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Introduction: Bathing in the Blue Lagoon (BL) in Iceland benefits patients with psoriasis. Accordingly, the BL water contains algae with biological activities that improve skin barrier function and affect T-cell responses relevant for psoriasis. Bathing in the BL is also becoming increasingly popular among healthy individuals and anecdotal evidence suggests positive effects on uneven skin pigmentation. Objective: The aim of the study was to address the impact of BL algae on skin pigmentation. Methods: In this work, in vitro gene expression studies in melanocytes and a noninvasive in vivo study were conducted. Results: We here report that normal human epidermal melanocytes, which had been treated with nontoxic concentrations of BL algae, show a significantly reduced expression of α melanocyte-stimulating hormone-induced expression of genes important for melanin synthesis, such as tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, melan A protein, and pre-melanosome protein. This in vitro observation prompted us to conduct a randomized, double-blind, intra-individual, comparative split-face in vivo study, in which 60 volunteers with pre-existing facial pigment spots were treated twice daily with a BL algae containing serum or a vehicle control. We found that constitutive skin pigmentation as determined by colorimetry (individual typology angle and luminescence) did not differ significantly between vehicle- and serum-treated skin sites. In marked contrast, digital photography under cross-polarized lighting and RBX technology (VISIA CR) revealed that the number of pigment spots in the serum-treated face decreased significantly compared to the vehicle-treated side. Conclusion: Thus, BL algae can affect human melanocyte function in vitro and reduce uneven facial skin pigmentation in vivo.

Mammalian sirtuins are involved in the control of metabolism and life-span regulation. Here, we link the mitochondrial sirtuin SIRT4 with cellular senescence, skin aging, and mitochondrial dysfunction. SIRT4 expression significantly increased in human dermal fibroblasts undergoing replicative or stress-induced senescence triggered by UVB or gamma-irradiation. In-vivo, SIRT4 mRNA levels were upregulated in photoaged vs. non-photoaged human skin. Interestingly, in all models of cellular senescence and in photoaged skin, upregulation of SIRT4 expression was associated with decreased levels of miR-15b. The latter was causally linked to increased SIRT4 expression because miR-15b targets a functional binding site in the SIRT4 gene and transfection of oligonucleotides mimicking miR-15b function prevented SIRT4 upregulation in senescent cells. Importantly, increased SIRT4 negatively impacted on mitochondrial functions and contributed to the development of a senescent phenotype. Accordingly, we observed that inhibition of miR-15b, in a SIRT4-dependent manner, increased generation of mitochondrial reactive oxygen species, decreased mitochondrial membrane potential, and modulated mRNA levels of nuclear encoded mitochondrial genes and components of the senescence-associated secretory phenotype (SASP). Thus, miR-15b is a negative regulator of stress-induced SIRT4 expression thereby counteracting senescence associated mitochondrial dysfunction and regulating the SASP and possibly organ aging, such as photoaging of human skin.

