NAD(P)H oxidase contributes to the pathogenesis of cancer and cardiovascular diseases such as hypertension, atherosclerosis, restenosis, cardiac hypertrophy and heart failure. Plumbagin, a plant-derived naphthoquinone, has been shown to exert anticarcinogenic and anti-atherosclerosis effects in animals. However, the molecular mechanisms underlying these effects remain unknown. It is possible that the beneficial effect of plumbagin is due to the inhibition of NAD(P)H oxidase. Human embryonic kidney 293 (HEK293) and brain tumour LN229 cells express mainly Nox-4, a renal NAD(P)H oxidase. We have examined the effect of plumbagin on Nox-4 activity in HEK293 and LN229 cells using lucigenin-dependent chemiluminescence assay. Plumbagin inhibited the activity of Nox-4 in a time- and dose-dependent manner in HEK293 and LN229 cells. Production of superoxide in HEK293 cells was inhibited by diphenyleneiodonium (DPI), a NAD(P)H oxidase inhibitor. The superoxide production in HEK293 cells was NADPH- and NADH-dependent indicating that the superoxide was generated by a NAD(P)H oxidase in HEK293 cells, but not by the redox-cycling of lucigenin. Furthermore, plumbagin inhibited the superoxide production in Nox-4 transfected COS-7 cells. These results indicated that plumbagin directly interacted with Nox-4 and inhibited its activity.

Abstract-Cyclosporin A (CsA) is used to reduce transplant rejection rates. Chronic use, however, has a destructive toxic effect on the kidney, resulting in hypertension. In this study, we investigated the effects of CsA treatment on the bradykinin/soluble guanylate cyclase signaling cascade and the involvement of superoxide in LLC-PK1 porcine kidney proximal tubule cells. Treatment with 1 mol/L CsA for 24 hours increased basal cGMP levels by 41%, whereas CsA inhibited bradykinin-stimulated cGMP production by 26%. Western blotting showed increased expression of eNOS, but no other protein in the bradykinin/soluble guanylate cyclase (sGC) pathway was affected. Using lucigenin-dependent chemiluminescence, we found that CsA treatment significantly increased superoxide production. Production of O 2 Ϫ was not significantly reduced by 10 mol/L oxypurinol or 30 mol/L ketoconazole. However, it was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium chloride (10 mol/L) as well as the O 2 Ϫ scavenger superoxide dismutase (SOD) (100 U). On treatment with 50 mol/L quercetin, 10 mmol/L N-acetyl-cysteine, both antioxidants, as well as the O 2 Ϫ scavenger Tiron (10 mmol/L), concomitant with 1 mol/L CsA for 24 hours the activation of cGMP production, was restored in combination with a reduction in O 2 Ϫ . Incubation with 100 mol/L menadione, a reactive oxygen generator, and 10 nmol/L bradykinin showed similar effects on the level of cGMP as with CsA. CsA treatment was found to increase nitrotyrosine levels. Key Words: cyclosporin Ⅲ bradykinin Ⅲ nitric oxide Ⅲ cyclic GMP Ⅲ antioxidants C yclosporin (CsA) is an important immunosuppressant used in improving the chances of whole organ transplant and graft survival. 1,2 However, cyclosporin treatment has been linked to several significant nephrotoxic side effects. The side effects range from afferent arteriolar constriction 3 and a reduction in glomerular filtration rate 4 to interstitial fibrosis 5 and ultimately hypertension. Although the toxic effects are well established, the exact mechanisms that lead to the pathology and hypertension are not agreed on. The proposed mechanisms for the development of hypertension focus on induction of vasoconstrictive pathways as well as obstruction of vasodilative pathways examined in patients, rat models, and endothelial cells. The pathways examined as possibly affected by CsA include the renal sensory nerve endings, 6 the renin-angiotensin system, 7 endothelin-1, 8,9 thromboxane, 9,10 and the renal kallikrein-kinin system. 11,12 Bradykinin is an important vasodilating peptide involved in the renal kallikrein-kinin system. The bradykinin peptide exerts its effects by binding to its receptor, activating a heterotrimeric G protein complex, phospholipase C, and then nitric oxide synthase (eNOS), which generates nitric oxide (NO). NO then binds to the heme group of soluble guanylate cyclase (sGC), thereby activating the enzyme to produce cGMP. 13,14 The NO synthase family and the NO radical have been shown to play an important role in many ...

No abstract

Yes-associated protein (YAP) is a transcriptional coactivator in the Hippo pathway that regulates cell proliferation, differentiation, and apoptosis. The MEK5/ERK5 MAPK cascade is essential for the early step of myogenesis. In this study, we generated C2C12 stable cell lines that expressed YAP (C2C12-YAP cells) and found that ERK5 and MEK5 were activated in C2C12-YAP cells compared with control C2C12 (C2C12-vector) cells. C2C12-YAP stable cells also differentiated into myotubes better than C2C12-vector cells, and expressed elevated levels of myogenin, a transcription factor that regulates myogenesis, as well as elevated levels of myosin heavy chain, a skeletal muscle marker. Western blot analysis revealed that Src and c-Abl (Abelson murine leukemia viral oncogene homolog 1) activation were enhanced in C2C12-YAP cells. Conversely, treatment of inhibitors of c-Abl, Src, or MEK5 inhibited activation of MEK5 and ERK5 and myogenesis of C2C12 myoblasts. Specific interactions between YAP and proteins in the ERK5 pathway, such as MEK kinase 3 (MEKK3) and ERK5, were illustrated by coimmunoprecipitation experiments. MEKK3 contains the PPGY motif (aa 178-181), which may interact with YAP. Site-directed mutagenesis experiments revealed that expression of MEKK3 Y181F mutant inhibited MEK5/ERK5 activation and myogenic differentiation. These results suggest that YAP promotes muscle differentiation by activating the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade.-Chen, T.-H., Chen, C.-Y., Wen, H.-C., Chang, C.-C., Wang, H.-D., Chuu, C.-P., Chang, C.-H. YAP promotes myogenic differentiation the MEK5-ERK5 pathway.