Casper G. Schalkwijk

Interleukin-1β, tumor necrosis factor and forskolin stimulate the synthesis and secretion of group II phospholipase A2 in rat mesangial cells

Abstract Treatment of rat glomerular mesangial cells with interleukin-1β, tumor necrosis factor or forskolin resulted in the release of phospholipase A2 activity in the culture medium. Essentially all of this phospholipase A2 activity was bound to immobilized monoclonal antibodies raised against rat liver mitochondrial 14 kDa group II phospholipase A2. Gelfiltration confirmed the absence of higher molecular weight phospholipases A2 in the culture medium. Immunoblot experiments showed the virtual absence of this 14 kDa group II phospholipase A2 in unstimulated mesangial cells. The time-dependent increase of phospholipase A2 activity in both cells and culture medium upon stimulation with interleukin-1β plus forskolin is accompanied with elevated 14 kDa phospholipase A2 protein levels. These results indicate that the increased phospholipase A2 activity upon treatment of mesangial cells with these stimulators is due to increased synthesis of group II phospholipase A2. Over 85 % of this newly synthesized phospholipase A2 appears to be secreted from the cells.

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.

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.

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).