Immunomodulated signaling in macrophages: Studies on activation of Raf-1, MAPK, cPLA_2 and secretion of IL-12

Little is known about the mechanism and signal transduction by LPS-mediated immunomodulation of murine peritoneal macrophages. It is found that the signal molecules of the down-stream of Ras, Raf-1, MAPK p44, and MAPK p42 are phosphorylated, and cPLA2 is activated with a significant increase of the release of [ H3 ] AA by macrophages in response to LPS and PMA. Compared with the very recent finding that LPS and PMA trigger the activation and translocation of PKC-α and PKC-ε, these findings suggest that there is a connection between PKC signaling pathway and the Raf-1/MAPK pathway and that the activation of these main signaling events may be closely related to the secretion of IL-12 during LPS-induced modulation of macrophages.     

Differential contributions of protein kinase C isoforms in the regulation of group IIA secreted phospholipase A2 expression in cytokine-stimulated rat fibroblasts

Protein kinase C (PKC) is a family of serine/threonine kinases involved in various signal transduction pathways. We investigated the roles of PKC in the regulation of group IIA secreted phospholipase A₂ (sPLA₂-IIA) expression in cytokine-stimulated rat fibroblastic 3Y1 cells. Here we show that the induction of sPLA₂-IIA by proinflammatory cytokines was under the control of both classical cPKCα and atypical aPKCλ/ι pathways by using PKC inhibitors, a PKC activator, and PKC knockdowns. Treatment of 3Y1 cells with PKC selective inhibitors having broad specificity, such as chelerythrine chloride and GF109203X, blocked IL-1β/TNFα-dependent induction of sPLA₂-IIA protein in a dose-dependent manner. Treatment with the PKC activator phorbol 12-myristate 13-acetate (PMA), which activates cPKC and novel nPKC isoforms, markedly attenuated the cytokine-dependent induction of sPLA₂-IIA expression. In comparison, 24-h pretreatment with PMA, which down-regulates these PKC isoforms, markedly enhanced sPLA₂-IIA expression. Results with short hairpin RNA (shRNA)-mediated knockdown of PKC isoforms revealed that the cytokine-induced sPLA₂-IIA expression was markedly enhanced in cPKCα knockdown cells compared to those in replicate control cells. In contrast, knockdown of the aPKCλ/ι isoform reduced the cytokine-induced expression of sPLA₂-IIA. These results suggest that the aPKCλ/ι pathway is required for the induction of sPLA₂-IIA expression and that the cPKCα pathway acts as a negative regulator of sPLA₂-IIA expression in cytokine-stimulated rat fibroblasts.     

Differential Distribution of Both IL-12Rβ Chains in the Plasma Membrane of Human T Cells

IL-12 is a cytokine that stimulates the expression of CD26, a T cell– and raft-associated ectopeptidase. IL-12 also enhances the interaction between CD26 and CD45RO, which removes the phosphatase CD45RO from raft microdomains. Since Janus kinases are known CD45 substrates, our hypothesis was that this relocation of CD45RO in nonraft areas of the membrane could be important to switch off the signaling via cytokine receptors, e.g., the IL-12 receptor (IL-12R). Accordingly, both IL-12R and CD45RO should be equally positioned in the cell membrane upon IL-12R ligation. However, there were no data available on the membrane distribution of IL-12R on human T cells. Working with phytohemagglutinin (PHA) lymphoblasts, we tried to fill that gap. The high-affinity IL-12R is made of two chains: IL-12Rβ1 and IL-12Rβ2. Using flow cytometry, Western blot and confocal microscopy, we obtained data suggesting that IL-12Rβ1 is mainly associated to phospholipid-rich membrane areas, a location even enhanced upon IL-12 incubation of PHA blasts. Instead, IL-12Rβ2 is found more segregated into membrane rafts, which could explain why two IL-12-triggered events, T-cell proliferation and ERK1/2 activation, are both methyl-β-cyclodextrin-sensitive events. Ligation of IL-12R with IL-12 seems to induce a partial enrichment of IL-12Rβ2 in phospholipid-rich areas, where according to our data IL-12Rβ1 is already present. Therefore, although new data will be required, the present results support the initial hypothesis.     

p44/42(ERK1/2) MAPK and PLD activation by PGD2 preserves papillary phosphatidylcholine homeostasis

Previous works from our laboratory demonstrated that PGD(2) modulates phosphatidylcholine (PC) biosynthesis in renal papillary tissue. In the present work, we have evaluated the mechanism by which PGD(2) exerts this action. PGD(2) caused two stimulatory waves in PC synthesis which were reproduced by its full-agonist BW245C. At 1min stimulation, PGD(2) increased PC synthesis by 131%; this increase was blocked by neomycin and ethanol, cheleritrine and U0126, PLD, PKC, and MEK1/2 inhibitors, respectively. A second PC synthesis increase (100%) was observed after 15min, which was blocked by PLD inhibitors. PGD(2) also increased phospho-ERK1/2 MAPK in a biphasic-fashion, which was abolished by PLC and PKC inhibitors but not by ethanol, which overincreased phospho-ERK1/2, suggesting that PGD(2)-induced ERK1/2 activation requires previous PLC-PKC activation while PLD down-regulates it. Our results indicate that PGD(2) stimulatory effect involves both PLD and ERK1/2-MAPK activation, and both pathways operate independently of PC synthesis homeostasis.     

