HEV one for 47%, the Green one for 37% and the Green+Red one for 36%, confirming no significant effect for Red light. CONCLUSIONS: Altogether, these results underline the need for UVA1 photoprotection up to 400 nm and highlight the importance of protecting the skin from solar VL wavelengths and especially from HEV, Blue and Green light, to limit induced pigmentation.

The Damaging Effects of Long UVA (UVA1) Rays: A Major Challenge to Preserve Skin Health and Integrity.

Within solar ultraviolet (UV) light, the longest UVA1 wavelengths, with significant and relatively constant levels all year round and large penetration properties, produce effects in all cutaneous layers. Their effects, mediated by numerous endogenous chromophores, primarily involve the generation of reactive oxygen species (ROS). The resulting oxidative stress is the major mode of action of UVA1, responsible for lipid peroxidation, protein carbonylation, DNA lesions and subsequent intracellular signaling cascades. These molecular changes lead to mutations, apoptosis, dermis remodeling, inflammatory reactions and abnormal immune responses. The altered biological functions contribute to clinical consequences such as hyperpigmentation, inflammation, photoimmunosuppression, sun allergies, photoaging and photocancers. Such harmful impacts have also been reported after the use of UVA1 phototherapy or tanning beds. Furthermore, other external aggressors, such as pollutants and visible light (Vis), were shown to induce independent, cumulative and synergistic effects with UVA1 rays. In this review, we synthetize the biological and clinical effects of UVA1 and the complementary effects of UVA1 with pollutants or Vis. The identified deleterious biological impact of UVA1 contributing to clinical consequences, combined with the predominance of UVA1 rays in solar UV radiation, constitute a solid rational for the need for a broad photoprotection, including UVA1 up to 400 nm.

Sunscreens with the New MCE Filter Cover the Whole UV Spectrum: Improved UVA1 Photoprotection In Vitro and in a Randomized Controlled Trial.

BACKGROUND: UVA1 rays (340-400 nm) contribute to carcinogenesis, immunosuppression, hyperpigmentation, and aging. Current sunscreen formulas lack sufficient absorption in the 370-400 nm wavelengths range. Recently, a new UVA1 filter, Methoxypropylamino Cyclohexenylidene Ethoxyethylcyanoacetate (MCE) exhibiting a peak of absorption at 385 nm, was approved by the Scientific Committee on Consumer Safety for use in sunscreen products. These studies evaluated, in a three-dimensional skin model and in vivo, the protection afforded by state-of-the-art sunscreen formulations enriched with MCE. TRIAL DESIGN: This study is a monocentric, double-blinded, randomized, and comparative trial. This study is registered at ClinicalTrials.gov with the identification number NCT04865094. METHODS: The efficacy of sunscreens with MCE was compared with that of reference formulas. In a three-dimensional skin model, histology, protein, and gene expression were analyzed. In the clinical trial, pigmentation was analyzed in 19 volunteers using colorimetric measurements and visual scoring. RESULTS: MCE addition in reference formulas enlarged the profile of absorption up to 400 nm; reduced UVA1-induced dermal and epidermal alterations at cellular, biochemical, and molecular levels; and decreased UVA1-induced pigmentation. CONCLUSIONS: Addition of MCE absorber in sunscreen formulations leads to full coverage of UV spectrum and improved UVA1 photoprotection. The data support benefits in the long term on sun-induced consequences, especially those related to public health care issues.

Photo-aging evaluation - In vitro biological endpoints combined with collagen density assessment with multi-photon microscopy.

BACKGROUND: Ultraviolet exposure has profound effect on the dermal connective tissue of human skin. OBJECTIVE: We aimed to develop and validate an evaluation method/methodology using a full-thickness reconstructed skin model, to assess the anti-photoaging efficacy of cosmetic ingredients and sunscreen formulas by blending multi relevant biological endpoints including the newly developed dermal collagen quantification method with Multi-photon microscopy. METHODS: The response of ex vivo human skin to UVA exposure was first characterized with multiphoton microscopy. Reconstructed full-thickness skin models was then used to reproduce the data and to create a proof-of-concept study by treating the models with sunscreen prototypes A or B, which differ on their UVA absorption properties, and systemic Vitamin C (Vit C). After exposure to UVA, the collagen density was quantified via multiphoton microscopy with automatic imaging processing. Histology, fibroblasts number, metalloprotease 1 (MMP1) secretion were also assessed. RESULTS: UVA exposure induced pronounced reduction in collagen density and increased MMP1 secretion within both ex vivo human skin and reconstructed skin. Histological damage and fibroblast disappearance was observed with reconstructed skin. Within the proof-of-concept study prototype B, possessing higher UVA filtration, gave better protection than prototype A on the UV associated biological markers, and association with Vit C boosted sunscreen formula efficacy. CONCLUSIONS: The photoaging evaluation method, consists of multi biological markers as well as dermal collagen quantification, is a relevant mean to assess the pre-clinical efficacy of anti-photoaging ingredients and sunscreen products. This approach is also beneficial for evaluating the efficacy of sunscreens and photoprotective ingredients.

Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA-induced metabolic stress in reconstructed human skin.

Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.

In Vitro Skin Models for the Evaluation of Sunscreen-Based Skin Photoprotection: Molecular Methodologies and Opportunities.

Identifying and understanding the biological events that occur following ultraviolet (UV) exposure are mandatory to elucidate the biological and clinical consequences of sun exposure, and to provide efficient and adequate photoprotection strategies. The main UVinduced biological features (markers related to sunburn, cancer, photoaging immunosuppression, pigmentation), characterized in human skin in vivo, could be reproduced in adapted models of reconstructed skin in vitro, attesting their high relevance in the field of photobiology. In turn, 3D skin models were useful to discover precise biological pathways involved in UV response and were predictive of in vivo situation. Although they did not follow a strict validation process for the determination of protection factors, they enabled to evidence important concepts in photoprotection. Indeed, the use of reconstructed skin model highlighted the importance of broad spectrum sunscreen use to protect essential cellular functions, and biologically proved that SPF value was not predictive of the level of protection in the UVA wavelength domain. New biological approaches, such as transcriptomic or proteomic studies as well as quantitative and qualitative determination of DNA damage, will indisputably increase the added value of such systems for sunscreen efficiency evaluation.

Identification of chronological and photoageing-associated microRNAs in human skin.

MicroRNAs are short non-coding RNAs that play key roles in regulating biological processes. In this study, we explored effects of chronological and photoageing on the miRNome of human skin. To this end, biopsies were collected from sun-exposed (outer arm, n = 45) and sun-protected (inner arm, n = 45) skin from fair-skinned (phototype II/III) healthy female volunteers of three age groups: young, 18-25 years, middle age, 40-50 years and aged, > 70 years. Strict inclusion criteria were used for photoageing scoring and for chronological ageing. Microarray analysis revealed that chronological ageing had minor effect on the human skin miRNome. In contrast, photoageing had a robust impact on miRNAs, and a set of miRNAs differentially expressed between sun-protected and sun-exposed skin of the young and aged groups was identified. Upregulation of miR-383, miR-145 and miR-34a and downregulation of miR-6879, miR-3648 and miR-663b were confirmed using qRT-PCR in sun-exposed skin compared with sun-protected skin. qRT-PCR analysis revealed that miR-383, miR-34a and miR-134 were differentially expressed in all three age groups both in chronological and photoageing, suggesting a synergetic effect of intrinsic and extrinsic ageing on their expression. In conclusion, our study identifies a unique miRNA signature which may contribute to skin ageing.

Analysis of gene expression dynamics revealed delayed and abnormal epidermal repair process in aged compared to young skin.

With aging, epidermal homeostasis and barrier function are disrupted. In a previous study, we analyzed the transcriptomic response of young skin epidermis after stratum corneum removal, and obtained a global kinetic view of the molecular processes involved in barrier function recovery. In the present study, the same analysis was performed in aged skin in order to better understand the defects which occur with aging. Thirty healthy male volunteers (67 ± 4 years old) were involved. Tape-strippings were carried out on the inner face of one forearm, the other unstripped forearm serving as control. At 2, 6, 18, 30 and 72 h after stripping, TEWL measurements were taken, and epidermis samples were collected. Total RNA was extracted and analyzed using DermArray(®) cDNA microarrays. The results highlighted that barrier function recovery and overall kinetics of gene expression were delayed following stripping in aged skin. Indeed, the TEWL measurements showed that barrier recovery in the young group appeared to be dramatically significant during the overall kinetics, while there were no significant evolution in the aged group until 30 h. Moreover, gene expression analysis revealed that the number of modulated genes following tape stripping increased as a function of time and reached a peak at 6 h after tape stripping in young skin, while it was at 30 h in aged skin, showing that cellular activity linked to the repair process may be engaged earlier in young epidermis than in aged epidermis. A total of 370 genes were modulated in the young group. In the aged group, 382 genes were modulated, whose 184 were also modulated in the young group. Only eight genes that were modulated in both groups were significantly differently modulated. The characterization of these genes into 15 functional families helped to draw a scenario for the aging process affecting epidermal repair capacity.