TO THE EDITOR The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons, and related environmental contaminants (Abel and Haarmann-Stemmann, 2010). The unligated AhR is trapped in a cytosolic multiprotein complex, which rapidly dissociates upon ligand binding. Subsequently, the AhR shuttles into the nucleus, dimerizes with ARNT, and binds to xenobiotic-responsive elements (XREs) in the promoter of target genes, e.g., encoding cytochrome P450 (CYP) 1 monooxygenases, to enforce transcription (Abel and Haarmann-Stemmann, 2010). Furthermore, AhR-triggered activation of c-src tyrosine kinase stimulates EGFR and downstream mitogen-activated protein kinase signaling, resulting in the induction of XRE-independent genes, such as cyclooxygenase-2 (COX-2; Abel and Haarmann-Stemmann, 2010). We have previously shown that the AhR in keratinocytes is not only activated by anthropogenic chemicals but also by UVB irradiation, which leads to the intracellular formation of the tryptophan photoproduct and high-affinity AhR ligand 6-formylindolo[3,2-b]carbazole (FICZ; Rannug et al., 1995; Fritsche et al., 2007). Indeed, UVB exposure enhances AhR/XRE binding (Supplementary Figure 1 online) and accompanied CYP1A1/1B1 expression (Katiyar et al., 2000), as well as XRE-independent COX-2 expression (Fritsche et al., 2007). Because (i) overexpression of a constitutively active AhR causes inflammatory skin lesions (Tauchi et al., 2005), (ii) an increase in CYP activity leads to reactive oxygen species formation (Puntarulo and Cederbaum, 1998), (iii) CYP1 enzymes are critical for chemical-induced skin carcinogenesis (Shimizu et al., 2000), and (iv) COX-2 is involved in UV-induced inflammation and carcinogenesis (Elmets et al., 2010), it was postulated that a transient inhibition of AhR may protect human skin against the detrimental effects of UVB irradiation (Agostinis et al., 2007; Haarmann-Stemmann et al., 2012). Moreover, we have shown that the expression of matrix metalloproteinase-1 (MMP-1), which is critically involved in extrinsic skin aging, is upregulated in an AhR-dependent manner in tobacco smoke extract–exposed keratinocytes (Ono et al., 2013). Therefore, we decided to develop an AHR antagonist that is suitable for topical UV-protection. We screened a library of compounds that possess the structural prerequisites to interact with AhR and identified E/Z-2-benzylidene-5,6-dimethoxy-3,3-dimethylindan-1-one (BDDI; Figure 1a) as the most promising candidate. Figure 1 Characterization of antagonistic capacities of E/Z-2-benzylidene-5,6-dimethoxy-3,3-dimethylindan-1-one (BDDI) in normal human epidermal keratinocytes (NHEKs) In concentrations from 0.33 to 33 µM, BDDI did not negatively affect cell viability or cause cytotoxicity in normal human epidermal keratinocytes (NHEKs; Figure 1b; for description of methods see Supplementary Material online). It is noteworthy that exposure to higher concentrations of BDDI enhanced cell viability (Figure 1b) without increasing the proliferation rate (data not shown). Exposure of NHEKs to 0.33 to 33 µM BDDI or 10 µM of the specific AhR inhibitor 3′-methoxy-4′-nitroflavone (MNF; Lu et al., 1995) resulted in a concentration-dependent decrease of basal CYP1A1 expression (Figure 1c). AhR activation by 10nM FICZ or 250 nM benzo(a)pyrene [B(a)P] resulted in 12- to 14-fold induction of CYP1A1 transcription after 4 h, whereas irradiation with 100 Jm−2 UVB led to a fourfold increase in CYP1A1 expression after 8 hours (Figure 1c). Pretreatment with 10µM MNF or 33 µM BDDI significantly attenuated CYP1A1 induction. Interestingly, a dose of 3.3 µM BDDI was sufficient to repress UVB- and FICZ-stimulated, but not B(a)P-induced, CYP1A1 expression, which was probably due to the different half-lives of the inducing agents (Figure 1c). To confirm the inhibitory effect of BDDI on CYP1A1, we treated NHEKs for 24 h with 250 nM B(a)P alone or in combination with BDDI and measured CYP1A-mediated 7-O-ethoxyresorufin-deethylase (EROD) activities. A 1 hour pretreatment with 0.33 to 33 µM BDDI resulted in a dose-dependent decline of B(a)P-induced EROD activity (Figure 1d), thereby confirming the AhR antagonistic properties of BDDI. Importantly, BDDI only transiently represses AhR function. Whereas a BDDI pretreatment for 1 hour attenuated UVB-mediated CYP1A1 induction in NHEKs, a pretreatment for 24 hours was not effective (Figure 1e). BDDI treatment of NHEKs directly after irradiation also decreased UVB-mediated CYP1A1 induction, providing evidence that BDDI does not act as a UVB-filter (Figure 1f). To elucidate the mode of action of BDDI, we performed an electrophoretic mobility shift assay (EMSA) that is well established to detect a direct binding of AhR/ARNT to a XRE consensus oligonucleotide (Denison et al., 1988; Vogel et al., 2004). Upon exposure of human HaCaT keratinocytes to 10 nM TCDD or 100 nM FICZ, we observed a strong binding of the AhR/ARNT complex to its DNA target motif (Figure 2a). Co-exposure of HaCaT cells to 3.3 µM BDDI or 10 µM MNF clearly blocked TCDD-and FICZ-triggered AhR/XRE binding (Figure 2a), providing evidence that BDDI acts as a true competitive AhR antagonist. Figure 2 BDDI disturbs XRE binding of aryl hydrocarbon receptor (AhR)/ARNT and represses UVB-induced gene expression in a human in vivo study To investigate whether BDDI is suitable for UV-protection of human skin, we treated defined skin areas of 10 healthy volunteers once daily on 4 consecutive days with a formulation containing 0.5% BDDI or a placebo formulation (Figure 2b). On day 4, 2 hours after the application of the substances, volunteers were irradiated with 1.5 MED (minimal erythema dose) UVB, and 24 hours later skin biopsies were taken. Quantitative expression analyses revealed a significantly increased expression of CYP1A1, COX-2, and MMP-1 in UVB-irradiated compared with sham-irradiated skin. Topical application of BDDI, but not the placebo formulation, significantly reduced the UVB-induced expression of all these genes, indicating that BDDI penetrates human skin and blocks AhR-dependent signaling. This experiment also revealed that the AhR is activated upon UVB irradiation in human skin in vivo. Importantly, the erythema response of the volunteers was not significantly affected during the study. As CYP1A1, COX-2, and MMP-1 are critically involved in cutaneous inflammatory diseases, skin cancer, and skin aging, we propose that the topical application of this chemical inhibitor presents a promising strategy to protect human skin against UVB-induced damage. In contrast to MNF (structural safety alert), BDDI has the clinical advantage of being suitable for dermal applications in humans. Our in vitro data further indicate that BDDI may protect against the adverse effects of polycyclic aromatic hydrocarbons, which are frequently found on airborne particulate matter (Vierkotter et al., 2010). Finally, BDDI may serve as a tool to study the involvement of AhR signaling in human skin (patho)physiology.

