Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

Microarray profiling of gene expression in human keratinocytes suggests a new protective activity against UV-induced DNA damage for a compound previously known to interact with SCF-KIT signalling pathway.

The stem cell factor (SCF) and its protein-tyrosine kinase receptor KIT are together implicated in the regulation of diverse biological processes and particularly in melanogenesis. Indeed, this signalling pathway controls melanoblast migration from the neural crest during embryogenesis and allows the communication between keratinocytes and melanocytes in the adult. In melanocytes, the binding of SCF to its transmembrane receptor leads to the activation of signalling pathways implicating protein kinases which finally control the expression of pigmentation-related genes. We have developed a biological compound called IV09.007, which we previously described as a modulator of the SCF/KIT signalling pathway with a pro-pigmenting effect. In the present work, we have studied the expression and localization of both SCF and KIT mRNAs and proteins in the skin or skin-derived cell lines. Then, we explored with a microarray approach the ability of IV09.007 to modulate the expression of genes in human keratinocytes and melanocytes in culture. Thereby, we observed the regulation of genes implicated in DNA repair, mainly related to base/nucleotides excision pathways. A modulated transcriptional response was also observed for some genes implicated in the response against oxidative stress, in apoptosis inhibition and in lowering inflammatory immune response. These microarray results predicted a conferred protective effect of IV09.007 and we verified this hypothesis by performing comet assays on UVB-irradiated keratinocytes or melanocytes, to demonstrate the efficacy of IV09.007 on preventing DNA damage.

Maintenance of the ubiquitin-proteasome system activity correlates with visible skin benefits.

Researches on longevity and anti-ageing molecules have clearly evidenced the potential to increase lifespan of the cells. These recent scientific data raise interests and questions on the capacity of the cells to live longer and maintain their fundamental mechanisms of protection, reparation or degradation of abnormal proteins to maintain their capital of healthy and functional cellular activity. In this concern, this study was focused on the ubiquitin-proteasome system as an essential cellular tool to maintain the pool of functionally active proteins allowing renewal of proteins and degradation of damaged proteins. As the proteasome keeps the 'cells health capital', it should be particularly interesting to associate the maintenance of the proteasome activity with increasing longevity. Indeed, although oxidative stress damage increases with ageing leading to collagen and cellular membrane alterations, it also leads to a reduction in the proteasome activity which is critical for the cells. The aim of this study was to better understand the cellular role of the proteasome and to provide new data showing the skin beneficial effects in activating the overall system of ubiquitination and proteasomal degradation. For this purpose, in vitro, ex vivo and in vivo experiments were performed to evaluate the effects of maintaining the ubiquitin-proteasome activity in basal and stress conditions on young versus aged cells. Experiments have included evaluation of a newly developed dimerized tripeptide targeting specifically the ubiquitin-proteasome pathway. Our results have demonstrated that maintenance of this essential mechanism that participates in abnormal protein elimination and protein renewal allows maintaining cellular integrity that correlates with visible skin benefits.

Cotton honeydew (Gossypium hirsutum L.) extract offers very interesting properties for hair cosmetics and care products.

Cotton honeydew extract is composed of a unique combination of oligosaccharides, including fructose, glucose, inositol, melezitose, saccharose, trehalose and trehalulose. Studies have shown that these oligosaccharides exhibit a protective effect. Therefore, we were interested in studying the effect of these oligosaccharides on normal and damaged human hair. Both clinical and scanning electron microscopy (SEM) studies were performed. Standardized human hair samples were used to determine the effect of a rinse-off mask with 1% cotton honeydew extract on the ultrastructure of hair. In addition, hair samples were submitted to different aggressions, following various experimental protocols. SEM showed that, without extra aggression, the cuticle scales appeared to lie more smoothly in the hair in cotton honeydew extract-treated samples than in untreated samples. The extract-treated hair samples were also less prone to chipping. In contrast, the control, untreated hair samples retained a dry and damaged appearance and were prone to chipping and progressive splitting. In a clinical study, 15 volunteers had half of their hair treated with a formula with 1% honeydew extract and the other half was left untreated as a control. Pictures and visual evaluation of the hair showed that the honeydew extract formula left the hair with a smoothness that was far superior to the control side and this result was confirmed by SEM. In addition, mRNA studies on epidermal cells were performed and confirmed the stimulating effect of honeydew extract on keratin synthesis. These results demonstrate that cotton honeydew extract can be of great use in hair care products and cosmetics.

