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-1 beta and transforming growth factor-beta 2 enhance cytosolic high-molecular-mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells.

Interleukin-1 beta induces gene expression and secretion of group-II phospholipase A2 and release of prostaglandin E2 from rat mesangial cells. The interleukin-1 beta-induced synthesis of group-II phospholipase A2 is prevented by transforming growth factor-beta 2, whereas transforming growth factor-beta 2 potentiated the interleukin-1 beta-evoked prostaglandin E2 production. Transforming growth factor-beta 2 itself did not induce synthesis of group-II phospholipase A2, although it stimulated prostaglandin E2 formation. Here we describe the effect of interleukin-1 beta and transforming growth factor-beta 2 on a cytosolic phospholipase A2 activity and prostaglandin E2 formation in rat mesangial cells. Based on the resistance to dithiothreitol and migration profiles on a Mono-Q anion-exchange column and a Superose 12 gel-filtration column, the cytosolic phospholipase A2 activity was assigned to a high-molecular-mass phospholipase A2. Measured with 1-stearoyl-2-[1-14C]arachidonoylglycero-phosphocholine as substrate, both interleukin-1 beta and transforming growth factor-beta 2 enhanced the high-molecular-mass phospholipase A2 activity. The stimulation of rat mesangial cells with interleukin-1 beta and transforming growth factor-beta 2 was time- and dose-dependent with maximal cytosolic phospholipase A2 activities at 10 nM and at 10 ng/ml respectively, after 24 h of stimulation. Under these conditions, interleukin-1 beta and transforming growth factor-beta 2 enhanced the cytosolic phospholipase A2 activity 2.2 +/- 0.6-fold and 2.5 +/- 0.6-fold, respectively. These results strongly suggest that an enhanced cytosolic high-molecular-mass phospholipase A2 activity is involved in the formation of prostaglandin E2 mediated by transforming growth factor-beta 2. Whether interleukin-1 beta induced group-II phospholipase A2 and/or interleukin-1 beta-enhanced cytosolic phospholipase A2 activity is involved in prostaglandin E2 formation in rat mesangial cells is discussed.     

Transforming growth factor beta 2 differentially modulates interleukin-1 beta- and tumour-necrosis-factor-alpha-stimulated phospholipase A2 and prostaglandin E2 synthesis in rat renal mesangial cells.

Isolated hepatocytes from streptozotocin-diabetic rats failed to respond to a glucose load with an activation of glycogen synthase. This lesion was associated with severely decreased activities of glycogen-synthase phosphatase and of glucokinase. All these defects were abolished after consumption for 13-18 days of drinking water containing Na3VO4 (0.7 mg/ml), and they were partially restored after 3.5 days, when the blood glucose concentration was already normalized. In all conditions the maximal extent of activation of glycogen synthase in cells closely parallelled the activity of glycogen-synthase phosphatase.     

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

Treatment of rat glomerular mesangial cells with interleukin-1 beta, 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-1 beta 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.     

Involvement of a pertussis toxin-sensitive G-protein-coupled phospholipase A2 in lipopolysaccharide-stimulated prostaglandin E2 synthesis in cultured rat mesangial cells

E. coli lipopolysaccharide (LPS) stimulated a dose- and time-dependent release of prostaglandin E2 (PGE2) in cultured rat glomerular mesangial cells. Pertussis toxin, an inhibitor of several GTP-binding proteins (G proteins), blocked nearly 80% of the LPS-stimulated PGE2 formation, while having virtually no effect on calcium ionophore-stimulated PGE2 production. We tested the possibility that a G protein-coupled activation of phospholipase A2 mediated the LPS-stimulated PGE2 production. Evidence for LPS activation of phospholipase A2 included a time-dependent LPS-induced generation of [32P]lysophosphatidylcholine and the inhibitory effects of a phospholipase A2 inhibitor, mepacrine, on LPS-induced PGE2 formation. Possible roles for phospholipase C-dependent activation of PGE2 synthesis by LPS seemed unlikely, as LPS did not elevate the cytosolic free calcium concentration or augment the appearance of water-soluble inositol phosphates. We conclude that LPS-induced PGE2 synthesis in rat glomerular mesangial cells is mediated through a G-protein-coupled phospholipase A2 activation. The activation of phospholipase A2 releases arachidonic acid and stimulates PGE2 synthesis preferentially, thereby improving glomerular hemodynamic events in endotoxemia.     

