IDENTIFICATION OF A 27-BP 5'-FLANKING REGION ELEMENT RESPONSIBLE FOR THE LOW-LEVEL CONSTITUTIVE EXPRESSION OF THE HUMAN CYTOSOLIC PHOSPHOLIPASE-A(2) GENE

The cytosolic phospholipase A(2) (cPLA(2)) gene codes for an enzyme th at liberates arachidonic acid from membrane phospholipids, and thus pl ays a pivotal role in the production of the prostaglandin and leukotri ene mediators of inflammation, as well as in a variety of cell signall ing pathways. After preliminary studies demonstrated the cPLA(2) gene is expressed in a variety of human tissues and was localized to the q arm of chromosome 1 between markers F13B and D1S74, we cloned and char acterized the 5'-flanking region of this gene in order to identify the elements controlling its low revel constitutive expression. The 5'-fl anking region has features typical of a housekeeping gene with no TATA box or CAAT box, although atypical in that it is not GC rich, has no SP1 sites, and has a long run of CA repeats. Analysis of fragments of the 5'-flanking region demonstrated that 541 bp 5' to exon 1 supported reporter gene activity at a level 30% of the SV40 promoter. Interesti ngly, similar activity was observed by deleting most of the B-flanking region down to a 27 bp region containing a sequence with homology to the initiator sequence in the terminal deoxynucleotidyl transferase ge ne and a polypyrimidine tract similar to the initiator element of the mouse ribosomal protein gene. Within this 27 bp region, a 10 bp fragme nt (-17 to -8 bp) within the polypyrimidine tract is critical for the baseline expression of the human cPLA(2) gene. While the 5'-flanking r egion contains a putative composite AP-1 and glucocorticoid response e lement, this region does not respond to tumor necrosis factor-proporti onal to (TNF) and/or glucocorticoids in a cell line (HEp-2) that exhib its upregulation of cPLA(2) mRNA transcript levels by TNF. The observa tions that the expression of the cPLA(2) gene is tightly controlled at a relatively low level is consistent with the evolving concept that m odulation of expression of this critical enzyme is primarily at the po sttranslational level.