Activation of TNF-transcription utilizes distinct MAP kinase pathways in ...

Activation of TNF-a transcription utilizes distinct MAP kinase pathways in different macrophage populations Terry K. Means,*† Ryan P. Pavlovich,† Dominic Roca,* Mary W. Vermeulen,‡ and Matthew J. Fenton*† *Pulmonary Center and †Department of Pathology, Boston University School of Medicine, Massachusetts; and ‡Eisai Research Institute, Andover, Massachusetts

Abstract: Stimulation of macrophages by lipopolysaccharide (LPS) leads to the rapid activation of MAP kinases (MAPK) and the subsequent induction of cytokine gene expression. We sought to determine whether LPS-inducible cytokine genes were differentially regulated in macrophages derived from different tissues. Our studies revealed that PD98059, an inhibitor of the extracellular-regulated kinase (ERK) pathway, blocked LPS-induced activation of tumor necrosis factor a (TNF-a) gene expression in a murine cell line derived from alveolar macrophages but not in a nonpulmonary macrophage cell line. These findings were confirmed using primary murine alveolar and peritoneal macrophages. This suggests that the TNF-a promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell type-specific manner. We also found that differences in MAPK-regulated signaling were not mediated by NF-kB, LITAF, Egr-1, CREB, or ATF2/ c-Jun. Together, these studies demonstrate that transcriptional activation of the TNF-a gene requires the ERK signaling cascade in selected macrophage populations. J. Leukoc. Biol. 67: 885–893; 2000. Key Words: lipopolysaccharide · cytokines · signal transduction · alveolar macrophages

INTRODUCTION Gram-negative bacterial lipopolysaccharide (LPS) is released during bacterial infection, and induces macrophages to produce a variety of proinflammatory cytokines [1]. One of the most important cytokine mediators is tumor necrosis factor a (TNFa), which is rapidly secreted in large quantities by macrophages following LPS stimulation. Passive immunization against TNF-a protects against the lethal effects of LPS, and direct administration of purified TNF-a causes tissue injury and shock, similar to that observed in bacterial sepsis [2, 3]. The macrophage receptor for LPS is CD14, a glycosylphosphatidylinositol (GPI)-linked membrane protein whose constitutive expression is limited to monocytic cells and neutrophils. Because CD14 lacks transmembrane and intracellular domains, the mechanism by which an intracellular signal is

generated following LPS binding remains unclear [4]. Recent data support the hypothesis that Toll-like receptor proteins mediate the generation of intracellular signals following engagement of CD14 by LPS [5–9]. Although the details of this mechanism remain to be determined, it is clear that LPS activates multiple intracellular signaling pathways. The LPS signaling pathway leading to TNF-a biosynthesis is unknown but has been reported to depend on the activation of protein tyrosine kinases, the proto-oncogene product Ras, and the serine-threonine kinase Raf-1 (reviewed in [10]). These signaling events lead to the activation of several mitogenactivated protein kinase (MAPK) signaling pathways. Three major MAPK families have been found to be activated by LPS. These are the extracellular-regulated kinases (ERK), p38 kinases, and c-Jun N-terminal kinases (JNK). These kinases are activated via phosphorylation by a family of MAPK kinases (MEK). Although LPS activates all three major MAPK families, these kinases can also be differentially activated in response to other stimuli. The ERK are selectively activated by growth factors and phorbol esters, whereas JNK and p38 are selectively activated by cellular stress, exposure to UV light, or exogenous TNF-a [11–13]. MAPK are known to phosphorylate, and thereby activate, many transcription factors, although the connection between these signaling kinases and transcription factors that control TNF-a gene expression has not been determined. Several transcription factors have been shown to regulate the TNF-a promoter in macrophages, including Ets, Egr-1, CREB, ATF2/c-Jun, and LITAF [14–16]. Our studies were directed at determining whether the MEK/ERK pathway is required for LPS-induced TNF-a transcription and whether TNF-a expression in distinct macrophage populations differs in the requirement for MAPKs. We used PD98059, a specific inhibitor of the MEK/ERK pathway, to address this question. PD98059 acts by blocking activation of ERK by the upstream MAPK kinases MEK-1 and MEK-2 [17]. We found that MAPKs appear to mediate TNF-a expression in alveolar macrophages but did not appear to be required for TNF-a expression in peritoneal or monocyte-derived macrophages. Our data suggest that LPS activates TNF-a transcrip-

Correspondence: Dr. Matthew J. Fenton, Pulmonary Center, Boston University School of Medicine, 80 East Concord Street, Boston MA 02118-2394. E-mail: [email protected] Received November 14, 1999; revised January 14, 2000; accepted January 18, 2000.

