Project description:Ultraviolet light is the dominant environmental oxidative skin stressor and a major skin aging factor. We studied which oxidized phospholipid (OxPL) mediators Ultraviolet A (UVA) would generate in primary human keratinocytes (KC). Mass spectrometric analysis of the oxidized phospholipidome of KC immediately or 24h post stress revealed dynamic changes in abundance of 174 oxidized phosphocholine species. Exposure to UVA and to in vitro UVA - oxidized phospholipids both activated, on transcriptome and proteome level, NRF2/antioxidant response signaling and lipid metabolizing enzyme expression, whereas UVA additionally initiated the unfolded protein response (UPR). We identified Nupr1 as an upstream transcriptional regulator of UVA/OxPL mediated gene expression that is itself transcriptionally regulated by reactive lipids, which also aggregate and crosslink recombinant Nupr1 protein. Nupr1 governs the basal and stress regulated expression of cell cycle, redox reactive, autophagy- and lipid metabolizing genes in epidermal keratinocytes, making it a potential key factor in skin ROS responses, -aging and -pathology.
Project description:E. coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure. Keywords: stress response
Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin.
Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed âantioxidant responseâ or âPhase II detoxificationâ. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental âimageâ of UV generated lipid mediators. Experiment Overall Design: we profiled global mRNA expression levels in human dermal fibroblasts that had been treated with either UVA-1 or oxidized lipids. To investigate the effect of oxidized phospholipids on gene regulation, we used two preparations, which differed in their degree of oxidation; the minimally oxidized UV-PAPC resulting from UVA-1 irradiation of PAPC, and air-oxidized PAPC (OxPAPC), which represents the full spectrum of oxidation products (Gruber 07) (Reis et al., 2005). We irradiated dermal fibroblasts with UVA-1 (40J/cm²) or treated them with UV-PAPC, OxPAPC or native PAPC (100µg/ml each). We analyzed global gene expression four hours after stimulation with gene arrays (Affymetrix U133A Plus 2.0 Gene Chips).
Project description:E. coli growing in continuous culture under continuous UVA irradiation exhibits growth inhibition with a subsequent adaptation to the stress. Transcriptome analysis was performed during transient growth inhibition and in the UVA light-adapted growth state. The results indicate that UVA light induces stringent response and an additional response that includes the upregulation of the synthesis of valine, isoleucine, leucine, phenylalanine, histidine and glutamate. The induction of several SOS response-genes strongly points to DNA damage as a result of UVA exposure. The involvement of oxidative stress was observed with the induction of ahpCF. Taken together it supports the hypothesis of the production of reactive oxygen species by UVA light. In the UVA-adapted cell population strong repression of the acid tolerance response was found. We identified the enzyme chorismate mutase as a possible chromophore for UVA light-inactivation and found strong repression of the pyrBI operon and the gene mgtA encoding for an ATP dependent Mg2+ transporter. Furthermore, our results indicate that the role of RpoS may not be as important in the adaptation of E. coli to UVA light as it was implicated by previous results with starved cells, but that RpoS might be of crucial importance for the resistance under transient light exposure. Slide microarrays were purchased from MWG-Biotech AG (Ebersberg, Germany). The MWG E. coli Array contains 4,288 gene specific oligonucleotide probes representing the complete E. coli K12 genome. Microarray slides were scanned using the Affymetrix 428TM Array Scanner (High Wycombe, UK). Spot intensities and corresponding background signals were quantified with the Affymetrix JaguarTM software version 2.0. Further data analysis was performed with the program GeneSpring from Silicon Genetics (Redwood City, CA, USA). Induction factors (IF) were calculated from the Cy3 and Cy5 signal intensities of the spot. Spots with signal intensity below a value of 50 were excluded from the analysis and the minimal induction factor was set to 0.01. The normalization was performed with the 50th percentile distribution of remaining spots after background correction. The mean value of the IF of a specific gene was calculated from three replicates. Biological experiments were carried out three times, which provided three biological repeats. Dye swapping was performed for one replicate and did not alter the result.
