p63-microRNA feedback in keratinocyte senescence.

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated ß-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture.

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high-passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high-passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti-skin-ageing strategies.

Reduced expression of the adhesion protein tensin1 in cultured human dermal fibroblasts affects collagen gel contraction.

As revealed by immunohistochemistry and RT-QPCR, the focal adhesion protein tensin1 is expressed in cultured human dermal fibroblasts and reduced by 60% after transfection with tensin1 siRNA. Tensin1 silenced fibroblast exhibited a strongly reduced capacity to contract collagen gels. Aged fibroblasts, generated with the Hayflick replicative senescence model, exhibit as siRNA silences fibroblasts, a reduced tensin1 expression and an impaired gel contraction capacity. Based on these results, we speculate that in human dermal fibroblasts, tensin1 plays an important role in cell-matrix interaction and that a reduced expression might contribute to the dermal alterations observed during skin ageing.

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

Stress chaperone mortalin regulates human melanogenesis.

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.

Heat shock protein 47 expression in aged normal human fibroblasts: modulation by Salix alba extract.

Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.

Algae extract protection effect on oxidized protein level in human stratum corneum.

Modification of proteins by reactive oxygen species is implicated in different disorders. The proteasome is a multicatalytic proteinase in charge of intracellular protein turnover and of oxidized proteins degradation. Consequently, proteasome function is very important in controlling the level of altered proteins in eukaryotic cells. Evidence for a decline in proteasome activity during skin photo-aging has been provided in Bulteau et al. in 2002. The ability of a lipid algae extract (Phaeodactylum tricornutum) to stimulate 20S proteasome peptidase activities was described by Nizard et al. in 2001. Furthermore, keratinocytes treated with Phaeodactylum tricornutum extract and then UVA and UVB irradiated, exhibited a sustained level of proteasome activity comparable to the one of nonirradiated cells. The level of modified proteins can be quantified by measurement of protein carbonyl content (Oxyblot technique), which has been shown to increase with aging and other disorders. In this paper, it is described that, in the presence of this lipid algae extract, the level of oxidized proteins is reduced, as assessed by the Oxyblot technique. These results are obtained both with culture of human keratinocytes and stratum corneum skin cells (obtained by stripping) from human volunteers. Altogether, these results argue for the presence of compounds in this algae extract that have a stimulating and/or protective effect on proteasome activity, resulting in a decreased level of protein oxidation.

TGF-ß1 induces differentiation of papillary fibroblasts to reticular fibroblasts in monolayer culture but not in human skin equivalents.

UNLABELLED: Fibroblasts isolated from the papillary and reticular dermis are different from each other in vitro. If papillary fibroblasts are subjected to prolonged serial passaging they will differentiate into reticular fibroblasts. Reticular fibroblasts have been shown to resemble myofibroblasts in several ways. TGF-ß1 is the most important factor involved in myofibroblast differentiation. AIMS: we investigated if TGF-ß1 can induce differentiation of papillary fibroblasts into reticular fibroblasts, in monolayer cultures and in human skin equivalents. METHOD: Monolayer cultures of and human skin equivalents generated with papillary fibroblasts were stimulated with TGF-ß1. The expression of markers specific for reticular and papillary fibroblasts was measured by qPCR and immunohistochemical analysis in monolayer cultures. In human skin equivalents, the morphology and the expression of several markers was analysed and compared to untreated papillary and reticular human skin equivalents. RESULTS: Monolayer cultures of papillary fibroblasts started to express a reticular marker profile after stimulation with TGF-ß1. Human skin equivalents generated with papillary fibroblast and stimulated with TGF-ß1 were similar to papillary control equivalents and did not obtain reticular characteristics. Expression of reticular markers was only found in the lower layers of TGF-ß1-stimulated papillary skin equivalents. CONCLUSIONS: TGF-ß1 can induce differentiation to reticular fibroblasts in monolayer cultures of papillary fibroblasts. In skin equivalents no such effects were found. The major difference between these experiments is the presence of extracellular matrix in skin equivalents. Therefore, we hypothesize that the matrix secreted by papillary fibroblasts protects them from TGF-ß1 induced differentiation.

MicroRNAs in human skin ageing.

The skin protects humans from the surrounding environment. Tissues undergo continuous renewal throughout an individual's lifetime; however, there is a decline in the regenerative potential of tissue with age. The accumulation of senescent cells over time probably reduces tissue regenerative capacity and contributes to the physiological ageing of the tissue itself. The mechanisms that govern ageing remain unclear and are under intense investigation, and insight could be gained by studying the mechanisms involved in cellular senescence. In vitro, keratinocytes and dermal fibroblasts undergo senescence in response to multiple cellular stresses, including the overproduction of reactive oxygen species and the shortening of telomeres, or simply by reaching the end of their replicative potential (i.e., reaching replicative senescence). Recent findings demonstrate that microRNAs play key roles in regulating the balance between a cell's proliferative capacity and replicative senescence. Here, we will focus on the molecular mechanisms regulated by senescence-associated microRNAs and their validated targets in both keratinocytes and dermal fibroblasts.

