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• W2039471809 abstract "Hypoxic stress activates various signal transduction pathways including posttranslational modification with the ubiquitin-like SUMO protein (SUMOylation). However, the molecular mechanisms by which SUMOylation regulates hypoxic responses remain unclear. Here, we investigated the ability of rat salivary Pa-4 epithelial cells to resist cell injury elicited by 1% O2- or hypoxia-mimetic desferroxamine (DFO)-stimulated SUMOylation processes. By using Pa-4 cells stably transduced with lenti-SUMO-1 and a cell-permeant peptide harboring SUMO-binding motif to interfere with SUMO-dependent protein-protein interactions, we demonstrate that SUMOylation augments cell survival against DFO treatment. This appeared to be partly mediated through attenuation of Protein Kinase C (PKC)-δ activation and caspase-3 cleavage, hallmarks of pro-apoptotic signaling. Intriguingly, DFO-induced phosphorylation of DNA damage marker ataxia-telangiectasia-mutated protein S1981 preceded activation of PKCδ and caspase-3. Constitutive SUMOylation facilitated 1% O2- or DFO-induced nuclear factor κB transactivation, possibly via activation of genotoxic signaling cascade. In addition, we observed transient preservation of transepithelial electrical resistance during the early stage of hypoxia (1% O2) as well as enhanced transepithelial electrical resistance recovery after prolonged hypoxia in SUMO-1-expressing cell monolayers. In conclusion, our results unveil a previously unrecognized mechanism by which SUMOylation and activation of ataxia-telangiectasia-mutated protein, PKCδ, caspase-3, and nuclear factor κB signaling pathways modulate salivary adaptive responses to stress in cells exposed to either 1% O2 or DFO. Hypoxic stress activates various signal transduction pathways including posttranslational modification with the ubiquitin-like SUMO protein (SUMOylation). However, the molecular mechanisms by which SUMOylation regulates hypoxic responses remain unclear. Here, we investigated the ability of rat salivary Pa-4 epithelial cells to resist cell injury elicited by 1% O2- or hypoxia-mimetic desferroxamine (DFO)-stimulated SUMOylation processes. By using Pa-4 cells stably transduced with lenti-SUMO-1 and a cell-permeant peptide harboring SUMO-binding motif to interfere with SUMO-dependent protein-protein interactions, we demonstrate that SUMOylation augments cell survival against DFO treatment. This appeared to be partly mediated through attenuation of Protein Kinase C (PKC)-δ activation and caspase-3 cleavage, hallmarks of pro-apoptotic signaling. Intriguingly, DFO-induced phosphorylation of DNA damage marker ataxia-telangiectasia-mutated protein S1981 preceded activation of PKCδ and caspase-3. Constitutive SUMOylation facilitated 1% O2- or DFO-induced nuclear factor κB transactivation, possibly via activation of genotoxic signaling cascade. In addition, we observed transient preservation of transepithelial electrical resistance during the early stage of hypoxia (1% O2) as well as enhanced transepithelial electrical resistance recovery after prolonged hypoxia in SUMO-1-expressing cell monolayers. In conclusion, our results unveil a previously unrecognized mechanism by which SUMOylation and activation of ataxia-telangiectasia-mutated protein, PKCδ, caspase-3, and nuclear factor κB signaling pathways modulate salivary adaptive responses to stress in cells exposed to either 1% O2 or DFO. Small ubiquitin-like modifier (SUMO) is the best characterized member of a growing family of ubiquitin-related proteins. SUMO is conjugated to target proteins using an enzyme conjugation system similar to but distinct from that of ubiquitin.1Dohmen RJ SUMO protein modification.Biochim Biophys Acta. 2004; 1695: 113-131Crossref PubMed Scopus (209) Google Scholar, 2Johnson ES Protein modification by SUMO.Annu Rev Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1401) Google Scholar Importantly, SUMO-1, −2, −3, and −4 have emerged as important posttranslational modifiers that regulate diverse cellular functions including intracellular targeting, DNA repair, cell cycle progression, and responses to extracellular stimuli.1Dohmen RJ SUMO protein