Format:

Author:

Gummadidala, Phani Madhuri

Title:

Role of nuclear factor κB in regulation of hypoxia inducible factor-1α and angiogenesis

Dept./Program:

Pharmacology

Year:

2009

Degree:

MS

Abstract:

The transcription factor hypoxia inducible factor-lα (HIF-1α) plays a vital role in the transcription of genes related to erythropoiesis, energy metabolism, angiogenesis (primarily vascular endothelial growth factor; VEGF) and cell survival. Studies show that HIF-1α expression increases as cancer advances and its expression can be regulated by cytokines. Tumor necrosis factor a (TNFα), an inflammatory cytokine, has been linked to regulation of HIF-1α protein and tumor angiogenesis but the cellular mechanisms involved are yet to be identified.
We hypothesize that TNFα dose dependently regulates HIF-1α transcriptional activity through the induction of the NFκB pathway and that NFκB regulates angiogenesis.
To test the first half of our hypothesis, we treated cells with TNFα and studied HIF-1α protein levels, transcriptional activity and mRNA expression. Contrary to the existing literature, our preliminary experimentation in SKOV-3 and HEK 293 cells treated with various concentrations of TNFα revealed that TNFα does not affect HIF-1α protein levels. But, interestingly, we observed that TNFα decreased HIF-1α transcriptional activity by 25% in hypoxia. As TNFα acts primarily through the NFκB pathway, we modulated the activity of NFκB by transfecting HEK293 cells with IKKβ constitutively active plasmid (NFκB activity stimulation) and IκBα dominant negative plasmid (NFκB activity inhibition) in vitro. Neither plasmid changed HIF-1α protein levels. IκBα reversed the inhibitory affect of TNFα on hypoxic HIF-1α transcriptional activity and IKKβ significantly decreased both normoxic and hypoxic HIF-1α transcriptional activity. But, the mRNA expressions, as detected by RT-qPCR, of HIF-1α and VEGF were unaffected. We conclude that TNFα inhibits HIF-1α transcriptional activity in hypoxia, through the activation of the NFκB pathway, but further experimentation is required to understand the precise method in which NFκB affects HIF-1α regulation.
To test the second half of our hypothesis, we studied angiogenic gene profiling in IKKβ and IκBα transfected HEK293 cells and performed in vitro angiogenesis assays to understand the effect of NFKB on angiogenesis. Superarray analysis revealed that many of the chemokines and their receptors (CCL25, CCRl), angiogenesis inhibitors (interferon β1) regulated by NFκB were downregulated and angiogenic promoters (CXCL14) were upregulated by hypoxia and IKKβ. Imaging of rat aortic explants treated with conditioned media from transfected HEK 293 cells showed that conditioned media encouraged growth of vascular smooth muscle cells but not endothelial cells. Osteopotin (marker of cell migration) was more expressed in IKKβ treated explants towards the growing edge of the explant. We conclude that NFκB regulates angiogenesis by regulating genes responsible for angiogenesis and also encourages cell migration facilitating tumor metastasis.