title: VEGF-A expression in osteoclasts is regulated by NF-kappaB induction of HIF-1alpha.
abstract: Large osteoclasts (10+ nuclei), predominant in rheumatoid arthritis and periodontal disease, have higher expression of proteases and activating receptors and also have increased resorptive activity when compared to small (2-5 nuclei) osteoclasts. We hypothesized that large and small osteoclasts activate different signaling pathways. A Signal Transduction Pathway Finder Array was used to compare gene expression of large and small osteoclasts in RAW 264.7-derived osteoclasts. Expression of vascular endothelial growth factor A (Vegfa) was higher in large osteoclasts and this result was confirmed by RT-PCR. RT-PCR further showed that RANKL treatment of RAW cells induced Vegfa expression in a time-dependent manner. Moreover, VEGF-A secretion in conditioned media was also increased in cultures with a higher proportion of large osteoclasts. To investigate the mechanism of Vegfa induction, specific inhibitors for the transcription factors NF-kappaB, AP-1, NFATc1, and HIF-1 were used. Dimethyl bisphenol A, the HIF-1alpha inhibitor, decreased Vegfa mRNA expression, whereas blocking NF-kappaB, AP-1, and NFATc1 had no effect. Furthermore, the NF-kappaB inhibitor gliotoxin inhibited Hif1alpha mRNA expression. In conclusion, VEGF-A gene and protein expression are elevated in large osteoclasts compared to small osteoclasts and this increase is regulated by HIF-1. In turn, Hif1alpha mRNA levels are induced by RANKL-mediated activation of NF-kappaB. These findings reveal further differences in signaling between large and small osteoclasts and thereby identify novel therapeutic targets for highly resorptive osteoclasts in inflammatory bone loss.
authors.affiliation: Faculty of Medicine, Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. diana.trebec@utoronto.ca
authors.forename: Diana P
authors.initials: DP
authors.lastname: Trebec-Reynolds
authors.forename: Irina
authors.initials: I
authors.lastname: Voronov
authors.forename: Johan N M
authors.initials: JN
authors.lastname: Heersche
authors.forename: Morris F
authors.initials: MF
authors.lastname: Manolson
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D016328:NF-kappa B / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D010010:Osteoclasts / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A