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Low Dose Cadmium Stimulates Myofibroblast Differentiation and Lung Fibrosis by Activation of SMAD and Nuclear Thioredoxin-1
61 Molecular Basis for the Redox Regulation of the Src Kinase David E. Heppner1, Christopher M. Dustin1, Chenyi Liao1, Milena Hristova1, Bin Deng1, Ying-Wai Lam1, Jianing Li1, and Albert van der Vliet1 1 University of Vermont, Burlington, USA The non-receptor tyrosine kinase Src facilitates signal transduction processes that govern a range of cellular functions. While it is well established that Src activity is regulated by conformational changes of the Src protein in tyrosine phosphorylation-dependent mechanisms, recent evidence supports the notion that Src is also redox regulated involving oxidation of Src cysteine residues. However, current reports are inconsistent with respect to the identity of the redox-sensitive cysteines and their consequence on Src kinase activation. Our previous studies show that the NADPH oxidase DUOX1 generates H2O2 to promote Src activation as a critical mechanism in lung epithelial wound responses, which involves DUOX1-dependent oxidation of Src cysteines. This present study aims to characterize the molecular-level details of Src redox regulation. Mass spectrometry using dimedone-trapping approaches indicates that of the nine Src cysteines C185 and C277 are oxidized to sulfenic acids (-SOH) by H2O2. Interestingly, while earlier reports implicate C277 oxidation in inactivation of Src, oxidation of C185 has not been previously observed. Importantly, Src kinase activity is enhanced by H2O2 in WT but not for the C185A variant confirming a role for C185 in Src redox regulation. Molecular dynamics simulations reveal that sulfenic acids at C185 and C277 induce significant structural changes within Src consistent with kinase activation. Specifically, compared to the reduced thiol, sulfenic acids introduce new electrostatic interactions that weaken the regulatory phosphotyrosine-SH2 binding and induce unfolding of the Src activation loop. These results present a more complete understanding of oxidative mechanisms of Src-mediated signaling relevant to physiology and in pathological conditions. Supported by R01HL085646, F32HL129706, P20GM103449, PSCA15029P.
doi: 10.1016/j.freeradbiomed.2016.10.102 62 Molecular Mechanism of PI 3-Kinase-Akt Signaling Stimulated by 1,2-Naphthoquinone (NQ) Hideki Hiraoka1, Kyohei Hamada1, Kengo Nakahara1, Yoshito Kumagai2, and Takashi Uehara1 1 Dept. Medicinal Pharmacol., Grad. Sch. Med. Den. Pharmaceutical Sci., Okayama Univ., Japan, 2Biomed Sci., Grad. Sch. Comprehensive Human Sci., Univ. Tsukuba, Japan The redox-mediated chemical modification occurs via oxidative reaction between electrophile and cysteine thiol in the presence of an electron acceptor. It is widely recognized that long exposure to or high concentration of 1,2-NQ, an electrophile in cigarette ingredients/smoke or metabolite of naphthalene, results in cytotoxic effects. Whereas, low concentrations of 1,2-NQ specifically stimulates Akt signaling involving cell proliferation and anti-apoptosis possibly via protein oxidation. In the present study, we attempted to identify the target protein of 1,2-NQ and to clarify the protective role of 1,2-NQ on apoptotic cell death.
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The levels of phosphorylated Akt were increased in 1,2-NQ-treated cells in a concentration- and time-dependent manner in human lung adenocarcinoma A549 cells. This phosphorylation induced by 1,2-NQ was sensitive to PI 3-kinase and PDK1 inhibitors. Then, we found that 1,2-NQ triggers phosphorylation of tyrosine kinase receptor. Akt activation contributes to protect the apoptotic cell death of A549 cells induced by serum deprivation possibly via Bad phosphorylation. Our findings show that treatment with appropriate amount of 1,2NQ leads to anti-apoptotic action through PI 3-kinase–Akt signaling.