Interleukin-1 alpha stimulates prostaglandin biosynthesis in serum-activated mesangial cells by induction of a non-pancreatic (type II) phospholipase A2

Enhanced prostaglandin (PG) biosynthesis is a hallmark of inflammation, and interleukin-1 (IL), a proinflammatory cytokine, is a potent stimulus of PG production. We investigated the mechanisms of IL-1 alpha-enhanced PG synthesis in serum-stimulated mesangial cells. The rIL-1-stimulated increase in PGE2 synthesis was dose- and time-dependent and inhibited by both cycloheximide and actinomycin D. Phospholipase (PL) activity was increased 5- to 10-fold in acid extracts of rIL-1-treated cells as measured by arachidonate release from exogenous [14C]arachidonyl-phosphatidyl-ethanolamine. This induced phospholipase activity was Ca(2+)-dependent and inhibited by the PLA2 inhibitors, aristocholic acid, 7,7-dimethyl-5,8-eicosadienoic acid, and p-bromophenacylbromide, but not by the 1,2-diacylglycerol lipase inhibitor RHC 80267. The rIL-1-stimulated PLA2 had an alkaline pH optimum, and phosphatidylethanolamine was preferred over phosphatidylcholine as substrate. The PLA2 activity increased by rIL-1 was inhibited in cells coincubated with cycloheximide and was measurable after 6 h. A sensitive and specific solution hybridization assay demonstrated a coordinate time-dependent induction of non-pancreatic PLA2 mRNA expression which was increased at least 6-fold by 24 h. In whole cells, IL-1 had no effect on basal [3H]arachidonic acid release but vasopressin (1 microM)-stimulated release was potentiated 2- to 3-fold, suggesting that IL-1 may prime cells for increased PG synthesis via increased PLA2 activity. Thus IL-1 directly stimulates, as well as primes cells for, enhanced PG synthesis, in part, by increasing PLA2 activity through new synthesis of a non-pancreatic (Type II) PLA2.     

Protein phosphatase 2A is a negative regulator of transforming growth factor-beta1-induced TAK1 activation in mesangial cells

TAK1 (transforming growth factor (TGF)-beta-activated kinase 1) is a serine/threonine kinase that is rapidly activated by TGF-beta1 and plays a vital function in its signal transduction. Once TAK1 is activated, efficient down-regulation of TAK1 activity is important to prevent excessive TGF-beta1 responses. The regulatory mechanism of TAK1 inactivation following TGF-beta1 stimulation has not been elucidated. Here we demonstrate that protein phosphatase 2A (PP2A) plays a pivotal role as a negative regulator of TAK1 activation in response to TGF-beta1 in mesangial cells. Treatment with okadaic acid (OA) induces autophosphorylation of Thr-187 in the activation loop of TAK1. In vitro dephosphorylation assay suggests that Thr-187 in TAK1 is a major dephosphorylation target of PP2A. TGF-beta1 stimulation rapidly activates TAK1 in a biphasic manner, indicating that TGF-beta1-induced TAK1 activation is tightly regulated. The association of PP2A(C) with TAK1 is enhanced in response to TGF-beta1 stimulation and closely parallels TGF-beta1-induced TAK1 activity. Attenuation of PP2A activity by OA treatment or targeted knockdown of PP2A(C) with small interfering RNA enhances TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3 (MAPK kinase 3). Endogenous TAK1 co-precipitates with PP2A(C) but not PP6(C), another OA-sensitive protein phosphatase, and knockdown of PP6(C) by small interfering RNA does not affect TGF-beta1-induced phosphorylation of TAK1 at Thr-187 and MKK3. Moreover, ectopic expression of phosphatase-deficient PP2A(C) enhances TAK1-mediated MKK3 phosphorylation by TGF-beta1 stimulation, whereas the expression of wild-type PP2A(C) suppresses the MKK3 phosphorylation. Taken together, our data indicate that PP2A functions as a negative regulator in TGF-beta1-induced TAK1 activation.     

