MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1β Production through HIF-1α Expression

Abstract

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1β. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1β as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1β production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1β production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1β production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1β expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1β production through modulating HIF-1α expression.

FIGURE 1.. MEK1 and MEK2 isoform regulate LPS mediated IL-1β and IL-6 production in BMDMs.

BMDMs derived from WT, Mek1^d/dSox2^Cre and Mek2^−/− mice were cultured under similar conditions and treated with LPS (100 ng/mL) for 24 h. (A) Nitric Oxide production. After LPS challenge for 24h the generation of NO in the conditioned medium was determined by measuring nitrite accumulation using Griess reaction. Conditioned media were analyzed for (B) TNF-α, (C) IL-10, (D) IL-12, (E) IL-6 and (F) IL-1β using ELISA. Data presented as mean of three independent experiments. Using ANOVA Mann-Whitney U test, * signifies a p value <0.05, while ** signifies a p value <0.001, and error bars indicate SEM.

FIGURE 2.. MEK2 regulates IL-1β and HIF-1α expression at the transcript and protein level.

BMDMs derived from WT and MEK2^−/− and Mek1^d/d Sox2^Cre mice were treated with LPS (100 ng/mL) for 1h. Total RNA was extracted and reverse-transcribed using the Reverse Transcription System. (A) The primers targeting IL-1β were used to amplify cDNA using iQSYBR Green Supermix. Relative mRNA levels were calculated by normalizing to GAPDH. Data were analyzed using the paired, two-tailed Student’s t test, and the results were expressed as fold change. (B) BMDMs derived from WT and MEK2^−/− and Mek1^d/d Sox2^Cre mice were treated with LPS (100 ng/mL) for 3h. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using specific antibodies against pro IL-1β. (C) Equal loading was determined using antibodies against β-actin. (D) BMDMs derived from WT and MEK2^−/− and Mek1^d/d Sox2^Cre were treated with LPS (100 ng/mL) for different time points as indicated. Whole cell extracts were prepared and subjected to SDS-PAGE and Western analysis using specific antibodies to NLRP3. (E) Densitometric values expressed as fold changes of the ratio NLRP3/ β-actin. (F) HIF-1α mRNA expression. RNA was isolated from cells and expression assessed using qRT-PCR. Values were normalized to GAPDH. Results represent mean values of 4 independent experiments. Using ANOVA Mann-Whitney U test a p value of <0.05 was considered significant and error bars indicate SEM. (G) ARNT (HIF-1 β) mRNA expression. Total RNA was extracted from WT and MEK2^−/− and Mek1^d/d Sox2^Cre BMDMs treated with LPS (100 ng/mL) for 1h. The primers targeting ARNT were used to amplify cDNA. Relative mRNA levels were calculated by normalizing to GAPDH. Data were analyzed using the paired, two-tailed Student’s t test, and the results were expressed as fold change. (H) HIF-1α expression. Whole cell extracts were subjected to SDS-PAGE and Western blot analysis using specific antibodies against HIF-1α. Equal loading was determined using antibodies against β-actin. (I) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio HIF-1α/ β-actin.

Figure 3.. Despite higher pVHL HIF-1α accumulates in nuclear extract of MEK2 deficient BMDMs and co-immunoprecipitates with p300/CBP.

WT, MEK2^−/−, and MEK1^d/dSox2^cre BMDMs were cultured and challenged with LPS (100 ng/mL) for 3h. Nuclear and cytosolic extracts were prepared and subjected to SDS-PAGE. (A) Western blot analysis was performed using specific antibodies against, VHL, HIF-1α and against β-actin. MEK2^−/− BMDMs exhibit higher HIF-1α in nuclear extracts. Protein lysates prepared from untreated WT and MEK2^−/− BMDMs were immunoprecipitated with HIF-1α specific antibody and equal amount of immunoprecipitates were subjected to SDS-PAGE. (Band C) Western blot analysis was performed using specific antibodies to HIF-1α and p300/CBP. MEK2^−/− BMDMs exhibit higher p300/CBP protein in the lysates immunoprecipitated with HIF-1α specific antibody. (D) GLUT1 expression. BMDMs derived from WT and MEK2^−/− and Mek1^d/d Sox2^Cre mice were treated with LPS (100 ng/mL) for 6 h and 24h. Whole cell extracts were subjected to SDS-PAGE and Western blot analysis using specific antibody against GLUT1. Equal loading was determined using antibody against β-actin. (E) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio Glut1/ β-actin. MEK2 deficiency exhibited higher Glut1 levels in response to LPS. (F) VEGF mRNA expression. Total RNA was extracted from WT and MEK2^−/− and Mek1^d/d Sox2^Cre BMDMs treated with LPS (100 ng/mL) for 1h and VEGF expression was assessed using qRT-PCR. Values were normalized to GAPDH. Results represent mean values of 4 independent experiments. Using ANOVA Mann-Whitney U test, * signifies a p value <0.05, while ** signifies a p value <0.001, and error bars indicate SEM. MEK2 deficient BMDMs showed highly significant expression of VEGF in response to LPS.

FIGURE 4.. Targeted downregulation of HIF-1α in MEK2 deficient macrophages leads to a decreased IL-1β production.