doi: 10.1016/j.freeradbiomed.2016.10.103 63 Redox-Regulation of Ca2+-Independent Phospholipase A2γ: From Computational Predictions to Experimental Procedures Blanka Holendová1, Petr Ježek1, and Martin Jabůrek1 1 Department of Mitochondrial Physiology, Institute of Physiology CAS, Prague, Czech Republic Redox reactions of cysteine residues lead to a range of reversible post-translational modifications of proteins, regulating their functions. Ca2+-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes which catalyze the hydrolysis of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids, contributing to cellular signaling, membrane homeostasis and energy metabolism. The activity of iPLA 2γ is regulated by a redox-sensitive process, suggesting a thiol-based modification. Using recent computational methods, we searched the primary sequence of human iPLA2γ for potential cysteine residues that could be involved in a thiol-based redox regulation of the protein. Two cysteines, Cys233 and Cys329, were pointed out as highly probable S-sulfenylation target sites. In addition, we used protein structure homology modeling to create a three-dimensional structure of iPLA2γ to visualize the topology of the predicted cysteine residues. The primary sequence was aligned to search for suitable templates and the model was optimized by energy minimization using the GROMOS96 parameter set and checked for errors in the 3D protein structure. The final 3D structure with visualized cysteine residues was designed using molecular graphic system PyMOL. Using isolated recombinant human iPLA2γ, both Cys233Ser and Cys329Ser targeted mutations revealed altered regulation of the catalytic activity by H2O2. These results are consistent with the participation of iPLA2γ in cellular redox homeostasis and serve as preliminary tools leading to a direct identification of cysteine oxidative modifications of the enzyme. Supported by grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.
doi: 10.1016/j.freeradbiomed.2016.10.104 64 Low Dose Cadmium Stimulates Myofibroblast Differentiation and Lung Fibrosis by Activation of SMAD and Nuclear Thioredoxin-1 Xin Hu1, Jolyn Fernandes1, Dean P Jones1, and Young-Mi Go1 Emory University, Atlanta, USA
1
SfRBM / SFRRI 2016
Background: Toxic effects of cadmium (Cd) from cigarette smoke and occupational sources are well studied, yet low-level Cd also occurs in human diet. Cd accumulates in vivo due to a twenty-year biological half-life. Increasing evidence suggests that exposure to dietary Cd can impact human health by potentiating inflammatory mechanism. Submicromolar Cd can alter cellular redox control mechanism, contributing to stimulation of pulmonary diseases. The pathogenesis of idiopathic pulmonary fibrosis involves excessive myofibroblast activation which leads to increased contractures in lung. Substantial evidence supports that myofibroblast differentiation is associated with disruption of redox homeostasis, yet the mechanistic effects of exogenous exposure remains unknown. Objectives: To test our hypothesis that low dietary levels of Cd stimulate pulmonary fibrosis via activation of SMAD transcription factor and stimulation of myofibroblast differentiation. Results: Using human fetal lung fibroblasts (HFLF), we found that two differentiation markers, α-Smooth-Muscle-actin (α-SM-actin) and Extra-Domain-A-containing fibronectin (ED-A FN), were significantly elevated in transcriptional and translational levels by low-dose Cd (0.5, 1.0 and 2.0 μM). Under the same Cd treatment, an increased formation of stress fibers was observed in HFLF. To examine the effects of low-dose Cd on SMAD activation and redox regulatory mechanism, HFLF was transfected with nuclear exporting or nuclear localized signal conjugated thioredoxin (Trx)1 to express in cytoplasm or nucleus, respectively. The results show that Cd increased SMAD activity by 3 fold and stimulated translocation of Trx1 from cytoplasm to nucleus. Overexpression of Trx1 in cytoplasm diminished Cd induced SMAD activation, while overexpression of Trx1 in nuclei mimicked the Cd effect. This suggests that low-dose Cd stimulates differentiation of HFLF to myofibroblast by mechanism involving nuclear translocation of Trx1 followed by activation of SMAD and subsequent gene expression of α-SM-actin and ED-A FN for differentiation and fibrosis. Conclusion: Low dietary exposure to Cd presents a potential risk for pulmonary fibrosis by affecting redox signaling mechanism. The improved understanding of redox regulation mechanism in differentiation and fibrosis can provide therapeutic targets to maintain pulmonary health.
