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The Role of Nrf2 Signaling in the Regulation of Vascular BK Channels in Diabetes
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Yong Li1, Xiao-Li Wang1, Qiang Chai1, Tong Lu1, Jingchao Li1, and Hon-Chi Lee1 1 Mayo Clinic, USA Background and Objective: The nuclear factor E2-related factor-2 (Nrf2) signaling pathway has emerged as a master regulator of cellular redox status. The large conductance calcium-activated potassium (BK) channels, which are major determinants of vasodilation, are impaired in diabetes mellitus (DM) due to the down-regulation of BK-ȕE\UHDFWLYHR[\JHQVSHFLHV-dependent mechanisms. The goal of this study is to test the hypothesis that Nrf2 signaling plays a central role in the regulation of BK channel function in DM. Methods and Results: Studies were performed combining cellular, molecular, vascular, and electrophysiological techniques. In type 2 diabetic db/db mouse aorta, Nrf2 protein expression was significantly reduced, associated with significant down-regulation of BK-ȕ DQG Keme oxygenase 1 (HO-1), a known Nrf2 downstream target. Also, the muscle ring finger protein 1 (MuRF1), a known E-3 ligase targeting BK-ȕ ZDV VLJQLILFDQWO\ upregulated. These findings were reproduced by knockdown of Nrf2 by siRNA in cultured human coronary smooth muscle cells (HCSMC), whereas adenoviral transfer of Nrf2 gene in these cells was associated with down-regulation of MuRF1 and upregulation of BK-ȕ DQG +2-1 expression. Activation of Nrf2 by dimethyl fumarate in high glucose-cultured HCSMCs or in diabetic db/db mouse coronary arteries preserved BK-ȕ H[SUHVVLRQ VKRZQ E\ Western blot, BK channel activities using patch clamp recordings, and vascular function assessed by shear stress-mediated vasodilation in isolated mouse coronary arteries. Conclusions: Expression of BK-ȕ LV FORVHO\ UHJXODWHG E\ 1UI through its effect on MuRF1, which regulates BK-E1 degradation, and vascular BK channel function can be restored by activation of Nrf2. Nrf2 should be considered a novel therapeutic target in the treatment of diabetic vasculopathy.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.140 101 5HGR['HSHQGHQW,QWHUDFWLRQ%HWZHHQ3URWHLQ 'LVXOILGH,VRPHUDVHDQG3SKR[DFWLYDWHV1R[ 1$'3+2[LGDVHLQ9DVFXODU'LVHDVH
Marcela Gimenez1,2, Brandon Schickling2, Ilka Wittig3, Francis Miller2, and Lucia Rossetti Lopes1 1 University of São Paulo, Brazil, 2University of Iowa, USA, 3 University of Frankfurt, Germany We previously showed that Protein disulfide isomerase (PDI) expression and NADPH oxidase-dependent ROS generation are increased in resistance arteries in hypertension. Here, we sought to investigate the molecular mechanisms through which PDI regulates Nox1 activation. Recombinant proteins were expressed in Escherichia coli transformed with plasmids encoding p47phox, PDI wild type (PDIwt) and PDI mutated (PDImut) to serine in all four cysteines of the catalytic motifs (C36, 39, 383, 386S). Incubation of PDIwt with p47phox demonstrated intermolecular disulfide bonding of the two proteins, as detected by non-reducing western blot, only after addition of arachidonic acid, confirming this interaction is dependent on p47phox activation and unfolding. This bonding was not detected when p47phox was incubated with
S54
PDImut. The overexpression of PDTwt, but not PDImut, increased superoxide generation in vascular smooth muscle cells and ERK phosphorylation in thrombin and TNF-Į-stimulated HEK293 cells. Next, Cos phox cells constitutively expressing p22phox were transfected with Nox1, p47phox, p67phox, and PDIwt or PDImut. PDIwt, but not PDImut, increased superoxide generation and recruitment of p47phox to the membrane in resting conditions and after thrombin stimulation. Furthermore, recruitment of PDI to the oxidase complex was impaired with the PDImut. These data implicate a role for the redox cysteines of PDI in Nox1 complex assembly and activation. Mass spectrometry analysis identified PDI/p47phox homo and hetero-dipeptides and revealed several disulfide bonds between PDI and p47phox. Mutation of p47phox cysteine 196 to alanine decreased superoxide generation and Nox1 complex assembly by PDI in resting and stimulated cells. These findings suggest that PDI facilitates redox-mediated recruitment of Nox1 regulatory subunits to the plasma membrane and contributes to Nox1 signaling in vascular disease.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.141 102 0RGXODWLRQRI0DFURSKDJH)XQFWLRQE\2[LGL]HG 3KRVSKROLSLGVLQ/RZ'HQVLW\/LSRSURWHLQVLQWKH &RQWH[WRI$WKHURJHQHVLV
