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Originally published In Press as doi:10.1074/jbc.M110675200 on April 11, 2002

J. Biol. Chem., Vol. 277, Issue 24, 21123-21129, June 14, 2002
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Regulation of T Cell Receptor CD3zeta Chain Expression by L-Arginine*

Paulo C. RodriguezDagger , Arnold H. ZeaDagger , Kirk S. CulottaDagger , Jovanny ZabaletaDagger , Juan B. Ochoa§, and Augusto C. OchoaDagger

From the Dagger  Tumor Immunology Program, Stanley S. Scott Cancer Center and Department of Pediatrics, Louisiana State University, Health Sciences Center, New Orleans, Louisiana 70112 and the § Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received for publication, November 6, 2001, and in revised form, March 21, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

L-Arg plays a central role in the normal function of several organ systems including the immune system. L-Arg can be depleted by arginase I produced by macrophages and hepatocytes in several disease states such as trauma and sepsis and following liver transplantation. The decrease in L-Arg levels induces a profound decrease in T cell function through mechanisms that have remained unclear. The data presented here demonstrate that Jurkat T cells cultured in medium without L-Arg (L-Arg-free RPMI) have a rapid decrease in the expression of the T cell antigen receptor zeta  chain (CD3zeta ), the principal signal transduction element in this receptor, and a decrease in T cell proliferation. This phenomenon is completely reversed by the replenishment of L-Arg but not other amino acids. These changes are not caused by cell apoptosis; instead, the diminished expression of CD3zeta protein is paralleled by a decrease in CD3zeta mRNA. This change in CD3zeta mRNA expression is not caused by a decrease in the transcription rate but rather by a significantly shorter CD3zeta mRNA half-life. This mechanism is sensitive to cycloheximide. Therefore, the regulation of L-Arg concentration in the microenvironment could represent an important mechanism to modulate the expression of CD3zeta and the T cell receptor and consequently of T cell function.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

L-Arg plays a central role in several functions of the immune system (1-3). It is metabolized in macrophages by two independent enzymatic pathways (4), the inducible nitric-oxide synthetase and arginase I, leading to different effects on the immune system (4-11). L-Arg is metabolized by inducible nitric-oxide synthetase to produce nitric oxide, one of the principal cytotoxic mechanisms in macrophages (12-15). Alternatively, arginase I metabolizes L-Arg to L-ornithine and urea, the first being the precursor for the production of polyamines that are essential for cell proliferation and fibroblast function (5, 6, 11). The depletion of L-Arg by an increased production of arginase I following liver transplantation (16-18), severe trauma (9, 20), or sepsis (21) coincides with a major decrease in T cell proliferation. Furthermore, the infusion of high doses of L-Arg results in a recovery of T cell function and an increase in the number of CD4+ cells (22-24), suggesting that L-Arg may play an important role in regulating the T cell function by mechanisms that have remained unclear.

The T cell receptor zeta  chain (CD3zeta ) is the principal signal transduction element of the T cell antigen receptor (TCR) (25-27). A decreased expression of CD3zeta has been described in T cells from patients with cancer (28-32), lupus (33), and chronic infectious diseases such as leprosy (34) and tuberculosis (35). The mechanisms mediating the CD3zeta decrease are poorly understood. We tested the effect of the absence of L-Arg on T cell signal transduction. The results show that Jurkat T cells cultured in tissue culture medium without L-Arg had a rapid decrease in the expression of CD3zeta but not of other chains of the TCR such as CD3epsilon (36). The absence of L-Arg did not impair the up-regulation of the IL-2 receptor chains nor the production of IL-2 after antigen stimulation (36). This effect was specific to L-Arg, since the depletion of other amino acids such as L-glutamine or L-leucine did not change the expression of CD3zeta (36). In addition, the work presented here suggests that the CD3zeta down-regulation induced by L-Arg starvation is not caused by apoptosis but rather through post-transcriptional mechanisms that decrease the half-life of the CD3zeta mRNA. This process is completely reversible by the replenishment of L-Arg.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Tissue Culture Medium-- Tissue culture medium included complete RPMI 1640 (C-RPMI),1 which contains 1140 µM L-Arg (BioWhitaker (Walkersville, MD)), L-Arg-free RPMI, and L-glutamine-free RPMI (L-Gln-free RPMI) (Invitrogen). All media were supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 25 mM HEPES (Invitrogen), 4 mM L-glutamine (Biowhitaker), and 100 units/ml penicillin/streptomycin (Invitrogen). RPMI from Invitrogen or RPMI from BioWhitaker showed similar effects in Jurkat CD3zeta expression.

