title: Osteoblast-derived TGF-beta1 stimulates IL-8 release through AP-1 and NF-kappaB in human cancer cells.
abstract: BACKGROUND
  The bone marrow microenvironment is further enriched by growth factors released during osteoclastic bone resorption. It has been reported that the chemokine interleukin (IL)-8 is a potent and direct activator of osteoclastic differentiation and bone resorption. However, the effect of bone-derived growth factors on the IL-8 production in human cancer cells and the promotion of osteoclastogenesis are largely unknown. The aim of this study was to investigate whether osteoblast-derived TGF-beta1 is associated with osteolytic bone diseases.
  
  
  METHODS
  IL-8 mRNA levels were measured using RT-PCR analysis. MAPK phosphorylation was examined using the Western blot method. siRNA was used to inhibit the expression of TGF-beta1, BMP-2, and IGF-1. DNA affinity protein-binding assay and chromatin immunoprecipitation assays were used to study in vitro and in vivo binding of c-fos, c-jun, p65, and p50 to the IL-8 promoter. A transient transfection protocol was used to examine IL-8, NF-kappaB, and activator protein (AP)-1 activity.
  
  
  RESULTS
  Osteoblast conditioned medium (OBCM) induced activation of IL-8, AP-1, and NF-kappaB promoter in human cancer cells. Osteoblasts were transfected with TGF-beta1, BMP-2, or IGF-1 small interfering RNA, and the medium was collected after 48 h. TGF-beta1 but not BMP-2 or IGF-1 siRNA inhibited OBCM-induced IL-8 release in human cancer cells. In addition, TGF-beta1 also directly induced IL-8 release in human cancer cells. Activation of AP-1 and NF-kappaB DNA-protein binding and MAPKs after TGF-beta1 treatment was shown, and TGF-beta1-induced IL-8 promoter activity was inhibited by the specific inhibitors of MAPK cascades.
  
  
  CONCLUSIONS
  In this study, we provide evidence to show that the osteoblasts release growth factors, including TGF-beta1, BMP-2, and IGF-1. TGF-beta1 is the major contributor to the activation of extracellular signal-related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to the activation of AP-1 and NF-kappaB on the IL-8 promoter and initiation of IL-8 mRNA and protein release, thereby promoting osteoclastogenesis.
authors.affiliation: Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan.
authors.forename: Yi-Chin
authors.initials: YC
authors.lastname: Fong
authors.forename: Ming-Chei
authors.initials: MC
authors.lastname: Maa
authors.forename: Fuu-Jen
authors.initials: FJ
authors.lastname: Tsai
authors.forename: Wen-Chi
authors.initials: WC
authors.lastname: Chen
authors.forename: Jaung-Geng
authors.initials: JG
authors.lastname: Lin
authors.forename: Long-Bin
authors.initials: LB
authors.lastname: Jeng
authors.forename: Rong-Sen
authors.initials: RS
authors.lastname: Yang
authors.forename: Wen-Mei
authors.initials: WM
authors.lastname: Fu
authors.forename: Chih-Hsin
authors.initials: CH
authors.lastname: Tang
mesh_terms: D045744:Cell Line, Tumor; D003470:Culture Media; D004789:Enzyme Activation; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D009369:Neoplasms / Q000378:metabolism*; D010006:Osteoblasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D003470:Culture Media; D016209:Interleukin-8; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; D018808:Transcription Factor AP-1; D053773:Transforming Growth Factor beta1; D020928:Mitogen-Activated Protein Kinases