Oxidative stress mediated Ca(2+) release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells.

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Oxidative stress mediated Ca2+ release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells Mufti R. Farrukh a, Ul A. Nissar a,b, Quadri Afnan a, Rather A. Rafiq a, Love Sharma a,b, Shajrul Amin c, Peerzada Kaiser a, Parduman R. Sharma d, Sheikh A. Tasduq a,b,* a

PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, Jammu and Kashmir, India Academy of Scientific and Innovative Research (AcSIR), New Delhi, India c Department of Biochemistry, University of Kashmir, Srinagar, Jammu and Kashmir, India d Cancer Pharmacology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu Tawi, Jammu and Kashmir, India b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 November 2013 Received in revised form 21 February 2014 Accepted 31 March 2014

Background: Exposure of skin to ultraviolet (UV) radiation, an environmental stressor induces number of adverse biological effects (photodamage), including cancer. The damage induced by UV-irradiation in skin cells is initiated by the photochemical generation of reactive oxygen species (ROS) and induction of endoplasmic reticulum (ER) stress and consequent activation of unfolded protein response (UPR). Objective: To decipher cellular and molecular events responsible for UV-B mediated ER stress and UPR activation in skin cells. Methods: The study was performed on human skin fibroblast (Hs68) and keratinocyte (HaCaT) cells exposed to UV-B radiations in lab conditions. Different parameters of UVB induced cellular and molecular changes were analyzed using Western-blotting, microscopic studies and flow cytometry. Results: Our results depicted that UV-B induces an immediate ROS generation that resulted in emptying of ER Ca2+ stores inducing ER stress and activation of PERK-peIF2a-CHOP pathway. Quenching ROS generation by anti-oxidants prevented Ca2+ release and subsequent induction of ER stress and UPR activation. UV-B irradiation induced PERK dependent G2/M phase cell cycle arrest in Hs68 and G1/S phase cell cycle arrest in HaCaT. Also our study reflects that UV-B exposure leads to loss of mitochondrial membrane potential, activation of apoptotic cascade as evident by AnnexinV/PI staining, decreased expression of Bcl-2 and increased cleavage of PARP-1 protein. Conclusion: UV-B induced Ca2+ deficit within ER lumen was mediated by immediate ROS generation. Insufficient Ca2+ concentration within ER lumen developed ER stress leading to UPR activation. These changes were reversed by use of anti-oxidants which quench ROS. ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Ultraviolet-B Skin Oxidative stress Intracellular calcium Endoplasmic reticulum stress Unfolded protein response

1. Introduction Important cellular functions critical for living systems like synthesis, folding, post translational modifications and quality control of secretory and transmembrane proteins are carried out within lumen of ER [1]. ER lumen is well suited to carry out these functions as it has an oxidizing environment, which is highly critical for disulphide bond formation and has the

highest concentration of Ca2+ ions within the cell due to the active transport by Ca2+-ATPases to ensure chaperone functioning and protein folding [2–5]. Any of the disturbance (intrinsic/ extrinsic) that compromises functioning of the ER including redox or Ca2+ regulation leads to the accumulation of unfolded or misfolded proteins within ER lumen, triggering an evolutionary conserved response termed as the unfolded protein response (UPR) [6].

Abbreviations: PERK, RNA dependent protein kinase like ER kinase; peIF2a, Phosphorylated eukaryotic initiation factor 2a; CHOP, C/EBP homologous protein; PARP1, poly ADP-ribose polymerase 1. * Corresponding author at: PK-PD and Toxicology Division, CSIR-Indian Institute of Integrative Medicine, Council of Scientific and Industrial Research (CSIR), Canal Road, Jammu Tawi, Jammu and Kashmir, India. Tel.: +91 191256900010x332; fax: +91 1912659333. E-mail addresses: [email protected], [email protected] (S.A. Tasduq). http://dx.doi.org/10.1016/j.jdermsci.2014.03.005 0923-1811/ß 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Farrukh MR, et al. Oxidative stress mediated Ca2+ release manifests endoplasmic reticulum stress leading to unfolded protein response in UV-B irradiated human skin cells. J Dermatol Sci (2014), http://dx.doi.org/10.1016/ j.jdermsci.2014.03.005