Cell Lines-- Jurkat T cells, a CD4+ cell line (clone E6-1) (ATCC, Manassas, VA), was used to test the role of L-Arg starvation on CD3zeta expression. Cells were counted and recultured every 2 days to 5 × 105 cells/ml in fresh C-RPMI (Bio Whitaker, Walkersville, MD). In each experiment, CD3zeta was tested by flow cytometry at time 0 (at the time of passage), and its value ranged between 55 and 63 mean fluorescence intensity. The flow cytometry parameters were kept constant, so data could be compared from one experiment to the next. COS-7 L African green monkey kidney cells (Invitrogen) were used for transfection experiments. These cells do not express detectable levels of CD3zeta mRNA or protein before the transfection.

Antibodies and Probes-- Anti-CD3epsilon -fluorescein isothiocyanate, anti-CD3zeta -phycoerythrin, and anti-APO2.7-fluorescein isothiocyanate (Beckman-Coulter, Miami, FL) were used for flow cytometry. Mouse IgG1-fluorescein isothiocyanate and mouse IgG-phycoerythrin (Beckman-Coulter) were used as isotype controls. Human cDNA for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1.6 kb) (CLONTECH) and CD3zeta (1.7 kb) (a kind gift from Dr. Cox Terhorst and Dr. Allan Weissman) were used to detect CD3zeta and GAPDH by Northern blot.

Flow Cytometry-- Flow cytometry analysis was performed as previously described (36). Briefly, 5 × 105 Jurkat cells were washed once with Dulbecco's phosphate-buffered saline (DPBS) and resuspended in 200 µl of DPBS containing 1 µg of anti-CD3epsilon or isotype control. Cells were incubated for 15 min at 4 °C, washed with DPBS, and resuspended in 200 µl of DPBS containing 500 µg/ml of digitonin plus 1 µg of anti-CD3zeta or 1 µg of isotypic control. To detect apoptosis, 1 µg of anti-APO2.7 was added. Cells were incubated for 8 min after which they were washed and resuspended in 400 µl of DPBS. Fluorescence acquisition and analysis were done in a Coulter-EPICS XL flow cytometer (Beckman-Coulter, Miami, FL) with a 488-nm argon laser. The data are expressed as mean channel fluorescence intensity.

Northern Blot-- 10 million Jurkat cells were used for RNA extraction. Total RNA was extracted by lysis with TRIzol (Invitrogen) and purified according to the manufacturer's specifications. Northern blot analysis was performed as previously described (37). Briefly, 10 µg of total RNA from each sample were electrophoresed under denaturing conditions, blotted onto Nytran membranes (Schleicher & Schuell) and cross-linked by UV irradiation. Membranes were prehybridized at 42 °C in ULTRAhyb buffer (Ambion, Austin, TX) and hybridized overnight with 1 × 106 cpm/ml of 32P-labeled probe. Human cDNA for GAPDH (CLONTECH, Palo Alto, CA) and CD3zeta were labeled by random priming using a RediPrime Kit (Amersham Biosciences) and [alpha -32P]dCTP (3000 Ci/mmol; PerkinElmer Life Sciences). Membranes were washed three times, once at 65 °C for 30 min, using a buffer containing 2× SSC and 0.1% SDS and twice at 65 °C for 30 min in 0.1× SSC and 0.1% SDS. Membranes were subjected to autoradiography at -70 °C using Eastman Kodak Co. Biomax-MR films and intensifying screens. To test mRNA transcription inhibition, cells were cultured in tissue culture medium containing actinomycin D (Act D) (Sigma) at a final concentration of 5 µg/ml. Cycloheximide (10 µg/ml) (Sigma) was used to study the role of de novo proteins in the mRNA stability. To determine the half-life of RNA, the CD3zeta /GAPDH ratio at time 0 was considered as 100% of expression and was used to calculate the half-life of CD3zeta mRNA at all other time points. A densitometer, alphaImager 2000 (Alpha Innotech Corp., San Leandro, CA) was used to analyze the band intensities. All signal intensities were normalized to GAPDH.