Journal of Leukocyte Biology

Volume 67, June 2000

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tion via distinct signaling pathways in these different macrophage populations.

MATERIALS AND METHODS Animals and cell culture C57Bl/6 and BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, ME). The murine macrophage cell line RAW264.7 (TIB-71) was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The alveolar macrophage cell line AMJ2C-8 (C-8) was a gift of Dr. Alicia Palleroni (Hoffman LaRoche, Nutley, NJ) and has been previously described [18]. Resident peritoneal macrophages were recovered from female C57Bl/6 and BALB/c mice by instilling and withdrawing 10 ml of sterile, serum-free RPMI-1640 media from the peritoneal cavity. Primary alveolar macrophages were collected by instilling and withdrawing 1 ml of sterile phosphate-buffered saline (PBS) from the lungs via intratracheal incubation. Peritoneal and alveolar macrophages were allowed to adhere for 2 h to the culture dishes, the nonadherent cells were removed, and the cells were cultured for 2–3 days before they were used in any experiments. RAW264.7, C-8, and primary murine macrophages were cultured in RPMI-1640 culture medium (Biowhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), 10 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ·g/ml streptomycin (Biowhittaker). LPS levels in all media and reagents were less than 10 pg/ml as determined using a quantitative Limulus lysate assay (Biowhittaker). Cells were cultured at 37°C in the presence of 5% CO2 using a humidified incubator. LPS (purified from Escherichia coli 055:B5) was purchased from Sigma (St. Louis, MO). The MEK-1 inhibitor PD98059 was purchased from New England Biolabs (Beverly, MA).

Plasmids The luciferase reporter plasmids pTNF(21311)Luc, pTNF(21185)Luc, and pTNF(2615)Luc, containing portions of the human TNF-a promoter, were provided by Dr. James Economou (University of California, Los Angeles) and have been previously described [19]. The luciferase reporter plasmids pTNF(Egr-1 mut)Luc, pTNF(CRE mut)Luc, and pTNF(kB3 mut)Luc were generously provided by Dr. Nigel Mackman (Scripps Research Institute, La Jolla, CA) and were prevoiusly described [15]. A luciferase reporter plasmid containing a 180 bp fragment of the TNF-a promoter [pTNF(2180)Luc] was generated from the pTNF(21311)Luc construct using polymerase chain reaction (PCR). The luciferase reporter plasmids p-mTNF(21059)Luc and p-mTNF(2200)Luc, containing portions of the murine TNF-a promoter, were subcloned from chloramphenicol acetyltransferase (CAT) reporter plasmids provided by Dr. Steven Taffet (State University of New York, Syracuse). The endothelial leukocyte adhesion molecule (ELAM)-Luc reporter plasmid was generated by subcloning a portion of the nuclear factor (NF)-kB-dependent ELAM-1 promoter into the promoterless pGL3 luciferase reporter plasmid (Promega, Madison, WI) [20]. The pXT-Luc reporter plasmid contains a 3.7-kb portion of the human interleukin (IL)-1b promoter (positions 23757 to 1 11) ligated to the luciferase gene in the pGL3 reporter plasmid and was previously described [21]. Plasmids were prepared using Qiagen (Valencia, CA) plasmid DNA purification columns. DNA was eluted from the columns using LPS-free buffers, and contaminating LPS levels were found to be less than 10 pg/ml.

Transfection and reporter assays Transient transfections were performed using Super-Fect reagent (Qiagen), as recommended by the manufacturer. Briefly, cells (53105) were plated in 6-well dishes, cultured for 1–2 days until the cells were 80% confluent, and then transfections were performed. Plasmid DNA (3 µg total) was added to 100 µl of OPTI-MEM reduced-serum media (GIBCO-BRL, Grand Island, N.Y.). SuperFect (10 µl) was added to the DNA-media mixture and incubated for 10 min at ambient temperature. Subsequently, 600 µl of serum-containing RPMI-1640 media was added to each DNA mixture, and the entire mixture was then added to the individual wells. Each DNA mixture was prepared individually, and each transfection was performed in triplicate. DNA-containing mixtures were incubated with the cells for 2–3 h, whereupon the mixtures were removed from

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the cells, and fresh serum-containing media was added. On the following day, individual wells were left untreated or pretreated with PD98059 for 2 h and then stimulated with LPS as indicated in the text. Cells were then incubated for an additional 5 h prior to harvesting. A single representative experiment is shown, although all transfection experiments were repeated at least three times using different plasmid preparations. Each single experiment represents triplicate independent transfections, and data are expressed as mean values 6 SD. Luciferase reporter activity was measured using the Luciferase Assay System (Promega), according to the manufacturer’s instructions. Briefly, cells were washed and scraped on ice in cold PBS, pelleted by centrifugation, and resuspended in 100 µl of lysis buffer. Samples were freeze-thawed once and centrifuged at 14,000 g for 10 min at 4°C to remove cellular debris. Supernatants were recovered and assayed for total protein using a Bio-Rad (Hercules, CA) protein assay kit according to the manufacturer’s instructions. Equal amounts of total protein from each lysate were assayed for luciferase activity, as measured by light emissions in a liquid scintillation counter.

