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Bioactive Antioxidants Regulate RAC-GTP Expression at the Leading Edge of Migrating Oral Fibroblasts
epidermal growth factor receptor (EGFR) phosphorylation. Inhibition of EGFR phosphorylation also inhibited H2O2 induced migration as well as strongly reducing H2O2 induced ERK 1/2 phosphorylation but not p38 MAPK phosphorylation. This shows that H2O2 induced ERK 1/2 phosphorylation is largely due to the activation of EGFR phosphorylation. While the concentration of H2O2 used was non-cytotoxic and had a mild proliferative effect, inhibition of EGFR, ERK 1/2 and p38 phosphorylation sensitizes the cells to H2O2. This implied that activation of these kinases not only induces cell migration but also ensures that the cell survives the insult. doi: 10.1016/j.freeradbiomed.2010.10.368
360 Comparison of Antioxydant Defenses in Human Epidermal Melanocytes and Keratinocytes Suggests that Pigment Cells are Adapted to Oxidative Stress Induced by Melanin Related Compounds. 1,2
1
doi: 10.1016/j.freeradbiomed.2010.10.370
362
1
Laurent Marrot , Jean-Philippe Belaïdi , Laurence Denat , Daniel Duche1, Christophe Jones1, Philippe Perez1, Jérémie Sœur1, and JeanRoch Jean-Roch Meunier1 1 2 L'Oreal, Phototoxicity Melanin and its chemical intermediates can both generate and scavenge reactive species. Melanocyte have thus to manage a specific situation towards oxidative stress, especially when exposed to sunlight. For instance oxidative stress induced by UV radiation from a solar simulator (SSUV: 300-400 nm or UVA: 320400 nm) in cultured human melanocytes was stronger when melanogenesis was stimulated. In fact, photoinduced DNA breakage detected using the comet assay and fluorescence of the specific redox probe Dihydro-Rodhamine123 were enhanced upon UV exposure when melanin content increased. By comparing antioxidant status in human melanocytes and keratinocytes from same donors, we could show that (i) reduced glutathione content was higher in keratinocytes, (ii) basal expression of NQO1 (mRNA and protein) was higher in melanocytes, (iii) when Nrf2 was stimulated (by sulforaphane, lipoïc acid or by silencing of Keap1), HO1 (mRNA and protein) and modulatory subunit of γ-glutamyl-cysteine-ligase (GCLm, mRNA) were mainly induced in melanocytes whereas catalytic subunit of GCL (GCLc) was over expressed in keratinocytes, (iii) in a microarray assay, HO1, ferritin, catalase as well as genes from NQO or GST family displayed a stronger basal expression in melanocytes whereas genes from GPX family where mainly expressed in keratinocytes. Melanogenesis could influence such differences through endogenous generation of quinones and hydrogen peroxide. These data are of importance in order to better understand how skin can cope with environmental oxidative stress. doi: 10.1016/j.freeradbiomed.2010.10.369
361 Multistep Regulation of TXNIP Expression under Oxidative Stress Conditions: A role for microRNAs Fernando Toshio Ogata1, Wagner Luiz Batista1, Tarsis Gestira Ferreira1, Roberto Jun Arai1, Junji Yodoi2, and Hugo Pequeno Monteiro1 1 2 UNIFESP, Kyoto University Our group recently demonstrated that oxidative/nitrosative stress conditions mediated ERK1/2 MAPks activation which triggered TRX-1 nuclear translocation. These findings regarding TRX-1 are affected by ERK1/2 spatial distribution within cells, when activated by oxidants, these kinases migrate to the nucleus. This is associated with down-regulation of the physiological inhibitor of
S134
TRX-1, TXNIP. mRNA and protein levels of TXNIP are regulated by hyperglycemic conditions and elevated levels of reactive oxygen and nitrogen species – ROS and RNS. We also established that ROS/RNS mediate such control through ERK 1/2 MAPKs activity and spatial localization. In addition, we described a stress responsive domain on TXNIPs promoter rich in AP-1 homologous sequences. In the present communication, a further step was taken. We described another participant on the regulation of TXNIP expression under stress conditions, microRNAs. According to a database generated in our lab, we selected those microRNAs with the highest scores and test their ability to target TXNIPs mRNA. Three microRNAs were tested and the H2O2-modulated oncomir 93 expression correlated negatively with TXNIP expression at the protein level. In conclusion, regulation of TXNIP expression under stress conditions operates at different levels and microRNAs play an important role in this process. Acknowledgments:.FTO is a FAPESP fellow (2009/50708-4).