Nuclear Run-on-- Nuclear run-on experiments were performed as previously described (38). Briefly, nuclei from 50 × 107 cells/sample were isolated by lysing cells in 4 ml of lysis buffer (10 mM Tris-HCl, pH 7.4, 3 mM MgCl2, 10 mM NaCl, 150 mM sucrose, and 0.5% Nonidet P-40 (Sigma)) for 5 min on ice. Nuclei were centrifuged at 2000 rpm for 5 min at 4 °C, and pellets were resuspended in lysis buffer without Nonidet P-40. Nuclei were pelleted again as described above and resuspended in 150 µl of freezing buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA). Run-on assays were performed by adding 150 µl of 2× transcription buffer (20 mM Tris-HCl, pH 8.0, 300 mM KCl, 10 mM MgCl2, 200 mM sucrose, 20% glycerol, 1 mM dithiothreitol, 0.5 mM ATP, GTP, CTP) and 100 µCi of 800 Ci/mmol [alpha -32P]uridine triphosphate (PerkinElmer Life Sciences). Samples were incubated at 29 °C for 30 min. Labeled transcripts were isolated using TRIzol (Invitrogen) as described before and purified according to the manufacturer's specifications. Equal amounts of radioactivity (2 × 106 cpm of labeled RNA) or the same number of nuclei were added in 2 ml of ULTRAhyb buffer (Ambion) to Nytran membranes onto which 500 ng of denatured full-length human CD3zeta cDNA and human GAPDH cDNA were immobilized using a slot blot apparatus (Invitrogen) and a UV cross-linker (Fisher). Hybridization was performed at 42 °C for 48 h. Filters were washed three times at 42 °C for 15 min with 2× SSC, 0.1% SDS and twice at 65 °C for 20 min with 0.2× SSC, 0.1% SDS. Filters were then autoradiographed at -70 °C. Data were normalized for the content of GAPDH present in each sample using an alphaImager 2000 (Alpha Innotech Corp.).

Transfection of COS 7 L Cells with CD3zeta cDNA-- Coding region CD3zeta cDNA (1.5 kb) was excised from pGEM/zeta and joined by T4 DNA ligase (Promega, Madison, WI) to pCI mammalian expression vector (cytomegalovirus promoter) (Promega), which had been previously linearized with XbaI and EcoRI. COS-7 L African green monkey kidney cells (Invitrogen) were cultured to 90% confluence according to the manufacturer's specifications, after which they were transfected with PCI/CD3zeta or empty vector using LipofectAMINE 2000 reagent (Invitrogen), following the manufacturer's recommendations. The cells were then cultured and studied for mRNA stability. Briefly, COS-7 L cells were cultured for 48 h, after which 3 × 106 cells were cultured in C-RPMI or L-Arg-free RPMI in presence of Act D (5 µg/ml) for 2, 4, and 8 h. Northern blot was used to study the CD3zeta and GAPDH half-life, as described above.

