Les microARNs sont de petits ARN non codants régulant négativement l'expression génique au niveau post-transcriptionnel. Ils interviennent dans de nombreux processus biologiques et leur rôle dans la régulation de l'homéostasie cutanée est clairement démontrée. Cependant, leur fonction durant le vieillissement épidermique n'a jamais été étudiée. Nous avons donc réalisé une analyse exhaustive du miRnome épidermique durant son vieillissement afin d'identifier les microARNs différentiellement exprimés avec l'âge dans ce tissu. Plusieurs microARNs significativement modulés dans des kératinocytes âgés, nous ont permis d'établir une signature du vieillissement épidermique. Parmi eux, les deux brins du microARN miR-30a sont induits dans les épidermes âgés. La construction d'un lentivirus permettant la surexpression stable et inductible de ce microARN a facilité son étude fonctionnelle dans un modèle organotypique d'épiderme reconstruit. Nous avons observé que la surexpression de ce microARN dans un modèle de culture tridimensionnelle induit un phénotype épidermique présentant des similitudes avec celui observé durant son vieillissement chronologique et caractérisé par une forte altération de la différenciation des kératinocytes, par une perturbation de sa fonction barrière et par une augmentation de l'abondance des cellules apoptotiques. Ce projet de thèse a permis l'identification de 3 cibles directes de miR-30a dans les kératinocytes. Il s'agit de LOX, codant pour la lysyl oxydase qui intervient dans la balance prolifération/différenciation des kératinocytes, d'AVEN, un inhibiteur de caspase et d'IDH1, codant pour l'isocitrate déshydrogénase, enzyme du métabolisme énergétique. Ainsi, ce projet de thèse a révélé un nouveau microARN acteur du vieillissement épidermique et a permis de de mettre à jour de nouveaux mécanismes moléculaires expliquant certaines altérations phénotypiques observées dans l'épiderme avec l'âge / MicroRNAs are small non-coding RNA that negatively regulate gene expression at the post-transcriptional level. There are involved in many biological processes and play a key role in the regulation of skin homeostasis. However, their function during epidermal aging has never been studied. We performed an exhaustive analysis of the epidermal miRnome during its aging in order to identify microRNAs differentially expressed with age in this tissue. Several microRNAs significantly modulated in elderly keratinocytes, allowed us to establish a signature of epidermal aging. Among them, the two strands of the microRNA miR-30a are induced in aged epidermis. The construction of a lentivirus allowing inducible and stable overexpression of this microRNA facilitated its functional study in an organotypic model of reconstructed epidermis. We observed that the overexpression of this microRNA in a three-dimensional culture model induces an epidermal phenotype similar of those observed during its chronological aging characterized by a strong alteration of keratinocyte differentiation, by a disturbance of its barrier function and by an increase in the abundance of apoptotic cells. This thesis project allowed the identification of three miR-30a targets in keratinocytes : LOX encoding lysyl oxidase, which plays a role in proliferation/differentiation balance of keratinocytes, AVEN encoding a caspase inhibitor and IDH1 encoding isocitrate deshydrogenase, a key enzyme of cellular metabolism.Our work revealed a new miRNA actor and deciphered new molecular mechanisms to explain some alterations observed in epidermis during aging, especially those concerning keratinocytes differentiation and apoptotic death

Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. Reepithelialisation and epidermal wound coverage counteract excessive scar formation. We have previously shown that interleukin-1α (IL-1α) derived from keratinocytes conteracts TGF-β-stimulated CTGF-expression. The aim of this thesis was to further explore the effects of keratinocytes and IL-1α on gene and protein expression, as well as pathways, in TGF-β stimulated fibroblasts. Fibroblasts were studied in vitro by conventional two dimensional cell culture models and in a three dimensional keratinocyte-fibroblast organotypic skin culture model. The results showed that IL-1 suppresses basal and TGF-β-induced CTGF mRNA and protein, involving a possible TAK1 mechanism. Keratinocytes regulate the expression of fibroblast genes important for the turnover of the extracellular matrix. Most of the genes analysed (11/13) were regulated by TGF-β and counter regulated by keratinocytes. The overall results support a view that keratinocytes regulate fibroblasts to act catabolically (anti-fibrotic) on the extracellular matrix. Transcriptional microarray and gene set enrichment analysis showed that antagonizing effects of IL-1α on TGF-β were much more prominent than the synergistic effects. The most confident of these pathways was the interferon signaling, which were inhibited by TGF-β and activated by IL-1α. A proteomics study confirmed that IL-1α preferentially conteracts TGF-β effects. Six new fibroblast proteins involved in synthesis/ regulation were identified, being regulated by TGF-β and antagonized by IL-1α. Pathway analysis confirmed counter-regulation of interferon signaling by the two cytokines. These findings have implications for understanding the role of fibroblasts for inflammatory responses and development of fibrosis in the skin.

