Cot Kinase Activates Tumor Necrosis Factor-α Gene Expression in a Cyclosporin A-resistant Manner*

Cot kinase is a protein serine/threonine kinase, classified as a mitogen-activated protein kinase kinase kinase, implicated in T lymphocyte activation. Here we show that an increase in Cot kinase expression promotes tumor necrosis factor-α (TNF-α) production in Jurkat T cells stimulated by soluble anti-CD3 or by low concentrations of phorbol 12,13-dibutyrate (PDBu) and calcium ionophore. Overexpression of Cot kinase in Jurkat cells activates TNF-α gene expression. Cot kinase promotes TNF-α promoter activation to a similar extent as calcium ionophore and PDBu or soluble anti-CD28 and PDBu. Neither phorbol esters nor calcium ionophore can replace Cot kinase on TNF-α promoter-driven transcription. Expression of a dominant negative form of Cot kinase inhibits TNF-α promoter activation induced by stimulation with either calcium ionophore and PDBu, soluble anti-CD28 and PDBu, or soluble anti-CD3 and PDBu. TNF-α promoter-driven transcription by Cot kinase is partially mediated by MAPK/ERK kinase and is cyclosporin A-resistant. Cot kinase increases at least the AP-1 and AP-2 response elements. These data indicate that Cot kinase plays a critical role in TNF-α production.

Tpl-2/Cot kinase is a mitogen-activated protein kinase kinase kinase that activates both the ERK 1 and JNK signal transduction pathways (1)(2)(3)(4). The COT kinase gene was first cloned in a truncated form in transformed foci induced in SHOK cells by transfection of the genomic DNA of a human thyroid carcinoma cell line (5). The human COT gene is unique in the genomic sequence (5). The normal human cellular homologue has an open reading frame encoding 467 amino acids, being the first 397 identical to the truncated form, whereas the 69 amino acids from the C terminus are replaced by 18 amino acids in the truncated form (6 -8). The rat homologue gene was identified as an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats (Tpl-2) (9,10). The provirus insertion occurs in the last intron of the Tpl-2 gene. Transgenic mice expressing the truncated oncogenic protein in thymocytes develop T cell lymphomas (11). As occurs with the human homologue, the disruption of the last coding exon of Tpl-2 appears to unmask the oncogenic potential of the protein (6, 10 -12). However, overexpression of the human normal gene is also capable of conferring the transformed phenotype in established cell lines (6,8).
At the amino acid level the identity between the human Cot kinase and the rat Tpl-2 homologue is Ͼ95% (1). Transient expression of Cot/Tpl-2 kinase activates ERK1 (1,13,14). Overexpression of the rat homologue in human Jurkat T cells also phosphorylates and activates JNKK, and consequently JNK is also phosphorylated and activated (1). This activation of JNK by Cot kinase leads to the phosphorylation of c-jun in its Nterminal region (1).
The data shown here demonstrate that overexpression of Cot kinase enhances TNF-␣ secretion in Jurkat cells. Transfection of Cot kinase in Jurkat T cells promotes TNF-␣ gene expression. We also demonstrate that a dominant negative form of Cot kinase inhibits TNF-␣ promoter-driven transcription. Our data also further demonstrate that Cot kinase activates TNF-␣ promoter-driven transcription in a CSA-resistant way and that Cot kinase regulates at least the AP-1 and AP-2 response elements.

EXPERIMENTAL PROCEDURES
DNA Constructs-The DraI fragment (222-1871 nt) of cot (8) was subcloned in the eukaryotic expression vector pEF-BOS, previously digested with BamHI, treated with CIP, and subsequently with Klenow enzyme. A pEF-BOS-cot construct, in the 5Ј-3Ј orientation relative to the EF promoter, was selected. A similar pEF-BOS-trunc-cot construct, in the 5Ј-3Ј orientation relative to the EF promoter, was obtained by digesting truncated cot with DraI (30 -1302 nt) (5).