Statistical Analysis-- Comparison in CD3zeta expression between Jurkat cells cultured in C-RPMI and L-Arg-free RPMI was done by Student's t test using the Graph Pad statistical program (Graph Pad, San Diego, CA).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Absence of L-Arginine Induces a Decrease of CD3zeta Expression in Jurkat Cells-- Jurkat cells cultured in RPMI 1640 without L-Arg (L-Arg-free RPMI) have a rapid decrease in the expression of CD3zeta by 24 h that becomes more pronounced after 48 h (Fig. 1A). A careful study of the kinetics of this phenomenon shows a gradual decrease of CD3zeta detectable after 2 h of culture in L-Arg-free RPMI, followed by a plateau of 24 h and a final and more pronounced decrease by 48 h. In contrast, cells cultured in C-RPMI or L-glutamine-free RPMI (L-Gln-free RPMI) do not show changes in the expression of CD3zeta (Fig. 1B). The flow cytometry data was confirmed using Western blots (data not shown). We previously reported that the down-regulation of the CD3zeta induced by L-Arg starvation was specific, since the depletion of other amino acids such as L-glutamine and L-leucine did not change the expression of this protein (36). In addition, we showed that the absence of L-Arg does not produce a decrease in other TCR proteins such as CD3epsilon . Furthermore, it does not prevent the up-regulation of other receptors such as the IL-2 receptor nor the production of IL-2 (36). The decrease in CD3zeta induced by the absence of L-Arg was completely reversed by replenishment of L-Arg (Fig. 2). Jurkat cells cultured in L-Arg-free RPMI for 48 h and transferred to RPMI with 100 µM L-Arg recover the CD3zeta expression to normal levels within 24 h. In contrast, cells kept in L-Arg-free RPMI and transferred to fresh L-Arg-free RPMI continued to have a decreased expression of CD3zeta (Fig. 2).


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  		Fig. 1.   The absence of L-Arg induces the down-regulation of the CD3zeta in Jurkat cells. A, cells were cultured in the presence (C-RPMI) or absence of L-Arg (L-Arg-free RPMI) for 24 and 48 h, during which changes in CD3zeta were measured using flow cytometry. B, Jurkat T cells were cultured in C-RPMI, L-Arg-free RPMI, and L-Gln-free RPMI. Changes in CD3zeta expression were measured at 2, 4, 8, 12, 24, and 48 h. All experiments were repeated at least three times. Error bars show the S.D.


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  		Fig. 2.   Replenishment of L-Arg induces the recovery of the CD3zeta expression. Jurkat cells were cultured in L-Arg-free RPMI for 48 h, after which they were washed and cultured in L-Arg-free RPMI plus 100 µM L-Arg (black-diamond ) or L-Arg-free RPMI () for an additional 24 h. Cells were cultured in C-RPMI as control (black-square). CD3zeta expression was measured by flow cytometry.

An increase in T cell apoptosis has been suggested as one of the mechanisms that could cause a decreased expression of CD3zeta (39, 40). However, Jurkat cells cultured in L-Arg free medium did not show an increase in the percentage of APO 2.7-positive cells in the first 72 h of culture, compared with cells cultured in C-RPMI (Table I). Similar results were observed using annexin V and propidium iodide as markers of apoptosis/necrosis (data not shown).

                              
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Table I
Percentage of apoptotic cells in Jurkat cells cultured in the presence or absence of L-Arg

The Absence of L-Arg Leads to a Decrease in CD3zeta mRNA Expression-- We studied the expression of mRNA encoding for CD3zeta as a possible explanation for the decreased expression on CD3zeta protein. As shown in Fig. 3A, Jurkat cells cultured in L-Arg-free RPMI for 24 and 48 h showed a marked decrease in CD3zeta mRNA expression (2.3- and 4.2-fold decrease, respectively), compared with control cells cultured in C-RPMI. The kinetics of the phenomena showed a gradual decrease in CD3zeta mRNA expression starting at 4 h of culture in L-Arg-free RPMI, which continued throughout the 48 h of culture (Fig. 3, B and C). In contrast, cells cultured in C-RPMI had a constant expression of the CD3zeta RNA.


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  		Fig. 3.   The absence of L-Arg induces a down-regulation of the CD3zeta mRNA in Jurkat cells. Cells were cultured in C-RPMI or L-Arg-free RPMI for 24 and 48 h (A) or for 2, 4, 8, 12, or 24 h (B and C). 10 µg of total RNA was used for Northern blot analysis. CD3zeta and GAPDH mRNA detection were always done on the same membrane. The band intensities were measured by densitometry, and values are expressed as CD3zeta /GAPDH ratio (C). Data shown are from one representative experiment of three performed.

