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The Journal of Immunology, 2000, 164: 6349-6358.
Copyright © 2000 by The American Association of Immunologists

Regulation of TNF Expression by Multiple Mitogen-Activated Protein Kinase Pathways1

Wei Zhu2,*, Jocelyn S. Downey2,*, Jun Gu{dagger}, Franco Di Padova{ddagger}, Hermann Gram{ddagger} and Jiahuai Han3,*

* Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; {dagger} Center of Molecular Medicine, Sun Yat-Sen University of Medical Science, Guangzhou, China; and {ddagger} Novartis Pharma, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stimulating macrophages with bacterial endotoxin (LPS) activates numerous intracellular signaling pathways that lead to the production of TNF. In this study, we show that four mitogen-activated protein (MAP) kinase pathways are activated in LPS-stimulated macrophages: the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase, p38, and Big MAP kinase (BMK)/ERK5 pathways. Although specific activation of a single MAP kinase pathway produces only a modest effect on TNF promoter activation, activation of each MAP kinase pathway is important for full induction of the TNF gene. Interestingly, a dramatic induction of TNF promoter-driven gene expression was observed when all of the four MAP kinase pathways were activated simultaneously, suggesting a cooperative effect among these kinases. Unexpectedly, cis elements known to be targeted by MAP kinases do not play a major role in multiple MAP kinase-induced TNF gene expression. Rather, a 40-bp sequence harboring the TATA box, is responsible for the gene up-regulation induced by MAP kinases. The proximity of the MAP kinase-responsive element to the transcriptional initiation site suggested that MAP kinases regulate the transcriptional initiation complex. Utilizing {alpha}-amanitin-resistant RNA polymerase II mutants with or without a C-terminal domain (CTD) deletion, we found that deleting the CTD to 31 tandem repeats ({Delta}31) led to >90% reduction in MAP kinase-mediated TNF production. Thus, our data demonstrate coordination of multiple MAP kinase pathways in TNF production and suggest that the CTD of RNA polymerase II is required to execute MAP kinase signaling in TNF expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor necrosis factor (TNF or TNF-{alpha}/cachectin) is a proinflammatory cytokine that acts as a mediator of host defense against both neoplasia and infection and is principally expressed in macrophages (1, 2, 3, 4), where its secretion may be increased 10,000-fold after exposure to bacterial endotoxin (LPS) (5). Along with numerous beneficial roles in immune regulation, TNF has been implicated in the pathogenesis of both acute and chronic inflammatory disease (6), and therefore it is of great interest to dissect the molecular mechanisms of TNF gene expression.

Eukaryotic gene expression is heavily controlled by enhancer/promoter elements, which act in conjunction with the RNA polymerase II (pol II)4 holoenzyme to mediate transcription. Mammalian RNA pol II contains a characteristic C-terminal domain (CTD) in its largest subunit, consisting of 52 repeats of the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (7, 8, 9). This CTD has been shown to interact with other RNA pol II subunits, including the TATA-binding protein (8, 10), and it is known to be involved in regulating gene transcription because partial deletions in the CTD modulate the regulatory properties of distinct promoters in different ways (11, 12, 13, 14, 15, 16). In addition, the CTD is phosphorylated by cellular kinases, and this phosphorylation appears to play a role in regulating initiation and elongation of transcription (17, 18, 19, 20). Despite the importance of RNA pol II and its CTD in transcriptional control, the regulatory role that this holoenzyme plays in LPS-induced TNF transcription has not been studied to date.

Sequence analysis has revealed a number of cis elements present in the TNF promoter, including several NF-{kappa}B-like motifs, which are thought to play a primary role in TNF gene transcription (21). However, there is evidence from studies employing deletion constructs in the promoter to indicate that other elements are involved in TNF regulation (22, 23). In addition, AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of TNF mRNA are thought to play a role in mediating mRNA stability and efficiency of translation (24, 25). Therefore, TNF production is controlled at both transcriptional and posttranscriptional levels.

Exposure of cells to LPS activates a number of signaling pathways, including NF-{kappa}B, phosphatidylinositol 3-kinase, and protein kinase C (26). Previous work from this and other laboratories has found that LPS activates the extracellular signal-regulated kinase (ERK) (27), the c-Jun N-terminal kinase (JNK) (28, 29), and the p38 mitogen-activated protein (MAP) kinase pathways (30, 31). Selective inhibition of the p38 and ERK pathways inhibits LPS-stimulated TNF production (32, 33), and it has also been reported that JNK kinases are required for translation of TNF mRNA after LPS induction (34). However, attempts to induce TNF with Raf I, an upstream activator of ERK pathway, indicate that although ERK activation is sufficient to produce small amounts of TNF, the levels produced are 20 times smaller than those produced in response to LPS stimulation of cells (35, 36, 37). Studies in glial cells have also shown that although inhibiting either the ERK pathway using the selective drug PD98059 or the p38 pathway using SB203580 suppresses TNF biosynthesis, inhibiting both ERK and p38 together results in almost complete abolition of TNF biosynthesis (38). Therefore, it is clear that multiple signals must converge on TNF synthesis to elicit its full response.

