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The Journal of Immunology, 2004, 173: 5095-5102.
Copyright © 2004 by The American Association of Immunologists

Novel Negative Regulator of Expression in Fas Ligand (CD178) Cytoplasmic Tail: Evidence for Translational Regulation and against Fas Ligand Retention in Secretory Lysosomes1

Sheng Xiao*, Umesh S. Deshmukh*, Satoshi Jodo{dagger}, Takao Koike{dagger}, Rahul Sharma*, Akiro Furusaki{dagger}, Sun-sang J. Sung* and Shyr-Te Ju2,*

* Department of Internal Medicine, Division of Rheumatology and Immunology, University of Virginia, Charlottesville, VA 22908; and {dagger} Department of Medicine II, Hokkaido University Graduate School of Medicine, Kita-ku, Sapporo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Fas ligand ((FasL) CD178), a type II transmembrane protein, induces apoptosis of cells expressing the Fas receptor. It possesses a unique cytoplasmic tail (FasLCyt) of 80 aa. As a type II transmembrane protein, the early synthesis of FasLCyt could affect FasL translation by impacting FasL endoplasmic reticulum translocation and/or endoplasmic reticulum retention. Previous studies suggest that the proline-rich domain (aa 43–70) in FasLCyt (FasLPRD) inhibits FasL membrane expression by retaining FasL in the secretory lysosomes. This report shows that deletion of aa 2–33 of FasLCyt dramatically increased total FasL levels and FasL cell surface expression. This negative regulator of FasL expression is dominant despite the presence of FasLPRD. In addition, retention of proline-rich domain-containing FasL in the cytoplasm was not observed. Moreover, we demonstrated that FasLCyt regulates FasL expression by controlling the rate of de novo synthesis of FasL. Our study demonstrated a novel negative regulator of FasL expression in the FasLCyt region and its mechanism of action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Fas (CD95) is a type I transmembrane protein expressed by many nucleated cells (1, 2, 3). The physiological Fas ligand (FasL)3 is a type II transmembrane protein expressed by activated T cells and non-T cells under a variety of conditions (4, 5, 6). The extracellular domain of FasL (FasLext) on effector cells binds to Fas on target cells and cross-linking of Fas induces target cells to undergo apoptosis (7, 8, 9). The Fas/FasL-mediated apoptosis pathway has been implicated in immune response regulation (9, 10), peripheral tolerance (5, 11, 12, 13, 14, 15), graft rejection (16, 17, 18), tumor surveillance (19), tissue pathology (20, 21, 22), chemotaxis (17, 18, 23), and maintenance of the immune privileged sites (24, 25).

Regulation of FasL expression has been demonstrated at the transcriptional and posttranslational levels. At the transcriptional level, the FasL gene is regulated by different transcription factors, depending on cell types and experimental conditions (25, 26, 27, 28, 29, 30, 31, 32, 33). At the posttranslational level, cell surface FasL can be removed by metalloproteinase cleavage that generates soluble FasL, which is a poor mediator of cytotoxicity (34, 35). Recent studies also showed that FasL are released from cells in the form of vesicles (36, 37, 38). In contrast to soluble FasL, these vesicles contain full-length FasL and express potent cytotoxic activity (37).

FasL is a member of the TNF family but it possesses a unique 80-aa cytoplasmic tail (FasLCyt) that is highly conserved among species (39). As a type II transmembrane protein, FasLCyt may have motifs that can regulate FasL translation and processing soon after its de novo protein synthesis begins. Recent studies suggest that the proline-rich domain (PRD) of FasLCyt regulates FasL cell surface expression by retaining FasL in the secretory lysosomes (40, 41). Moreover, cells lacking secretory lysosomes strongly expressed cell surface FasL upon transfection with the fasl gene (41). However, these studies were conducted with constructs whose 5' ends were attached to GFP gene. Thus, the effects of GFP, a large protein with ~220 aa, on FasLCyt functions in FasL translation, translocation, processing, and trafficking cannot be ruled out. In addition, the use of GFP-based fluorescence microscopy and flow cytometry makes an accurate quantitative analysis of total FasL expression by transfected cells difficult.

