title: TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats.
abstract: TNF-alpha is known to inhibit osteoblast differentiation in vitro and yet it is essential for bone fracture repair. Roles of TNF-alpha in the bony repair of injured growth plate were examined in young rats treated with a TNF-alpha antagonist. The results show that TNF-alpha mediates p38 activation, which influences the recruitment, proliferation, and osteoblast differentiation of mesenchymal cells and negatively regulates bone formation at the injured growth plate.
  
  
  BACKGROUND
  TNF-alpha inhibits expression of osteoblast differentiation factor cbfa1 and osteoblast differentiation in vitro and yet TNF-alpha signaling is essential for bone fracture healing. Roles of TNF-alpha in the bony repair of injured growth plate cartilage are unknown.
  
  
  METHODS
  Roles of TNF-alpha in the activation of p38 mitogen activated protein (MAP) kinase and the subsequent bony repair of the injured growth plate were examined in young rats receiving the TNF-alpha inhibitor ENBREL or saline control. Activation of p38 was determined by Western blot analysis and immunohistochemistry. Inflammatory cell counts on day 1, measurements of repair tissue proportions, and counting of proliferative mesenchymal cells on day 8 at growth plate injury site were carried out (n = 6). Expression of inflammatory cytokines TNF-alpha and IL-1beta, fibrogenic growth factor (FGF)-2, cbfa1, and bone protein osteocalcin at the injured growth plate was assessed by quantitative RT-PCR. Effects of TNF-alpha signaling on proliferation, migration, and apoptosis of rat bone marrow mesenchymal cells (rBMMCs) and the regulatory roles of p38 in these processes were examined using recombinant rat TNF-alpha, ENBREL, and the p38 inhibitor SB239063 in cultured primary rBMMCs.
  
  
  RESULTS
  p38 activation was induced in the injured growth plate during the initial inflammatory response, and activated p38 was immunolocalized in inflammatory cells at the injury site and in the adjacent growth plate. In addition, activation of p38 was blocked in rats treated with TNF-alpha antagonist, suggesting a role of TNF-alpha in p38 activation. Whereas TNF-alpha inhibition did not alter inflammatory infiltrate and expression of TNF-alpha and IL-1beta at the injured growth plate on day 1, it reduced mesenchymal infiltrate and cell proliferation and FGF-2 expression on day 8. Consistently, TNF-alpha increased proliferation and migration of rBMMCs in vitro, whereas p38 inhibition reduced rBMMC proliferation and migration. At the injured growth plate on day 8, TNF-alpha inhibition increased expression of cbfa1 and osteocalcin and increased trabecular bone formation at the injury site. There was a significant inverse correlation between TNF-alpha and cbfa1 expression levels, suggesting a negative relationship between TNF-alpha and cbfa1 in this in vivo model.
  
  
  CONCLUSIONS
  These observations suggest that TNF-alpha activates p38 MAP kinase during the inflammatory response at the injured growth plate, and TNF-alpha-p38 signaling seems to be required for marrow mesenchymal cell proliferation and migration at the growth plate injury site and in cell culture. Furthermore, TNF signaling has an inhibitory effect on bone formation at the injured growth plate by suppressing bone cell differentiation and bone matrix synthesis at the injury site.
authors.affiliation: Department of Orthopaedic Surgery, Women's and Children's Hospital, North Adelaide, South Australia.
authors.forename: Fiona H
authors.initials: FH
authors.lastname: Zhou
authors.forename: Bruce K
authors.initials: BK
authors.lastname: Foster
authors.forename: Xin-Fu
authors.initials: XF
authors.lastname: Zhou
authors.forename: Allison J
authors.initials: AJ
authors.lastname: Cowin
authors.forename: Cory J
authors.initials: CJ
authors.lastname: Xian
mesh_terms: D000818:Animals; D001861:Bone Regeneration / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050723:Fractures, Bone / Q000378:metabolism* / Q000473:pathology; D006132:Growth Plate / Q000378:metabolism* / Q000473:pathology; D008297:Male; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D010012:Osteogenesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D048051:p38 Mitogen-Activated Protein Kinases