Heparin stimulation of plasminogen activator secretion by macrophage-like cell line RAW264.7: role of the scavenger receptor

Secretion of urokinase-type plasminogen activator (uPA) by RAW264.7 cells was stimulated by heparin in a dose- and time-dependent manner. Secretion of uPA was not detected when cells were exposed to heparin at 4 degrees C, indicating that heparin was not simply releasing receptor-bound uPA. Furthermore, prior removal of membrane-associated uPA did not influence heparin's ability to stimulate the release of uPA from the macrophage-like line. Low molecular weight and weakly anticoagulant heparins stimulated uPA secretion but less effectively than other heparin fractions. The observed stimulation in macrophage uPA secretion by heparin is similar to that previously reported for polyanions recognized by the scavenger receptor including fucoidan, polyinosinic acid, dextran sulfate, and acetyl-LDL (Falcone and Ferenc: J. Cell. Physiol., 135:387-396, 1988). Evidence that heparin's binding to RAW264.7 cells is mediated by the scavenger receptor is derived from experiments in which fucoidan blocked the specific binding of [3H]-heparin to RAW264.7 cells. However, heparin partially inhibited the stimulation of cholesteryl [3H]-oleate synthesis observed in these cells upon incubation with acetyl-LDL and weakly inhibited cellular binding of 125I-acetyl-LDL at 4 degrees C. These data indicate that heparin's binding to RAW264.7 cells is mediated, only in part, by the scavenger receptor. Nonetheless, neither heparin nor fucoidan was able to stimulate the release of plasminogen activator activity from monocyte-like U937 cells which are devoid of scavenger receptor activity.     

Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors

It has been reported that ligands of the macrophage scavenger receptor (MSR) induce a range of cellular responses including urokinase-type plasminogen activator and the production of inflammatory cytokines. Although nitric oxide (NO) is an important regulatory molecule in physiological functions such as vascular homeostasis, neurotransmission, and host defense, the effect of MSR ligands on NO production from macrophages was unknown. Here, we demonstrate that the MSR ligand, fucoidan, but neither oxidized low-density lipoprotein, acetylated LDL, maleylated bovine serum albumin nor dextran sulfate induces activation of inducible nitric oxide synthase (iNOS) promoter or NO production in RAW264.7 cells. Furthermore, we investigated the molecular mechanism by which fucoidan induces iNOS promoter activation. Using different inhibitors, we showed that the stimulation of fucoidan was mediated by both the p38 mitogen-activated protein kinase and the NF-kappaB-dependent pathways. Although these two pathways were independent, heat shock protein 90 (HSP90) played a significant role in both pathways. Our previous study showed that HSP90 directly interacts with the cytoplasmic domain of MSR. These results provide the evidence that HSP90 bound to the cytoplasmic domain of MSR is implicated in MSR-mediated signal transduction. Moreover, fucoidan-induced NO production by peritoneal macrophages from MSR-knockout (MSR-/-) mice significantly decreases compared with those from wild-type mice. This is the first indication that MSR transduces the signal of fucoidan to iNOS gene expression.     

Secretion of macrophage urokinase plasminogen activator is dependent on proteoglycans.

The importance of proteoglycans for secretion of proteolytic enzymes was studied in the murine macrophage cell line J774. Untreated or 4beta-phorbol 12-myristate 13-acetate (PMA)-stimulated macrophages were treated with hexyl-beta-d-thioxyloside to interfere with the attachment of glycosaminoglycan chains to their respective protein cores. Activation of the J774 macrophages with PMA resulted in increased secretion of trypsin-like serine proteinase activity. This activity was completely inhibited by plasminogen activator inhibitor 1 and by amiloride, identifying the activity as urokinase plasminogen activator (uPA). Treatment of both the unstimulated or PMA-stimulated macrophages with xyloside resulted in decreased uPA activity and Western blotting analysis revealed an almost complete absence of secreted uPA protein after xyloside treatment of either control- or PMA-treated cells. Zymography analyses with gels containing both gelatin and plasminogen confirmed these findings. The xyloside treatment did not reduce the mRNA levels for uPA, indicating that the effect was at the post-translational level. Treatment of the macrophages with xylosides did also reduce the levels of secreted matrix metalloproteinase 9. Taken together, these findings indicate a role for proteoglycans in the secretion of uPA and MMP-9.     

