J Physiol Biochem (2014) 70:769–779 DOI 10.1007/s13105-014-0345-0
ORIGINAL PAPER
Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase Kyaw Thu Moe & Katwadi Khairunnisa & Nwe Oo Yin & Jaye Chin-Dusting & Philip Wong & Meng Cheong Wong
Received: 15 December 2013 / Accepted: 1 July 2014 / Published online: 25 July 2014 # University of Navarra 2014
Abstract An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2’, 7’dichlorodihydrofluorescein diacetate assay. TNF-αK. T. Moe (*) : K. Khairunnisa : N. O. Yin : P. Wong Research and Development Unit, National Heart Centre Singapore, 7 Hospital Drive SingHealth Research Facilities Block A, #03-05, Singapore 169611, Singapore e-mail: [email protected] J. Chin-Dusting Baker IDI Heart and Diabetes Institute, Melbourne, Australia M. C. Wong Department of Pharmacology, National University of Singapore, Singapore, Singapore
induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-αinduced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NFkappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes. Keywords TNF-α . NADPH oxidase . NF-kappa B . Cardiomyocytes . Signaling mechanism
Introduction Tumor necrosis factor (TNF)-α plays an important role in many cardiovascular diseases. TNF-α induces a wide range of biological effects including cell differentiation, proliferation, apoptosis, and inflammation in cardiac cells [3]. It interacts with one of two TNF-α receptors, TNFR1 and TNFR2, and regulates the expression of a
770
variety of proteins, including interleukin (IL)-1β, IL-6, vascular cell adhesion molecule (VCAM)-1, plateletderived growth factor, and transforming growth factor-β [17]. Binding of TNF-α to its receptors leads to recruitment of adaptor proteins and downstream signaling events that result in the activation of nuclear factor (NF)-κB family of transcription factors [29]. NF-κB proteins are a family of transcriptional factors that are crucially important in mediation of inflammation and immunity. The mammalian NF-κB proteins consist of three Rel family members (RelA or p65, RelB, and cRel), p50, and p52. In resting cells, the activity of NF-κB is controlled through its cytoplasmic sequestration by a family of proteins, inhibitors of NF-κB (IκB). In response to stimuli, IκB kinase (IKK) is activated and phosphorylates IκB that is then targeted for ubiquitination and proteasome-dependent degradation [23]. The released NF-κB localizes to the nucleus, and the activation of NF-κB induces the transcription of a subset of genes [21]. Although IκBα phosphorylation is a critical step in the activation of NF-κB, regulatory mechanisms exist at different levels in the NF-κB pathway including phosphorylation of MEK kinase (MEKK), IKK, and RelA or p65 and direct oxidation of p50 [11, 40]. In the regulation of NF-κB, IKKs are the primary target of reactive oxygen species (ROS) [40]. Sources of ROS include enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport chain. Among them, NADPH oxidase plays a major role in several cardiovascular diseases. NADPH oxidases are a family of enzyme complexes that catalyze the transfer of electrons from NADPH to molecular oxygen, thereby generating superoxide and hydrogen peroxide. It is present in both phagocytic and non-phagocytic cells, including cardiomyocytes [8]. NADPH oxidase-mediated ROS generation in the heart increases in response to various stimuli, e.g., angiotensin II and TNF-α [30, 41, 44]. Pharmacological inhibitors of NF-κB are identified in suppressing TNF-α-induced upregulation of NADPH subunits p47phox, p67phox, and Nox2 in human monocytic cells, suggesting that NF-κB partly mediates TNF-α-induced upregulation of NADPH oxidase subunits [18]. However, little is known whether NADPH oxidase mediates TNF-α-induced NF-κB activation in human cardiomyocytes. Recently, we demonstrated that TNF-α administration in mice induced ventricular remodeling, and
K.T. Moe et al.
treatment of cardiomyocytes with Nox2 and Nox4 siRNA resulted in attenuation of TNF-α-induced upregulation of IL-1β and IL-6 [37]. In the present study, we demonstrated that NADPH oxidase-mediated TNF-αinduced phosphorylation of IKK and p65, degradation of IκBα, and binding of activated NF-κB to its binding motif; blockade of NADPH oxidase attenuated TNF-αinduced NF-κB activation and upregulation of IL-1β and VCAM-1.