We then tested whether the decrease in CD3zeta mRNA caused by L-Arg starvation was due to a decrease in the transcription rate of the CD3zeta mRNA. Run-on experiments were done using the nuclei from Jurkat cells cultured for 24 h in the presence or absence of L-Arg. Results consistently showed a lower [alpha -32P]UTP incorporation (data not shown) and lower transcription rates for both CD3zeta and GAPDH in the nuclei of Jurkat cells cultured in L-Arg-free RPMI (Fig. 4A), suggesting a general decrease in RNA synthesis in the absence of L-Arg. However, there were not significant differences in the CD3zeta /GAPDH ratio when compared with cells cultured in C-RPMI (Fig. 4A). In addition, when run-on experiments were normalized based on the amount of radiolabeled RNA (2 × 106 cpm), there were no measurable differences in either CD3zeta or GAPDH mRNA expression in Jurkat cells cultured in C-RPMI and L-Arg-free RPMI (Fig. 4B). Therefore, although the absence of L-Arg induced a general decrease in the transcriptional rate, it was not specific for CD3zeta mRNA.


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  		Fig. 4.   Down-regulation of CD3zeta mRNA induced by L-Arg starvation is not due to a specific decrease in CD3zeta gene transcription. Jurkat cells (5 × 107) were cultured in C-RPMI or L-Arg-free RPMI for 24 h. Nuclei were labeled, and the rate of transcription of CD3zeta and GAPDH was assessed by nuclear run-on analysis. Data presented are from two experiments. The relative values are expressed as CD3zeta and GAPDH ratio.

L-Arg Starvation Diminishes the Half-life of CD3zeta mRNA in Jurkat Cells-- The diminished expression of CD3zeta mRNA could also be explained by a decrease in RNA stability. It has been previously reported that the absence of L-Arg induces changes in post-transcriptional mechanisms in yeast (41). Furthermore, L-Arg starvation induced changes in the expression of genes associated with its own metabolic pathway (41). To test whether L-Arg starvation induced changes in the CD3zeta RNA stability, Jurkat cells were cultured in C-RPMI and L-Arg-free RPMI in the presence of Act D (5 µg/ml), an inhibitor of transcription, followed by measurement of the expression of CD3zeta mRNA at various time points. Jurkat cells cultured in C-RPMI displayed a constant expression of CD3zeta mRNA for at least 8 h. In contrast, cells cultured in L-Arg-free RPMI had a significantly lower CD3zeta mRNA stability (p < 0.0001), which was seen as early as 4 h after culture (Fig. 5A). The half-life of CD3zeta mRNA cultured in L-Arg-free RPMI was 3.8 h as compared with 11.2 h of Jurkat cells cultured in C-RPMI (Fig. 5B). Therefore, the data suggest that the decreased half-life of CD3zeta mRNA could be induced by post-transcriptional mechanisms.


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  		Fig. 5.   The absence of L-Arg reduces the half-life of the CD3zeta mRNA in Jurkat cells. A, cells were cultured in C-RPMI or L-Arg-free RPMI plus Act D (5 µg/ml). Total RNA was extracted at 2, 4, 8, 12, and 24 h, electrophoresed, transferred, and hybridized with specific probes for CD3zeta and GAPDH. B, kinetics of CD3zeta mRNA half-life was done based on the densitometry values. CD3zeta /GAPDH ratio at time 0 was considered as 100% expression and was used to calculate the mRNA half-life at all other time points. Data shown are from one of three experiments.

