title: Pioglitazone inhibition of lipopolysaccharide-induced nitric oxide synthase is associated with altered activity of p38 MAP kinase and PI3K/Akt.
abstract: BACKGROUND
  Previous studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-gamma)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-gamma agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.
  
  
  METHODS
  Mesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-gamma, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.
  
  
  RESULTS
  Our results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-gamma, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.
  
  
  CONCLUSIONS
  We demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-gamma activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.
authors.affiliation: Department of Anatomy and Neurobiology, 310 Davis Mills Building, University of Kentucky, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA. bxing3@email.uky.edu
authors.forename: Bin
authors.initials: B
authors.lastname: Xing
authors.forename: Tao
authors.initials: T
authors.lastname: Xin
authors.forename: Randy Lee
authors.initials: RL
authors.lastname: Hunter
authors.forename: Guoying
authors.initials: G
authors.lastname: Bing
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D001921:Brain / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D018920:Coculture Techniques; D004298:Dopamine / Q000378:metabolism; D004660:Encephalitis / Q000188:drug therapy* / Q000201:enzymology* / Q000503:physiopathology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D018836:Inflammation Mediators; D048031:JNK Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides; D017628:Microglia / Q000187:drug effects / Q000201:enzymology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000187:drug effects / Q000378:metabolism*; D047495:PPAR gamma / Q000037:antagonists & inhibitors / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism*; D000077205:Pioglitazone; D051381:Rats; D017207:Rats, Sprague-Dawley; D045162:Thiazolidinediones / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*
chemical_list: D000893:Anti-Inflammatory Agents; D007004:Hypoglycemic Agents; D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D047495:PPAR gamma; D045162:Thiazolidinediones; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D019869:Phosphatidylinositol 3-Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D004298:Dopamine; D000077205:Pioglitazone