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Reactive oxygen species (ROS) are produced as by-product of metabolism within the cellular organelles like ER and mitochondria and due to exposure to toxic agents such as radiations and environmental pollutants [7–9]. Accumulating evidence suggest severe damaging effects due to higher concentrations of ROS following ultraviolet (UV) irradiation of the skin leading to photo-damage [10– 12]. UV-B irradiation is known to induce ROS production at two distinct stages; immediately and hours after UV-B exposure [13]. The ROS generated immediately after UV-B exposure is due to the activity of NADPH-oxidase [14,15]. The changes in the intracellular redox environment are known to have an impact on many cellular processes, including gating properties of ion-channels and the activity of iontransporters like sarcoplasmic reticulum Ca2+ release channels [16]. ROS has also been implicated in inducing ER stress, a condition manifested with accumulation of the unfolded or the misfolded proteins within the ER lumen as a consequence of imbalance between the load of the unfolded proteins that enter the lumen and the capacity of ER to deal with this load [17,18]. ER stress is increasingly being recognized as a basic factor responsible for many human pathologies including diabetes, neurodegeneration disorders, cancers and aging [19,20].The cell responds to ER stress by activating evolutionary conserved signaling events collectively termed as UPR [21]. The three main players defining the distinct arms of UPR are IRE-1 (inositolrequiring kinase 1), ATF-6 (activating transcription factor 6) and PERK (RNA dependent protein kinase (PKR) like ER kinase). Each of these proteins is an ER trans-membrane protein, sensing the protein folding status within ER through their luminal domain and transmitting the information to the cytosol via their cytosolic domains. These proteins set up three distinct responses in motion within a cell, the first two (IRE-1 and ATF-6) which are rectifying, try to re-establish ER homeostasis. If ER homeostasis in not reinstated, a third player (PERK) is activated leading to cell death presumably to protect the tissue from the rogue cells that display the misfolded proteins [4,22]. The PERK branch of the UPR elicits pro-apoptotic effects after activation [23]. Also PERK pathway is the major pathway responsible for attenuation of mRNA translation under ER stress thereby preventing influx of new proteins into already stressed ER compartment. The attenuation of translation is mediated by phosphorylation of eukaryotic translation initiation factor 2a (eIF2a). Phosphorylation of eIF2a allows preferential translation of UPR genes such as ATF4 leading to the induction of CHOP/GADD153 and GADD34 [24]. CHOP plays a convergent role in UPR and has been identified as one of the most important mediators of ER stress induced apoptosis [25]. Activation of GADD34 by CHOP promotes dephosphorylation of eIF2a and hence reversing translational attenuation. Restoring translation leads to the accumulation of misfolded proteins in ER and permits the translation of pro-apoptotoic proteins [26]. Another possible mechanism by which CHOP induces apoptosis is down regulation of Bcl2 [27] and up regulation of Bim [28]. UV-B irradiated skin cells have been well studied for various cellular signaling pathways leading to photodamage. However, the association between UV-B mediated enhanced oxidative burden and ER stress is still not clear [29]. The current study was designed to assess the cellular and molecular basis of ER stress after UV-B irradiation leading to cell death. This is the first study to demonstrate in detail the linkage between UV-B irradiation induced ROS generation and depletion of ER Ca2+ stores leading to the development of ER stress and UPR activation. 2. Materials and methods 2.1. Cell culture and UV-B irradiation of fibroblasts (Hs68) and keratinocytes (HaCaT) The immortalized human keratinocytes cell line, HaCaT (Cell Lines Service GmbH, Germany) and human foreskin fibroblast cell