To better define the role of FasLCyt in the translational regulation of FasL expression, we engineered various deletion constructs of human fasl gene without a GFP tag, used these constructs to generate stable transfectants of various cell lines, conducted quantitative ELISA for FasL, and determined their de novo synthesis rates and their homeostatic expression levels. We found that FasLCyt negatively regulates FasL cell surface expression by limiting its total cellular expression level. The responsible region was located between aa 2–33 (FasL2–33). In addition, we observed that fully expressed FasL containing the PRD was not selectively retained in the cytoplasm. Instead, FasLCyt regulates FasL expression by controlling the rate of FasL de novo synthesis. Our study demonstrates the presence of a novel negative regulator of FasL expression in the FasLCyt region and identifies its mechanism of action.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Neuro-2a (mouse neuroblastoma), NIH-3T3 (mouse fibroblast), B16F1 (mouse melanoma), rat basophilic leukemia (RBL), and COS-7 (monkey kidney fibroblast) cell lines were obtained from American Type Culture Collection (Manassas, VA). Culture medium was prepared by supplementing high glucose (4.5 g/L) DMEM (Cellgro; Mediatech, Herndon, VA) with 10% heat-inactivated FCS (Invitrogen Life Technologies, Carlsbad, CA), 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM L-glutamine, 1 mM sodium pyruvate, and 5 µl/L of 2-ME. PE-Alf-2.1 anti-human FasL mAb was purchased from Caltag Laboratories (Burlingame, CA). PE-NOK-1 and FITC-NOK-1 anti-human FasL mAb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Unlabeled G247.4 and NOK-1 anti-FasL mAb were obtained from BD Biosciences (San Diego, CA). All restriction endonucleases were obtained from New England Biolabs (Beverly, MA). The prokaryotic expression vector pBlueScript II KS was obtained from Stratagene (La Jolla, CA). The human FasL cDNA construct and the mammalian expression vector BCMGSneo (14.5 kb) were kindly provided by Dr. S. Nagata (Osaka University Medical Center, Osaka, Japan) (39).

Construction of FasL deletion mutants

The full-length hfasl cDNA cloned in the pBlueScript II KS was used to generate deletion mutants by PCR using different 5' primers and the same 3' primer. All 5' primers used contain the translation start sequence ATG (shown in bold) that codes for methionine. Because methionine is the first amino acid of wild-type (WT) and deletion mutants, deletion begins with amino acid residue 2 of FasL. All primers were obtained from Integrated DNA Technologies (Coralville, IA). The sequences of the 5' primers are 5'-ATGACCTCTGTGCCCAGAAGGCC-3' (for {Delta}33 in which FasL2–33 is deleted), 5'-ATGCTGAAGAAGAGAGGGAACCACAGC-3' (for {Delta}70 in which FasL2–70 is deleted), and 5'-ATGCAGCTCTTCCACCTACAGAAGGAGC-3' (for {Delta}102 in which FasL2–102 is deleted). The sequence of the 3' primer is 5'-GTAAAACGACGGCCAGTGAGCG-3'. The PCR products were subcloned into pBlueScript II KS. These inserts were excised with NotI and XhoI and cloned into BCMGSneo vector (39). Gene sequences of each construct were confirmed by DNA sequencing.

Transfection

Multiple cell lines were transfected with the expression constructs using PolyFect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Briefly, cells (8 x 105 per 60-mm dish) were seeded in 5 ml of culture medium the day before transfection. Culture medium was replaced with 3 ml of DMEM before transfection. To prepare transfection mixtures, 2.5 µg of plasmid DNA were diluted with DMEM to 150 µl, and 15 µl of PolyFect transfection reagent was added. After incubation for 10 min at room temperature, the transfection mixtures were mixed with 1 ml of DMEM and immediately transferred to dishes containing seeded cells. Dishes were gently swirled, cultured for 24 h, and then replaced with culture medium containing 0.4 mg/ml G418 (Invitrogen Life Technologies). Cell populations that survived the G418 selection were expanded in G418-containing culture medium and examined. Typically, the selection process takes 3–4 wk to complete. Expanded cells were analyzed and aliquots were stored in liquid nitrogen tank.