The inactive kinase form of full-length cot was generated by PCR. The AvaI-HindIII fragment of cot was cloned into the AvaI-HindIII sites of a normal pUC19 vector. A PCR with the mutagenic primers AGAA-TGGCGTGTGCACTGATCCCA and TAGTCTACCGAATTTAACTAG-ATG encompassing the substitution Lys-168 to Ala-168 was performed using this construct as template. Separately, cot cDNA (8) was ligated to a pUC19 vector devoid of AvaI and HindIII sites. The AvaI-HindIII fragment of cot was replaced by the mutated AvaI-HindIII fragment obtained by PCR, yielding inactive cot (inac-cot), which was subse-* This work was supported in part by Plan Nacional, Comunidad de Madrid, and Europharma. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
quently cloned in the pEF-BOS vector as described for cot kinase and obtaining pEF-BOS-inac-cot(5Ј-3Ј). DNA sequencing of inac-cot was performed to verify the construct.
Generation of Antibodies-The anti-CD3 monoclonal antibody was obtained by injecting 10 6 T3b hybridoma cells (41,42), generously provided by Dr. F. Sanchez-Madrid in Balb/c mice, previously pristanized. Immunoglobulin from the ascitic fluid was purified by Sepharose-protein A chromatography.
Cells and Medium-The human leukemia T cell line Jurkat was obtained from Dr. Abelardo López-Rivas and maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (Life Technologies, Inc.), gentamycin (50 g/ml), and L-glutamine (2 mM) (complete medium).
TNF-␣ Production Assay-DNA-mediated gene transfer into Jurkat cells was accomplished by electroporation (43). Exponentially growing cells were washed and resuspended in complete medium at a concentration of 2 ϫ 10 7 /ml, and 1 ml of the cell suspension was transferred into a 0.4-cm electroporation cuvette (Bio-Rad), and unless otherwise indicated, 10 g of the different pEF-BOS constructs and 5 g of the different pTNF␣-Luc constructs were added. Electroporation was accomplished with a Gene Pulser apparatus (Bio-Rad) with a capacitance of 960 microfarads and an electrical field of 300 V. The electroporated cells were transferred into tissue culture flasks (Costar) in complete medium at a concentration of 10 6 cells/ml. After 2 h in culture, transfected cells were stimulated with calcium ionophore A23187 (Boehringer Mannheim) and different concentrations of PDBu (Sigma) or with soluble anti-CD3 for 24 h. Culture supernatants were tested for TNF-␣ production with the human TNF-␣ quantification enzymelinked immunosorbent assay kit (Genzyme), following the manufacturer's instructions. The detection limit according to the standard curve was over 73 pmol.
The transfection efficiency of electroporation, as tested by expression of the fluorescent protein from the pTR-UF2 construct (44), was about 35%.
To perform the RT reaction (45), 2 g of total RNA from nonstimulated or stimulated cells were heated at 37°C for 30 min in the presence of 100 mM dithiothreitol, 0.5 mM dNTPs, 50 units of ribonuclease inhibitor (Life Technologies, Inc.), 2.5 units of DNase (Life Technologies, Inc.) and RT buffer (Life Technologies, Inc.), and then treated at 95°C for 5 min. Ten M random primers (Life Technologies, Inc.) were subsequently added, and the mixture was heated at 70°C for 10 min. After chilling on ice, 200 units of reverse transcriptase (Superscript RNase H Ϫ , Life Technologies, Inc.) were added, and the reaction was continued at 42°C for 60 min. The reaction was finished by heating at 99°C for 5 min.
To perform the PCR the specific oligonucleotides, 5Ј AGCCTCT-TCTCCTTCCTGAT (277-297 nt) and 3Ј AGTAGATGAGGGTCCAG-GAG (575-595 nt), deduced from the human TNF-␣ cDNA, and 5Ј AGCACAATGAAGATCAAGAT (1292-1311 nt) and 3Ј TGTAACG-CAACTAAGTCATA (1460 -1479 nt), deduced from the human ␤-actin cDNA were used. The first round was performed for 25 cycles, with 10 M TNF-␣ primers, 1 M ␤-actin primers, and with 1 l of the RT, and at an annealing temperature of 57°C. The second round was performed in the same conditions, using 0.2 l of the first PCR as template, except that 10 M of the 4 primers were used. The reactions were analyzed in 1% agarose gels.
