Margriet J. B. M. Vervoordeldonk

Cytokine-and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.

Interleukin‐1β‐induced cytosolic phospholipase A2 activity and protein synthesis is blocked by dexamethasone in rat mesangial cells

Interleukin‐1β induces gene expression and secretion of the secretory phospholipase A2 (sPLA2) and prostaglandin E2 (PGE2) release from rat mesangial cells. We have previousy shown that prolonged treatment of rat mesangial cells with interleukin‐1β (IL‐1β) also enhances the cytosolic phospholipase A; (cPLA2) activity. This effect of IL‐1β on the cPLA2 activity is inhibited by actinomycin D and cycloheximide, indicating that both transcription and translation are involved. Here, we describe that IL‐1β increases mRNA levels and protein synthesis of cPLA2 itself. In parallel with the effect of dexamethasone on the sPLA2, this glucocorticoid inhibits the IL‐1β‐enhanced cPLA2 activity as a result of the suppression of IL‐1β‐induced cPLA2 gene expression. This report suggests that the pro‐inflammatory action of interleukin‐1β may, in part, be mediated by its effects on cPLA2 activity.

Aspirin inhibits expression of the interleukin-1β-inducible group II phospholipase A2

Nonsteroidal anti‐inflammatory drugs (NSAIDs) clearly inhibit the synthesis and release of prostaglandins. However, these actions are not sufficient to explain all the anti‐inflammatory effects of these drugs. Recently, it has been shown that aspirin and sodium salicylate inhibit the activation of the transcription factor NF‐κB. Group II phospholipase A2 (sPLA2) is expressed in rat glomerular mesangial cells upon exposure to the inflammatory cytokine interleukin‐1β (IL‐1β) and this induction is attenuated by the NF‐κB inhibitor pyrrolidine dithiocarbamate (PDTC). We now report that aspirin inhibits the IL‐1β‐induced sPLA2 activity in rat mesangial cells in a dose‐dependent manner. The IC50 value of aspirin for sPLA2 inhibition was 6.5 mM. This decrease in sPLA2 activity was not due to direct inhibition of enzymatic activity but rather to the fact that aspirin inhibits the expression of IL‐1β‐induced sPLA2 protein and mRNA. Furthermore, by electrophoretic mobility shift analysis we demonstrate reduced DNA binding of the nuclear factor κB, an essential component of the IL‐1β‐dependent upregulation of sPLA2 gene transcription, after treatment of the cells with aspirin. The study described in this report indicates that the inhibition of sPLA2 expression as induced by pro‐inflammatory cytokines potentially represents an additional mechanism of action for aspirin.

Levels and localization of group II phospholipase A2 and annexin I in interleukin- and dexamethasone-treated rat mesangial cells: evidence against annexin mediation of the dexamethasone-induced inhibition of group II phospholipases A2

The mechanism by which glucocorticosteroids inhibit the synthesis and secretion of pro-inflammatory arachidonate metabolites is still controversial. Initially it was postulated that glucocorticoids can induce the formation of PLA2 inhibitory proteins termed annexins. We have previously shown that the cytokine-induced 14 kDa PLA2 activity and the synthesis of prostaglandin E2 in rat mesangial cells is dose-dependently blocked by pretreatment of the cells with dexamethasone (Schalkwijk et al. (1991) Biochem. Biophys. Res. Commun. 180, 46-52). Concurrently, the synthesis of 14 kDa group II PLA2 is suppressed. The regulation of PLA2 activity is complex and may well involve superimposable mechanisms. Thus, although the decrease in PLA2 protein levels could in itself explain the dexamethasone-induced decrease in PLA2 activity, a contribution of the glucocorticoid-induced anti-phospholipase A2 protein annexin cannot be ruled out a priori. To investigate this possibility we analyzed the level of annexin I by Western blotting and immunostaining in mesangial cells treated with interleukin-1 beta and/or dexamethasone. Under conditions where 14 kDa group II PLA2 activity and protein levels were dramatically affected by interleukin-1 and dexamethasone, the level of annexin I in the cells remained constant. Dexamethasone also did not induce the secretion of annexin I. In addition, no evidence for dexamethasone-induced translocation of annexin I from the cytosol to membranes, thereby possibly sequestering the substrates for PLA2, was obtained. Immunofluorescence studies localized the cytokine-induced PLA2 to the Golgi area and punctate structures in the cytoplasm. We have also studied the subcellular localization of annexin I in rat mesangial cells using confocal microscopy. These studies located annexin I mainly in the cytoplasma and the nucleus. We conclude from these experiments that the dexamethasone-induced inhibition of 14 kDa group II PLA2 in rat mesangial cells is not mediated by annexin I and is solely due to the suppression of PLA2 gene expression.

Prevention of the induced synthesis and secretion of group II phospholipase A2 by brefeldin A.

Brefeldin A (BFA) has previously been shown to block protein secretion and to cause dismantling of the Golgi cisternae in many cultured cell lines. BFA was found to prevent the induced synthesis and secretion of 14 kDa group II phospholipase A2 (PLA2) in rat mesangial cells. Furthermore, BFA inhibited total protein synthesis although PLA2 appeared to be more sensitive to the effect of this compound than total protein synthesis assessed by amino acid incorporation. BFA was unable to block protein synthesis or PLA2 activity in the cell completely but secretion of enzymatic activity and PLA2 protein into the cell culture media was totally inhibited.