Flow cytometric analysis

To determine cell surface expression of FasL, cells (0.3 x 106) were suspended in 100 µl of PBS containing 4% BSA and incubated with 1 µg of PE-Alf-2.1 mAb or PE-conjugated isotype control for 45 min at 4°C. Reaction mixtures were gently mixed periodically. Cells were washed twice with cold PBS and then analyzed. To determine both cell surface and intracellular expression of FasL, cells were first stained with 2 µg of FITC-NOK-1 mAb for 45 min at 4°C. After washing, labeled cells were fixed for 20 min at room temperature in 2% paraformaldehyde, permeated with 0.1% saponin, and then stained with PE-NOK-1 mAb. Using the same anti-FasL mAb prevented cell surface staining by PE-NOK-1 mAb. At least 104 stained cells were analyzed using FACScan equipped with CellQuest software (BD Biosciences).

Confocal microscopic analysis

Various transfectants were first stained with 2 µg of FITC-NOK-1 mAb at 4°C. After washing, labeled cells were fixed for 20 min in 2% paraformaldehyde, permeated with 0.1% saponin, and then stained with PE-NOK-1 mAb. The stained cells were examined using a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss, Thornwood, NY).

Cell-mediated cytotoxicity

Cell-mediated cytotoxicity was conducted as previously described using the 51Cr-labeled, Fas+ A20 B lymphoma target cells (38). Transfectants were incubated with 2 x 104 target cells at various E:T ratios for 5 h at 37°C in a 10% CO2 incubator. Cell-free supernatants were collected and counted with a gamma counter (LKB, Turku, Finland). Cytotoxicity, expressed as percent of specific Cr-release, was calculated by the formula: 100 x (experimental release – background release)/(total release – background release). Background release was obtained by culturing target cells with medium and total release was determined by lysing target cells with 2% Triton X-100. Experiments were performed in duplicate and repeated at least twice.

FasL-specific ELISA

Cells (107) were treated with Ag-extraction buffer provided with the FasL ELISA kit (Oncogene, Boston, MA). All samples were diluted in sample dilution buffer (provided with the kit) and immediately assayed. Standard curves were generated with various molar concentrations of recombinant soluble FasL. The amount of FasL in each sample was calculated and converted to picomoles based on the molecular weights of the engineered FasL proteins.

FasL mRNA analysis

Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies). FasL and {beta}-actin mRNA was measured by RT-PCR essentially as previously described (42), but our re-designed primers detected FasL mRNA irrespective of their introduced deletion. The sequences of the forward and reverse primers for FasL were 5'-AACTCCGAGAGTCTACCAGCCAG-3' and 5'-GATACTTAGAGTTCCTCATGTAGACC-3', respectively. FasL mRNA was also determined by RNase protection assays using the customized RiboQuant Multiprobe Template set (BD Biosciences). This template set was designed to specifically quantitate mouse L32, mouse GAPDH, and human FasL transcripts.

[35S]Methionine labeling of FasL

Various transfectants were [35S]methionine-labeled ([35S]Express; PerkinElmer, Boston, MA) for the indicated times as previously described (43). Cells were subsequently lysed with 0.05 M Tris-HCl buffer (pH 8.0) containing 0.3 M NaCl, protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO), 2 mM EDTA, and 0.5% Nonidet P-40. NOK-1 mAb (10 µg) was adsorbed onto Protein A/G Plus-agarose (20 µl) (Amersham Biosciences, Piscataway, NJ) for 1 h at room temperature, followed by incubation with cell lysates for 2 h at 4°C. As an internal control, aliquots of lysates were also incubated with a mouse polyclonal anti-small nuclear ribonucleoproteins (snRNP) Abs adsorbed onto Protein A/G Plus-agarose. After extensive washing, bound proteins were released from beads by boiling in SDS-PAGE loading buffer and analyzed by 12% SDS-PAGE. Gels were dried and autoradiographed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
FasLCyt regulates FasL cell surface expression