doi: 10.1016/j.freeradbiomed.2016.10.105 65 Nrf2 in Keratinocytes Modulates UVB-Induced DNA Damage and Apoptosis in Melanocytes through ROSDependent Mechanism Saowanee Jeayeng1, Adisak Wongkajornsilp1, Andrzej T Slominski2, Siwanon Jirawatnotai1, Somponnat Sampattavanich1, and Uraiwan Panich1 1 Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand, 2 Department of Dermatology, University of Alabama at Birmingham, USA Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be regulated by their neighbouring keratinocytes (KC). Aims: We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC co-cultured with primary human KC using a trans-well co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Results: Co-culture of MC with siNrf2-transfected KC enhanced
UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation in correlation with phosphorylation of ERK, JNK and p38 in MC. MC damage induced by UVB was increased in MC treated with conditioned medium (CM) derived from Nrf2-depleted KC, suggesting that KC modulated UVBmediated MC responses via paracrine effects. Additionally, at 30 min post-irradiation, depletion of Nrf2 in KC suppressed UVBinduced α-MSH levels, although pre-treatment with Nacetylcysteine (NAC) elevated its levels in CM from siNrf2transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis in MC. Conclusion: Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC responses, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of reactive oxygen species (ROS) levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate paracrine effect of KC probably by affecting levels of the paracrine factor α-MSH via a ROSdependent mechanism.
doi: 10.1016/j.freeradbiomed.2016.10.106 66 Copper Transporter, Antioxidant-1 (Atox-1), Modulates Redox Homeostasis during Phorbol-EsterInduced THP-1 Cell Differentiation Tetsuro Kamiya1, Kosuke Takeuchi1, Saki Fukudome1, Hirokazu Hara1, and Tetsuo Adachi1 1 Gifu Pharmaceutical University, Japan Copper (Cu), an essential micronutrient, is involved in vascular remodeling, and its bioavailability regulated by Cu transporter proteins, such as ATP7A and antioxidant-1 (Atox-1). Superoxide dismutase 3 (SOD3), which has a Cu ion in the active site, plays a pivotal role in the redox homeostasis, and its loss exacerbates vascular diseases. We have reported that SOD3 expression induces in phorbol ester (TPA)-treated human monocytic THP-1 cells; however, the involvement of Cu ion in its induction is entirely unknown. TPA stimulation in THP-1 cells induced ATP7 expression and Atox-1 nuclear translocation through PKC and MEK/ERK signaling. According to the previous report, which shows that Atox1 functions as a transcription factor for SOD3, we demonstrated the role of Atox-1 in TPA-elicited SOD3 induction. Overexpression of Atox-1 protein enhanced TPA-elicited SOD3 expression, and knockdown of Atox-1 with siRNA suppressed its expression, suggesting that Atox-1 plays a pivotal role in SOD3 regulation in THP-1 cells. Additionally, knockdown of Atox-1 suppressed mRNA expression of gp91phox, an essential component of NOX2. Overall, we have determined that intracellular Cu modulation via its transporter might regulate redox homeostasis in THP-1 cells by the alteration in expression of SOD3 and gp91phox.
doi: 10.1016/j.freeradbiomed.2016.10.107
SfRBM / SFRRI 2016
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doi: 10.1016/j.freeradbiomed.2016.10.102 62 Molecular Mechanism of PI 3-Kinase-Akt Signaling Stimulated by 1,2-Naphthoquinone (NQ) Hideki Hiraoka1, Kyohei Hamada1, Kengo Nakahara1, Yoshito Kumagai2, and Takashi Uehara1 1 Dept. Medicinal Pharmacol., Grad. Sch. Med. Den. Pharmaceutical Sci., Okayama Univ., Japan, 2Biomed Sci., Grad. Sch. Comprehensive Human Sci., Univ. Tsukuba, Japan The redox-mediated chemical modification occurs via oxidative reaction between electrophile and cysteine thiol in the presence of an electron acceptor. It is widely recognized that long exposure to or high concentration of 1,2-NQ, an electrophile in cigarette ingredients/smoke or metabolite of naphthalene, results in cytotoxic effects. Whereas, low concentrations of 1,2-NQ specifically stimulates Akt signaling involving cell proliferation and anti-apoptosis possibly via protein oxidation. In the present study, we attempted to identify the target protein of 1,2-NQ and to clarify the protective role of 1,2-NQ on apoptotic cell death.