line, Hs68 (ATCC1, 1635, Manassas, VA, USA) were grown in DMEM medium, supplemented with 10% fetal bovine serum, 200 U/mL penicillin and 270 mg/mL streptomycin (Sigma–Aldrich, St. Louis, MO, USA). The cells were maintained in a humidified chamber at 37 8C and 5% CO2. The UV chamber (Daavlin, UVA/UVB Research Irradiation Unit, Bryan, OH, USA) equipped with a digital control to regulate UV doses at a fixed distance of 24 cm from the lamps to the surface of cell culture dishes was used. Majority of the resulting wavelengths (>90%) were in UV-B range (290–320 nm). UV-B doses of 10, 20 and 30 mJ/cm2 were used for experiments. For UV-B exposure, the cells (about 80% confluence) were first washed with phosphate buffered saline (PBS), irradiated with UV-B under a thin cover of PBS, after which cells were again washed with PBS and incubated in fresh medium or fresh medium supplemented with antioxidant combinations (i) trolox 200 mM + ascorbic acid 10 mM and (ii) trolox 200 mM + ascorbic acid 20 mM (18 h before UV-B exposure). 2.2. Cell viability The cell viability was determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole) assay as described earlier [30]. The cells (1 106) were seeded in 35 mm dishes, incubated overnight and subjected to UV-B exposure. The cells were further incubated for 24 h post UV-B irradiation. The cell viability was evaluated by assaying for the ability of functional mitochondria to catalyze the reduction of MTT (0.25 mg/mL, at 37 8C for 2.5 h, Sigma–Aldrich, St. Louis, MO, USA) to form formazan salt by mitochondrial dehydrogenases, as determined by ELISA reader at 570 nm (Multiskan Spectrum; Thermo Electron Corporation, Waltham, MA, USA). Untreated control cells were considered as 100% viable. 2.3. ROS measurement using fluorescence microscopy The ROS generation was measured using 20 ,70 -Dichlorofluorescin diacetate (H2DCFDA, Sigma–Aldrich St. Louis, MO, USA) as described [31]. The cells (1.5 106) seeded in 60 mm dishes were incubated overnight. 30 min prior to UV-B exposure the cells were incubated with 5 mM H2DCFDA at 37 8C, after which cells were washed with PBS and irradiated with UV-B. The ROS generation was measured immediately after UV-B exposure using fluorescence microscope (10 lens), Nikon eclipse TE 2000-U. Source of light was a high pressure mercury lamp and blue filter was used for excitation of the dye. 2.4. Determination of intracellular Ca2+and mitochondrial membrane potential (Dcm) using confocal microcopy. The accumulation of Ca2+ ions in the cytoplasm was studied by fluorescent dye Fluo3-AM (Molecular Probes, Eugene, OR, USA), as described with some modifications [32]. Dcm was studied by JC-1 (Molecular Probes, Eugene, OR, USA) staining as described [33]. Briefly, cells (1.5 106) were seeded on sterile cover slips in 60 mm dishes. At 40–50% confluence, cells were washed with PBS and irradiated with UV-B. Control dishes were not subjected to UVB exposure. To confirm that the Ca2+ release is mediated by ROS, 100 mM H2O2 was used as a positive control (for 45 min in serum free media). For studying intracellular Ca2+, cells were incubated with 5 mM Fluo3-AM and to study the Dcm, cells were incubated with 5 mg/mL JC-1 stain for 30 min at 37 8C. The cells were washed with PBS to remove any unbound dye. The cover slips were then mounted in PBS on the glass slides and edges were sealed. Live imaging of the cells was done using a Laser scan confocal microscope (Olympus fluoview FV-1000). The fluorescence emitted by the Fluo3-AM when bound to Ca2+ ions was recorded using