To define the role of FasLCyt in regulating FasL expression, we generated various deletion constructs of human fasl gene (Fig. 1a). The insert sizes were confirmed in digests using restriction endonucleases NotI and XhoI (Fig. 1a), Their sequences were confirmed by DNA sequence analyses (see Materials and Methods). These expression vectors were transfected into various cell types and G418-resistant cell lines were selected. FasL cell surface expression on the selected cell lines was assessed by flow cytometry using the anti-FasLext mAb (Fig. 1b). Although WT FasL transfectants displayed a relatively low level of membrane FasL, deletion of aa 2–33 from FasL ({Delta}33 FasL) resulted in a significant increase in FasL cell surface expression. This trend was observed in all of the cell lines examined. However, there was variability in expression among individual experiments. Deleting aa 2–70 ({Delta}70 FasL) that contained the PRD further increased the percentage of FasL expression in Neuro-2a, RBL, and B16F1 transfectants (Fig. 1b), but not appreciably in the NIH-3T3 or COS-7 transfectants. In the latter case, the mean fluorescence intensity was slightly increased. Membrane FasL was not detected in vector control (Vc) or {Delta}102 FasL transfectants. {Delta}102 FasL was not expressed on cell membrane because it lacked an intact transmembrane domain.



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  		FIGURE 1. Sequence map of various FasL constructs used for transfection and FasL cell surface expression on various transfected cell lines. a, Deletion mutants used in this study. Deletion mutants of FasL cytoplasmic tail ({Delta}33, {Delta}70, and {Delta}102) were made as described in Materials and Methods. Because methionine residue is the first amino acid of WT and deletion mutants, {Delta}33, {Delta}70, and {Delta}102 represent mutants in which FasL2–33, FasL2–70 and FasL2–102 were deleted, respectively. The PRD is underlined. TM indicates transmembrane domain. The insert sizes of FasL deletion constructs were confirmed by excising the inserts from vectors using restriction endonucleases XhoI and NotI. The digests were analyzed by agarose gel electrophoresis. The sizes of WT, {Delta}33, {Delta}70, and {Delta}102 inserts are ~1000, ~850, ~750, and ~650 bp, respectively. The Vc contains a stuffer of ~350 bp between XhoI and NotI. b, N-terminal deletion of FasL2–33 and FasL2–70 resulted in an increase in FasL cell surface expression. Expression vectors of WT, {Delta}33, {Delta}70, {Delta}102 FasL, and Vc were transfected into various cell lines as indicated. FasL cell surface expression by stable transfectants was detected by flow cytometric analysis using PE-Alf-2.1 anti-FasL mAb. The number in each panel indicates the percentage of cells expressing cell surface FasL. Values below 1.5% are considered undetectable based on staining with isotype-matched control mAb (data not shown).

 
Cell-mediated cytotoxicity of various transfectants

FasL expression on various transfectants was determined with a mAb reactive with FasLext that is presumably not modified by the deletion. To determine whether FasL transfectants were functional, we conducted cell-mediated cytotoxicity using FasL-sensitive A20 B lymphoma cells as targets. We first examined our WT transfectants. FasL-mediated cytotoxicity was detected for each of the five cell lines (data not shown). We then determined the cell-mediated cytotoxicity of transfectants of various deletion mutants of the Neuro-2a and NIH-3T3 cell lines (Fig. 2). In both series, FasL-mediated cytotoxicity was detected in a dose-dependent manner for transfectants of WT, {Delta}33, and {Delta}70 FasL. Cytotoxicity was not detected for {Delta}102 FasL or Vc transfectants in similar conditions. Thus, cell-mediated cytotoxicity apparently correlated with FasL cell surface expression. However, the cytotoxic potentials of WT, {Delta}33, and {Delta}70 transfectants did not correlate well with FasL cell surface expression levels. WT transfectants that expressed significantly lower surface FasL than {Delta}33 or {Delta}70 FasL transfectants displayed cytotoxicity that is either similar to (in the case of Neuro-2a) or slightly stronger than (in the case of NIH-3T3) {Delta}33 and {Delta}70 FasL transfectants. The data strongly suggest that FasLCyt can influence FasL bioactivity across a membrane barrier (S. Jodo and S.-T. Ju, manuscript in preparation).