RNase protection assay The RiboQuant multiprobe ribonuclease protection assay system by Pharmingen (San Diego, CA) was used to measure the expression of several mRNA species simultaneously. Assays were performed according to the manufacturer’s specifications. Briefly, total RNA was purified from cells using TriReagent (Molecular Research Center, Inc., Cincinnati, OH), according to the manufacturer’s protocol. Target RNA (10 µg) was hybridized overnight to the selected [a32P]UTP-radiolabeled, antisense RNA probes. The hybrids were subjected to RNase digestion, to remove unbound probe, and other single-stranded RNA. The samples were then fractionated on polyacrylamide sequencing gels and visualized using autoradiography.

Cytokine assays TNF-a and IL-1b protein levels in the culture supernatants were measured by sandwich ELISA (Biosource, Camarillo, CA). Samples were assayed in triplicate, and the data are expressed as average values 6 SD.

RESULTS PD98059 differentially affects LPS-induced TNF-a mRNA production in C-8 and RAW264.7 cells We tested whether the MEK/ERK pathway was necessary for LPS-induced activation of proinflammatory cytokine expression in different macrophage populations. RNase protection assays were performed using RNA purified from the murine macrophage cell line RAW264.7 and the murine alveolar macrophagederived cell line C-8. A portion of the cells were pretreated for 2 h with 20 µM PD98059. Untreated and pretreated cells were then stimulated with 100 ng/ml LPS for 4 h. As shown in Figure 1, PD98059 was able to block LPS-induced TNF-a mRNA expression in the C-8 cells. In contrast, the MEK/ERK inhibitor did not block TNF-a gene activation by LPS in the RAW264.7 cell line. PD98059 blocked IL-1b mRNA expression in both macrophage lines. However, PD98059 failed to block LPS-induced IL-6 mRNA expression in either macrophage lines. To confirm that PD98059 could block ERK activity in both cell lines, we measured the activation of ERK-1 and ERK-2 in these cells by western blotting using an antiphosphoERK antibody. We found that PD98059, at the concentration used to block TNF-a expression, did block LPS-inducible ERK phosphorylation without affecting cell viability (data not shown). The above data suggested that TNF-a is regulated http://www.jleukbio.org

the presence and absence of PD98059, as described above. Following stimulation for 4 h, culture supernatants were collected, and TNF-a protein levels were measured by ELISA. As shown in Figure 2, TNF-a protein levels were readily induced in both cell lines following LPS stimulation. However, PD98059 was able to block TNF-a production in the C-8 cells in a dose-dependent fashion but not in the RAW264.7 cell line. Thus, the sensitivity of C-8 alveolar macrophages to the MEK/ERK inhibitor at the level of protein secretion correlated with TNF-a mRNA levels in these cells. In contrast, induction of IL-1b protein was blocked by PD98059 in a dose-dependent manner in both cell lines, thus correlating with the effects of PD98059 on IL-1b mRNA expression in these cells. Fig. 1. The MEK inhibitor PD98059 selectively blocks LPS-induced TNF-a mRNA expression in different macrophage cell lines. The murine alveolar macrophage cell line AMJ2C-8 (C-8) and the murine macrophage cell line RAW264.7 were stimulated with LPS (100 ng/ml). A portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Four hours later, these cells were harvested, and total RNA was purified. As a control, a portion of the cells was pretreated with 20 µM PD98059 alone for 6 h prior to harvesting and lysis. An RNase protection assay was used to measure the steady-state levels of murine TNF-a, IL-1b, IL-6, and GAPDH mRNA.

differently in these two cell lines representing distinct macrophage populations. Thus, these results support the hypothesis that activation of the TNF-a gene appears to require the ERK signaling cascade in LPS-stimulated alveolar macrophages.