Half-life of interleukin-1β-induced group II phospholipase A2 in rat mesangial cells

Group II phospholipase A2 (sPLA2) has been implicated as an important agent involved in a number of inflammatory processes. Potent pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor (TNF) have been found to induce sPLA2 synthesis and release from many cell types among which mesangial cells. Although considerable research has been devoted to unravelling the mechanisms underlying the induction of sPLA2 not much is known about the time scale at which the cytokine elicited signals for sPLA2 induction persist in target cells. In this study we addressed that question by using rat renal mesangial cells as a model target cell. We found that after removal of IL-1 beta from the culture medium, the induced-sPLA2 synthesis continues at gradually decreasing rates for approximately 8 h. This is accompanied by a decrease in sPLA2 mRNA levels. Furthermore, with pulse-chase experiments we investigated the half-life of sPLA2 disappearance from the cells. This disappearance was found to be biphasic. A rapidly disappearing pool, constituting approx. 74% of the total, exhibited a half-life of 1.6 +/- 0.2 h. The remaining pool of the induced enzyme was much more stable and its level remained constant for at least 24 h. Analysis of the appearance of newly synthesized enzyme in the culture medium indicated this process to be completed in an hour.

Regulation of 14 kDa Group II PLA2 in Rat Mesangial Cells

Phospholipase A2 (PLA2) is believed to play an essential role in inflammation through the release of arachidonic acid from membrane phospholipids for the production of important lipid mediators such as eicosanoids and platelet activating factor (Van den Bosch, 1980; Glaser et al, 1993). The last years it has become clear that PLA2S are a heterogeneous family of enzymes that can be classified in two classes based on their molecular weight. There is a class of low molecular weight PLA2S (14 kDa) and one of the more recently discovered high molecular mass enzymes (85 kDa). The high molecular weight PLA2, also referred to as cPLA2, is mainly located in the cytosolic fraction of cells and tissues including human platelets (Takayamo et al, 1991), rat renal mesangial cells (Gronich et al, 1988; Bonventre et al, 1990), and the human monoblast U937 cell line (Clark et al., 1990; Kramer et al, 1991). Although the enzyme has been shown to be 85 kDa by sequence and cloning (Clark et al, 1991; Sharp et al, 1991), it shows a molecular weight on SDS-PAGE of about Mr 110,000 (Leslie et al, 1988; Clark et al, 1990; Kramer et al, 1991). This enzyme preferentially hydrolyzes arachidonic acid from the sn-2-position of phospholipids (Clark et al, 1990), is insensitive for dithiotreitol and has optimal activity at micromolar Ca2+-concentrations (Gronich et al, 1990). cPLA2 also comprises multiple phosphorylation sites, among which a MAP-kinase phosphorylation site (serine-505) that appears to play an important role in enzyme activation (Liscovitch and Cantley, 1994). The 14 kDa PLA2S can be further divided in two groups, based on their amino acid sequence (Heinrikson et al, 1977). Mammalian group I PLA2 comprises the pancreatic type of PLA2 and is characterized by the presence of cys 11. Homologous non-pancreatic group II phospholipase A2 is lacking cys 11. Type II PLA2 is often found as a membrane-bound, enzyme (Aarsman et al, 1989; Ono et al., 1988) which has an optimal activity at millimolar Ca2+-concentrations (Mizushima et al., 1989) and has no selectivity for arachidonic acid (Schalkwijk et al, 1990).

Phospholipases A2 and Prostaglandin Formation in Rat Glomerular Mesangial Cells

Phospholipases A2 are believed to play important roles in the production of bioactive mediators by regulating the release of precursors for these compounds from structural membrane phosphoglycerides. It has become clear during the last years that several and distinct cellular phospholipases A2 exist. These can be distinguished in low molecular weight 14 kDa group I (pancreatic-type) and group II (non-pancreatic-type) enzymes and in high molecular weight 85 kDa or cytosolic phospholipase A2. These require Ca2+ for activity, either in their catalytic mechanism as in the case of 14 kDa phospholipases or for translocation from the cytosol to membranes as in the case of cytosolic phospholipase A2. In addition, Ca2+-independent enzymes exist. Each of these phospholipase A2 activities appears to be subject to multiple regulation mechanisms, both at the transcriptional and post-translational level, depending on the cell type and the cell stimulators. This paper summarizes our data on the regulation of phospholipase A2 activities in relation to prostaglandin formation in rat glomerular mesangial cells.

Phospholipases A2 and the Production of Bioactive Lipids

Phospholipases A2 and their regulation have received much attention during the last decades because they have been implicated in the release of precursors for bioactive lipid production from structural membrane phospholipids. As such they are considered to be the rate limiting enzymes in the pathways leading to the formation of various eicosanoids and plateletactivating factor from released arachidonate and lyso-platelet-activating factor, respectively. These bioactive lipids are usually secreted from cells and activate specific receptors on neighbouring cells (Negishi et al, 1993). As such they serve in many physiological processes and after excessive formation also in pathophysiological processes such as inflammation, fever, pain and allergic conditions. Hence, not only lipid mediators but also phospholipase A2 has been implicated in inflammation (Pruzanski and Vadas, 1991). Arguments in favour of this view are several fold. In the early stages of inflammatory conditions activated monocytes/macrophages secrete pro-inflammatory cytokines such as interleukin-lp (IL-lp) and tumor necrosis factor (TNF). These in turn activate phospholipases A2 in many target cells and frequently lead to secretion of phospholipases A2. In line with this notion, highly elevated levels of circulating phospholipase A2 have been found under inflammatory conditions such as rheumatoid arthritis, peritonitis and septic shock (Pruzanski and Vadas, 1991). Recently, these observations have been extended to typhoid infections (Keuter et al, 1995), meningitis and acute phases of sickle cell anaemia (unpublished observations).