Cyclooxygenases (COXs) catalyze the rate-limiting step in the synthesis of prostaglandins, prostacyclin, and thromboxanes. Derived from arachidonate, these biomediators act as local hormones affecting inflammation, pain, and a host of other physiological processes in the cell and organism. There are presently two known COXs. COX-1 is typically expressed constitutively like a housekeeping protein. COX-2, in contrast, is highly inducible by growth factors and hormones. Many cells contain both COX-1 and COX-2. In these cells arachidonate is delivered selectively by an unknown mechanism to COX-2 following mitogen stimulation. Also, COX-1 and COX-2 can have very different sensitivities to NSAIDs in vivo than in their purified states in vitro. These data suggest that, in spite of the colocalization of COX-1 and COX-2 to the lumen of the endoplasmic reticulum and nuclear envelope, unknown factors confer unique, isoenzyme-specific properties on these structurally and enzymatically similar proteins. While in search of proteins which might confer these properties, our laboratory determined that the calcium binding protein, nucleobindin (Nuc), associates with cyclooxygenases in the yeast 2-hybrid system and in other in vitro and in vivo assays. Extracellular release of Nuc into the blood stream has been associated with systemic lupus erythematosus-like symptoms in mice. Indeed, Nuc was first isolated as a protein responsible for the generation of anti-DNA antibodies in a genetic mouse model for lupus. Moreover, injection of purified Nuc into normal mice elicits some of the symptoms of lupus. Similar to COXs, Nuc is widely expressed in tissues; however, its function in these tissues is unknown. Our hypothesis is that Nuc functions in COX/Nuc complexes to regulate prostaglandin synthesis and that COXs may function in the release of Nuc in autoimmune disorders. In these proposed studies, Nuc and COX isoenzymes will be co-expressed at high levels in insect cells and the enzymatic and physical properties of COX/Nuc complexes relative to unbound COX- 1 and COX-2 will be studied. Pharmacologically important aspects to be analyzed include the effect of Nuc on the rate of prostaglandin synthesis and on the inhibitory action of non-steroidal antiinflammatory drugs (NSAIDs). Additionally, a new variant of Nuc expressed in fibroblasts that lacks 33 amino acids in its COX binding domain will be examined.
