NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha

Abstract

The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose alpha subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1alpha, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-kappaB plays a central role. NF-kappaB activation is controlled by IkappaB kinases (IKK), mainly IKK-beta, needed for phosphorylation-induced degradation of IkappaB inhibitors in response to infection and inflammation. IKK-beta is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-kappaB and HIF-1alpha has proven elusive. Using in vitro systems, it was reported that HIF-1alpha activates NF-kappaB, that NF-kappaB controls HIF-1alpha transcription and that HIF-1alpha activation may be concurrent with inhibition of NF-kappaB. Here we show, with the use of mice lacking IKK-beta in different cell types, that NF-kappaB is a critical transcriptional activator of HIF-1alpha and that basal NF-kappaB activity is required for HIF-1alpha protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-beta deficiency results in defective induction of HIF-1alpha target genes including vascular endothelial growth factor. IKK-beta is also essential for HIF-1alpha accumulation in macrophages experiencing a bacterial infection. Hence, IKK-beta is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

Figure 1. IKKβ is required for microbial-induced HIF-1α expression in macrophages

a) BMDM from either Ikkβ^F/F (IKKβ^+/+) or poly(IC)-injected Ikkβ^F/F/Mx1-Cre (Ikkβ^Δ; IKKβ^−/−) mice were incubated with either with GAS or P. aeruginosa (MOI of 10 for 4 hrs). HIF-1α expression was analyzed by immunoblotting. b) RNA was extracted from BMDM incubated with GAS and gene expression was analyzed by quantitative (Q) RT-PCR. Results are averages of 3 separate experiments done in triplicate. Values were normalized relative to 18S rRNA. c) ChIP was performed with an anti-RelA antibody using fixed and sheared chromatin isolated from RAW264.7 mouse macrophages incubated with or without LPS. The HIF-1α promoter fragment, which contains a κB site at −197/−188 bp, was detected by PCR amplification.

Figure 2. Hypoxia activates the NF-kB pathway in macrophages

RAW264.7 mouse macrophages were incubated with or without LPS or cultured under hypoxia (O₂ = 0.5 %). a) At the indicated time points of LPS stimulation or hypoxia, IKK activity was measured by an immunocomplex kinase assay using GST-IκBα as a substrate. b) Cell lysates were prepared and IKKβ and IκBα phosphorylation and abundance were analyzed by immunoblotting. c) Nuclear extracts were prepared at 2 hrs post-LPS or -hypoxia and NF-κB DNA binding activity was examined by a mobility shift assay. Antibody inhibition was performed using an anti-RelA antibody.

Figure 3. IKKβ regulates hypoxia-induced HIF-1α and target genes in mouse macrophages

a) BMDM from Ikkβ^F/F (IKKβ^+/+) or Ikkβ^Δ (IKKβ^−/−) mice were incubated with desferrioxamine (DFX) for 4 hrs. HIF-1α, HIF-1β and IKKβ expression were analyzed by immunoblotting. b) BMDM were obtained as above and cultured under hypoxia (O₂ = 0.5% for 4 hrs). HIF-1α expression was analyzed by immunoblotting. c) BMDM were treated as above and mRNA expression was analyzed by Q-RT-PCR. Results are averages of three separate experiments done in triplicates. p<0.05: *, vs normoxic Ikkβ^+/+ cells; #, vs hypoxic Ikkβ^+/+ cells. d) MEF from either Ikkβ^+/+ or Ikkβ^−/− embryos were transfected with a luciferase reporter gene driven by the HIF-1α promoter. After 36 hrs the cells were incubated for 3 hrs with DFX. p<0.05: *, vs normoxic Ikkβ^+/+ cells; #, vs hypoxic Ikkβ^+/+ cells.

Figure 4. IKKβ regulates HIF-1α expression in hypoxic mice

Ikkβ^F/F (CRE-) or Ikkβ^Δ (CRE+) mice were treated with DFX (600 mg/Kg). After 15 hrs, livers were removed for protein (a) and RNA (b) analysis. a) HIF-1α and IKKβ expression was analyzed by immunoblotting. b) Expression of HIF-1α and VEGF mRNA was examined by Q-RT- PCR (n=3). p<0.05: *, vs normoxic CRE- mice; #, vs DFX-treated CRE- mice. c,d) Ikkβ^F/F and Ikkβ^Δ mice were kept under normoxia or hypoxia (O₂ = 8%) for 24 hrs and HIF-1α expression was analyzed by immunoblotting of liver (c) or brain (d) extracts. e) VEGF expression in brain of mice from above experiment was analyzed by ELISA. p<0.05: *, vs normoxic CRE- mice; #, vs hypoxic CRE- mice. f) VEGF and HIF-1α mRNA expression was analyzed by Q-RT-PCR of total brain RNA. p<0.05: *, vs normoxic CRE- mice; #, vs hypoxic CRE- mice (n=3).

Figure 5. IKKβ deficiency results in increased astrogliosis in brains of hypoxic mice

Mice of the indicated genotypes were kept under normoxia or hypoxia (O₂ = 8%) for 24 hrs. After this period the mice were perfused with a fixative and the brain was collected and frozen. Brain sections at the cerebellar region (10 μm) were stained with an antibody against GFAP (an astrocyte marker). Magnification x20.