Inhibition of CDKS by roscovitine suppressed LPS-induced *NO production through inhibiting NFkappaB activation and BH4 biosynthesis in macrophages.
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 IkappaB kinase beta (IKKbeta), IkappaB, and p65 but enhanced the phosphorylation of ERK, p38, and c-Jun NH(2)-terminal kinase (JNK). In addition, roscovitine dose dependently inhibited LPS-induced expression of cyclooxygenase-2 (COX)-2, IL-1beta, and IL-6 but not tumor necrosis factor (TNF)-alpha. Tetrahydrobiopterin (BH(4)), an essential cofactor for iNOS, is easily oxidized to 7,8-dihydrobiopterin (BH(2)). Roscovitine significantly inhibited LPS-induced BH(4) biosynthesis and decreased BH(4)-to-BH(2) ratio. Furthermore, roscovitine greatly reduced the upregulation of GTP cyclohydrolase-1 (GCH-1), the rate-limiting enzyme for BH(4) 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 NFkappaB, and induction of GCH-1 by LPS. Together, our data indicate that roscovitine abolishes LPS-induced (*)NO production in macrophages by suppressing nuclear factor-kappaB activation and BH(4) 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.
Du
Jianhai
J
Division of Pediatric Surgery, Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. jhdu@mcw.edu
Wei
Na
N
Guan
Tongju
T
Xu
Hao
H
An
Jianzhong
J
Pritchard
Kirkwood A
KA
Shi
Yang
Y
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis; D002460:Cell Line; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006136:GTP Cyclohydrolase / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D011687:Purines / Q000494:pharmacology*; D000077546:Roscovitine
D008070:Lipopolysaccharides; D016328:NF-kappa B; D011687:Purines; D000077546:Roscovitine; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; D052247:Nitric Oxide Synthase Type II; D018844:Cyclin-Dependent Kinases; D006136:GTP Cyclohydrolase; C003402:sapropterin