modification.Biochim Biophys Acta. 2004; 1695: 113-131Crossref PubMed Scopus (209) Google Scholar, 2Johnson ES Protein modification by SUMO.Annu Rev Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1401) Google Scholar, 3Chen XL Reindle A Johnson ES Misregulation of 2 microm circle copy number in a SUMO pathway mutant.Mol Cell Biol. 2005; 25: 4311-4320Crossref PubMed Scopus (61) Google Scholar A large number of proteins are modified by SUMO, and recent proteomic studies have shown that as many as 400 yeast proteins are modified by yeast SUMO homolog, and 2683 potential SUMO substrates are conserved in both humans and mice.4Panse VG Hardeland U Werner T Kuster B Hurt E A proteome-wide approach identifies sumoylated substrate proteins in yeast.J Biol Chem. 2004; 279: 41346-41351Crossref PubMed Scopus (225) Google Scholar, 5Wohlschlegel JA Johnson ES Reed SI Yates III, JR Global analysis of protein sumoylation in Saccharomyces cerevisiae.J Biol Chem. 2004; 279: 45662-45668Crossref PubMed Scopus (278) Google Scholar, 6Zhou F Xue Y Lu H Chen G Yao X A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus.FEBS Lett. 2005; 579: 3369-3375Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar The precise functional differences of various SUMO paralogs remain to be established. However, given the importance of SUMOylation, it is not surprising that SUMO plays important roles in the development of various diseases.7Bohren KM Nadkarni V Song JH Gabbay KH Owerbach D A M55V polymorphism in a novel SUMO gene (SUMO-4) differentially activates heat shock transcription factors and is associated with susceptibility to type I diabetes mellitus.J Biol Chem. 2004; 279: 27233-27238Crossref PubMed Scopus (294) Google Scholar, 8Li Y Wang H Wang S Quon D Liu YW Cordell B Positive and negative regulation of APP amyloidogenesis by sumoylation.Proc Natl Acad Sci USA. 2003; 100: 259-264Crossref PubMed Scopus (129) Google Scholar, 9Steffan JS Agrawal N Pallos J Rockabrand E Trotman LC Slepko N Illes K Lukacsovich T Zhu YZ Cattaneo E Pandolfi PP Thompson LM Marsh JL SUMO modification of Huntingtin and Huntington's disease pathology.Science. 2004; 304: 100-104Crossref PubMed Scopus (563) Google Scholar Although the significance of SUMOylation in modulating cellular adaptive responses is well established, it is still not clear how SUMO modification regulates specific key cellular functions. Accumulating evidence suggests the potential importance of SUMOylation in governing cellular hypoxic responses in that hypoxia up-regulates the steady-state level of SUMO-1 by as much as 100-fold.10Comerford KM Leonard MO Karhausen J Carey R Colgan SP Taylor CT Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.Proc Natl Acad Sci USA. 2003; 100: 986-991Crossref PubMed Scopus (156) Google Scholar, 11Shao R Zhang FP Tian F Anders Friberg P Wang X Sjoland H Billig H Increase of SUMO-1 expression in response to hypoxia: direct interaction with HIF-1alpha in adult mouse brain and heart in vivo.FEBS Lett. 2004; 569: 293-300Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar Hypoxia is a (patho)physiological condition that arises when cellular oxygen demand exceeds supply. Regions of hypoxia occur not only in disease states but also during normal development. For example, mammalian embryos are, for significant periods of time, in an almost entirely hypoxic environment before a blood circulatory system is established.12Chen EY Fujinaga M Giaccia AJ Hypoxic microenvironment within an embryo induces apoptosis and is essential for proper morphological development.Teratology. 1999; 60: 215-225Crossref PubMed Scopus (91) Google Scholar Hypoxia is also a physiological inducer of the p53 tumor suppressor and provides selective pressure during tumor growth for the elimination of cells with wild-type p53 and the clonal expansion of cells with mutated p53.13Koumenis C Alarcon R Hammond E Sutphin P Hoffman W Murphy M Derr J Taya Y Lowe SW Kastan M Giaccia A Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation.Mol Cell Biol. 2001; 21: 1297-1310Crossref PubMed Scopus (309) Google Scholar It has been established that hypoxic cells acquire genetic and adaptive changes to survive and proliferate in a hypoxic microenvironment, enabling