Transforming growth factor beta1 rescues serum deprivation-induced apoptosis via the mitogen-activated protein kinase (MAPK) pathway in macrophages

Cell death and cell survival are central components of normal development and pathologic states. Transforming growth factor beta1 (TGF-beta1) is a pleiotropic cytokine that regulates both cell growth and cell death. To better understand the molecular mechanisms that control cell death or survival, we investigated the role of TGF-beta1 in the apoptotic process by dominant-negative inhibition of both TGF-beta1 and mitogen-activated protein kinase (MAPK) signaling pathways. Murine macrophages (RAW 264.7) undergo apoptosis following serum deprivation, as determined by DNA laddering assay. However, apoptosis is prevented in serum-deprived macrophages by the presence of exogenous TGF-beta1. Using stably transfected RAW 264.7 cells with the kinase-deleted dominant-negative mutant of TbetaR-II (TbetaR-IIM) cDNA, we demonstrate that this protective effect by TGF-beta1 is completely abrogated. To determine the downstream signaling pathways, we examined TGF-beta1 effects on the MAPK pathway. We show that TGF-beta1 induces the extracellular signal-regulated kinase (ERK) activity in a time-dependent manner up to 4 h after stimulation. Furthermore, TGF-beta1 does not rescue serum deprivation-induced apoptosis in RAW 264.7 cells transfected with a dominant-negative mutant MAPK (ERK2) cDNA or in wild type RAW 264.7 cells in the presence of the MAPK kinase (MEK1) inhibitor. Taken together, our data demonstrate for the first time that TGF-beta1 is an inhibitor of apoptosis in cultured macrophages and may serve as a cell survival factor via TbetaR-II-mediated signaling and downstream intracellular MAPK signaling pathway.     

Excretory/secretory products from plerocercoids of Spirometra erinaceieuropaei suppress interleukin-1beta gene expression in murine macrophages

The present study shows that ES products from plerocercoids of Spirometra erinaceieuropaei suppressed interleukin-1beta mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages in the absence or presence of a cyclic AMP analogue, dibutyryl cyclic AMP. Investigation using the inhibitors of mitogen-activated protein kinase (MAPK) pathways revealed that extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways are crucial for full induction of interleukin-1beta mRNA expression. ES products additionally suppressed interleukin-1beta mRNA expression in the cells treated with p38 mitogen-activated protein kinase inhibitor (SB203580) or extracellular signal-regulated protein kinase 1/2 inhibitor (PD98059). Western blot analysis showed that dibutyryl cyclic AMP enhanced lipopolysaccharide-induced phosphorylation of extracellular signal-regulated protein kinase 1/2, p38 mitogen-activated protein kinase and cyclic AMP responsive element binding protein (CREB) and, in turn, we demonstrated that ES products reduced the lipopolysaccharide and dibutyryl cyclic AMP-induced phosphorylation of extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase, but not cyclic AMP responsive element binding protein. These data demonstrate that ES products from the plerocercoids of S. erinaceieuropaei may evade induction of interleukin-1beta mRNA by inhibiting extracellular signal-regulated protein kinase 1/2 and p38 mitogen-activated protein kinase pathways in lipopolysaccharide and/or dibutyryl cyclic AMP-stimulated macrophages.     

Flavopiridol inhibits lipopolysaccharide-induced TNF-α production through inactivation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway

Flavopiridol is a cyclin-dependent kinase inhibitor and inhibits the growth of various cancer cells. The effect of flavopiridol on lipopolysaccharide (LPS)-induced proinflammatory mediator production was examined in RAW 264.7 macrophage-like cells. Flavopiridol significantly reduced the production of tumor necrosis factor-α and, to a lesser extent, nitric oxide in LPS-stimulated cells. Flavopiridol inhibited the activation of nuclear factor-κB and IκB kinase in response to LPS. Flavopiridol also inhibited the activation of a series of mitogen-activated protein kinases, such as p38, stress-activated protein kinase/c-Jun N-terminal kinase and extracellular signal-regulated kinase 1/2 in response to LPS. However, flavopiridol did not alter the expression of tumor necrosis factor receptor-associated factor 6, myeloid differentiation factor 88 (MyD88) or CD14/toll-like receptor (TLR) 4. Flavopiridol inhibited nitric oxide production induced by a MyD88-dependent TLR2 ligand, but not a MyD88-independent TLR3 ligand. Further, flavopiridol did not alter the phosphorylation of interferon regulatory factor 3 in the MyD88-independent pathway. Therefore, it was suggested that flavopiridol exclusively inhibited the activation of nuclear factor-κB and mitogen-activated protein kinases in the MyD88-dependent pathway. Flavopiridol might be useful for the prevention of LPS-induced inflammatory response.     

Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors

ADP-ribosylation is involved in nuclear factor kappaB (NF-kappaB)-dependent gene expression induced by lipopolysaccharide in murine macrophages. Here we have investigated the mechanism by which ADP-ribosylation inhibitors block signaling pathways induced in macrophages. In RAW264.7 macrophages the inducers of NF-kappaB activate the production of reactive oxygen species and three mitogen-activated protein kinases (MAPK), the extracellular signal regulated kinase (ERK), the c-jun N-terminal kinase/stress-activated protein kinase (JNK), and p38. We demonstrate that ADP-ribosylation inhibitors specifically inhibit ERK MAPK activation and reduce the release of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha), IL-6 and nitrite.     

Piceatannol prevents lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nuclear factor (NF)-kappaB activation by inhibiting IkappaB kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.     

Glycogen synthase kinase 3- and extracellular signal-regulated kinase-dependent phosphorylation of paxillin regulates cytoskeletal rearrangement

Paxillin is a 68-kDa focal adhesion-associated protein that plays an important role in controlling cell spreading and migration. Phosphorylation of paxillin regulates its biological activity and thus has warranted investigation. Serine 126 and serine 130 were previously identified as two major extracellular signal-regulated kinase (ERK)-dependent phosphorylation sites in Raf-transformed fibroblasts. Here serine 126 is identified as a phosphorylation site induced by lipopolysaccharide (LPS) stimulation of RAW264.7 cells. A number of other stimuli, including adhesion and colony-stimulating factor, induce serine 126 phosphorylation in RAW264.7 cells, and nerve growth factor (NGF) treatment induces serine 126 phosphorylation in PC12 cells. The kinase responsible for phosphorylation of this site is identified as glycogen synthase kinase 3 (GSK-3). Interestingly, this GSK-3-dependent phosphorylation is regulated via an ERK-dependent priming mechanism, i.e., phosphorylation of serine 130. Phosphorylation of S126/S130 was required to promote spreading in paxillin null cells, and LPS-induced spreading of RAW264.7 cells was inhibited by expression of the paxillin S126A/S130A mutant. Furthermore, this mutant also retarded NGF-induced PC12 cell neurite outgrowth. Hence, phosphorylation of paxillin on serines 126 and 130, which is mediated by an ERK/GSK-3 dual-kinase mechanism, plays an important role in cytoskeletal rearrangement.     

The function of mitogen-activated protein kinase phosphatase-1 in peptidoglycan-stimulated macrophages

Mitogen-activated protein (MAP) kinases play a pivotal role in the macrophages in the production of proinflammatory cytokines triggered by lipopolysaccharides. However, their function in the responses of macrophages to Gram-positive bacteria is poorly understood. Even less is known about the attenuation of MAP kinase signaling in macrophages exposed to Gram-positive bacteria. In the present study, we have investigated the regulation of MAP kinases and the role of MAP kinase phosphatase (MKP)-1 in the production of pro-inflammatory cytokines using murine RAW264.7 and primary peritoneal macrophages after peptidoglycan stimulation. Treatment of macrophages with peptidoglycan resulted in a transient activation of JNK, p38, and extracellular signal-regulated kinase. Most interestingly, MKP-1 expression was potently induced by peptidoglycan, and this induction was concurrent with MAP kinase dephosphorylation. Triptolide, a diterpenoid triepoxide, potently blocked the induction of MKP-1 by peptidoglycan and prolonged the activation of JNK and p38. Overexpression of MKP-1 substantially attenuated the production of tumor necrosis factor (TNF)-alpha induced by peptidoglycan, whereas knockdown of MKP-1 by small interfering RNA substantially increased the production of both TNF-alpha and interleukin-1 beta. Finally, we found that in primary murine peritoneal macrophages, MKP-1 induction following peptidoglycan stimulation also coincided with inactivation of JNK and p38. Blockade of MKP-1 induction resulted in a sustained activation of both JNK and p38 in primary macrophages. Our results reveal that MKP-1 critically regulates the expression of TNF-alpha and interleukin-1 beta in RAW264.7 cells and further suggest a central role for this phosphatase in controlling the inflammatory responses of primary macrophages to Gram-positive bacterial infection.     

