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 Ca(2+) influx. Verapamil, an inhibitor of L-type calcium channels, inhibited 1,25D3-induced Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) 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.

Immunostimulatory effect of fermented red ginseng in the mouse model.

In this study, Woongjin fermented red ginseng extract (WFRG) was evaluated for its potential ability to act as an adjuvant for the immune response of mice. For the in vitro study, macrophages were treated with serial concentrations (1 µg/mL, 10 µg/mL, and 100 µg/mL) of WFRG. For in vivo studies, mice were administered different concentrations (10 mg/kg/day, 100 mg/kg/day, and 200 mg/kg/day) of WFRG orally for 21 days. In vitro, the production of nitric oxide and TNF-α by RAW 264.7 cells increased in a dose-dependent manner. In vivo, WFRG enhanced the proliferation of splenocytes induced by two mitogens (i.e., concanavalin A and lipopolysaccharide [LPS]) and increased LPS-induced production of TNF-α and IL-6, but not IL-1ß. In conclusion, WFRG has the potential to modulate immune function and should be further investigated as an immunostimulatory agent.

Mycophenolic acid regulates spleen tyrosine kinase to repress tumour necrosis factor-alpha-induced monocyte chemotatic protein-1 production in cultured human aortic endothelial cells.

Atherosclerosis develops from cascades of inflammatory processes. Spleen tyrosine kinase (Syk) and monocyte chemotatic protein-1 (MCP-1) play important roles in the pathogenesis of atherosclerosis. Mycophenolic acid (MPA) has an anti-inflammatory effect. We have investigated whether MPA regulates Syk to repress tumour necrosis factor-α (TNF-α)-induced MCP-1 production in cultured human aortic endothelial cells. Expression of MCP-1 mRNA and its protein were measured by real time RT-PCR and ELISA, respectively. Reactive oxygen species (ROS) production were measured using 2'7'-dichlorofluorescein diacetate. Activation of AP-1 and NF-κB were assessed by electrophoretic mobility shift assay. Tyrosine phosphorylation of Syk was examined by Western blot analysis. TNF-α increased MCP-1 at both mRNA and protein levels. TNF-α-induced MCP-1 mRNA expression was inhibited by N-acetylcysteine (NAC), Syk inhibitor, Syk-siRNA and MPA. TNF-α-induced MCP-1 protein production was also inhibited by Syk inhibitor and MPA. TNF-α increased DNA binding activity of AP-1 and NF-κB, whereas both AP-1 and NF-κB decoy oligodeoxynucleotides downregulated TNF-α-induced MCP-1 mRNA expression. TNF-α increased ROS generation, which was inhibited by NAC and MPA, but not by Syk inhibitor. TNF-α increased tyrosine phosphorylation of Syk, which was attenuated by NAC and MPA. MPA and Syk inhibitor attenuated TNF-α-induced DNA binding activity of NF-κB and AP-1. TNF-α induced MCP-1 expression via activation of AP-1 and NF-κB. AP-1 and NF-κB were mediated through ROS, followed by Syk. MPA exerts anti-inflammatory effect by inhibiting MCP-1 expression via suppression of ROS and Syk.

Mycophenolic acid attenuates tumor necrosis factor-alpha-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.