Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-kappaB

Abstract

Inflammatory mediators activate the transcriptional complex HIF-1 (hypoxia-inducible factor-1), the key regulator of hypoxia-induced gene expression. Here we report that bacterial LPS (lipopolysaccharide) induces HIF-1alpha mRNA expression and HIF-1alpha protein accumulation in human monocytes as well as in non-differentiated and differentiated cells of the human monocytic cell line THP-1 under normoxic conditions. LPS and hypoxia synergistically activated HIF-1. Whereas LPS increased HIF-1alpha mRNA expression through activation of a NF-kappaB (nuclear factor kappaB) site in the promoter of the HIF-1alpha gene, hypoxia post-translationally stabilized HIF-1alpha protein. HIF-1alpha activation was followed by increased expression of the HIF-1 target gene encoding ADM (adrenomedullin). Knocking down HIF-1alpha by RNA interference significantly decreased ADM expression, which underlines the importance of HIF-1 for the LPS-induced ADM expression in normoxia. Simultaneously with HIF-1 activation, an increase in p44/42 MAPK (mitogen-activated protein kinase) phosphorylation was observed after incubation with LPS. In cells pretreated with the p44/42 MAPK inhibitor PD 98059 or with RNAi (interfering RNA) directed against p44/42 MAPK, LPS-induced HIF-1alpha accumulation and ADM expression were significantly decreased. From these results we conclude that LPS critically involves the p44/42 MAPK and NF-kappaB pathway in the activation of HIF-1, which is an important transcription factor for LPS-induced ADM expression.

Figure 1. Undifferentiated THP-1 cells accumulate HIF-1α in response to hypoxia and LPS

(A) THP-1 cells were incubated under normoxic (NOX) and hypoxic (HOX) conditions for up to 16 h in the presence or absence of LPS (1μg/ml). A 70 μg portion of total cell lysate was resolved by SDS/PAGE. HIF-1α protein was detected with an anti-HIF-1α antibody, and anti-α-tubulin antibody was used to demonstrate equal gel loading. (B) THP-1 cells were incubated for 3 h with LPS. Total RNA was prepared, reverse-transcribed into cDNA, and HIF-1α cDNA was quantified by real-time PCR. Amounts of HIF-1α cDNA were normalized to 1 μg of total RNA. Shown are the means±S.D. from four separate experiments, *P<0.05. (C) LPS and hypoxia increase HIF-1 DNA-binding activity. An 80 μg portion of nuclear extract from treated THP-1 cells was incubated with a biotin-labelled double stranded oligonucleotide containing a specific HIF-1 binding sequence or a mutated (mut) HRE for 30 min at room temperature. Detection of DNA-bound HIF-1 complexes was performed using streptavidin–HRP conjugate. Binding activity of HIF-1 from LPS- and hypoxia-treated cells were calculated as a percentage of that exhibited by untreated normoxic controls. n=3, ***P<0.001. (D) LPS induces the expression of the HIF-1 target gene. THP-1 cells were incubated under normoxic and hypoxic condtions in the presence and absence of LPS (1 μg/ml) for 6 h. Total RNA was isolated and reverse-transcribed into cDNA. ADM cDNA was quantifed by real-time PCR. Shown are the means±S.D. from four separate experiments; *P<0.05.

Figure 2. LPS induces HIF-1α in differentiated THP-1 cells and in primary human macrophages

(A) THP-1 cells were treated for 5 days with 10 nM PMA to achieve differentiation from a monocytic to a macrophage-like phenotype. (B) Differentiated cells were incubated with LPS (1μg/ml) under normoxic (NOX) or hypoxic HOX) conditions. Whole-cell lysates were prepared and subjected to SDS/PAGE. HIF-1α and α-tubulin proteins were detected by immunoblotting. In addition, total RNA was extracted and HIF-1α cDNA was quantifed by real-time PCR. Shown are the means±S.D. from four independent experiments, *P<0.05. (C) Primary human macrophages were incubated with LPS (1 μg/ml) under normoxic or hypoxic conditions. Whole-cell lysates were prepared and subjected to SDS/PAGE. A representative immunoblot of HIF-1α and α-tubulin proteins is shown (n=3). After a 3 h treatment with LPS in normoxia and hypoxia, total RNA was extracted and HIF-1α cDNA was quantified by real-time PCR. Shown are the means±S.D. from four independent experiments. *P<0.05.