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  		FIGURE 2. WT, {Delta}33, and {Delta}70 FasL transfectants express cell-mediated cytotoxicity. Cell-mediated cytotoxicity of various transfectants of Neuro-2a (a) and NIH-3T3 (b) were conducted using 51Cr-labeled A20 B lymphoma cells (2 x 104) as target. Cytotoxicity assays and the determination of cytotoxicity (expressed as % specific Cr-release) were conducted as described in Materials and Methods.

 
Effect of FasLCyt deletion on total FasL level

The observation that {Delta}33 FasL transfectants strongly expressed membrane FasL in five different cell lines demonstrated that FasL2–33 was a negative regulator of FasL membrane expression. In addition, it was dominant over FasLPRD because FasL membrane expression was increased even in the presence of intact FasLPRD as observed in {Delta}33 FasL transfectants. Previous studies concluded that FasLPRD prevents FasL from being expressed on the cell surface by retaining FasL in the secretory lysosomes (41). Because WT transfectants expressed low levels of cell surface FasL in our study, the possibility that FasL was retained inside the cells was addressed. For this purpose, FasL distribution at the cell surface and in the cytoplasm was determined using both confocal microscopy and flow cytometry. Confocal microscopic analysis showed that {Delta}33 FasL and {Delta}70 FasL transfectants of Neuro-2a cells have more cell surface and intracellular FasL than WT FasL transfectants (Fig. 3a). Indeed, flow cytometric analysis showed that 7.8, 35, and 75% of WT, {Delta}33, and {Delta}70 FasL transfectants, respectively, express cell surface FasL (Fig. 3b). Similarly, analysis on permeated cells indicated that {Delta}33 and {Delta}70 FasL transfectants have more intracellular FasL (4.4% for {Delta}33 and 15.6% for {Delta}70) than WT FasL transfectants (1.8%). In both analyses, FasL was not detected in Vc or {Delta}102 FasL transfectants. These data indicate that FasLCyt can negatively regulate FasL expression and that the PRD-containing transfectants (WT and {Delta}33) did not retain FasL in the cytoplasm. The total amount of FasL and its cell surface expression were both increased as a result of deletion of these regulators.



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  		FIGURE 3. Expression of intracellular and cell surface FasL by various transfectants. a, Confocal microscopic analysis of intracellular and cell surface FasL. Stable transfectants of Neuro-2a cells were stained with FITC-NOK-1 mAb for cell surface expression of FasL, fixed, permeated, and then stained with PE-NOK-1 mAb for intracellular FasL expression. b, Cells were analyzed with a flow cytometer.

 
Determine the absolute FasL expression level of various transfectants

To provide quantitative determination on the effect of FasLCyt on total FasL expression levels, the total FasL levels of Neuro-2a and NIH-3T3 transfectants were measured using FasL-specific ELISA (Table I). For Neuro-2a transfectants, deleting FasL2–33 resulted in a significant increase in FasL expression compared with WT FasL transfectant. Further deleting the FasL34–70 segment caused more expression of total FasL. For NIH-3T3 transfectants, deleting FasL2–33 also significantly increased total FasL level. However, only a modest increase in FasL expression was observed with the {Delta}70 FasL transfectant. As with confocal microscopic and flow cytometric analyses, ELISA measurements did not detect FasL expression in Vc or {Delta}102 FasL transfectants.


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  		Table I. FasL production by various transfected cell linesa

 
Mechanism of action of the FasLCyt negative regulator on FasL expression

Because FasLCyt did not retain FasL in the cytoplasm and the cell membrane FasL expression was proportional to total FasL levels of transfectants, we asked whether FasL expression was controlled at the transcriptional and/or translational levels. We used both RT-PCR (Neuro-2a and RBL) and RNase protection assays (Neuro-2a and NIH-3T3) to determine the FasL transcription efficiencies of the various transfectants (Fig. 4). No significant differences in FasL-specific RT-PCR products were observed for any of the transfectants except Vc, which lacked FasL mRNA (Fig. 4a). Likewise, protected FasL mRNA was detected in all transfectants except Vc. The levels of protected FasL mRNA among various FasL transfectants of Neuro-2a and NIH-3T3 were similar (Fig. 4b). The data suggest that the increase in FasL expression levels was not caused by transcription efficiency differences.