Materials and methods Materials Human recombinant TNF-α was purchased from Miltenyi Biotec (Auburn, CA, USA). 5 (and 6)Chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate-acetyl ester (CM-H2DCFDA) was purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). Diphenyleneiodonium (DPI), 4-hydroxy-3methoxyacetophenone (apocynin), superoxide dismutase (SOD), nitro-L-arginine methyl ester (L-NAME), allopurinol, and rotenone were purchased from SigmaAldrich (St. Louis, MO, USA). Anti-human IL-1β, VCAM-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antihuman IκB kinase-alpha (IKK-α), phospho-IKK-α/β (Ser176/180) which detects IKK-α when phosphorylated at Ser176/180, and IKK-β when phosphorylated at Ser177/181, IκBα, NF-κB p65, and phospho-NF-κB p65 (Ser276) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell culture Adult human cardiomyocytes were obtained from PromoCell GmbH (Heidelberg, Germany) and maintained in myocyte growth medium according to the manufacturer’s instructions. Cells were used between the third and sixth passage and at 85–90 % confluence. NADPH oxidase activity assay by measuring intracellular ROS production ROS production was measured in cardiomyocytes using CM-H2DCFDA (5 μM), as previously described [7, 19, 38]. Cardiomyocytes were treated with human
TNF-α induces NF-kappaB activation
recombinant TNF-α (20 ng/mL) at 37 °C with or without NADPH (100 μM) [32]. Probe-free cells were used as a blank control. Fluorescence was measured at 15min intervals, up to 1 h, in a 1420 Wallac Victor3 multilabel counter (Perkin-Elmer Life Sciences, Cambridge, UK) using excitation and emission wavelengths of 490 and 605 nm, respectively. All measurements were performed in triplicate. In some experiments, ROS measurement was performed in the presence of the NADPH oxidase inhibitors (DPI, 10−5 M, and apocynin, 1 mM), xanthine oxidase inhibitor allopurinol (1 mM), nitric oxide synthase inhibitor, LNAME (100 μM), mitochondrial respiration inhibitor rotenone (1 μM), or SOD (60 units/mL). Real-time PCR Total RNA was extracted from cardiomyocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) using previously described methods [18, 39] and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA). First-strand cDNA synthesis was performed using 700-ng total RNA and the SuperScriptII first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA). For realtime PCR, 3 μL first-strand reaction was used for each 25-μL PCR reaction using TaqMan® Universal PCR Master Mix (Applied Biosystems International, Foster City, CA, USA), together with 2.5 μL Taqman® Gene Expression Assays primers. Real-time quantitative PCR analysis was conducted to examine the expression of human IL-1β, VCAM-1, and GAPDH genes using an Applied Biosystems 7300 RealTime PCR System (Applied Biosystems International, Foster City, CA, USA). After an initial incubation step for 2 min at 50 °C and denaturation for 10 min at 95 °C, PCR was performed using 40 cycles (95 °C for 15 s and 60 °C for 60 s). Equal amounts of input RNA were used for all RT-PCR reactions; reactions were performed in duplicate, and GAPDH was used as an internal control. Total RNA with no reverse transcription (−RT) was used as a negative control. Differential gene expression analysis was calculated using the comparative (ΔΔCt) method, as described previously [35, 39]. Luciferase assay NF-κB reporter activity was measured using a Cignal™ reporter assay kit (SABiosciences, Frederick, MD,
771
USA) using dual-luciferase plasmids following previously described methods [24, 31]. Briefly, the NF-κB transcription response element (TRE) containing construct was diluted in unsupplemented cardiomyocyte growth medium, and cells were transfected using SureFact reagent (SABiosciences, Frederick, MD) for 24 h. Cells were then treated with TNF-α (20 ng/mL) at 37 °C for 1 h. The constitutively expressed Renilla luciferase acted as an internal control. Luciferase activities were measured using the 1420 Wallac Victor3 multilabel counter and the Dual-Luciferase Assay System (Promega, Madison, USA). Three independent transfections were carried out in triplicate for each reporter assay. In a separate experiment, cells were pretreated with apocynin (1 mM) for 30 min and treated with TNF-α (20 ng/mL) for 1 h. EMSA Cardiomyocytes were treated with human recombinant TNF-α (20 ng/mL) for 1 h at 37 °C with and without pretreatment with apocynin (1 mM) for 30 min following previously described methods [38]. Nuclear protein was extracted from whole cells using NE-PER nuclear extraction reagents (Pierce, Rockford, IL, USA). Electrophoretic