eventual evasion from hypoxia-induced cell death.14Nelson DA Tan TT Rabson AB Anderson D Degenhardt K White E Hypoxia and defective apoptosis drive genomic instability and tumorigenesis.Genes Dev. 2004; 18: 2095-2107Crossref PubMed Scopus (219) Google Scholar As an activator of pro-inflammatory and anti-apoptotic genes, the transcription factor nuclear factor κB (NF-κB) is a key factor in determining whether cells survive after being subjected to genotoxic stress. Hypoxia-elicited phenotypic manifestations have been reported to be a consequence of the induction of tumor necrosis factor-α (TNF-α), a known activator of NF-κB signaling.15Ma TY Iwamoto GK Hoa NT Akotia V Pedram A Boivin MA Said HM TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G367-G376Crossref PubMed Scopus (721) Google Scholar, 16Taylor CT Dzus AL Colgan SP Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha.Gastroenterology. 1998; 114: 657-668Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar NF-κB transcriptional activity is stimulated by hypoxia.10Comerford KM Leonard MO Karhausen J Carey R Colgan SP Taylor CT Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.Proc Natl Acad Sci USA. 2003; 100: 986-991Crossref PubMed Scopus (156) Google Scholar, 15Ma TY Iwamoto GK Hoa NT Akotia V Pedram A Boivin MA Said HM TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-kappa B activation.Am J Physiol Gastrointest Liver Physiol. 2004; 286: G367-G376Crossref PubMed Scopus (721) Google Scholar, 17Chau CH Clavijo CA Deng HT Zhang Q Kim KJ Qiu Y Le AD Ann DK Etk/Bmx mediates the expression of stress-induced adaptive genes, VEGF, PAI-1, and iNOS via multiple signaling cascades in different cell systems.Am J Physiol Cell Physiol. 2005; 289: C444-C454Crossref PubMed Scopus (36) Google Scholar Hypoxia also appears to be a key factor involved in the development of genetic instability.18Bindra RS Schaffer PJ Meng A Woo J Maseide K Roth ME Lizardi P Hedley DW Bristow RG Glazer PM Down-regulation of Rad51 and decreased homologous recombination in hypoxic cancer cells.Mol Cell Biol. 2004; 24: 8504-8518Crossref PubMed Scopus (298) Google Scholar Recent studies have suggested that hypoxia elicits increased DNA damage, enhanced mutagenesis, and functional impairment in DNA repair pathways (reviewed in Ref. 19Hammond EM Giaccia AJ The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation.DNA Repair (Amst). 2004; 3: 1117-1122Crossref PubMed Scopus (70) Google Scholar,20Harris AL Hypoxia: a key regulatory factor in tumour growth.Nat Rev Cancer. 2002; 2: 38-47Crossref PubMed Scopus (4379) Google Scholar). Because SUMOylation has been demonstrated to be involved in governing DNA repair and genomic stability,1Dohmen RJ SUMO protein modification.Biochim Biophys Acta. 2004; 1695: 113-131Crossref PubMed Scopus (209) Google Scholar we postulated that SUMO-1 functions as a central player in the generalized hypoxic response. SUMOylation of Inhibitor of NF-κB (IκB) has been previously demonstrated to act in an anti-NF-κB fashion by attenuating the activation of NF-κB by various cytokines.21Desterro JM Rodriguez MS Hay RT SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.Mol Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (926) Google Scholar In contrast, reports from a recent study by Huang et al22Huang TT Wuerzberger-Davis SM Wu ZH Miyamoto S Sequential modification of NEMO/IKKgamma by SUMO-1 and ubiquitin mediates NF-kappaB activation by genotoxic stress.Cell. 2003; 115: 565-576Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar suggest that both SUMOylation and ataxia-telangiectasia-mutated protein (ATM) activation enhance genotoxic stress-mediated NF-κB activation. Hence, SUMO-1 can function in both anti-NF-κB and pro-NF-κB manners, depending on the individual stimuli and specific pathway used for NF-κB activation. However, questions remain regarding the exact role of SUMOylation in modulating NF-κB transactivation in response to stress by hypoxia (1% O2) or hypoxia-mimetic desferroxamine (DFO). In this report, we demonstrate that treatment with 1% O2 or DFO induces global SUMOylation, disrupts epithelial barrier function and ZO-1 assembly, is cytotoxic, and activates genotoxic signaling cascade. We