Jianhong Lu1, Shuyuan Guo1, and Huiyong Yin1 1 Shanghai Institute for Biological Sciences (SIBS), CAS, People's Republic of China Cardiovascular diseases caused by atherosclerosis are the leading causes for mortality and morbidity in developed countries. Chronic inflammation and oxidative stress play an important role in atherosclerosis progression. Recent studies have demonstrated that oxidized phospholipids (oxPLs) on oxidized low density lipoprotein (oxLDL) play a critical role in atherosclerosis. However, the exact components that may be responsible for these effects have not been clearly elucidated. We identified a novel class of oxPLs in vitro and in human atherosclerotic plaques termed deoxy-A2/J2-IsoP-PC which contains a cyclopentenone moiety. We chemically synthesized a representative compound 15d-PGJ2-PC for this novel class of oxPLs and our preliminary experiments showed that 15d-PGJ2-PC can active Nrf2 and 33$5V LQFOXGLQJ 33$5Į 33$5į DQG 33$5Ȗ ZKLOH it inhibites 1)ț% activation, suggesting that 15d-PGJ2-PC may regulate inflammatory response and oxidative stress. Interestingly, this compound can induce a new phenotype of polarized macrophage, Mox (induced by oxPAPC). Our lipidomic and metabolomic studies demonstrated that Mox phenotype had distinct metabolic features from M1 and M2 phenotypes including arachidonic acid pathways and glycolysis. In summary, our studies demonstrate that these novel phospholipid oxidation products modulate macrophage functions in response to inflammation and oxidative stress in the context of atherosclerosis.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.142
SFRBM 2015
Yong Li1, Xiao-Li Wang1, Qiang Chai1, Tong Lu1, Jingchao Li1, and Hon-Chi Lee1 1 Mayo Clinic, USA Background and Objective: The nuclear factor E2-related factor-2 (Nrf2) signaling pathway has emerged as a master regulator of cellular redox status. The large conductance calcium-activated potassium (BK) channels, which are major determinants of vasodilation, are impaired in diabetes mellitus (DM) due to the down-regulation of BK-ȕE\UHDFWLYHR[\JHQVSHFLHV-dependent mechanisms. The goal of this study is to test the hypothesis that Nrf2 signaling plays a central role in the regulation of BK channel function in DM. Methods and Results: Studies were performed combining cellular, molecular, vascular, and electrophysiological techniques. In type 2 diabetic db/db mouse aorta, Nrf2 protein expression was significantly reduced, associated with significant down-regulation of BK-ȕ DQG Keme oxygenase 1 (HO-1), a known Nrf2 downstream target. Also, the muscle ring finger protein 1 (MuRF1), a known E-3 ligase targeting BK-ȕ ZDV VLJQLILFDQWO\ upregulated. These findings were reproduced by knockdown of Nrf2 by siRNA in cultured human coronary smooth muscle cells (HCSMC), whereas adenoviral transfer of Nrf2 gene in these cells was associated with down-regulation of MuRF1 and upregulation of BK-ȕ DQG +2-1 expression. Activation of Nrf2 by dimethyl fumarate in high glucose-cultured HCSMCs or in diabetic db/db mouse coronary arteries preserved BK-ȕ H[SUHVVLRQ VKRZQ E\ Western blot, BK channel activities using patch clamp recordings, and vascular function assessed by shear stress-mediated vasodilation in isolated mouse coronary arteries. Conclusions: Expression of BK-ȕ LV FORVHO\ UHJXODWHG E\ 1UI through its effect on MuRF1, which regulates BK-E1 degradation, and vascular BK channel function can be restored by activation of Nrf2. Nrf2 should be considered a novel therapeutic target in the treatment of diabetic vasculopathy.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.140 101 5HGR['HSHQGHQW,QWHUDFWLRQ%HWZHHQ3URWHLQ 'LVXOILGH,VRPHUDVHDQG3SKR[DFWLYDWHV1R[ 1$'3+2[LGDVHLQ9DVFXODU'LVHDVH