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the laser line 480/525 nm, 40 dry lens and for Dcm the excitation of JC-1 was done using laser line 488 nm, 60 oil lens and emission was recorded at 525 nm as green fluorescence and 590 nm as red fluorescence. Thapsigargin (1 mM) and camptothecin (5 mM) were used as positive controls for studying intracellular Ca2+ and Dcm respectively. Quantitative analysis of the images was performed by fluoview FV-1000 software. 2.5. Cell cycle and apoptosis assays by flow cytometry The cell cycle analysis was done as described [34]. Briefly the cells (2 106) were seeded in 60 mm dishes and incubated overnight. The cells were washed with PBS and irradiated with UVB. After the required time durations the cells were trypsinized, collected and washed with PBS. For cell cycle analysis cells were fixed overnight in 70% ethanol at 4 8C, incubated with RNase (10 mg/mL) for 90 min at 37 8C followed by 30 min of incubation with PI (5 mg/mL) at room temperature. Apoptosis was detected by AnnexinV/PI staining as per manufacturer’s instructions (Invitrogen, FITC Annexin V/dead cell apoptosis kit). The cells were analyzed for DNA analysis and apoptosis by BD-FACS calibur cytometer. To calculate the cells in various phases of cell cycle, the Mod Fit LT software (Verity Software House, Topsham, ME, USA) was used. Camptothecin (5 mM) was used as positive control for the study. 2.6. Cell lysate preparation and western blotting The cells after mentioned treatments were lysed in ice cold RIPA buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% IGEPAL-CA, 1 mM Na3VO4, 1 mM PMSF) with freshly added 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail for 45 min at 4 8C. The cell suspension was centrifuged at 14,000 rpm for 30 min at 4 8C and the supernatant was collected and stored at 20 8C for future use. Protein estimation was done using Bradford Reagent. All the biochemicals used were purchased from Sigma–Aldrich, St. Louis, USA. For western blotting 40 mg of protein was resolved on 7–12% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane (EMD-Millipore, Billerica, MA, USA). The membrane was blocked for 2 h in 5% non fat dry milk in TBST (Tris 50 mM, pH 8.0, NaCl 150 mM, KCl 2.6 mM, Tween-20 0.05%) at room temperature followed by an overnight incubation with appropriate primary antibody (rabbit: anti-GRP-78, anti-PERK, anti-eIF2a, anti-ATF-4, anti-CHOP, anti-GADD-34, anti-Bcl-2, anti-PARP-1, mouse: anti-a-tubulin (Santa Cruz Biotechnologies, Santa Cruz, CA, USA), mouse anti-b-actin (Sigma–Aldrich, St. Louis, USA) and rabbit anti-peIF2a (Cell Signaling Technology, Danvers, MA, USA) at 4 8C. This was followed by an incubation of 90 min at room temperature with horse raddish peroxidase conjugated anti-rabbit and anti-mouse secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The chemilumniscence was detected by Immobilon western chemilumniscent HRP substrate (EMD-Millipore, Billerica, MA, USA) and visualized by Molecular Image ChemiDocTM XRS+ (Bio-Rad, Hercules, CA, USA). Densitometric analysis of the blots was performed by Image Lab TM software, version 3.0 (Hercules, CA, USA). 2.7. Statistical analysis Data are presented as mean SD from three independent experiments with triplicates in each experiment. Statistical comparisons between the groups were determined by using one-way ANOVA followed by Dunnett’s test using Primer of Biostatistics, version 4.0 (McGraw Hill).