next examined the effect of the augmented SUMOylation process on modulating the stress response in salivary epithelial Pa-4 cells by investigating the following biological events. Is enhanced SUMOylation capacity beneficial for cell survival under DFO treatment? Will SUMOylation elicit an inhibitory or stimulatory effect on 1% O2- or DFO-dependent NF-κB activation? Does SUMOylation protect cells against hypoxia-mediated decreases in transepithelial electrical resistance (TER) and alter tight-junctional protein distribution? To achieve this goal, we stably overexpressed SUMO-1 in rat salivary epithelial Pa-4 cells by lentivirus-mediated transduction to examine the effect of SUMOylation on governing epithelial homeostatic control mechanisms against exposure to 1% O2 or DFO. Together, we conclude that SUMOylation attenuates activation of pro-apoptotic protein kinase-Cδ and caspase-3 while promoting genotoxicity-induced NF-κB transactivation and facilitating TER restoration and assembly of tight junction-associated proteins in response to exposure to reduced oxygen tension or DFO. The rat parotid epithelial cell lines Pa-4 and SUMO-1-transduced Pa-4 were cultured as previously described.23Chau CH Chen KY Deng HT Kim KJ Hosoya K Terasaki T Shih HM Ann DK Coordinating Etk/Bmx activation and VEGF upregulation to promote cell survival and proliferation.Oncogene. 2002; 21: 8817-8829Crossref PubMed Scopus (51) Google Scholar, 24Li D Lin HH McMahon M Ma H Ann DK Oncogenic raf-1 induces the expression of non-histone chromosomal architectural protein HMGI-C via a p44/p42 mitogen-activated protein kinase-dependent pathway in salivary epithelial cells.J Biol Chem. 1997; 272: 25062-25070Crossref PubMed Scopus (37) Google Scholar, 25Wen X Lin HH Shih HM Kung HJ Ann DK Kinase activation of the non-receptor tyrosine kinase Etk/BMX alone is sufficient to transactivate STAT-mediated gene expression in salivary and lung epithelial cells.J Biol Chem. 1999; 274: 38204-38210Crossref PubMed Scopus (62) Google Scholar, 26Zentner MD Lin HH Deng HT Kim KJ Shih HM Ann DK Requirement for high mobility group protein HMGI-C interaction with STAT3 inhibitor PIAS3 in repression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) transcription by Ras activation in salivary epithelial cells.J Biol Chem. 2001; 276: 29805-29814Crossref PubMed Scopus (36) Google Scholar Human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) plus 1% penicillin/streptomycin and maintained at 37°C. HeLa cells were grown in Dulbecco's modified Eagle's medium (4.5 g/L glucose) supplemented with 10% FBS, 1 mmol/L sodium pyruvate, and 1% penicillin/streptomycin. DFO was purchased from Sigma (St. Louis, MO). The enhanced green fluorescent protein (EGFP)-SUMO-1 fragment obtained from digestion of pEGFP-C1-SUMO-1 was cloned into the PinAI and HincII sites of the pRRLsin.hCMV156 vector to construct pRRLsin.hCMV-EGFP-SUMO-1 (Figure 1A). HEK 293T cells (70% confluence) in 175-mm flasks were cotransfected by calcium phosphate precipitation with 10 μg of pRRLsin.hCMV-EGFP (a human immunodeficiency virus-based self-inactivating replication-defective lentiviral transfer vector expressing EGFP), -EGFP-SUMO-1, or -EGFP-SUMO-1aa (unconjugatable SUMO-1); 10 μg of pΔ8.7 (for viral packaging); and 6 μg of pVSV-G (for VSV-G pseudotyping). Chloroquine was added to a final concentration of 25 μmol/L, and cells were incubated in a 5% CO2 incubator at 37°C for 16 hours. Chloroquine-containing medium was replaced with culture medium containing 10 mmol/L sodium butyrate, and cells were incubated for at least another 8 hours before the addition of fresh culture medium and then incubated for an additional 16 hours. Viral supernatant was collected, centrifuged at 2500 rpm for 10 minutes, and stored immediately at 4°C. Supernatants were pooled and concentrated using a Macrosep Centrifuge Device with a 300-kd molecular mass cut-off (Millipore, Billerica, MA) and a 0.45-μm syringe filter. Aliquots of concentrated virus were stored at −80°C. Titers of concentrated (2 to 4 × 108 transducing units/ml) lentiviral stocks (pRRLsin.hCMV-EGFP and -EGFP-SUMO-1) were determined by infecting HEK 293T cells in the presence of 6 μg/ml polybrene with serial