Unique Role of Nuclear HO-1 in the Nuclear Transport of Nrf2: Implications for Oxidative Cytoprotection Chhanda Biswas1, Guang Yang2, Phyllis A. Dennery1,2, Manasa Muthu2, Nidhi Shah2, and Ping La2 1University of Pennsylvania, United States, 2Children's Hospital of Philadelphia, United States The nuclear factor E2-related factor 2 (Nrf2) is a

of the antioxidant response. Oxidative stress promotes Nrf2 stabilization and nuclear translocation. Although Nrf2 activation enhances antioxidant defenses, it also regulates genes such as glucose-6-phosphate dehydrogenase (G6PD), which is essential for reprogramming energy metabolism, allowing for improved survival in adverse environments. Activation of Nrf2 also induces heme oxygenase-1 (HO-1), an important cytoprotective molecule. HO-1 can be truncated at the C-terminus, facilitating its nuclear translocation. We hypothesized that HO-1 cooperates with Nrf2 to promote downstream events, which facilitate cell survival in oxidative stress. To this effect, WT and HO-1 null MEF cells stably transfected with either a FLAG tagged nuclear (TR), cytoplasmic (FL) HO-1 cDNA or empty vector as well as HO-1 KO and WT cells transiently transfected with an ARE/Luc reporter construct, were exposed to 95%O2, 5%CO2 (hyperoxia) for 18 hours. Controls were exposed to 95%Air/5%CO2 (air). in some experiments, shRNA was used to silence HO-1 expression. Hyperoxia resulted in induction and nuclear translocation of HO-1 as well as enrichement of Nrf2 in the nucleus of WT MEF cells. Additionally, HO-1 KO MEF cells rescued with HO-1 TR showed enrichment of Nrf2 in the nucleus compared to HO-1 (FL) and vector infected cells. Immunoprecipitation with FLAG retrieved Nrf2 and immunoprecipitation with Nrf2 antibodies retrieved HO-1 TR, suggesting a physical interaction of HO-1 and Nrf2 in the nucleus. Photon emission was significantly higher in hyperoxia exposed HO-1 WT vs KO cells after ARE/Luc transfection. After hyperoxic exposure, the viability of TR cells was significantly higher. Lastly, silencing HO-1 reduced the expression of G6PDH and also reduced G6PDH enzymatic activity. We conclude that nuclear HO-1 promotes nuclear transport of Nrf2 and induction of specific Nrf2 target genes involved in metabolic reprogramming. We speculate that this provides a survival advantage.

Role of PIX in PDGF-Induced Lamellipodia Dynamics in VSMC Charity Duran1, Holly C Williams1, Bernard Lassegue1, Kathy K Griendling1, and Alejandra San Martin1 1Emory University School of Medicine, United States During atherosclerosis and postangioplasty restenosis, the release of growth factors, such as the platelet derived growth factor (PDGF), results in the migration of vascular smooth muscle cells (VSMCs) into the intima. We demonstrated previously that PDGF-induced ROS production by Nox1 is indispensable for VSMC migration. Cell migration is a tightly coordinated process that starts with the extension of the lamellipodia, a surface-attached protrusion at the leading edge of the migrating cell. However, the intracellular signaling cascades involved in

lamellipodia formation are unclear. Because Nox1 activation requires the small GTPase Rac1, we sought to investigate the role of the in PDGF-induced lamellipodia formation in VSMC. as expected, treatment of VSMCs with PDGF induced the activation of Rac1 between 1 and 5 min with a peak at 2 min. Likewise, we were able to pull down

from PDGF-treated cell lysates using purified GST-Rac1 protein, suggesting the participation of in PDGF-induced Rac1 activation in VSMC. Lucigenin assays demonstrated that down-regulation of with siRNA abrogated PDGF-induced superoxide formation (2.5 ± 0.7 vs. 0.6 ± 0.1 RLU p=0.04). with regards to lamellipodium dynamics, live cell imaging revealed that in of inducing lamellipodia formation. However, the knockdown of -induced lamellipodia protraction (2.8 ± 0.7 vsp=0.02) and p=0.04). in addition, knockdown of the protrusion

unchanged. in total, our work demonstrates the critical role of in mediating PDGF-induced Rac1/Nox1 activation and

specific lamellipodium features, thus aiding PDGF-induced VSMC migration.