TNF-␣ Promoter-driven Transcription Assay-DNA-mediated gene transfer into Jurkat cells was performed as explained above. Jurkat cells were cotransfected with different TNF-␣ promoter-Luc reporter constructs (5 g/ml) together with control construct (pEF-BOS) (10 g/ml) or different pEF-BOS-cot constructs (10 g/ml). After 30 min in culture, cells were stimulated for 12 h with different stimuli as follows: soluble anti-CD3, soluble anti-CD28 9.3 antibody (generously donated by Dr. C. June), calcium ionophore A23187, or PHA (Sigma) in the presence or absence of different concentrations of PDBu (Sigma). 8-Br-cAMP (Boehringer Mannheim), Dex (Sigma), CSA, or PD 98059 (MEK inhibitor) (Calbiochem) were added to the cells 30 min after electroporation and then the cells were stimulated 2 h later. Twelve hours after stimulation cells were collected by centrifugation, and Luc activity was determined by the luciferase assay kit (Promega), according to the manufacturer's instructions. Cell extracts were normalized by protein measurements with the D c protein assay (Bio-Rad). Table I illustrates the effect of Cot expression in Jurkat T cells on TNF-␣ production. Jurkat cells stimulated with 0.25 M calcium ionophore and 50 ng/ml PDBu (maximum stimulation) produced about 240 pmol/5 ϫ 10 5 cells of TNF-␣, independently of whether cells were electroporated with pEF-BOS-cot(5Ј-3Ј), pEF-BOS, or no plasmid. Stimulation with suboptimal concentrations of calcium ionophore and PDBu only increased TNF-␣ production in cells overexpressing Cot kinase (Table I). Soluble anti-CD3 (10 g/ml) alone did not stimulate TNF-␣ production in pEF-BOS or non-plasmid electroporated cells. However, with this stimulus TNF-␣ production was detected in pEF-BOS-cot(5Ј-3Ј)-electroporated cells. In the absence of any stimuli pEF-BOS-cot(5Ј-3Ј)-transfected cells were not able to produce TNF-␣, indicating that overexpression of Cot kinase by itself was not able to induce TNF-␣ production.

Cot Kinase Promotes TNF-␣ Production in Submaximally Stimulated Jurkat T Cells-
Cot Kinase Induces TNF-␣ Gene Expression in Jurkat T Cells-We then decided to investigate whether Cot kinase regulated TNF-␣ gene expression in Jurkat cells. RNA from pEF-BOS, pEF-BOS-cot(5Ј-3Ј), or non-plasmid electroporated cells stimulated or not with different concentrations of calcium ionophore and PDBu was isolated to perform RT-PCR assays. In the different electroporated cells stimulated with 0.25 M calcium ionophore and 50 ng/ml PDBu (maximum stimulation), TNF-␣ mRNA was detected (Fig. 1). A similar amount of TNF-␣ PCR product was obtained in Cot-transfected cells stimulated with 0.1 M calcium ionophore and 5 ng/ml PDBu (submaximal stimulation). However, in pEF-BOS or no plasmid-transfected cells incubated with these concentrations of stimuli, a significant decrease in the intensity of the band corresponding to the TNF-␣ PCR product was detected. Without stimuli, the TNF-␣ PCR product was only detected in cells overexpressing Cot TABLE I Regulation of TNF-␣ production by Cot kinase Jurkat cells (2 ϫ 10 7 ) were electroporated with 10 g/ml pEF-BOS-cot(5Ј-3Ј), 10 g/ml pEF-BOS, or no DNA (-) as described under "Experimental Procedures" and stimulated with calcium ionophore A23187 (0.25 M) and PDBu (50 ng/ml) (maximum stimulation), with calcium ionophore A23187 (0.1 M) and PDBu (5 ng/ml), with calcium ionophore A23187 (0.1 M) and PDBu (0.5 ng/ml), or with soluble anti-CD3 (10 g/ml) for 24 h, and TNF-␣ production was measured in the supernatant. The data expressed in pmol/5 ϫ 10 5 cells show the mean of three experiments performed in duplicate.
Cot Kinase Activates Ϫ1185 TNF-␣ Promoter-driven Transcription in Jurkat T Cells-Next, we decided to determine whether Cot kinase enhanced TNF-␣ promoter-driven transcription. Jurkat cells electroporated with Ϫ1185 pTNF␣-Luc alone or together with pEF-BOS-cot(5Ј-3Ј) or pEF-BOS were subjected to different stimuli, and Luc activity was measured.