Overexpression of protein kinase C isoforms protects RAW 264.7 macrophages from nitric oxide-induced apoptosis: involvement of c-Jun N-terminal kinase/stress-activated protein kinase, p38 kinase, and CPP-32 protease pathways

Nitric oxide (NO) induces apoptotic cell death in murine RAW 264.7 macrophages. To elucidate the inhibitory effects of protein kinase C (PKC) on NO-induced apoptosis, we generated clones of RAW 264.7 cells that overexpress one of the PKC isoforms and explored the possible interactions between PKC and three structurally related mitogen-activated protein (MAP) kinases in NO actions. Treatment of RAW 264.7 cells with sodium nitroprusside (SNP), a NO-generating agent, activated both c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38 kinase, but did not activate extracellular signal-regulated kinase (ERK)-1 and ERK-2. In addition, SNP-induced apoptosis was slightly blocked by the selective p38 kinase inhibitor (SB203580) but not by the MAP/ERK1 kinase inhibitor (PD098059). PKC transfectants (PKC-beta II, -delta, and -eta) showed substantial protection from cell death induced by the exposure to NO donors such as SNP and S-nitrosoglutathione (GSNO). In contrast, in RAW 264.7 parent or in empty vector-transformed cells, these NO donors induced internucleosomal DNA cleavage. Moreover, overexpression of PKC isoforms significantly suppressed SNP-induced JNK/SAPK and p38 kinase activation, but did not affect ERK-1 and -2. We also explored the involvement of CPP32-like protease in the NO-induced apoptosis. Inhibition of CPP32-like protease prevented apoptosis in RAW 264.7 parent cells. In addition, SNP dramatically activated CPP32 in the parent or in empty vector-transformed cells, while slightly activated CPP32 in PKC transfectants. Therefore, we conclude that PKC protects NO-induced apoptotic cell death, presumably nullifying the NO-mediated activation of JNK/SAPK, p38 kinase, and CPP32-like protease in RAW 264.7 macrophages.     

Lipopolysaccharide activation of the TPL-2/MEK/extracellular signal-regulated kinase mitogen-activated protein kinase cascade is regulated by IkappaB kinase-induced proteolysis of NF-kappaB1 p105

The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-kappaB1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IkappaB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-kappaB1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.     

IFN-gamma + LPS induction of iNOS is modulated by ERK, JNK/SAPK, and p38(mapk) in a mouse macrophage cell line

Nitric oxide (NO.) produced by inducible nitric oxide synthase (iNOS) mediates a number of important physiological and pathophysiological processes. The objective of this investigation was to examine the role of mitogen-activated protein kinases (MAPKs) in the regulation of iNOS and NO. by interferon-gamma (IFN-gamma) + lipopolysaccharide (LPS) in macrophages using specific inhibitors and dominant inhibitory mutant proteins of the MAPK pathways. The signaling pathway utilized by IFN-gamma in iNOS induction is well elucidated. To study signaling pathways that are restricted to the LPS-signaling arm, we used a subclone of the parental RAW 264.7 cell line that is unresponsive to IFN-gamma alone with respect to iNOS induction. In this RAW 264.7gammaNO(-) subclone, IFN-gamma and LPS are nevertheless required for synergistic activation of the iNOS promoter. We found that extracellular signal-regulated kinase (ERK) augmented and p38(mapk) inhibited IFN-gamma + LPS induction of iNOS. Dominant-negative MAPK kinase-4 inhibited iNOS promoter activation by IFN-gamma + LPS, also implicating the c-Jun NH(2)-terminal kinase (JNK) pathway in mediating iNOS induction. Inhibition of the ERK pathway markedly reduced IFN-gamma + LPS-induced tumor necrosis factor-alpha protein expression, providing a possible mechanism by which ERK augments iNOS expression. The inhibitory effect of p38(mapk) appears more complex and may be due to the ability of p38(mapk) to inhibit LPS-induced JNK activation. These results indicate that the MAPKs are important regulators of iNOS-NO. expression by IFN-gamma + LPS.     

