MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression

Abstract

Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that plays a major role in inflammatory diseases as well as cancer. The inflammatory response after Toll-like receptor (TLR) 4 activation is tightly regulated through phosphorylation of MAP kinases, including p38 and JNK pathways. The activation of MAP kinases is negatively regulated by MAPK phosphatases (MKPs). MKP-1 preferentially dephosphorylates p38 and JNK. IL-1β is regulated through the activation of MAPK, including p38 as well as several transcription factors. The oxygen-sensitive transcription factor HIF-1α is a known transcription factor for several inflammatory cytokines including IL-1β and IL-6. Here, we report that MKP-1 regulates HIF-1α expression in response to LPS. MKP-1 deficient bone marrow derived macrophages (BMDMs) exhibited increased reactive oxygen species (ROS) production and higher HIF-1α expression. In contrast, the expression of all three isoforms of prolyl hydroxylases (PHDs), which are important in destabilizing HIF-1α through hydroxylation, were significantly decreased in MKP-1 deficient BMDMs. LPS challenge of MKP-1 deficient BMDMs led to a substantial increase in IL-1β production. An inhibitor of HIF-1α significantly decreased LPS mediated IL-1β production both at the transcript and protein levels. Similarly, inhibition of p38 MAP kinase reduced LPS mediated pro-IL-1β and HIF-1α protein levels as well as ROS production in MKP-1 deficient BMDMs. These findings demonstrate a regulatory function for MKP-1 in modulating IL-1β expression through p38 activation, ROS production and HIF-1α expression.

Keywords: BMDMs; DUSPs; HIF-1 α; IL-1β; MKP-1; Macrophages; p38.

Figure 1. MKP-1 deficient BMDMs exhibit enhanced pro-IL-1β expression in response to LPS

(A) BMDMs isolated from WT and MKP-1^−/− mice were challenge with LPS (100 ng/mL) for 3h, 6h, and 24h. Whole cell extracts were prepared and 15 μg of proteins were subjected to SDS-gel electrophoresis and Western blot analysis using antibodies against pro-IL-1β. Equal loading was con rmed using an antibody against β-actin. (B) Densitometric values expressed as fold increase of the ratio of pro-IL-1β/β-actin (n=4). (C) BMDMs derived from WT and MKP-1^−/− mice were treated with LPS for 24 h. Conditioned media were analyzed for IL-1β via ELISA. Data are presented as mean ± SEM (n=3), p< 0.001. (D) BMDMs obtained from WT and MKP-1^−/− mice were cultured under similar conditions and activated with LPS for 1h. RNA was isolated and subjected to qRT-PCR to measure IL-1β gene expression. Values were normalized to GAPDH and are shown as fold changes. Data presented as mean of at least three independent experiments. Using ANOVA Mann-Whitney U test, a p-value <0.05 was considered significant and error bars indicate SEM.

Figure 2. MKP-1^−/− BMDMs exhibit higher p38 and JNK activation in response to LPS

BMDMs from WT and MKP-1^−/− mice were cultured and challenged with LPS (100 ng/mL) for 30 min, 1h, 3h, 6h, and 24h. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using phospho-specific antibodies against p38 (Thr180/Tyr182), ERK (Thr202/Tyr204) and JNK (Thr183/Tyr185). Equal loading was con rmed using the corresponding non-phosphorylated antibodies. As shown in (A) MKP-1^−/− BMDMs exhibited higher activation of p38 after LPS stimulation as compared to WT. (B) Densitometric values expressed as fold changes of the ratio of phosphorylated p38/total values of p38. (C) Activation of JNK at baseline and after LPS stimulation in MKP-1 deficient mice. MKP-1^−/− BMDMs exhibited higher phospho-JNK at baseline with an increase in phosphorylation in response to LPS challenge. (D) Densitometric values expressed as fold increase of the ratio of phosphorylated JNK/total values of JNK. (E) Activation of ERK after LPS stimulation was similar both in WT and MKP-1^−/− BMDMs. (F) Densitometric values expressed as fold increase of the ratio of phosphorylated ERK/total values of ERK. All densitometric data represents mean+ SEM of at least 4 independent experiments. * Represents a p value < 0.05.