To define their role in TNF biosynthesis, we have studied the regulation of the TNF promoter by various MAP kinase pathways. We have evaluated the activation and requirement for different MAP kinase pathways, including the Big MAP kinase (BMK)/ERK5 pathway, in LPS responsiveness of the macrophage (M{Phi}) cell line RAW264.7. Selective activation of one, two, three, or four MAP kinase pathways in different combinations was used to determine the biological consequences of MAP kinase activation in terms of TNF expression. Although activation of any individual MAP kinase had little effect on TNF gene expression, activation of all four MAP kinase pathways produced a dramatic synergistic effect on TNF promoter-driven gene expression. It is further shown that known cis elements such as AP-1 and NF-{kappa}B are not the major target of MAP kinases, whereas the MAP kinases’ responsiveness is mediated by an element located in -43 to -1 bp immediately upstream of the transcriptional initiation site. We further demonstrated that the MAP kinase-induced TNF gene expression is, at least in part, mediated by a regulation of RNA pol II CTD. Our results demonstrate a convergence of different MAP kinase pathways to one regulatory site that may play a key role in TNF expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation of recombinant proteins

Escherichia coli BL21(DE3) was transformed with the vector pETM1 containing cDNAs encoding myocyte-enhancer factor 2C (MEF2C). The BL21 strain of E. coli was transformed with the vector pGEX containing cDNAs encoding ELK1 and activating transcription factor 2 (ATF2). The transformed bacteria were grown at 37°C in Luria-Bertani broth until the A600 was 0.5, at which time isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 1 mM for 5 h. Cells were collected by centrifuging at 8000 x g for 10 min, and the bacterial pellet was resuspended in 10 ml of buffer A (30 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl, and 2 mM PMSF) for every 100 ml of original bacterial culture. The cell suspension was sonicated, and cellular debris was removed by centrifuging at 10,000 x g for 30 min. Recombinant proteins were purified from the cleared lysate using nickel-nitriloacetic acid (Ni-NTA) purification system (Qiagen, Valencia, CA) or glutathione-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) following the manufacturer’s instruction.

Protein kinase assays

In vitro kinase assays were conducted at 37°C for 30 min using purified immunoprecipitate as the kinase, 5 mg of kinase substrate, 250 µM ATP, and 10 µCi of [{gamma}-32P]ATP in 20 ml of kinase reaction buffer as described previously (39). Reactions were terminated by the addition of Laemmli sample buffer. Reaction products were resolved by 12% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography and were quantified by phosphoimaging.

Plasmid preparation

All plasmid DNA used in transfection experiments was prepared using CsCl2-gradient ultracentrifugation. Possible LPS and other bacterial sugar/lipid contamination was subsequently removed with Endotoxin Removal Affinity Resin (Associates of Cape Cod, Falmouth, MA). Because differences in transfection efficiency may result if DNA quality is not well controlled, we have evaluated several methods and have determined this procedure to give the best reproducibility in transfection efficiency and results when between different batches of plasmids.

Transfection

RAW264.7 cells were maintained in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1% nonessential amino acids. With the exception of the drug-inhibition assay, transfections were performed on 2 x 106 cells seeded into wells in six-well dishes. However, to investigate the effects of MAP kinase inhibitors on a TNF reporter gene, 1 x 107 cells were transfected, and these cells were then subdivided onto six-well plates 24 h after transfection. Cells were transfected with 0.6 µg of each plasmid per well using calcium phosphate precipitation before glycerol shock (24). Empty pcDNA3 vector was used to normalize the amount of total DNA used in each transfection to 3 µg/well. An exception to this was the transfection of pol II expression plasmids, in which we used 6 µg/well for transfection because of the much larger size of the plasmids. Pol II-transfected cells were selected with 2.5 mg/ml {alpha}-amanitin 16 h after transfection. The other transfected cells were processed 24 h after transfection. Some cells either were left unstimulated or were treated with LPS (1 ng/ml) for 8 h. The cells were washed in PBS, harvested, and resuspended in 100 µl of a reporter lysis buffer (Promega, Madison, WI). Lysed cells were briefly centrifuged, and the relative strength of reporter induction was calculated by measuring the luciferase activity of the supernatant by luminometer in a luciferase assay reagent (Promega). Transfection efficiency was normalized by cotransfecting cells with 0.6 µg of an expression plasmid containing a CMV promoter-driven ß-galactosidase reporter. ß-Galactosidase activity was measured by using the chemiluminescent assay Galacto-Light (Tropix, Bedford, MA) or by using O-nitrophenyl-ß-D-galactopyranoside (ONPG) as follows: 20 µl of lysis supernatant was added to 80 µl 3.5 mM ONPG solution, incubated at 37°C for 30 min, and measured at 405 nm.

Reporter constructs

Murine TNF (mTNF) luciferase reporter was generated from a chloramphenicol acetyltransferase (CAT) reporter described previously (40). A 1-kb mTNF promoter was taken from Pro-CAT construct by digesting with BamHI and HindIII and was inserted into pGL2 basic using BglII and HindIII sites. A progressive series of deletions was generated in the murine 5'-TNF promoter using PCR. A series of human TNF (hTNF) reporter constructs were generously provided by Dr. J. S. Economou (University of California, Los Angeles, CA). 5'UTR of hTNF was removed by digesting with BamHI and KpnI and was replaced with a synthesized oligonucleotide linker. Deletion constructs of -75, -65, and -55 and chimeric constructs C1, C2, C3, and C4 were constructed by oligonucleotide replacement. The constructs were sequenced to confirm their integrity. Oligonucleotide sequences used in PCR and oligonucleotide replacement are available upon request. NF-{kappa}B and AP-1 luciferase reporters containing a basic promoter element (TATA box) joined to a NF-{kappa}B or AP-1 site were obtained from Stratagene (La Jolla, CA). The nonrelated minimal promoter (nmp) reporter was constructed using the same backbone but was joined to -55 to -43 bp of human TNF promoter sequence. Sp1 luciferase reporter was constructed using the same backbone inserted with three repeats of Sp1 site.