Transforming growth factor-β1 stimulates macrophage urokinase expression and release of matrix-bound basic fibroblast growth factor

Macrophage expression of urokinase-type plasminogen activator (uPA) appears to play a role in their release of matrix-bound basic fibroblast growth factor (bFGF) and transforming growth factor-β (TGF-β). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF-β1 on macrophage uPA expression. TGF-β1 stimulated in a dose- and time-dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin-1α, tumor necrosis factor-α, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF-β1 led to a rapid and sustained increase in the steady-state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF-β1-induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitior H7 markedly reduced the ability of TGF-β1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF-β1 to upregulate macrophage uPA expression. TGF-β1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane-bound plasmin than control cells. Preincubation of TGF-β1 with either serum or methylamine-modified α2-macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF-β1-primed macrophages were cultured on matrices containing bound¹²⁵I-bFGF, their release of ¹²⁵I-bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF-β to induce macrophage uPA expression and the plasmin-dependent release of matrix-bound bFGF may provide an indirect mechanism by which TGF-β stimulates angiogenesis. © 1993 Wiley-Liss, Inc.     

Urokinase plasminogen activator receptor promotes macrophage infiltration into the vascular wall of ApoE deficient mice

The urokinase plasminogen activator receptor (uPAR) regulates macrophage adhesion and migration by binding directly to matrix proteins and signaling through integrin complexes. In this study, we examined the role of uPAR on macrophage infiltration into the vascular wall. Stable murine macrophage (Raw264.7) cell lines expressing high levels of human uPAR, human urokinase plasminogen activator (uPA), or both were established using expression vectors driven by the human CD68 promoter. Stimulation with human uPA specifically induced phosphorylation of early response regulated kinase (ERK) in cells expressing human uPAR but not in sham transfected cells. The human uPAR expressing Raw264.7 cells showed increased adhesion to both human uPA and vitronectin (Vn). Raw264.7 cells expressing human uPAR or both human uPAR and uPA, but not uPA alone, were detected in the aortic wall of ApoE(-/-) mice, and no cells were detected in that of age-matched C57BL/6J mice after intravenous infusion of the cells. Blocking of Mac-1/ICAM-1 interaction by anti-alphaM antibody (M1/70) significantly reduced the infiltration of huPAR-expressing Raw264.1 cells into aorta of ApoE(-/-) mice. Treatment of C57BL/6J mice with angiotensin II resulted in infiltration of Raw264.7 cells expressing human uPAR. These data demonstrate that uPAR plays a key role in promoting macrophage infiltration into the arterial wall of ApoE(-/-) mice.     

THP-1 macrophage membrane-bound plasmin activity is up-regulated by transforming growth factor-β1 via increased expression of urokinase and the urokinase receptor

Receptors for urokinase (uPA) and plasminogen provide a mechanism to direct the cellular activation of plasminogen. The regulation of these receptors is important for several macrophage functions. In these studies, the effect of transforming growth factor-b?1 (TGF-b?1) on uPA, uPA receptor, and plasminogen receptor expression by human THP-1 macrophage was examined. TGF-b?1 induction of uPA expression by THP-1 cells was differentiation dependent. Suspension and adherent cultures expressed similar constitutive levels of uPA. Exposure of adherent cells to TGF-b?1 led to a dose- and time-dependent increase in uPA activity which was paralleled by an increase in uPA antigen and uPA mRNA. In contrast, uPA expression by suspension cultures was unresponsive to TGF-b?1. The differential response exhibited by suspension and adherent THP-1 cells may reflect differences in their expression of TGF-b?1 receptors, since when assayed by crosslinking techniques, suspension cells primarily expressed a 65 kDa receptor; whereas, the adherent cells expressed 65 and 100 kDa receptors. TGF-b?1-induced alterations in uPA receptor expression by adherent THP-1 cells were examined by quantitating membrane-bound uPA activity. Membrane-bound uPA activity increased three-fold when cells were incubated with TGF-b?1. The increase in membrane-uPA activity expressed by TGF-b?1-treated cells was not due to increased uPA receptor occupancy since incubation of either control or TGF-b?1 primed cells with exogenous uPA did not lead to an increase in membrane-bound uPA activity. Furthermore, immunoreactive uPA receptor was increased in TGF-b?1-treated cells. Following incubation with plasminogen, membrane-bound plasmin activity increased three-fold in TGF-b?1-treated cells. However, no change in immunoreactive membrane-bound plasmin(ogen) was observed. In addition, binding of ¹²⁵I-Lys-plasminogen to THP-1 cells was not affected by TGF-b?1 treatment. We conclude that TGF-b?1 stimulates membrane-bound plasmin activity, without affecting plasminogen receptor expression, through the up-regulation of uPA and the uPA receptor expression. © 1995 Wiley-Liss, Inc.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