mobility shift assay (EMSA) was performed using a LightShift chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) following the methods previously described [12, 42] with slight modification. Briefly, the same amount of protein from each nuclear extract was incubated with a double-stranded oligonucleotide containing a consensus κB binding site (5′AGTTGAGGGGACTTTCCCAGGC-3′; the underlined sequence represents the consensus κB region) [46] that had been labeled with biotin using biotin the 3’-end DNA labeling kit (Pierce, Rockford, IL, USA). Protein–DNA binding was evaluated using 2.5 ng biotin probe, 3 μg nuclear extract, 1 μg poly(dI-dC), 2.5 % glycerol, 5 mM MgCl 2 , and 0.05 % NP-40 in a 20-μL reaction for 20 min at room temperature followed by 1 h on ice. A competitor assay was performed by adding 100-fold excess of unlabeled NF-κB probe. The reaction mixture was electrophoresed using a 6 % native polyacrylamide gel transferred onto an immobilon Ny+ (Millipore) membrane and cross-linked immediately at 120 mJ/cm 2 using a UV cross-linker (UV Stratalinker 1800, Stratagene, La Jolla, CA, USA). Biotin–streptavidin–HRP immunodetection was performed and images developed
772
using AlphaEase® FC software and FluorChem HD2 (Alpha Innotech Corp., San Leandro, CA, USA). Western blot analysis Whole cell extracts were prepared using the M-PER® mammalian protein extraction reagent and mixed with the Halt™ protease inhibitor cocktail kit (Pierce, Rockford, IL, USA) per manufacturer’s instruction. Protein concentration was determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific Inc. Wilmington, DE, USA). Equal amounts (25 μg) of protein were subjected to 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Proteins were detected using anti-human IL-1β, VCAM-1, IKK-α, phospho-IKK-α/β, IκBα, NF-κB p65, and phospho-NF-κB p65 antibodies following the procedures previously described [38]. After incubation with HRP-conjugated secondary antibody, proteins were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). For cytoplasmic proteins, each membrane was re-probed with anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) after clearing the membrane with Restore™ Western blot stripping buffer (Pierce, Rockford, IL, USA). Signals were detected using AlphaEase® FC software and FluorChem HD2 (Alpha Innotech Corp., San Leandro, CA, USA). In a separate experiment, cells were pretreated with DPI (10−5 M) and apocynin (1 mM) for 15 and 30 min and treated with TNF-α.
Statistical analysis Data are expressed as mean±SD. Statistical analysis was performed using ANOVA and Student’s t test with Bonferroni correction for multiple comparisons.
Results TNF-α-induced ROS production is mediated by NADPH oxidase
K.T. Moe et al.
cells were treated with human recombinant TNF-α (20 ng/mL) for 1 h with or without NADPH. ROS production was measured using the CM-H2DCFDA fluorescence probe at 15-min intervals. A significant increase in TNF-α-induced ROS production was detected starting at 15 min (Fig. 1a, b). A time-dependent increase in ROS was observed up to 60 min after treatment. TNF-α-induced ROS production was observed only with the addition of the NADPH substrate at all time points. Moreover, inhibitors of NADPH oxidase, apocynin (Fig. 1a), and DPI (Fig. 1b) significantly attenuated TNF-α-induced ROS production (P
ORIGINAL PAPER
Tumor necrosis factor-α-induced nuclear factor-kappaB activation in human cardiomyocytes is mediated by NADPH oxidase Kyaw Thu Moe & Katwadi Khairunnisa & Nwe Oo Yin & Jaye Chin-Dusting & Philip Wong & Meng Cheong Wong
Received: 15 December 2013 / Accepted: 1 July 2014 / Published online: 25 July 2014 # University of Navarra 2014
Abstract An elevated level of tumor necrosis factor (TNF)-α is implicated in several cardiovascular diseases including heart failure. Numerous reports have demonstrated that TNF-α activates nuclear factor (NF)kappaB, resulting in the upregulation of several genes that regulate inflammation, proliferation, and apoptosis of cardiomyocytes. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a major source of reactive oxygen species (ROS), is also activated by TNF-α and plays a crucial role in redox-sensitive signaling pathways. The present study investigated whether NADPH oxidase mediates TNF-α-induced NF-kappaB activation and NF-kappaB-mediated gene expression. Human cardiomyocytes were treated with recombinant TNF-α with or without pretreatment with diphenyleneiodonium (DPI) and apocynin, inhibitors of NADPH oxidase. TNF-α-induced ROS production was measured using 5-(and-6)-chloromethyl-2’, 7’dichlorodihydrofluorescein diacetate assay. TNF-αK. T. Moe (*) : K. Khairunnisa : N. O. Yin : P. Wong Research and Development Unit, National Heart Centre Singapore, 7 Hospital Drive SingHealth Research Facilities Block A, #03-05, Singapore 169611, Singapore e-mail: [email protected] J. Chin-Dusting Baker IDI Heart and Diabetes Institute, Melbourne, Australia M. C. Wong Department of Pharmacology, National University of Singapore, Singapore, Singapore
