Hypothalamic-pituitary-adrenal (HPA) axis is the main regulatory algorithm maintaining body homeostasis after stress challenge. Ultraviolet B (UVB) is the skin-specific stressor, which is able to induce neuroendocrine changes not only in the skin, but also in distant organs. The goal of this project was to evaluate a role of UVB radiation in regulation of the HPA axis activity with the use of hypophysectomized (Hypox) mice. Intact and Hypox females of C57 BL/6 in telogen were shaved and

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... ed with 400 mJ/cm 2 of UVB waveband. Following 12 and 24 h time of exposure, plasma, skin, brain and adrenals were collected and extracted for RNA, peptides/proteins, and steroids or fixed for immunohistochemistry (IHC). Expression of the crucial components for HPA axis activation [corticotropin-releasing hormone (CRH), urocortin (Ucn), β-endorphin (β-END), adrenocorticotropin (ACTH) and corticosterone (CORT)] has been measured. Cutaneous UVB radiation led to increased CRH, Ucn, β-END, ACTH and CORT plasma levels in intact animals. Hypox mice followed this pattern but only partially. There was no increase in ACTH and CORT plasma levels in Hypox mice after UVB treatment, except of increased CRH in paraventricular nucleus of hypothalamus and of CRH with Ucn in plasma. Only intact animals showed increased expression of mRNAs coding melanocortin receptor 2 (Mc2R) as well as of Star and Cyp11b1 in adrenal glands. However, the "cutaneous HPA axis" was maintained and stimulated in both animal groups after UVB exposure. UVB irradiated skin expressed higher levels of CRH (by IHC), and of Ucn, β-END and CORT both at gene and peptide levels. In conclusion, UVB radiation stimulates both local and central HPA axis activity in mice leading to increased concentrations of immunosuppressive CORT in plasma. The UVB-activation of central HPA axis is dependent on pituitary, since Hypox animals showed lack of stimulation of plasma levels of ACTH and CORT. The skin is a multilayered epithelium that protects organisms from external challenges such as UV radiation. UV-induced persistent DNA damage is a primary cause for carcinogenic mutations and tumour permissive systemic immune suppression. Previously we have shown that epidermal loss of IGF-1R signalling reduced proliferative potential and antagonized hyperplasia induced by a carcinogenic stimulus. Here we asked how epidermal IGF-1R signalling regulates acute and long term UVB response using mice with an epidermal specific deletion of the IGF-1R (IGF-1R epi-/-). Although these mice have a thinner epidermis, UVB irradiation did not result in an increased amount of DNA damage. However, IGF-1R epi-/mice were more susceptible to UVB-induced apoptosis. Surprisingly, despite this initial increase in cell death, UVB radiation induced an enhanced and sustained hyperproliferative response in IGF-1R epi-/mice compared to control, as judged by interfollicular epidermal thickness and BrdU positive cells. This is in contrast to the requirement of IGF-1R during epidermal development when loss of IGF-1R counteracts growth and proliferative potential. Further investigation showed an increased and prolonged inflammatory response as judged by increased numbers of macrophages and neutrophils in the dermis. Since newborn IGF-1R epi-/mice showed increased total P53 protein levels and P53 is known to regulate inflammation in other systems, we next asked whether adult IGF-1R epi-/also had increased P53 levels. Immunofluorescence analysis revealed an increase in nuclear P53 levels in IGF-1R epi-/mice. To examine if the increased nuclear P53 is responsible for the enhanced hyperproliferation and inflammation in IGF-1R epi-/mice, we crossed P53 floxed and IGF-1R floxed mice with the K14-Cre mice. Combined epidermal inactivation of P53 and IGF-1R -/counteracted the prolonged epidermal hyperproliferative and inflammatory response. Our data indicate that loss of epidermal IGF-1R signalling controls the UVB induced hyperproliferative and inflammatory response via regulation of p53 activity. 