Nam Jeong Han

High glucose-induced NF-κB activation occurs via tyrosine phosphorylation of IκBα in human glomerular endothelial cells : involvement of Syk tyrosine kinase

Activation of nuclear factor-kappaB (NF-kappaB) occurs by dissociation from IkappaB after serine or tyrosine phosphorylation of IkappaBalpha, but the way of NF-kappaB activation by high glucose has not been defined. High glucose is known to activate NF-kappaB via protein kinase C and reactive oxygen species (ROS). In this study, we investigated how high glucose activates NF-kappaB for CC chemokine ligand 2 production in cultured human glomerular endothelial cells. High glucose increased nuclear translocation of p65 and also increased NF-kappaB DNA binding activity. High glucose-induced NF-kappaB activation occurred without degradation of IkappaBalpha. In agreement with this, there was no increase in serine phosphorylation of IkappaBalpha, while tyrosine phosphorylation of IkappaBalpha was increased by high glucose. High glucose increased the generation of ROS, whereas both alpha-lipoic acid and N-acetylcysteine scavenged the ROS and decreased high glucose-induced tyrosine phosphorylation of IkappaBalpha, nuclear translocation of p65, and NF-kappaB DNA binding activity. Protein kinase C pseudosubstrate inhibited high glucose-induced ROS production, tyrosine phosphorylation of IkappaBalpha, and nuclear translocation of p65. Both BAY 61-3606, a specific inhibitor of Syk protein-tyrosine kinase, and small interfering RNA directed against Syk inhibited high glucose-induced tyrosine phosphorylation of IkappaBalpha as well as p65 nuclear translocation. High glucose increased tyrosine phosphorylation of Syk, while it was inhibited by alpha-lipoic acid and protein kinase C pseudosubstrate. In summary, high glucose-induced NF-kappaB activation occurred not by serine phosphorylation of IkappaBalpha. Our data suggest that ROS-mediated tyrosine phosphorylation of IkappaBalpha is the mechanism for high glucose-induced NF-kappaB activation, and Syk may play a role in tyrosine phosphorylation of IkappaBalpha.

Mycophenolic acid attenuates tumor necrosis factor-α-induced endothelin-1 production in human aortic endothelial cells

AIMS Atherosclerotic cardiovascular disease is the major cause of morbidity and mortality in solid organ transplant recipients. Endothelin-1 (ET-1) is implicated in the pathogenesis of atherosclerosis and is one of the potential therapeutic targets. This study was conducted to evaluate the effect of mycophenolic acid (MPA), an immunosuppressant for the transplant recipients, on tumor necrosis factor-alpha (TNF-alpha)-induced ET-1 production in aortic endothelial cells. METHODS AND RESULTS In cultured human aortic endothelial cells, TNF-alpha increased ET-1 through AP-1 and NF-kappaB, whereas MPA attenuated it by reducing both AP-1 and NF-kappaB DNA-binding activities. TNF-alpha increased ET-1 via c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), but not extracellular signal-regulated kinase. N-acetylcysteine that downregulated TNF-alpha-induced reactive oxygen species (ROS) inhibited JNK activation, but not p38 MAPK. N-acetylcysteine, SP600125 (JNK inhibitor) and SB203580 (p38 MAPK inhibitor) attenuated TNF-alpha-induced DNA-binding activities of both AP-1 and NF-kappaB. MPA inhibited JNK and p38 MAPK activations as well as ROS generation. N-acetylcysteine, SP600125, SB203580 and MPA had no effect on either TNF-alpha-induced IkappaBalpha degradation or p65 nuclear translocation, but attenuated p65 Ser276 phosphorylation. CONCLUSION MPA attenuated TNF-alpha-induced ET-1 production through inhibitions of ROS-dependent JNK and ROS-independent p38 MAPK that regulated NF-kappaB as well as AP-1. These findings suggest that MPA could have an effect of amelioration of atherosclerosis.

Darbepoetin alfa suppresses tumor necrosis factor-α-induced endothelin-1 production through antioxidant action in human aortic endothelial cells: role of sialic acid residues.

