Regulation of hypoxia-inducible factor-1alpha by NF-kappaB

Abstract

HIF (hypoxia-inducible factor) is the main transcription factor activated by low oxygen tensions. HIF-1alpha (and other alpha subunits) is tightly controlled mostly at the protein level, through the concerted action of a class of enzymes called PHDs (prolyl hydroxylases) 1, 2 and 3. Most of the knowledge of HIF derives from studies following hypoxic stress; however, HIF-1alpha stabilization is also found in non-hypoxic conditions through an unknown mechanism. In the present study, we demonstrate that NF-kappaB (nuclear factor kappaB) is a direct modulator of HIF-1alpha expression. The HIF-1alpha promoter is responsive to selective NF-kappaB subunits. siRNA (small interfering RNA) studies for individual NF-kappaB members revealed differential effects on HIF-1alpha mRNA levels, indicating that NF-kappaB can regulate basal HIF-1alpha expression. Finally, when endogenous NF-kappaB is induced by TNFalpha (tumour necrosis factor alpha) treatment, HIF-1alpha levels also change in an NF-kappaB-dependent manner. In conclusion, we find that NF-kappaB can regulate basal TNFalpha and, in certain circumstances, the hypoxia-induced HIF-1alpha.

Figure 1. NF-κB subunits can activate the HIF-1α promoter

(a) Schematic diagram of the HIF-1α promoter–luciferase constructs. (b) HEK-293 cells were co-transfected with 1 μg of HIF-1α promoter–luciferase constructs and 1 μg of each of the NF-κB subunits. Luciferase activity was measured 48 h post-transfection. Values are means+S.D. for a minimum of three independent experiments performed in duplicate. The y-axis shows the fold activation above control plasmid. (c) NF-κB subunits bind to the κB site in the HIF-1α promoter. HEK-293 cells were transfected with 2 μg of each of the NF-κB subunits, nuclear extracts were prepared, and the DNA-binding activity was measured by EMSA using a specific probe for the HIF-1α promoter. (d) Nuclear extracts were prepared as in (c), and the DNA-binding activity was measured using a canonical NF-κB target promoter, HIV. (e) Nuclear extracts were analysed by Western blot for the individual NF-κB subunits.

Figure 2. Endogenous NF-κB subunits control basal HIF-1α mRNA levels

(a) HEK-293 cells were transfected with the indicated siRNA oligonucleotides, and qRT-PCR was performed. The histogram depicts the relative levels of HIF-1α mRNA normalized to actin mRNA levels. The mean+S.D. were calculated from a minimum of three independent experiments. A Student's t test was performed and *P<0.050 and **P<0.010 when compared with the control. Actual P values are: RelA, P=0.012; RelB, P=0.012; c-Rel, P=0.076; p50, P=0.348; p52, P=0.035; HIF-1α, P=0.007. (b) ChIP analysis using the indicated antibodies and PCR analysis using specific primers for the HIF-1α promoter and HIF-1α control region was performed.

Figure 3. NF-κB subunits control basal levels of HIF-1α protein

(a) HEK-293 cells were transfected with the indicated DNA constructs, and whole cell lysates were obtained 48 h post-transfection. At 3 h prior to harvest, half of the samples were incubated with 50 μM MG132. Western blot analysis for the indicated proteins was then performed on these extracts. (b) HEK-293 cells were transfected with the indicated siRNA oligonucleotides, and whole cell lysates were obtained 48 h post-transfection. At 3 h prior to harvest, half of the samples were incubated with 50 μM MG132. Western blot analysis for the indicated proteins was then performed on these extracts.

Figure 4. TNFα treatment induces NF-κB and HIF-1α

(a) HEK-293 cells were treated with 10 ng/ml TNFα for the indicated times, nuclear extracts were prepared, and these extracts were analysed by Western blot for the indicated proteins. PCNA was used as a loading control. (b) Cells were treated as in (a), and cell extracts were analysed by Western blot for the indicated HIF-1α targets. (c) Cells were treated as in (a), but mRNA was extracted and qRT-PCR was performed for the indicated gene transcripts. The histogram depicts relative levels of specific mRNA transcripts normalized to actin mRNA levels. The mean+S.D. were calculated from a minimum of three independent experiments. (d) Cells were transfected with the indicated siRNA oligonucleotides and treated with 10 ng/ml TNFα, 24 h prior to total mRNA extraction. qRT-PCR was performed as in (c).

Figure 5. TNFα induces HIF-1α activity in an NF-κB-dependent manner

(a) HEK-293 cells were transfected with the indicated siRNA oligonucleotides, treated with 10 ng/ml TNFα 24 h prior to harvest, and whole cell lysates were prepared. Extracts were analysed by Western blot using the indicated antibodies. (b) ChIP analysis using the indicated antibodies and PCR of specific regions of the HIF-1α target genes, VEGF, CA9 and DEC1. HRE, hypoxia-response element. (c) As in (b) but the HIF-1α promoter and control regions were analysed. (d) Cells were treated as in (a), and Western blot analysis was performed using the indicated antibodies. PCNA was used as a loading control.

Figure 6. Complete inhibition of NF-κB impairs hypoxia induced HIF-1α levels

(a) U2OS cells were transfected with the IKKα or IKKβ siRNA oligonucleotides and exposed to 1% O₂ for the indicated times prior to harvest. Whole cell lysates were analysed by Western blot. (b) Wild-type, IKKα^−/− and IKKβ^−/− mouse embryonic fibroblasts were exposed to 1% O₂ for the indicated periods of time, and whole cell lysates were prepared. Extracts were analysed by Western blot. (c) Wild-type and IKKα/β^−/− mouse embryonic fibroblasts were exposed to 1% O₂ for the indicated periods of time, and whole cell lysates were prepared. Extracts were analysed by Western blot. (d) U2OS cells were left untreated or treated for 4 h with 1% O₂ prior to harvest, and ChIP analysis using the indicated antibodies was performed. PCR analysis using specific primers for the HIF-1α promoter and HIF-1α control region was performed.