Other constructs

The expression constructs for different MAP kinase kinase mutants were cloned in pcDNA3 expression vector as described previously (41, 42, 43, 44, 45). The expression constructs of {alpha}-amanitin-resistant RNA pol II and its CTD deletion mutants were kindly provided by Hans-Peter Gerber (Genentech, South San Francisco, CA).

Quantification of reporter mRNA

An RNA isolation kit from Qiagen was used to isolate total RNA from cultured cells according to manufacturer’s instruction. Luciferase mRNA was quantified by Taqman (Perkin-Elmer, Foster City, CA). Cotransfected ß-galactosidase was used as an internal control.

Metabolic labeling and immunoprecipitation of transfected pol II and pol II {Delta}31

Pol II or pol II {Delta}31 was cotransfected into 5 x 107 RAW cells with or without the expression plasmids of MAP/ERK kinase 1 (MEK1)(E), MEK5(D), MKK6(E), and MKK7(D) using calcium phosphate precipitation. Sixteen hours after transfection, these cells were selected with 2.5 µg/ml {alpha}-amanitin for 3 days, after which time surviving cells were replated onto 10-cm dishes. Cells were metabolically labeled as previously described (46). Briefly, the ATP pool of cells will be labeled using [32P]orthophosphate (1 mCi/ml for 2 h), and recombinant RNA pol II proteins were immunoprecipated using anti-hemagglutinin (anti-HA) Ab (Covance, Richmond, CA). SDS-PAGE was performed on the immunoprecipitates, and the dried gel was exposed on a phosphoimaging cassette for 2 days.

Western blotting

Cells were rapidly chilled on ice, washed twice with ice-cold washing buffer (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1 mM Na3VO4), and then lysed in 250 µl (per 106 cells) lysis buffer (20 mM Tris-HCl (pH 7.5), 120 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride). The proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. mAb H14 (Covance) was used to detect phosphorylation of pol II CTD. The H14 Ab was developed against a purified phosphorylated form of RNA pol II and recognizes the phosphorylation of serine 5 in the heptapeptide repeat YSPTSpPS at the C-terminal domain.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of M{Phi} with LPS can lead to activation of multiple MAP kinase pathways

Previous studies by others and us have revealed that LPS activates each of the ERK, JNK, and p38 MAP kinase pathways in M{Phi} (27, 28, 31). By using immunokinase assays and Western blotting, we now know that LPS can also activate BMK1/ERK5 in RAW264.7 cells, a murine macrophage cell line. Fig. 1Go shows the kinetics of the activation of the four different MAP kinases: ERK2, JNK1, p38 (p38{alpha}), and BMK1. Activation of all MAP kinases can be detected within 5 min of LPS stimulation; however, the time at which the activation reaches a maximum is different: both ERK2 and BMK1 were maximally activated in 5 min, whereas maximal activation of JNK1 and p38 did not occur until 15 and 30 min, respectively. In addition, along with the differences in the time taken to activate each of the MAP kinases, there were significant differences in the intensity of activation, such that although p38, ERK2, and JNK1 were strongly activated (~10-, 6-, and 9-fold, respectively), BMK1 demonstrated only an ~3-fold level of induction. Furthermore, studies by several investigators have shown that LPS-mediated MAP kinase activation is not confined to the activation of a single member MAP kinase in each pathway: ERK1 and ERK2 (27), JNK1 and JNK2 (28), p38, p38ß, p38{gamma}, and p38{delta} (our unpublished results) are all activated by LPS stimulation in M{Phi}. Activation of all known MAP kinases by a single stimulus has not been reported by any other stimulus. This may be important and may be one possible reason for the phlogistic effects of LPS in initiating innate immune responses. Although different signal pathways may be responsible for different cellular changes initiated by LPS, they may also ultimately converge onto one cellular reaction, such as TNF production. Therefore, we performed the experiments described below to address the role of these MAP kinase pathways in regulating TNF gene expression.



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  		FIGURE 1. Activation of multiple MAP kinases in LPS-stimulated RAW264.7 cells. RAW cells were stimulated with LPS (1 ng/ml) for different periods of time and then the cells were harvested. BMK1, ERK2, p38, and JNK1 were immunoprecipitated from cell lysates using specific polyclonal Abs. Immunokinase assays were performed using the immunoprecipitates as kinase. Recombinant His-MEF2C, GST-ELK1(307–428), GST-ATF2(1–109), and GST-ATF2(1–109) were used as substrate for BMK1, ERK2, p38, and JNK1, respectively. The kinase reactions were stopped by SDS-sample buffer, and the reaction products were resolved by SDS-PAGE. Phosphorylated proteins were visualized by autoradiography. Comparable results were obtained in two experiments.

 
Activation of each of the four MAP kinase pathways is required for the full induction of TNF gene

Because of the availability of specific inhibitors for the p38 and ERK pathways, inhibition of these two pathways has been shown to inhibit TNF production in several cell systems (32, 33, 38). Here we used specific inhibitors of the ERK and p38 pathways to test whether a luciferase reporter gene driven by a 1-kb human TNF promoter can mimic the responsiveness of the endogenous TNF gene to MAP kinases in RAW264.7 cells. RAW cells were transiently transfected with TNF reporter gene and then were replaced onto 6-well plates. Twenty-four hours after transfection, the cells were pretreated with different concentrations of SB203580 or U0126 for 30 min and then were stimulated with LPS (1 ng/ml) for 8 h before a luciferase assay was performed on the lysed cell extract. As shown in Fig. 2GoA, the inhibitor of p38{alpha}ß, SB203580, inhibited LPS-induced TNF reporter gene expression in a dose-dependent manner to give an IC50 of ~18 µM. U0126, an inhibitor of MEK1/2 (47), also inhibited TNF reporter gene expression (IC50 = 18 µM). These IC50 are similar to those for endogenous TNF in this cell line (data not shown). A further inhibition of the reporter synthesis was observed when both inhibitors were present (Fig. 2GoA), the same as previously reported for endogenous TNF (38).