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  		FIGURE 4. Transcription efficiency of FasL of various transfectants. a, Total RNA was isolated from various transfectants of Neuro-2a and RBL cells. FasL and control {beta}-actin mRNA were measured by RT-PCR as described in Materials and Methods. The sizes of the PCR products are ~300 and 540 bp for FasL and {beta}-actin, respectively. b, Total RNA was isolated from various transfectants of Neuro-2a and NIH-3T3 cells. FasL mRNA was measured by RNase protection assay using a customized kit. In this assay, a 3' fragment of FasL mRNA (encoding a FasLext fragment) was protected. The sizes of protected human FasL, mouse L32, and mouse GAPDH mRNA are 351, 112, and 97 bp, respectively.

 
The FasL translation efficiencies of our transfectants were determined by using [35S]methionine labeling that detects the de novo synthesis of FasL. First, we conducted a 16-h labeling experiment in Neuro-2a transfectants to determine whether the expression of labeled FasL correlated with FasL expression determined in earlier experiments. In this case, a very strong expression was observed with the {Delta}70 FasL transfectant, a strong expression was observed with the {Delta}33 FasL transfectant and a weak expression was observed with the WT transfectant (Fig. 5a, upper panel). The specificity of the assay system was demonstrated by the expected sizes of labeled FasL and by the fact that no detectable incorporation of [35S]methionine into FasL was observed for the {Delta}102 FasL or Vc transfectant. This expression hierarchy was similar to that described earlier using flow cytometric and ELISA measurements. In addition, incorporation of [35S]methionine into snRNP, a normal constituent of Neuro-2a cells, was comparable among transfectants (lower panel). These data indicate that the 16-h [35S]methionine labeling experiment detects the homeostatic state of FasL expression.



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  		FIGURE 5. Translational regulation of FasL expression in various transfectants. a, Determination of the homeostatic expression of FasL based on long-term [35S]methionine incorporation. Various FasL transfectants of Neuro-2a cells were cultured in labeling medium containing [35S]methionine for 16 h. Labeled FasL and labeled snRNP were detected as described in Materials and Methods. The multiple bands of snRNP are due to the snRNP complex formed by various snRNP components. b, Determination of the de novo synthesis rate of FasL. Various FasL transfectants of COS-7 cells (WT, {Delta}33, and {Delta}70) were cultured in [35S]methionine-containing labeling medium for 5 and 10 min, respectively. Labeled FasL and labeled snRNP were detected as described in Materials and Methods. The positions of m.w. standards are indicated on the left side of the gel.

 
To determine whether FasLCyt regulates the rate of FasL de novo synthesis, we conducted short-term (5- or 10-min) [35S]methionine labeling experiments in the COS-7 transfectants of WT, {Delta}33, and {Delta}70 FasL (Fig. 5b). In both cases, the amount of labeled FasL as measured by autoradiography correlated with FasL total expression levels determined in earlier experiments using flow cytometry and ELISA. The incorporation of [35S]methionine into FasL in both the {Delta}33 and the {Delta}70 FasL transfectants was significantly higher than that in the WT FasL transfectant. A slightly higher level of [35S]methionine-labeled FasL was observed in the {Delta}70 FasL transfectant than the {Delta}33 FasL transfectant (Fig. 5b, upper panel). The experiment determined the rate of de novo protein synthesis because a clear increase in [35S]methionine incorporation was observed between 5- and 10-min labeling, and this increase was observed for both the transfected FasL and the control snRNP (Fig. 5b, lower panel). This increase in translation was specific because the de novo synthesis of snRNP proteins, under both the short-term (5- and 10-min) and the long-term (16-h) labeling conditions, was comparable among the transfectants. We also noted that the patterns for WT, {Delta}33, and {Delta}70 FasL were the same for COS-7 and Neuro-2a cells but the snRNP patterns were somewhat different (Fig. 5b). The results indicate that the increase in {Delta}33 and {Delta}70 FasL was due to an increase in the rate of de novo synthesis of the {Delta}33 and {Delta}70 FasL deletion mutants.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Numerous specific motifs that target proteins to certain organelles have been identified. Motifs that regulate the total amount of transmembrane proteins have been described in a number of cases (44). The present study demonstrates that FasLCyt regulates the total level as well as the cell surface expression of FasL. We have identified FasL2–33 as a negative regulator of FasL expression. Presence of this region in FasL prevented strong expression of FasL. Deletion of FasL2–33 increased both the total FasL expression and the membrane FasL expression. The observation of this activity in five cell lines of distinct origin indicated that this regulator functions in a cell type-independent manner. Based on the results obtained from {Delta}70 transfectants, FasL34–70 also appears to negatively regulate FasL level. FasL34–70 contained the PRD that has been shown to negatively regulate cell surface expression of GFP-FasL by retaining FasL in the secretory lysosomes (40, 41). However, our data indicated that the increase in FasL cell surface expression was mainly caused by the increase in total FasL expression. In addition, we demonstrated that FasLCyt regulates FasL expression by limiting the rate of de novo synthesis of FasL.

