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)

The 85 kDa cytosolic phospholipase A 2 (cPLA 2 ) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA 2 undergoes calciumdependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA 2 activation, we constructed chimeras of cPLA 2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA 2 ) or the C-terminal targeting signal of K-Ras4B (cPLA 2 -Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the a 2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [ 3 H]arachidonic acid, indicating that constitutive association of cPLA 2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA 2 increased [ 3 H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA 2 -Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA 2 . The lack of stimulation of cPLA 2 -Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

The 85 kDa Ca2+-sensitive cytosolic phospholipase A 2 (cPLA2) specifically hydrolyzes arachidonic acid-containing phospholipids, thus initiating the biosynthesis of inflammatory mediators under the stimulation of membrane receptors. Two main mechanisms regulate cPLA 2 activation: its calcium-dependent transtocation to membrane phospholipids and its phosphorylation by several SertThr kinases, including MAPKinases. In order to investigate the respective importance of these two mechanisms in the activation of cPLA 2 by G protein-coupled receptors, we constitutively targeted cPLA 2 to plasma membrane, therefore bypassing calcium-dependent translocation.We constructed a chimeric cPLA 2 constituted by the fulllength cPLA 2 and containing at its C-terminal end the 18 last amino acids of K-Ras4B. This motif targeted cytosolic proteins to plasma membrane. Chinese Hamster ovary (CHO) cells permanently expressing ~2~-adrenergic receptor were stably transfected with the cDNA encoding for the chimeric cPLA2-Ras. Immunofluorescence of one stable transfectant, the 2B7 clone, demonstrated that cPLAz-Ras localizes to the plasma membrane. We demonstrated by gas chromatography/mass spectrometry that cPLAz-Ras is enzymatically active on endogenous membrane phosphotipids. Stimulation of the 2B7 clone with epinephrine induces a gel shift of cPLA2-Ras, resulting from its phosphorylation on Ser505 by MAPKinases. However, treatment of 2B7 clone with epinephrine, calcium ionophore A23187, or both agonists leads to an inhibition of stimulation of arachidonic acid release in comparison with parental CHO-@2B cells. In contrast with this inhibition, overexpression of wild-type cPLA 2 at the same level has no effect on arachidonic acid release induced by epinephrine and/or calcium ionophore.These results suggest that the presence of cPLAz-Ras at the plasma membrane inhibits the activation of endogenous cPLA 2 in CHO-c~2B cells.In order to verify the level of activation of cPLA2-Ras, we expressed the chimera in the baculovirus-Sf9 cell system. Sf9 cells do not contain endogenous cPLA 2 and perform the same posttranslational modifications as in mammalian cells. We constructed two recombinant viruses, allowing the expression of either wild-type cPLA 2 or cPLA2-Ras.In these cells, the expression of wild-type cPLA 2 leads to an important stimulation of arachidonic acid release from prelabeled cells in response to A23187 or to okada'l"C acid, a Ser/Thr phosphatase inhibitor. In contrast, the expression of cPLA2-Ras leads to a dramatic decrease of arachidonic acid release in response to these agonists. This lack of activation of cPLA2-Ras is not due to a lack of phosphorylation. Indeed, okada'fc acid induces the same increase of [32p] incorporation in cPLA2-Ras as compared to wild-type cPLA 2.These results indicate that, in spite of constitutive membrane localization and agonist-induced phosphorylation, the chimeric cPLA2-Ras is enzymatically active but is not activated in response to stimuli. Moreover its plasma membrane targeting inhibits endogenou...

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