Hydrogen Peroxide Modulates the Transcriptional Regulation of the 3’IgH Regulatory Region via the NFKappa B Pathway Eric J Romer1, and Courtney E.W. Sulentic1 1 Wright State University Transcription of the immunoglobulin heavy chain (IgH) in B lymphocytes is governed by a 3'IgH Regulatory Region (3'IgHRR) and is an essential step in antibody production. Lymphocytes are known to be functionally affected by exposure to reactive oxygen species (ROS) such as H2O2. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in IgH transcriptional activity. This study demonstrates that μM concentrations of H2O2 induces a biphasic effect on 3'IgHRR activity and that expression of a degradation resistant Inhibitory κB (IκBαAA) can suppress H2O2-mediated activation of the 3’IgHRR. Furthermore, H2O2 concentrations that suppressed 3’IgHRR activity appeared to stabilize the NFκB family member p50 expression, increase IκBα expression, and induce the formation of a proteolytic cleavage product of IκBα which has been shown to exhibit a stronger inhibitory function than the noncleaved native IκBα. These observations suggest that exposure of B lymphocytes to H2O2 can modulate IgH transcriptional activity in a concentration-dependent manner via the 3'IgHRR and modulation of the NFκB pathway (Supported by NIEHS R01ES014676). doi: 10.1016/j.freeradbiomed.2010.10.371
363 Bioactive Antioxidants Regulate RACGTP Expression at the Leading Edge of Migrating Oral Fibroblasts Symone San Miguel1, Lynne Opperman1, Edward Allen1, Jan Zielinski2, and Kathy Svoboda1 1 2 TAMHSC Baylor College of Dentistry, Dallas, Texas, Zielinski Research, Vista, California The need for antioxidants (AO) becomes even more critical with increased exposure to free radicals generated by pollution, cigarette smoke, drugs, illness, stress and exercise. Smoking is associated with increased risk for oral health and dental problems. We tested the hypothesis that AOs could counteract the effects of nicotine on cell migration. To test the effects of nicotine and AO compositions on cell migration, human gingival fibroblast (HGF) or human periodontal ligament (HPDL) cells were
SFRBM/SFRRI 2010
grown to confluence in 4 quadrant dishes. They were then placed in 0.1% FBS medium and 6 mM nicotine for two hours. Thereafter, a scratch wound assay was performed using a sterile 10 µl pipet tip on confluent cell monolayers. Pure AO compounds, ferulic acid (F), tetrahydrocurcuminoids CG (T), and -5 resveratrol (R) in single, double or triple combinations (10 M) were tested. The migratory behavior of the wounded cells with or without nicotine in the presence of AO combinations was recorded every 15 min. for ten hours, using a Nikon Biostation. Immunohistochemical analysis of nicotine-treated cells had decreased signaling protein RacGTP at the leading edge of migrating cells. Treatment with single, double and triple combinations of antioxidants increased the level of Rac-GTP activation in HGF and HPDL cells at the leading edge. These results clearly demonstrated that combinations of AOs promote cell migration and counteract the effects of nicotine in cultured oral fibroblasts via the RacGTP signal transduction pathway. doi: 10.1016/j.freeradbiomed.2010.10.372
364 Modulation of 4Hydroxy2Nonenal Induced Signaling by Glutathione STransferase A44 Rajendra Sharma1, Pankaj Chaudhary1, Abha Sharma1, Rit Vatsyayan1, Sanjay Awasthi1, and Yogesh C Awasthi1 1 University of North Texas HLTH Sci. Center The lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE) has been recognized as an important second messenger in cell cycle signaling. Here we demonstrate that 4-HNE induces signaling for apoptosis via Fas mediated extrinsic and p53 mediated intrinsic pathway in HepG2 cells. 4-HNE induces apoptosis through p53 pathway by activating Bax, p21, JNK, and caspase-3. 4-HNE also induces Fas mediated DISC-independent apoptosis pathway by activating ASK1, JNK and caspase-3. 4-HNE exposure to HepG2 cells leads to the activation of both Fas and Daxx and promotes the export of Daxx from the nucleus to cytosol, where it binds to Fas and inhibits apoptosis. Depletion of Daxx by siRNA results in potentiation of apoptosis indicating that Daxx inhibits apoptosis by binding to Fas. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear translocation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE can be attenuated by transfection of cells with hGSTA4-4, the isozyme of glutathione S-transferase with high activity for 4HNE. Through immunoprecipitation and liquid chromatography– tandem mass spectrometry, we have demonstrated covalent binding of 4-HNE to Daxx. We have also provided evidence that 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 in HepG2 cells to facilitate its cytoplasmic export. These results indicate that while 4-HNE contributes to oxidant toxicity through several mechanisms, in parallel it evokes signaling for defense mechanisms to self regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/ repressors. Supported in part by NIH grants ES012171 (YCA) and CA77495 (SA) doi: 10.1016/j.freeradbiomed.2010.10.373
365 Thioredoxin 1 Downregulates but Thioredoxin Reductase Upregulates ROS Generation and Secretion/expression of MCP1 in Endotheliallike cells Xun Shen1, Beidong Chen1, Zhen-Bo Liu1, Dandan Guan1, and Dandan Guan1 1 Institute of Biophysics, Chinese Academy of Sciences To know if the high expression of thioredoxin 1（Trx1）and thioredoxin reductase 1 (TrxR1) in atherosclerotic plaques suggests a role of the enzymes in antioxidant defense mechanisms against atherosclerosis, the effect of Trx1 and TrxR1 on expression of MCP-1 and ROS generation was investigated in human endothelial cells. Opposite role of Trx1 and TrxR1 was observed: (1) Trx1 suppressed but TrxR1 enhanced ROS generation in the cells. The mechanism involves regulation of the phox expression of p22 , the subunit of NADPH oxidase. (2) Trx1 suppressed but TrxR1 enhanced the ox-LDL-stimulated MCP-1 release and expression. The former greatly promoted nuclear translocation of Ref-1 and subsequently reduced DNA-binding activity of AP-1, while the latter promoted nuclear translocation and DNA-binding activity of NF-κB. It seems that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis. However, TrxR1 appears a dualistic enzyme which catalyzes reduction of disulfide in oxidized thioredoxin, but enhances ROS generation and subsequent MCP1 expression in endothelium, thus may promote rather than prevent vascular endothelium from forming atherosclerotic plaque. doi: 10.1016/j.freeradbiomed.2010.10.374
366 The Majority of Nox1 Resides at Internal Membranes and Traffics Differentially with Stimulation Jennifer Streeter1, and Francis Miller1 1 University of Iowa The Nox1 subunit of NADPH oxidase is a membrane protein that generates superoxide and activates redox-dependent signaling pathways in multiple cell types. Localization appears to be a regulatory mechanism for Nox1 activation. However, little is known regarding the relative membrane distribution of Nox1 and whether it traffics in response to agonist stimulation. Previous studies of Nox1 localization are limited by the use of heterologous over-expression systems. The goal of this study was to characterize endogenous Nox1 localization and trafficking under basal and stimulated conditions. Studies utilized cultured murine and rat aortic smooth muscle cells (SMCs). Biotinylation of plasma membrane (PM) proteins reveal that at basal conditions ~5% of total cellular Nox1 resides at the PM. Having previously shown that tumor necrosis factor (TNF)-α and thrombin activate Nox1 in SMCs, we found that treatment of SMCs with TNF-α protects biotinylated Nox1 from surface biotin cleavage, indicating internalization of PM Nox1 in response to TNF-α. In contrast, treatment of cells with thrombin does not protect Nox1 from surface biotin cleavage, signifying the absence of Nox1 internalization in response to thrombin. Stimulation of SMCs with TNF-α or thrombin in the presence of biotin increased the amount of biotinylated Nox1, suggesting possible recruitment of intracellular Nox1 to the PM. However, inhibiting endocytosis with a dominant-negative dynamin or 4°C prevents this increase in biotinylated Nox1 in response to TNF-α or thrombin. We interpret this finding to indicate that Nox1 is not recruited to the PM after agonist stimulation, but instead, agonist-induced endosomes fuse with intracellular Nox1-containing vesicles. These novel findings confirm differential activation of Nox1 in response to different