Project description:Ultraviolet (UV) wavebands in sunlight are immunomodulatory. About half the amount of UVA within a minimum erythemal dose of sunlight is systemically immunosuppressive, while higher doses protect from UVB immunosuppression in mice. We have previously shown that these responses to UVA are genetically restricted as they occur in C57BL/6 but not Balb/c mice. We used gene set enrichment analysis of microarray data and real-time RT-PCR confirmation to determine the molecular mechanisms associated with UVA immunomodulation. We found up-regulation of mRNA for the alternative complement pathway. The core-enriched genes complement component 3, properdin and complement factor B were all activated by the immunosuppressive dose of UVA only in UVA-responsive C57BL/6 but not unresponsive BALB/c mice. This therefore matched the genetic restriction and dose responsiveness of UVA immunosuppression. The immune-protective higher UVA dose prevented UVB from down regulating chemokine receptor 7 and IL-12B, and decreased IL-10, supporting previous identification of IL-12 and IL-10 in high dose UVA protection from UVB immunosuppression. Our study has identified activation of the alternative complement pathway as a trigger of UVA-induced systemic immunosuppression and suggests that this pathway is likely to be an important sensor of UVA-induced damage to the skin. 24 hours after UVA, UVB and ssUV irradiation, a 1 cm2 uniform section of skin was excised from the dorsal surface of irradiated and control mice. Total RNA was then extracted from the whole skin using TRIzol reagent (Gibco Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturerâ??s instructions, purified, DNase treated and reverse transcribed into cDNA. For the microarray study a direct incorporation of Cyanine 3-dCTP and Cyanine 5-dCTP fluorescent dyes (Perkin Elmer Life Sciences, Inc. Boston, MA, USA) was used for cDNA synthesis. For each UV dose, a reference design was used to compare an unirradiated control against an irradiated sample. Microarray experiments used compugen 22k mouse oligonucleotide microarray slides (The Clive and Vera Ramaciotti Centre for Gene Function Analysis, Sydney Australia (http://www.ramaciotti.unsw.edu.au). Lower and higher UVA doses were used. C57BL/6 mice were irradiated with lower UVA, higher UVA, UVB, or ssUV; Balb/C mice were irradiated with lower or higher UVA. Experiments were replicated 6 times for each UV dose. A fluorescent dye swap was done for each alternate hybridisation to reduce systematic dye bias of incorporated fluorescent dyes.
Project description:Primary human epidermal keratinocytes were exposed to in-vitro UVA-oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or to UVA in presence and absence of a commercial UVA filter.
Project description:Long wavelength Ultraviolet (UVA-1) radiation causes oxidative stress that leads to the formation of noxious substances within the skin. As a defensive mechanism skin cells produce detoxifying enzymes and antioxidants when they detect modified molecules. We have recently shown that UVA-1 irradiation oxidizes the abundant membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which then induced the synthesis of the stress response protein heme oxygenase 1 (HO-1) in dermal fibroblasts. Here we examined the effects of UVA-1 and (UV-) oxidized phospholipids on the global gene expression in human dermal fibroblasts. We identified a cluster of genes that were co-induced by UVA-1-oxidized PAPC and UVA-1 radiation. The cluster included HO-1, glutamate-cysteine ligase modifier subunit (GCLM), aldo-keto reductases-1-C1 and -C2 (AKR1C1, AKR1C2), and interleukin 8 (IL8). These genes are members of the cellular stress response system termed “antioxidant response” or “Phase II detoxification”. Accordingly, the regulatory regions of all these genes contain binding sites for NF-E2-related factor 2 (Nrf2), a major regulator of the antioxidant response. Both UVA-1 irradiation and treatment with oxidized lipids led to increased nuclear accumulation of Nrf2. Silencing expression of Nrf2 using siRNA or using cells and tissue from Nrf2-deficient mice, we show that the induction of the co-regulated genes was suppressed. Expression of other canonical UVA-1-induced genes, including cyclooxygenase 2 (Cox2) and interleukin 6 (IL6) was unaltered in the absence of Nrf2. Together, our data show that UVA-1-mediated lipid oxidation induces induction of antioxidant response genes, which is dependent on the redox-regulated transcription factor Nrf2. To activate Nrf2 is a major strategy for novel antioxidant drugs, the skin photo-adaptation (SPA) inducers. Our finding that specific uv-oxidized lipids act similar sheds a new (ultraviolet) light on the usually detrimental “image” of UV generated lipid mediators.