Recombinant human erythropoietin (r-HuEPO) is widely used to correct anemia in end-stage renal disease patients, who commonly suffer from atherosclerosis. Endothelin-1 (ET-1) has been implicated in the pathogenesis of atherosclerosis. Here, we tested whether darbepoetin alfa, a hypersialylated analogue of r-HuEPO, regulates tumor necrosis factor-α (TNF-α)-induced ET-1 production in human aortic endothelial cells, and sought to identify the signal pathways involved. Darbepoetin alfa attenuated TNF-α-induced ET-1 production. It also diminished TNF-α-induced reactive oxygen species (ROS) accumulation and subsequent activation of c-Jun NH2-terminal kinase (JNK), which regulates the DNA-binding activities of both AP-1 and NF-κB required for ET-1 gene transcription. Like a JNK inhibitor, darbepoetin alfa did not affect IκBα degradation or p65 nuclear translocation, but did inhibit mitogen- and stress-activated protein kinase 1 (MSK1) activation and attenuated p65 phosphorylation (serine 276), effects that may account for the reduction in NF-κB DNA-binding activity. Desialylation completely abolished darbepoetin alfas inhibitory effects on TNF-α-induced ROS accumulation, MSK1 activation, and ET-1 gene expression, without affecting its stimulation of STAT5 activity. These data demonstrate that darbepoetin alfa suppresses TNF-α-induced ET-1 production through its antioxidant action and suggest that the sialic acid residues of darbepoetin alfa are essential for its antioxidant effect, possibly by scavenging ROS.

Activation of spleen tyrosine kinase is required for TNF-α-induced endothelin-1 upregulation in human aortic endothelial cells.

Endothelin‐1 (ET‐1) promotes atherosclerosis. We tested whether spleen tyrosine kinase (Syk) mediates tumor necrosis factor‐α (TNF‐α)‐induced ET‐1 upregulation in human aortic endothelial cells (HAECs) and sought to identify the signal pathways involved. TNF‐α‐induced reactive oxygen species (ROS) activated Syk and phosphatidylinositol 3‐kinase (PI3K), which was required for the activation of AP‐1 and subsequent ET‐1 gene transcription. ROS mediated c‐Jun NH2‐terminal kinase (JNK) is also required for AP‐1 activation, but Syk and PI3K regulated AP‐1 activation independently of JNK. Through regulation of ET‐1 production, Syk could be implicated in atherosclerosis.

TNF-α Activates High-Mobility Group Box 1 - Toll-Like Receptor 4 Signaling Pathway in Human Aortic Endothelial Cells

Background/Aims: Toll-like receptor 4 (TLR4) interacts with endogenous substances as well as lipopolysaccharide. We explored whether TLR4 is implicated in tumor necrosis factor-α (TNF-α) signal transduction in human aortic endothelial cells. Methods: The pathway was evaluated by transfection of siRNAs, immunoprecipitation and Western blot analysis. Results: TNF-α activated spleen tyrosine kinase (Syk) within 10 min, which led to endothelin-1 (ET-1) production. TLR4 was also rapidly activated by TNF-α stimulation, as shown by recruitment of interleukin-1 receptor-associated kinase 1 to TLR4 and its adaptor molecule, myeloid differentiation factor 88 (MyD88). siRNA depletion of TLR4 markedly attenuated TNF-α-induced Syk activation and ET-1 production. TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody and siRNA depletion of MyD88 also attenuated TNF-α-induced Syk activation. Syk was co-immunoprecipitated with TLR4, and TNF-α activated Syk bound to TLR4. High-mobility group box 1 (HMGB1) was rapidly released and associated with TLR4 after TNF-α stimulation with a peak at 5 min, which was prevented by N-acetylcysteine, an antioxidant. Glycyrrhizin (HMGB1 inhibitor), HMGB1-neutralizing antibody and siRNA depletion of HMGB1 all suppressed TNF-α-induced Syk activation and ET-1 production. Conclusion: Upon TNF-α stimulation, TLR4 is activated by HMGB1 that is immediately released after the generation of reactive oxygen species, and plays a crucial role in the signal transduction.

1,25-Dihydroxyvitamin D3 Causes ADAM10-Dependent Ectodomain Shedding of Tumor Necrosis Factor Receptor 1 in Vascular Smooth Muscle Cells