PD98059 differentially affects LPS-induced TNF-a protein secretion in C-8 and RAW264.7 cells Regulation of TNF-a production is tightly controlled at the transcriptional, post-transcriptional, translational, and posttranslational levels [22, 23]. Also, TNF-a mRNA can be detected within cells in the absence of TNF-a protein production or release [24]. Thus, we sought to determine whether the differential effect of PD98059 on TNF-a mRNA production by LPS-stimulated C-8 and RAW264.7 cells was also observed at the level of protein secretion. Cells were stimulated with LPS in

PD98059 differentially affects LPS-induced TNF-a protein secretion in primary alveolar and peritoneal macrophages We subsequently sought to determine whether the differential effect of PD98059 on TNF-a production could also be observed in primary murine peritoneal and alveolar macrophages. Macrophages were obtained from BALB/c and C57Bl/6 mice as described in Materials and Methods. These two strains were selected to determine whether the sensitivity of macrophages to PD98059 was strain-specific. In addition, these strains were selected because the RAW264.7 and C-8 cell lines were derived from BALB/c and C57Bl/6 mice, respectively. As shown in Table 1, TNF-a protein levels were readily induced by LPS in alveolar and peritoneal macrophages. However, PD98059 was able to block TNF-a production only in the alveolar macrophages and not in the peritoneal macrophages. In contrast, LPS-inducible IL-1b protein production was blocked by PD98059 in the alveolar and peritoneal macrophages. Thus, the effects of PD98059 on cytokine production were cytokine- and cell-type-specific but not strain-specific. The above data demonstrate the validity of using the C-8 and RAW264.7 cell lines as a model for the responses of these primary macrophage populations to LPS.

Fig. 2. PD98059 selectively blocks LPS-induced TNF-a secretion in an alveolar macrophage cell line. The alveolar macrophage cell line AMJ2C-8 (C-8, A) and the macrophage cell line RAW264.7 (B) were stimulated with LPS (100 ng/ml). A portion of the cells was pretreated with 20 µM PD98509 for 2 h prior to LPS stimulation. Sixteen hours later, these culture supernatants were collected, and a specific murine TNF-a and IL-1b ELISA was used to determine TNF-a and IL-1b protein levels. As a control, a portion of the cells was pretreated with 20 µM PD98059 alone for 18 h. TNF-a and IL-1b production is expressed as the mean of three independent supernatants (6SD). Basal levels of TNF-a and IL-1b produced by unstimulated C-8 and RAW264.7 cells were , 4 ng/ml.

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TABLE 1.

PD98059 Selectively Blocks LPS-Induced TNF-a Secretion in Primary Alveolar Macrophages

PD98059 µM LPS ng/ml

0 0

0 100

1 100

10 100

20 100

C57Bl/6 PM C57Bl/6 AM BALB/c PM BALB/c AM

2.1 6 0.3 1.3 6 0.1 1.5 6 0.5 0.9 6 0.3

22.3 6 1.8 8.1 6 0.8 18.1 6 1.3 6.5 6 0.9

TNF-a (ng/ml) 21.1 6 1.1 6.3 6 1.0 18.2 6 2.1 4.5 6 1.2

23.4 6 1.3 3.7 6 0.7 17.6 6 1.9 2.7 6 0.4

24.9 6 1.6 1.8 6 0.4 18.5 6 0.8 1.1 6 0.3

C57Bl/6 PM C57Bl/6 AM BALB/c PM BALB/c AM

4.3 6 0.8 5.1 6 0.7 7.1 6 1.1 5.5 6 1.3

56.3 6 6.8 28.1 6 3.8 47.3 6 0.3 34.5 6 7.9

IL-1b (ng/ml) 42.6 6 5.1 22.3 6 4.1 32.4 6 0.9 16.5 6 2.9

21.1 6 3.7 9.7 6 2.9 14.5 6 0.9 5.4 6 1.4

14.9 6 3.6 5.8 6 1.6 3.5 6 0.6 3.4 6 2.1

Pooled peritoneal macrophages and alveolar macrophages were obtained from C57B1/6 mice by peritoneal and bronchoalveolar lavage, respectively. Cells were plated in media containing 10% FBS and allowed to adhere for 2 hr prior to the experiment. Adherent cells were stimulated with LPS (100 ng/ml). A portion of the cells were pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Sixteen hours later, these culture supernatants were collected, and a specific murine TNF-a and IL-1b ELISA was used to measure TNF-a and IL-b protein levels. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 18 h. TNF-a and IL-1b levels are expressed as the mean of three independent supernatants 6 SD.