Project description:This study demonstrates the cooperation of rutin and ascorbic acid in cytoprotective action against UVA induced changes in the context of proteins involved in gene expression, catalytic processes, antioxidant pathways, as well as signalling molecules including transmembrane channels. The main advantages of the work is application in research a three-dimensional (3D) cell culturing system providing the most epidermal-like conditions.
Project description:The aim of the measurements was to investigate RNA damage and RNA-protein crosslinks (RPC) formation in the context of aldehyde-induced toxicity. For this, a photoactivatable-ribonucleoside-enhanced crosslinking (PAR-CL) strategy using 4-thiouridine (4-SU) and UVA-radiation was used. The submitted dataset contains data from LC-MS/MS measurements of crosslinks between proteins and poly-adenylated mRNAs (mRPCs). The measurements included the following six samples sets: Sample set 1 (9 runs = 3 conditions in 3 replicates): RPCs after formaldehyde (FA) and 4-SU + UVA treatment isolated using the protein-x-linked RNA extraction (XRNAX) protocol (doi:10.1016/j.cell.2018.11.004) Conditions: RPCs from untreated HAP1 =Ctrl RPCs from HAP1 treated with FA RPCs from HAP1 treated with 4-SU + UVA Sample set 2 (12 runs = 4 conditions in 3 replicates): mRPCs after 4-SU + UVA treatment using poly-A pulldown Conditions: mRPCs from untreated HAP1=Ctrl mRPCs from HAP1 treated with UVA but without 4-SU (0h after irradiation) mRPCs from HAP1 treated with 4-SU but without UVA mRPCs from HAP1 treated with 4-SU + UVA (0h after irradiation) Sample set 3 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and Ub-E1i treatment using poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and Ub-E1i (0h, 0.5h and 1h after irradiation) Sample set 4 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and MG132 treatment isolated using poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and MG132 (0h, 0.5h and 1h after irradiation) Sample set 5 (24 runs = 6 conditions in 4 replicates): mRPCs after 4-SU + UVA and ANS treatment using isolated poly-A pulldown Conditions: mRPCs from HAP1 treated with 4-SU + UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 treated with 4-SU + UVA and ANS (0h, 0.5h and 1h after irradiation) Sample set 6 (24 runs = 6 conditions in 4 replicates ): mRPCs after 4-SU+UVA in AAVS1 control and RNF14 KO cells using poly-A pulldown mRPCs from HAP1 AAVS1 control cells (clone #6) treated with 4-SU+UVA (0h, 0.5h and 1h after irradiation) mRPCs from HAP1 RNF15 KO cells (clone #15) treated with 4-SU+UVA (0h, 0.5h and 1h after irradiation) Abbreviations: 4-SU: 4-thiouridine ANS: Anisomycine, a translation elongation inhibitor FA: Formaldehyde MG132: proteasome-inhibitor mRPCs: crosslinks between proteins and poly-adenylated mRNAs isolated by Poly-A pulldown RPCs: crosslinks between proteins and RNA isolated by XRNAX Ub-E1i: ubiquitin E1 inhibitor (E1i, TAK-243) UVA: Ultraviolet A