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  		FIGURE 2. Requirement of MAP kinase pathways in LPS-induced TNF reporter gene expression. A, RAW cells were transfected with hTNF reporter, and the cells were replated into 6-well plates to ensure equal transfection in each sample. LPS (1 ng/ml) stimulation was applied 24 h after transfection. Samples that were pretreated for 30 min with a different dose of SB203580 or U0126 or both inhibitors are as indicated. Luciferase activity was measured 8 h after LPS stimulation. B, RAW cells were cotransfected with hTNF reporter and MEK1(A), MKK4(A), MEK5(A), MKK6(A), or empty vector pcDNA3. LPS stimulation was applied 24 h later after transfection for 8 h. Luciferase activity was measured. Cotransfected ß-galactosidase was used to normalize the transfection efficiency. The fold of induction was calculated by dividing the luciferase value for the LPS-treated sample with that from the sample without LPS treatment. Comparable results were obtained in two experiments.

 
To test the importance of each of the MAP kinase pathways in TNF induction, especially the MAP kinase pathways that do not have a specific drug inhibitor, another approach was adopted in which dominant negative MAP kinase kinase molecules were used to suppress individual MAP kinase pathways and to examine their effect on TNF reporter gene synthesis.

Normal activation of MAP kinase kinase family members is achieved by the dual phosphorylation of two serine/threonine residues located between kinase domains VII and VIII in the proteins. Substituting these residues with alanine (A) produces a dominant-negative form of the molecules. Dominant negative forms of MEK1, MEK5, MKK6, and MKK4, the MAP kinase kinases for the ERK, BMK/ERK5, p38, and JNK/stress-activated protein kinase pathways, respectively, were created and have been used in previous experiments by us (41). RAW cells were then cotransfected with a TNF reporter gene, and MEK1(A), MEK5(A), MKK4(A), or MKK6(A), and LPS stimulation was applied for 8 h, 1 day after transfection. As shown in Fig. 2GoB, suppressing any one of the MAP kinase pathways led to a reduction in LPS-induced TNF reporter gene expression. Because a great excess of an individual MKK(A) is needed to competitively inhibit endogenous MKKs, we have estimated that this approach may inhibit an endogenous MAP kinase by less than 50% (data not shown). This finding agreed with the inhibition observed in Fig. 2GoB. Simultaneously expressing all MKK(A) led to a stronger reduction in the reporter gene expression (Fig. 2GoB), which may be due to the additive effect of the partial inhibition of each MKK. Because the endogenous level of expression of different MEK/MKKs is different and because the competitive inhibitory effect of different MKKs may also be different, the inhibition data presented in Fig. 2GoB may not represent the relative contribution of the different MAP kinase pathways. It is clear that all four MAP kinase pathways are involved in TNF gene expression induced by LPS.

Cooperative effect of different MAP kinase pathways on TNF gene expression

In contrast to substituting the dual phosphorylation sites with alanine, substituting the residues with either glutamic acid (E) or aspartic acid (D) produces dominant-active forms of MAP kinase kinases (41). Because all MAP kinase pathways are involved in LPS-induced TNF gene transcription, we sought to determine the role of each MAP kinase and the combinatorial effect of these MAP kinases in the gene expression of TNF. Sole activation of a given MAP kinase pathway was achieved by transiently expressing dominant-active mutants of different MKKs, and the level of induction of TNF transcription was evaluated using a luciferase reporter driven by either the mouse or human TNF promoters. Almost the same activation profiles of hTNF and mTNF reporter genes were observed (Fig. 3Go), suggesting that MAP kinases regulate the TNF promoter via a conserved mechanism. When individual MAP kinase pathways were activated, the folds of induction of the human TNF reporter expression by the ERK, BMK, p38, and JNK activation were 1.1, 1.3, 2.8, and 1.3, respectively, indicating that the p38 pathway produced the strongest effect on TNF promoter activation (Fig. 3Go). When two different MAP kinase pathways were activated simultaneously, the BMK and p38 pathways together resulted in the highest activation of the TNF promoter. Simultaneous activation of ERK, BMK, and p38 pathways led to the highest expression of TNF reporter in comparison with the other three MAP kinase combinations, and activation of all four MAP kinase pathways had the highest induction of TNF reporter gene expression. Therefore, it is clear that all MAP kinase pathways participate in regulating TNF gene expression, with the p38 pathway seeming to contribute to a greater extent than other pathways. In addition, activation of multiple pathways induced the TNF promoter to a much greater extent than the sum of the induction produced by individual MAP kinases, and, as such, the combinatorial effect of these MAP kinase pathways cannot be explained by an additive effect. For example, the ERK, BMK, and p38 pathways together led to an ~75-fold induction of hTNF reporter gene expression, whereas the additive effect of these three MAP kinase pathways is only ~5-fold (1.1 x 1.3 x 2.8). Thus, MAP kinase pathways seem to act through a coordinated mechanism to produce the high expression of TNF observed in M{Phi} stimulated with LPS.



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  		FIGURE 3. Individual and combination effects of different MAP kinase pathways on TNF reporter gene expression. hTNF or mTNF reporter with different dominant-active MKKs or their combinations were transfected into RAW cells. The empty vector pcDNA3 was used to normalize the amount of total DNA used in the transfection. CMV-ß-galactosidase was cotransfected, and ß-galactosidase activity was used to normalize the transfection efficiency. Luciferase activity was used to calculate the induction. Comparable results were obtained in three experiments.