Distinct mechanisms of taxol-induced serine phosphorylation of the 66-kDa Shc isoform in A549 and RAW 264.7 cells

Nanomolar concentrations of Taxol, and other antimitotic agents that interact with microtubules, mediate serine phosphorylation of the 66-kDa Shc isoform (p66shc) in A549 human lung carcinoma cells, 9-18 h after drug treatment. This event coincides with the release of PARP cleavage fragments that are early indicators of apoptosis. Taxol-induced serine phosphorylation of p66shc results from a MEK-independent signaling pathway that is activated in A549 cells that have a prolonged or abnormal mitotic phase of the cell cycle [Cancer Res. 60 (2000) 5171]. In contrast, in murine macrophage RAW 264.7 cells, micromolar concentrations of Taxol but not other microtubule-interacting agents induced serine phosphorylation of p66shc that correlated with the phosphorylation of Raf-1 and extracellular signal-regulated kinase (ERK1/2), within 15-30 min after Taxol treatment. This event also was induced by lipopolysaccharide (LPS). The MEK-inhibitor, U0126, that specifically inhibits the activation of ERK also blocked the phosphorylation of p66shc and Raf-1, suggesting that these processes were MEK-dependent, quite different from that which was observed in A549 cells. Taxol also induced phosphorylation of p38 and JNK MAP kinases within 8-15 min after drug treatment. It is known that Taxol, but not other microtubule-interacting agents, induces the production of cytokines, such as tumor necrosis factor alpha (TNF-alpha) in mouse macrophages. The time course of Taxol-induced TNF-alpha expression coincides with that of Taxol-induced p66shc phosphorylation, and U0126 inhibits significantly Taxol-induced TNF-alpha expression in RAW 264.7 cells. Our data indicate that the Taxol-induced serine phosphorylation of p66shc in RAW 264.7 cells is microtubule-independent and may be related to increased TNF-alpha expression after Taxol and LPS treatment. It is concluded that the mechanisms involved in Taxol-induced p66shc phosphorylation are distinct in A549 and RAW 264.7 cells.     

MnTBAP, a synthetic metalloporphyrin, inhibits production of tumor necrosis factor-alpha in lipopolysaccharide-stimulated RAW 264.7 macrophages cells via inhibiting oxidative stress-mediating p38 and SAPK/JNK signaling

Antioxidants are able to inhibit inflammatory gene expression in response to lipopolysaccharide via down-regulating generation of intracellular reactive oxygen species (ROS) as second messengers. The effect of manganese (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a synthetic metalloporphyrin with antioxidant activity, on tumor necrosis factor (TNF)-alpha production in lipopolysaccharide-stimulated RAW 264.7 macrophage cells was examined. MnTBAP prevented the generation of intracellular ROS in lipopolysaccharide-stimulated RAW 264.7 cells and further inhibited lipopolysaccharide-induced TNF-alpha production. MnTBAP exclusively prevented the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK/JNK) whereas it did not affect the phosphorylation and activation of nuclear factor-kappaB and extracellular signal regulated kinase 1/2. MnTBAP was suggested to inhibit lipopolysaccharide-induced TNF-alpha production by the prevention of intracellular ROS generation and subsequent inactivation of p38 MAPK and SAPK/JNK.     