Marcela Gimenez1,2, Brandon Schickling2, Ilka Wittig3, Francis Miller2, and Lucia Rossetti Lopes1 1 University of São Paulo, Brazil, 2University of Iowa, USA, 3 University of Frankfurt, Germany We previously showed that Protein disulfide isomerase (PDI) expression and NADPH oxidase-dependent ROS generation are increased in resistance arteries in hypertension. Here, we sought to investigate the molecular mechanisms through which PDI regulates Nox1 activation. Recombinant proteins were expressed in Escherichia coli transformed with plasmids encoding p47phox, PDI wild type (PDIwt) and PDI mutated (PDImut) to serine in all four cysteines of the catalytic motifs (C36, 39, 383, 386S). Incubation of PDIwt with p47phox demonstrated intermolecular disulfide bonding of the two proteins, as detected by non-reducing western blot, only after addition of arachidonic acid, confirming this interaction is dependent on p47phox activation and unfolding. This bonding was not detected when p47phox was incubated with
S54
PDImut. The overexpression of PDTwt, but not PDImut, increased superoxide generation in vascular smooth muscle cells and ERK phosphorylation in thrombin and TNF-Į-stimulated HEK293 cells. Next, Cos phox cells constitutively expressing p22phox were transfected with Nox1, p47phox, p67phox, and PDIwt or PDImut. PDIwt, but not PDImut, increased superoxide generation and recruitment of p47phox to the membrane in resting conditions and after thrombin stimulation. Furthermore, recruitment of PDI to the oxidase complex was impaired with the PDImut. These data implicate a role for the redox cysteines of PDI in Nox1 complex assembly and activation. Mass spectrometry analysis identified PDI/p47phox homo and hetero-dipeptides and revealed several disulfide bonds between PDI and p47phox. Mutation of p47phox cysteine 196 to alanine decreased superoxide generation and Nox1 complex assembly by PDI in resting and stimulated cells. These findings suggest that PDI facilitates redox-mediated recruitment of Nox1 regulatory subunits to the plasma membrane and contributes to Nox1 signaling in vascular disease.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.141 102 0RGXODWLRQRI0DFURSKDJH)XQFWLRQE\2[LGL]HG 3KRVSKROLSLGVLQ/RZ'HQVLW\/LSRSURWHLQVLQWKH &RQWH[WRI$WKHURJHQHVLV
Jianhong Lu1, Shuyuan Guo1, and Huiyong Yin1 1 Shanghai Institute for Biological Sciences (SIBS), CAS, People's Republic of China Cardiovascular diseases caused by atherosclerosis are the leading causes for mortality and morbidity in developed countries. Chronic inflammation and oxidative stress play an important role in atherosclerosis progression. Recent studies have demonstrated that oxidized phospholipids (oxPLs) on oxidized low density lipoprotein (oxLDL) play a critical role in atherosclerosis. However, the exact components that may be responsible for these effects have not been clearly elucidated. We identified a novel class of oxPLs in vitro and in human atherosclerotic plaques termed deoxy-A2/J2-IsoP-PC which contains a cyclopentenone moiety. We chemically synthesized a representative compound 15d-PGJ2-PC for this novel class of oxPLs and our preliminary experiments showed that 15d-PGJ2-PC can active Nrf2 and 33$5V LQFOXGLQJ 33$5Į 33$5į DQG 33$5Ȗ ZKLOH it inhibites 1)ț% activation, suggesting that 15d-PGJ2-PC may regulate inflammatory response and oxidative stress. Interestingly, this compound can induce a new phenotype of polarized macrophage, Mox (induced by oxPAPC). Our lipidomic and metabolomic studies demonstrated that Mox phenotype had distinct metabolic features from M1 and M2 phenotypes including arachidonic acid pathways and glycolysis. In summary, our studies demonstrate that these novel phospholipid oxidation products modulate macrophage functions in response to inflammation and oxidative stress in the context of atherosclerosis.
doi: xxxxx doi: 10.1016/j.freeradbiomed.2015.10.142
SFRBM 2015