 
To assess whether the MAP kinases acted at the level of transcriptional activation, the relative amount of mRNA produced from the luciferase reporter was measured when the four MAP kinase pathways were activated, using an RT-PCR-based instrument, Taqman (Perkin-Elmer). After normalization of the transfection efficiency using cotransfected ß-galactosidase mRNA, we detected an ~30-fold induction of luciferase mRNA in comparison with that of the control (data not shown). We also measured the half-life of reporter mRNA with or without induction. The luciferase mRNA is very stable and its half-life (~240 min) was not altered by LPS stimulation (data not shown). Therefore, the regulatory effect of MAP kinases observed in our reporter gene experiments probably occurs at a transcriptional level. However, these data do not deny additional posttranscriptional processes in the regulation of endogenous TNF by MAP kinases.

Because the hTNF reporter contained ~106 bp of the 5'-UTR of TNF mRNA, we removed the hTNF 5'-UTR from the reporter construct and compared hTNF reporter with or without the 5'-UTR when different MAP kinases were activated. In agreement with the data achieved using the murine TNF reporter (which lacked the 5'-UTR from TNF), no differences were observed (data not shown), suggesting that MAP kinase activation does not act on the 5'-UTR of TNF.

Mapping the cis element targeted by the MAP kinases

Because there are several transcription factor binding sites that have been suggested to be involved in LPS-induced TNF production (21, 22, 23), we examined whether MAP kinases require these sites to function. Reporter constructs with specific site mutations were obtained from Dr. J. S. Economou and were coexpressed with dominant-active MKKs. Mutations in Egr-1, cAMP response element, NF-{kappa}B, AP-1, or AP-2 produced no significant effect on MAP kinase-mediated TNF reporter gene expression, indicating that these sites are not targeted by MAP kinases (Fig. 4GoA). Two cis reporter constructs, NF-{kappa}B and AP-1 reporter, were used to directly test whether these cis elements were regulated by MAP kinases. As shown in Fig. 4GoB, only a modest induction was achieved when the MAP kinases were activated.



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  		FIGURE 4. The known cis elements in TNF promoter are not the major sites regulated by MAP kinases. A, The hTNF promoter point mutation series was cotransfected with or without dominant-active mutants of MEK1, MEK5, MKK6, and MKK7 as described in Fig. 3Go. B, The NF-{kappa}B or AP-1 luciferase reporter with different combinations of dominant MKKs was transfected into RAW cells as described in Fig. 3Go. The induction of the reporters was determined, and comparable results were obtained in three experiments.

 
To localize MAP kinase-responsive elements in the TNF promoter, RAW cells were transiently transfected with a series of luciferase reporter constructs containing progressive deletions in the 5'-TNF promoter sequence. Deleting sequence upstream of position -55 did not reduce MAP kinase-mediated gene expression (Fig. 5Go). However, deletions to and beyond position -43 reduced the basal expression of the reporter to a level below the detection limit of the luciferase assay and prevented the determination of the relative induction of the reporter gene. Thus, the role of the cis element within -55 to -43 bp (which contains a Sp1-like sequence) in MAP kinase-mediated gene induction was not resolved from these experiments. To overcome the problem of low expression, we constructed a reporter containing the -55 to -43 sequence placed 5' to an unrelated sequence derived from cis reporter backbone plasmid (termed nmp). The nmp had the same length as -55 reporter. When these two constructs were compared, nmp was much less responsive to MAP kinases than the -55 TNF reporter was (Fig. 6Go), suggesting the importance of the sequence harbored -43 to -1 in the TNF promoter in responding to multiple MAP kinase activation. To further map the sequence within the -43 element controlling MAP kinase-mediated TNF gene transcription, we created a series of chimeric promoter elements and evaluated the effect of the sequence within this element on its responsiveness to MAP kinases. C1 was created by replacing the sequence between the Sp1 site and TATA box of -55 with nmp sequence. C2 was created by further replacing the C1 TATA box with the nmp TATA box. C3 contained the Sp1 and TATA box sequence from -55 and the rest from nmp. C4 is a hybrid of -55 and nmp joined 3' to the TATA box. As shown in Fig. 6Go, swapping the -43 sequence with a nonresponsive sequence derived from a cis reporter backbone plasmid did not change the profiles in response to different combinations of MAP kinases. However, an ~2-fold general reduction was observed for the chimeras in comparison with native sequence. Analysis of the results presented in Fig. 6Go did not further narrow down the location of the response element but did confirm the importance of -43 sequence in response to MAP kinases and suggested that the integrity of endogenous gene is also important. Taken together, our data indicated that -43 to -1 is the region in TNF promoter that is regulated by MAP kinases.



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  		FIGURE 5. The induction of a deletion series of 5'-hTNF-luciferase reporters by the four MAP kinases. A series of deletions were made in the 5'-hTNF promoter, and these deletion mutants were cotransfected with or without dominant-active mutant of MEK1, MEK5, MKK6, and MKK7 as described in Fig. 3Go. The induction was determined by luciferase expression, and each experiment was performed three times.

 


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  		FIGURE 6. The -43 to -1 TNF promoter is required for TNF transcription mediated by the four MAP kinases. A, Luciferase reporter genes were constructed by fusing the -55 to -1 human TNF (-55 hTNF) and -55 to -43 TNF promoter fragments to a nmp sequence. These constructs comprised various elements of the -55 region of the promoters including the TNF TATA box ({blacksquare}), the TATA box from the nmp ({square}), the sequence from surrounding TNF DNA (), and nmp DNA (solid line). B, RAW cells were then transfected with cDNA from different combinations of dominant MKKs, along with luciferase reporters containing the -55 hTNF promoter, the nmp promoter, or a TNF-nmp chimeric promoter, as described in Fig. 3Go. Reporter induction was determined. Comparable results were obtained in three experiments.