Lipid synthesis is required for cell growth and is subject to pharmacologic regulation. Keratinocyte growth factor (KGF) stimulates proliferation and lipogenesis in H292 cells, a pulmonary epithelial cancer cell line, but the signaling pathways are not known. KGF stimulated the expression of the transcription factors sterol-regulatory element binding protein-1 (SREBP-1), CCAAT/enhancer binding protein α (C/EBPα), and C/EBPδ and two key enzymes involved in lipogenesis, FAS and stearoyl coenzyme A desaturase-1 (SCD-1). We found that KGF induced rapid activation of Akt, p70 S6K, JNK, and extracellular signal-regulated (ERK). Induction of SREBP-1, SCD-1, and FAS by KGF was inhibited by the JNK inhibitor SP600125 and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the ERK inhibitor PD98059. Using FAS and SCD-1-luciferase promoter constructs, we observed that KGF stimulated the transcription of these promoters and that exogenous cholesterol inhibited the induction. Mutation of the SREBP-1 binding site in the SCD-1 promoter abolished the effect of KGF on SCD-1 transcription. In addition, overexpression of active SREBP-1 directly stimulated SCD-1 and FAS. Conversely, adenovirus-mediated overexpression of a dominant negative form of SREBP-1 inhibited the KGF effect on FAS and SCD-1 expression. In summary, we conclude that KGF requires both PI3K and JNK signaling pathways to induce SREBP-1, which in turn induces SCD-1 and FAS expression in H292 cells.

TGF-β isoforms are important signalling molecules in wound repair in the skin. Transforming growth factor β3 (TGF-β3) has been implicated in scarless healing. In both animal and human models the application of exogenous TGF-β3 causes a reduction in the inflammatory response and improves the architecture of the neodermis. Research into the influence of TGF-β on scarring has tended to focus on fibroblasts. However, keratinocytes play a major role in scarring both indirectly, as a result of their influence over the behaviour of fibroblasts and also by directly influencing wound contraction. Thus, experiments were carried out to investigate the influence of TGF-β3 on the behaviours of a keratinocyte cell line (HaCaT). Incubation with TGF-β3 increased cell spreading and appeared to reduce cell-surface contacts indicated by both SPR imaging and a detachment assay. TGF-β3 also caused a decreased cell alignment response to microcontact printed protein patterns, in part due to the deposition of laminin which is associated with the TGF-β induced cell migration. There is evidence that TGF-β isoforms differentially influence the outcome of wound healing. Similar to the results produce following addition of exogenous TGF-β3, the neutralisation of TGF-β1 and 2 has been shown to reduce scar formation in the adult wounds. During reepithelialisation keratinocytes experience a dynamic environment. Both extracellular matrix proteins and growth factors influence the progression of wound repair which includes both cell migration and proliferation. Few studies have examined collective cell behaviour in response to TGF-β isoforms and ECM coated substrates. Thus both wound closure and cell proliferation assays were conducted for different ECM proteins fibronectin, laminin and collagen type I and for TGF-β1, 2 and 3. Rates of wound closure were significantly reduced on laminin coated substrates while cell proliferation rates were increased. TGF-β2 and 3 induced significant increases in wound closure rates. This appeared to correspond with an increase in the number of cells independently migrating out from the wound margins. Only TGF-β3 caused a significant decrease in cell proliferation over a 4 day period. Laminin332 deposition is central to the reepithelialisation process and is known to be induced in response to TGF-β. Thus experiments were carried out to investigate HaCaT cell laminin332 deposition in response to TGF-β1, 2 and 3. Both an immunofluorescence staining technique and an ELISA based semi-quantification method was used. Following 4 day incubation all TGF-β isoforms significantly increased laminin332 deposition; however TGF-β2 and 3 caused the most significant increases. Integrin receptors enable cell-matrix interactions during wound repair. TGF-β is known to influence the expression of integrin subunits. Thus, experiments were carried out to compare the influence of each TGF-β isoform on the expression of subunits α3, α2, α5, β1 and β4. All TGF-β isoforms significantly increased all subunit expression. TGF-β3 caused the most significant increase in β4 and both TGF-β2 and 3 caused the most significant increase in α2. While there were differences in cell responses to each isoforms, TGF-β3 did not stand out from the other two isoforms. Interestingly, TGF-β2 shared more similarities with TGF-β3 than it did with TGF-β1, in its role in enhancing wound closure and LN332 deposition. These comparative studies have shown that differences exist in the way TGF-β isoforms influence HaCaT cell behaviour, namely migration, laminin deposition and integrin expression. / EPSRC and DTA grant