In Ϫ1185 pTNF␣-Luc alone or together with pEF-BOS electroporated cells, it is necessary to add both calcium ionophore (0.25 M) and PDBu (50 ng/ml) (maximum stimulation) to observe an increase (about 25-fold) in TNF-␣ promoter-driven transcription ( Fig. 2A). Cells transfected with Cot kinase together with the Ϫ1185 pTNF␣-Luc exhibited a similar Luc activity in the absence of any stimulus ( Fig. 2A). Jurkat cells overexpressing Cot kinase treated with calcium ionophore (0.25 M) and PDBu (50 ng/ml) (maximum stimulation) or calcium ionophore (0.25 M) alone resulted in a further increase in the Luc activity. Stimulation of these cells with 50 ng/ml PDBu, or with suboptimal doses of calcium ionophore and PDBu (0.1 M and 5 ng/ml respectively), or with 2 g/ml of PHA did not further enhance Cot kinase-induced TNF-␣ promoter activation ( Fig. 2A).
Similar results were obtained when Jurkat cells were transfected with pEF-BOS-trunc-cot(5Ј-3Ј) instead of pEF-BOScot(5Ј-3Ј) (data not shown). Jurkat cells overexpressing the kinase-inactive form of Cot, by transfection of pEF-BOS-inaccot(5Ј-3Ј), did not enhance the Ϫ1185 pTNF␣-driven transcription of the Luc gene (data not shown). These data indicate that the kinase activity of Cot is necessary for TNF-␣ promoter activation. CSA added at a concentration of 100 ng/ml inhibited the TNF-␣ promoter-driven transcription in cells electroporated with pEF-BOS and Ϫ1185 pTNF␣-Luc or only with pTNF␣-Luc by 90%. CSA, used at the same concentration, did not significantly inhibit the TNF-␣ promoter transcription activity in Jurkat cells overexpressing truncated Cot kinase or Cot kinase (Fig. 2B).
Soluble Anti-CD28 or Soluble Anti-CD3 Does Not Cooperate with Cot Kinase for Transactivation of the TNF-␣ Promoter-To determine whether stimulation with soluble anti-CD28 (1 g/ ml) or with soluble anti-CD3 (10 g/ml) further enhanced Cot kinase activation of the TNF-␣ promoter, cells were electroporated with either pEF-BOS-cot(5Ј-3Ј) or pEF-BOS, and Ϫ1185 pTNF␣Luc or only with the Ϫ1185 pTNF␣ ϪLuc, and different stimuli were added.
Cells electroporated with the Ϫ1185 pTNF␣-Luc alone or together with pEF-BOS exhibited a significant activation of the TNF-␣ promoter-driven transcription when stimulated with anti-CD28 and anti-CD3 (about a 9-fold induction) or when activated with anti-CD28 and PDBu (about a 20-fold induction) (Fig. 3). In these cells CSA inhibited a 30% TNF-␣ promoter activation by soluble anti-CD3 and anti-CD28 and did not regulate the stimulation by anti-CD28 and PDBu. One of the hallmarks of the anti-CD28 stimulation of cytokine production is its insensitivity to CSA (49).

Expression of Kinase-inactive Cot Inhibits TNF-␣ Promoter Activation in Stimulated Jurkat Cells-Jurkat cells electroporated with
Ϫ1185 pTNF␣-Luc alone or together with pEF-BOSinac-cot(5Ј-3Ј) or pEF-BOS were subjected to stimulation with different additives, and TNF-␣ promoter-driven transcription of the Luc gene was measured.
pEF-BOS-inac-cot-transfected cells exhibited a significant reduction in the Luc activity, when compared with cells electroporated with pEF-BOS and Ϫ1185 pTNF␣-Luc or with cells electroporated only with Ϫ1185 pTNF␣-Luc. Inhibition of the TNF-␣ promoter-driven transcription in kinase-inactive Cot electroporated cells was observed with all the different stimuli: soluble anti-CD28 and PDBu, or soluble anti-CD3 and PDBu, or calcium ionophore and PDBu, although the percentage of inhibition varied with the different stimuli and different con-
Cot kinase also activated the Ϫ36 pTNF␣-Luc construct; this plasmid only contains an AP-2 response element (25). This increase in the Ϫ36 TNF-␣ promoter-driven transcription with Cot kinase was similar to that observed in pEF-BOS electroporated cells stimulated with 1 g/ml anti-CD28 and 50 ng/ml PDBu (Fig. 5). Activation of the different TNF-␣ promoter-Luc constructs by Cot kinase was CSA-insensitive (data not shown). Truncated Cot kinase activated the different TNF-␣ promoter-Luc constructs described above in a similar manner as Cot kinase (data not shown).