There are two major differences between our findings and those described previously (27, 28). First, we have identified FasL2–33 as the major negative regulator for FasL membrane expression, whereas Bossi and Griffiths (40, 41) reported that FasL1–37 does not have a role in regulating FasL membrane expression. We observed that WT transfectants, irrespective of their cell types, express low levels of FasL unless FasL2–33 or FasL2–70 were deleted. In contrast, they reported high levels of GFP-FasL expression by WT transfectants that lack secretory lysosomes (40, 41). The second major difference was that we did not detect cytoplasmic retention of WT FasL. This was demonstrated by the low FasL levels in five distinct WT transfectants, irrespective of cell types or of whether they contained secretory lysosomes. In addition, confocal and fluorescent staining failed to reveal a strong retention of WT FasL in the cytoplasm.

There are several possible explanations for these discrepancies. First, Bossi and Griffiths (41) fused GFP to the N terminus of WT FasL and its deletion mutants. This could have modified the function of the N-terminal region of FasL. Because of the close proximity of GFP to the region examined and because of the large size of GFP relative to the small FasLCyt, functions such as regulation of FasL translation efficiency or FasL trafficking could have been affected. Our observations support the former possibility. In contrast, GFP fused in close proximity to PRD (position 37) apparently did not inhibit the regulatory function of FasLPRD in their studies (40, 41). Second, we conducted all of our experiments with stable transfectants. Transient transfection used in their experiments may not permit sufficient time for the full execution of the regulatory mechanisms for FasL expression. In addition, transient transfection may not be influenced by FasL-mediated deletion of Fas+ transfectants. Third, the fasl gene used in their study had a leucine codon instead of cysteine in position 32. The amino acid they reported is different from those published in the literature (26) and those reported in the databanks. The importance of Cys32 residue was suggested by its conservation among humans, mice, and rats (39), and its potential involvement in acetylation, palmitoylation, or disulfide bonding. Fourth, Bossi and Griffiths deleted FasL1–37 and we deleted FasL2–33 (41). It is possible that FasL33–37 contained the critical amino acids responsible for the observed difference. Finally, it is important to emphasize the dominant role of FasL2–33 over FasL33–70 (that contains the PRD) in mediating FasL expression in the present study. This dominance could have been lost in GFP-fused FasL. In support of this, we have generated {Delta}PRD FasL transfectants for NIH-3T3, Neuro-2a, and COS-7 cells, and increases in cell surface FasL expression on the transfectants were not observed (V. Pidiyar and S.-T. Ju, unpublished observation).

Regulation of FasL expression has been extensively studied in T cells. Activated T cells produce FasL as a result of increased fasl gene activation. Despite the inhibitory effect of FasLCyt negative regulatory elements implicated by the present study, activated T cells express FasL on the cell surface. Activation of T cells may induce other mechanisms that overcome the negative regulation by FasLCyt. One of the proposed mechanisms is based on activation-induced increases in secretory vesicle trafficking (45). However, activated T cells from Ashen mice that have a defect in activation-dependent lysosomal secretion were shown to express normal levels of FasL-mediated cytotoxicity (46). It is important to note that our findings are entirely consistent with the FasL expression on activated T cells because we demonstrated that the cell surface expression of FasL was directly proportional to the total FasL produced by cells and that WT FasL was not retained in the cytoplasm.