CEL-I, an invertebrate N-acetylgalactosamine-specific C-type lectin, induces TNF-alpha and G-CSF production by mouse macrophage cell line RAW264.7 cells

Our previous studies demonstrated that CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin purified from the marine invertebrate Cucumaria echinata (Holothuroidea) showed potent cytotoxicity to several cell lines such as HeLa, MDCK and XC cells. In this study, we found that CEL-I induced increased secretion of tumour necrosis factor-alpha (TNF-alpha) and granulocyte colony stimulation factor (G-CSF) by mouse macrophage cell line RAW264.7 cells in a dose-dependent manner, whereas this cell line was highly resistant to CEL-I cytotoxicity. The cytokine-inducing activity of CEL-I was stronger than that of phytohaemagglutinin (PHA-L). A binding study using FITC-labelled CEL-I (F-CEL-I) indicated that the amount of bound F-CEL-I on RAW264.7 cells was greater than that of F-PHA-L, suggesting that the greater activity of CEL-I to induce cytokine secretion by RAW264.7 cells is partly due to the higher binding ability. Since the cell binding and cytokine-inducing activity of CEL-I were partly but significantly inhibited by the specific sugar (GalNAc), it is considered that the binding of CEL-I to cell-surface-specific saccharide moieties, which may be recognized by CEL-I with higher affinity than GalNAc, is essential for the induction of cytokine secretion. The secretion of TNF-alpha and G-CSF from CEL-I-treated RAW264.7 cells were almost completely prevented by brefeldin A (BFA), whereas increase in mRNA levels of these cytokines were not affected by BFA. Bio-Plex beads assay suggested that temporal increase in phosphorylation of extracellular-regulated kinase (ERK), c-jun NH(2)-terminal kinase (JNK) and p38 MAP kinase occurred at relatively early time following CEL-I treatment. Furthermore, the secretion of TNF-alpha and G-CSF were inhibited by specific inhibitors for these MAP kinases. These results suggest that the intracellular signal transduction through the activation of MAP kinase system is involved in CEL-I-induced cytokine secretion.     

uPA Attenuated LPS-induced Inflammatory Osteoclastogenesis through the Plasmin/PAR-1/Ca2+/CaMKK/AMPK Axis

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis-caused bone destruction, results from an increase of bone-resorbing osteoclasts (OCs) induced by inflammation. However, the detailed mechanisms underlying this disorder remain unclear. We herein investigated that the effect of urokinase-type plasminogen activator (uPA) on inflammatory osteoclastogenesis induced by lipopolysaccharide (LPS), which is a potent stimulator of bone resorption in inflammatory diseases. We found that the uPA deficiency promoted inflammatory osteoclastogenesis and bone loss induced by LPS. We also showed that LPS induced the expression of uPA, and the uPA treatment attenuated the LPS-induced inflammatory osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells. Additionally, we showed that the uPA-attenuated inflammatory osteoclastgenesis is associated with the activation of plasmin/protease-activated receptor (PAR)-1 axis by uPA. Moreover, we examined the mechanism underlying the effect of uPA on inflammatory osteoclastogenesis, and found that uPA/plasmin/PAR-1 activated the adenosine monophosphate-activated protein kinase (AMPK) pathway through Ca2+/calmodulin dependent protein kinase kinase (CaMKK) activation, and attenuated inflammatory osteoclastogenesis by inactivation of NF-κB in RAW264.7 cells. These data suggest that uPA attenuated inflammatory osteoclastogenesis through the plasmin/PAR-1/Ca2+/CaMKK/AMPK axis. Our findings may provide a novel therapeutic approach to bone loss caused by inflammatory diseases.     

Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.     