Cot Kinase Activation of the AP-1 Response Element in Jurkat Cells-In Cot kinase-transfected cells the Ϫ105 pTNF␣-Luc construct exhibited a much higher Luc activity than the Ϫ36 pTNF␣-Luc construct. Several response elements have been identified in the Ϫ105to Ϫ36-bp region of the human TNF-␣ promoter (19,20,25,37), including the Ϫ60 bp AP-1 response element. Rhoades et al. (25) have shown, in different cell systems, that this AP-1 response element plays an important role in the transcriptional regulation of the human TNF-␣ promoter. On the other hand, Cot/Tpl-2 kinase is a mitogen-activated protein kinase kinase kinase that activates the ERK1 and the JNK pathways and consequently induces JNK to phosphorylate c-jun (1). All these data indicated that Cot/Tpl-2 kinase could regulate the AP-1 response element. To test this hypothesis we decided to study the regulation of the AP-1 element by Cot kinase in Jurkat cells. To perform this study two different constructions were used. One was the Ϫ73 col promoter linked to the Luc gene that contains only an AP-1 site as response element (39,50). The other construct was the NFAT-AP-1 composite element from the IL-2 promoter linked to the Luc gene (40,51,52).
To determine whether the expression of Cot and truncated Cot could contribute to the activation of the Ϫ73 col promoter, we examined the effect of transfection of pEF-BOS-trunccot(5Ј-3Ј), pEF-BOS-cot(5Ј-3Ј), pEF-BOS, or no additional plasmid on the Ϫ73 col promoter-driven transcription of the Luc gene.
Cells transfected with truncated Cot or Cot kinase exhibited, respectively, a 15-or 12-fold higher Luc activity than cells electroporated with Ϫ73 pcol-Luc alone or together with pEF-BOS (Fig. 6A). Addition of PDBu (50 ng/ml) or calcium ionophore (0.25 M) to cells electroporated with Ϫ73 pcol-Luc alone or together with pEF-BOS did not increase the value of the Luc activity; however, an increase of about 7-fold was observed when both stimuli were added together (Fig. 6A) (50). Addition of 0.25 M calcium ionophore alone or 0.25 M calcium ionophore and 50 ng/ml PDBu further increased the Ϫ73 col promoter-driven transcription in cells overexpressing truncated Cot or Cot. Addition of PDBu alone to these cells did not further enhance the Ϫ73 col promoter-driven transcription (Fig. 6A). As expected, Cot kinase did not activate the Ϫ63 col promoter linked to the Luc gene (data not shown). In this construct the AP-1 element is deleted (39,50).
Jurkat cells were transfected with the NFAT-AP-1 composite element from the IL-2 promoter linked to the Luc gene alone or together with pEF-BOS-trunc-cot(5Ј-3Ј), pEF-BOS-cot(5Ј-3Ј), or pEF-BOS. In the absence of any stimuli truncated Cot or Cot activated about 20-or 15-fold, respectively, the Luc activity when compared with cells electroporated with the NFAT-AP-1-Luc construct alone or together with pEF-BOS plasmid (Fig.   FIG. 5. Cot kinase regulation of TNF-␣ promoter constructs in Jurkat cells. Jurkat cells were transfected with pEF-BOS-cot(5Ј-3Ј) (10 g/ml) or with pEF-BOS (10 g/ml) and different constructs of the TNF-␣ promoter (Ϫ1185, Ϫ615, Ϫ305, Ϫ106, or Ϫ36 bp) (5 g/ml) linked to the Luc gene. Cells electroporated with pEF-BOS and the different TNF-␣ promoter-Luc constructs were stimulated with 0.25 M calcium ionophore and 50 ng/ml PDBu or with anti-CD28 (1 g/ml) and PDBu (50 ng/ml). To cells electroporated with pEF-BOS-cot(5Ј-3Ј) and the different TNF-␣ promoter-Luc constructs no stimuli were added. 100% of Luc activity is given to the value obtained with Ϫ1185 TNF-␣ promoter-Luc-transfected cells. I, calcium ionophore. The graph shows the mean of three different experiments. 6B). Stimulation with 0.25 M calcium ionophore and 50 ng/ml PDBu increased the Luc activity by about 1000-fold, independently of whether cells were electroporated with the NFAT-AP-1-Luc alone or together with pEF-BOS-trunc-cot(5Ј-3Ј), pEF-BOS-cot(5Ј-3Ј), or pEF-BOS. Addition of calcium ionophore alone only increased the Luc activity to this extent in cells overexpressing truncated Cot kinase or Cot kinase. Addition of 50 ng/ml PDBu did not stimulate the Luc activity in any of the transfected cells (Fig. 6B).
