Characterization of a novel prostaglandin endoperoxide H synthase-1 transcript and examination of prostaglandin endoperoxide H synthase-1 expression.

Abstract

The prostaglandin endoperoxide H synthase (PGHS) enzyme plays a pivotal role in the prostanoid biosynthetic pathway because it catalyzes the formation of prostaglandin H2 (PGH2), the common precursor for prostanoids. The object of my research is to characterize a novel PGHS transcript and to examine the mechanisms that regulate PGHS-1 gene expression. By Northern blotting with the entire hPGHS-1 coding region, 2.8 kb and 5.1 kb transcripts as well as a novel 4.5 kb transcript were detected in the human megakaryoblastic cell line, MEG-01. A non-canonical polyadenylation signal (AAGAAA) and a cleavage site (CA) were found and could generate the 4.5 kb PGHS-1 transcript. A commercial RNA dot blot with 50 different human tissues, showed a strong signal for the 4.5 kb PGHS-1 transcript in the bladder and appendix. Subsequent hybridization of a multiple tissue Northern blot detected 2.8, 4.5 and 5.1 kb transcripts in the bladder, indicating the 4.5 kb PGHS-1 transcript is not an artifact of the MEG-01 cell line. Treatment of MEG-01 cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) elevated PGHS-1 gene expression. The pattern of PGHS-1 mRNA and protein expression in MEG-01 cells after TPA treatment did not match. From our results, increased mRNA stability could not be determined because control and TPA treated PGHS-1 mRNA levels remained elevated for at least 8 hrs after treatment with inhibitors of transcription (Actinomycin-D and 5,6-Dichlorobenzimidazole Riboside). The effect of TPA of treatment on PGHS-1 mRNA levels disappeared after treatment with the protein synthesis inhibitor cycloheximide, indicating the increases in PGHS-1 mRNA levels involves de novo protein synthesis. (Abstract shortened by UMI.)