Two opposing effects of non-steroidal anti-inflammatory drugs on the expression of the inducible cyclooxygenase. Mediation through different signaling pathways

The efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) is considered to be a result of their inhibitory effect on cyclooxygenase (COX) activity. Here, we report that flufenamic acid shows two opposing effects on COX-2 expression; it induces COX-2 expression in the colon cancer cell line (HT-29) and macrophage cell line (RAW 264.7); conversely, it inhibits tumor necrosis factor alpha (TNFalpha)- or lipopolysaccharide (LPS)-induced COX-2 expression. This inhibition correlates with the suppression of TNFalpha- or LPS-induced NFkappaB activation by flufenamic acid. The inhibitor of extracellular signal-regulated protein kinase, p38, or NFkappaB does not affect the NSAID-induced COX-2 expression. These results suggest that the NSAID-induced COX-2 expression is not mediated through activation of NFkappaB and mitogen-activated protein kinases. An activator of peroxisome proliferator-activated receptor gamma, 15-deoxy-Delta(12,14)-prostaglandin J(2), also induces COX-2 expression and inhibits TNFalpha-induced NFkappaB activation and COX-2 expression. Flufenamic acid and 15-deoxy-Delta(12,14)-prostaglandin J(2) also inhibit LPS-induced expression of inducible form of nitric-oxide synthase and interleukin-1alpha in RAW 264.7 cells. Together, these results indicate that the NSAIDs inhibit mitogen-induced COX-2 expression while they induce COX-2 expression. Furthermore, the results suggest that the anti-inflammatory effects of flufenamic acid and some other NSAIDs are due to their inhibitory action on the mitogen-induced expression of COX-2 and downstream markers of inflammation in addition to their inhibitory effect on COX enzyme activity.     

Caffeic acid phenethyl ester protects mice from lethal endotoxin shock and inhibits lipopolysaccharide-induced cyclooxygenase-2 and inducible nitric oxide synthase expression in RAW 264.7 macrophages via the p38/ERK and NF-kappaB pathways

Caffeic acid phenethyl ester has been shown to have anti-inflammatory and anti-cancer effects. We examined the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced production of nitric oxide and prostaglandin E(2), and expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 macrophages. We also investigated the effects of caffeic acid phenethyl ester on lipopolysaccharide-induced septic shock in mice. Our results indicate that caffeic acid phenethyl ester inhibits lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production in a concentration-dependent manner and inhibits inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7 cells, without significant cytotoxicity. To further examine the mechanism responsible for the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by caffeic acid phenethyl ester, we examined the effect of caffeic acid phenethyl ester on lipopolysaccharide-induced nuclear factor-kappaB activation and the phosphorylation of mitogen-activated protein kinases. Caffeic acid phenethyl ester treatment significantly reduced nuclear factor-kappaB translocation and DNA-binding in lipopolysaccharide-stimulated RAW 264.7 cells. This effect was mediated through the inhibition of the degradation of inhibitor kappaB and by inhibition of both p38 mitogen-activated protein kinase and extracellular signal-regulated kinase phosphorylation, at least in part by inhibiting the generation of reactive oxygen species. Furthermore, caffeic acid phenethyl ester rescued C57BL/6 mice from lethal lipopolysaccharide-induced septic shock, while decreasing serum levels of tumor necrosis factor-alpha and interleukin-1beta. Collectively, these results suggest that caffeic acid phenethyl ester suppresses the induction of cytokines by lipopolysaccharide, as well as inducible nitric oxide synthase and cyclooxygenase-2 expression, by blocking nuclear factor-kappaB and p38/ERK activation. These findings provide mechanistic insights into the anti-inflammatory and chemopreventive actions of caffeic acid phenethyl ester in macrophages.     

TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-kappaB in lipopolysaccharide-stimulated macrophages

Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-kappaB (NF-kappaB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-beta activated kinase 1 (TGF-TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), in the LPS-induced signaling cascade. A kinase-negative mutant of TAK1 inhibited the LPS-induced NF-kappaB activation both in a macrophage-like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll-like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS-induced signaling pathway.