dilutions based on results of fluorescence-activated cell sorting (FACS). Pa-4, Pa-4/SUMO-1, or HEK 293T cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) with 0.5 μg of pGL2-NF-κB luciferase reporter construct harboring an interleukin-6 promoter and two NF-κB binding sites (a generous gift of Dr. Yun Yen, City of Hope, Duarte, CA). For normalization of transfection efficiency, 0.1 μg of the Renilla luciferase pRL-TK plasmid was cotransfected. Six hours after the start of transfection, cells were recovered overnight in 0.05% stripped-serum medium and subsequently exposed to normoxic or hypoxic conditions (1% O2 or DFO treatment [see below]) for 24 hours before luciferase assays. Relative luciferase activity from the firefly luciferase reporter gene was determined and normalized to Renilla luciferase activity using the Dual Luciferase Reporter Assay System (Promega, Madison, WI). Fold induction by hypoxia was calculated after normalizing the pGL2-NF-κB reporter activities with the activities from cotransfected pRL-TK. The nuclear protein preparation and TransBinding NF-κB Assays (Panomics, Redwood City, CA) were performed according to manufacturer's instructions. Pa-4 cells (1 × 105) were plated to reach 50% confluence before transduction (multiplicity of infection [MOI] of 0.1 to 40) with lentivirus encoding SUMO-1 in the presence of 6 μg/ml polybrene to establish the optimal transduction efficiency. Twenty-four hours after transduction, the poly-brene-containing medium was replaced with fresh culture medium and incubated for an additional 16 hours before analyses. Seventy-two hours after transduction, cells were washed and trypsinized for further propagation. An aliquot of these cells was analyzed by FACS for determination of transduction efficiency. For FACS analyses, trypsinized cells were re-suspended in 0.5 ml of phosphate-buffered saline (PBS) (4 × 105 cells/ml). For generation of Pa-4/SUMO-1aa (unconjugatable form of SUMO-1) and Pa-4/EGFP, Pa-4 cells were transduced as described above at an MOI of 10. For hypoxic exposure, cells were incubated in medium with reduced serum (0.05% FBS) for 24 hours before exposure to 1% O2 for time-course studies. At appropriate time points, cells maintained under normoxic and hypoxic conditions were washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, and quenched with 50 mmol/L NH4Cl in PBS. Fixed cells were then permeabilized with 0.5% Triton X-100 in PBS for 15 minutes, blocked with 1% BSA in PBS, and incubated with rhodamine-phalloidin (1:100; Chemicon International, Temecula, CA) for 1 hour for detection of F-actin and an anti-ZO-1 antibody (1:200; Zymed, South San Francisco, CA) to detect ZO-1, respectively. Processed cells were mounted with Prolong Antifade (Molecular Probes, Eugene, OR) and examined. Cells were seeded into 24-well plates to obtain a confluency of 35 to 50% on the day of the experiment. Cells were then treated with increasing concentrations of DFO (up to 100 μmol/L), and medium was changed daily for 2 days. Twenty-four to 48 hours after the start of DFO treatment (depending on cell types), 0.2 ml of 0.1 mg/ml MTT (Sigma) in OptiMEM I (Invitrogen, Carlsbad, CA) was added to each well, and the plate was incubated at 37°C for an additional 1.5 hours. The MTT solution was then aspirated, and 0.2 ml of isopropanol was added to each well to dissolve the formazan crystals. Absorbance was read immediately at 540 nm in a scanning multiwell spectrophotometer. The results were depicted as percentage cell viability and reported as the mean ± SD of three independent experiments performed in triplicate. Cells were washed twice with ice-cold 1× PBS, lysed in 2× sodium dodecyl sulfate lysis buffer, boiled for 5 minutes, and spun at 13,000 rpm for 10 minutes before being snap-frozen and stored at −80°C. For SUMOylation assays, 25 mmol/L N-ethylmaleimide (Sigma) was included in the lysis buffer. Cell lysates were collected, and protein concentrations were determined using the Bradford protein assay. Twenty to 40 μg of protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses. Phosphorylated ATM protein extraction and Western analyses were performed according to manufacturer's instructions. Western analyses were