Quercus suber cork extract displays a tensor and smoothing effect on human skin: an in vivo study.

Recently, it has become indispensable for anti-aging active ingredients to provide a visible and immediate smoothing antiwrinkle effect. In Quercus suber, suberin is the most important structural component of cork cell walls. Studies have shown that suberin is made up mostly of hydroxycarboxylic acids and that it is endowed with many special mechanical and chemical properties that evoke a possible smoothing effect on the surface of the skin. Therefore, we were interested in investigating the effect of this cork extract on the skin's surface in a double-blind clinical study. The study was conducted in 15 healthy volunteers, aged 22 to 52 years. The volunteers applied a gel formula with 3% of cork extract, or placebo gel, on each forearm. Skin surface roughness was evaluated visually by pictures and by silicone replicas 1 and 2 h after application, followed by statistical analysis using the matched-pairs McNemar statistical test. McNemar analysis of the pictures revealed that application of cork extract on the skin resulted in a highly significant reduction of roughness 1 h after application. This effect was observed in 73.3% of volunteers. Two hours after cork extract application, a highly significant improvement of skin roughness was found in 78.6% of volunteers. Moreover, silicone replica treatment confirmed significant improvement in average of roughness at 2 h. These results demonstrate that cork extract provides a remarkable and highly significant tensor and smoothing effect on the skin, which could be of great use in anti-aging skin care products.

Stimulating effect of collagen-like peptide on the extracellular matrix of human skin: histological studies.

Recent studies of our newly developed synthetic collagen-like hexapeptide have shown that it enhances cultured cell adhesion and differentiation and improves the morphology of ex vivo skin. Consequently, we were interested in further investigating the effects of the collagen-like peptide on the skin. We performed different immunostaining studies on ex vivo human skin samples treated with the collagen-like peptide at 1% in time course studies. Our research also included comparative studies with vitamin C (often used as a positive control for enhancing collagen synthesis). The results showed that application of the collagen-like peptide to the skin enhanced synthesis of many extracellular matrix (ECM) molecules and that this effect was observed very early in some ECM molecules such as laminin 5, collagen 111, and collagen IV The expression of the other molecules was increased after different times of application of the collagen-like peptide. Interestingly, comparative studies with vitamin C showed that the synthesis response of some ECM molecules such as laminin 5, collagen 111 and collagen IV was more rapid after the administration of the collagen-like peptide than through vitamin C administration. Our results also revealed that after a longer treatment period, both active ingredients stimulated ECM molecule synthesis to a similar degree, with the exception of some molecules that remained superiorafterpeptide administration, such as collagen IV and beta 1 integrin. These histological studies demonstrate the remarkable and rapid effect of the collagen-like peptide on stimulating ECM molecule synthesis and suggest wide application for the peptide in antiaging and photoaging skin care products.

The HET-CAM test combined with histological studies for better evaluation of active ingredient innocuity.

