15-PGDH-derived 15-keto-PGE₂ from hepatocytes inhibits Kupffer cell activation via PPAR-γ.

<p>(A) PGE₂ metabolites concentration in CM of hepatocyte as measured by the Prostaglandin E Metabolite assay. We observed that 15-PGDH overexpression enhanced PGE₂ metabolite production and this effect was more apparent in the presence of arachidonic acid substrate (1μM); this effect was blocked by treatment with the 15-PGDH inhibitor (1μM). (B) mRNA levels of pro-inflammatory cytokines (IL1β, IL6, TNF-α, MCP1 and CXCL2) in WT Kupffer cells treated with DMSO/15-keto-PGE₂ (10μM) followed by LPS. The results are shown as fold changes compared to Kupffer cells treated with DMSO. (C) mRNA levels of PPAR-γ downstream genes (CD36, ADRP, CPT1α and ABCG1) in WT Kupffer cells treated with CM of hepatocytes (upper panel) or DMSO/15-keto-PGE₂ (10μM) (lower panel). The data are shown as fold changes compared to Kupffer cells treated CM of wild type hepatocytes (upper panel) or Kupffer cells treated with DMSO (lower panel). (D) DNA binding ability of PPAR-γ from mouse macrophages (RAW264.7) treated with CM of hepatocytes (upper panel) or DMSO/15-keto-PGE₂ (10μM) (lower panel), as determined by EMSA assay using PPRE dsDNA probe. (E) mRNA levels of pro-inflammatory cytokines (IL1β, IL6, TNF-α, MCP1 and CXCL2) in WT Kupffer cells treated with CM of hepatocytes (upper panel) or DMSO/15-keto-PGE₂ (10μM) (lower panel) (with or without GW9662 [10μM] or LPS treatment). For the upper panel, the results are shown as fold changes compared to Kupffer cells treated with CM of 15-PGDH Tg hepatocytes. For the lower panel, the results are shown as fold changes compared to Kupffer cells treated with GW9662. The quantitative data in this figure were obtained from three independent experiments and are expressed as means ± SE (*p<0.05, **p<0.01, ***p<0.001; N.S—no statistical significance).