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3. Results 3.1. Exposure to UV-B radiation induces cytotoxicity in fibroblast (Hs68) and keratinocytes (HaCaT) To assess the cytotoxic potential of UV-B irradiation, Hs68 and HaCaT cells were exposed to different doses of UV-B radiation (10, 20 and 30 mJ/cm2). The cell viability was determined after 24 h. UV-B induced a dose dependent increase in the cytotoxic effect in both the cell types. At UV-B doses of 10, 20 and 30 mJ/cm2, a cytotoxic effect of 13%, 29% and 33% and 19.5%, 33% and 51% was recorded in Hs68 and in HaCaT respectively (Fig. 1a). 3.2. UV-B irradiation induces increased ROS generation which is efficiently quenched by combination of trolox and ascorbic acid ROS levels were assessed immediately after UV-B exposure of Hs68 and HaCaT cells, and were found to be elevated in a dose dependent manner (10, 20 and 30 mJ/cm2) as determined by fluorescence microscopy. Various combinations of trolox, a water soluble analog of vitamin E and ascorbic acid were used to scavenge the enhanced levels of UV-B induced ROS (data not shown). Following two combination therapies showed most prominent results (i) 200 mM of trolox and 10 mM of ascorbic acid and (ii) 200 mM of trolox and 20 mM of ascorbic acid (Fig. 1b). These anti-oxidants combinations reduced the ROS levels equal to or less than control sample that was not exposed to UV-B radiation. 3.3. Depletion of ER Ca2+ stores by UV-B irradiation is mediated by intracellular ROS generation To determine the effect of UV-B irradiation on ER Ca2+ stores, Hs68 and HaCaT cells were exposed to 10, 20 and 30 mJ/cm2 of UVB radiation and the change in cytoplasmic Ca2+ levels was studied using Fluo3-AM. Our results clearly indicated that UV-B irradiation caused an increase in the cytoplasmic Ca2+ levels in a dose dependent fashion. To determine whether intra-cellular ROS generation had any effect on depletion of ER Ca2+ stores, 100 mM H2O2 was used as a positive control for ROS generation and antioxidant in two combinations (i) Trolox 200 mM + ascorbic acid 10 mM and (ii) Trolox 200 mM + ascorbic acid 20 mM were used to quench the ROS generation. The results showed antioxidant treatments in both the combinations decreased the leakage of Ca2+ ions from ER into cytoplasm due to UV-B exposure or H2O2 treatment, indicating that UV-B modulation of ER Ca2+ stores is actually mediated by intra-cellular ROS generation and ROS quenching by antioxidant treatment directly inhibits Ca2+ mobilization from ER stores (Fig. 2). 3.4. Alterations of the ER Ca2+ stores following UV-B exposure develops ER stress. Anti-oxidant treatment exerts strong protective effect and reverts ER stress To evaluate the effect of UV-B irradiation on induction of ER stress, western blotting was employed to check the activation of UPR as it acts as a marker of ER stress. Our results clearly indicate that exposure of Hs68 cells to UV-B radiation induced ER stress and activation of PERK-peIF2a pathway. The expression of glucose regulated protein 78 (GRP78) increased in response to UV-B irradiation. Also the phosphorylation of eukaryotic initiation factor 2a (p-eIF2a) increased by 1.3-, 1.6- and 1.9-fold following 10, 20 and 30 mJ/cm2 exposure to UV-B radiation respectively and the


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Fig. 1. UV-B irradiation induced loss of cell viability and generation of ROS in Hs68 and HaCaT cells. The cells exposed to different doses of UV-B (a) Post 24 h of UV-B exposure the cells were incubated with MTT dye (0.25 mg/mL) for 2.5 h. The formazan crystals formed were dissolved in DMSO and the absorbance was recorded at 570 nm. (b) UV-B irradiation induced ROS generation was quenched by anti-oxidant combinations (i) Trolox 200 mM and ascorbic acid 10 mM, (ii) Trolox 200 mM and ascorbic acid 20 mM. Cells pretreated with antioxidant combinations for 18 h were incubated with H2DCFDA (5 mM), washed and irradiated with UV-B. ROS generation was measured immediately after UV-B exposure. Five random microscopic fields were selected. The images shown are representative images. An asterisk indicates a significant statistical difference (p < 0.05) compared to control sample.

expression of other proteins downstream of p-eIF2a like activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) increased, confirming the activation of PERK-peIF2a pathway in Hs68. This effect was not evident in HaCaT cells (Fig. 3a, b and e). We have already mentioned that ROS quenching attenuates the effect of UV-B irradiation on ER Ca2+ stores (Fig. 2). To further strengthen our claim that UV-B mediated ROS generation develops ER stress, the effect of ROS quenching on development of ER stress and UPR activation was checked. Our results show that the activation of PERK-peIF2a pathway following UV-B exposure can effectively be reversed by relieving oxidative stress as the phosphorylation of eIF2a decreased by 1.6- and 2.6-folds

upon treatment with anti-oxidant combinations of (i) Trolox 200 mM + ascorbic acid 10 mM and (ii) Trolox 200 mM + ascorbic acid 20 mM respectively. Also the expression of other proteins downstream of p-eIF2a like ATF4, CHOP and growth arrest and DNA damage 34 (GADD34) decreased on treatment with similar antioxidant combinations (Fig. 3d and f). Further we also checked the effect of antioxidant alone on the expression of PERK-peIF2a pathway and our results indicating that antioxidants alone do not modulate the expression of proteins involved in this pathway (Fig. 3c). This observation strongly suggests the central role of oxidative stress mediated depletion of ER Ca2+ stores in the development of ER stress following UV-B irradiation in fibroblasts.


