title: A dominant function of p38 mitogen-activated protein kinase signaling in receptor activator of nuclear factor-kappaB ligand expression and osteoclastogenesis induction by Aggregatibacter actinomycetemcomitans and Escherichia coli lipopolysaccharide.
abstract: OBJECTIVE
  Lipopolysaccharide from gram-negative bacteria is one of the microbial-associated molecular patterns that initiate the immune/inflammatory response, leading to the tissue destruction observed in periodontitis. The aim of this study was to evaluate the role of the p38 mitogen-activated protein kinase (MAPK) signaling pathway in lipopolysaccharide-induced receptor activator of nuclear factor-kappaB ligand (RANKL) expression by murine periodontal ligament cells.
  
  
  METHODS
  Expression of RANKL and osteoprotegerin mRNA was studied by reverse transcription-polymerase chain reaction upon stimulation with lipopolysaccharide from Escherichia coli and Aggregatibacter actinomycetemcomitans. The biochemical inhibitor SB203580 was used to evaluate the contribution of the p38 MAPK signaling pathway to lipopolysaccharide-induced RANKL and osteoprotegerin expression. Stable cell lines expressing dominant-negative forms of MAPK kinase (MKK)-3 and MKK6 were generated to confirm the role of the p38 MAPK pathway. An osteoclastogenesis assay using a coculture model of the murine monocytic cell line RAW 264.7 was used to determine if osteoclast differentiation induced by lipopolysaccharide-stimulated periodontal ligament was correlated with RANKL expression.
  
  
  RESULTS
  Inhibiting p38 MAPK prior to lipopolysaccharide stimulation resulted in a significant decrease of RANKL mRNA expression. Osteoprotegerin mRNA expression was not affected by lipopolysaccharide or p38 MAPK. Lipopolysaccharide-stimulated periodontal ligament cells increased osteoclast differentiation, an effect that was completely blocked by osteoprotegerin and significantly decreased by inhibition of MKK3 and MKK6, upstream activators of p38 MAPK. Conditioned medium from murine periodontal ligament cultures did not increase osteoclast differentiation, indicating that periodontal ligament cells produced membrane-bound RANKL.
  
  
  CONCLUSIONS
  Lipopolysaccharide resulted in a significant increase of RANKL in periodontal ligament cells. The p38 MAPK pathway is required for lipopolysaccharide-induced membrane-bound RANKL expression in these cells.
authors.affiliation: Department of Diagnosis and Surgery, School of Dentistry at Araraquara, State University of Sao Paulo, Araraquara, SP, Brazil.
authors.forename: C
authors.initials: C
authors.lastname: Rossa
authors.forename: M
authors.initials: M
authors.lastname: Liu
authors.forename: K L
authors.initials: KL
authors.lastname: Kirkwood
mesh_terms: D016976:Aggregatibacter actinomycetemcomitans; D000818:Animals; D002454:Cell Differentiation; D002461:Cell Line, Transformed; D004926:Escherichia coli; D015870:Gene Expression; D008070:Lipopolysaccharides; D048371:MAP Kinase Kinase 3 / Q000037:antagonists & inhibitors; D048669:MAP Kinase Kinase 6 / Q000037:antagonists & inhibitors; D020935:MAP Kinase Signaling System; D051379:Mice; D010010:Osteoclasts; D053244:Osteoprotegerin / Q000096:biosynthesis; D010513:Periodontal Ligament / Q000166:cytology / Q000378:metabolism*; D053245:RANK Ligand / Q000096:biosynthesis*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D053244:Osteoprotegerin; D053245:RANK Ligand; D048051:p38 Mitogen-Activated Protein Kinases; D048371:MAP Kinase Kinase 3; D048669:MAP Kinase Kinase 6