Suppression of urokinase expression and invasiveness by urinary trypsin inhibitor is mediated through inhibition of protein kinase C- and MEK/ERK/c-Jun-dependent signaling pathways

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, interacts with cells as a negative modulator of the invasive cells. Human ovarian cancer cell line, HRA, was treated with phorbol ester (PMA) to evaluate the effect on expression of urokinase-type plasminogen activator (uPA), since the action of uPA has been implicated in matrix degradation and cell motility. Preincubation of the cells with UTI reduced the ability of PMA to trigger the uPA expression at the gene level and at the protein level. UTI-induced down-regulation of PMA-stimulated uPA expression is irreversible and is independent of a cytotoxic effect. Down-regulation of uPA by UTI is mediated by its binding to the cells. We next asked whether the mechanism of inhibition of uPA expression by UTI was due to interference with the protein kinase C second messenger system. An assay for PKC activity demonstrated that UTI does not directly inhibit the catalytic activity of PKC and that PMA translocation of PKC from cytosol to membrane was inhibited by UTI, indicating that UTI inhibits the activation cascade of PKC. PMA could also activate a signaling pathway involving MEK1/ERK2/c-Jun-dependent uPA expression. When cells were preincubated with UTI, we could detect suppression of phosphorylation of these proteins. Like several types of PKC inhibitor, UTI inhibited PMA-stimulated invasiveness. We conclude that UTI markedly suppresses the cell motility possibly through negative regulation of PKC- and MEK/ERK/c-Jun-dependent mechanisms, and that these changes in behavior are correlated with a coordinated down-regulation of uPA which is likely to contribute to the cell invasion processes.     

Fucoidan Isolated from Laminaria angustata var. longissima Induced Macrophage Activation

We investigated macrophage activation by fucoidan from Laminaria angustata var. longissima in a murine macrophage cell line, RAW 264.7. The ratio of the chemical composition of the fucoidan was L-fucose:D-galactose:D-glucose:D-xylose:uronic acid:sulfate = 1.00:0.54:0.08:0.08:0.64:0.87. The fucoidan induced production of nitric oxide, tumor necrosis factor-α, and interleukin-6 in RAW 264.7 cells. These results indicate that the fucoidan induced macrophage activation.     

Ligands of macrophage scavenger receptor induce cytokine expression via differential modulation of protein kinase signaling pathways

Our previous works demonstrated that ligands of macrophage scavenger receptor (MSR) induce protein kinases (PKs) including protein-tyrosine kinase (PTK) and up-regulate urokinase-type plasminogen activator expression (Hsu, H. Y., Hajjar, D. P., Khan, K. M., and Falcone, D. J. (1998) J. Biol. Chem. 273, 1240--1246). To continue to investigate MSR ligand-mediated signal transductions, we focus on ligands, oxidized low density lipoprotein (OxLDL), and fucoidan induction of the cytokines tumor necrosis factor-alpha (TNF) and interleukin 1 beta (IL-1). In brief, in murine macrophages J774A.1, OxLDL and fucoidan up-regulate TNF production; additionally, fucoidan but not OxLDL induces IL-1 secretion, prointerleukin 1 (proIL-1, precursor of IL-1) protein, and proIL-1 message. Simultaneously, fucoidan stimulates activity of interleukin 1-converting enzyme. We further investigate the molecular mechanism by which ligand binding-induced PK-mediated mitogen-activated protein kinase (MAPK) in regulation of expression of proIL-1 and IL-1. Specifically, fucoidan stimulates activity of p21-activated kinase (PAK) and of the MAPKs extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38. Combined with PK inhibitors and genetic mutants of Rac1 and JNK in PK activity assays, Western blotting analyses, and IL-1 enzyme-linked immunosorbent assay, the role of individual PKs in the regulation of proIL-1/IL-1 was extensively dissected. Moreover, tyrosine phosphorylation of pp60Src as well as association between pp60Src and Hsp90 play important roles in fucoidan-induced proIL-1 expression. We are the first to establish two fucoidan-mediated signaling pathways: PTK(Src)/Rac1/PAK/JNK and PTK(Src)/Rac1/PAK/p38, but not PTK/phospholipase C-gamma 1/PKC/MEK1/ERK, playing critical roles in proIL-1/IL-1 regulation. Our current results indicate and suggest a model for MSR ligands differentially modulating specific PK signal transduction pathways, which regulate atherogenesis-related inflammatory cytokines TNF and IL-1.     

