Thierry Oddos

Antiaging Action of Retinol: From Molecular to Clinical

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.

Human collagen Krox up-regulates type I collagen expression in normal and scleroderma fibroblasts through interaction with Sp1 and Sp3 transcription factors.

Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a –112/–61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The –112/–61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Background: NF-κB regulation of COL1A1 in physiopathological situations is largely unknown. Results: NF-κB down-regulates COL1A1 in normal and scleroderma fibroblasts, through its recruitment on COL1A1 by protein interactions with the Sp1/Sp3/c-Krox trans-activators, which are different in fibrotic fibroblasts. Conclusion: Our findings highlight a new mechanism for COL1A1 regulation. Significance: These data could allow the development of new antifibrotic strategies. Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the −112/−61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

ORAI1 calcium channel orchestrates skin homeostasis

Significance The epidermis of the skin is composed of keratinocytes that are organized in several layers. Basal cells divide and produce cells moving outwards the epidermis while undergoing the process of terminal differentiation, crucial for the barrier function of the skin. Calcium is an indispensible ion for differentiation, and calcium channels are of primary importance. Unexpectedly, we discovered that the Orai1 calcium channel is mainly expressed in the basal layer, functioning to negatively control differentiation. The Orai1 channel supplies calcium to sustain proliferation and, in particular, to drive migration of keratinocytes, both processes being the feature of basal keratinocytes. 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 Ca2+ channel that is a major molecular counterpart for Ca2+ 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.

A microarray to measure repair of damaged plasmids by cell lysates

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.

Evaluation of the efficacy of a topical cosmetic slimming product combining tetrahydroxypropyl ethylenediamine, caffeine, carnitine, forskolin and retinol, In vitro, ex vivo and in vivo studies

Three studies were performed to investigate the mechanism of action and evaluate the efficacy of a topical cosmetic slimming product combining tetrahydroxypropyl ethylenediamine, caffeine, carnitine, forskolin and retinol. The Ex vivo study on skin explants showed that caffeine and forskolin both stimulated glycerol release and demonstrates for the first time that retinol and carnitine in combination synergistically stimulated keratinocyte proliferation, which leads to an increase epidermal thickness. The double‐blind, randomized, placebo‐controlled clinical study associating circumference measurements on five selected parts of the body, cutaneous hydration measurements as well as blinded expert grading of skin aspect was conducted on 78 women who applied the product or placebo twice daily for 12 consecutive weeks. After 4 weeks of twice‐daily application of the product, significant reductions in circumference of abdomen, hips–buttocks and waist were already observed. Improvements concerned all the measured body parts after 12 weeks. Orange peel and stubborn cellulite decreased significantly from 4 weeks of treatment and tonicity improved from 8 weeks, demonstrating that the product improved skin aspect. At the end of the study, eight parameters of the thirteen evaluated were significantly improved in the active group and compared with placebo.

NF-κB Accumulation Associated with COL1A1 Transactivators Defects during Chronological Aging Represses Type I Collagen Expression through a –112/–61-bp Region of the COL1A1 Promoter in Human Skin Fibroblasts

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

Age-associated modifications of Base Excision Repair activities in human skin fibroblast extracts

Base Excision Repair (BER) is the predominant repair pathway responsible for removal of so-called small DNA lesions such as abasic sites (AP site), uracil (U), 8-oxo-7,8-dihydroguanine (8oxoG), thymine glycol (Tg). In this study, we investigated effect of aging on excision efficacy of several endogenous base lesions and AP sites using an in vitro multiplexed fluorescent approach on support (parallelized oligonucleotide cleavage assay). Human fibroblasts nuclear extracts from 29 donors of different ages were characterized in their ability to simultaneously excise the different lesions. Clearly, three different groups of lesions emerged according to the efficiency of their cleavage: one exhibited very high cleavage efficiency (AP sites and U paired with G), one showed intermediate cleavage efficiency (U paired with A and Tg). The third group included 8oxoG, A paired with 8oxoG, T at CpG site and hypoxanthine (Hx) and displayed poor repair. Aging was significantly associated with modification of excision efficiency for AP sites, uracil, Tg and 8oxoG. Repair rate decreased for the first three lesions and the most drastic effects were observed for repair of U:A. Surprisingly, excision of 8oxoG increased with aging suggesting a completely different regulation or adaptation for the initiation step of this related specific repair pathway.

A purified Feverfew extract protects from oxidative damage by inducing DNA repair in skin cells via a PI3-kinase-dependent Nrf2/ARE pathway

BACKGROUND Environmental factors such as solar ultraviolet (UV) radiation and other external aggressors provide an oxidative challenge that is detrimental to skin health. The levels of endogenous antioxidants decrease with age, thus resulting in less protection and a greater potential for skin damage. The NF-E2-related factor-2 (Nrf2) - antioxidant response element (ARE) pathway is a primary defense mechanism against oxidative stress, and induces the expression of antioxidant, detoxification and repair genes. Activation of ARE-Nrf2 can help restore oxidative homeostasis of the skin and play a role in inflammatory response and DNA repair mechanisms. OBJECTIVE To evaluate the role of a purified parthenolide-depleted Feverfew (PD-Feverfew) extract on the ARE-Nrf2 pathway and DNA repair in skin cells. METHODS These studies were undertaken in primary human keratinocytes or KB cells using Luciferase Promoter assay, siRNA transfection studies, Western blot analyses, Immunofluorescence microscopy, comet assay and quantitative real-time PCR. RESULTS PD-Feverfew was found to induce Nrf2 nuclear translocation and to increase ARE activity in a dose dependent manner. Furthermore, knockdown of Nrf2 resulted in suppression of PD-Feverfew-induced ARE activity. PD-Feverfew was also found to induce phosphorylation of Akt, a kinase downstream of PI3K. Inhibition of PI3K via pre-treatment with the selective pharmacological inhibitor, LY294002, abolished PD-Feverfew-induced Nrf2/ARE activation. PD-Feverfew also reduced UV-induced DNA damage in a PI3K and Nrf2-dependent manner. CONCLUSIONS Therefore, by increasing endogenous defense mechanisms and aid in DNA repair of damaged skin cells via activation of a PI3K-dependent Nrf2/ARE pathway, PD-Feverfew may help protect the skin from numerous environmental aggressors.