TGF- Mediates Focal Adhesion Maturation by a Smad/Nox4-Dependent Mechanism that Involves Regulation of Hsp27 and Hic5 Isabel Fernandez1, Abel Martin-Garrido1, Roza E Clempus1, Bonnie Seidel-Rogol1, Angelica Amanso1, Bernard Lassegue1, Kathy K Griendling1, and Alejandra San Martin1 1Department of Medicine, Division of Cardiology, Emory University, United States Focal adhesions (FAs) link the cytoskeleton to the extracellular matrix and as such play a critical role in growth, migration and contractile properties of vascular smooth muscle cells. Recently, it has been shown that NADPH oxidase Nox4, a TGF- -induced, H2O2-producing enzyme which localizes to FA, is critical for FA maturation. However, its downstream effectors are unknown. Studies have identified FA resident protein Hic5 as H2O2 and TGF-

-inducible and a potential binding partner to heat shock protein (Hsp) 27. Therefore, we hypothesize that Hic5 and Hsp27 participate in Nox4-mediated FA maturation. Indeed, TGF-treatment promotes the formation of mature, Hic5-containing FAs, while Hic5 knockdown prevents TGF- -induced FAs. in addition, TGF- and Hsp27 (9.8±0.01 vs. 2.5±0.4 AU;p<0.001) expression which are blocked in siNox4 treated cells (9.8±0.01 vs. 3.5±1.1 AU;p<0.001). Interestingly, downregulation of the TGF-canonical effector, smad4, blocked Nox4 expression (23.8±0.8 vs 17.1±1.8 AU;p<0.01) while siNox4 impeded smad2/3 phosphorylation (1.8±0.1 vs. 1±0.1 AU; p<0.001) suggesting a forward feedback loop for smad activity. Thus, similar to siNox4 treated cells, Hsp27 and Hic5 upregulation was blocked in smad4 deficient cells (27.2±1.7 vs. 10.9±1.6 AU;p<0.001 and 2.8±0.5 vs. 1.5±0.1 AU;p<0.01 respectively). Interestingly, TGF- induced Hsp27 and Hic5 co-localization in stress fibers and cytosol (5.5±0.2 vs. 1 AU;p<0.01), suggesting that Hsp27 aids Hic5 trafficking to FAs. in fact, in Hsp27-deficient cells, Hic5 fails to localize to FA with no effect on paxillin localization. Taken together, our work identifies Hic5 as a novel effector of Nox4 and supports the idea that a smad/Nox4/smad pathway mediates TGF- -induced FA maturation by upregulation and interaction of Hic5 and Hsp27 that specifically targets Hic5 to FA.

doi: 10.1016/j.freeradbiomed.2013.10.790

doi: 10.1016/j.freeradbiomed.2013.10.791

Differential Regulation of B-crystallin by Pro-Oxidant Agents in Skeletal Muscle Cells Simona Fittipaldi1, Neri Mercatelli1, Ivan Dimauro1, Maria Paola Paronetto1, and Daniela Caporossi1 1University of Rome "Foro Italico", Italy

- -cry) plays an important role in muscle homeostasis displaying an active role in the redox metabolism. Here, we investigated the signalling pathways underlying -cry modulation in myogenic cells exposed to different oxidants. C2C12 myoblasts were treated for 2h with either sodium meta-arsenite NaAsO2 or hydrogen peroxide H2O2 (100 and changes in the -cry phosphorylation and expression, along with the modifications in signal transduction pathways and thioredoxin (Trx-1 and Trx-2) status were analysed at different time post-treatment (0h, 6h, 18h). A rapid onset of -cry phosphorylation at ser59 occurred immediately after treatment with either NaAsO2 or H2O2 persisting up to 18h. NaAsO2 treatment induced a 4-fold and 8-fold increase of -cry mRNA at time 0h and 6h, while the -cry protein increased by 2-fold and 6-fold at 6h and 18h post-treatment, respectively. on the contrary, H2O2 treatment showed a 1.5-fold and 3-fold increase in mRNA and protein levels, respectively, at 18h only. Activation of c-Jun N-terminal kinases (JNK) and AKT were evident from 30 min up to 2 h exposure for both oxidants. Interestingly, the AKT phosphorylation induced by NaAsO2 was always much less intense than those resulting by H2O2, while a stronger activation of p38 kinase was observed after NaAsO2 compared to H2O2 treatment. NaAsO2 exposure induced an almost complete oxidation of both Trx-1 and Trx-2, with a complete recovery of Trx redox status at 6h from treatment. the redox status of both Trx-1 andTrx-2 was not affected by H2O2. These results suggest that NaAsO2 and H2O2 have a differential effect on cellular redox state and signalling, leading to differential

-cry mRNA expression. a computational analysis of the mouse -cry promoter region has revealed the presence of prominent

consensus sequences for redox-sensitive transcription factors (RSTFs) other than HSF-1, like AP-1, Nrf- and p53. To verify their role in -cry induction by oxidants, we are going to verify differential binding activity of candidate RSTFs on -cry promoter after NaAsO2 or H2O2 exposure.