performed with the following antibodies: anti-GMP (SUMO-1) (Zymed), anti-phospho-ATM (Ser1981) (Upstate Biotechnology, Lake Placid, NY), anti-phospho-H2AX (Ser139) (Upstate), anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA), anti-caspase-3 (Cell Signaling Technology, Beverly, MA), anti-cyclin A (Santa Cruz Biotechnology), anti-IκBα (Santa Cruz Biotechnology), anti-green fluorescent protein (Santa Cruz Biotechnology), anti-actin (Chemicon), and anti-tubulin (Santa Cruz Biotechnology), as indicated. Images were visualized using an enhanced chemiluminescence detection kit (ECL Plus; Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer's instructions and the Versadoc 5000 Imaging System (BioRad, Hercules, CA). Quantitative analyses were performed using Quantity One software (BioRad) and normalized with the respective steady-state level of tubulin. Cells were seeded at 50 to 80% confluency in 35-mm dishes and serum-starved overnight to synchronize cell cycle. After the desired treatment, cells were fixed in 70% ethanol overnight and stained with propidium iodide (20 μg/ml). Flow cytometry was performed at the Norris Cancer Center Flow Cytometry Core Facility using FACSCaliber (Becton Dickinson, Franklin Lakes, NJ). Parental Pa-4 and transduced cells were seeded onto tissue culture-treated, 12-mm polycarbonate filters (Costar-Corning, Corning, NY) and cultured for 2 days at 35°C and 5% CO2 in air before being transferred to an exposure chamber, which was flushed with 1% O2 balanced with 5% CO2 and 94% N2. The chamber was then sealed airtight and kept at 35°C for the duration of hypoxia treatment. For NF-κB reporter assays, transiently transfected cells were exposed to 1% O2 for 24 hours as described above. Bioelectrical parameters of epithelial cell monolayers were monitored using a MilliCell ERS screening device (Millipore). Spontaneous potential difference (expressed in millivolts [apical side as a reference]) and TER (expressed in kilo-ohms/centimeter2) were measured with a set of chopstick-style electrodes. Offset potential differences generated by the voltage-sensing electrodes and background electrical resistance contributed by both the bathing medium and the filter membrane were measured at the beginning and end of each set of bioelectric measurements using blank filters and corrected for. Spontaneous potential difference and TER were measured up to 6 days. Data are presented as mean ± SD from at least three separate experiments performed in triplicate. The peptide that noncovalently binds to SUMO (PIASX) as well as a control peptide with scrambled amino acid sequence with identical composition27Song J Durrin LK Wilkinson TA Krontiris TG Chen Y Identification of a SUMO-binding motif that recognizes SUMO-modified proteins.Proc Natl Acad Sci USA. 2004; 101: 14373-14378Crossref PubMed Scopus (474) Google Scholar was fused with human immunodeficiency virus-1 TAT nuclear localization signal. Peptides were synthesized by the Peptide Synthesis Core Facility at the City of Hope, purified by high performance liquid chromatography (HPLC), and verified by mass spectrometry. Pa-4/SUMO-1 cells were pre-incubated with SBM (10 μmol/L) for 24 hours, followed by treatment with varying concentrations of DFO in the presence or absence of 10 μmol/L SBM and incubated for 72 hours before determining cell viability by MTT assays. Experiments were independently performed in duplicate at least three times, unless stated otherwise. One representative data set from these three independent experiments is presented where appropriate. Reporter activity shown is the mean ± SD based on at least three independent transfection experiments. Error bars represent the SD of the mean. Statistical analyses were performed using one-way analysis of variance, followed by posthoc comparisons based on modified Newman-Keuls-Student procedure with P < 0.05 considered significant. Where appropriate, unpaired Student's t-tests were also performed to determine the difference between two data groups. Because the exact role of hypoxia-induced SUMO-1 overexpression in modulating hypoxic response is still unclear, we sought to develop a cell model system to examine the effect of augmented SUMO-1 expression in