Loss of subcutaneous fat is a hallmark of ageing usually starting in the face. Attempts to ameliorate cosmetically the appearance of subcutaneous fat loss have been of limited success as they fail to rebuild the missing subcutaneous tissue. Ageing-driven loss of subcutaneous fat results from (i) the reduced capacity of pre-adipocytes to differentiate into adipocytes and (ii) the fact that adipocytes of the elderly secrete increased amounts of TNFα, that in turn enhances lipolysis, inhibits pre-adipocyte differentiation and induces dedifferentiation of adipocytes. The neolignan dihydrodehydrodiisoeugenol (DDE) caused a 30% increase in lipid accumulation in murine 3T3-L1 cells. This effect was accompanied by an induction of the differentiation-associated transcription factors peroxisome proliferator-activated receptorγ (PPARγ2), CAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protein 4 and adiponectin, and a loss of the pre-adipocyte marker Pref1. In addition, DDE diminished both basal and TNFα-induced lipolysis. Similar results were obtained in human subcutaneous (hsc) pre-adipocytes cultured in an age-adapted hormone mix with reduced levels of insulin and dexamethasone. In this system, DDE significantly increased lipid accumulation by 71% and 94% and was associated with an induction of PPARγ2 and adiponectin mRNA expression. DDE also reduced basal lipolysis in mature hsc adipocytes. DDE acted as a partial PPARγ agonist because (i) DDE displaced PPARγ ligand from the human PPAR ligand-binding site, (ii) DDE-induced lipid accumulation and (iii) DDE-induced adiponectin secretion could be overcome by the addition of PPARγ antagonists. Taken together, these studies identify DDE as a compound well suited to prevent and reverse loss of subcutaneous fat.