LPS-induced transcription of TNF-a requires MEK-1 activation in alveolar macrophages Having confirmed that PD98059 has a selective effect on LPSinduced TNF-a production at the mRNA and protein levels, we next sought to determine whether the MEK/ERK inhibitor blocked TNF-a expression at the transcriptional level. This was done using a luciferase reporter plasmid under the control of the full-length TNF-a promoter (21311 to 115). C-8 and RAW264.7 cells were transiently transfected with the reporter plasmid pTNF(1311)Luc and then stimulated with LPS in the presence or absence of PD98059. As shown in Figure 3, LPSinducible reporter expression was observed in both cell lines, but this induction could be blocked by PD98059 only in the C-8 cells (Fig. 3A). This finding is consistent with the RNase protection and protein data presented above and demonstrates that the MEK/ERK inhibitor acts by blocking TNF-a expression at the transcriptional level. Although these transfections were performed using the human TNF-a promoter to direct

luciferase reporter gene expression, identical results were obtained using the murine TNF-a promoter (data not shown). To confirm the inhibition of TNF-a in C-8 cells by PD98059 was specific for this cytokine, we also measured the effect of PD98059 on LPS-inducible IL-1b promoter activity. This was done using a luciferase reporter plasmid under the control of the full-length IL-1b promoter (23757 to 111). C-8 and RAW264.7 cells were transiently transfected with the reporter plasmid pXT-Luc and then stimulated with LPS in the presence or absence of PD98059. As shown in Figure 3, LPS-inducible reporter expression was observed in both cell lines, and this induction was blocked by PD98059 in both cell lines. This finding is consistent with the RNase protection data presented above (Fig. 1) and suggests that the IL-1b promoter is regulated in both cell lines by the same MAPK-dependent mechanisms. In contrast, the TNF-a promoter appears to be regulated by a MAPK-dependent pathway in C-8 cells and by a MAPKindependent pathway in RAW264.7 cells.

Fig. 3. PD98059 blocks LPS-induced TNF-a promoter activity only in alveolar macrophages. (A) The alveolar macrophage cell line AMJ2C-8 (C-8) and the macrophage cell line RAW264.7 were transiently transfected with 3 µg of the TNF-a reporter plasmid pTNF(21311)Luc. Eighteen hours later, a portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Cells were stimulated with LPS (100 ng/ml) for 5 h. The cells were then harvested and lysed, and equal amounts of lysate were assayed for luciferase activity. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 7 h. The reporter plasmid used contained a portion of the human TNF-a promoter (21311 to 115) ligated into the vector pGL3 upstream of the luciferase reporter gene. The mean luciferase activity obtained from triplicate independent transfections is shown (6SD). (B) RAW264.7 and C-8 cells were transiently transfected with 3 µg of the IL-1b reporter plasmid pXT- Luc. Eighteen hours later, a portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Cells were stimulated with LPS (100 ng/ml) for 5 h. The cells were then harvested and lysed, and equal amounts of lysate were assayed for luciferase activity. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 7 h. The reporter plasmid used contained a portion of the human IL-1b promoter (23757 to 111) ligated into the vector pGL3 upstream of the luciferase reporter gene. The mean luciferase activity obtained from triplicate independent transfections is shown (6SD).

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The PD98059-sensitive element is located within 180 bp of the TNF-a promoter cap site To define the 58 boundary of the PD98059-sensitive element within the TNF-a promoter, C-8 and RAW264.7 cells were transiently transfected with a series of luciferase reporter plasmids containing progressive truncations at the 58 end of the promoter. These truncations generated promoter fragments with 58 termini at positions 21311, 21185, 2615, and 2180, relative to the start of transcription. Reporter plasmids containing fewer than 180 bp of the TNF-a promoter were not evaluated because they were not LPS-inducible [15]. C-8 and RAW264.7 cells were transiently transfected with these reporter plasmids and then stimulated with LPS in the presence or absence of PD98059. As shown in Figure 4, LPS-inducible reporter expression was observed in both cell lines, although this induction was blocked by PD98059 only in the C-8 cells (Fig. 4A). This finding demonstrates that the cell-type-specific, MAPK-dependent, regulatory element is located between positions 2180 and 115 on the human TNF-a promoter. Similar data were obtained using murine TNF-a promoter fragments with 58 termini at positions 21059 and 2200 (Fig. 4, C and D). Although the shorter promoter fragment contains several regulatory elements required for LPS-inducible transcription (see below), it does not contain the upstream NF-kB binding sites [25, 26] and the region known to bind the distinct factor LITAF [16]. Thus, it appears that neither NF-kB nor LITAF mediates the PD98059-sensitive activation of the TNF-a promoter in C-8 cells and alveolar macrophages. Furthermore, our data demonstrate that the upstream NF-kB sites are not required for LPS-inducible activation of the TNF-a promoter in these two macrophage cell lines.