469 Nicotinamide replenishes cellular energy and enhances DNA repair in UV-irradiated human melanocytes and keratinocytes Nicotinamide (vitamin B3) prevents UV-induced immunosuppression and reduces premalignant keratoses and possibly nonmelanoma skin cancer in Phase 2 clinical trials. As a precursor of nicotinanide adenine dinucleotide (NAD + ), nicotinamide plays a central role in cellular energy pathways. To determine nicotinamide's effects on DNA repair, we incubated human HaCaT keratinocytes and normal human keratinocytes and melanocytes with and without nicotinamide and then exposed the cells to solar-simulated UV radiation. Using an unscheduled DNA synthesis (UDS) assay, we found enhanced DNA repair after irradiation in all cell types (each n=6; p<0.03; one way ANOVA). Cells were also irradiated with and without nicotinamide, then allowed to undergo DNA repair before being fixed and stained for oxidative DNA damage (8-oxo-2'-deoxyguanosine; 8oxoG). Nicotinamide significantly increased 8oxoG repair in irradiated HaCaTs, normal keratinocytes and melanocytes (each n=6, p<0.05, repeated measures ANOVA). We also assessed the effect of a highly selective inhibitor of NAD + production, FK866, on DNA repair (UDS) in keratinocytes and primary human melanocytes. FK866 significantly reduced the ability of nicotinamide to enhance DNA repair in each cell type (both p=0.02, n=6, one way ANOVA), suggesting that its protective effects occur by preventing UV-induced ATP loss. To confirm this, we used a Seahorse Bioscience Glycolysis Stress Test to monitor the effects of nicotinamide and UV irradiation on cellular metabolism. HaCaT keratinocytes were incubated for 24h with these treatments, and then irradiated. Glycolysis, represented by extracellular acidification rate, was monitored in real time 2 hours post-irradiation. UV reduced glycolysis by 33% (p<0.001), whilst nicotinamide increased glycolysis by 24% (UV vs Nicotinamide+UV p<0.01; n=20, two-way ANOVA). Nicotinamide is nontoxic and inexpensive and is a promising agent for skin cancer chemoprevention. 470 Lipid oxidation patterns and -kinetics in keratinocytes under senescence-promoting stress Oxidation of lipids and proteins is a manifestation of aged skin and potentially causative for age-related aesthetic decline and pathology. Extrinsic oxidative stress generates reactive lipid oxidation products. Polyunsaturated fatty acids of phospholipids (PL) are easily oxidized by stressors that promote skin aging, and the resulting lipid mediators elicit stress responses. To study in keratinocytes, which oxidation products are generated upon UV stress and to study kinetics of intrinsic and extrinsically added oxidized PL, we performed lipidomic analysis. We applied a novel HPLC-tandem-MS method and quantified over 500 PUFA-PL oxidation products in KC immediately and 24 hours after irradiation with 40J/cm 2 UVA-1. We also performed analysis of global mRNA expression and of selected cyto/chemokines and stress response enzymes on protein level. Studying the PL kinetics, we found,unexpectedly, that immediately after UVA-1 radiation PL containing esterified dicarboxylic acids show higher accumulation than PL hydroperoxides and -hydoxides. Levels of dicarboxylic acid containing PL returned to baseline after 24h, while significantly increased PL-hydroxide levels persisted. Exogenously added UV-oxidized PL initially underwent rapid oxidation and chain shortening, whereas after 24 hours a massive increase of F and E,I,D class PL-Isoprostanes was detected. The transcriptomic and proteomic profiling of the stressed KC performed in parallel indicated that part of the observed changes in the lipid profile after 24h would be compatible with the transcriptional modulation of enzymes regulating prostanoid metabolism, like prostaglandin F synthase. As isoprostanes and isoprostane containing PL are correlated to aging (also of the skin), the modulation of isoprostane levels by UVA may be a novel mechanism contributing to photoageing. In this study we validated our 3D-human dermis equivalent (3D-HDE), as an in vitro biological platform to study ECM modification under simulated solar exposure and as screening platform for testing cosmetic agents. 3D-HDE has been realized by means of a tissue engineering bottom-up approach. Due to endogenous and three-dimensional features of this tissue model is possible to analyze structure, composition and ECM assembly, tissue remodeling due to damage and photo-protective action of active compound. Samples have been investigated by Multiphoton Microscopy (MPM), immunofluorescence analyses (IF) and histological analyses. Second harmonic generation (SHG) imaging has been exploited to assess modification in endogenous collagen assembly with and without UV irradiation. Texture analysis has been performed by Grey Level Co-occurrence Matrix on the SHG images in order to assess the correlation length (L C ) a parameters indicating the coarsening degree