Effect of Aging on DNA Excision/Synthesis Repair Capacities of Human Skin Fibroblasts

TO THE EDITOR A key factor in the skin aging process is the cumulative effects of chronological aging and environmental-based assaults. Endogenous cellular oxidative processes generate reactive oxygen species and reactive polyunsaturated fatty acid derivatives (Lindahl, 1993; Marnett and Plastaras, 2001). These attacks on DNA cause substantial base and sugar damage, and the persistence of such lesions leads to mutations and genome instability. Skin may also suffer from chronic exposure to sun; UV radiation causes oxidative DNA damage and induces photoproducts (mainly cyclobutane pyrimidine dimers and 6-4 photoproducts (Moriwaki and Takahashi, 2008)). Age-related accumulation of somatic damage can thus be worsened by sun exposure, leading to an increased incidence of skin disorders and dramatic acceleration of skin aging (Niedernhofer, 2008). Mammalian cells have evolved several DNA-repair pathways to remove all the categories of DNA base lesions, relying in particular on DNA excision mechanisms. One of these, nucleotide excision repair, removes bulky adducts and is thus an essential mechanism for correcting UV-induced DNA damage (Sarasin, 1999). The base excision repair pathway corrects small base modifications such as oxidized and alkylated bases (Almeida and Sobol, 2007). The importance of repair mechanisms is demonstrated by the hazardous consequences of genetic defects in DNA repair (Friedberg, 2001), but investigating DNA repair with respect to aging remains a challenge. This is due to the complexity of the underlying repair mechanisms as well as to the varying approaches in terms of assays and end points measured (Vijg, 2008). To better understand the relationship between aging and DNA repair, we took advantage of our newly developed multiplexed excision/synthesis assay (Millau et al., 2008) to examine simultaneously, using nuclear extracts, the base excision repair and nucleotide excision repair capacities of human primary fibroblasts derived from healthy donors of different ages. In addition, we investigated changes in DNA repair attributed to chronic sun exposure. A total of 33 healthy Caucasian women were recruited by the Dermscan Group (Lyon, France). Biopsy removal was performed in accordance with the Declaration of Helsinki Principles Guidelines after approval for the study had been given by a medical ethics committee and written consent obtained from the donors. The volunteers were classified into three groups by age (group 1: mean age1⁄425 years, range 20–33, n1⁄49; group 2: mean age1⁄446 years, range 40–50, n1⁄4 9; group 3: mean age1⁄4 65 years, range 61–68, n1⁄415). All subjects were nonsmokers, had phototype II or III skin, declared no excessive exposure to sun or UVA, had no cutaneous pathology, and were not receiving medical treatment. Fibroblast cultures were established from outgrowth of two 3 mm punches taken on the volar forearm (photoexposed area) and the upper inner arm (photoprotected area). Cells were harvested during the exponential phase of growth and stored frozen in liquid nitrogen at passage 5. Nuclear extracts were prepared as described by Millau et al. (2008). For each sample, excision/ synthesis repair reactions were run for 2.5 hours at 30 1C at a final protein concentration of 0.15 mg ml , along with 1 mM adenosine triphosphate and 1.25 mM dCTP-Cy5 (Amersham, Little Chalfont, England), on damaged plasmid microarrays prepared as described by Millau et al. (2008) (see Supplementary Data and Supplementary Figures online for experimental details and nuclear extract features). The microarrays were prepared using Hydrogel slides (Perkin Elmer, Courtaboeuf, France) spotted with unmodified control plasmids together with five plasmid families containing serial amounts of typical lesions (three dilutions per plasmid family). Lesions present on the support were formed by specific physical and chemical treatments: photoproducts (cyclobutane pyrimidine dimers and (6–4)photoproducts), 8-oxoguanine, alkylated bases, abasic sites, and pyrimidine glycols. Total fluorescence intensity related to the fluorescence incorporated into plasmids was the parameter used for calculations (Genepix 4200A scanner, Axon GenePix, Molecular Devices, Sunnyvale, CA). A total fluorescence intensity value was calculated for each lesion type by adding the values for the corresponding replicates. Hence, each microarray generated one value per lesion type. The mean of the two values obtained per sample and per lesion type were used for statistical purposes. Assessment of the mean fluorescence intensity (shown with the corresponding standard error for each lesion type in Figure 1) revealed a decrease in excision/ synthesis activity with age, irrespective of the repair pathway considered. Statistical analysis (general linear model (Minitab V14 software, The MathWorks, Natick, MA) was performed on these latter data from 31 samples: photoprotected and photoexposed cells from the same subject. The general linear model was used to estimate the influence of age