Exposure to non-extreme solar UV daylight: spectral characterization, effects on skin and photoprotection.

The link between chronic sun exposure of human skin and harmful clinical consequences such as photo-aging and skin cancers is now indisputable. These effects are mostly due to ultraviolet (UV) rays (UVA, 320-400 nm and UVB, 280-320 nm). The UVA/UVB ratio can vary with latitude, season, hour, meteorology and ozone layer, leading to different exposure conditions. Zenithal sun exposure (for example on a beach around noon under a clear sky) can rapidly induce visible and well-characterized clinical consequences such as sunburn, predominantly induced by UVB. However, a limited part of the global population is exposed daily to such intense irradiance and until recently little attention has been paid to solar exposure that does not induce any short term clinical impact. This paper will review different studies on non-extreme daily UV exposures with: (1) the characterization and the definition of the standard UV daylight and its simulation in the laboratory; (2) description of the biological and clinical effects of such UV exposure in an in vitro reconstructed human skin model and in human skin in vivo, emphasizing the contribution of UVA rays and (3) analysis of photoprotection approaches dedicated to prevent the harmful impact of such UV exposure.

New insights in photoaging, UVA induced damage and skin types.

UVA radiation is the most prevalent component of solar UV radiation; it deeply penetrates into the skin and induces profound alterations of the dermal connective tissue. In recent years, the detrimental effects of UVA radiation were more precisely demonstrated at cellular and molecular levels, using adequate methods to identify biological targets of UVA radiation and the resulting cascade impairment of cell functions and tissue degradation. In particular gene expression studies recently revealed that UVA radiation induces modulation of several genes confirming the high sensitivity of dermal fibroblasts to UVA radiation. The major visible damaging effects of UVA radiation only appear after years of exposure: it has been clearly evidenced that they are responsible for more or less early signs of photoageing and photocarcinogenesis. UVA radiation appears to play a key role in pigmented changes occurring with age, the major sign of skin photoaging in Asians. Skin susceptibility to photoaging alterations also depends on constitutive pigmentation. The skin sensitivity to UV light has been demonstrated to be linked to skin color type.

Diversity of biological effects induced by longwave UVA rays (UVA1) in reconstructed skin.

Despite their preponderance amongst the ultraviolet (UV) range received on Earth, the biological impacts of longwave UVA1 rays (340-400 nm) upon human skin have not been investigated so thoroughly. Nevertheless, recent studies have proven their harmful effects and involvement in carcinogenesis and immunosuppression. In this work, an in vitro reconstructed human skin model was used for exploring the effects of UVA1 at molecular, cellular and tissue levels. A biological impact of UVA1 throughout the whole reconstructed skin structure could be evidenced, from morphology to gene expression analysis. UVA1 induced immediate injuries such as generation of reactive oxygen species and thymine dimers DNA damage, accumulating preferentially in dermal fibroblasts and basal keratinocytes, followed by significant cellular alterations, such as fibroblast apoptosis and lipid peroxidation. The full genome transcriptomic study showed a clear UVA1 molecular signature with the modulation of expression of 461 and 480 genes in epidermal keratinocytes and dermal fibroblasts, respectively (fold change> = 1.5 and adjusted p value<0.001). Functional enrichment analysis using GO, KEGG pathways and bibliographic analysis revealed a real stress with up-regulation of genes encoding heat shock proteins or involved in oxidative stress response. UVA1 also affected a wide panel of pathways and functions including cancer, proliferation, apoptosis and development, extracellular matrix and metabolism of lipids and glucose. Strikingly, one quarter of modulated genes was related to innate immunity: genes involved in inflammation were strongly up-regulated while genes involved in antiviral defense were severely down-regulated. These transcriptomic data were confirmed in dose-response and time course experiments using quantitative PCR and protein quantification. Links between the evidenced UVA1-induced impacts and clinical consequences of UVA1 exposure such as photo-aging, photo-immunosuppression and cancer are discussed. These early molecular events support the contribution of UVA1 to long term harmful consequences of UV exposure and underline the need of an adequate UVA1 photoprotection.

Modulations of gene expression induced by daily ultraviolet light can be prevented by a broad spectrum sunscreen.