Elucidation of Nuclear Trx1 Interactions Following Hyperoxic Injury Miranda Jo Floen1, Shanna Baack2, and Peter F Vitiello1,2 1Sanford School of Medicine, University of South Dakota, United States, 2Sanford Research/USD, United States When reactive species are present in supra-physiological concentrations, proteins may undergo oxidative modifications altering signaling pathways via protein-protein interactions, DNA binding, and protein activities. Thioredoxins (Trx) are oxidoreductases that have evolved to maintain the redox homeostasis of the reactive thiol proteome through the reduction

of disulfides. Trx1, a cytosolic member, is implicated in cell proliferation, redox homeostasis, redox signaling, and antioxidant induction. Previous studies have shown that Trx1 oxidoreductase activity mediates cytoprotection against hyperoxic injury. Additionally, H1299 and A549 cells exposed to 95% oxygen for 3 days demonstrated a 4-fold increase in nuclear Trx1 compared to control cells shown by immunoblots of nuclear and cytoplasmic enrichments. While general Trx1 interactions have been elucidated, nuclear specific interactions potentially mediating hyperoxic cytoprotection have not been described. Therefore, a nuclear localized Trx1 mass trapping approach was utilized whereby Trx1 C35S and Trx1 C32/35S constructs were targeted to the nuclear compartment using a carboxy-terminal nuclear localization sequence and an amino-terminal Flag-tag. Nuclear localization of the transgenes were confirmed by immunoblotting and immunocytochemistry. Nuclei were isolated from control cells and cells cultured in 95% oxygen for 2 days. Trx1 and bound substrates were subsequently immunopurified for the flag epitope. Redox-sensitive interactions were eluted by DTT reduction and the redox non-specific interactions were boiled off in Laemmli buffer. Elutions were analyzed by silver stain, immunoblotting, and 1D LC/MS/MS protein identification. Altogether, a system to elucidate compartmentalized redox-dependent Trx1 interactions during hyperoxic injury has been developed and implemented.

Expression Profiling of microRNAs Related to Heme Oxygenase 1 in a Mouse Model of Hyperoxic Lung Injury Hayato Go1, Ping La1, Fumihiko Namba2, Patrick Amal Fernando1, Guang Yang1, and Phyllis A. Dennery1,3 1Children's Hospital of Philadelphia, United States, 2Saitama Medical Center, Japan, 3University of Pennsylvania, United States Neonatal rodents tolerate hyperoxia better than adults perhaps due to enhanced antioxidant defenses such as heme oxygenase (HO-1), which is regulated by the transcriptional repressor, Bach1. We have previously demonstrated that neonatal mice are less able to induce HO-1 mRNA in hyperoxia vs adults. Perhaps developmental differences in micro RNA expression explain this effect. Neonatal (postnatal day 0) and adult (8 week old) C57BL/6 mice were exposed to room air or >95% oxygen for 3 days. Lung expression of 682 miRNAs was measured using TaqMan Low Density Arrays. Target miRNAs associated to HO-1 related gene were identified if 2 of 3 programs (TargetScan and miRanda or PicTar) demonstrated an interaction between the and the miRNA seed sequence. In neonates exposed to hyperoxia, 50 miRNAs increased more than 2-fold, and 17 decreased by more than half whereas in adults, 21 increased more than 2-fold, and 12 decreased by more than half. No miRNAs that directly regulate HO-1 were found, however, the Bach1 targeted miR-196a increased 250-fold in neonatal lung compared with adults. during early postnatal development, lung miR-196a significantly decreased whereas Bach1 mRNA significantly increased. After hyperoxia, lung miR-196a further decreased whereas Bach1 mRNA significantly increased in the neonatal lung whereas no changes were observed in adults. to verify the effects of hyperoxia on Bach1 mRNA regulation, MEF cells transfected with a Bach1 3 UTR/luciferase construct were exposed to 95% O2/ 5% CO2 for 18 hours. Relative light intensity increased whereas it did not in

doi: 10.1016/j.freeradbiomed.2013.10.792

doi: 10.1016/j.freeradbiomed.2013.10.793

doi: 10.1016/j.freeradbiomed.2013.10.794