Positive and Negative Hepatic Regulation of the Human Type II Phospholipase A2 Gene

Abstract

To identify the elements which regulate the liver transcription of the human type II phospholipase A2 gene and its stimulation by interleukin 6, the 5' flanking region from -1614 to +806 and several 3' and 5' deleted fragments have been analyzed in CAT assays. Negative regulatory 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. Footprinting assays have been performed on this region and showed four protected elements, A [-35;-6], B [-125;-86], C [-209;-176], and D [-247;-211]. Deletion of element D enhanced the transcription of the reporter gene 10.5-fold compared to the [-326;+20]-CAT construct. Further deletions up to position -87 which removed both the elements B and C or the substitution of element C by a nonspecific sequence lowered the promoter activity to 23% and 70% of the control, respectively. These results indicate that element C binds positive regulatory factors and element D binds a negative regulatory factor. Furthermore, stimulation by interleukin 6 is lost when element C is substituted or deleted. As shown by the footprinting and band shift assays, the transcription factors C/EBP alpha and C/EBP beta can bind to elements C and D but the dissociation constant (Kd) 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 rat liver nuclear extracts showed that element C formed four heat stable complexes, some of which could be supershifted by anti C/EBP alpha antibodies. The binding of C/EBP factors to element C was confirmed by competition with previously described oligonucleotide and nucleotide substitution of element C. Band shift experiments using rat liver nuclear 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 the binding site of C/EBP. However, anti-CREB antibodies did not supershift this complex. Methylation interference experiments showed the involvement of a G nucleotide upstream to the sequence homologous to CRE in the binding of the hepatic nuclear factors.(ABSTRACT TRUNCATED AT 400 WORDS)

Posttranscriptional effect of insulin-like growth factor-I on interleukin-1beta-induced type II-secreted phospholipase A2 gene expression in rabbit articular chondrocytes.

“…The transcriptional regulatory activity of IL-1␤ has been partly linked to activation of nuclear factor-B/I-B (50) and activator protein-1 (51). However, responsive elements for nuclear factor-B or activator protein-1 action were not found in the Ϫ1614/ϩ806 part of the human type II sPLA 2 promoter (52). At least part of the effect of IL-1␤ on type II sPLA 2 gene expression is probably indirect via the induction of other regulatory genes.…”

Suppressive effect of ursodeoxycholic acid on type IIA phospholipase A2 expression in HepG2 cells†‡

“…[-326; ϩ20] resulted in greater transcriptional activity than that of the whole region. 29 We tested the effect of UDCA on the binding ability of the proteins to these different promoter element sequences by electrophoretic mobility shift assays ( Fig. 5C.…”

“…In addition, previous reports have supported GR activation by UDCA in a GR ligand independent manner. 29,30 Therefore, the role of GR in the PLA 2 IIA inhibition by UDCA was investigated. One hundred nanomolar DEX showed a maximum suppressive effect on the PLA 2 IIA concentration (Fig.…”

Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.

“…The 6.2-kb HindIII fragment also contains 1.6 kb upstream and 0.35 kb downstream of the PLA 2 gene. A number of transcriptional response element consensus sequences have been identified within the 1.6 kb upstream region including response elements for IL-6 (54), interferon, hepatocyte NF-3 (55), AP1 (56), AP2 (56), C/EBP (56), and CRE (56). A CCAAT box and TATA box sequence are also present immediately upstream of the proposed RNA start site.…”