Introduction and Objectives: In recent years there has been an increasing interest in the use of nutritional supplements to benefit human skin. Molecular evidence substantiating such effects, however, is scarce. In the present study we investigated whether nutritional supplementation of women with the standardized pine bark extract Pycnogenol® will improve their cosmetic appearance and relate these effects to expression of corresponding molecular markers of their skin. Materials and Methods: For this purpose 20 healthy postmenopausal women were supplemented with Pycnogenol for 12 weeks. Before, during and after supplementation, their skin condition was assessed (i) by employing non-invasive, biophysical methods including corneometry, cutometry, visioscan and ultrasound analyses and (ii) by taking biopsies and subsequent PCR for gene expression analyses related to extracellular matrix homeostasis. Results: Pycnogenol supplementation was well tolerated in all volunteers. Pycnogenol significantly improved hydration and elasticity of skin. These effects were most pronounced in women presenting with dry skin conditions prior to the start of supplementation. The skin-physiological improvement was accompanied by a significant increase in the mRNA expression of hyaluronic acid synthase-1 (HAS-1), an enzyme critically involved in the synthesis of hyaluronic acid, and a noticeable increase in gene expression involved in collagen de novo synthesis. Conclusions: This study provides skin-physiological and for the first time molecular evidence that Pycnogenol supplementation benefits human skin by increasing skin hydration and skin elasticity. These effects are most likely due to an increased synthesis of extracellular matrix molecules such as hyaluronic acid and possibly collagen. Pycnogenol supplementation may thus be useful to counteract the clinical signs of skin aging.

UVA(Ultraviolet A)-induced gene expression is supposed to be a hallmark for inflammation, for immunosuppression and in long-term cancer formation. In previous studies, we have shown for keratinocytes that physiological doses of UVA radiation result in the upregulation of gene expression mediated by ceramide formation from sphingolipids/cholesterol-rich microdomains (rafts), which can be blocked by preloading keratinocytes with cholesterol or plant sterols. Here, we show that besides stigmasterol and s-sitosterol, also sterols like 14-dehydroergosterol, ergosterol-peroxide and 29-norcycloartenol inhibit the UVA response. Moreover, we present evidence that natural material-derived triterpenoids such as oleanolic acid can abrogate UVA-induced gene expression by raft stabilization. This effect depends on the structure of the molecule, because its isomer ursolic acid also integrates within the rafts without inhibiting ceramide formation and upregulation of gene expression.

Compatible organic osmolytes, such as betaine, myo-inositol and taurine, are involved in cell protection. Human dermal fibroblasts accumulate these osmolytes and express mRNA specific for their transporting systems betaine-/gamma-amino-n-butyric acid (GABA) transporter (BGT-1), sodium-dependent myo-inositol transporter (SMIT) and taurine transporter (TAUT). Taurine uptake was about sixfold higher than that of betaine and myo-inositol. Compared with normoosmotic (305 mOsm/l) control, hyperosmotic exposure (405 mOsm/l) led to a twofold induction of osmolyte uptake. Ultraviolet A (UVA) upregulated osmolyte transporter mRNA levels and increased osmolyte uptake. Taurine inhibited UVA-induced interleukin-6 (Il-6) mRNA expression by 40%. Furthermore, Il-6 accumulation in the supernatants of UVA-irradiated dermal fibroblasts was much slower when cells were preincubated with taurine. These data indicate that taurine accumulation seems to be part of the fibroblast response to UVA radiation and may protect against UVA-induced Il-6 overexpression.

Bathing in the Blue Lagoon, a specific geothermal biotope in Iceland has been known for many years to be beneficial for human skin in general and for patients with psoriasis and atopic dermatitis in particular. The scientific rationale for this empirical observation, however has remained elusive. We now report that extracts prepared from silica mud and two different microalgae species derived from the Blue Lagoon are capable of inducing involucrin, loricrin, transglutaminase-1 and filaggrin gene expression in primary human epidermal keratinocytes. The same extracts also affects primary human dermal fibroblasts, because extracts from silica mud and one type of algae inhibited UVA radiation-induced upregulation of matrix metalloproteinase-1 expression and both algae, as well as silica mud extracts induced collagen 1A1 and 1A2 gene expression in this cell type. These effects were not restricted to the in vitro situation because topical treatment of healthy human skin (n = 20) with a galenic formulation containing all three extracts induced identical gene regulatory effects in vivo, which were associated with a significant reduction of transepidermal water loss. In aggregate, these results suggest that the bioactives in Blue Lagoon have the capacity to improve skin barrier function and to prevent premature skin ageing. These observations explain at least some of the beneficial effects of bathing in the Blue Lagoon and provide a scientific basis for the use of Blue Lagoon extracts in cosmetic and/or medical products.