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No / This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Arsenic is a universal environmental toxicant associated mostly with skin related diseases in people exposed to low doses over a long term. Low dose arsenic trioxide (ATO) with long exposure will lead to chronic exposure. Experiments were performed to provide new knowledge on the incompletely understood mechanisms of action of chronic low dose inorganic arsenic in keratinocytes. Cytotoxicity patterns of ATO on long-term cultures of HaCaT cells on collagen IV was studied over a time course of 14 days. DNA damage was also assessed. The percentages of viable cells after exposure were measured on Day 2, Day 5, Day 8, and Day 14. Statistical and visual analytics approaches were used for data analysis. In the result, a biphasic toxicity response was observed at a 5 μg/ml dose with cell viability peaking on Day 8 in both chronic and acute exposures. Furthermore, a low dose of 1 μg/ml ATO enhanced HaCaT keratinocyte proliferation but also caused DNA damage. Global gene expression study using microarray technique demonstrated differential expressions of genes in HaCaT cell exposed to 0.5 μg/ml dose of ATO up to 22 passages. Four of the up-regulated and 1 down-regulated genes were selected and confirmed with qRT-PCR technique. These include; Aldo-Keto Reductase family 1, member C3 (AKR1C3), Insulin Growth Factor-Like family member 1 (IGFL1), Interleukin 1 Receptor, type 2 (IL1R2) and Tumour Necrosis Factor [ligand] Super-Family, member 18 (TNFSF18), and down-regulated Regulator of G-protein Signalling 2 (RGS2). The decline in growth inhibiting gene (RGS2) and increase in AKR1C3 may be the contributory path to chronic inflammation leading to metaplasia. This pathway is proposed to be a mechanism leading to carcinogenesis in skin keratinocytes. The observed over expression of IGFL1 may be a means of triggering carcinogenesis in HaCaT keratinocytes. In conclusion, it was established that at very low doses, arsenic is genotoxic and induces aberrations in gene expression though it may appear to enhance cell proliferation. The expression of two genes encoding membrane proteins IL1R2 and TNFSF18 may serve as possible biomarkers of skin keratinocytes intoxication due to arsenic exposure. This research provides insights into previously unknown gene markers that may explain the mechanisms of arsenic-induced dermal disorders including skin cancer / Environmental Sciences / D. Phil. (Environmental science)

La peau et son vieillissement sont un enjeu de santé publique. Les modèles expérimentaux disponibles pour l'étude du vieillissement cutané restent perfectibles. Dans ce contexte, nos objectifs étaient simultanément d'utiliser les modèles de peaux reconstruites (PR) développés dans notre laboratoire afin i) de mieux comprendre les mécanismes du vieillissement cutané, ii) de démontrer l'efficacité et le mécanisme d'action du sélénium comme « anti âge » et enfin iii) de les faire évoluer en utilisant le support poreux ou auto-assemblé avec des fibroblastes du même donneur prélevés à des âges différents. Ainsi, le modèle de PR cultivé sur une longue période a montré une surexpression du microARN miR30-a par RT qPCR dans les PR « âgées » avec une altération de la fonction barrière mesurée par la perte insensible en eau et une perturbation de la différenciation terminale (baisse d'expression de la loricrine et de l'involucrine). Avec le même modèle in vitro, nos résultats démontrent que la supplementation en sélénium retarde la sénescence des kératinocytes souches. Cette efficacité passe non pas par un effet antioxydant comme attendu mais par l'activation de leur adhésion à la lame basale, qui participe à les conserver souche et donc à préserver le renouvellement épidermique. Enfin, nous avons eu la chance exceptionnelle de préparer des PR avec des fibroblastes provenant d'un donneur unique prélevé à 36 et 72 ans. Les résultats immunohistologiques montrent que l'âge induit une augmentation de l'expression de l'élastine et de la fibrilline ainsi que leur co-expression. L'augmentation de LTBP1 et aSMA suggère que cette augmentation inattendue est due à une dérégulation de la voie TGF-ß et une différenciation des fibroblastes en myofibroblastes. En conclusion l'utilisation de différents modèles de PR a permis d'explorer les mécanismes conduisant au vieillissement cutané et de démontrer l'efficacité du sélénium comme anti âge / Skin and its aging is a public health issue. In vitro skin models available for the study aging remain perfectible. In this context, our objectives were simultaneously to use skin equivalent (SE) developed in our laboratory i) to better understand mechanisms of skin aging, ii) to demonstrate the effectiveness of selenium as “anti-aging” and finally iii) to improve SE using the porous or scaffold free model with fibroblasts from the same donor at different ages. Thus, the model of SE mimicking senescence showed an overexpression of microRNA miR30-a by RT qPCR in old SE with an alteration of the barrier function measured by the transepidermal water loss and a deficiency of epidermal terminal differentiation (decreased expression of loricrin and involucrin). With the same SE model, our results demonstrate that selenium supplementation delays the senescence of keratinocytes stem cells. This effectiveness does not involve antioxidant effect as expected but the activation of their adhesion to the basement membrane, which participates in preserving stemness and epidermal renewal. Finally, we had the opportunity to prepare SE with fibroblasts from a single donor at 36 and 72 years old. The histological results show that age induces an increase in the expression of elastin and fibrillin as well as their co-expression. The increase of LTBP1 and aSMA suggests that this unexpected increase is due to deregulation of the TGF-ß pathway and fibroblasts differentiation into myofibroblasts. In conclusion, the use of different models of SE helps us to explore some mechanisms leading to skin aging and to demonstrate the efficacy of selenium as “anti-aging”