Identification and characterization of murine IRAK-2

Interleukin-1 receptor-associated kinases (IRAKs) are pivotal signaling elements of the Toll/IL-1 receptor (TIL) family, which play a role in innate immune responses by coordinating host defence mechanisms. Presently four different forms of human IRAK molecules are cloned (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, and hu-IRAK-4). In the murine system, only three genes have been identified so far, mouse Pelle-Like Kinase (mPLK), which corresponds to human IRAK-1, mu-IRAK-M, and mu-IRAK-4. Here we report the molecular cloning and characterization of murine IRAK-2 (mu-IRAK-2), a mouse homolog to human IRAK-2 (hu-IRAK-2). Murine and human IRAK-2 molecules show 67% sequence identity, they are ubiquitiously expressed, and both practically lack autophoshorylation kinase activity. The murine molecule reveals two remarkable differences to its human counterpart: it shows a C-terminal extension and it has no stimulatory effect on IL-1 induced NF-kappa B activation when compared to hu-IRAK-2, suggesting subtle functional differences in signaling by IRAK-2 in human and mouse cells.     

Prostaglandin E2 suppresses β1-integrin expression via E-prostanoid receptor in human monocytes/macrophages

β₁-Integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E₂ (PGE₂) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE₂ on the expression of β₁-integrin remains unknown. In this study, we investigated the effects of PGE₂ on the expression of β₁-integrin in the human monocytic cell line THP-1 and in CD14⁺ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE₂ receptors and E-prostanoid (EP) receptors on PGE₂-mediated inhibition. We found that PGE₂ significantly inhibited the expression of β₁-integrin, mainly through EP₄ receptors in THP-1 cells and CD14⁺ monocytes/macrophages in human peripheral blood. We suggest that PGE₂ has anti-inflammatory effects, leading to the inhibited expression of β₁-integrin in human monocytes/macrophages, and that the EP₄ receptor may play an important role in PGE₂-mediated inhibition.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

Identification and characterization of murine IRAK-M

Interleukin-1 receptor-associated-kinases (IRAKs) are signal transduction mediators of the Toll/IL-1 receptor family, which comprise several transmembrane proteins involved in host defense mechanisms. Today four different human IRAKs (hu-IRAK-1, hu-IRAK-2, hu-IRAK-M, hu-IRAK-4) and two murine IRAKs (mouse pelle like kinase (mPLK) and mu-IRAK-4) have been described. Here we report the identification and characterization of murine IRAK-M (mu-IRAK-M), a mouse homologue to human IRAK-M (hu-IRAK-M). These IRAK-M molecules show 71% sequence identity, a comparable cellular expression, and functional similarities with respect to signal transduction capacity and kinase activity, suggesting functional homology in signalling in human and mouse cells.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

PKCdelta and MAPK mediate G(1) arrest induced by PMA in SKBR-3 breast cancer cells

The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G(1). Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCdelta inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCdelta, WTPKCdeltaAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCdeltaAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH(2)-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCdelta) in the G(1) arrest induced by PMA and suggested PKC as a target for breast cancer treatment.     

Different pathways leading to activation of extracellular signal-regulated kinase and p38 MAP kinase by formyl-methionyl-leucyl-phenylalanine or platelet activating factor in human neutrophils

Summary The signaling pathways leading to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation by N-formyl-Met-Leu-Phe (fMLP) or platelet activating factor (PAF) in human neutrophils were examined. Previously, we found that changes of intracellular Ca²⁺ ([Ca ) stimulated by PAF and fMLP were due to Ca²⁺ influx and internal Ca²⁺ release, respectively. To further determine the mechanism of MAPK activation and its relation with Ca²⁺ influx, blood from healthy human volunteers was taken by venous puncture. Human polymorphonuclear cells (PMNs) were isolated and incubated with protein kinase C (PKC) inhibitor Calphostin C, PKC- isoform inhibitor GF109203X, phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002, phospholipase C (PLC) inhibitor U73122, phospholipase A₂ (PLA₂) inhibitor Aristolochic acid, store-operated calcium (SOC) channel inhibitor SKF96365, or extracellular calcium chelator EGTA followed by fMLP or PAF treatment. Phosphorylation of ERK p38 was determined by immunoblotting analysis. Our data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK phosphorylation is mediated by PI3K, PKC, PLA₂, PLC, and extracellular calcium, whereas fMLP-induced ERK phosphorylation does not involve the PKC- isoform and extracellular calcium. PAF-induced p38 phosphorylation involves PLA2, whereas fMLP-induced p38 activation is PLC dependent.     

Dual role of the p38 MAPK/cPLA2 pathway in the regulation of platelet apoptosis induced by ABT-737 and strong platelet agonists

p38 Mitogen-activated protein (MAP) kinase is involved in the apoptosis of nucleated cells. Although platelets are anucleated cells, apoptotic proteins have been shown to regulate platelet lifespan. However, the involvement of p38 MAP kinase in platelet apoptosis is not yet clearly defined. Therefore, we investigated the role of p38 MAP kinase in apoptosis induced by a mimetic of BH3-only proteins, ABT-737, and in apoptosis-like events induced by such strong platelet agonists as thrombin in combination with convulxin (Thr/Cvx), both of which result in p38 MAP kinase phosphorylation and activation. A p38 inhibitor (SB202190) inhibited the apoptotic events induced by ABT-737 but did not influence those induced by Thr/Cvx. The inhibitor also reduced the phosphorylation of cytosolic phospholipase A₂ (cPLA2), an established p38 substrate, induced by ABT-737 or Thr/Cvx. ABT-737, but not Thr/Cvx, induced the caspase 3-dependent cleavage and inactivation of cPLA2. Thus, p38 MAPK promotes ABT-737-induced apoptosis by inhibiting the cPLA2/arachidonate pathway. We also show that arachidonic acid (AA) itself and in combination with Thr/Cvx or ABT-737 at low concentrations prevented apoptotic events, whereas at high concentrations it enhanced such events. Our data support the hypothesis that the p38 MAPK-triggered arachidonate pathway serves as a defense mechanism against apoptosis under physiological conditions.