Harnstoffhaltige Externa werden seit Langerem erfolgreich zur Verbesserung der Barrierefunktion der Haut eingesetzt. In der vorliegenden Studie sollte untersucht werden, ob die Wirksamkeit eines harnstoffhaltigen Externums durch den Zusatz von Vitaminen und Ceramiden verbessert werden kann. Hierzu wurde an 10 hautgesunden Probanden eine intraindividuelle Vergleichsuntersuchung durchgefuhrt. Es zeigte sich, dass nach einer 14-tagigen Applikation das Kombinationspraparat aus Harnstoff, Vitaminen und Ceramiden dem Harnstoffmonopraparat hinsichtlich einer Reduktion des transepidermalen Wasserverlustes und einer Erhohung der Hautfeuchtigkeit signifikant uberlegen war. Diese verbesserte Wirksamkeit war mit einer verstarkten transkriptionellen Expression von Keratinozytendifferenzierungsgenen in den behandelten Hautarealen assoziiert. So fuhrte die Applikation beider Praparate zu einer vermehrten mRNS-Expression von Transglutaminase, Involucrin, Loricrin und Filaggrin, die vergleichend fur die mit dem Kombinationspraparat behandelten Hautareale signifikant starker ausfiel. Diese Untersuchung zeigt, dass die Wirksamkeit harnstoffhaltiger Externa durch die Zugabe von Ceramiden und Vitaminen gesteigert werden kann.

Photoaged skin is characterized by a crease of dermal collagen fiber, exculting from an increased breakdowb and a diminished de novo synthesis. The increased breakdown results from an increased expression of matrix metalloproteinases (MMPs). The main building blocks involved in de novo synthesis of collagen fibers are collagen 1A1 and 1A2, the expression of which is reduced in photoaged skin. We studied the effect of topical application of vitamins, phytosterols and ceramides on UV-induced up-regulation of the expression of MMP - and on UV-induced down-regulation of COL 1A 1 and COL 1A2. The study was conducted with 10 subjects with healthy skin who were comparatively treated for 10 days with (i) a basic preparation containing jojoba oil, (ii) the basic preparation supplemented with vitamins, (iii) the basic preparation supplemented with and (iv) the basic preparation supplemented with vitamins, phytoeterols and oeramides, All four preparations inhibited the UV induced up-regulation ot MIMP- 1. Neither the basic product nor that supplemented with vitamins inhibited down-regulation of COL 1A 1 and COL 1A2, but addition of phytosterols and ceramides caused a decreased down-regulation of the expression of these genes. Our results indicate that phytosterols and ceramides are effective in blocking the reduced collagen synthesis after UV irradiation and even stimulating synthesis. They may be useful additions to anti-aging products.

Solar ultraviolet A (UVA) (320-400 nm) radiation-induced gene expression in keratinocytes is initiated at the level of the cell membrane via generation of singlet oxygen and subsequent formation of ceramide from sphingomyelin. We now report that the UVA response also involves raft signaling and that ceramide and raft signaling are linked with each other. Upon UVA irradiation, the lipid composition of rafts decreased 40% in sphingomyelin and 60% in cholesterol (Chol). Also, decrease of Chol increased the susceptibility towards UVA-induced gene expression, whereas increase of Chol completely abolished their capacity to generate signaling ceramides and to mount the subsequent UVA response. This inhibition was not associated with UVA-induced Chol oxidation and was also seen after treatment of cells with plant sterols. The UVA responsiveness depended on the ratio of Chol versus ceramide in rafts. A ratio smaller than 1 permitted initiation and transduction of the signaling response, whereas a ratio greater than 1, for example, upon sterol pretreatment, abolished this response, indicating that UVA radiation-induced ceramide signaling is controlled by the lipid composition of rafts.

