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Effects of dexamethasone and transforming growth factor-beta 2 on group II phospholipase A2 mRNA and activity levels in interleukin 1 beta- and forskolin-stimulated mesangial cells.

ABSTRACT: The expression of 14 kDa group II phospholipase A2 [also referred to as secretory PLA2 (sPLA2)] is induced in rat glomerular mesangial cells by exposure to inflammatory cytokines and forskolin, a cAMP elevating agent. Previously we have shown that dexamethasone and transforming growth factor-beta 2 (TGF-beta 2) suppress sPLA2 protein synthesis and enzyme activity induced by cytokines and forskolin. The regulation of sPLA2 by pro-inflammatory cytokines suggests that the enzyme may play a role in glomerular inflammatory reactions. In order to understand the regulation of sPLA, in more detail, we investigated whether dexamethasone and TGF-beta 2 also suppress sPLA, mRNA after its induction by either interleukin-1 beta (IL-1 beta) or forskolin. We found that IL-1 beta-induced sPLA2 mRNA in rat mesangial cells is not down-regulated by pretreatment of the cells with dexamethasone, even at a concentration of 10 microM, which dramatically decreases sPLA2 protein levels and activity. Metabolic labelling experiments indicated that the decreased sPLA2 levels under these conditions can be explained by inhibition of the rate of sPLA2 synthesis from the elevated mRNA levels. In contrast, the forskolin-induced elevation of sPLA, mRNA is inhibited by dexamethasone in a concentration-dependent manner. Likewise, TGF-beta 2 inhibits the elevation of sPLA, mRNAs induced by either IL-1 beta or forskolin. The decrease in sPLA2 mRNA caused by TGF-beta 2 corresponds with the decrease in sPLA2 enzyme levels and activity. These data suggest that cytokine- and forskolin-induced sPLA2, expression is tightly controlled via both transcriptional and post-transcriptional mechanisms. Furthermore, we show that pretreatment of mesangial cells with epidermal growth factor prior to stimulation with IL-1 beta or forskolin had no suppressing effect on sPLA2 levels or enzyme activity, as has been reported previously for osteoblasts.

SUBMITTER: Vervoordeldonk MJ

PROVIDER: S-EPMC1217214 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Project description:We have previously shown that recombinant interleukin 1 (IL-1) and recombinant tumour necrosis factor (TNF) synergistically stimulate phospholipase A2 release from mesangial cells. We now report that treatment of mesangial cells with the beta-agonist salbutamol, prostaglandin E2 (PGE2), cholera toxin or forskolin, which all activate adenylate cyclase, increased release of phospholipase A2 activity. Likewise, addition of a membrane-permeant cyclic AMP (cAMP) analogue or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine enhanced release of phospholipase A2 activity from mesangial cells. There was a lag period of about 8 h before a significantly enhanced secretion could be detected. Furthermore, actinomycin D or cycloheximide completely suppressed cAMP-stimulated secretion of phospholipase A2. Angiotensin II, the phorbol ester phorbol 12-myristate 13-acetate, the Ca2+ ionophore A23187 and a membrane-permeant cGMP analogue did not stimulate phospholipase A2 release from the cells. Treatment with indomethacin completely inhibited IL-1 beta- and TNF-stimulated PGE2 synthesis, without having any effect on phospholipase A2 secretion, thus excluding cytokine-induced PGE2 synthesis as the mediator of phospholipase A2 release. Neither IL-1 beta nor TNF induced any increase in intracellular cAMP in mesangial cells. Furthermore, incubation of the cells with 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not block cytokine-stimulated phospholipase A2 secretion. In addition, IL-1 beta and TNF synergistically interacted with forskolin to stimulate phospholipase A2 release from the cells. The protein kinase inhibitors H-8, staurosporine, K252a and amiloride inhibited IL-1 beta- and TNF-stimulated phospholipase A2 secretion. However, high concentrations that inhibit other protein kinases were needed. These observations suggest that IL-1 beta and TNF cause secretion of phospholipase A2 by a mechanism independent of cAMP. The signalling pathways used by IL-1 beta and TNF may involve a protein kinase that is probably different from protein kinase A or protein kinase C.

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Effects of dexamethasone and transforming growth factor-beta 2 on group II phospholipase A2 mRNA and activity levels in interleukin 1 beta- and forskolin-stimulated mesangial cells.

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