MEK2^−/− BMDMs were transfected with HIF-1α or scrambled siRNA. 24 h post-transfection, cells were incubated with/without LPS for 6 h or 24. (A) Total cellular protein (of siRNA transfected cells) were subjected to Western blot analysis using antibodies against HIF-1α, pro IL-1β or β-actin. (B) Densitometric values expressed as fold increase of the ratio of HIF-1α/β-actin. (C) Densitometric values expressed as fold increase of the ratio of pro IL-1β/β-actin. (D) Conditioned media were collected for IL-1β analyses via ELISA. Targeted down regulation of HIF-1α resulted in a significant decrease of IL-1 β in response to LPS (p < 0.05). Data are presented as relative gene expression levels from three independent transfections each performed in triplicates. Data are representative results of three independent experiments each performed in triplicates. Using ANOVA Mann-Whitney U test, * signifies a p value <0.05, while ** signifies a p value <0.001, and error bars indicate SEM.

FIGURE 5.. MEK2 is dispensable for LPS mediated MAP kinase activation including ERK phosphorylation.

Murine BMDMs derived from WT, and Mek2^−/− mice were treated with LPS (100 ng/mL) for different time points as indicated. (A) Detection of MEK1 and MEK2 isoforms. Whole cell extracts were prepared and subjected to SDS-gel electrophoresis and Western blot analysis using antibodies against MEK1 and MEK2. Equal loading was confirmed using β-actin antibodies. As shown, MEK2^−/− BMDMs lack expression of MEK2. (B) LPS induced phosphorylation of MEK1/2 in WT and MEK2^−/− BMDMs. Murine BMDMs were challenged with LPS for different time points as indicated. Whole cell extracts were prepared and 15μg of proteins were subjected to SDS-gel electrophoresis and Western blot analysis was performed using antibodies against the phosphorylated forms of MEK1/2 (Ser217/221). Equal loading was confirmed using β-actin antibodies. MEK2^−/− BMDMs showed a lower phosphorylation of MEK1/2 which is mainly due to phosphorylation of MEK1. (C) LPS induced phosphorylation of ERK. Whole cell extracts were subjected to SDS-gel electrophoresis and Western blot analysis performed using antibodies against the phosphorylated forms of ERK (Thr202/Tyr204) and total ERK. (D) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total ERK. (E) LPS induced phosphorylation of JNK. Western blot analysis was performed with antibodies against the phosphorylated form of SAPK/JNK (Thr183/Tyr185) and equal loading was determined measuring total JNK. (F) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total JNK. (G) LPS induced phosphorylation of p38. Western blot analysis was performed with antibody against the phosphorylated form of p38 (Thr180/Tyr182) and equal loading was determined using antibody against total p38. (H) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total p38. Using ANOVA Mann-Whitney U test, a p value <0.05 was considered significant and error bars indicate SEM.

FIGURE 6.. MEK2 overexpression decreases IL-1β production with no significant effect on ERK activation.

RAW264.7 cells were transfected with a Mek2-Myc construct (CMV-MEK2) for 24h or cultured without plasmid (NT). MEK2 overexpressed or NT cells were treated with LPS (100 ng/mL) for 30 minutes or 3h. (A) Whole cell lysates were subjected to SDS-PAGE followed by Western blot analysis using antibodies against MEK2 and phospho-specific antibody against MEK1/2. Equal loading was determined using antibody against β-actin. (B) Densitometric values expressed as fold change of the ratio pMEK1/2/β-actin. (C) Western blot analysis was performed using antibodies against phospho-specific ERK1/2 and total ERK. (D) Densitometric values expressed as fold change of the ratio pERK1/2/total ERK. (E) Western blot analysis was performed using antibody against pro IL-1β, equal loading was determined using antibody against β-actin. (F) Densitometric values expressed as fold increase of the ratio pro IL-1β/β-actin. Data presented for all experiments are representative of at least 3 independent experiments. Using ANOVA Mann-Whitney U test for all results, a p value <0.05 was considered significant and error bars indicate SEM.

FIGURE 7.. MEK2 expression determines IL-1β and HIF-1α expression in response to LPS.

Murine BMDMs derived from WT, MEK2^−/−, one MEK1 left (1M1L), and one MEK2 left (1M2L) were treated with LPS (100 ng/mL) for 30 minutes. (A) Whole cell lysates were subjected to SDS-PAGE followed by Western blot analysis using specific antibody against phospho ERK. Equal loading was determined using antibody against total ERK. (B) Densitometric analysis expressed as fold increase of the ratio pERK1/2/ERK. (C) Western blot analysis was performed using antibodies against HIF-1α and against β-actin. (D) Densitometric analysis expressed as fold increase of the ratio of HIF-1α/β-actin. (E) BMDMs were isolated from WT, MEK1-deficient mice, one MEK1 left (1M1L), and one MEK2 left (1M2L) and challenged with LPS (100 ng/mL) for 24h. Conditioned media were analyzed for IL-1β via ELISA. (F) Densitometric quantifications of three immunoblots of MEK2 protein expression (x-axis) plotted against pro-IL-1β (y-axis). Data presented for all experiments are representative of at least 4 independent experiments. Using ANOVA Mann-Whitney U test, a p value <0.05 was considered significant and error bars indicate SEM.