Effect of 8-Br-cAMP, Dex, CSA, and MEK Inhibitor on the AP-1 Response Element Activated by Cot Kinase in Jurkat T Cells-To study whether overexpression of Cot kinase and calcium ionophore stimulated the AP-1 response element through the same mechanism as PDBu and calcium ionophore, several inhibitors were added to electroporated cells prior to stimulation.
Cells transfected with pEF-BOS-cot(5Ј-3Ј) and Ϫ73 pcol-Luc were stimulated only with calcium ionophore (0.25 M). Cells electroporated with pEF-BOS and Ϫ73 pcol-Luc, or only with the Ϫ73 pcol-Luc were activated with calcium ionophore (0.25 M) and PDBu (50 ng/ml). Addition of 8-Br-cAMP (0.5 mM) or Dex (10 Ϫ7 M) did not inhibit the Luc activity in any of the transfected cells (Fig. 7A). MEK inhibitor reduced the Ϫ73 col promoter-driven transcription by about 60% independently of whether Jurkat cells were transfected with Cot kinase or not. CSA inhibited the Luc activity in Cot-transfected cells by about 15% and the Luc activity in cells electroporated with pEF-BOS and Ϫ73 pcol-Luc by about 50% (Fig. 7A).
When the NFAT-AP-1 composite element from the IL-2 promoter was tested, CSA reduced almost completely the Luc activity in cells electroporated with pEF-BOS-cot(5Ј-3Ј) and stimulated with calcium ionophore (0.25 M) (Fig. 7B). In cells transfected with the NFAT-AP-1 composite site linked to the Luc gene alone or together with pEF-BOS, and stimulated with calcium ionophore (0.25 M) and PDBu (50 ng/ml), the inhibition by CSA was complete (Fig. 7B). Addition of 8-Br-cAMP (0.5 mM) or Dex (10 Ϫ7 M) did not inhibit the Luc activity in either pEF-BOS-cot(5Ј-3Ј) or pEF-BOS-transfected cells. MEK inhibitor reduced (about 50%) the Luc activity of the NFAT-AP-1 composite linked to the Luc gene to the same extent in all the electroporated cells (Fig. 7B). DISCUSSION Cot/Tpl-2 kinase is a protein serine/threonine kinase that belongs to the mitogen-activated protein kinase kinase kinase family (2)(3)(4). Cot/Tpl-2 kinase has been involved in IL-2 production (43) and lymphocyte activation (1,9,10). This paper demonstrates that Cot promotes TNF-␣ production in Jurkat cells stimulated by soluble anti-CD3 or by low concentrations of PDBu and calcium ionophore. In addition, the data also show that Cot kinase, by itself, promotes TNF-␣ mRNA expression. The data also show that Cot kinase induces TNF-␣ promoter activation as it enhanced transcription of a reporter gene linked to the TNF-␣ promoter in Jurkat T cells. Here, we also demonstrate that expression of a kinase-inactive form of Cot inhibits TNF-␣ promoter activation. Together these data indicate that Cot kinase plays a critical role in T cell TNF-␣ production.
Overexpression of Cot kinase in Jurkat cells is sufficient to induce TNF-␣ gene expression (Fig. 1) and TNF-␣ promoter activation ( Fig. 2) but not for TNF-␣ secretion (Table I), confirming that TNF-␣ production is not only regulated at transcriptional levels but that some additional events in TNF-␣ production are regulated. Supporting this view, calcium appears to control pre-TNF-␣ processing and TNF-␣ secretion (53)(54)(55), and the data shown here demonstrate that Cot kinase does not replace calcium-generated signals (see below).
We have recently shown that Cot kinase enhances IL-2 promoter activation, although, in contrast with the TNF-␣ promoter, Cot kinase alone could not regulate IL-2 promoter activation in Jurkat T cells (43). All these data also demonstrate that in the activation of TNF-␣ and IL-2 promoters some common intracellular pathways are shared, but other(s) are cytokine-specific.