SFRBM/SFRRI 2010
S135
360 Comparison of Antioxydant Defenses in Human Epidermal Melanocytes and Keratinocytes Suggests that Pigment Cells are Adapted to Oxidative Stress Induced by Melanin Related Compounds. 1,2
1
doi: 10.1016/j.freeradbiomed.2010.10.370
362
1
Laurent Marrot , Jean-Philippe Belaïdi , Laurence Denat , Daniel Duche1, Christophe Jones1, Philippe Perez1, Jérémie Sœur1, and JeanRoch Jean-Roch Meunier1 1 2 L'Oreal, Phototoxicity Melanin and its chemical intermediates can both generate and scavenge reactive species. Melanocyte have thus to manage a specific situation towards oxidative stress, especially when exposed to sunlight. For instance oxidative stress induced by UV radiation from a solar simulator (SSUV: 300-400 nm or UVA: 320400 nm) in cultured human melanocytes was stronger when melanogenesis was stimulated. In fact, photoinduced DNA breakage detected using the comet assay and fluorescence of the specific redox probe Dihydro-Rodhamine123 were enhanced upon UV exposure when melanin content increased. By comparing antioxidant status in human melanocytes and keratinocytes from same donors, we could show that (i) reduced glutathione content was higher in keratinocytes, (ii) basal expression of NQO1 (mRNA and protein) was higher in melanocytes, (iii) when Nrf2 was stimulated (by sulforaphane, lipoïc acid or by silencing of Keap1), HO1 (mRNA and protein) and modulatory subunit of γ-glutamyl-cysteine-ligase (GCLm, mRNA) were mainly induced in melanocytes whereas catalytic subunit of GCL (GCLc) was over expressed in keratinocytes, (iii) in a microarray assay, HO1, ferritin, catalase as well as genes from NQO or GST family displayed a stronger basal expression in melanocytes whereas genes from GPX family where mainly expressed in keratinocytes. Melanogenesis could influence such differences through endogenous generation of quinones and hydrogen peroxide. These data are of importance in order to better understand how skin can cope with environmental oxidative stress. doi: 10.1016/j.freeradbiomed.2010.10.369
361 Multistep Regulation of TXNIP Expression under Oxidative Stress Conditions: A role for microRNAs Fernando Toshio Ogata1, Wagner Luiz Batista1, Tarsis Gestira Ferreira1, Roberto Jun Arai1, Junji Yodoi2, and Hugo Pequeno Monteiro1 1 2 UNIFESP, Kyoto University Our group recently demonstrated that oxidative/nitrosative stress conditions mediated ERK1/2 MAPks activation which triggered TRX-1 nuclear translocation. These findings regarding TRX-1 are affected by ERK1/2 spatial distribution within cells, when activated by oxidants, these kinases migrate to the nucleus. This is associated with down-regulation of the physiological inhibitor of
S134
TRX-1, TXNIP. mRNA and protein levels of TXNIP are regulated by hyperglycemic conditions and elevated levels of reactive oxygen and nitrogen species – ROS and RNS. We also established that ROS/RNS mediate such control through ERK 1/2 MAPKs activity and spatial localization. In addition, we described a stress responsive domain on TXNIPs promoter rich in AP-1 homologous sequences. In the present communication, a further step was taken. We described another participant on the regulation of TXNIP expression under stress conditions, microRNAs. According to a database generated in our lab, we selected those microRNAs with the highest scores and test their ability to target TXNIPs mRNA. Three microRNAs were tested and the H2O2-modulated oncomir 93 expression correlated negatively with TXNIP expression at the protein level. In conclusion, regulation of TXNIP expression under stress conditions operates at different levels and microRNAs play an important role in this process. Acknowledgments:.FTO is a FAPESP fellow (2009/50708-4).
