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