Effects of transforming growth factor-beta and IL-1 alpha on prostaglandin synthesis in serum-deprived osteoblastic cells.

We investigated the effects that the combination of IL-1 alpha and transforming growth factor-beta (TGF-beta) had on PGE2 production in a murine clonal osteoblastic cell line MC3T3-E1 and primary rat calvarial osteoblast-like cells. In serum-supplemented medium, IL-1 alpha was a potent stimulator of PGE2 production in MC3T3-E1 cells (50-fold increase with 0.1 ng/ml). TGF-beta (10 ng/ml) had only a small effect alone and no additional effect on IL-1 alpha-induced responses. In serum-deprived MC3T3-E1 cells, PGE2 responses to IL-1 alpha were either absent or markedly reduced. TGF-beta alone had small effects. However, simultaneous addition of TGF-beta with IL-1 alpha to MC3T3-E1 cells partially restored the ability of IL-1 alpha to generate a PGE2 response (10-fold increase in PGE2 with 0.1 ng/ml of both IL-1 alpha and TGF-beta). As with MC3T3-E1 cells, serum-deprived primary fetal rat calvarial osteoblastic cells also did not respond to IL-1 alpha, unless TGF-beta was present in the medium (sixfold increase in PGE2 with 0.1 ng/ml IL-1 alpha and 10 ng/ml TGF-beta). The synergistic effect of TGF-beta and IL-1 alpha was specific for PGE2 responses, because these factors did not synergistically affect cell proliferation, collagen and noncollagen protein synthesis, or alkaline phosphatase activity. The observed synergy was not associated with changes in the steady state cyclooxygenase (PGH synthase) mRNA levels. However, it did correlate with increased release of [3H]arachidonic acid from prelabeled serum-depleted MC3T3-E1 cells. Hence, the synergistic interactions of IL-1 alpha and TGF-beta on PGE2 appear to occur through an increase in the release of arachidonic acid substrate from phospholipid pools. These effects may be important for both normal bone turnover and the responses of bone to inflammatory and immune stimuli.     

Increased toxicity by transforming growth factor-beta 1 in liver cells overexpressing CYP2E1

Ethanol treatment causes an increase in expression of TGF-beta1 and CYP2E1 in the centrilobular area. Alcoholic liver disease is usually initiated in the centrilobular region of the liver. We hypothesized that the combination of TGF-beta1 and CYP2E1 produces increased oxidative stress and liver cell toxicity. To test this possibility, we studied the effects of TGF-beta1 on the viability of HepG2 E47 cells that express human CYP2E1, and C34 HepG2 cells, which do not express CYP2E1. E47 cells underwent greater growth inhibition and enhanced apoptosis after TGF-beta1 treatment, as compared to the C34 cells. There was an enhanced production of reactive oxygen species (ROS) and a decline in reduced glutathione (GSH) levels in the TGF-beta1-treated E47 cells and the enhanced cell death could be prevented by antioxidants. The CYP2E1 inhibitor diallyl sulfide prevented the potentiated cell death in E47 cells validating the role of CYP2E1. Mitochondrial membrane potential declined in the TGF-beta1-treated E47 cells, prior to developing toxicity, and cell death could be prevented by trifluoperazine, an inhibitor of the mitochondrial membrane permeability transition. TGF-beta1 also produced a loss of cell viability in hepatocytes from pyrazole-treated rats with elevated levels of CYP2E1, compared to control hepatocytes. In conclusion, increased toxic interactions by TGF-beta1 plus CYP2E1 can occur by a mechanism involving increased production of intracellular ROS and depletion of GSH, resulting in mitochondrial membrane damage and loss of membrane potential, followed by apoptosis. Potentiation of TGF-beta1-induced cell death by CYP2E1 may contribute to mechanisms of alcohol-induced liver disease.     

