Inhibition of CDKS by roscovitine suppressed LPS-induced •NO production through inhibiting NFκB activation and BH4 biosynthesis in macrophages

Du J, Wei N, Guan T, Xu H, An J, Pritchard KA, Jr., Shi Y. Inhibition of CDKS by roscovitine suppressed LPS-induced (NO)-N-center dot production through inhibiting NF kappa B activation and BH4 biosynthesis in macrophages. Am J Physiol Cell Physiol 297: C742-C749, 2009. First published June 24, 2009; doi:10.1152/ajpcell.00138.2009.-In inflammatory diseases, tissue damage is critically associated with nitric oxide ((NO)-N-center dot) and cytokines, which are overproduced in response to cellular release of endotoxins. Here we investigated the inhibitory effect of roscovitine, a selective inhibitor of cyclin-dependent kinases (CDKs) on (NO)-N-center dot production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of (NO)-N-center dot induced by lipopolysaccharide (LPS). Moreover, roscovitine significantly inhibited LPS-induced inducible nitric oxide synthase (iNOS) mRNA and protein expression. Our data also showed that roscovitine attenuated LPS-induced phosphorylation of I kappa B kinase beta (IKK beta), I kappa B, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH2-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1 beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH4), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH2). Roscovitine significantly inhibited LPS-induced BH4 biosynthesis and decreased BH4-to-BH2 ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH4 biosynthesis. Using other CDK inhibitors, we found that CDK1, CDK5, and CDK7, but not CDK2, significantly inhibited LPS-induced (NO)-N-center dot production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited (NO)-N-center dot production, iNOS, and COX-2 upregulation, activation of NF kappa B, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (NO)-N-center dot production in macrophages by suppressing nuclear factor-kappa B activation and BH4 biosynthesis, which might be mediated by CDK1, CDK5, and CDK7. Our results also suggest that roscovitine may inhibit inflammation and that CDKs may play important roles in the mechanisms by which roscovitine attenuates inflammation.