Arachidonic acid metabolites, some of which may activate thromboxane A 2 receptors (TPr) and contribute to the development of diabetes complications, including nephropathy, are elevated in diabetes. This study determined the effect of blocking TPr with S18886 or inhibiting cyclooxygenase with aspirin on oxidative stress and the early stages of nephropathy in streptozotocin-induced diabetic apolipoprotein E ؊/؊ mice. Diabetic mice were treated with S18886 (5 mg ⅐ kg ؊1 ⅐ day ؊1 ) or aspirin (30 mg ⅐ kg ؊1 ⅐ day ؊1 ) for 6 weeks. Neither S18886 nor aspirin affected hyperglycemia or hypercholesterolemia. There was intense immunohistochemical staining for nitrotyrosine in diabetic mouse kidney. In addition, a decrease in manganese superoxide dismutase (MnSOD) activity was associated with an increase in MnSOD tyrosine-34 nitration. Tyrosine nitration was significantly reduced by S18886 but not by aspirin. Staining for the NADPH oxidase subunit p47phox , inducible nitric oxide synthase, and 12-lipoxygenase was increased in diabetic mouse kidney, as were urine levels of 12-hydroxyeicosatetraenoic acid and 8-iso-prostaglandin F 2␣ . S18886 attenuated all of these markers of oxidant stress and inflammation. Furthermore, S18886 significantly attenuated microalbuminuria in diabetic mice and ameliorated histological evidence of diabetic nephropathy, including transforming growth factor-␤ and extracellular matrix expression. Thus, in contrast to inhibiting cyclooxygenase, blockade of TPr may have therapeutic potential in diabetic nephropathy, in part by attenuating oxidative stress. Diabetes 55:110 -119, 2006

Activation of NF-kappaB requires the phosphorylation and degradation of its associated inhibitory proteins, IkappaB. Previously, we reported that the extracellular signal-regulated kinase (ERK) is required for IL-1beta to induce persistent activation of NF-kappaB in cultured rat vascular smooth muscle cells (VSMCs). The present study examined the mechanism by which the ERK signaling cascade modulates the duration of NF-kappaB activation. In cultured rat VSMCs, IL-1beta activated ERK and induced degradation of both IkappaBalpha and IkappaBbeta, which was associated with nuclear translocation of both ribosomal S6 kinase (RSK)1 and NF-kappaB p65. RSK1, a downstream kinase of ERK, was associated with an IkappaBbeta/NF-kappaB complex, which was independent of the phosphorylation status of RSK1. Treatment of VSMCs with IL-1beta decreased IkappaBbeta in the RSK1/IkappaBbeta/NF-kappaB complex, an effect that was attenuated by inhibition of ERK activation. Knockdown of RSK1 by small interference RNA attenuated the IL-1beta-induced IkappaBbeta decrease without influencing ether ERK phosphorylation or the earlier IkappaBalpha degradation. By using recombinant wild-type and mutant IkappaBbeta proteins, both active ERK2 and RSK1 were found to directly phosphorylate IkappaBbeta, but only active RSK1 phosphorylated IkappaBbeta on Ser19 and Ser23, two sites known to mediate the subsequent ubiquitination and degradation. In conclusion, in the ERK signaling cascade, RSK1 is a key component that directly phosphorylates IkappaBbeta and contributes to the persistent activation of NF-kappaB by IL-1beta.

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Activation of thromboxane receptors (TPr) may promote atherosclerosis by enhancing oxidative stress and the inflammation. This study examined the role of Nox1, an NADPH-oxidase subunit, in the enhancement of interleukin (IL)-1β-induced monocyte adhesion by TPr. In cultured rat aortic vascular smooth muscle cells (VSMCs), U46619, a stable thromboxane A2 mimetic, together with interleukin-1β significantly enhanced Nox1 mRNA expression, as well as adhesion of THP-1 monocytes. Activation of TPr also enhanced IL-1β-induced vascular cell adhesion molecule (VCAM)-1 expression, but inhibited inducible nitric oxide synthase (iNOS) expression. Silencing Nox1 expression by siRNA prevented the U46619 enhancement of IL-1β-induced monocyte adhesion, but had no significant effect on VCAM-1 or iNOS expression. Furthermore, monocyte adhesion was inhibited by superoxide dismutase, enhanced by a specific iNOS inhibitor, L-N6-(1-iminoethyl)-lysine, but not influenced by catalase. U46619 inhibited IL-1β-induced cyclic GMP production, and the inhibition was partially prevented by superoxide dismutase. In conclusion, activation of TPr enhances IL-1β-induced Nox1 expression in VSMCs, which is responsible for the up-regulation of monocyte adhesion. The effect of Nox1 is independent of the changes in VCAM-1 and iNOS expression, but depends upon the inactivation of nitric oxide via generation of superoxide anion.