MicroRNA-152 and -181a participate in human dermal fibroblasts senescence acting on cell adhesion and remodeling of the extra-cellular matrix.

Ageing of human skin is associated with phenotypic changes in the cutaneous cells; the major functional markers of ageing occur as consequences of dermal and epidermal cell senescence and of structural and compositional remodeling of normally long-lived dermal extracellular matrix proteins. Understanding the contribution of the dermal cells in skin ageing is a key question, since this tissue is particularly important for skin integrity and its properties can affect the epidermis. Several microRNAs have been shown to be involved in the regulation of pathways involved in cellular senescence and exerted important effects on tissues ageing. In this study, we demonstrate that the expression of miR-152 and miR-181a increased during the human dermal fibroblasts senescence and that their overexpression, is sufficient to induce cellular senescence in early-passage cells. The increase of these miRNAs during cells senescence was accompanied by a decrease in integrin α5 and collagen XVI expression at mRNA and/or protein levels resulting in reduced cellular adhesion and suggesting extracellular matrix remodeling. These findings indicate that changes in miRNAs expression, by modulating the levels of adhesion proteins and extra-cellular matrix components, such as integrin α5 and collagen XVI, could contribute to the compositional remodelling of the dermis and epidermis occurring during skin aging.

Different gene expression patterns in human papillary and reticular fibroblasts.

The dermis contains two distinct layers: the papillary and the reticular layers. In vitro cultures of the fibroblasts from these layers show that they are different. However, no molecular markers to differentiate between the two subtypes of fibroblasts are known. We performed gene expression analysis on cultured fibroblasts isolated from the papillary and reticular dermis. In all, 116 genes were found to be expressed differentially. Of these, 13 were validated by quantitative reverse transcriptase-PCR analysis and two markers could be validated at the protein level in monolayer cultures. Three markers showed differential expression in in vivo skin sections. The identified, characteristic markers of the two fibroblast subpopulations provide useful tools to perform functional studies on reticular and papillary fibroblasts.

miR-146a is modulated in human endothelial cell with aging.

BACKGROUND: Increasing evidence has demonstrated that the senescence of vascular endothelial cells has critical roles in the pathogenesis of vascular dysfunction such as atherosclerosis and thrombosis. MicroRNA (miR) are small non-coding RNAs that inhibit gene expression by binding to complementary sequences in the 3'UTR of their target mRNAs. MiRs modulate a variety of biological functions such as cell development, cell differentiation, and apoptosis. Moreover, several miRs involved in endothelial cell function have been identified. METHODS AND RESULTS: Through a microarray approach, we have identified a miR-146a that is progressively modulated in endothelial cells with aging. In young human umbilical vein endothelial cells, this miR is involved in a premature senescence-like phenotype through direct targeting of the NOX4 protein, implicated in cell senescence and aging. CONCLUSIONS AND GENERAL SIGNIFICANCE: Finding important factors that regulate endothelial cell senescence, like miR-146a, will help provide novel therapeutic strategies for vascular disorders.

Development of a new sensor based on micro-electro-mechanical systems for objective in vivo measurement of the cutaneous temperature: application to foundations.

BACKGROUND/AIMS: The purpose of this work was to develop a new sensor for objective in vivo measurement of the cutaneous temperature based on micro-electro-mechanical systems (MEMS), and to compare these performances with those of a classical thermocouple. Research on this new sensor was carried out to allow the quantification of the thermal properties of the made-up skin. METHODS: Sixteen female subjects divided into two different age groups (18-35 and >50 years old) were recruited for this study. Several zones of the face and forearms were made up at random with foundations containing or not a thermoregulator raw material. The quantity of foundation applied on the skin was standardized and measurements were carried out first before make-up, and then 10 s and 5 min after make-up. The new sensor and the thermocouple were used successively on each zone. The cutaneous temperature was expressed in degrees celsius. RESULTS/CONCLUSION: The two systems are similar in terms of repeatability and reproducibility, with some differences in sensibility. The data measured by the MEMS sensor appear lower than those measured by the thermocouple. After make-up, the MEMS sensor detects a progressive increase of the temperature in time whereas the thermocouple detects a decrease. We found the same evolution on the face but in a more attenuated way. These results tend to show that the devices do not measure the same phenomenon. The thermocouple appears more sensitive to the thermal response of the made-up surface whereas the MEMS sensor appears more sensitive to the heat transfers in the interface between the skin and make-up.