Prostanoid Receptors of Murine NIH 3T3 and RAW 264.7 Cells

Prostaglandin endoperoxide H synthase-1 (PGHS-1) is expressed constitutively in murine NIH 3T3 cells and RAW 264.7 cells. PGHS-2 is inducibly expressed in these cells following stimulation with serum or bacterial lipopolysaccharide (LPS), respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis established that a variety of G protein-linked and peroxisomal proliferator-activated prostanoid receptors are expressed in both of these cell types. The levels of the EP2 and EP4 prostaglandin E2 (PGE2) receptors and the prostaglandin I2 receptor were changed in these cells by serum or LPS stimulation. Quantitative RT-PCR indicated that the mRNA for the murine EP4 receptor, the butaprost-insensitive PGE2 receptor that couples to Gs, increases 1.5-3-fold in response to serum (NIH 3T3) or LPS (RAW 264.7) with a time course approximating the induction of PGHS-2 expression. To study expression of the EP4 receptor we isolated the mouse EP4 receptor gene; the gene is 10 kilobase pairs (kb) in length and, like other known prostanoid receptor genes, contains three exons and two introns. The first intron is 0.5 kb and is located 16 base pairs (bp) downstream of the translational start site. This is a different location than that of the first introns of other prostanoid receptor genes. The second intron is located immediately following the sixth transmembrane domain at the same position as the second intron of the thromboxane A2 receptor, prostaglandin D2 receptor, prostaglandin I2 receptor, and one of the PGE2 (EP1) receptor genes. A major transcriptional start was detected at −142 bp upstream of the translational start. There are a variety of putative cis-acting elements within 1.5 kb upstream of the translational start site and within the first intron. Promoter analyses of the EP4 receptor gene promoter in RAW 264.7 cells indicated that there is a constitutive negative regulatory region between −992 and −928 bp, a constitutive positive region between −928 and −554 bp, and an LPS/serum-responsive region between −554 and −116 bp.