 
C-terminal domain of RNA pol II is involved in MAP kinase-mediated TNF transcription

Because -43 to -1 encompasses the TATA box binding site and the RNA pol II complex assembles around this region to initiate transcription, it is possible that RNA pol II complex is responsible for MAP kinase-mediated up-regulation of TNF synthesis; and because phosphorylation of the CTD of RNA pol II has been implicated in transcriptional regulation, we thought that a possible role of the CTD in MAP kinase-induced TNF gene activation should be examined. Transient transfections utilizing {alpha}-amanitin-resistant CTD deletion mutants of pol II’s largest subunit have been successfully used to examine the function of CTD (11). We adopted this established method to study the possible functional relations between CTD and MAP kinase activation in TNF gene expression. To demonstrate the involvement of RNA pol II in LPS-induced TNF expression, pol II- and pol II {Delta}31- (a pol II mutant with a CTD containing only 31 repeats) transfected cells cotransfected with TNF reporter genes were stimulated with LPS (1 ng/ml). Cells were cultured in the presence of 2.5 µg/ml {alpha}-amanitin, which inhibited >95% endogenous pol II activity in RAW cells (data not shown). The expression levels of the full-length pol II and pol II {Delta}31 were shown to be comparable by Western blotting using anti-HA Ab (data not shown). The basal expression levels of the TNF-reporter, mediated either by the full-length pol II or by pol II {Delta}31, were similar (Fig. 7Go, lanes 1 and 3). Measuring the luciferase activity produced by the reporter promoters demonstrated that LPS caused a significant induction of the TNF reporter in full-length pol II-transfected cells (Fig. 7Go, lanes 1 and 2). However, the induction of the TNF reporter in cells containing the RNA pol II {Delta}31 showed a reduced level of induction (around one-third of the full-length RNA pol II; Fig. 7Go). These data suggested that the CTD of RNA pol II may participate in TNF promoter activation by LPS. The reporter vector pGL2 promoter (Promega, Madison, WI) containing a 195-bp SV40 minimal promoter sequence is almost LPS unresponsive (Fig. 7Go, lanes 5 and 6). The CTD deletion did not alter the expression profile of pGL2 in the presence or absence of LPS (Fig. 7Go, lanes 5–8), suggesting that RNA pol II promotion of SV40 minimal promoter differs from that of TNF. To determine whether CTD-dependent TNF induction by LPS is mediated by MAP kinases, we cotransfected pol II or pol II {Delta}31 with or without the four active MKKs/MEKs. As in the observation using endogenous pol II, individual MAP kinase pathways did not lead to significant induction of the TNF reporter gene when {alpha}-amanitin-resistant pol II was employed (Figs. 3Go and 8GoA, lanes 1–5). Significant induction of the TNF reporter by the four MAP kinase pathways was observed when {alpha}-amanitin-resistant full-length pol II was used (Fig. 8GoA, lanes 1 and 5). Induction of the TNF reporter by the four MAP kinase pathways was much lower when pol II {Delta}31 was used (Fig. 8GoB, lanes 1 and 5), with ~90% reduction compared with full-length pol II. We compared the activity of pol II and pol II {Delta}31 on the pGL2 reporter when the dominant-active MKKs were expressed. Activation of the four MAP kinase pathways modestly up-regulated the reporter expression regardless of whether full-length pol II or pol II {Delta}31 was used (Fig. 8Go, A and B, lanes 7–12), indicating that the pol II CTD may be involved in the selective enhancement of transcription. Collectively, our data suggest that the CTD of RNA pol II participates in LPS-induced TNF gene activation and that MAP kinase pathways are involved in the regulation of pol II.



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  		FIGURE 7. CTD of RNA pol II is involved in LPS-mediated TNF transcription. TNF or pGL2 reporters were cotransfected with or without {alpha}-amanitin-resistant pol II possessing either the full-length CTD (pol II) or a shortened CTD with 31 repeats (pol II {Delta}31). The cells were grown for 16 h to allow the transfected pol II gene to be expressed, and {alpha}-amanitin (2.5 µg/ml) was added at this point to inhibit endogenous pol II. Seventy-two hours later, cells were stimulated with LPS (1 ng/ml) for 8 h, after which time luciferase activity was measured. Comparable results were obtained in two experiments.

 


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  		FIGURE 8. CTD of RNA pol II is involved in MAP kinase-mediated TNF transcription. TNF or pGL2 reporter was cotransfected with or without {alpha}-amanitin-resistant pol II that contained the full-length CTD (pol II) (A) or the shortened CTD with 31 repeats (pol II {Delta}31) (B) and dominant-active mutants of MEK1, MEK5, MKK6, and MKK7 into RAW cells as described in Fig. 3Go. The cells were grown for 16 h to allow for expression of the transfected pol II gene. {alpha}-amanitin (2.5 µg/ml) was added at this point to inhibit endogenous pol II. Luciferase activity was measured 72 h later. Comparable results were obtained in two experiments.

