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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.

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.

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.