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

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.

MKP-1 HIF-1α p300 IL-1β Bone marrow derived macrophages a b s t r a c t 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. &

Value of Data
This data shows an enhanced expression of HIF-1α in MKP-1 deficient macrophages [1]. Nuclear extracts of MKP-1 deficient macrophages exhibited higher accumulation of HIF-1α along with its transcriptional co-activator p300 [2,3] as compared to WT macrophages.
Presented data has the potential to guide future exploration of the mechanisms involved in the stabilization of HIF-1α in absence of MKP-1or through increased activation of p38 MAPK.

Data
This dataset extends the reproducibility of the recent published data [1]. BMDMs derived from WT and MKP-1 -/mice exhibit higher p38 and JNK activation in response to LPS (Fig. 1). HIF-1α expression is significantly increased at baseline and in response to LPS challenge in nuclear extracts of MKP-1 deficient BMDMs under normoxic condition as compared to WT BMDMs (Fig. 2). In nuclear extracts of MKP-1 deficient BMDMs expression of p300, a transcriptional coactivator that binds with HIF-1α at the promoter regions of targeted genes, is significantly increased at baseline and in response to LPS treatment (Fig. 2). LPS-induced HIF-1α expression is significantly inhibited by SB203850, a specific inhibitor of p38 but not by SP600125, a specific inhibitor of JNK ( Fig. 3) LPS-induced IL-1β, TNF-α, and IL-6 production in MKP-1 deficient BMDMs are significantly inhibited by SB203850, a specific inhibitor of p38 MAPK (Fig. 4).

Chemicals and antibodies
LPS was purchased from Invivogen (San Diego, CA). Phospho-specific antibodies against the phosphorylated form of ERK1/2, p38, JNK, as well as total ERK1/2, JNK, and β-actin were purchased from Cell Signaling Technology (Beverly, MA). Total p38 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The IL-1β antibody was purchased from R&D Systems (Minneapolis, MN). The HIF-1α antibody was purchased from Bioss Inc (Woburn, MA). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG secondary antibodies were purchased from Cell Signaling Technology, and horseradish peroxidase (HRP)-conjugated anti-goat antibody was purchased from Santa Cruz Biotechnology.

Mice and isolation of bone marrow derived macrophages (BMDMs)
Wild-type (WT), MKP-1 knockout mice were generated as previously described [4]. Animal studies were approved by the Institutional Committee on Animal Use and Care of the Research Institute at Nationwide Children's Hospital. BMDMs from mice were prepared as described previously [5]. Briefly, femurs and tibias from 6-to 12-week-old mice were dissected and the bone marrow was flushed out. Macrophages were cultured with IMDM media containing glutamine, sodium pyruvate, 10% heatinactivated fetal FBS, 30% L929 conditioned medium, and antibiotics for 5-7 days. BMDMs were replated at a density of 2×10 6 cells/well the day before the experiment.

Protein extraction and immunoblotting
After the appropriate treatments, cells were washed with PBS, and harvested in RIPA buffer (Millipore, Billerica, MA) containing protease inhibitor and anti-phosphatase cocktails, as previously described [6]. Equal amounts of proteins (15 μg) were mixed with the same volume of 2x sample buffer, separated on 10% SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene di-fluoride (PVDF) membrane (Bio-Rad, Hercules, CA) at 18 V for 1 h using a semi dry transfer cell (Bio-Rad) as previously described [3]. The PVDF membrane was blocked with 5% dry milk in TBST (Tris-buffered saline with 0.1% Tween-20), rinsed, and incubated with primary antibody overnight. The blots were washed and incubated with HRP-conjugated secondary anti-IgG antibody. Membranes were washed and immunoreactive bands were visualized using a chemiluminescent substrate (ECL-Plus, GE Healthcare, Pittsburgh, PA). Images were captured on Hyblot CL film (Denville Scientific Inc, Metuchen, NJ). Optical density analysis of signals was performed using ImageQuant software (version 5, GE Healthcare).

Fractionation of cytoplasmic and nuclear proteins
The cytoplasmic and nuclear fractions were separated as described previously [6]. Briefly, after treatment the cells were resuspended in a hypotonic buffer (10 mm HEPES, pH 7.9, 0.5% Igepal, 2 mm MgCl 2 , 10 mm KCl, 0.1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, 1.0 μg/ml leupeptin, and 1.0 μg/ml aprotinin) and incubated on ice for 10 min. After centrifugation at 14,000×g for 1 min at 4°C , the supernatant (cytoplasmic) and the pellets (nuclear fraction) were collected.