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Fig. 2. UV-B irradiation induced ER calcium store depletion in Hs68 and HaCaT and effect of externally provided anti-oxidant combinations (i) Trolox 200 mM and ascorbic acid 10 mM, (ii) Trolox 200 mM and ascorbic acid 20 mM. After 45 min of UV-B irradiation or H2O2 treatment (100 mM), the cells were loaded with Fluo3-AM dye and incubated for another 30 min. The cells were washed and mounted in PBS on glass slides for live cell imaging using confocal microscopy. The cells treated with antioxidants were processed as mentioned above except for pretreatment with anti-oxidant combinations for 18 h before UV-B irradiation or H2O2 treatment (100 mM). Five random microscopic fields were selected. The images shown are representative images (g) Quantification of (a and c). Thapsigargin (1 mM) was used as a positive control. An asterisk indicates a significant statistical difference (p < 0.05) compared to control sample.


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Fig. 3. Effect of UV-B irradiation on ER stress and activation of PERK pathway and ameliorating effect of antioxidant combinations (i) Trolox 200 mM and ascorbic acid 10 mM, (ii) Trolox 200 mM and ascorbic acid 20 mM. (a and b) 6 h post UV-B irradiation, Hs68 and HaCaT cells (c) Hs68 cells incubated with antioxidant combinations only for 18 h and (d) cells pretreated with the anti-oxidant combination for 18 h before UV-B irradiation were lysed in RIPA buffer for protein extraction. Equal amounts of proteins were subjected to SDS-PAGE and transferred to PVDF membrane and probed with desired antibody. Chemilumniscence was used to detect the signal. Total e-IF2a, a- tubulin and b-actin were used as loading controls. (e and f) densitometric analysis of indicated proteins in Hs68 cells. Thapsigargin (1 mM) was used as a positive control for inducing ER stress.


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Fig. 4. Cell cycle arrest induced by UV-B irradiation in human skin cells. (a) Hs68 and (b) HaCaT cells were exposed to UV-B radiation at 10, 20 and 30 mJ/cm2. 6 h post UV-B exposure, cells were harvested and processed as mentioned in materials and methods section. Cells were analyzed by flow cytometry on BD-FACS calibur cytometer. Camptohtecin (5 mM) was used as a positive control for the study.


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Fig. 5. Loss of mitochondrial membrane potential (Dcm) induced by UV-B irradiation was effectively restored by antioxidants combinations (i) Trolox 200 mM and ascorbic acid 10 mM, (ii) Trolox 200 mM and ascorbic acid 20 mM. For antioxidant treatment cells were incubated with the above mentioned antioxidant combinations for 18 h before UV-B irradiation. 6 h post UV-B irradiation, (a) Hs68 and (b) HaCaT cells were incubated with JC-1 stain for 30 min. Cells were washed and mounted on glass slides in PBS. Live cell images were acquired by Olympus fluoview FV-1000 confocal microscope. Five random microscopic fields were selected. The images shown are representative images (c) quantification of Dcm loss in Hs68 and HaCaT cells. Camptohtecin (5 mM) was used as a positive control for the study. An asterisk indicates a significant statistical difference (p < 0.05) compared to control sample.


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Fig. 6. UV-B irradiation causes apoptosis in Hs68 and HaCaT. (a) Hs 68 (c) HaCaT post UV-B irradiation, cells were harvested and analyzed for apoptosis using Invitrogen, FITC Annexin V/PI dead cell apoptosis kit as per the manufacturer’s instructions. Camptohtecin (5 mM) was used as a positive control for the study. (b and d) Expression of pro and anti-apoptotic proteins as evaluated by western blotting. (e) Densitometric analysis of cleaved PARP1 in Hs68 and HaCaT cells.


