The dataset describes: HIF-1 α expression and LPS mediated cytokine production in MKP-1 deficient bone marrow derived murine macrophages

Abstract

The data presented in this article are related to the research article entitled "MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression" (Talwar et al., 2017) [1]. This data describes that LPS-mediated p38 and JNK phosphorylation is enhanced in MKP-1 deficient macrophages. HIF-1α expression and its nuclear accumulation are significantly increased in the nuclear extracts of MKP-1 deficient BMDMs. MKP-1 deficient BMDMs exhibited higher expression of the coactivator p300 of HIF-1α both at baseline and after LPS challenge. Inhibition of p38 MAP kinase decreased LPS mediated HIF-1α protein levels and its nuclear translocation in MKP-1 deficient BMDMs. Inhibition of p38 MAP kinase inhibited LPS induced pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α.

Keywords: Bone marrow derived macrophages; HIF-1α; IL-1β; MKP-1; p300.

Fig. 1

MKP-1^-/- BMDMs exhibit higher p38 and JNK activation in response to LPS. BMDMs derived from WT and MKP-1^-/- mice were cultured and challenged with LPS (100 ng/mL) for 30 min. 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 confirmed using the corresponding non-phosphorylated antibodies. (A) MKP-1^-/- BMDMs exhibited higher activation of pp38 by LPS as compared to WT. (B) Densitometric values expressed as fold changes of the ratio of phosphorylated p38/total p38. (C) 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 (54 kDa and 46 kDa)/total JNK. (E) Phosphorylation of ERK after LPS stimulation in WT and MKP-1^-/- BMDMs. (F) Densitometric values expressed as fold increase of the ratio of phosphorylated ERK/total ERK. All densitometric data represent mean±SEM of at least 4 independent experiments. * Represents a p value<0.05.

Fig. 2

MKP-1^-/- BMDMs exhibit higher HIF-1α and p300 expression in nuclear extracts: WT and MKP-1 deficient macrophages were cultured and challenged with LPS (100 ng/mL) for 3 h. Nuclear extracts were prepared and subjected to SDS-PAGE. Western blot analysis was performed using specific antibodies against HIF-1α (A) and p300 (C). Equal loading was confirmed using antibody against proliferating cell nuclear antigen (PCNA). Densitometric values expressed as fold change of the ratio of HIF-1α /PCNA (B) and p300/PCNA (D). Data represent mean±SEM of at least 3 independent experiments. * Represents a p value<0.05. MKP-1 deficient macrophages show an increased translocation of HIF-1α and p300 to the nucleus as compared to WT.

Fig. 3

SB203850 inhibits LPS-induced HIF-1α nuclear accumulation: (A) WT and MKP-1 deficient macrophages were cultured and challenged with LPS (100 ng/mL) for 3 h in the presence and absence of SB 203850 (10 µM), a specific inhibitor of p38 and SP 600125 (20 µM), a specific inhibitor of JNK. Nuclear extracts were prepared and subjected to SDS-PAGE. Western blot analysis was performed using specific antibodies against HIF-1α. Equal loading was confirmed with PCNA. MKP-1 deficient macrophages show a decreased translocation of HIF-1α to the nucleus after specific inhibition of p38 but not JNK. (B) Densitometric values expressed as fold change of the ratio of HIF-1α/PCNA. Data represents mean±SEM of at least 3 experiments. * Represents a p value<0.05.

Fig. 4

Inhibition of p38 MAP kinase blocks LPS induced IL-1β, IL-6, and TNF-α production in WT and MKP-1 deficient BMDMs: BMDMs derived from WT and MKP-1^-/- mice were treated with LPS (100 ng/mL) for 24 h in the presence and absence of SB 203850 (specific inhibitor of p38) and SP 600125 (specific inhibitor of JNK). Conditioned media were analyzed via ELISA for IL-1β (1:1 sample dilution) (A), IL-6 (1:40 sample dilution) (B), and TNF-α (1:20 sample dilution) (C). Data are presented as mean±SEM (n=3) p<0.05. Production of IL-1β, IL-6 and TNF-α were decreased by inhibition of p38 but not JNK.