Interleukin 1 and tumor necrosis factor synergistically stimulate prostaglandin synthesis and phospholipase A2 release from rat renal mesangial cells

Treatment of rat glomerular mesangial cells with recombinant human interleukin 1 alpha (rIL-1 alpha), recombinant human interleukin 1 beta (rIL-1 beta) or recombinant human tumor necrosis factor (rTNF) induces prostaglandin E2 (PGE2) synthesis and the release of a phospholipase A2 (PLA2) activity. rIL-1 beta is significantly more potent than rIL-1 alpha or rTNF in stimulating PGE2 as well as PLA2 release from mesangial cells. When given together, rTNF interacts in a synergistic fashion with rIL-1 alpha and rIL-1 beta to enhance both, PGE2 synthesis and PLA2 release. The released PLA2 has a neutral pH optimum and is calcium-dependent. Pretreatment of cells with actinomycin D or cycloheximide inhibits basal and cytokine-stimulated PGE2 and PLA2 release.     

Epidermal growth factor stimulates phospholipase A2 in vasopressin-treated rat glomerular mesangial cells.

Epidermal growth factor (EGF) enhances vasopressin- and ionophore-A23187-induced prostaglandin production and arachidonate release by rat glomerular mesangial cells in culture. The purpose of the present study was to delineate the phospholipid pathways involved in this effect. In cells labelled with [14C]arachidonate, EGF significantly enhanced the free arachidonate released in response to A23187 or vasopressin without enhancing the production of [14C]arachidonate-labelled diacylglycerol. EGF increased the [14C]arachidonate-labelled phosphatidic acid formed in response to vasopressin, but to a much smaller extent than it increased free arachidonate release. These results indicate that activation of phospholipase C is not sufficient to explain the increase in free arachidonate release observed on addition of EGF. To examine if EGF enhanced phospholipase A2 activity, mesangial cells were labelled with [2-2H]glycerol and [14C]-arachidonate, and the formation of arachidonate-poor lysophospholipids was studied. When combined with vasopressin, EGF significantly enhanced the formation of arachidonate-poor lysophospholipids as compared with vasopressin alone. The fate of exogenously added lysophosphatidylcholine was not altered after stimulation with vasopressin plus EGF, indicating that decreased deacylation or reacylation of the lysophospholipids was not responsible for their accumulation. Taken together, these results indicate that EGF enhances free arachidonate release by activation of phospholipase A2. The signalling mechanism responsible for the change in phospholipase A2 activity is not known, but could conceivably involve phosphorylation of modulating proteins such as lipocortin or G-proteins.     

Interaction of transforming growth factor-beta 1 with alpha 2-macroglobulin. Role in transforming growth factor-beta 1 clearance.

It has been widely assumed that the interaction of transforming growth factor-beta 1 (TGF-beta 1) with its serum-binding protein, alpha 2-macroglobulin (alpha 2M), mediates the rapid clearance of TGF-beta 1 from the circulation. To test this, we have analyzed the effect of TGF-beta 1 binding on the conformational state of alpha 2M. Our results demonstrate that the binding of TGF-beta 1 to alpha 2M does not lead to the conformational change in the alpha 2M molecule that is required for the clearance of the alpha 2M.TGF-beta 1 complex via the alpha 2M receptor. Furthermore, endogenous TGF-beta 1 is associated with the conformationally unaltered slow clearance form of alpha 2M. Clearance studies in mice show that the half-life of 125I-TGF-beta 1 in the circulation (1.6 +/- 0.71 min) is not affected by blocking the alpha 2M receptor with excess conformationally altered alpha 2M. These results suggest that TGF-beta 1 is rapidly cleared from the circulation after injection by a pathway not involving alpha 2M.     