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Fig. 6. (Continued ).

3.5. UV-B irradiation induces cell cycle arrest differentially in Hs68 and HaCaT UV-B irradiation at doses 10, 20 and 30 mJ/cm2 induced G2/M phase arrest in Hs68. It is due to the activation of PERK protein which induces p53/47 mRNA translation and p53/47 homooligomerization [35]. In HaCaT G1/S phase arrest in HaCaT cells as determined by flow cytometry (Fig. 4).

population of apoptotic cells as compared to cells that were not subjected to UV-B irradiation (Fig. 6a and c). Also the expression profile of PARP-1 and Bcl-2 revealed that UV-B exposure induced apoptosis in both Hs68 and HaCaT with PARP-1 cleavage increasing by 3.8-, 3.5- and 3-folds at 10, 20 and 30 mJ/cm2 respectively in Hs68 cells. In case of HaCaT cells the PARP-1 cleavage increased by 2.3-, 4- and 5.3-folds following exposure to 10, 20 and 30 mJ/cm2 doses of UV-B radiation respectively (Fig. 6b, d and e).

3.6. UV-B irradiation induces loss of mitochondrial membrane potential (Dcm) and apoptosis

4. Discussion

Exposures of Hs68 and HaCaT to UV-B radiation lead to the establishment of apoptotic cascade. Our results indicated that UVB doses of 10, 20 and 30 mJ/cm2 resulted in loss of Dcm as indicated by an increase in the green fluorescence (low Dcm) of JC1 dye as compared to the red fluorescence of dye emitted by the Jaggregates formed within intact mitochondria. The imaging was done using confocal microscopy (Suppl. Fig. S(S1–S3)). Since loss of Dcm is considered as the ‘point of no return’ as this event is responsible for engaging the cellular apoptotic machinery in cell death pathway [36], so we evaluated the effect of ROS quenching on loss of Dcm and our results indicated that quenching the ROS generation by antioxidant combination resulted in restoring the loss induced by UV-B irradiation (Fig. 5). The Annexin V/Propidium Iodide staining also revealed that UVB irradiation of Hs68 and HaCaT cells lead to an increase in the

Both UV-A and UV-B radiations have the ability to induce the generation of ROS near or within the cell surface membrane in human skin [37]. The ultraviolet radiation from the sun damages the human skin, causing premature aging and severe skin pathologies, including cancer [38]. Chronic exposure to UV radiation has profound effects on both the epidermis and the dermis of the skin, the epidermis exhibits atrophy as compared to the sun protected areas of the same individual often with the disruption of the normal keratinocyte maturation [39]. Hisological features of photoaging are most apparent in the dermis exhibiting overall dermal atrophy and reduced amounts of fibrillar collagen and elastic fibers [40,41]. The results of the present study suggest that exposure of Hs68 and HaCaT cells to UV-B radiation (10– 30 mJ/cm2) resulted in increased intracellular ROS generation with a dose dependent decrease in cell viability (Fig. 1a and b).


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Fig. 7. Postulated mechanism of ROS mediated ER stress induced by UV-B irradiation in human skin cells. Exposure of human skin cells to UV-B radiation generates ROS within these cells which act on ER Ca2+ stores, causing leakage of Ca2+ ions from ER lumen, generating ER stress and so activating UPR. These effects of UV-B radiation can be reversed by use of efficient antioxidant combinations that quench the ROS and prevent its further effects on other cellular/subcellular events.