微进化对阿萨希毛孢子菌与小鼠巨噬细胞RAW264.7相互作用的影响

目的研究微进化对阿萨希毛孢子菌(T.asahii)与巨噬细胞RAW264.7相互作用的影响。方法将微进化前后的T.asahii与RAW264.7细胞共培养,检测RAW264.7对两株菌的吞噬和杀伤能力以及两株菌对RAW264.7产生的细胞毒性的差异,同时分析RAW264.7自身细胞因子分泌情况的变化。结果巨噬细胞对原代菌株(TO)的吞噬能力以及杀伤率均明显高于微进化株(TEVO);TO菌株对巨噬细胞的细胞毒性要强于TEVO;当巨噬细胞与菌株按1∶3或1∶6共培养24 h时,与TO共培养的巨噬细胞TNF-α和IL-6的分泌量要高于TEVO组,而按1∶9共培养时TEVO组细胞因子的分泌量却高于TO组。结论微进化后的TEVO菌株与巨噬细胞的相互作用明显弱于原代株TO,这也为TEVO菌株在宿主体内长期共存提供了良好的基础。     

Different Suppressive Effects of Fucoidan and Lentinan on IL-8 mRNA Expression in in Vitro Gut Inflammation

A system for assessing the anti-inflammatory effects of food factors was developed by establishing a co-culture system with intestinal epithelial Caco-2 cells and macrophage RAW264.7 cells. The results indicate that fucoidan and lentinan exhibited different suppressive effects on interleukin-8 (IL-8) mRNA expression in Caco-2 through tumor necrosis factor-α (TNF-α) production from RAW264.7 stimulated with lipopolysaccharide (LPS).     

Stimulation of macrophage by enzymatically synthesized glycogen: the relationship between structure and biological activity

Glycogen is exclusively known as an energy and carbon reserve in animal cells and micro-organisms. We synthesized glycogens of varying molecular weight by using three enzymes, and investigated the relationship between the structure and immunostimulating activity of glycogen. These results indicated that glycogens with a molecular weight of more than 1.0×10⁷ hardly activated RAW264.7, a murine macrophage cell line, whereas glycogens of 5.0–6.5×10⁶ strongly stimulated RAW264.7 in the presence of interferon-γ, leading to augmented production of nitric oxide, tumour necrosis factor-α and interleukin-6. Additionally, the number-average unit chain length and the exterior and interior chain lengths of the glycogens showed a minor correlation between active and less-active glycogen derivatives. On the other hand, the binding activity of glycogen toward RAW264.7 did not depend on the molecular weight of glycogen. The available evidence suggests that the macrophage-stimulating activity of glycogen is strictly related to its molecular weight rather than to fine structural properties.     

Phosphorylation of tumor necrosis factor receptor 1 (p55) protects macrophages from silica-induced apoptosis

Macrophages play a fundamental role in silicosis in part by removing silica particles and producing inflammatory mediators in response to silica. Tumor necrosis factor alpha (TNFalpha) is a prominent mediator in silicosis. Silica induction of apoptosis in macrophages might be mediated by TNFalpha. However, TNFalpha also activates signal transduction pathways (NF-kappaB and AP-1) that rescue cells from apoptosis. Therefore, we studied the TNFalpha-mediated mechanisms that confer macrophage protection against the pro-apoptotic effects of silica. We will show that exposure to silica induced TNFalpha production by RAW 264.7 cells, but not by IC-21. Silica-induced activation of NF-kappaB and AP-1 was only observed in RAW 264.7 macrophages. ERK activation in response to silica exposure was only observed in RAW 264.7 macrophages, whereas activation of p38 phosphorylation was predominantly observed in IC-21 macrophages. No changes in JNK activity were observed in either cell line in response to silica exposure. Silica induced apoptosis in both macrophage cell lines, but the induction of apoptosis was significantly larger in IC-21 cells. Protection against apoptosis in RAW 264.7 cells in response to silica was mediated by enhanced NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNFalpha receptor. Inhibition of these two protective mechanisms by specific pharmacological inhibitors or transfection of dominant negative mutants that inhibit IkappaBalpha or ERK phosphorylation significantly increased silica-induced apoptosis in RAW 264.7 macrophages. These data suggest that NF-kappaB activation and ERK-mediated phosphorylation of the p55 TNF receptor are important cell survival mechanisms in the macrophage response to silica exposure.