of the collagen matrix. The results revealed that 3D-HDE UVA irradiated suffered a strong disappearance of fibroblasts; nuclei with irregular morphology and ECM alterations. Furthermore, IF test showed an up-regulation of both MMP-1 and MMP-9 and histological analyses by Picro Sirius Red staining showed a decrease of both mature and immature collagen. According to literature data on photoaged skin, 3D-HDE UVA irradiated showed an increase in Lc respect to control, it means that collagen degeneration causing change in textural features of collagen matrix. Moreover, active compound tested as photoprotectans show reduce the damage due to UV irradiation both at cellular and ECM level. Taken together these results highlight that our model is unique in the capability to recapitulate in vitro relevant phenomena occurring in the native dermis during human daily life such as chronic exposure to sunlight. The photoaging of the skin is characterized by wrinkles, mottled pigmentation, telangiectasia and malignancies due to the effects of long-term UV exposure. Photodynamic therapy (PDT) is a therapeutic modality for cutaneous malignant and premalignant condition. Recently, several clinical studies reported that the PDT has photorejuvenation effects on the aged skin. In the previous report, which is examined direct effect of PDT on fibroblast (FB), PDT induced increase of matrix metalloproteinase (MMP)-3 and collagen type Iα through prolonged activation of extracellular signal-regulated kinase (ERK). Increased MMP-3 may induce breakage of old collagen and allow new collagen synthesis. In this study, we investigated molecular mechanism of photorejuvenation after PDT focused on interaction between keratinocyte (KC) and FB. The "low level PDT" condition was used for PDT therapy to the KC and FB. FBs were stimulated with the KC-conditioned medium taken after PDT. Various kinds of cytokines in the supernatants of KC were evaluated by enzyme-linked immunosorbent assay (ELISA). The mRNA level of MMPs, transforming growth factor (TGF)-β and collagen type Iα in the FB were determined by real-time polymerase chain reaction (PCR). After stimulating FB with KC-conditioned medium taken after PDT, the mRNA of MMP-1 was decreased and the mRNA of collagen type Iα were increased compare to control. However, MMP-3 and TGF-β were not significantly changed. Among the FB stimulating cytokines, a significant elevation of interleukin (IL)-1α, IL-6, tumor necrosis factor (TNF)-α level in KC supernatants was noted after PDT compared with the untreated controls. TGF-β was not significantly altered after PDT. In conclusion, PDT induced new collagen synthesis may be mediated not only by direct effect of PDT on FB but also by indirect effect of PDT on FB through cytokines from KC such as IL-1α, IL-6 and TNF-α. ABSTRACTS | Photobiology Xeroderma pigmentosum (XP) is a rare photosensitive genodermatosis with defective post-UV DNA repair. Clinically XP patients have pigmentary abnormalities and a very high incidence of skin cancers. There are genetically different seven nucleotide excision repair (NER) deficient complementation groups (A through G) and translesion DNA synthesis (TLS) deficient form (XP variant). More than 50% of the Japanese XP patients are the severest form of XP, XP group A (XP-A) and all of the XP-A patients have XP -related neurological abnormalities caused by unknown pathogenesis. Here we found a very unusual Japanese case of XP-A. The patient, 69-year-old female (XP46HM) was referred to our department for an assessment of a SCC on her lip with mild cutaneous XP features. Neither neurological nor ocular abnormalities were detected. Cultured cells from the patient are hypersensitive to killing by UV and the level of the cells of post-UV unscheduled DNA synthesis was 11% of normal cells. The impaired host cell reactivation was corrected only by expression of wild type XPA. Genetic analysis showed a novel homozygous c.529G>A mutation (p.D177N) in exon4 of XPA gene in XP46HM. Interestingly, this genetic change resulted in 29 bp deletion in XPA mRNA in 3' site of exon 4 causing missplicing, nonsense-mediated XPA mRNA decay (NMD) and no detectable XPA protein by Western blot analysis, while a very little existence of intact XPA mRNA was confirmed by XPA cDNA RFLP analysis. These findings suggest that the very mild form XP symptoms without neurological problems in the XP-A patient (XP46HM) may be associated with extremely small quantity of functional XPA mRNA escaped from missplicing and NMD.