The increased ROS production alters the gene and the protein structure and function as the excited oxygen electrons of ROS have the ability to interact with various cellular molecules that regulate many biochemical processes like activation of various cytoplasmic signal transduction pathways that are related to growth, differentiation, replicative senescence and connective tissue degradation [42–45]. Besides altering various growth and differentiation pathways UV radiation has been reported to be an inducer of ER stress and an activator of UPR [29]. However, current literature lacks the information on UV-B irradiation mediated generation of ER stress and underlying mechanism involved. Our data now with high degree of clarity is indicative of the fact that ROS generation is the prime and immediate event post UV-B exposure (Fig. 1b). A significant increase in ROS production after UV-B exposure is likely to play an important role in inducing ER stress. The ROS generated after UV-B exposure facilitates the depletion of Ca2+ ions from the ER lumen (Fig. 2a, c and g), causing the malfunctioning of the ER chaperones and other proteins leading to the accumulation of unfolded/misfolded proteins within ER, generating ER stress and activation of UPR [6,46,47]. Our results also suggested that UV-B irradiation induces ER stress and PERK-peIF2a pathway in Hs68 cells. However, the expression of all the proteins downstream of PERK remained unaltered in HaCaT cells (Fig. 3a, b and e). Combinations of strong antioxidants, used in clinical dermatological/cosmetic practices (trolox and ascorbic acid) were used to inhibit UV-B induced oxidative stress in order to determine if this can prevent depletion of ER Ca2+ stores. The ROS quenching secured the ER Ca2+ stores (Fig. 2b, d–f) and thus prevented the development of ER stress which was further confirmed by studying the expression of the UPR proteins by western blotting (Fig. 3d and f). Further it was confirmed by western blotting that antioxidant combinations alone do not have any effect on the expression of proteins involved in PERK-peIF2a pathway (Fig. 3c). The results clearly shows that ROS quenching prevented the depletion of Ca2+

ions from ER lumen and so there was decreased induction of ER stress and suppressed activation of UPR. The UV radiation is known to be a potent inducer of DNA damage in the cells. In response to such genotoxic stresses the cell activates the cellular recovery mechanisms like the cell cycle arrest and apoptosis which are essential to maintain the integrity of the genome during cellular proliferation [48–50]. Consistent with the previous reports our data demonstrated that irradiation of Hs68 and HaCaT with UV-B resulted in cell cycle arrest and finally establishment of apoptotic cascade; loss of Dcm and activation of caspases to cause death to the damaged cells (Fig. 6). The loss of Dcm, which is considered as the ‘point of no return’ in cell death program [36] was also restored by quenching the ROS using antioxidant combinations (Fig. 5), confirming that apoptosis induced by UV-B irradiation was ROS mediated. In Hs68 cells, G2/M phase arrest was observed after UV-B irradiation (Fig. 4a). This is in agreement with the previous report that shows, ER stress promotes PERK dependent induction of P53/47mRNA translation and P53/47 homo-oligomerization and induces G2/M phase arrest of the cell cycle [35]. This p53/47 isoform is a product of alternate mRNA translation that can be synthesized by cap-independent mechanism in response to various stresses like serum deprivation or ER stress [51,52]. In HaCaT cells, G1/S phase arrest was observed (Fig. 4b). With regard to the results of the present study, G1/S phase arrest can be attributed to depletion of intracellular Ca2+ stores due to UV-B irradiation. This is in agreement with previous reports showing Ca2+ content from intracellular pools exerts a profound control over cell growth and progression through cell cycle. Depletion of Ca2+ from intracellular stores causes cell to enter a growth arrest in G1 phase [53,54]. We conclude that UV-B exposure mediated photodamage is a direct and immediate consequence of oxidative stress that leads to disturbance of Ca2+ homeostasis in ER which develops ER stress leading to UPR activation (Fig. 7). Apoptosis remains the hallmark of cell death due


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to UV-B irradiation in both keratinocytes and fibroblasts. Further, this molecular framework may help in devising therapies for pathological states involving oxidative stress manifested depletion of ER Ca2+ stores leading to the development of ER stress and UPR activation. Acknowledgements This study was supported by Council of Scientific and Industrial Research (CSIR), New Delhi, India, under its 12th Five Year Plan Project titled, ‘‘Toward Understanding Skin Cell Homeostasis’’ (TOUCH), Project No. BSC 0302. Financial assistance to first two author (FRM and NUA) by University Grants Commission (UGC), New Delhi, India, is acknowledged

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