Hydrogen Peroxide Modulates the Transcriptional Regulation of the 3’IgH Regulatory Region via the NFKappa B Pathway Eric J Romer1, and Courtney E.W. Sulentic1 1 Wright State University Transcription of the immunoglobulin heavy chain (IgH) in B lymphocytes is governed by a 3'IgH Regulatory Region (3'IgHRR) and is an essential step in antibody production. Lymphocytes are known to be functionally affected by exposure to reactive oxygen species (ROS) such as H2O2. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in IgH transcriptional activity. This study demonstrates that μM concentrations of H2O2 induces a biphasic effect on 3'IgHRR activity and that expression of a degradation resistant Inhibitory κB (IκBαAA) can suppress H2O2-mediated activation of the 3’IgHRR. Furthermore, H2O2 concentrations that suppressed 3’IgHRR activity appeared to stabilize the NFκB family member p50 expression, increase IκBα expression, and induce the formation of a proteolytic cleavage product of IκBα which has been shown to exhibit a stronger inhibitory function than the noncleaved native IκBα. These observations suggest that exposure of B lymphocytes to H2O2 can modulate IgH transcriptional activity in a concentration-dependent manner via the 3'IgHRR and modulation of the NFκB pathway (Supported by NIEHS R01ES014676). doi: 10.1016/j.freeradbiomed.2010.10.371
363 Bioactive Antioxidants Regulate RACGTP Expression at the Leading Edge of Migrating Oral Fibroblasts Symone San Miguel1, Lynne Opperman1, Edward Allen1, Jan Zielinski2, and Kathy Svoboda1 1 2 TAMHSC Baylor College of Dentistry, Dallas, Texas, Zielinski Research, Vista, California The need for antioxidants (AO) becomes even more critical with increased exposure to free radicals generated by pollution, cigarette smoke, drugs, illness, stress and exercise. Smoking is associated with increased risk for oral health and dental problems. We tested the hypothesis that AOs could counteract the effects of nicotine on cell migration. To test the effects of nicotine and AO compositions on cell migration, human gingival fibroblast (HGF) or human periodontal ligament (HPDL) cells were
SFRBM/SFRRI 2010
grown to confluence in 4 quadrant dishes. They were then placed in 0.1% FBS medium and 6 mM nicotine for two hours. Thereafter, a scratch wound assay was performed using a sterile 10 µl pipet tip on confluent cell monolayers. Pure AO compounds, ferulic acid (F), tetrahydrocurcuminoids CG (T), and -5 resveratrol (R) in single, double or triple combinations (10 M) were tested. The migratory behavior of the wounded cells with or without nicotine in the presence of AO combinations was recorded every 15 min. for ten hours, using a Nikon Biostation. Immunohistochemical analysis of nicotine-treated cells had decreased signaling protein RacGTP at the leading edge of migrating cells. Treatment with single, double and triple combinations of antioxidants increased the level of Rac-GTP activation in HGF and HPDL cells at the leading edge. These results clearly demonstrated that combinations of AOs promote cell migration and counteract the effects of nicotine in cultured oral fibroblasts via the RacGTP signal transduction pathway. doi: 10.1016/j.freeradbiomed.2010.10.372
364 Modulation of 4Hydroxy2Nonenal Induced Signaling by Glutathione STransferase A44 Rajendra Sharma1, Pankaj Chaudhary1, Abha Sharma1, Rit Vatsyayan1, Sanjay Awasthi1, and Yogesh C Awasthi1 1 University of North Texas HLTH Sci. Center The lipid peroxidation product, 4-hydroxy-2-nonenal (4-HNE) has been recognized as an important second messenger in cell cycle signaling. Here we demonstrate that 4-HNE induces signaling for apoptosis via Fas mediated extrinsic and p53 mediated intrinsic pathway in HepG2 cells. 4-HNE induces apoptosis through p53 pathway by activating Bax, p21, JNK, and caspase-3. 4-HNE also induces Fas mediated DISC-independent apoptosis pathway by activating ASK1, JNK and caspase-3. 