Interleukin-2 Promoter Activation in T-Cells Expressing Activated Ha-ras*

Antigen triggering of the T-cell receptor results in an accumulation of activated GTP-bound p21'" pro- tein. To assess the role of ras protein in T-cell activation we have cotransfected the murine thymoma line EL4 with a construct capable of expressing a consti- tutively active, oncogenic form of Ha-ras and a re-porter construct containing the human interleukin-2 promoter fused upstream of the bacterial gene for chloramphenicol acetyltransferase. We show that the ras oncoprotein contributes to interleukin-2 promoter activation. Its pattern of synergism with a calcium ionophore or the lymphokine interleukin- 1 indicates that it replaces a signal mediated by protein kinase C. Interleukin-2 promoter activity in the presence of ras oncoprotein was inhibited by H7, a potent inhibitor of protein kinase C, but not by HA1004, an inhibitor of cyclic nucleotide-dependent kinase, suggesting that protein kinase C mediates the ras effect. In addition, we show that in these cells, expression of activated ras results in activation of a synthetic promoter containing several copies of an NFKB binding site.

EL4 with a construct capable of expressing a constitutively active, oncogenic form of Ha-ras and a reporter construct containing the human interleukin-2 promoter fused upstream of the bacterial gene for chloramphenicol acetyltransferase. We show that the ras oncoprotein contributes to interleukin-2 promoter activation. Its pattern of synergism with a calcium ionophore or the lymphokine interleukin-1 indicates that it replaces a signal mediated by protein kinase C. Interleukin-2 promoter activity in the presence of ras oncoprotein was inhibited by H7, a potent inhibitor of protein kinase C, but not by HA1004, an inhibitor of cyclic nucleotide-dependent kinase, suggesting that protein kinase C mediates the ras effect. In addition, we show that in these cells, expression of activated ras results in activation of a synthetic promoter containing several copies of an NFKB binding site.
Proliferation and subsequent differentiation of resting Tcells is initiated by antigen receptor (TCR)' triggering in combination with accessory signals such as IL-1. Prolonged TCR stimulation is necessary for commitment to proliferation after which activating signals can be removed without affecting the capacity of the cells to complete the differentiation process. Commitment to proliferation coincides with activation of interleukin-2 (IL-2) and IL-2 receptor expression. Signals derived from the TCR induce several known second messenger pathways including limited activation of protein kinase C (PKC), mobilization of calcium and activation of protein tyrosine kinases (reviewed by Clevers et al. (1988) and Crabtree (1989). In vitro, phorbol esters and calcium ionophores can mimick, at least in part, these signals and induce expression of IL-2 or an IL-2 promoter/reporter gene con-" 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.
The proto-oncogene product p21H"'"" is a GTP-binding protein related to the low molecular weight GTPases which are involved in coupling transmembrane receptors to membrane associated enzymes such as adenylate cyclase and phospholipase C (reviewed by Barbacid (1987)). The oncogenicity of ras results from mutations which block GTPase activity or increase the rate of exchange of bound nucleotide such that the GTP-bound active form of the protein accumulates in the cell (Satoh et al., 1987(Satoh et al., , 1988Field et al., 1987). Its location at the inner surface of the plasma membrane and its relatedness to G proteins suggests that normal p21Ha"a8 may mediate extracellular mitogenic stimuli. In support of this, activation of T-cells via the TCR results in an increase in the GTPbound active form of p21Ha"" (Downward et al., 1990). However, a direct role for ras in T-cell activation has not been shown.
We have addressed the question of the role of p21H""ae in T-cell activation using a gene coding for an oncogenic form of the protein transfected into T-cell culture lines.

MATERIALS AND METHODS
Cell Culture, Transfections, and CAT Assays-Transfections, activations, protein determinations, and CAT assays were carried out as described (Macchia et al., 1990;Baldari et al., 1991). Transfection efficiency varied between experiments. For this reason, in each experiment, activations, for 12 h beginning 30 h after transfection, were done on equal aliquots of a single pool of transfected cells. When rastransfected cells were compared with control cells, the transfection mix, including the indicator plasmid, was prepared, then divided into equal aliquots to which were added either the T24 ras plasmid or the vector lacking only T24 sequences. Autoradiograms were scanned using an UltrascanXL enhanced laser densitometer and evaluated using GSXL software. The results of representative experiments are shown.
Phmids-The ras expression plasmid was constructed by insertion of the 1400-bp CluI/DraI restriction fragment containing the Moloney leukemia virus long terminal repeat from pDOL- (Korman et al., 1987) into the Hind111 site into the plasmid pT24C3 (Santos et al., 1982) after filling of the overhanging ends with Klenow polymerase. IL-2/CAT, which contains approximately 2000 bp of the human IL-2 promoter upstream of the bacterial chloramphenicol acetyltransferase (CAT) coding sequence, has been described elsewhere (Macchia et al., 1990). The NFKB-containing plasmid was described as PRDII (Fan and Maniatis, 1989).
Analysis of RNA-RNA was prepared from transfected cells as described (Macchia et al., 1990). Polymerase chain reaction amplification was carried out using the Invitrogen cDNA cycle kit for reverse transcriptiofi-polymerase chain reaction on 2 fig of purified RNA according to the manufacturers' instructions. The oligonucleotide primers were 17-mers designed to be complementary to sequences in the 5'-and 3"nontranslated regions of the human Ha-ras mRNA.

RESULTS AND DISCUSSION
In cells of the murine thymoma line EL4, an IL-2 promoter/ CAT fusion gene responds to treatment with high concentrations (>5 ng/ml) of phorbol myristate acetate (PMA) or suboptimal concentrations (0.5-1 ng/ml) of PMA in combination with either IL-1 or the calcium ionophore A23187. In the absence of PMA, IL-1 and A23187 either alone or in combination have no effect (Macchia et al., 1990) (Fig. 1A).
PMA can replace diacyl glycerol (DAG), the natural activator of PKC, and cause activation and translocation of PKC  3. A synthetic multimeric NFKB binding site is activated by ras. CAT assays of extracts of EL4 cells cotransfected with a plasmid containing eight copies of an NFKB binding site upstream of the CAT gene and either the ras plasmid or the vector without the ras insert. The -fold stimulation compared with untreated cells in the absence of ras is shown above each track. Symbols are as in Fig. 1. H7 (20 p~) was included during the activation where indicated. cells was found to be similar to stimulation of control cells by PMA. Treatment of ras-transfected cells with IL-1 resulted in an approximately &fold increase in CAT activity, whereas treatment with 0.7 ng/ml PMA did not augment the CAT activity. ras-induced activation of the NFKB promoter was also inhibited by H7. In the presence of H7 expression of the NFKB/CAT construct was reduced almost to constitutive level, whereas IL-1 activation was reduced to the level obtained by IL-1 activation in the absence of ras (Fig. 3).
We conclude that TCR-induced accumulation of GTPbound p21Ha"an plays an important role in activation of IL-2 gene expression and consequently T-cell activation. In addition, we suggest that p21Ha"'* controls PKC activation of both AP-1 and NFKB like transcription factors. Since in some cells, transformation by oncogenic ras results in release of DAG (Wolfman and Macara, 1987) it is possible that p21Hacouples the TCR to a phospholipase. However the mechanism by which the TCR controls accumulation of the GTPbound form is as yet still unclear.