Realistic non-zenithal solar ultraviolet (UV) exposure, obtained using standard ultraviolet daylight spectrum (DUVR), has deleterious impact on epidermal and dermal compartments of human skin. The present study was designed to assess gene expression in human reconstructed skin following exposure to DUVR and the protective effect of a broad spectrum sunscreen. Reconstructed skins were exposed to a realistic daily UV dose of 12 J/cm(2) DUVR in the presence of a sunscreen product (Sun(burn) Protection Factor (SPF)=13 and UVA protection factor UVAPF (PPD) 10.5) or its vehicle. Six hours post exposure, gene expression was investigated in fibroblasts (225 genes) and keratinocytes (244 genes) separately using quantitative PCR arrays. DUVR exposure led to significant modulation of 35 and 66 genes in fibroblasts and keratinocytes, respectively. These genes were involved in extracellular matrix homeostasis, oxidative stress response, cell growth, inflammation and epidermal differentiation. Sunscreen use significantly reduced DUVR-induced gene modulation. Hierarchical clustering showed that gene expression profiles in protected and DUVR-exposed samples were very close to those of unexposed samples. The number of DUVR-modulated genes was significantly decreased by tested sunscreen (zero and four modulated genes in fibroblasts and keratinocytes, respectively). Our results demonstrate that a broad-spectrum sunscreen product is highly effective in protecting reconstructed human skin against DUVR-induced changes in gene expression.

Solar ultraviolet radiation induces biological alterations in human skin in vitro: relevance of a well-balanced UVA/UVB protection.

Cutaneous damages such as sunburn, pigmentation, and photoaging are known to be induced by acute as well as repetitive sun exposure. Not only for basic research, but also for the design of the most efficient photoprotection, it is crucial to understand and identify the early biological events occurring after ultraviolet (UV) exposure. Reconstructed human skin models provide excellent and reliable in vitro tools to study the UV-induced alterations of the different skin cell types, keratinocytes, fibroblasts, and melanocytes in a dose- and time-dependent manner. Using different in vitro human skin models, the effects of UV light (UVB and UVA) were investigated. UVB-induced damages are essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA radiation-induced damages are mostly located within the dermal compartment. Pigmentation can also be obtained after solar simulated radiation exposure of pigmented reconstructed skin model. Those models are also highly adequate to assess the potential of sunscreens to protect the skin from UV-associated damage, sunburn reaction, photoaging, and pigmentation. The results showed that an effective photoprotection is provided by broad-spectrum sunscreens with a potent absorption in both UVB and UVA ranges.

A broad-spectrum sunscreen prevents UVA radiation-induced gene expression in reconstructed skin in vitro and in human skin in vivo.

The efficacy of sunscreens to protect against ultraviolet (UV) A radiation is usually assessed by measuring erythema formation and pigmentation. The biological relevance of these endpoints for UVA-induced skin damage, however, is not known. We therefore carried out two complementary studies to determine UVA protection provided by a broad-spectrum sunscreen product at a molecular level by studying UVA radiation-induced gene expression. One study was performed on human reconstructed skin in vitro with a semi-global gene expression analysis of 227 genes in fibroblasts and 244 in keratinocytes. The second one was conducted in vivo in human volunteers and focused on genes involved in oxidative stress response and photo-ageing (haeme oxygenase-1, superoxide dismutase-2, glutathione peroxidase, catalase, matrix metalloproteinase-1). In-vitro UVA radiation induced modulation of genes involved in extracellular matrix homeostasis, oxidative stress, heat shock responses, cell growth, inflammation and epidermal differentiation. Sunscreen pre-application abrogated or significantly reduced these effects, as underlined by unsupervised clustering analysis. The in vivo study confirmed that the sunscreen prevented UVA radiation-induced transcriptional expression of the five studied genes. These findings indicate the high efficacy of a broad-spectrum sunscreen in protecting human skin against UVA-induced gene responses and suggest that this approach is a biologically relevant complement to existing methods.

Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation.

Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as "daily UV radiation" (DUVR) with a higher UVA (320-400 nm) to UVB (280-320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure.

Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response.

The stratum corneum (SC) is a superficial skin compartment that protects the body from the outside environment. Any disturbance of this function induces cascading steps of molecular and cellular repair in the whole epidermis. The aim of this study was to investigate epidermal gene expression following SC removal by tape stripping. Twenty-nine healthy male volunteers were included (27 +/- 4 years old). Tape stripping was processed on one inner forearm, the other unstripped forearm served as a control. Epidermis samples were collected at 2, 6, 19, 30 and 72 h after tape stripping. Trans-epidermal water loss measurements were performed at each step to monitor barrier restoration. Total RNA was extracted from collected epidermis samples and analysed by using DermArray cDNA microarrays. Among 4000 genes under investigation, we found that the expression of 370 genes varied significantly at least once during the time following stripping. Using an original clustering method, the modulated genes were gathered into eight groups. A functional characterization of the clusters enabled us to get a dynamic and global view of the main molecular processes taking place during epidermal recovery.