4-HNE exposure to HepG2 cells leads to the activation of both Fas and Daxx and promotes the export of Daxx from the nucleus to cytosol, where it binds to Fas and inhibits apoptosis. Depletion of Daxx by siRNA results in potentiation of apoptosis indicating that Daxx inhibits apoptosis by binding to Fas. 4-HNE-induced translocation of Daxx is also accompanied by the activation and nuclear translocation of HSF1 and up-regulation of heat shock protein Hsp70. All these effects of 4-HNE can be attenuated by transfection of cells with hGSTA4-4, the isozyme of glutathione S-transferase with high activity for 4HNE. Through immunoprecipitation and liquid chromatography– tandem mass spectrometry, we have demonstrated covalent binding of 4-HNE to Daxx. We have also provided evidence that 4-HNE modification induces phosphorylation of Daxx at Ser668 and Ser671 in HepG2 cells to facilitate its cytoplasmic export. These results indicate that while 4-HNE contributes to oxidant toxicity through several mechanisms, in parallel it evokes signaling for defense mechanisms to self regulate its toxicity and can simultaneously affect multiple signaling pathways through its interactions with membrane receptors and transcription factors/ repressors. Supported in part by NIH grants ES012171 (YCA) and CA77495 (SA) doi: 10.1016/j.freeradbiomed.2010.10.373
365 Thioredoxin 1 Downregulates but Thioredoxin Reductase Upregulates ROS Generation and Secretion/expression of MCP1 in Endotheliallike cells Xun Shen1, Beidong Chen1, Zhen-Bo Liu1, Dandan Guan1, and Dandan Guan1 1 Institute of Biophysics, Chinese Academy of Sciences To know if the high expression of thioredoxin 1（Trx1）and thioredoxin reductase 1 (TrxR1) in atherosclerotic plaques suggests a role of the enzymes in antioxidant defense mechanisms against atherosclerosis, the effect of Trx1 and TrxR1 on expression of MCP-1 and ROS generation was investigated in human endothelial cells. Opposite role of Trx1 and TrxR1 was observed: (1) Trx1 suppressed but TrxR1 enhanced ROS generation in the cells. The mechanism involves regulation of the phox expression of p22 , the subunit of NADPH oxidase. (2) Trx1 suppressed but TrxR1 enhanced the ox-LDL-stimulated MCP-1 release and expression. The former greatly promoted nuclear translocation of Ref-1 and subsequently reduced DNA-binding activity of AP-1, while the latter promoted nuclear translocation and DNA-binding activity of NF-κB. It seems that Trx1 inherently suppresses MCP-1 expression in vascular endothelium and may prevent atherosclerosis. However, TrxR1 appears a dualistic enzyme which catalyzes reduction of disulfide in oxidized thioredoxin, but enhances ROS generation and subsequent MCP1 expression in endothelium, thus may promote rather than prevent vascular endothelium from forming atherosclerotic plaque. doi: 10.1016/j.freeradbiomed.2010.10.374
366 The Majority of Nox1 Resides at Internal Membranes and Traffics Differentially with Stimulation Jennifer Streeter1, and Francis Miller1 1 University of Iowa The Nox1 subunit of NADPH oxidase is a membrane protein that generates superoxide and activates redox-dependent signaling pathways in multiple cell types. Localization appears to be a regulatory mechanism for Nox1 activation. However, little is known regarding the relative membrane distribution of Nox1 and whether it traffics in response to agonist stimulation. Previous studies of Nox1 localization are limited by the use of heterologous over-expression systems. The goal of this study was to characterize endogenous Nox1 localization and trafficking under basal and stimulated conditions. Studies utilized cultured murine and rat aortic smooth muscle cells (SMCs). Biotinylation of plasma membrane (PM) proteins reveal that at basal conditions ~5% of total cellular Nox1 resides at the PM. Having previously shown that tumor necrosis factor (TNF)-α and thrombin activate Nox1 in SMCs, we found that treatment of SMCs with TNF-α protects biotinylated Nox1 from surface biotin cleavage, indicating internalization of PM Nox1 in response to TNF-α. In contrast, treatment of cells with thrombin does not protect Nox1 from surface biotin cleavage, signifying the absence of Nox1 internalization in response to thrombin. Stimulation of SMCs with TNF-α or thrombin in the presence of biotin increased the amount of biotinylated Nox1, suggesting possible recruitment of intracellular Nox1 to the PM. However, inhibiting endocytosis with a dominant-negative dynamin or 4°C prevents this increase in biotinylated Nox1 in response to TNF-α or thrombin. We interpret this finding to indicate that Nox1 is not recruited to the PM after agonist stimulation, but instead, agonist-induced endosomes fuse with intracellular Nox1-containing vesicles. These novel findings confirm differential activation of Nox1 in response to different
SFRBM/SFRRI 2010
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