To test this possibility COS-7 L African green monkey kidney cells were transfected with a mammalian expression vector containing the coding region for CD3zeta . The transfected COS-7 L cells were then cultured in C-RPMI or L-Arg-free RPMI and tested for mRNA stability. As shown in Fig. 6, CD3zeta mRNA was detected in COS-7 L cells transfected with the plasmid containing the coding region CD3zeta cDNA (lane 3), but it was not detectable in cells not transfected or transfected with the empty vector (lanes 1 and 2). CD3zeta mRNA expression was similar in transfected COS-7 L cells cultured in presence or absence of L-Arg for 2, 4, and 8 h (lanes 4-9). However, when cells were cultured in the presence of Act D, an inhibitor of transcription, COS-7 L cultured in L-Arg-free RPMI had a decreased CD3zeta mRNA half-life (lanes 13-15) when compared with cells cultured in C-RPMI (lanes 10-12). These results confirm that the absence of L-Arg induces a decrease in CD3zeta mRNA stability by post-transcriptional mechanisms.


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  		Fig. 6.   L-Arg starvation induces a down-regulation in the CD3zeta mRNA stability by post-transcriptional mechanisms. COS-7 L African green monkey kidney cells (Invitrogen) were cultured to 90% confluence. They were then transfected with an empty PCI mammalian expression vector (Promega) or PCI vector plus coding region CD3zeta cDNA. Cells were then cultured in C-RPMI or L-Arg-free RPMI in the presence or absence of Act D (5 µg/ml). Northern blot analysis was used to detect the CD3zeta mRNA stability.

Decrease in mRNA Half-life Is Associated with de Novo Protein Synthesis-- Because little is known about the mechanisms regulating CD3zeta mRNA under these conditions, we tested whether the decreased in RNA stability was associated with the de novo synthesis of proteins. Jurkat cells were cultured in C-RPMI or L-Arg-free RPMI in the presence of cycloheximide (a protein synthesis inhibitor) and Act D. As shown in Fig. 7, cells cultured in C-RPMI had a longer CD3zeta mRNA half-life than cells cultured in L-Arg-free RPMI (lanes 2 and 3 versus lanes 4 and 5). There were no differences in CD3zeta mRNA when cycloheximide was added to cells cultured with and without L-Arg (lanes 6-9), since, as previously shown, there were no differences in transcriptional rate. In contrast, when cells were cultured in the presence of Act D and cycloheximide, there was a significant increase in the CD3zeta mRNA level in cells cultured in L-Arg-free RPMI (lanes 4 and 5 versus lanes 12 and 13). This suggests that the decrease in CD3zeta mRNA stability could be associated with the synthesis of a new protein such as a ribonuclease or other protein regulating CD3zeta mRNA stability.


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  		Fig. 7.   The decrease in CD3zeta mRNA half-life is sensitive to actinomycin D. To determine whether a newly synthesized protein was associated with the decrease in CD3zeta RNA stability, Jurkat cells were cultured in C-RPMI or L-Arg-free RPMI for 2, 4, and 8 h in the presence of 5 µg/ml Act D (lanes 2-5 and 10-13) and/or 10 µg/ml cycloheximide (lanes 6-13). RNA was isolated, and Northern blot analysis for the CD3zeta and GAPDH was done. B, densitometric value analysis. Data shown are from one representative experiment of two performed.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of T cells is initiated by the binding of antigen to the alpha beta chains of the TCR in the context of major histocompatibility complex and the delivery of co-stimulatory signals (42). The TCR is a multiple subunit complex made of eight polypeptide subunits including TCRalpha beta and CD3epsilon gamma , -epsilon delta , and -zeta zeta . CD3zeta generally exists within the TCR·CD3 complex as a disulfide-linked homodimer, which is mostly intracellular and plays a central role in initiating the signal transduction cascade that leads to T-cell activation (25, 27). CD3zeta is also the rate-limiting step in the assembly and membrane expression of the TCR (26). Regulation of CD3zeta expression is mostly mediated by antigen stimulation (43). Binding of antigen to the TCRalpha beta chains leads to the phosphorylation of the CD3zeta immunoreceptor tyrosine-based activation motifs, which starts T cell activation. It also triggers TCR internalization and the subsequent degradation of CD3zeta in lysosomes or proteasomes (43-46). T cell activation eventually leads to the up-regulation of CD3zeta mRNA, resulting in the recovery of CD3zeta expression to normal levels by 24-48 h and an increase in the total number of TCR expressed on the cell membrane (43, 46).