In the search for alternative methods to animal testing, the Hen's egg test on chorioallantoic membrane (HET-CAM) plays a central role in evaluating the innocuity of active ingredients. Therefore, in the following studies we combined the HET-CAM test with histological evaluation in order to increase the sensitivity of evaluation. Twenty active ingredients from four different categories of origin (vegetal, marine, biotechnological and chemical synthetic) were subjected to innocuity evaluation at two different concentrations (pure and 10%). We performed the HET-CAM test and histological evaluation after trypan blue and hematoxylin-eosin staining of the chorioallantoic membrane to microscopically evaluate its state of damage after application of each active ingredient. These studies showed that when the active ingredient was diluted (10%), no discrepancy was seen between the classical HET-CAM evaluation and the histological reading of the chorioallantoic membrane. The histological findings corresponded with the visual observation of the CAM. When the active ingredients were tested at pure concentration, 7 out of 20 tested products demonstrated discrepancy between the two tests. In six cases, the histological examination revealed signs of irritation, such as hyperemia, while visual HET-CAM evaluation was negative. In another case, the histological examination revealed a slight hemorrhage whereas the HET-CAM reading showed only hyperemia. Moreover, the results of trypan blue staining corroborated the histological evaluation of the CAM. These results strongly suggest that the combination of histological and visual HET-CAM tests is of interest for a more sensitive evaluation of the innocuity of cosmetic active ingredients. This additional sensitivity may help to prevent some cases of in vivo intolerance reactions.

Date palm kernel extract exhibits antiaging properties and significantly reduces skin wrinkles.

Hormones play a central role in skin appearance and are implicated in skin aging. Recently, along with the remarkable increase in interest in natural products, the application of phytohormones in antiaging products has become very important. In this context, we developed date palm kernel extract. Date palm kernel is rich in phytohormones and we investigated the antiaging properties of date palm kernel in this in vivo study on wrinkles. Ten healthy women volunteers, between the ages of 46 and 58 years, applied the cream formula with 5% date palm kernel or placebo on the eye area twice a day for 5 weeks. The evaluation was made both clinically and by silicon replica analysis followed by statistical analysis using the Wilcoxon test. Silicon replica results showed that topical application of date palm kernel reduced the total surface of wrinkles by 27.6% (p = 0.038). Moreover, date palm kernel reduced the depth of wrinkles by 3.52% (p = 0.0231). These results are statistically significant and were clinically confirmed where visual improvement was seen in 60% of the volunteers treated. This in vivo study demonstrates that date palm kernel exhibits a significant antiwrinkle effect and is therefore of interest in antiaging skin care products.

Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines.

The expression of the 3 currently known neurotensin receptors was studied in human cancer cells of prostatic, colonic or pancreatic origin by means of RT-PCR analysis and binding experiments. All the cells selected for this work have been shown to exhibit a growth response to neurotensin. We found that the 7 transmembrane domain, levocabastine insensitive receptor (NTR1) is expressed in most but not all of the cells studied whereas the 7 transmembrane domain, levocabastine sensitive receptor (NTR2) is present in none of these cells. The 100 kDa-type I neurotensin receptor (NTR3) is expressed in all the cells assayed. Moreover, we demonstrated that neurotensin can stimulate the growth of CHO cells stably transfected with the NTR3. Taken together, our results strongly suggest that the NTR3 subtype could be involved in the growth response of human cancer cells to neurotensin.

Receptor-mediated internalization of somatostatin in rat cortical and hippocampal neurons.

Binding of neuropeptides to their receptors usually results in internalization of receptor-ligand complexes. This process serves a crucial role in receptor downregulation, resensitization, and transmembrane signaling. It has mainly been investigated in cells ectopically expressing recombinant receptors. In the present study, we investigated whether rat central neurons and astrocytes naturally expressing somatostatin (SRIF) receptors internalized this neuropeptide. We demonstrated that 29% of cortical and 45% of hippocampal neurons in culture expressed the SRIF receptor sst(2A) and that 40-50% of the neurons internalized fluorescent SRIF. Similarly, an important proportion of astrocytes expressed sst(2A) (up to 60% in cortical cultures) and internalized fluo-SRIF. Competition experiments using the sst(2)/sst(5)-preferring agonist SMS 201-995 (octreotide) showed that a subpopulation of neurons internalized fluo-SRIF via sst(2) and/or sst(5) receptors, but that others also did so via other subtypes. Fluo-SRIF labeling was barely competed for by the sst(1)-selective agonist CH-275, indicating that sst(1) was unlikely to be mediating SRIF internalization in hippocampal and cortical neurons. Given the paucity of sst(5) receptors in cerebral cortex and hippocampus and the poor yield of sst(4) internalization in transfected cells, we conclude that sst(2) and sst(3) subtypes are the most likely to be responsible for SRIF internalization in our culture systems.