induced NF-kappaB activation was also examined using immunoblot; NF-kappaB binding to its binding motif was determined using a Cignal reporter luciferase assay and an electrophoretic mobility shift assay. TNF-αinduced upregulation of interleukin (IL)-1β and vascular cell adhesion molecule (VCAM)-1 was investigated using real-time PCR and immunoblot. TNF-α-induced ROS production in cardiomyocytes was mediated by NADPH oxidase. Phosphorylation of IKK-α/β and p65, degradation of IkappaBα, binding of NFkappaB to its binding motif, and upregulation of IL-1β and VCAM-1 induced by TNF-α were significantly attenuated by treatment with DPI and apocynin. Collectively, these findings demonstrate that NADPH oxidase plays a role in regulation of TNF-α-induced NF-kappaB activation and upregulation of proinflammatory cytokines, IL-1β and VCAM-1, in human cardiomyocytes. Keywords TNF-α . NADPH oxidase . NF-kappa B . Cardiomyocytes . Signaling mechanism
Introduction Tumor necrosis factor (TNF)-α plays an important role in many cardiovascular diseases. TNF-α induces a wide range of biological effects including cell differentiation, proliferation, apoptosis, and inflammation in cardiac cells [3]. It interacts with one of two TNF-α receptors, TNFR1 and TNFR2, and regulates the expression of a
770
variety of proteins, including interleukin (IL)-1β, IL-6, vascular cell adhesion molecule (VCAM)-1, plateletderived growth factor, and transforming growth factor-β [17]. Binding of TNF-α to its receptors leads to recruitment of adaptor proteins and downstream signaling events that result in the activation of nuclear factor (NF)-κB family of transcription factors [29]. NF-κB proteins are a family of transcriptional factors that are crucially important in mediation of inflammation and immunity. The mammalian NF-κB proteins consist of three Rel family members (RelA or p65, RelB, and cRel), p50, and p52. In resting cells, the activity of NF-κB is controlled through its cytoplasmic sequestration by a family of proteins, inhibitors of NF-κB (IκB). In response to stimuli, IκB kinase (IKK) is activated and phosphorylates IκB that is then targeted for ubiquitination and proteasome-dependent degradation [23]. The released NF-κB localizes to the nucleus, and the activation of NF-κB induces the transcription of a subset of genes [21]. Although IκBα phosphorylation is a critical step in the activation of NF-κB, regulatory mechanisms exist at different levels in the NF-κB pathway including phosphorylation of MEK kinase (MEKK), IKK, and RelA or p65 and direct oxidation of p50 [11, 40]. In the regulation of NF-κB, IKKs are the primary target of reactive oxygen species (ROS) [40]. Sources of ROS include enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, nitric oxide synthase, and mitochondrial electron transport chain. Among them, NADPH oxidase plays a major role in several cardiovascular diseases. NADPH oxidases are a family of enzyme complexes that catalyze the transfer of electrons from NADPH to molecular oxygen, thereby generating superoxide and hydrogen peroxide. It is present in both phagocytic and non-phagocytic cells, including cardiomyocytes [8]. NADPH oxidase-mediated ROS generation in the heart increases in response to various stimuli, e.g., angiotensin II and TNF-α [30, 41, 44]. Pharmacological inhibitors of NF-κB are identified in suppressing TNF-α-induced upregulation of NADPH subunits p47phox, p67phox, and Nox2 in human monocytic cells, suggesting that NF-κB partly mediates TNF-α-induced upregulation of NADPH oxidase subunits [18]. However, little is known whether NADPH oxidase mediates TNF-α-induced NF-κB activation in human cardiomyocytes. Recently, we demonstrated that TNF-α administration in mice induced ventricular remodeling, and
K.T. Moe et al.
treatment of cardiomyocytes with Nox2 and Nox4 siRNA resulted in attenuation of TNF-α-induced upregulation of IL-1β and IL-6 [37]. In the present study, we demonstrated that NADPH oxidase-mediated TNF-αinduced phosphorylation of IKK and p65, degradation of IκBα, and binding of activated NF-κB to its binding motif; blockade of NADPH oxidase attenuated TNF-αinduced NF-κB activation and upregulation of IL-1β and VCAM-1.