MAPK-regulated signaling is not mediated by NF-kB, Egr-1, CREB, or ATF2/c-Jun To determine whether selected transcription-factor binding sites within the TNF-a promoter were responsible for the PD98059 sensitivity observed in C-8 cells and alveolar macrophages, luciferase reporter plasmids containing specific mutations at these sites were examined. These reporter plasmids contain specific mutations within the Egr-1 site (2169 bp), the CRE site (2106 bp), and the k3 site (297 bp). The Egr-1 binding site and the region containing a CRE were previously reported to participate in LPS-inducible activation of the TNF-a promoter [15]. Further analysis of the CRE revealed that this site bound CREB or a complex containing c-Jun and ATF-2 [27]. The k3 site bears only a weak resemblance to an NF-kB binding site, although mutation of this site reduces LPS and superantigen inducibility of the promoter in transient transfection assays [15, 25, 28]. Together, each of these sites contributes to the total LPS inducibility of the TNF-a promoter. We reasoned that mutations that prevent binding of a transcription factor responsible for the PD98059 sensitivity observed in C-8 cells would result in a reporter plasmid that would still be LPS-inducible (albeit more weakly) and not affected by PD98059 in the C-8 cells. As shown in Figure 5, mutations at the individual sites did reduce LPS inducibility of the reporter plasmids in C-8 cells and RAW264.7. Furthermore, these mutations had no effect on the ability of PD98059 to block LPS

induction of the TNF-a promoter in C-8 cells (Fig. 5A). Thus, we conclude that Egr-1, CREB, and c-Jun/ATF-2 are not the transcription factors that mediate MAPK-dependent activation of the TNF-a promoter in LPS-stimulated C-8 cells and alveolar macrophages. The data reported in Figure 5 provide indirect evidence that NF-kB was not responsible for the PD98059 sensitivity of the TNF-a promoter in C-8 cells and alveolar macrophages. To assess the effect of PD98059 on LPS-inducible, NF-kBdependent transcription directly, C-8 and RAW264.7 cells were transiently transfected with a luciferase reporter gene under the control of the NF-kB-dependent E-selectin (ELAM-1) promoter. As shown in Figure 6, activation of NF-kB-dependent transcription by LPS was not blocked by PD98059 in the C-8 or the RAW264.7 cell. This demonstrates that NF-kB is not the transcription factor whose function is affected by PD98059 and, thus, is unlikely to mediate MAPK-dependent activation of the TNF-a promoter.

DISCUSSION This study reports that the MEK/ERK pathway is required for activation of TNF-a transcription in alveolar macrophages but not in peritoneal macrophages. This conclusion is a result of the sensitivity of these cells to the MEK-1/2 inhibitor PD98059. We have found that this PD98059-sensitive element is located within the cap-site proximal region of the TNF-a promoter (positions 2180 to 115). This suggests that the TNF-a promoter contains MAPK-dependent and -independent regulatory elements that are used in a cell-type-specific manner. Although PD98059 could suppress IL-1b production in both cell types, it could only block TNF-a production in alveolar macrophages. This finding demonstrated that PD98059 is fully capable of blocking the MEK/ERK pathway in these cells. We also found that differences in TNF-a transcription activated by the MEK/ERK pathway were not mediated by NF-kB, LITAF, Egr-1, CREB, or ATF2/c-Jun. In addition, upstream NF-kB elements within the TNF-a promoter were not required for maximal LPS-inducible transcription in macrophages. Together, these studies suggest that a novel, yet unidentified MAPK-dependent, factor is required for activation of the TNF-a gene in a cell-type-specific manner. Activation of MAPK by LPS has been shown to be mediated by activation of the upstream signaling factors Ras and Raf-1. Geppert et al. [29] reported that dominant-negative mutants of Ras and Raf-1 could block LPS-induced activation of the TNF-a promoter in transfected RAW264.7 cells. Based on these findings, we would have anticipated that blocking signals downstream of Raf-1, such as MEK activation, would also block TNF-a transcription. Instead we found that the MEK inhibitor PD98059 failed to block LPS-inducible TNF-a transcription in the RAW264.7 cells. The study by Geppert et al. did not directly assay Raf-1 kinase activity in the macrophages, therefore it cannot be determined whether the dominantnegative Ras and Raf-1 were acting directly or indirectly in their system. More recent publications by other investigators have reported Ras/Raf-independent pathways for MAPK activation by LPS in RAW264.7 cells [30, 31]. Furthermore, LPS was Means et al.


