POSITIVE AND NEGATIVE HEPATIC REGULATION OF THE HUMAN TYPE-II PHOSPHOLIPASE A(2) GENE

To identify the elements which regulate the liver transcription of the human type II phospholipase A(2) gene and its stimulation by interleu kin 6, the 5' flanking region from -1614 to +806 and several 3' and 5' deleted fragments have been analyzed in CAT assays. Negative regulato ry elements have been located in the regions -1614 to -326 and +20 to +806. The fragment -326 to +20 contains the main elements required for the transcription as well as for the stimulation by interleukin 6. Fo otprinting assays have been performed on this region and showed four p rotected elements, A [-35;-6], B [-125;-86], C [-209;-176], and D [-24 7;-211]. Deletion of element D enhanced the transcription of the repor ter gene 10.5-fold compared to the [-326;+20]-CAT construct. Further d eletions up to position -87 which removed both the elements B and Cor the substitution of element C by a nonspecific sequence lowered the pr omoter activity to 23% and 70% of the control, respectively. These res ults indicate that element C binds positive regulatory factors and ele ment D binds a negative regulatory factor. Furthermore, stimulation by interleukin 6 is lost when element C is substituted or deleted. As sh own by the footprinting and band shift assays, the transcription facto rs C/EBP alpha and C/EBP beta can bind to elements C and D but the dis sociation constant (K-d) of C/EBP alpha is 10 times lower for element C (0.6 nM) than for element D (5.8 nM). Band shift experiments using r at liver nuclear extracts showed that element C formed four heat stabl e complexes, some of which could be supershifted by anti C/EBP alpha a ntibodies. The binding of C/EBP factors to element C was confirmed by competition with previously described oligonucleotide and nucleotide s ubstitution of element C. Band shift experiments using rat liver nucle ar extracts showed that element D formed one major DNA-protein complex . This complex could be competed out by oligonucleotides containing a cAMP responsive element (CRE) but not by oligonucleotides containing t he binding site of C/EBP. However, anti-CREB antibodies did not supers hift this complex. Methylation interference experiments showed the inv olvement of a G nucleotide upstream to the sequence homologous to CRE in the binding of the hepatic nuclear factors. Further studies are req uired to determine the nature of the different factors which bind to e lement D and generated the major and minor complexes observed in band shift experiments as well as their regulatory role in the transcriptio n of PLA(2) gene and their putative interaction with C/EBP factors bou nd to element C.