CD3zeta expression has been found decreased in T cells and NK cells of patients with cancer, autoimmune diseases, and chronic infectious diseases. The mechanisms that can lead to a down-regulation of CD3zeta include T cell apoptosis (39, 40), the production of peroxide by macrophages and polymorphonuclear cells (47), and chronic T cell stimulation (48). Here we describe a new mechanism by which the expression of CD3zeta may be modulated in several diseases. Mechanisms including transport, synthesis, and recycling help maintain serum concentrations of L-Arg between 80 and 120 µM (4). L-Arg is metabolized by nitric-oxide synthase to produce NO and citrulline and by arginase I and II to produce urea and ornithine (1, 4, 9, 12, 49). High concentrations of arginase I and the resulting low levels of L-Arg have been reported in the serum of patients undergoing liver transplantation (16-18) or trauma (21) and patients with certain tumors (50-54), who also have a significant impairment in T cell function (24). We therefore studied whether L-Arg could modulate the expression of CD3zeta . Recently, we showed that the absence of L-Arg induced a decrease in T cell proliferation and down-regulated the expression of CD3zeta but did not alter the production of IL-2 or the up-regulation of the IL-2 receptor chains (36). Thus, the deficiency of L-Arg selectively alters the expression of certain proteins essential to T cell activation. In this report, we have elucidated some of the mechanisms by which the absence of L-Arg induces the down-regulation of CD3zeta in a T cell line. The decrease of CD3zeta expression is seen as early as 2 h, followed by a more significant drop after 24 h of L-Arg starvation. The mechanisms mediating the initial decrease on CD3zeta (first 2 h of L-Arg starvation) are unclear but do not appear to be associated with a decrease in RNA expression, since the initial changes in the CD3zeta mRNA levels are only seen at 4 h. This consistent decrease in CD3zeta mRNA is more likely to be associated with the second and more profound decrease in CD3zeta protein seen by 24 h, since the mean transcription time for CD3zeta is around 16 h.

The molecular mechanisms involved in the control of gene expression by amino acid deprivation have been extensively studied in yeast (41). In mammalian cells, the effect of starvation of different amino acids is less clear. The lack of L-Arg has been associated with the induction of certain genes regulating L-Arg metabolism, such as arginosuccinate synthase (41, 55), probably as a pathway for the synthesis of arginine from citrulline. Gazzola et al. (56) and Hyatt et al. (57-59) have reported that the absence of L-Arg induces the transcription of genes encoding for the several amino acid transport system, including CAT-1 (57, 58), which transport cationic amino acids such as L-Arg from the extracellular space into the cytoplasm. Moreover, Diah et al. (60) recently identified the TA1/LAT-1/CD98 light chain gene encoding a protein associated to lymphocyte activation, integrin signaling and amino acid transport including L-Arg, which is also increased by L-Arg starvation. The absence of leucine and L-Arg have also been associated with an increase in the amount and stability of mRNA for the CHOP gene (61-64). This gene encodes a transcription factor that blocks the action of the CCAAT/enhancer-binding protein beta , which in turn inhibits the normal proliferation of cells (62). Therefore, there is clear evidence of amino acids causing changes in the expression of specific genes.

In the model presented here, a diminished expression of CD3zeta appears to be clearly related to a decrease in mRNA. The CD3zeta gene is directed by a TATA-less promoter that extends from +58 to -307. Rellahan et al. (65) have shown that the nuclear transcription factor Elf-1 is associated with CD3zeta transcription. Elf-1 binds two sequences in the CD3zeta promoter (-35 and -135) maintaining the constitutive expression of CD3zeta mRNA. A decrease in cytoplasmic Elf-1 has been described in the T cells of patients with systemic lupus erythematosus, who also have a concomitant decrease in CD3zeta expression (66). In our model, we found that Jurkat cells cultured without L-Arg displayed a decrease in nuclear Elf-1 expression and a decrease in the Elf-1 binding to Ets-1 binding site in the CD3zeta promoter (data not shown). However, we did not find a specific down-regulation in the transcriptional rate of CD3zeta (Fig. 4). It is possible, however, that this gene is regulated by multiple nuclear transcription factors, which could maintain the CD3zeta transcription in the absence of Elf-1. More recently, the consensus amino acid response element (ATTGCATCA) has been described in several genes (41). Several reports have suggested that amino acids such as methionine, histidine, asparagine, cysteine, and arginine function as nuclear transcription factors, increasing the transcription and stability of several genes (41). However, the promoter of CD3zeta does not contain this particular amino acid response element, and lack of L-Arg did not induce a specific decrease in the transcription level of CD3zeta , although it induced a general decrease in transcriptional rate.

