HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride.
We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.
Tsoyi
Konstantin
K
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju, Republic of Korea.
Kim
Hye Jung
HJ
Shin
Jae-Soo
JS
Kim
Dal-Hyun
DH
Cho
Hee-Jeong
HJ
Lee
Sung Sook
SS
Ahn
Sun Kil
SK
Yun-Choi
Hye Sook
HS
Lee
Jae Heun
JH
Seo
Han Geuk
HG
Chang
Ki Churl
KC
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D051546:Cyclooxygenase 2 / Q000235:genetics*; D015232:Dinoprostone / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D051547:Heme Oxygenase-1 / Q000378:metabolism*; D053614:Janus Kinase 2 / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D050794:STAT1 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D044005:Tetrahydroisoquinolines / Q000737:chemistry / Q000494:pharmacology*
D000470:Alkaloids; D008070:Lipopolysaccharides; D016328:NF-kappa B; D050794:STAT1 Transcription Factor; D044005:Tetrahydroisoquinolines; C090309:YS 49; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D051547:Heme Oxygenase-1; D051546:Cyclooxygenase 2; D053614:Janus Kinase 2; D020928:Mitogen-Activated Protein Kinases; D015232:Dinoprostone