Korean J Blood Transfus.  2013 Apr;24(1):21-32.

Functional Interaction of HIF-1 and NF-kappaB Increasing the Transcriptional Activation of TNF-alpha Gene in Monocytes

• Park MJ
• Lee SM
• Ok SJ
• Kim HR
• Kim HH
• Cheong JH

Abstract

BACKGROUND
Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine fulfilling a broad variety of immunoregulatory functions. Monocytes and macrophages play a pivotal role in inflammation and immune regulation. NF-kappaB and HIF-1 are known to increase expression of the TNF-alpha gene in a separate way.
METHODS
Human monocytic leukemia, U937 cells, were transfected using the standard electroporation method for intracellular expression of NF-kappaB and HIF-1. We performed analysis using the mammalian two-hybrid assay and co-immunoprecipitation assay for detection of protein interaction of both proteins. In addition, chromatin immunoprecipitation analysis was performed for examination of NF-kappaB and HIF-1 binding on the TNF-alpha gene promoter.
RESULTS
Here we show that NF-kappaB and HIF-1 cooperatively induced an increase in expression of the TNF-alpha gene dependent on promoter activity by the direct protein interaction of these two transcription factors. Hypoxia signaling induced marked enhancement of the transactivation of TNF-alpha promoter by HIF-1 and NF-kappaB. A tandem NF-kappaB/HIF-1 binding site was identified within the TNF-alpha promoter, which acted as a strong enhancer element. Physical association of the Rel domain of NF-kappaB and the N-TD domain of HIF-1 was required. Hypoxia treatment also resulted in a significant increase in the protein interaction of NF-kappaB and HIF-1 in vivo. Both transcription factors were recruited on the chromatin TNF-alpha promoter dependent on hypoxia stimuli.
CONCLUSION
The results of this study indicate that a variety of extracellular signals for activation of TNF-alpha gene expression might converge on the transcriptional regulation through the NF-kappaB/HIF-1 signaling pathway.

Keyword

HIF-1; NF-kappaB; Hypoxia; TNF-alpha; Transactivation

MeSH Terms

Anoxia
Binding Sites
Chromatin
Chromatin Immunoprecipitation
Electroporation
Enhancer Elements, Genetic
Gene Expression
Humans
Immunoprecipitation
Inflammation
Leukemia
Macrophages
Monocytes
NF-kappa B
Proteins
Transcription Factors
Transcriptional Activation
Tumor Necrosis Factor-alpha
Two-Hybrid System Techniques
U937 Cells
Chromatin
NF-kappa B
Proteins
Transcription Factors
Tumor Necrosis Factor-alpha