Abstract
Inducible expression of group IIA secretory phospholipase A2 (sPLA2-IIA) by interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNFalpha) is under the control of group IVA cytosolic PLA2alpha and 12/15-lipoxygenase (12/15-LOX) in rat fibroblastic 3Y1 cells. We show here that this cytokine induction of sPLA2-IIA mRNA requires de novo protein synthesis. By means of cDNA array analysis, we found that the level of the CXC chemokine MIP-2 (macrophage inflammatory protein-2) was significantly elevated in 12/15-LOX-transfected cells compared with control cells. IL-1beta/TNFalpha-stimulated induction of endogenous MIP-2 preceded that of sPLA2-IIA, and exogenous MIP-2 induced sPLA2-IIA dose-dependently. Moreover, a MIP-2-specific antisense oligonucleotide and small interfering RNA attenuated the IL-1beta/TNFalpha-induced expression of sPLA2-IIA, suggesting that MIP-2 is an absolute intermediate requirement for optimal induction of sPLA2-IIA. In addition, the expression of c-jun and fra-1, which are components of the transcription factor AP-1, was elevated in 12/15-LOX-transfected cells, in which cytokine-dependent binding of AP-1 to the sPLA2-IIA promoter was increased significantly. Conversely, the receptors for transforming growth factor-beta and platelet-derived growth factor, which contributed to down-regulation of sPLA2-IIA expression, were decreased following 12/15-LOX overexpression. Taken together, 12/15-LOX-dependent up-regulation of sPLA2-IIA expression may result from the interplay between accelerated MIP-2 signaling, AP-1 activation, and attenuated transforming growth factor-beta and platelet-derived growth factor signaling.
Highlights
SPLA2-IIA mRNA Expression in IL-1/TNF␣-treated 3Y1 Cells Requires de Novo Protein Synthesis—We have shown previously that delayed PGE2 generation induced by IL-1 and TNF␣ in 3Y1 cells is regulated by functional coupling between two inducible enzymes, secretory PLA2s (sPLA2)-IIA and COX-2, and that the induction of sPLA2-IIA, but not COX-2, is suppressed by cPLA2 and LOX inhibitors at the transcriptional level [20]
In addition to these observations, we found that de novo protein synthesis was required for full induction of sPLA2-IIA, but not COX-2, mRNA, because adding CHX, a protein synthesis inhibitor, to 3Y1 cells greatly decreased the amount of IL-1/TNF␣-induced sPLA2-IIA mRNA in mock- and 12/15-LOX-transfected cells (Fig. 1, lanes 3 and 6)
We hypothesized that stimulation by IL-1/TNF␣ induces certain regulatory factor(s), which in turn amplify the expression of sPLA2-IIA in a manner dependent upon the product(s) of the cPLA2-12/15LOX pathway
Summary
Materials—Mouse IL-1, human TNF␣, and human PDGF-BB were purchased from Genzyme. Nordihydroguaiaretic acid (NDGA), a LOX inhibitor, was purchased from BioMol. Transfection of Antisense Oligonucleotide for MIP-2—The antisense (5Ј-TGG CGA GTG GGA GGG GCC AT-3Ј) and sense (5Ј-ATG GCC CTC CCA CTC GCC CA-3Ј) oligonucleotides, which correspond to the translation initiation site for rat MIP-2, were each incubated at 200 nM with Oligofectamine reagent in 200 l of Opti-MEM for 15 min at room temperature and added to cells that had attained 60 – 80% confluence in 6-well plates and had been supplemented with 800 l of OptiMEM. Establishment of cPLA2␣-(1–522)-expressing Cells—The cDNA for cPLA2␣-(1–522), which had been subcloned into pcDNA3.1/Hygro, was transfected into 3Y1 cells with Lipofectamine 2000 according to the manufacturer’s instructions. A 50-fold excess of unlabeled oligonucleotide was added to the binding reaction prior to the addition of the radiolabeled probe These incubation mixtures were electrophoresed in 4.5% native polyacrylamide gels with Tris borate-EDTA buffer, pH 8.8. The gels were dried and analyzed with a BAS2000 (Fuji Photo Film)
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