To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca 2+ channel that is a major molecular counterpart for Ca 2+ influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis.calcium signaling | cell migration | orai1 KO mice
Dermal elastic fibres are extracellular matrix protein complexes produced by fibroblasts and involved in skin elasticity. Elastin fibres decrease with age as a result of reduced synthesis and increased degradation, resulting in skin sagging and reduced skin elasticity. In this study, we show that retinol (ROL), known to enhance dermal collagen production, is also enhancing elastin fibre formation. ROL induced elastin gene expression and elastin fibre formation in cultured human dermal fibroblasts. Topical treatment of cultured human skin explants with a low dose (0.04%) of ROL increased mRNA and protein levels of tropoelastin and of fibrillin-1, an elastin accessory protein, as documented by QPCR and immunohistochemistry staining. Luna staining confirmed the increased elastin fibre network in the ROL-treated skin explants, as compared with untreated controls. These data demonstrate that ROL exerts its anti-ageing benefits not only via enhanced epidermal proliferation and increased collagen production, but also through an increase in elastin production and assembly.
DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.
We have investigated the effects of all-trans retinoic acid (ATRA) on aquaporin 3 (AQP3) expression and function both in vitro and ex vivo. ATRA treatment provoked a rapid accumulation of AQP3 transcripts in cultured normal human epidermal keratinocytes (NHEK). This increase was still observed 24 hours after application of ATRA. The induction of AQP3 gene was accompanied by an augmentation of immunoreactivity. Using a selective agonist, we demonstrated that the effect of ATRA was predominantly mediated by retinoic acid receptor subtype gamma (RARgamma). Incubation of NHEK in ATRA for 24, 48, and 72 hours stimulated glycerol influx, suggesting that the increase in AQP3 gene and protein expression was followed by an enhancement of biological activity. Topical application of ATRA for 24 hours on skin explants induced significant epidermal expression of AQP3 and strong immunoreactivity in the epidermal basal layers. Collectively, the present results show that ATRA increased AQP3 expression and enhanced biological activity in human skin.
The antiaging efficacy of retinol (ROL) has been explored mainly clinically in photoprotected skin sites and for high doses of ROL (0.4–1.6%). The objective of the study was to demonstrate the antiaging action of a low and tolerable dose of ROL (0.1%) ex vivo by measuring the expression of cellular retinoic-acid-binding protein II (CRABP2) and heparin-binding epidermal growth factor (HBEGF) by a histological evaluation of the epidermis and in vivo by assessing major aging signs and performing three-dimensional profilometry and digital imaging during a 9-month double-blind placebo-controlled study involving 48 volunteers. Finally, epidermal cell proliferation was evaluated using tryptophan fluorescence spectroscopy. Our results demonstrate that 0.1% ROL induced CRABP2 and HBEGF gene expression and increased keratinocyte proliferation and epidermal thickness. In human volunteers, topical application of a ROL-containing product improved all major aging signs assessed in our study (wrinkles under the eyes, fine lines and tone evenness). Moreover, tryptophan fluorescence increased in the active-agent-treated group and not in the placebo-treated group, indicating that cell proliferation was accelerated in vivo. These data demonstrate that a product containing a low dose (0.1%) of ROL promotes keratinocyte proliferation ex vivo and in vivo, induces epidermal thickening ex vivo and alleviates skin aging signs, without any significant adverse reaction.
Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative-senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age-related phenotypic and functional alteration of fibroblasts from 'young' (<35 years) and 'old' (>50 years) donors. Our results brought evidence of the senescent status of 'old' fibroblasts by senescence associated β-galactosidase (SA-βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in 'old' fibroblasts as well as a subset of upregulated genes involved in senescence features. In 'old' fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than 'young' ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of 'old' fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.
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