Figure 8. IL-1β regulates COX-2 mRNA decay through the ARE-binding protein HuR.

(A) LUC/3'UTR-reporter constructs containing no 3'UTR (LUCΔ3'UTR, white bars) or COX-2 3'UTR (LUC+3'UTR, black bars) were transfected into U937 cells. LUC activity (RLU) was normalized to total protein and is the average of 3 experiments. (B) U937 cells were transfected with LUC/3'UTR reporter constructs and kept in suspension medium (co), incubated on immobilized P-selectin, kept in suspension in medium containing 10 ng/ml IL-1β, or incubated on immobilized P-selectin in medium containing IL-1β. Relative LUC activity was normalized to total protein, and values shown are based on LUC expression from control transfections. (C) U937 cells (10⁶ cells) were left untreated (co) or incubated for 18 hours with 10 ng/ml IL-1β. ARE-binding proteins HuR and TIA-1 were examined in 15 μg of total cell lysates and 25 μg of cytoplasmic lysates by immunoblot. β-actin was detected as a loading control. (D) Cytoplasmic lysates from untreated (co) or IL-1β–treated U937 cells were incubated with ³²P-labeled AREs based on COX-2 or GM-CSF mRNA sequences. Bound proteins were cross-linked to the ARE and immunoprecipitated, using anti-HuR Ab or control IgG. The arrowhead indicates the major immunoprecipitated species. (E) Platelets were incubated with monocytes for 1 hour and 18 hours in the presence of thrombin, then examined for HuR localization by immunocytochemical analysis. Immunofluorescence of HuR in platelet-monocyte aggregates is shown in green; platelet and monocyte cytoskeleton are shown in red. Thick arrows indicate monocyte nuclei; thin arrow indicates monocyte cytoplasm. Scale bar: 5 μm.