NF-κB in hypoxia: And now for something completely different

The transcription factor NF-κB induces the expression of HIF-1α, providing a link between innate immunity and the hypoxic response.

During bacterial infection, induction of angiogenic factors such as hypoxia-inducible transcription factor-1 (HIF-1α), and immune response factors such as nuclear factor-κB (NF-κB) prevents hypoxia and cell death. Previous studies indicated that IκB kinase (IKK-β) — which regulates NF-κB activity — and HIF-1α were both post-translationally degraded by oxygen-dependent prolyl hydroxylases (PHDs) under normoxic conditions, suggesting a link between hypoxia and the innate immune response. In Nature, Michael Karin and colleagues now provide evidence for a novel intersection between these two pathways, as they show that NF-κB induces HIF-1α transcription in vivo.

The relationship between the NF-κB and HIF-1α pathways was examined in bone marrow-derived macrophages (BMDMs) from IKK-β-deficient mice. These BMDMs had reduced basal levels of Hif1α mRNA, and HIF-1α and its target genes were not induced during hypoxia or bacterial infection. When evaluated in vivo, hypoxia induced Hif1α mRNA in the livers of wild-type, but not IKK-β-deficient mice. Depletion of IKK-β also correlated with a reduction in both HIF-1α protein and vascular endothelial growth factor (Vegf) mRNA.

One possible explanation for this observation is that NF-κB is required for Hif1α transcription. Indeed, hypoxia promoted nuclear accumulation of the NF-κB RelA subunit and subsequent accumulation of HIF-1α in wild-type macrophages, but not IKK-β-deficient BMDMs. Furthermore, RelA bound directly to the Hif1α promoter. RelA was required for Hif1α induction in fibroblasts under normoxic conditions, suggesting that NF-κB also facilitates basal Hif1α transcription.

Lipopolysaccharide (LPS)-mediated induction of NF-κB in hypoxic cells induced Hif1α promoter activity, leading to accumulation of HIF-1α protein and increased Vegf expression. In normoxic cells, however, LPS treatment caused accumulation of Hif1α mRNA, but not protein, which indicates that accumulation of HIF-1α protein in hypoxic conditions is nonetheless dependent on the concomitant inhibition of PHDs.

Previous studies suggested that HIF-1α induction during hypoxia is predominately achieved through post-transcriptional mechanisms — namely, through the inhibition of PHD-mediated degradation. Karin and colleagues now reveal that hypoxia also stimulates NF-κB-mediated transcription of Hif1α mRNA in vivo. It is interesting to note that NF-κB is necessary for Hif1α transcription in normal and hypoxic conditions. However, post-transcriptional regulation of HIF-1α protein is essential for the hypoxic response, as HIF-1α protein was found to accumulate only in hypoxic conditions. Together, these data document a new role for IKK-β–NF-κB signaling in the response to bacterial infection and hypoxia, as it not only induces cytokines and antimicrobial peptides, but also upregulates pro-angiogenic factors to maintain tissue homeostasis.

Emily J. Chenette
Signaling Gateway

Original Reference:
Rius J. et al.
NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α
Nature advance online publication 23 April 2008 (doi:10.1038/nature06905).
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Oncogenic transformation: Invasion of the microvesicles

Glioma cells that express the oncogenic receptor EGFRvIII form microvesicles that fuse with neighboring, non-transformed cells to promote their oncogenic transformation.

A significant proportion of human gliomas express a truncated, constitutively active mutant of the epidermal growth factor receptor (EGFR) known as EGFR variant III (EGFRvIII). However, only a subset of the transformed cells within the EGFRvIII-positive glioma actually contains a mutated EGFRvIII gene. In Nature Cell Biology, Al-Nedawi et al. now show that EGFRvIII-positive glioma cells shed microvesicles containing EGFRvIII. These microvesicles fuse with benign neighboring cells to promote their oncogenic transformation.

Microvesicles are small membranous vesicles that can contain lipid raft-associated proteins, such as transmembrane receptor kinases. Overexpression of EGFRvIII in cultured U373 glioma cells potentiated EGFRvIII-positive microvesicle formation in vitro. Glioma cells expressing EGFRvIII formed tumors in mice, which shed EGFRvIII-containing microvesicles into the circulatory system.

Microvesicles fuse with the membranes of neighboring cells in a phosphatidylserine-dependent manner. Parental U373 cells that were incubated with EGFRvIII-containing microvesicles showed surface expression of EGFRvIII within 24 hours. Microvesicle transfer caused a 2-fold increase in anchorage-independent growth — a hallmark of oncogenic transformation. EGFRvIII uptake increased Erk1/2 and Akt phosphorylation, and Erk activation was blocked by pharmacologic inhibition of EGFR. Furthermore, annexin V treatment, which inhibits microvesicle fusion, blocked phosphorylation of Erk and Akt, suggesting that microvesicle-mediated transfer of EGFRvIII causes oncogenic transformation by upregulating MAPK signaling.

Activation of canonical EGFR signaling pathways by fusion of EGFRvIII-containing microvesicles resulted in the increased production of pro-tumorigenic factors such as vascular endothelial growth factor (VEGF) and Bcl-x_L, and decreased expression of the cyclin-dependent kinase inhibitor p27/Kip1. Inhibition of vesicle fusion by annexin V blocked these effects, further supporting the importance of EGFRvIII-positive microvesicle fusion in promoting the oncogenic transformation of inert glioma cells.

The horizontal transfer of tumorigenic material has been described for apoptotic cancer cells that transfer oncogenic DNA fragments to neighboring non-transformed cells, and for tumor cells that secrete soluble ligands to induce transformation of proximate cells. Al-Nedawi et al. describe a third novel mechanism of paracrine signaling in a tumor environment in which EGFRvIII protein is shuttled to a neighboring cell via an 'oncosome' to promote oncogenic transformation. If this mechanism is found to be important for human tumorigenesis in vivo, it would help explain why glioma cells that lack an EGFRvIII mutation nonetheless contribute to the overall growth of the tumor. Whether this effect is conserved among other receptor kinases with known roles in human tumorigenesis — MET, cKIT, or other EGFR/ErbB family members — remains to be elucidated.

Emily J. Chenette
Signaling Gateway

Original Reference:
Al-Nedawi, K. et al.
Intercellular transfer of the oncogenic receptor EGFRvIII by microvesicles derived from tumour cells
Nature Cell Biology 10, 619-624 (2008)
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