 
Having ascertained this, we further investigated the involvement of the RNA pol II CTD by examining whether activation of the four MAP kinase pathways would induce increased levels of phosphorylation of RNA polymerase II mutants. To achieve this, RAW cells transfected with the RNA pol II constructs along with either pcDNA3 vector or dominant-active mutants of MEK1, MEK5, MKK6, and MKK7 were metabolically labeled using [32P]orthophosphate. The recombinant pol II proteins were then immunoprecipitated using anti-HA Ab, resolved on SDS-PAGE gels, and analyzed by phosphoimaging. Activation of the pol II-transfected cells with the four MAP kinases showed enhanced pol II phosphorylation compared with that of unstimulated cells (Fig. 9GoA). In contrast, the pol II {Delta}31 mutant showed little evidence of increased phosphorylation associated with MAP kinase activation. Therefore, it is evident that the CTD of RNA pol II is targeted for phosphorylation after activation of the four MAP kinase pathways. To confirm that this CTD phosphorylation occurs in response to LPS stimulation of RAW cells, we measured CTD phosphorylation before and after LPS stimulation in the presence or absence of SB203580 and/or U0126. A mAb, H14 (Covance), specifically targeting the heptapeptide repeat YSPTSpPS intrinsic to the CTD of pol II, containing a phosphoserine at position 5 was then used in Western analysis. LPS stimulation produced an ~2-fold increase in CTD phosphorylation (Fig. 9GoB, top panel). This LPS-induced CTD phosphorylation was completely inhibited by treating the cells with SB203580 or U0126 (Fig. 9GoB, two middle panels). Because SB203580 and U0126 had an additive effect on LPS-induced TNF reporter gene expression (Fig. 2Go) and because we did not observe an additive effect of these two drugs on CTD phosphorylation (Fig. 9GoB), CTD may not be the only target regulated by p38 or ERK. It is also possible that the measurement of CTD phosphorylation by Western blotting is not sensitive enough to show the combined effect of these two inhibitors. Nevertheless, our data assert that the p38 and ERK pathways are required for LPS-induced CTD phosphorylation. The observed pre-existence of CTD phosphorylation in unstimulated cells is consistent with reports in other cell lines (48). Because there are more than 50 heptapeptide repeats in CTD and because Western analysis cannot distinguish the position of phosphorylation, we do not know whether LPS-induced phosphorylation differs from the pre-existing phosphorylation.



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  		FIGURE 9. Phosphorylation of pol II in response to activation of MAP kinases or LPS stimulation. A, Pol II and pol II {Delta}31 were cotransfected with either pcDNA3 vector or dominant-active mutants of MEK1, MEK5, MKK6, and MKK7 into RAW cells. The cells were selected with {alpha}-amanitin for 72 h, and then were metabolically labeled with [32P]orthophosphate for 2 h. Recombinant pol II proteins were immunoprecipitated with anti-HA Ab. These samples were resolved by SDS-PAGE and exposed on phosphoimaging cassettes. B, RAW cells were stimulated with 1 ng/ml LPS in presence or absence of SB203580 and/or U0126 for different periods of time. The cells were lysed, and equal protein from different samples were separated on 6% SDS-PAGE. The proteins were transferred to nitrocellulose membrane, and the phosphorylated RNA pol II was detected using anti-YSPTSpPS mAb H14. Normal loading of protein in each lane was visualized by staining the membrane with Ponceau S (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Transcriptional activation of the TNF gene in response to LPS stimulation is controlled by various transcription factors in conjunction with the RNA pol II complex. By studying the mechanism through which MAP kinases operate in TNF transcription, we have found that multiple MAP kinase pathways coordinate with each other to regulate the gene. The cis elements found in the TNF promoter, such as Egr-1, cAMP response element-, {kappa}B-, AP-1-, and AP-2-like motifs, are not the targets of these MAP kinase pathways, whereas the TATA box and its flanking sequence is required for MAP kinase-mediated TNF gene induction. This MAP kinase-responsive site represents the region around which the RNA pol II complex assembles, and we have presented evidence to demonstrate that the CTD of RNA pol II participates in MAP kinase-induced TNF gene transcription.

There are a number of transcription factors, such as c-Jun, MEF2A/2C, ELK-1, serum response factor accessory protein-1, ATF1, ATF2, and cAMP response element binding protein, that have been suggested to be regulated by MAP kinase pathways (49). It was predicted that MAP kinases would act via the transcription factors mentioned above and that they would directly or indirectly regulate TNF gene transcription. It was a surprise to find that MAP kinase-mediated TNF gene induction is largely dependent on a 43-bp sequence surrounding the TATA box rather than on the rest of the TNF promoter. These data suggested that important physiological substrates of MAP kinases in TNF expression remain unidentified. It is interesting that the MAP kinase-responsive element seems to possess some level of redundancy in that replacing different regions of the element with unrelated sequences did not abolish its function. The importance of this element is clear because completely replacing this sequence with an unrelated sequence leads to almost nonresponsiveness. The precise determinant harbored within this 43 bp was not revealed by our experimental approaches. Sequence comparison between different species shows that the primary sequence in this region of the TNF promoter is poorly conserved outside of the TATA box, although careful comparison of this region among 16 reported TNF promoters does reveal a very similar AT content (50–60%) and a C-rich cluster in the nonconserved region between different species. Therefore, it is possible that MAP kinase responsiveness is dependent on a secondary structural feature, possibly associated with the AT content or C-rich cluster of the sequence. This structural feature may not necessarily be required for selective protein binding, but it may be required for specific chromatin remodeling. The mechanism by which this sequence determines gene regulation awaits further investigation.

