Hypoxia has emerged as a key determinant of osteogenesis. HIF-1 alpha is the transcription factor mediating hypoxia responses that include induction of VEGF and related bone induction. Inflammatory signals antagonize bone repair via the NF-kappa B pathway. The present investigation explored the functional relationship of hypoxia (HIF-1 alpha function) and inflammatory signaling (NF-kappa B) in stem like and osteoprogenitor cell lines. The potential interaction between HIF-1 alpha and NF-kappa B signaling was explored by co-transfection studies in hFOB with p65, HIF-1 alpha, and 9x-HRE-luc or HIF-1 alpha target genes reporter plasmids. Nuclear cross-talk was directly tested using the mammalian Gal4/VP16 two-hybrid, and confirmed by co-immunoprecipitation/western blotting assays. The results show that inflammatory stimulation (TNF-alpha treatment) causes a marked inhibition of HIF-1 alpha function at the HRE in all cell lines studied. Also, co-transfection with p65 expression vector leads to reduced hVEGFp transcription after DFO-induced hypoxia. However, TNF-alpha treatment had little effect on HIF-1 alpha mRNA levels. The functional interaction of Gal4-HIF-1 alpha and VP16-p300 fusion proteins is effectively blocked by expression of p65 in a dose dependent manner. It was concluded that NF-kappa B-mediated inflammatory signaling is able to block HIF-1 alpha transactivation at HRE-encoding genes by direct competition for p300 binding at the promoter. Inflammation may influence the stem cell niche and tissue regeneration by influencing cellular responses to hypoxia. (C) 2010 Elsevier Inc. All rights reserved.