Materials and methods Materials Human recombinant TNF-α was purchased from Miltenyi Biotec (Auburn, CA, USA). 5 (and 6)Chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate-acetyl ester (CM-H2DCFDA) was purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). Diphenyleneiodonium (DPI), 4-hydroxy-3methoxyacetophenone (apocynin), superoxide dismutase (SOD), nitro-L-arginine methyl ester (L-NAME), allopurinol, and rotenone were purchased from SigmaAldrich (St. Louis, MO, USA). Anti-human IL-1β, VCAM-1, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antihuman IκB kinase-alpha (IKK-α), phospho-IKK-α/β (Ser176/180) which detects IKK-α when phosphorylated at Ser176/180, and IKK-β when phosphorylated at Ser177/181, IκBα, NF-κB p65, and phospho-NF-κB p65 (Ser276) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Cell culture Adult human cardiomyocytes were obtained from PromoCell GmbH (Heidelberg, Germany) and maintained in myocyte growth medium according to the manufacturer’s instructions. Cells were used between the third and sixth passage and at 85–90 % confluence. NADPH oxidase activity assay by measuring intracellular ROS production ROS production was measured in cardiomyocytes using CM-H2DCFDA (5 μM), as previously described [7, 19, 38]. Cardiomyocytes were treated with human
TNF-α induces NF-kappaB activation
recombinant TNF-α (20 ng/mL) at 37 °C with or without NADPH (100 μM) [32]. Probe-free cells were used as a blank control. Fluorescence was measured at 15min intervals, up to 1 h, in a 1420 Wallac Victor3 multilabel counter (Perkin-Elmer Life Sciences, Cambridge, UK) using excitation and emission wavelengths of 490 and 605 nm, respectively. All measurements were performed in triplicate. In some experiments, ROS measurement was performed in the presence of the NADPH oxidase inhibitors (DPI, 10−5 M, and apocynin, 1 mM), xanthine oxidase inhibitor allopurinol (1 mM), nitric oxide synthase inhibitor, LNAME (100 μM), mitochondrial respiration inhibitor rotenone (1 μM), or SOD (60 units/mL). Real-time PCR Total RNA was extracted from cardiomyocytes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) using previously described methods [18, 39] and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific Inc., Wilmington, DE, USA). First-strand cDNA synthesis was performed using 700-ng total RNA and the SuperScriptII first-strand synthesis system for RT-PCR (Invitrogen, Carlsbad, CA, USA). For realtime PCR, 3 μL first-strand reaction was used for each 25-μL PCR reaction using TaqMan® Universal PCR Master Mix (Applied Biosystems International, Foster City, CA, USA), together with 2.5 μL Taqman® Gene Expression Assays primers. Real-time quantitative PCR analysis was conducted to examine the expression of human IL-1β, VCAM-1, and GAPDH genes using an Applied Biosystems 7300 RealTime PCR System (Applied Biosystems International, Foster City, CA, USA). After an initial incubation step for 2 min at 50 °C and denaturation for 10 min at 95 °C, PCR was performed using 40 cycles (95 °C for 15 s and 60 °C for 60 s). Equal amounts of input RNA were used for all RT-PCR reactions; reactions were performed in duplicate, and GAPDH was used as an internal control. Total RNA with no reverse transcription (−RT) was used as a negative control. Differential gene expression analysis was calculated using the comparative (ΔΔCt) method, as described previously [35, 39]. Luciferase assay NF-κB reporter activity was measured using a Cignal™ reporter assay kit (SABiosciences, Frederick, MD,
771