1,25-Dihydroxyvitamin D3 (1,25D3) has a potential antiatherosclerotic effect through anti-inflammatory actions. We investigated how 1,25D3 regulates tumor necrosis factor-α (TNF-α)–induced lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) expression in cultured human aortic smooth muscle cells. TNF-α activated Rac1/reactive oxygen species/spleen tyrosine kinase and transcriptional factors, activator protein-1, and nuclear factor κB, which led to LOX-1 expression. 1,25D3 inhibited TNF-α–induced LOX-1 expression by inhibiting Rac1 activation and thereby its downstream signals. 1,25D3 rapidly induced extracellular Ca2+ influx. Verapamil, an inhibitor of L-type calcium channels, inhibited 1,25D3-induced Ca2+ influx and counteracted the inhibitory effects of 1,25D3 on Rac1 activation, whereas Bay K8644 [1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid, methyl ester], an L-type calcium channel agonist, attenuated TNF-α–induced Rac1 activation, as 1,25D3 did. 1,25D3 induced the ectodomain shedding of TNF receptor 1 (TNFR1), which was abolished by verapamil and in Ca2+-free media. Like 1,25D3, Bay K8644 induced the ectodomain shedding of TNFR1. Both 1,25D3 and Bay K8644 caused the translocation of a disintegrin and metalloprotease (ADAM) 10 from the cytoplasm to the plasma membrane, which was dependent on extracellular Ca2+ influx. In contrast, depletion of ADAM10 by transfection of ADAM10–small interfering RNA prevented 1,25D3- or Bay K8644–induced ectodomain shedding of TNFR1 and abolished the suppressive effect of 1,25D3 on TNF-α–induced Rac1 activation. Taken together, these findings suggest that 1,25D3 induces extracellular Ca2+ influx via L-type calcium channel, triggering ADAM10-mediated ectodomain shedding of TNFR1, and it thereby decreases responsiveness to TNF-α. By shedding TNFR1 from the cell surface, 1,25D3 may regulate inflammation and atherogenesis, whereas this effect could be attenuated by calcium channel blockers.

Spleen tyrosine kinase mediates high glucose-induced transforming growth factor-β1 up-regulation in proximal tubular epithelial cells

The role of spleen tyrosine kinase (Syk) in high glucose-induced intracellular signal transduction has yet to be elucidated. We investigated whether Syk is implicated in high glucose-induced transforming growth factor-β1 (TGF-β1) up-regulation in cultured human proximal tubular epithelial cells (HK-2 cell). High glucose increased TGF-β1 gene expression through Syk, extracellular signal-regulated kinase (ERK), AP-1 and NF-κB. High glucose-induced AP-1 DNA binding activity was decreased by Syk inhibitors and U0126 (an ERK inhibitor). Syk inhibitors suppressed high glucose-induced ERK activation, whereas U0126 had no effect on Syk activation. High glucose-induced NF-κB DNA binding activity was also decreased by Syk inhibitors. High glucose increased nuclear translocation of p65 without serine phosphorylation of IκBα and without degradation of IκBα, but with an increase in tyrosine phosphorylation of IκBα that may account for the activation of NF-κB. Both Syk inhibitors and Syk-siRNA attenuated high glucose-induced IκBα tyrosine phosphorylation and p65 nuclear translocation. Depletion of p21-activated kinase 2 (Pak2) by transfection of Pak2-siRNA abolished high glucose-induced Syk activation. In summary, high glucose-induced TGF-β1 gene transcription occurred through Pak2, Syk and subsequent ERK/AP-1 and NF-κB pathways. This suggests that Syk might be implicated in the diabetic kidney disease.

Toll-Like Receptor 4/Spleen Tyrosine Kinase Complex in High Glucose Signal Transduction of Proximal Tubular Epithelial Cells

Background/Aims: High glucose activates spleen tyrosine kinase (Syk) in human proximal tubular epithelial cells (HK-2 cells), which leads to NF-κB activation and transforming growth factor-ß1 (TGF-ß1) production. We explored the signal transduction pathway from high glucose to Syk activation. Methods: The pathway was evaluated by siRNA transfection, immunoprecipitation and Western blot. Results: High glucose stimulated Syk activation within 10 min. Depletion of toll-like receptor 4 (TLR4) attenuated high glucose-induced Syk activation, NF-κB p65 nuclear translocation, and TGF-ß1 production. In addition, TLR4 inhibitor (CLI-095), TLR4-neutralizing antibody, and depletion of myeloid differentiation factor 88 (MyD88) all attenuated high glucose-induced Syk activation. As an evidence of TLR4 activation, interleukin-1 receptor-associated kinase 1 was recruited to MyD88 and TLR4 upon exposure to high glucose. Syk was co-immunoprecipitated with TLR4, and Syk bound to TLR4 was activated by high glucose. High-mobility group box-1 (HMGB-1), an endogenous activator of TLR4, rapidly increased in TLR4 immunoprecipitates upon high glucose stimulation, and this association was reduced by N-acetylcysteine, an antioxidant. An HMGB-1 inhibitor glycyrrhizin suppressed high glucose-induced Syk activation. Conclusion: Syk is constitutively associated with TLR4. High glucose induces an immediate, reactive oxygen species-dependent, extracellular release of HMGB-1 which binds to TLR4 and activates it, leading to Syk activation.