FK506, transforming growth factor-beta1 and mesangial matrix synthesis: parallels and differences compared with cyclosporine A

Cyclosporine A (CsA)-induced glomerulosclerosis is a well-described side effect of CsA treatment. Current evidence indicates that FK506 causes similar morphologic changes. Recently, we demonstrated that CsA up-regulates the expression of transforming growth factor-beta1 (TGF-beta1), its receptors type I (TbetaR-I) and type II (TbetaR-II), as well as related matrix protein synthesis in mesangial cells (MCs). Here, we assessed the effect of FK506 on the expression of TGF-beta1, TbetaR-I, TbetaR-II, fibronectin (FN) and plasminogen activator inhibitor type-1 (PAI-1) in MCs. Resting MCs were incubated with/without FK506. Time- and concentration-dependent expression was measured at the mRNA and protein level. Compared to untreated controls, FK506 stimulated TGF-beta1 mRNA (maximum at 8 h, 100 ng/mL: 2.13+/-0.15-fold, P<0.005) and protein expression (maximum at 96 h, 100 ng/mL: 1.96+/-0.29-fold, P<0.005). In contrast, TbetaR-I and TbetaR-II protein expression remained unchanged. Concerning matrix protein synthesis, FK506 slightly increased FN production (96 h, 100 ng/mL: 1.38+/-0.28-fold, P<0.05), but not PAI-1 production. These results indicate that, comparable to CsA, FK506 induced glomerulosclerosis is also due to a direct effect on mesangial matrix production, which is at least in part mediated via up-regulation of TGF-beta1 expression. The fact that, unlike CsA, FK506 does not increase the expression of TbetaR-I, TbetaR-II, and PAI-1, deserves further investigation.     

Interleukin 1 and tumor necrosis factor potentiate angiotensin II- and calcium ionophore-stimulated prostaglandin E2 synthesis in rat renal mesangial cells

In resting mesangial cells, angiotensin II and the calcium ionophore A23187 stimulated prostaglandin E2 (PGE2) formation. After pretreatment with interleukin 1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha), which are themselves potent stimuli for PGE2 synthesis, mesangial cells displayed an amplified response to angiotensin II and A23187. The cytokine-induced effects occurred in a time- and dose-dependent manner and were attenuated by actinomycin D, cycloheximide and dexamethasone. IL-1 beta and TNF alpha treatment also increased the amount of arachidonic acid released after stimulation of cells with angiotensin II and A23187. In addition, IL-1 beta but not TNF alpha treatment augmented the formation of PGE2 from exogenous arachidonic acid by mesangial cells. Furthermore, the conversion of prostaglandin H2 to PGE2 was not changed by IL-1 beta and TNF alpha. These results suggest that IL-1 beta and TNF alpha exert a priming effect on PGE2 production in mesangial cells.     

Interleukin-13 and transforming growth factor-beta1 inhibit spontaneous sleep in rabbits

Proinflammatory cytokines, including interleukin-1beta and tumor necrosis factor-alpha are involved in physiological sleep regulation. Interleukin (IL)-13 and transforming growth factor (TGF)-beta1 are anti-inflammatory cytokines that inhibit proinflammatory cytokines by several mechanisms. Therefore, we hypothesized that IL-13 and TGF-beta1 could attenuate sleep in rabbits. Three doses of IL-13 (8, 40, and 200 ng) and TGF-beta1 (40, 100, and 200 ng) were injected intracerebroventricularly 3 h after the beginning of the light period. In addition, one dose of IL-13 (200 ng) and one dose of TGF-beta1 (200 ng) were injected at dark onset. The two higher doses of IL-13 and the highest dose of TGF-beta1 significantly inhibited spontanenous non-rapid eye movement sleep (NREMS) when they were given in the light period. IL-13 also inhibited NREMS after dark onset administration; however, the inhibitory effect was less potent than that observed after light period administration. The 40-ng dose of IL-13 inhibited REMS duration during the dark period. TGF-beta1 administered at dark onset had no effect on sleep. These data provide additional evidence for the hypothesis that a brain cytokine network is involved in regulation of physiological sleep.     

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.