Figure 3. MKP-1^−/− BMDMs exhibit higher HIF-1α expression

(A) BMDMs derived from WT and MKP-1^−/− mice were challenged with LPS (100 ng/mL) for 1h. RNA was isolated and subjected to qRT-PCR to measure HIF-1α gene expression. Values were normalized to GAPDH and are shown as fold changes. Data presented as mean of at least three independent experiments. Using ANOVA Mann-Whitney U test, a p value <0.05 was considered significant and error bars indicate SEM. LPS pretreatment significantly induced HIF-1α m-RNA levels in MKP-1^−/− BMDMs as compared to WT. (B) WT and MKP-1 deficient macrophages were cultured under similar conditions and challenged with LPS for 1h, 3h, 6h, and 24h. Whole cell extracts were prepared and subjected to SDS-PAGE. Western blot analysis was performed using specific antibodies against HIF-1α, equal loading was confirmed using antibody against β-actin. (C) Densitometric values expressed as fold change of the ratio of HIF-1α/β-actin. Data represent mean + SEM of at least 4 independent experiments. * Represents a p value <0.05. (D–F) PHD 1–3 mRNA expression. Total RNA was isolated from cells and reverse-transcribed using the Reverse Transcription System. The primers targeting PHD1, PHD 2 and PHD3 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 ± SEM of at least 3 independent experiments * Represents a p value < 0.05.

Figure 4. MKP-1^−/− BMDMs exhibit higher ROS production as compared to WT BMDMs

(A&B) Basal ROS production was measured by H₂DCFDA and MitoSOX fluorescence intensity in WT and MKP-1^−/− BMDMs. ROS production is significantly higher in MKP-1^−/− macrophages compared to WT macrophages. (C&D) WT and MKP-1^−/− BMDMs were challenged with LPS (100 ng/mL) for 1 h followed by measurement of ROS production using H₂DCFDA and MitoSOX fluorescence intensity. LPS challenge induced ROS production to a similar extent in both WT and MKP-1^−/− macrophages.

Figure 5. HIF-1α inhibitor blocks LPS mediated IL-1β production in WT and MKP-1^−/− BMDMs

(A) BMDMs derived from WT and MKP-1^−/− mice were cultured in the presence or absence of Echinomycin (10 nM) and challenged with LPS (100 ng/mL) for 1h. Total RNA was isolated and subjected to qRT-PCR to measure IL-1β and HIF-1α gene expression. Relative mRNA levels were calculated by normalizing to GAPDH. Data were analyzed using the paired, two-tailed Student’s t test, and results expressed as fold change ± SEM of at least 3 independent experiments. LPS mediated IL-1β mRNA induction in MKP-1^−/− BMDMs is reduced to basal levels in the presence of echinomycin. (B) BMDMs derived from WT and MKP-1^−/− mice were cultured in the presence absence of Echinomycin (10 nM) and challenged with LPS (100 ng/mL) for 3h. Western blot analysis was performed using antibodies against pro-IL-1β and HIF-1α, and equal loading was confirmed using β-actin antibody. (C) Densitometric values expressed as fold increase of the ratio of pro-IL-1β/β-actin values. (D) Densitometry values expressed as fold increase of the ratio of HIF-1α/β-actin values. Data were analyzed using the paired, two-tailed Student’s t test, and the results were expressed as fold change ± SEM of at least 3 independent experiments and * represents a p value < 0.05.