STAT1-Independent Down-Regulation of Interferon-Gamma-Induced Class II Transactivator and HLA-DR Expression by Transforming Growth Factor Beta-1 in Human Glomerular Endothelial Cells

Background: The competition between STAT1 and Smad3 for a limiting amount of the nuclear protein p300, a transcriptional coactivator, was suggested to be a mechanism for the antagonism between interferon-γ (IFN-γ) and transforming growth factor-β1 (TGF-β1). We investigated the effect of TGF-β1 on IFN-γ-induced HLA-DR production in cultured human glomerular endothelial cells (HGECs), and the involvement of p300 in this process. Methods: Cell surface expression of HLA-DR and mRNA levels of HLA-DR and class II transactivator (CIITA), the master regulator of HLA-DR gene transcription, were measured by cellular ELISA and Northern blot, respectively. The levels of STAT1 and Smad3 protein were analyzed by Western blot. Nuclear binding activity of STAT1 was assessed by electrophoretic mobility shift assay. Results: IFN-γ increased the cell surface expression of HLA-DR along with increases in the mRNA levels of CIITA and HLA-DR, while these stimulatory effects of IFN-γ were down-regulated by TGF-β1. IFN-γ increased phosphorylation of STAT1 and this activation was not inhibited by TGF-β1. IFN-γ increased binding of p-STAT1 to p300, while TGF-β1 increased binding of Smad3 to p300. TGF-β1-induced Smad3 binding to p300 was inhibited by IFN-γ, whereas IFN-γ-induced p-STAT1 binding to p300 was not inhibited by TGF-β1. IFN-γ increased DNA binding activity of STAT1. Inhibition of interaction between STAT1 and p300 by addition of anti-p300 antibody to nuclear extract down-regulated DNA binding activity of STAT1. In contrast, TGF-β1 did not inhibit IFN-γ-induced STAT1 binding to DNA. Conclusions: TGF-β1 down-regulated IFN-γ-induced CIITA and HLA-DR expression in HGECs. Though there was an antagonism between IFN-γ and TGF-β1, the competition for p300 between p-STAT1 and Smad3 was not the mechanism for it.

1,25-Dihydroxyvitamin D3 Attenuates the Effects of Lipopolysaccharide by Causing ADAM10-Dependent Ectodomain Shedding of Toll-Like Receptor 4

Background/Aims: We investigated how 1,25-dihydroxyvitamin D3 (1,25D3) inhibits the effects of lipopolysaccharide (LPS) in human aortic endothelial cells. Methods: Cellular signaling was explored by determination of protein abundance with Western blot, measurement of cytosolic Ca2+ concentration and immunofluorescence staining for a disintegrin and metalloprotease 10 (ADAM10). Results: LPS stimulated the expression of intercellular adhesion molecule 1 (ICAM-1) through toll-like receptor 4 (TLR4) and subsequent activation of p38 mitogen-activated protein kinase (p38 MAPK). Pretreatment with 1,25D3 attenuated LPS-induced p38 MAPK activation and ICAM-1 expression by causing ectodomain shedding of TLR4. This effect of 1,25D3 depended on its ability to induce a rapid extracellular Ca2+ influx through L-type calcium channels because the ectodomain shedding was prevented by the absence of extracellular Ca2+ or the presence of verapamil. TLR4 ectodomain shedding was also induced by Bay K8644 (L-type calcium channel agonist). Both 1,25D3 and Bay K8644 caused extracellular Ca2+ influx-dependent ADAM10 translocation to the cell surface. Depletion of ADAM10 by siRNA transfection prevented 1,25D3- and Bay K8644-induced ectodomain shedding of TLR4, and abolished the inhibitory effect of 1,25D3 on LPS-induced ICAM-1 expression. Conclusion: 1,25D3 causes ectodomain shedding of TLR4 and thereby decreases the responsiveness of cells to LPS. ADAM10, activated by extracellular Ca2+ influx, was implicated in the ectodomain cleavage of TLR4.