USA) using dual-luciferase plasmids following previously described methods [24, 31]. Briefly, the NF-κB transcription response element (TRE) containing construct was diluted in unsupplemented cardiomyocyte growth medium, and cells were transfected using SureFact reagent (SABiosciences, Frederick, MD) for 24 h. Cells were then treated with TNF-α (20 ng/mL) at 37 °C for 1 h. The constitutively expressed Renilla luciferase acted as an internal control. Luciferase activities were measured using the 1420 Wallac Victor3 multilabel counter and the Dual-Luciferase Assay System (Promega, Madison, USA). Three independent transfections were carried out in triplicate for each reporter assay. In a separate experiment, cells were pretreated with apocynin (1 mM) for 30 min and treated with TNF-α (20 ng/mL) for 1 h. EMSA Cardiomyocytes were treated with human recombinant TNF-α (20 ng/mL) for 1 h at 37 °C with and without pretreatment with apocynin (1 mM) for 30 min following previously described methods [38]. Nuclear protein was extracted from whole cells using NE-PER nuclear extraction reagents (Pierce, Rockford, IL, USA). Electrophoretic mobility shift assay (EMSA) was performed using a LightShift chemiluminescent EMSA kit (Pierce, Rockford, IL, USA) following the methods previously described [12, 42] with slight modification. Briefly, the same amount of protein from each nuclear extract was incubated with a double-stranded oligonucleotide containing a consensus κB binding site (5′AGTTGAGGGGACTTTCCCAGGC-3′; the underlined sequence represents the consensus κB region) [46] that had been labeled with biotin using biotin the 3’-end DNA labeling kit (Pierce, Rockford, IL, USA). Protein–DNA binding was evaluated using 2.5 ng biotin probe, 3 μg nuclear extract, 1 μg poly(dI-dC), 2.5 % glycerol, 5 mM MgCl 2 , and 0.05 % NP-40 in a 20-μL reaction for 20 min at room temperature followed by 1 h on ice. A competitor assay was performed by adding 100-fold excess of unlabeled NF-κB probe. The reaction mixture was electrophoresed using a 6 % native polyacrylamide gel transferred onto an immobilon Ny+ (Millipore) membrane and cross-linked immediately at 120 mJ/cm 2 using a UV cross-linker (UV Stratalinker 1800, Stratagene, La Jolla, CA, USA). Biotin–streptavidin–HRP immunodetection was performed and images developed
772
using AlphaEase® FC software and FluorChem HD2 (Alpha Innotech Corp., San Leandro, CA, USA). Western blot analysis Whole cell extracts were prepared using the M-PER® mammalian protein extraction reagent and mixed with the Halt™ protease inhibitor cocktail kit (Pierce, Rockford, IL, USA) per manufacturer’s instruction. Protein concentration was determined using a NanoDrop 1000 Spectrophotometer (Thermo Scientific Inc. Wilmington, DE, USA). Equal amounts (25 μg) of protein were subjected to 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Proteins were detected using anti-human IL-1β, VCAM-1, IKK-α, phospho-IKK-α/β, IκBα, NF-κB p65, and phospho-NF-κB p65 antibodies following the procedures previously described [38]. After incubation with HRP-conjugated secondary antibody, proteins were detected using SuperSignal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA). For cytoplasmic proteins, each membrane was re-probed with anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) after clearing the membrane with Restore™ Western blot stripping buffer (Pierce, Rockford, IL, USA). Signals were detected using AlphaEase® FC software and FluorChem HD2 (Alpha Innotech Corp., San Leandro, CA, USA). In a separate experiment, cells were pretreated with DPI (10−5 M) and apocynin (1 mM) for 15 and 30 min and treated with TNF-α.
Statistical analysis Data are expressed as mean±SD. Statistical analysis was performed using ANOVA and Student’s t test with Bonferroni correction for multiple comparisons.
Results TNF-α-induced ROS production is mediated by NADPH oxidase
K.T. Moe et al.
cells were treated with human recombinant TNF-α (20 ng/mL) for 1 h with or without NADPH. ROS production was measured using the CM-H2DCFDA fluorescence probe at 15-min intervals. A significant increase in TNF-α-induced ROS production was detected starting at 15 min (Fig. 1a, b). A time-dependent increase in ROS was observed up to 60 min after treatment. TNF-α-induced ROS production was observed only with the addition of the NADPH substrate at all time points. Moreover, inhibitors of NADPH oxidase, apocynin (Fig. 1a), and DPI (Fig. 1b) significantly attenuated TNF-α-induced ROS production (P