Figure 6. Inhibition of JNK MAP kinase has no effect on LPS mediated pro-IL-1β, HIF-1α protein levels as well as ROS production in WT and MKP-1^−/− BMDMs

BMDMs derived from WT and MKP-1^−/− mice were challenged with LPS (100 ng/mL) for 3h in the absence and presence of SP600125 (20 μM) for 30 min. (A) Western blot analysis was performed using phospho-specific antibodies against JNK (Thr183/Tyr185) and equal loading was confirmed using total JNK antibody. LPS mediated JNK phosphorylation was completely blocked in the presence of SP600125 in both WT and MKP-1^−/− BMDMs. (B) Western blot analysis was performed using pro-IL-1β and HIF-1α antibodies and equal loading was confirmed with β-actin. SP600125 had no effect on LPS-induced pro-IL-1β and HIF-1α expression neither in WT nor MKP-1^−/− BMDMs (C) Densitometric values expressed as fold increase of the ratio of pro-IL-1β/β-actin. (D) Densitometric values expressed as fold increase of the ratio of and HIF-1α/β-actin values. Data were analyzed using the paired, two-tailed Student’s t test, and the results were expressed as fold change ± SEM of at least 3 independent experiments and * represents a p value < 0.05. (E&F) BMDMs derived from MKP-1^−/− mice were pretreated with SP600125 inhibitor for 30 min prior LPS challenge (100 ng/mL) for 1h followed by the measurement of ROS production using H₂DCFDA (E) and MitoSOX (F) fluorescence intensity. Results show that cytosolic and mitochondrial ROS production induced by LPS was not inhibited by SP600125 pretreatment in MKP-1^−/− BMDMs.

Figure 7. Inhibition of p38 MAP kinase effectively blocks LPS mediated pro-IL-1β, and HIF-1α protein levels as well as ROS production in WT and MKP-1 deficient BMDMs

(A) WT and MKP-1 deficient BMDMs were cultured in the presence or absence of SB203580 (10 μM, 30 min.) followed by a challenge with LPS (100 ng/mL) for 30 min. Whole cell extracts were prepared and 15 μg total proteins were subjected to SDS-PAGE and Western blot analysis using specific antibodies against phospho p38, equal loading was confirmed using an antibody against total p38. Pretreatment with SB203580 significantly decreased p38 phosphorylation in response to LPS both in WT and MKP-1^−/− BMDMs. (B) WT and MKP-1 deficient BMDMs were cultured in the presence or absence of SB203850 (10 μM, 30 min.) followed by a challenge with LPS (100 ng/mL) for 3h. Whole cell extracts were prepared and 15 μg total proteins were subjected to SDS-PAGE and Western blot analysis using specific antibodies against pro-IL-1β and HIF-1α. Equal loading was confirmed with β-actin. Pretreatment with SB203850 significantly decreased both LPS-induced pro-IL-1β and HIF-1α expression (C) Densitometric values expressed as fold increase of the ratio of pro-IL-1β/β-actin. (D) Densitometric values expressed as fold increase of the ratio of HIF-1α/β-actin. (E&F) BMDMs derived from MKP-1^−/− mice were pretreated with SB203850 inhibitor (10 μM) for 30 min prior to LPS (100 ng/mL) challenge for 1 h followed by the measurement of ROS production using (E) H₂DCFDA and (F) MitoSOX fluorescence intensity. Results show that the cytosolic and mitochondrial ROS production induced by LPS was inhibited by SB203850 pretreatment in MKP-1^−/− BMDMs (n=3).

Figure 8. Proposed model of the regulatory role of MKP-1 in LPS mediated HIF-1α and IL-1β production

Endotoxins such as LPS activate TLR 4 mediated signal transduction including phosphorylation of p38. This activation regulates ROS production and HIF-1α expression, which in turn regulates cytokine production including IL-1β. MKP-1 predominantly negatively regulates p38 phosphorylation on the TXY motifs. The lack of MKP-1 leads to an increase in p38 phosphorylation and higher mROS and cROS production. Additionally, the absence of hydroxylation due to PHD inhibition leads to increased HIF-1α stability and translocation to the nucleus through heterodimerization with HIF-1β and binds to hypoxia-response elements (HREs) in the regulatory regions of the IL-1β gene. In addition to increased HIF-1α stability, LPS increases HIF-1α mRNA levels leading to higher transcriptional activity of the HIF-1α gene. Inhibition of p38 or HIF-1α (Echinomycin) decreases IL-1β production in MKP-1 deficient BMDMs.