The 'matrikine' concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti-ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double-blind, randomized, placebo-controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three-dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.

The damaging effects of UVA radiation have been well-documented. UVA radiation is known to induce molecular, cellular, and clinical damage. Such harm may lead to photoaging, immune system depression, altered gene expression, or oncogene and tumor suppressor gene modulation, all of which are partly responsible for the development of skin cancer. In parallel to an increased understanding of the added damage caused by UVA radiation, progress has been made in sunscreen formulation. A variety of UVA filters are now available for formulators to combine with UVB filters to reach high-level photostable protection using a minimum concentration of active ingredients. The efficacy of products that contain these UV filter combinations usually is determined by noninvasive assessments, which cause either UVA-induced erythema or skin pigmentation. However, the biologic relevance of these end points for UVA radiation-induced skin damage is unknown. In our study, we confirm that the assessment of UVA radiation-induced gene expression in skin specimens obtained from UVA-irradiated human skin by quantitative real-time polymerase chain reaction is a sensitive, reliable, and robust method to prove the efficacy of 2 daily moisturizers containing broad-spectrum sunscreen. Specifically, we demonstrate in vivo that topical application of a daily moisturizer with broad-spectrum sunscreen prevents UVA radiation-induced transcriptional expression of genes that are directly linked to skin aging (ie, matrix metalloproteinase 1 [MMP-1]) and also reflect the skin's antioxidative stress defense response (ie, catalase [CAT], superoxide dismutase [SOD], glutathione peroxidase [GPx]). Furthermore, we demonstrate that the protection against UV-induced skin damage provided by products with different sun protection factor (SPF) but the same UVA protection factor (UVA-PF) is similar, which emphasizes the importance of high UVA protection to maintain unaltered essential biologic functions. These data indicate that the use of a daily moisturizer containing broad-spectrum sunscreen with a well-balanced SPF/UVA-PF ratio on a regular basis is beneficial for human skin.

Uneven skin pigmentation is a significant cosmetic concern, and the identification of topically applicable molecules to address this issue is of general interest. We report that the tetrapeptide PKEK (Pro-Lys-Glu-Lys) can exert skin whitening effects based on one in vitro and four double-blinded vehicle-controlled in vivo studies. (i) Treatment of human keratinocytes with PKEK significantly reduced UVB-stimulated mRNA expression of interleukin (IL)-6, IL-8 and TNF-α and, most importantly, proopiomelanocorticotropin (POMC), i.e. a gene encoding the pigmentation-inducing soluble mediator α- (α-MSH). (ii) PKEK treatment significantly inhibited UVB-induced upregulation of genes encoding for IL-1α, IL-6, IL-8, TNF-α as well as POMC and tyrosinase in 10 healthy volunteers pretreated with PKEK for 4 weeks once daily. (iii) In a study enrolling 39 Caucasian women, facial pigment spots significantly faded after 6 weeks when PKEK was combined with the skin whitener sodium ascorbyl phosphate (SAP), whereas PKEK or SAP alone led to less pronounced fading of the pigment spots. (iv) Addition of PKEK enhanced the skin whitening potency of a SAP-containing preparation if applied for 8 weeks to the back of hands of 19 Caucasians. (v) 27 Japanese women were treated on their faces twice daily with an SAP only or a PKEK+SAP-containing formulation for 8 weeks. Application of PKEK+SAP significantly reduced skin pigmentation by 26% and by 18% according to SCINEXA score. We demonstrate that PKEK has the capacity to reduce UVB-induced skin pigmentation and may be suited to serve as a skin tone-modulating agent in cosmetic products.