Different signals regulate TNF-␣ gene expression in different cell types (15,16,19,27). Calcium influx alone, by regulating at least the NFAT response elements in a CSA-dependent manner, is sufficient for the rapid gene induction in A20 B cells and Ar-5 T cells (19,26,27). On the other hand, stimulation of MLA 144 T cells, U937 macrophages, or 729-6 B cells by phorbol esters is sufficient to induce TNF-␣ promoter activation; the AP-1 site plays an important role in this transcriptional regulation (25). Activation of the AP-1 of the Ϫ73 col promoter site in Jurkat T cells requires phorbol esters and calcium ionophore ( Fig. 6) (50). The data shown here demonstrate that in Jurkat T cells stimulation with calcium ionophore or PDBu alone is not sufficient for induction of TNF-␣ transcription, which requires the addition of both stimuli together. Soluble anti-CD28 antibody or soluble anti-CD3 antibody are not sufficient either to stimulate expression of this gene, and in Jurkat T cells addition of PDBu is also needed.
Addition of PDBu to Cot-transfected cells does not further induce transactivation of the TNF-␣ promoter, the Ϫ73 col promoter, or the NFAT-AP-1 composite element, indicating that the effects of PDBu on the activation of the TNF-␣ promoter and the two AP-1 containing sequences tested can be replaced in Jurkat cells by overexpression of Cot kinase. However, Cot kinase does not mimic the effects of PDBu exactly, because Cot kinase overexpression alone activates the TNF-␣ promoter constructs, the collagenase promoter construct, and to a certain extent the NFAT-AP-1 composite element and PDBu does not.
Calcium influx activates TNF-␣ promoter through the CSAsensitive NFAT response element (26,27,38,56). Stimulation of TNF-␣ promoter by calcium and PDBu is CSA-sensitive, but Cot kinase TNF-␣ promoter activation is CSA-insensitive suggesting that Cot kinase activation of this promoter is independent of the calcium pathway. This hypothesis is reinforced by the fact that addition of calcium ionophore in Cot-transfected cells further stimulates the TNF-␣ promoter, the Ϫ73 col promoter, and also the NFAT-AP-1 composite element.
The results obtained with a kinase-inactive form of Cot (Fig.  4) and with MEK inhibitor and CSA (Fig. 2B) indicate that Cot kinase and stimulation by PDBu and calcium share some signal pathways (the ERK and probably the JNK transduction pathways), but not other(s), leading to TNF-␣ promoter activation.
To our knowledge this is the first time that TNF-␣ promoter has been shown to be activated by stimulation with anti-CD28 and PDBu. Cot kinase mimics better the activation of this promoter by anti-CD28 and PDBu than by calcium ionophore and PDBu, because activation of TNF-␣ promoter by Cot kinase or by anti-CD28 and PDBu is not regulated by CSA. On the other hand, we have also demonstrated that expression of a kinase-inactive form of Cot inhibits the TNF-␣ promoter-driven transcription stimulated by anti-CD28 and PDBu. Further-FIG. 7. 8-Br-cAMP, Dex, CSA, and MEK inhibitor on the regulation of AP-1 response element activated by Cot kinase. Cells were transfected with pEF-BOS-cot(5Ј-3Ј) (10 g/ml), pEF-BOS (10 g/ml), or without pEF-BOS construct and with the Ϫ73 col promoter-Luc (5 g/ml) (A) or the NFAT-AP-1-Luc (5 g/ml) (B). A and B, after the addition of 8-Br-cAMP (0.5 mM), Dex (10 Ϫ7 M), CSA (100 ng/ml), or MEK inhibitor (20 g/ml). Cells transfected with pEF-BOS or without pEF-BOS were stimulated with PDBu (50 ng/ml) and calcium ionophore (0.25 M), and cells electroporated with pEF-BOS-cot(5Ј-3Ј) were stimulated with calcium ionophore (0.25 M). The graphs show the mean induction of three different experiments, giving a value of 100% of Luc activity to the different transfected cells and without addition of inhibitors, C. more, unstimulated Cot-transfected cells or anti-CD28 and PDBu-stimulated pEF-BOS electroporated cells activated, through its AP-2 site, the Ϫ36 TNF-␣ promoter construct to a similar extent. Cot kinase-transfected cells exhibit an induction of about 20-fold in the NFAT-AP-1 composite element (Fig.  6B), a similar level of activation as that achieved by anti-CD28 and phorbol esters in Jurkat T cells (57). We have previously demonstrated that Jurkat cells express the cot gene prior to stimulation (43), but the mechanism by which Cot kinase is activated is still unknown. All these data indicate that anti-CD28 together with another stimulus could regulate Cot kinase activation in T cells.