Binding of a pure 125I-monoiodoleptin analog to mouse tissues: a developmental study.

The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.

Intracellular dynamics of sst5 receptors in transfected COS-7 cells: maintenance of cell surface receptors during ligand-induced endocytosis.

Internalization of G protein-coupled receptors is crucial for resensitization of phosphorylation-desensitized receptors, but also for their long term desensitization through sequestration. To elucidate the mechanisms regulating cell surface availability of the somatostatin (SRIF) receptor subtype sst5, we characterized its internalization properties in transfected COS-7 cells using biochemical, confocal microscopic, and electron microscopic techniques. Our results demonstrated rapid and efficient sequestration of specifically bound [125I]Tyr0-D-Trp8-SRIF (up to 45% of bound radioactivity). Combined immunocytochemical detection of sst5 and visualization of a fluorescent SRIF analog by confocal microscopy revealed that whereas the internalized ligand progressively clustered toward the cell center with time, immunoreactive receptors remained predominantly associated with the plasma membrane. The preservation of cell surface receptors was confirmed by binding experiments on whole cells revealing a lack of saturability of [125I]Tyr0-D-Trp8-SRIF binding at 37 C. Binding was rendered saturable by the drug monensin, showing that receptor recycling played a key role in the preservation of cell surface receptors. Electron microscopy demonstrated that in addition to receptor recycling, internalization of receptor-ligand complexes triggered a massive recruitment of sst5 receptor molecules from intracellular stores to the membrane. This combination of recycling and recruitment of spare receptors may protect sst5 from long term down-regulation through sequestration and, therefore, facilitate extended SRIF signaling.

Receptor-mediated internalization is critical for the inhibition of the expression of growth hormone by somatostatin in the pituitary cell line AtT-20.

The inhibitory effect of the neuropeptide somatostatin on the expression of growth hormone was measured by quantitative polymerase chain reaction in the pituitary cell line AtT-20. We demonstrate that this effect is dependent on the internalization of somatostatin-receptor complexes and that it is totally independent from the peptide-induced inhibition of adenylate cyclase. Indeed, the inhibitory effect of the peptide on growth hormone mRNA levels was totally insensitive to pertussis toxin treatment but was totally abolished under conditions which block somatostatin receptor internalization. Comparative confocal microscopic imaging of fluorescent somatostatin sequestration and fluorescence immunolabeling of sst1, sst2A, and sst5 receptors suggests that sst2A is most probably responsible of the inhibitory effect of somatostatin on growth hormone expression.

Fluorescent ligands for studying neuropeptide receptors by confocal microscopy.

This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

Receptor-induced internalization of selective peptidic mu and delta opioid ligands.

The binding and internalization of radioiodinated and fluorescent mu and delta opioid peptides in mammalian cells were quantitatively studied by biochemical techniques and directly visualized by confocal microscopy. The labeled peptides were prepared by inserting either a 125I-Bolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and [Lys7]dermorphin. The purified derivatives kept most of their specificity and selectivity toward delta and mu opioid receptors, respectively. Biochemical and confocal microscopy data showed that both mu and delta opioid peptides were internalized in mammalian cells transfected with the corresponding opioid receptor according to a receptor-mediated mechanism. The internalization process was time- and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both delta and mu ligands occurred from a single large cap at one pole of the cell, indicating that polymerization of ligand-receptor complexes preceeded internalization. Finally, green and red fluorescent analogues of deltorphin-I and [Lys7]dermorphin, respectively, were found to internalize through partly distinct endocytic pathways in cells co-transfected with mu and delta receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.