Several observations suggested that the four MAP kinase pathways converge onto one regulatory site. First of all, the MAP kinases exhibited effects over and above those which could be attributed to an additive mechanism, indicating that one regulatory mechanism may be the ultimate target for all four MAP kinase pathways. Second, a region can be localized in the TNF promoter that is responsive to the activation of the four MAP kinase pathways. Third, deleting the CTD of RNA pol II almost abolished TNF induction mediated by the four MAP kinases. The cooperative effect of these MAP kinase pathways is probably mediated by targeting a single substrate such as the RNA pol II holoenzyme at different sites or subunits. The CTD of pol II is a potential target of MAP kinases because there are a number of potential MAP kinase targeting sites there. Phosphorylation of pol II CTD has been intensively studied and, although its role in controlling pol II remains unresolved, a number of protein kinases are able to phosphorylate the CTD on different sites in vitro (50, 51, 52, 53, 54). Experiments in yeast suggest the involvement of several protein kinases in CTD phosphorylation because different CTD phosphorylation events have been implicated in different stages of pol II activity (including elongation) and RNA polymerase release (17, 18, 19, 20). Immunofluorescence studies and protein DNA cross-linking assays suggest that the phosphorylation state of CTD may vary depending on which gene is being transcribed (20, 55). It is possible that different enzymes are involved in modifying the CTD to specifically transcribe selected genes. Negative regulation of gene transcription has been observed upon CTD phosphorylated by Cdc2 or Cdk7 (17). It would be very interesting to address whether MAP kinases play a role in CTD phosphorylation, and if this phosphorylation has a positive function in initiation or elongation of specific gene transcripts. Because multiple MAP kinase pathways have to be activated to achieve a significant level of TNF transcription (Fig. 3Go), the CTD has to be modified by multiple MAP kinase pathways at once or the other component(s) in the pol II complex need to be modified at the same time. This is a complicated situation and there could be many possible mechanisms by which MAP kinases regulate the TNF gene. One possibility is that the CTD is modified by multiple MAP kinases and/or their downstream kinases, leading to a specific state of CTD phosphorylation that is optimal for an LPS-inducible gene such as TNF. Different phosphorylation states of CTD can be generated by different kinases and their combinations because CTD of mammalian pol II has hundreds of serines, threonines, and tyrosines that have the potential to be selectively phosphorylated by these kinases. These phosphorylatable amino acids can provide up to 1036 phosphorylation states that may provide a basis for the selective and quantitative control of gene expression in eukaryotic cells. As for the cooperative action of MAP kinases in TNF gene expression, an initial point from which we can understand the mechanism can be found by identifying the functional substrate(s) of these MAP kinases.

TNF biosynthesis is controlled at multiple levels. The ARE in the 3'-UTR is known to play a pivotal role in TNF mRNA stability and translational regulation (24, 25). The JNK pathway had been shown to be involved in the stability of some ARE-bearing mRNAs (56) and to be required for TNF translation. The studies based on the specific inhibition of p38 by SB203580 suggested that the primary site of p38 action is TNF translation (24, 57). Our unpublished results show that the 3'-UTR in the TNF reporter further up-regulates gene expression in response to the activation of MAP kinases, supporting the notion that posttranscription regulation is indeed an important site targeted by MAP kinase. However, the data presented here add TNF transcription to the list of MAP kinase regulatory sites. Thus, MAP kinase pathways act on multiple levels for the full regulation of TNF.

Although MAP kinase pathways are important, other signal transduction pathways may also play important roles. Studies have shown that the upstream sequence of the TNF promoter also contributes to LPS-induced TNF gene expression (21, 22, 23). Coordination of MAP kinases with other pathways must exist, and indeed, we have observed activation of the NF-{kappa}B pathway by inhibitory {kappa}B protein kinase-2 to further up-regulate MAP kinase-induced TNF gene expression.

It is believed that eukaryotic transcription requires a set of general transcription factors to be assembled with RNA poly II to form a transcription initiation complex, and the CTD of pol II has been reported to specifically interact with some of these factors (8, 10). Our finding that the CTD of RNA pol II is involved in multiple MAP kinase-mediated TNF gene transcriptions indicates that MAP kinase pathways may regulate the RNA pol II complex. The robust induction of the TNF reporter upon activation of the four MAP kinase pathways suggests that study of the regulation of TNF gene transcription could provide a model system for the function of the CTD in gene regulation. Our data provide an important role for the general transcriptional machinery in the control of cytokine synthesis. A future challenge for our study is to define each of the signal transduction pathways in regulating promoters as well as the RNA pol II complex.


   Acknowledgments
 
We thank Dr. J. S. Economou (University of California, Los Angeles, CA) and Hans-Peter Gerber (Genentech, South San Francisco, CA) for plasmids and J. V. Kuhns for excellent secretarial assistance.


   Footnotes
 
1 This work was supported by grants from the U.S. Public Health Service (National Institutes of Health nos. GM51470 and AI41637; to J.H.) and the China Natural Science Foundation (no. 39730140; to J.G.). W.Z. was supported by a fellowship from the Chinese Medical Board. J.H. is an established investigator of the American Heart Association. This is publication no. 12269-IMM from the Department of Immunology of The Scripps Research Institute. Back

2 W.Z. and J.S.D. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jiahuai Han, Department of Immunology IMM-9, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. Back

4 Abbreviations used in this paper: pol II, polymerase II; BMK, Big MAP kinase; CTD, C-terminal domain; ARE, AU-rich elements; UTR, untranslated region; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein; MEF, myocyte-enhancer factor; ATF, activating transcription factor; nmp, nonrelated minimal promoter; MEK, MAP/ERK kinase; M{Phi}, macrophage; m, murine; h, human; pol II {Delta}31, pol II mutant with a CTD containing only 31 repeats. Back

Received for publication June 18, 1999. Accepted for publication March 28, 2000.


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J. Wang, S. Wu, X. Jin, M. Li, S. Chen, J. L. Teeling, V. H. Perry, and J. Gu
Retinoic Acid-Inducible Gene-I Mediates Late Phase Induction of TNF-{alpha} by Lipopolysaccharide
J. Immunol., June 15, 2008; 180(12): 80