REGULATION OF THE PGH SYNTHASE GENES

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Prostaglandin endoperoxide H(PGH) synthase (cyclooxygenase) is a central enzyme in the prostaglandin biosynthetic pathway and also the primary site of action of aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs). Mammalian cells contain two related, but unique, forms of PGH synthase, referred to as PGH synthase-1 (PGHS-1) and PGH synthase-2 (PGHS02). These two enzymes appear to have specialized functions; PGHS-1 produces prostaglandins that act extracellularly s local hormones to coordinate short-term cellular responses to hormonal stimulation; while PGHS-2 produces prostaglandins that coordinate prolonged physiological events such as inflammation and ovulation. PGHS-2 produces prostaglandins that are directed into the nucleus where, we would predict, they also help regulate mitogenesis. The primary hypothesis that we propose to test in this application is that PGHS-1 and pGHS-2 form two separate prostaglandin biosynthetic pathways. Immunocytochemical localization by us indicates that PGHS-1 and PGHS-2 are located on different cellular membranes and produce prostaglandins that are shunted to different intracellular spaces. PGHS-1 is located predominantly in the endoplasmic reticulum and produces prostaglandins that are released into the cytoplasm where they presumably exit the cell to signal via extracellular receptors. PGHS-2 is located predominately on the nuclear membrane and releases prostaglandin on the nucleus and cytoplasm. How prostaglandin might signal in the nucleus is not known. Recently reported experiments indicate the PGHS-1 and PGHS-2 use separate arachidonate acid pools. To establish model systems with which to study the biochemical basis for segregation of the two prostaglandin synthase pathways, we are proposing to construct a set of 10 stably transfected cells which can inducibly express native and variously modified PGHS-1 and PGHS-2 proteins. These cell lines will be used to study the structure-function relationships that determine substate coupling, product channeling, and intracellular localization of PGHS-1 and PGHS-1 and PGHS-2 (Specific Aims 1-2), and also to examine the protein and mRNA sequences responsible for post-transcriptional regulation of PGHS-2 mRNA and protein stability (Specific Aims 3-4). If separate systems for prostaglandin synthesis do exist, with different mechanisms for arachidonate release and different effector pathways, these pathways may provide additional sites for therapeutic intervention of inflammation. in addition, a more complete understanding of the cellular roles for prostaglandin synthesis by PGHS-1 and PGHS-2 may help us to better understand the protective effects of NSAIDs on certain cancers.