title: TNFα contributes for attenuating both Y397FAK and Y416Src phosphorylations in osteoblasts.
abstract: OBJECTIVE
  Our poor understanding of how inflammatory mediators can affect osteoblast behavior led us to investigate the tumor necrosis factor (TNF)α-induced focal adhesion kinase (FAK) and Src phosphorylation.
  
  
  METHODS
  MC3T3-E1 pre-osteoblast cells were harvested at specific time points after either TNFα treatment or RAW267 stimulated conditioned medium, and thereafter cell extracts were prepared for Immunoblotting assay. ELISA detected TNFα content at conditioned medium. Tumor necrosis factor-α-neutralizing antibodies also were used.
  
  
  RESULTS
  It was possible to show that TNFα provokes attenuation at Y-phosphorylation of both FAK (at Y397 ) and Src (at Y416 ) proteins (P < 0.05), suggesting a decrease in their activities. The very similar profile was observed when osteoblasts were incubated with conditioned medium from lipopolysaccharide (LPS)-stimulated macrophages, it being significantly different than control (FAK and Src, P < 0.05). Nevertheless, in order to validate these findings, we decided to pre-incubate osteoblasts with anti-TNFα neutralizing antibody (2 μg ml(-1) ) prior exposing to conditioned medium. Importantly, our results revealed that there was a diminution on those conditioned medium effects when the same biological parameters were evaluated (P < 0.05). Moreover, we also showed that TNFα impairs osteoblast adhesion, suggesting an interesting role on osteoblast performance.
  
  
  CONCLUSIONS
  Altogether, these results suggest that LPS-stimulated macrophage mediators attenuate both FAK and Src activations in osteoblast, suggesting a novel role for TNFα on osteoblast performance.
authors.affiliation: Federal University of São Carlos (UFSCar), Sorocaba, Brazil.
authors.forename: Adm
authors.initials: A
authors.lastname: Cavagis
authors.forename: Er
authors.initials: E
authors.lastname: Takamori
authors.forename: Jm
authors.initials: J
authors.lastname: Granjeiro
authors.forename: Rc
authors.initials: R
authors.lastname: Oliveira
authors.forename: Cv
authors.initials: C
authors.lastname: Ferreira
authors.forename: Mp
authors.initials: M
authors.lastname: Peppelenbosch
authors.forename: Wf
authors.initials: W
authors.lastname: Zambuzzi
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D017077:Culture Media, Conditioned; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology*; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D014409:Tumor Necrosis Factor-alpha; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases
keywords: Lipopolysaccharide; Src; cell signaling; focal adhesion kinase; inflammation; macrophages; osteoblasts; periodontal disease; tumor necrosis factor