Figure 3. Mechanisms involved in LPS-induced HIF-1α activation

(A) THP-1 cells were incubated under hypoxic (HOX) conditions in the presence or absence of LPS (1 μg/ml) for 6 h, followed by reoxygenation for the indicated time periods. Whole-cell lysates were prepared and subjected to SDS/PAGE. HIF-1α and α-tubulin proteins were detected by immunoblotting. A representative immunoblot (n=4) is shown. (B) THP-1 cells were treated with MG132 (‘MG 132’;10 μM) and cycloheximide (CHX; 10 μM) 30 min before the end of 4 h incubation with LPS under normoxic (NOX) conditions. HIF-1α and polyubiquitinated forms of HIF-1α were detected with an anti-HIF-1α antibody; α-tubulin served as control for equal protein loading. (C) Cells were treated with actinomycin D (ActD; 5 μg/ml) immediately before a 4 h incubation with LPS under normoxic or hypoxic conditions. Total RNA was prepared and HIF-1α cDNA quantified by real-time PCR. Shown are the means±S.D. from four independent experiments, **P<0.01; ***P<0.001; # indicates statistically significant difference with respect to the untreated controls. (D) THP-1 cells were pretreated with ActD (5 μg/ml), followed by a 4 h incubation with LPS under normoxic or hypoxic conditions. Total cell lysates were prepared and 70 μg of the lysate were resolved by SDS/PAGE. HIF-1α protein was detected with an anti-HIF-1α antibody; anti-α-tubulin antibody was used to demonstrate equal gel loading.

Figure 4. THP-1 cells express CD14 and TLR4, members of the LPS signalling cascade, leading to phosphorylation of p44/42 MAPK after LPS stimulation

(A) Undifferentiated and differentiated THP-1 cells were treated with LPS under normoxic and hypoxic (HOX) conditions for 6 h. Total RNA was prepared, transcribed into cDNA and expression of the cDNAs for CD14 (‘CD 14’) and TLR4 (‘TLR 4’) were quantified by PCR. (B) THP-1 cells were incubated with LPS (1 μg/ml) under normoxic and hypoxic conditions for 3 h. A 50 μg portion of whole-cell lysate were submitted to SDS/PAGE. Phosphorylated as well as total p44/42 MAPK were detected by immunoblot.

Figure 5. Inhibition of p44/42 MAPK phosphorylation reduces the LPS induced HIF-1α mRNA expression and protein accumulation

(A) THP-1 cells were preincubated with the MEK1/2 inhibitor PD 98059 (50 μM) 30 min before LPS treatment. After 3 h of incubation under normoxic (NOX) or hypoxic (HOX) conditions, total RNA and whole-cell lysates were prepared. Amounts of HIF-1α cDNA were quantified by real-time PCR and normalized to 1 μg of total RNA. Means±S.D. from four independent experiments are shown. **P<0.01. (B) In addition, HIF-1α protein and p44/42 MAPK were analysed by immunoblot in 70 μg of total cell lysate; α-tubulin was used as loading control. (C) Inhibition of p4/42 MAPK activation significantly reduced the LPS-induced expression of the HIF-1 target ADM gene. Amounts of HIF-1α cDNA were quantified by real-time PCR and normalized to 1 μg of total RNA. The means±S.D. for four independent experiments are shown. **P<0.01.

Figure 6. LPS-induced HIF-1α expression depends on the activation of NF-κB

(A) MonoMac6 cells were transfected with a luciferase reporter vector under the control of the HIF-1α promoter. The HIF-1 promoter activity was dose-dependently increased by LPS. Luciferase activity was normalized to the expression of the corresponding control vector. n=6; P<0.05. The insert shows a schematic drawing of the luciferase reporter construct. (B) Nuclear extracts from LPS-treated THP-1 cells were incubated with an oligonucleotide corresponding to the NF-κB binding motif −130 nt in front of the transcription start site of the HIF-1α promoter. Abbreviation: anti-p65 AK, anti-NF-κB p65 antibody. (C) The LPS-induced expression of HIF-1α mRNA was abolished by the NF-κB inhibitor bortezomib. Total RNA was prepared, and amounts of HIF-1α cDNA were quantified by real-time PCR and normalized to 1 μg of total RNA. Means±S.D. from four independent experiments are shown; **P<0.01.

Figure 7. HIF-1α is critically involved in LPS- and hypoxia-induced expression of ADM

(A) Knocking down HIF-1α by RNAi abolished the LPS and hypoxia-induced HIF-1α protein accumulation. HIF-1α protein was detected by immunoblot in 70 μg of total cell lysate. A mutated HIF-1α RNAi (RNAi HIF 1α _1004 mut) served as control. (B) Treatment of THP-1 with RNAi directed against HIF-1α reduced the LPS induced ADM expression under normoxic (NOX; left panel) and hypoxic (HOX; right panel) conditions. The mutated HIF-1 RNAi has no significant effects on LPS-induced ADM expression. ADM cDNA was quantified by real-time PCR. Results are means±S.D. for four independent experiments; *P<0.05; **P<0.01.