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Fig. 4. LPS induction of the TNF-a promoter from a 58 deletion series. The alveolar macrophage cell line AMJ2C-8 (C-8, A) and the macrophage cell line RAW264.7 (B) were transiently transfected with various truncations of the human TNF-a promoter: pTNF(21311), pTNF(21185), pTNF(2615), and pTNF(2180). C-8 (C) and RAW264.7 (D) cells were also transiently transfected with two truncations of the murine TNF-a promoter: p-mTNF(21059) and p-mTNF(2200). Eighteen hours later, a portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Cells were stimulated with LPS (100 ng/ml) for 5 h. The cells were then harvested and lysed, and equal amounts of lysate were assayed for luciferase activity. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 7 h. All reporter plasmids used contained a portion of the TNF-a promoter ligated into the vector pGL3 upstream of the luciferase reporter gene. The mean luciferase activity obtained from triplicate independent transfections is shown (6SD). Also shown is a graphical representation of the human TNF-a reporter plasmids used (E).

found to activate MAPK in these cells without inducing Raf-1 kinase activity [31]. Irrespective of the roles of Ras/Rafdependent, and –independent pathways in LPS-stimulated RAW264.7 cells, our data clearly demonstrate that MEK/ERK activity is not required for TNF-a transcription. This contrasts 890



with LPS-inducible IL-1b transcription, which was blocked by PD98059 in all macrophage populations examined. Several laboratories have shown that alveolar macrophages respond to stimuli in a manner that is distinct from peritoneal macrophages [32–36]. This implies that the signaling pathways http://www.jleukbio.org

Fig. 5. LPS induction of the TNF-a promoter containing mutations in the Egr-1, CRE, or k3 motifs. The alveolar macrophage cell line AMJ2C-8 (C-8, A) and the macrophage cell line RAW264.7 (B) were transiently transfected with various TNF-a reporter plasmid mutants: pTNF (Egr-1 mut), pTNF (CRE mut), or pTNF (k3 mut). Eighteen hours later, a portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Cells were stimulated with LPS (100 ng/ml) for 5 h. The cells were then harvested and lysed, and equal amounts of lysate were assayed for luciferase activity. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 7 h. The site-directed mutations for Egr-1, CRE, or kB3 were engineered using the template plasmid containing a portion of the human TNF-a promoter (2615 to 1 15), ligated into the vector pGL3 upstream of the luciferase reporter gene. The mean luciferase activity obtained from triplicate independent transfections is shown (6SD). Also shown is a graphical representation of the plasmids used (C).

leading to cytokine expression in alveolar macrophages may differ from those used by other macrophage populations. One example of such differences comes from our earlier study that alveolar macrophages from LPS-hyporesponsive C3H/HeJ mice secrete TNF-a following stimulation with LPS in vitro, whereas peritoneal macrophages from the same mice did not secrete TNF-a following LPS treatment [33]. Similarly, a macrophage cell line derived from C3H/HeJ alveolar macrophages (C3HAMSV40) was also found to secrete TNF-a in response to

Fig. 6. LPS-inducible NF-kB function is not blocked by PD98059 in RAW264.7 or C-8 cells. The macrophage cell line RAW264.7 and the alveolar macrophage cell line AMJ2C-8 (C-8) were transiently transfected with a luciferase reporter plasmid under the control of the NF-kB-dependent Eselectin (ELAM-1) promoter (ELAM-LUC). Eighteen hours after transfection, a portion of the cells was pretreated with 20 µM PD98059 for 2 h prior to LPS stimulation. Cells were stimulated with LPS (100 ng/ml) for 5 h. The cells were then harvested and lysed, and equal amounts of lysate were assayed for luciferase activity. As a control, a portion of the cells was treated with 20 µM PD98059 alone for 7 h. The mean luciferase activity obtained from triplicate independent transfections is shown (6SD).

LPS stimulation in vitro [33]. The gene locus responsible for the LPS-hyporesponsive phenotype of macrophages from the C3H/ HeJ mouse (Lps†) was later found to be a missense mutation within the Tlr4 gene that encodes Toll-like receptor 4 (TLR4) [5, 37, 38]. We and others have shown that TLR4 serves as the primary signal transducing protein that mediates LPS responsiveness in CD141 cells [9, 38, 39]. Thus, the finding that alveolar macrophages from TLR4-deficient C3H/HeJ mice can respond to LPS suggested that an alternative signaling pathway exists in these cells (see [33]), whereas peritoneal macrophages from these mice lack this signaling pathway. Whether this alternative signaling pathway leads to activation of the PD98059sensitive element within the TNF-a promoter in alveolar macrophages remains to be determined. Other examples of how alveolar and peritoneal macrophages differ in their response to stimuli have been reported. Pue et al. [34] reported that acute-phase levels of C-reactive protein enhance IL-1b and IL-1ra production in blood monocytes but inhibit production of these cytokines by alveolar macrophages. Similarly, Becker et al. [32] demonstrated that LPS stimulation increased colony-stimulating factor (CSF-1) and IL-1b production in blood monocytes but failed to enhance secretion of these cytokines by alveolar macrophages. We have also found that primary murine alveolar macrophages fail to be activated by lipoarabinomannan (LAM, see [9]), a mycobacterial glycolipid that activates peritoneal macrophages in a CD14-dependent manner (unpublished results). Thus, these macrophage populations differ in their capacity to recognize and respond to distinct stimuli. Recent studies have begun to address the mechanistic basis for these observations. Monick et al. [35] reported that protein kinase C (PKC) isoforms are differentially expressed in Means et al.