We also studied whether post-transcriptional regulation was involved in the decrease of CD3zeta mRNA induced by L-Arg starvation. Cells cultured in the absence of L-Arg had a shorter half-life of CD3zeta mRNA compared with cells cultured in the presence of L-Arg (Fig. 5), suggesting that L-Arg starvation induced a post-transcriptional regulation of the CD3zeta mRNA. The available information on mRNA stability in states of amino acid deprivation is complex and often contradictory. Guerrini et al. (67, 68) have described a differential post-transcriptional regulation of asparagine synthase mRNA induced by starvation of amino acids. Bruhat et al. (61) have found that mRNA extracted from HeLa cells cultured in the absence of leucine have a longer half-life than mRNA of cells cultured in the presence of leucine (61, 62). However, the molecular mechanisms that affect the stability of mammalian genes in these settings remain to be characterized. Our data show that a decrease in the CD3zeta mRNA half-life was associated with de novo protein synthesis (Fig. 7), suggesting a possible role of new proteins, probably ribonucleases, in mediating the post-transcriptional regulation of CD3zeta mRNA. This could occur by modification of the mRNA turnover or by degradation (69-71). Furthermore, transfection experiments using the coding region CD3zeta cDNA under the control of a cytomegalovirus promoter showed that COS-7 L cells cultured in the absence of L-Arg displayed a decreased CD3zeta mRNA half-life, confirming the post-transcriptional regulation of the CD3zeta gene in L-Arg starvation conditions.

In summary, our findings suggest that the L-Arg starvation for longer than 24 h induces a decrease in CD3zeta expression in Jurkat cells, due in part to a decrease in mRNA stability. A similar decrease in CD3zeta expression can be observed in activated T cells cultured in the absence of L-Arg.2 These results could be a model to understand the decreased CD3zeta expression found in T cells from patients with cancer, chronic infections such as tuberculosis, or chronic inflammatory diseases such as lupus, where an increase in arginase I could result in a local or systemic decrease of L-Arg concentrations. Other diseases, including trauma and sepsis, also have an increased production of arginase I, low levels of L-Arg, and a decreased T cell response; however, the expression of CD3zeta has not been studied in these patients.

    ACKNOWLEDGEMENTS

We thank Dr. Cox Terhorst for providing the CD3zeta cDNA and Dr. Allan Weissman (NCI, National Institutes of Health, Bethesda, MD), Dr. Kevin Brown, and Dr. Ronald Luftig (Louisiana State University Health Science Center, New Orleans, LA) for the review and constructive comments on the manuscript. We are also grateful to Sandra Lee for assistance in the preparation of the manuscript.

    FOOTNOTES

* This work was supported in part by NCI, National Institutes of Health, Grant CA82689-01, CA88885-01, and R21 CA831198.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tumor Immunology Program, Stanley S. Scott Cancer Center and Dept. of Pediatrics, Louisiana State University, Health Sciences Center, 533 Bolivar St., Rm. 455, New Orleans, LA 70112. Tel.: 504-599-0914; Fax: 504-599-0864; E-mail: aochoa@lsuhsc.edu.

Published, JBC Papers in Press, April 11, 2002, DOI 10.1074/jbc.M110675200

2 A. Zea, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: C-RPMI, complete RPMI; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DPBS, Dulbecco's phosphate-buffered saline; Act D, actinomycin D; IL-2, interleukin-2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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