S40
The levels of phosphorylated Akt were increased in 1,2-NQ-treated cells in a concentration- and time-dependent manner in human lung adenocarcinoma A549 cells. This phosphorylation induced by 1,2-NQ was sensitive to PI 3-kinase and PDK1 inhibitors. Then, we found that 1,2-NQ triggers phosphorylation of tyrosine kinase receptor. Akt activation contributes to protect the apoptotic cell death of A549 cells induced by serum deprivation possibly via Bad phosphorylation. Our findings show that treatment with appropriate amount of 1,2NQ leads to anti-apoptotic action through PI 3-kinase–Akt signaling.
doi: 10.1016/j.freeradbiomed.2016.10.103 63 Redox-Regulation of Ca2+-Independent Phospholipase A2γ: From Computational Predictions to Experimental Procedures Blanka Holendová1, Petr Ježek1, and Martin Jabůrek1 1 Department of Mitochondrial Physiology, Institute of Physiology CAS, Prague, Czech Republic Redox reactions of cysteine residues lead to a range of reversible post-translational modifications of proteins, regulating their functions. Ca2+-independent phospholipase A2γ (iPLA2γ) belongs to a family of enzymes which catalyze the hydrolysis of membrane glycerophospholipids to liberate free fatty acids and lysophospholipids, contributing to cellular signaling, membrane homeostasis and energy metabolism. The activity of iPLA 2γ is regulated by a redox-sensitive process, suggesting a thiol-based modification. Using recent computational methods, we searched the primary sequence of human iPLA2γ for potential cysteine residues that could be involved in a thiol-based redox regulation of the protein. Two cysteines, Cys233 and Cys329, were pointed out as highly probable S-sulfenylation target sites. In addition, we used protein structure homology modeling to create a three-dimensional structure of iPLA2γ to visualize the topology of the predicted cysteine residues. The primary sequence was aligned to search for suitable templates and the model was optimized by energy minimization using the GROMOS96 parameter set and checked for errors in the 3D protein structure. The final 3D structure with visualized cysteine residues was designed using molecular graphic system PyMOL. Using isolated recombinant human iPLA2γ, both Cys233Ser and Cys329Ser targeted mutations revealed altered regulation of the catalytic activity by H2O2. These results are consistent with the participation of iPLA2γ in cellular redox homeostasis and serve as preliminary tools leading to a direct identification of cysteine oxidative modifications of the enzyme. Supported by grant GA15-02051S to M.J. from the Grant Agency of the Czech Republic.
doi: 10.1016/j.freeradbiomed.2016.10.104 64 Low Dose Cadmium Stimulates Myofibroblast Differentiation and Lung Fibrosis by Activation of SMAD and Nuclear Thioredoxin-1 Xin Hu1, Jolyn Fernandes1, Dean P Jones1, and Young-Mi Go1 Emory University, Atlanta, USA
1
SfRBM / SFRRI 2016
Background: Toxic effects of cadmium (Cd) from cigarette smoke and occupational sources are well studied, yet low-level Cd also occurs in human diet. Cd accumulates in vivo due to a twenty-year biological half-life. Increasing evidence suggests that exposure to dietary Cd can impact human health by potentiating inflammatory mechanism. Submicromolar Cd can alter cellular redox control mechanism, contributing to stimulation of pulmonary diseases. The pathogenesis of idiopathic pulmonary fibrosis involves excessive myofibroblast activation which leads to increased contractures in lung. Substantial evidence supports that myofibroblast differentiation is associated with disruption of redox homeostasis, yet the mechanistic effects of exogenous exposure remains unknown. Objectives: To test our hypothesis that low dietary levels of Cd stimulate pulmonary fibrosis via activation of SMAD transcription factor and stimulation of myofibroblast differentiation. Results: Using human fetal lung fibroblasts (HFLF), we found that two differentiation markers, α-Smooth-Muscle-actin (α-SM-actin) and Extra-Domain-A-containing fibronectin (ED-A FN), were significantly elevated in transcriptional and translational levels by low-dose Cd (0.5, 1.0 and 2.0 μM). Under the same Cd treatment, an increased formation of stress fibers was observed in HFLF. To examine the effects of low-dose Cd on SMAD activation and redox regulatory mechanism, HFLF was transfected with nuclear exporting or nuclear localized signal conjugated thioredoxin (Trx)1 to express in cytoplasm or nucleus, respectively. The results show that Cd increased SMAD activity by 3 fold and stimulated translocation of Trx1 from cytoplasm to nucleus. Overexpression of Trx1 in cytoplasm diminished Cd induced SMAD activation, while overexpression of Trx1 in nuclei mimicked the Cd effect. This suggests that low-dose Cd stimulates differentiation of HFLF to myofibroblast by mechanism involving nuclear translocation of Trx1 followed by activation of SMAD and subsequent gene expression of α-SM-actin and ED-A FN for differentiation and fibrosis. Conclusion: Low dietary exposure to Cd presents a potential risk for pulmonary fibrosis by affecting redox signaling mechanism. The improved understanding of redox regulation mechanism in differentiation and fibrosis can provide therapeutic targets to maintain pulmonary health.