Our study suggests that the increase in FasL cell surface expression was mostly due to the increase in total FasL levels. Further analyses suggest that the FasL expression level, controlled by FasL2–33, was not due to differences in transcription because all of the transfectants expressed comparable levels of FasL mRNA. This observation also indicated that the difference in FasL protein expression was not due to differences in transfection efficiency. The identical pattern of FasL expression by transfectants in both short- and long-term labeling experiments indicated that de novo synthesis of FasL was the major mechanism responsible for increased FasL expression in {Delta}33 and {Delta}70 transfectants (Fig. 5). As {Delta}33 and {Delta}70 contain, respectively, 90 and 75% of the amino acids of the WT FasL protein, the increase in the de novo synthesized FasL deletion mutants cannot be accounted for by their shortened peptide lengths. Other translational mechanisms must be responsible for the increase in total FasL expression.

Sequences of "short, nearly exact matches" to human FasL1–70 based on "blast hits" were not found in any other transmembrane proteins in the protein databank of NCBI (www.ncbi.nlm.nih.gov). Our data suggest FasL2–70 contains motifs that regulate FasL’s translation rate. FasL is a type II transmembrane protein and FasLCyt contains three positively charged residues, K72K73R74, near its transmembrane domain (Fig. 1a). Because translocation of de novo synthesized transmembrane proteins is regulated by charged amino acids near the transmembrane domain (internal start-transfer sequence), it is likely that these positively charged amino acids are important for the translocation of nascent FasL chains through the endoplasmic reticulum (ER) membrane during de novo protein synthesis (47). The remarkable increase in FasL de novo synthesis resulting from the deletion of FasL2–70 suggests that this early event of FasL translation is a rate-limiting step for FasL synthesis.

Deletion of FasL2–33 also increased the rate of de novo synthesis of FasL. FasL2–33 contains the sequence S17S18A19S20S21 and SXXS is a casein kinase I (CKI)-targeted motif. It has been suggested that this motif may provide a retrograde signaling during T cell activation (48). We have tested this motif for its ability to regulate total FasL expression and FasL cell surface expression. The CKI-specific inhibitor, CK-7 (49), used at optimal but nontoxic concentration, failed to exert a detectable effect on FasL expression by FasL WT transfectants of NIH-3T3 cells. Furthermore, no effect was observed with NIH-3T3 cells transfected with a mutant construct in which the S17S18A19S20S21 site was changed to AAAAA by site-directed mutagenesis (data not shown). These results provide strong evidence that the putative CKI motif in FasL2–33 was not responsible for the regulation of FasL expression levels. FasL2–33 also may have "dityrosine" motifs (Y7PY9PQIY13W, see Fig. 1a). Such dityrosine (YQ, YF) motifs in the CD3{gamma}-chain have been implicated in ER retention and their presence results in reduced membrane expression (44). Perhaps, by regulating both ER translocation and ER retention, FasLCyt could effectively control FasL translation rates and ultimately total FasL expression levels, including expression on the plasma membrane. Study is in progress using more refined deletion mutants and using alanine-based substitution mutants in a systematic manner to identify the critical amino acid residue(s) responsible for the negative regulatory function of FasLCyt.


   Footnotes
 
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.

1 This work was supported in part by National Institutes of Health Grant AI36938 (to S.-T.J.) and HL070065 (to S.-s.J.S.), and a grant from the American Heart Association (to U.S.D.). Back

2 Address correspondence and reprint requests to Dr. Shyr-Te Ju, Department of Internal Medicine, Division of Rheumatology and Immunology, University of Virginia, Charlottesville, VA 22908-0412. E-mail address: sj8r{at}virginia.edu Back

3 Abbreviations used in this paper: FasL, Fas ligand; PRD, proline-rich domain; snRNP, small nuclear ribonucleoproteins; Vc, vector control; WT, wild type; RBL, rat basophilic leukemia; ER, endoplasmic reticulum; CKI, casein kinase I. Back

Received for publication May 18, 2004. Accepted for publication August 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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