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human alveolar macrophages and blood monocytes and that these differences are associated with functional alterations in these cells. More recently, these investigators have shown that human alveolar macrophages do not express AP-1 DNA binding [35, 36]. Certainly, it is possible that differences in AP-1 activity in alveolar macrophages vs. blood monocytes could underlie their functional differences. Lastly, ERK and p38 appear to mediate LPS-inducible TNF-a expression in human alveolar macrophages [40]. The involvement of the latter kinase was demonstrated using the specific p38 inhibitor SB203580. We also assessed the role of p38 kinase in our murine system using SB203580. We confirmed that this inhibitor blocked TNF-a protein secretion, although SB203580 did not block TNF-a gene transcription in our hands (data not shown). The signaling pathway leading to MAPK activation in LPS-stimulated macrophages remains unclear. Recent data indicate that LPS binds to CD14 and initiates an intracellular signal via TLR4, which initiates a sequential cascade of signaling events involving the adapter protein MyD88 [39, 41, 42], the IL-1 receptor-associated kinase (IRAK, [42]), tumor necrosis factor receptor-associated factor 6 (TRAF-6) [42, 43], and the NF-kB-inducing kinase (NIK, [44]). MAPK may become activated at several points along this signaling pathway. Muzio et al. [43] demonstrated that MAPK activation occurs upstream of TRAF-6 but downstream of MyD88. In contrast, a newly discovered factor, termed ECSIT, has been shown to bridge TRAF-6 to MEKK-1 (the first step in the activation of JNK) in cells stimulated via a constitutively active form of TLR4 [45]. Lastly, a dominant-negative TAK-1 kinase has been shown to block IL-1b-induced JNK activation [46]. TAK-1 is a kinase that bridges TRAF-6 to NIK, but it may also initiate activation of the MAPK pathway. This is a relevant finding because the IL-1b and LPS signaling pathways share many components [47]. Irrespective of the pathway by which MAPK are activated in macrophages, we have observed normal activation of ERK, p38, and JNK in LPS-stimulated alveolar and peritoneal macrophages (data not shown). Thus, the target for the PD98059-sensitive pathway in C-8 cells and alveolar macrophages is likely to be a transcription factor substrate rather than a signaling pathway component. The transcriptional regulation of the TNF-a promoter has been extensively studied, and several (often conflicting) conclusions have been reported in the literature. It was initially proposed that NF-kB was an important regulatory factor for human and murine TNF-a gene transcription in LPSstimulated macrophages and monocytes [26, 48]. Subsequent studies using the human TNF-a promoter have argued for [49, 50] and against [15, 25] a predominant role for NF-kB in LPS-inducible TNF-a transcription. We observed nearmaximal LPS responsiveness using reporter constructs that lack these upstream NF-kB binding sites (Fig. 4), thus supporting the latter conclusion. Furthermore, our studies exclude LITAF, Egr-1, CREB, and ATF2/c-Jun as candidates for the PD98059-sensitive factor in C-8 cells and alveolar macrophages, leaving the identity of this factor unresolved. Ets-like transcription factors have been reported to bind a conserved site within the human TNF-a promoter (positions 892



2115 to 298), and to be required for basal and PMA-inducible activity of this promoter in transient transfection assays [14]. Some Ets family members expressed in macrophages may require ERK-dependent phosphorylation to activate their transactivation function. For example, we have shown that the Ets-like protein PU.1 is phosphorylated by protein kinase CK2, which is, in turn, activated in LPS-stimulated cells via a ERK-dependent mechanism [51, 52]. Alternatively, AP-1 may be the factor that indirectly defines the PD98059 sensitivity of C-8 and alveolar macrophages. Monick et al. [36] reported that human alveolar macrophages do not express AP-1 DNA binding activity. A putative AP-1 site has been identified within the TNF-a promoter, although mutation of this site did not affect LPS responsiveness in transiently transfected THP-1 cells [15]. Additional studies will be needed to resolve this complex and critical question.

ACKNOWLEDGMENTS This work was supported by National Institutes of Health grant GM57053 (M.J.F.). The authors thank Dr. Anne Goldfeld (Center for Blood Research and Harvard Medical School, Boston, MA) for helpful discussions. The authors also thank Dr. Lisa Ryan for sharing her unpublished data and Dr. Shuyan Wang for her excellent technical assistance.

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