doi: 10.1016/j.freeradbiomed.2016.10.105 65 Nrf2 in Keratinocytes Modulates UVB-Induced DNA Damage and Apoptosis in Melanocytes through ROSDependent Mechanism Saowanee Jeayeng1, Adisak Wongkajornsilp1, Andrzej T Slominski2, Siwanon Jirawatnotai1, Somponnat Sampattavanich1, and Uraiwan Panich1 1 Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand, 2 Department of Dermatology, University of Alabama at Birmingham, USA Responses of melanocytes (MC) to ultraviolet (UV) irradiation can be regulated by their neighbouring keratinocytes (KC). Aims: We investigated the role of Nrf2 in regulating paracrine effects of KC on UVB-induced MC responses through phosphorylation of MAPKs in association with oxidative stress in primary human MC co-cultured with primary human KC using a trans-well co-culture system and small-interfering RNA-mediated silencing of Nrf2 (siNrf2). The mechanisms by which Nrf2 modulated paracrine factors including α-melanocyte-stimulating hormone (α-MSH) and paracrine effects of KC on UVB-mediated apoptosis were also assessed. Results: Co-culture of MC with siNrf2-transfected KC enhanced
UVB-mediated cyclobutane pyrimidine dimer (CPD) formation, apoptosis and oxidant formation in correlation with phosphorylation of ERK, JNK and p38 in MC. MC damage induced by UVB was increased in MC treated with conditioned medium (CM) derived from Nrf2-depleted KC, suggesting that KC modulated UVBmediated MC responses via paracrine effects. Additionally, at 30 min post-irradiation, depletion of Nrf2 in KC suppressed UVBinduced α-MSH levels, although pre-treatment with Nacetylcysteine (NAC) elevated its levels in CM from siNrf2transfected KC. Furthermore, NAC reversed the effect of CM from Nrf2-depleted KC on UVB-induced apoptosis in MC. Conclusion: Our study demonstrates for the first time that KC provided a rescue effect on UVB-mediated MC responses, although depletion of Nrf2 in KC reversed its protective effects on MC in a paracrine fashion in association with elevation of reactive oxygen species (ROS) levels and activation of MAPK pathways in MC. Nrf2 may indirectly regulate paracrine effect of KC probably by affecting levels of the paracrine factor α-MSH via a ROSdependent mechanism.
doi: 10.1016/j.freeradbiomed.2016.10.106 66 Copper Transporter, Antioxidant-1 (Atox-1), Modulates Redox Homeostasis during Phorbol-EsterInduced THP-1 Cell Differentiation Tetsuro Kamiya1, Kosuke Takeuchi1, Saki Fukudome1, Hirokazu Hara1, and Tetsuo Adachi1 1 Gifu Pharmaceutical University, Japan Copper (Cu), an essential micronutrient, is involved in vascular remodeling, and its bioavailability regulated by Cu transporter proteins, such as ATP7A and antioxidant-1 (Atox-1). Superoxide dismutase 3 (SOD3), which has a Cu ion in the active site, plays a pivotal role in the redox homeostasis, and its loss exacerbates vascular diseases. We have reported that SOD3 expression induces in phorbol ester (TPA)-treated human monocytic THP-1 cells; however, the involvement of Cu ion in its induction is entirely unknown. TPA stimulation in THP-1 cells induced ATP7 expression and Atox-1 nuclear translocation through PKC and MEK/ERK signaling. According to the previous report, which shows that Atox1 functions as a transcription factor for SOD3, we demonstrated the role of Atox-1 in TPA-elicited SOD3 induction. Overexpression of Atox-1 protein enhanced TPA-elicited SOD3 expression, and knockdown of Atox-1 with siRNA suppressed its expression, suggesting that Atox-1 plays a pivotal role in SOD3 regulation in THP-1 cells. Additionally, knockdown of Atox-1 suppressed mRNA expression of gp91phox, an essential component of NOX2. Overall, we have determined that intracellular Cu modulation via its transporter might regulate redox homeostasis in THP-1 cells by the alteration in expression of SOD3 and gp91phox.
doi: 10.1016/j.freeradbiomed.2016.10.107
SfRBM / SFRRI 2016
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