governing various hypoxia-elicited cellular responses, such as cell survival, NF-κB activation, and barrier integrity. Because there is a very limited (almost none) choice of pharmacological reagents that can be used to modulate SUMOylation capacity in cells, we used an EGFP-SUMO-1 (Figure 1A) lentivirus-mediated transduction system to enhance SUMOylation. First, by using FACS analyses to monitor EGFP expression, a dose-dependent transduction was established in Pa-4 cells by increasing MOIs from 0.1 to 40 to yield 21 to 99% EGFP-SUMO-1-positive cells (data not shown). To assess the stability of transgene expression, transduced cells were passaged, stored, and revived for FACS analyses, which showed that Pa-4/SUMO-1 cells remained >99% of EGFP-SUMO-1 positive after repeated passage at least eight times (data not shown). Western analyses were performed to confirm that the transduced EGFP-SUMO-1 was functionally conjugated to the target proteins by using whole-cell lysates prepared from Pa-4/SUMO-1 cells (data not shown). Consistent with previous reports in other cell types,10Comerford KM Leonard MO Karhausen J Carey R Colgan SP Taylor CT Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia.Proc Natl Acad Sci USA. 2003; 100: 986-991Crossref PubMed Scopus (156) Google Scholar, 11Shao R Zhang FP Tian F Anders Friberg P Wang X Sjoland H Billig H Increase of SUMO-1 expression in response to hypoxia: direct interaction with HIF-1alpha in adult mouse brain and heart in vivo.FEBS Lett. 2004; 569: 293-300Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar 1% O2 treatment stimulated overall SUMOylation profile by about 1.5-fold in both Pa-4 and Pa-4/SUMO-1 cells at 6 hours after treatment (Figure 1B). We next examined whether iron chelator DFO is able to mimic low O2 tension to induce SUMOylation process by treating both Pa-4 and Pa-4/SUMO-1 cells with 50 and 100 μmol/L DFO, respectively. As shown in Figure 1C, Pa-4 cells treated with 100 μmol/L DFO displayed an enhanced SUMOylation profile similar to that detected in Figure 1B, whereas both 50 and 100 μmol/L DFO treatment elicited a more sustained increase of overall SUMOylation pattern in Pa-4/SUMO-1 cells. After verifying that treatment of either 1% O2 or DFO increases global SUMOylation in both Pa-4 and Pa-4/SUMO-1 cells (Figure 1, B and C), we next investigated whether augmented SUMOylation process is (patho)physiologically relevant. Previously, we have demonstrated that hypoxic treatment causes a weakening of epithelial barrier properties in Pa-4 cells.28Hamm-Alvarez SF Chang A Wang Y Jerdeva G Lin HH Kim KJ Ann DK Etk/Bmx activation modulates barrier function in epithelial cells.Am J Physiol Cell Physiol. 2001; 280: C1657-C1668PubMed Google Scholar To search for functional consequence of increased SUMOylation, TER was measured in Pa-4 and Pa-4/SUMO-1 cell monolayers under both normoxic and hypoxic conditions. When TER changes over time in normoxic Pa-4 and Pa-4/SUMO-1 cells were measured, a notably higher baseline TER was observed in Pa-4/SUMO-1 cells (Figure 2A). Based on our previous experience that TER increases at day 2 after seeding in our experimental system,28Hamm-Alvarez SF Chang A Wang Y Jerdeva G Lin HH Kim KJ Ann DK Etk/Bmx activation modulates barrier function in epithelial cells.Am J Physiol Cell Physiol. 2001; 280: C1657-C1668PubMed Google Scholar studies on TER time courses under parallel normoxic or hypoxic treatments were performed on cells starting from 48 to 96 hours after seeding to investigate the effect of SUMOylation on TER maintenance. Under normoxic conditions, TER of Pa-4/SUMO-1 monolayers at 96 hours displayed a more robust increase (up to almost fivefold) than that (∼twofold) observed in Pa-4 monolayers (Figure 2B), indicating that augmented SUMOylation strengthens pa" @default.
• W2039471809 created "2016-06-24" @default.
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• W2039471809 date "2006-05-01" @default.
• W2039471809 modified "2023-10-12" @default.
• W2039471809 title "SUMOylation Attenuates Sensitivity toward Hypoxia- or Desferroxamine-Induced Injury by Modulating Adaptive Responses in Salivary Epithelial Cells" @default.
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