Study of cell responses after lipid raft disruption by cholesterol depletion in epidermal keratinocytes

The skin assumes a fascinating protection role by producing a barrier between the environment and the organism. This barrier, formed by the differentiation of epidermal keratinocytes, is vital for the organism as the skin is constantly exposed to external aggressions. In this study, we investigated the relevance of membrane cholesterol and lipid rafts in normal epidermal keratinocytes by extracting cholesterol with methyl-beta-cyclodextrin. Actually, the scientific interest in plasma membranes has strongly increased since the discovery that cholesterol-enriched membrane microdomains domains, lipid rafts, are directly involved in cell signaling. Our study identifies the heparin-binding EGF-like growth factor (HB-EGF) as a major actor after lipid raft disruption, HB-EGF is also shown to be expressed in wound-healing skin after burn injury. Parallelisms in the keratinocyte’s response to lipid raft disruption or to oxidative stress incited the investigation potential oxidative stress-mediated effects in cholesterol-depleted cells, but our results clearly demonstrate that lipid raft disruption does not initiate oxidative stress mechanisms. An unbiased microarray approach was then chosen to determine major actors, functions and pathways. HB-EGF, interleukin-8, urokinase-like plasminogen activator and its receptor are identified among the major transcriptional targets in lipid raft-disrupted keratinocytes. Moreover, bioinformatics analysis of differentially regulated genes permitted to establish a potential link with atopic dermatitis, an inflammatory skin disease. This hypothesis was investigated, and confirmed, in skin biopsies of atopic dermatitis patients. Thus, our study contributes to a better understanding of lipid raft biology and suggests that the functioning of keratinocyte lipid rafts could be compromised in atopic dermatitis.La peau assure un rôle de protection important en élaborant une barrière entre l’organisme et l’environnement. Cette barrière est essentiellement formée par la différentiation progressive de kératinocytes épidermiques. La barrière épidermique joue un rôle capital pour l’organisme puisque la peau est constamment exposée aux agressions externes. Dans cette étude, nous avons analysé l’importance du cholestérol membranaire et des lipid rafts dans des kératinocytes épidermiques normaux en déplétant le cholestérol à l’aide de la molécule methyl-beta-cyclodextrin. Depuis la découverte de l’implication directe de lipid rafts dans la signalisation cellulaire, l’intérêt scientitfique pour ces microdomaines membranaires enrichis en cholestérol a fortement augmenté. Notre étude identifie le facteur de croissance heparin-binding EGF-like growth factor (HB-EGF) comme un des acteurs majeurs suite à une perturbation des lipid rafts via extraction de cholestérol. Nous montrons également que HB-EGF est exprimé dans la peau en cicatrisation après blessure par brûlure cutanée. Les parallélismes dans la réponse des kératinocytes face à la perturbation des lipid rafts ou face au stress oxydatif nous ont incités à investiguer si des éventuels méchanismes de stress oxydatif sont impliqués dans les effets induits par une déplétion en cholestérol. Cependant, nos résultats démontrent clairement que la perturbation des lipid rafts n’induit pas de mécanismes de stress oxydatif. Par la suite, une approche transcriptomique sans a priori a été choisie pour déterminer les acteurs majeurs, fonctions et voies de signalisation. HB-EGF, interleukin-8, urokinase-like plasminogen activator et son récepteur sont identifiés parmi les cibles transcriptionnelles majeures dans les kératinocytes déplétés en cholestérol. De plus, des analyses bioinformatiques de gènes différentiellement régulés ont permis d’établir un lien potentiel avec la dermatite atopique, une maladie inflammatoire de la peau. Cette hypothèse a été investiguée, et confirmée, dans des biopsies de peau de patients atteints de dermatite atopique. Nos données contribuent donc à une meilleure compréhension de la biologie cutanée des lipid rafts et suggèrent que la fonctionnalité des lipid rafts de kératinocytes situés dans les lésions de dermatite atopique pourrait être compromise.(DOCMED02) -- FUNDP, 201

Method validation for preparing urine samples for downstream proteomic and metabolomic applications.

BACKGROUND: Formal validation of methods for biospecimen processing in the context of accreditation in laboratories and biobanks is lacking. A protocol for processing of a biospecimen (urine) was validated for fitness-for-purpose in terms of key downstream endpoints. METHODS: Urine processing was optimized for centrifugation conditions on the basis of microparticle counts at room temperature (RT) and at 4 degrees C. The optimal protocol was validated for performance (microparticle counts), and for reproducibility and robustness for centrifugation temperature (4 degrees C vs. RT) and brake speed (soft, medium, hard). Acceptance criteria were based on microparticle counts, cystatin C and creatinine concentrations, and the metabolomic profile. RESULTS: The optimal protocol was a 20-min, 12,000 g centrifugation at 4 degrees C, and was validated for urine collection in terms of microparticle counts. All reproducibility acceptance criteria were met. The protocol was robust for centrifugation at 4 degrees C versus RT for all parameters. The protocol was considered robust overall in terms of brake speeds, although a hard brake gave significantly fewer microparticles than a soft brake. CONCLUSIONS: We validated a urine processing method suitable for downstream proteomic and metabolomic applications. Temperature and brake speed can influence analytic results, with 4 degrees C and high brake speed considered optimal. Laboratories and biobanks should ensure these conditions are systematically recorded in the scope of accreditation

Heparin-binding EGF-like growth factor is induced by disruption of lipid rafts and oxidative stress in keratinocytes and participates in the epidermal response to cutaneous wounds

Epidermal homeostasis and repair of the skin barrier require that epidermal keratinocytes respond to alterations of their environment. We report that cellular stress with methyl-β-cyclodextrin (MBCD), a molecule that extracts membrane cholesterol and thereby disrupts the structure of lipid rafts, strongly induces the synthesis of heparin-binding EGF-like growth factor (HB-EGF) in keratinocytes through the activation of p38 mitogen-activated protein kinase. Interesting parallels between lipid raft disruption and oxidative stress can be drawn as hydrogen peroxide induces p38 activation and HB-EGF synthesis in keratinocytes. Consistent with other studies, we show increased HB-EGF expression in keratinocytes located at the margin of wounded skin areas. Analyzing cultured keratinocytes exposed to rhHB-EGF, we report increased HB-EGF mRNA levels and alterations in the expression of differentiation markers. Interestingly, identical alterations in differentiation markers are shown to occur in vivo at the wound margin and in HB-EGF-treated cultures. In addition, in vitro sectioning of skin samples also induces the expression of HB-EGF at the border of the incisions. Altogether, our data suggest that expression of HB-EGF is a marker of the keratinocyte's response to a challenging environment and demonstrate that this growth factor alters the phenotype of keratinocytes in a manner similar to that observed during epidermal repair