UVA(Ultraviolet A)-induced gene expression is supposed to be a hallmark for inflammation, for immunosuppression and in long-term cancer formation. In previous studies, we have shown for keratinocytes that physiological doses of UVA radiation result in the upregulation of gene expression mediated by ceramide formation from sphingolipids/cholesterol-rich microdomains (rafts), which can be blocked by preloading keratinocytes with cholesterol or plant sterols. Here, we show that besides stigmasterol and ß-sitosterol, also sterols like 14-dehydroergosterol, ergosterol-peroxide and 29-norcycloartenol inhibit the UVA response. Moreover, we present evidence that natural material-derived triterpenoids such as oleanolic acid can abrogate UVA-induced gene expression by raft stabilization. This effect depends on the structure of the molecule, because its isomer ursolic acid also integrates within the rafts without inhibiting ceramide formation and upregulation of gene expression.

The 'matrikine' concept claims that processing of the precursors for collagen results in the formation of peptides such as KTTKS which in turn augments extracellular matrix (ECM) production. In the present study, we show the development of an anti-ageing active from an in silico approach by molecular design resulting in the tetrapeptide GEKG derived from ECM proteins. The efficacy of the peptide to significantly induce collagen production of the protein level and mRNA level has been demonstrated in vitro in human dermal fibroblasts and in vivo in a double-blind, randomized, placebo-controlled study enroling 10 volunteers with an average age of 48.2 years. The effect of GEKG on facial wrinkles was studied in 30 volunteers using state of the art fringe projection, which allows determination of surface roughness in three-dimensions. Here, only GEKG but not the placebo was able to significantly decrease skin roughness as a measure for wrinkles.

Exposure of human keratinocytes to ultraviolet A (UVA) radiation at physiological doses leads to a biphasic activation of transcription factor activator protein-2 (AP-2) and subsequently to a biphasic increase in gene expression of, e.g. intercellular adhesion molecule-1 (ICAM-1). Both kinetics follow a pattern with a first peak between 0.5 and 2 h and a second, more sustained activation between 16 and 48 h. We have previously reported on a non-enzymatic triggering of the ceramide signaling cascade as the initiating step in UVA radiation-induced signaling. In this study, we report that this early (0.5–1 h) peak in ceramide content is followed by a second peak that (i) was associated with an increased expression and activity of serine palmitoyltransferase, the key enzyme of ceramide synthesis, (ii) could be prevented by inhibitors of this enzyme, and (iii) was of functional relevance because its inhibition abrogated the second, but not the first peak in UVA radiation-induced ICAM-1 gene expression. We hypothesize that this second peak most likely resulted from a ceramide-mediated autocrine loop, for (i) inhibition of the first ceramide peak resulted in inhibition of the second peak and (ii) cell-permeable ceramides-induced serine palmitoyltransferase expression, activity, and subsequently ceramide content.

The intracellular signaling pathway(s) through which second messenger ceramides induce gene expression in human cells has not yet been characterized. In the present study, ceramide-induced expression of intercellular adhesion molecule-1 (ICAM-1), which requires activation of transcription factor activator protein 2 (AP-2), was found to be mediated through a mitochondrial pathway. Inhibitors of mitochondrial electron transport chain (e.g. rotenone, thenoyltrifluoroacetone, and antimycin A) reduced ceramide-induced ICAM-1 expression. Stimulation of human keratinocytes with cell-permeant ceramides at concentrations that did not induce apoptosis (no activation of caspases 3, 8, and 9 and no nucleosomal fragmentation) but caused AP-2 activation and ICAM-1 induction released cytochrome c (cyt c) from mitochondria into the cytoplasm of cells. This cyt c release was an indispensable prerequisite for effective ceramide signaling, because its inhibition by modulating the mitochondrial megachannel with bonkrekic acid or carboxyatractyloside prevented ceramide-induced AP-2 activation and ICAM-1 expression. Analysis of the interaction between cyt c and AP-2 revealed that cyt c oxidized AP-2 and that this redox regulation greatly enhanced the DNA binding capacity of AP-2. Mitochondria thus have a previously unrecognized function in signaling ceramide-induced transcription factor activation and gene regulation.

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin.