Essential role of mitogen-activated protein kinase pathways in protease activated receptor 2-mediated nitric-oxide production from rat primary astrocytes.
Protease-activated receptors (PARs) play important roles in the regulation of brain function such as neuroinflammation by transmitting the signal from proteolytic enzymes such as thrombin and trypsin. We and others have reported that a member of the family, PAR-2 is activated by trypsin, whose involvement in the neurophysiological process is increasingly evident, and is involved in the neuroinflammatory processes including morphological changes of astrocytes. In this study, we investigated the role of PAR-2 in the production of nitric oxide (NO) in rat primary astrocytes. Treatment of PAR-2 agonist trypsin increased NO production in a dose-dependent manner, which was mediated by the induction of inducible nitric-oxide synthase. The trypsin-mediated production of NO was mimicked by PAR-2 agonist peptide and reduced by either pharmacological PAR-2 antagonist peptide or by siRNA-mediated inhibition of PAR-2 expression, which suggests the critical role of PAR-2 in this process. NO production by PAR-2 was mimicked by PMA, a PKC activator, and was attenuated by Go6976, a protein kinase C (PKC) inhibitor. PAR-2 stimulation activated three subtypes of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. NO production by PAR-2 was blocked by inhibition of ERK, p38, and JNK pathways. PAR-2 stimulation also activated nuclear factor-kappaB (NF-kappaB) DNA binding and transcriptional activity as well as IkappaBalpha phosphorylation. Inhibitors of NF-kappaB pathway inhibited PAR-2-mediated NO production. In addition, inhibitors of MAPK pathways prevented transcriptional activation of NF-kappaB reporter constructs. These results suggest that PAR-2 activation-mediated NO production in astrocytes is transduced by the activation of MAPKs followed by NF-kappaB pathways.
Park
Gyu Hwan
GH
Department of Pharmacolgy, College of Pharmacy, Seoul National University, San 56-1, Shillim-Dong, Kwanak-Gu, Seoul 151-742, Republic of Korea.
Jeon
Se Jin
SJ
Ryu
Jae Ryun
JR
Choi
Min Sik
MS
Han
Seol-Heui
SH
Yang
Sung-Il
SI
Ryu
Jong Hoon
JH
Cheong
Jae Hoon
JH
Shin
Chan Young
CY
Ko
Kwang Ho
KH
D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D002470:Cell Survival / Q000187:drug effects; D020672:I-kappa B Proteins / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D044464:Receptor, PAR-2 / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D009573:Nitrites; D044464:Receptor, PAR-2; D000072000:NF-KappaB Inhibitor alpha; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D014357:Trypsin