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RECEPTORS AND SIGNAL TRANSDUCTION

Inhibition of CDKS by roscovitine suppressed LPS-induced ^·NO production through inhibiting NFB activation and BH₄ biosynthesis in macrophages

¹Division of Pediatric Surgery, Department of Surgery, ²Children's Research Institution, ³Cardiovascular Research Center Medical College of Wisconsin, Milwaukee, Wisconsin; and ⁴Qilu Hospital of Shandong University, Jinan, China

Submitted 27 March 2009 ; accepted in final form 23 June 2009

In inflammatory diseases, tissue damage is critically associated with nitric oxide (^·NO) 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 production in mouse macrophages. In RAW264.7 cells, we found that roscovitine abolished the production of ^·NO 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 IB kinase β (IKKβ), IB, 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-1β, and IL-6 but not tumor necrosis factor (TNF)-. 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 production in macrophages. Similarly, in isolated peritoneal macrophages, roscovitine strongly inhibited ^·NO production, iNOS, and COX-2 upregulation, activation of NFB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced ^·NO production in macrophages by suppressing nuclear factor-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.

roscovitine; nitric oxide; tetrahydrobiopterin; nuclear factor B; macrophage