FasL Expression in Activated T Lymphocytes Involves HuR-mediated Stabilization*

A prolonged activation of the immune system is one of the main causes of hyperproliferation of lymphocytes leading to defects in immune tolerance and autoimmune diseases. Fas ligand (FasL), a member of the TNF superfamily, plays a crucial role in controlling this excessive lymphoproliferation by inducing apoptosis in T cells leading to their rapid elimination. Here, we establish that posttranscriptional regulation is part of the molecular mechanisms that modulate FasL expression, and we show that in activated T cells FasL mRNA is stable. Our sequence analysis indicates that the FasL 3′-untranslated region (UTR) contains two AU-rich elements (AREs) that are similar in sequence and structure to those present in the 3′-UTR of TNFα mRNA. Through these AREs, the FasL mRNA forms a complex with the RNA-binding protein HuR both in vitro and ex vivo. Knocking down HuR in HEK 293 cells prevented the phorbol 12-myristate 13-acetate-induced expression of a GFP reporter construct fused to the FasL 3′-UTR. Collectively, our data demonstrate that the posttranscriptional regulation of FasL mRNA by HuR represents a novel mechanism that could play a key role in the maintenance and proper functioning of the immune system.

Fas and Fas ligand (FasL) 3 are a transmembrane receptor ligand pair of the TNF receptor and TNF family, primarily involved in maintaining the homeostasis of the immune system by eliminating antigen-activated lymphocytes which consequently limits the magnitude and duration of the immune response (1). Fas/FasL mediates this effect by triggering an apoptotic response in these cells. This response involves recruiting the adaptor protein FADD to the intracellular tail of Fas via an interaction with a death domain. In turn, the FasL⅐Fas⅐FADD complex recruits procaspases 8 and 10 via homotypic death effector domain interactions leading to caspase cleavage and apoptosis (1). The importance of Fas and FasL is evidenced by the fact that defects in their expression trigger excessive lymphoproliferation resulting in loss of immune tolerance and autoimmune diseases (1)(2)(3)(4). Although the Fas receptor is constitutively expressed in most tissues, FasL is restricted to activated lymphocytes and sites of immune privilege (1). This supports the idea that FasL but not Fas is the limiting factor in the Fas/FasL-induced signaling pathways.
The disruption of Fas/FasL signaling pathways by spontaneous mutations in mice or in human patients has been associated with diseases such as systematic lupus erythematosus or autoimmune lymphoproliferative syndrome (1,3). Likewise, an increase in FasL-mediated apoptosis of normal, Fas-bearing, bystander cells causes certain immunopathologies such as hepatitis, which is linked to excessive T cell activation (1). Hence, a tight regulation of FasL expression during T cell activation is critical to maintain the homeostasis and the proper functioning of the immune system.
During the past decade, the majority of studies have focused on delineating the molecular mechanisms that modulate FasL expression at the transcriptional level. Several factors, such as NF-AT, NF-B, and IRF-1, have been shown to activate the transcription of the fasl gene directly (5)(6)(7). It is well established for other TNF family members such as TNF␣, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and several interleukins, that although transcription is tightly regulated, the amount of mRNA produced does not correlate with the protein expression levels (8). In many cases, although the steady-state levels of these messages remain unchanged, the protein levels increase significantly in response to extracellular stimuli (9,10). This increase is because the expression of these mRNAs is also regulated posttranscriptionally at the level of subcellular localization, mRNA turnover, and translational efficiency. These posttranscriptional effects are mediated mainly by AU-rich elements (AREs) in the 3Ј-untranslated regions (3Ј-UTRs) of these and other messages (11)(12)(13). AREs are known to regulate a variety of transiently expressed cytokines during T cell activation. These include IFN␥, GM-CSF, CD83, TNF␣, and CD40L (14 -16). This regulation is due to the stabilization of these messages by a mechanism that involves their association with ARE-binding proteins such as HuR (12,17,18).
HuR belongs to the ELAV (embryonic lethal abnormal vision) family of RNA-binding proteins that contains three other members, HuB, HuC, and HuD (19). Of the four ELAV family members, only HuR is ubiquitously expressed, and it is particularly well expressed in primary and secondary lymphoid tissues such as the thymus, spleen, and the gut (20,21).
Recently, it has been shown that in a tissue-specific knock-out mice, disrupting the hur gene in T lymphocytes causes a severe defect in their maturation (22). Indeed, although these mice have a wild type thymic microenvironment, the HuR Ϫ/Ϫ thymocytes of these mice are unable to undergo positive selection, negative selection, and thymic egress (22). The defect in positive selection is attributed to an alteration in the T cell receptor signaling pathway. Likewise, the defect in thymic egress can be explained by defects in chemokine signaling required in this process such as the TNF receptor family members including Fas (22).
The observations described above and the fact that FasL belongs to the TNF␣ family of cytokines raised the possibility that the expression of FasL could depend on posttranscriptional events involving ARE-binding proteins such as HuR. In this study we addressed this question and showed that the 3Ј-UTR of FasL mRNA contains AREs strikingly similar in structure to those of TNF␣. Our data demonstrate that via these AREs, FasL mRNA associates with HuR and that this association is absolutely required for its expression. We also discuss the functional relevance of posttranscriptional regulation of FasL and its impact on T lymphocyte maturation.

Constructs
The human FasL 3Ј-UTR was PCR-amplified, and a 5Ј-BamHI site followed by stop codon and a 3Ј-HindIII site were introduced (for primer sequences see supplemental Materials and Methods). This PCR fragment was inserted into a pEGFP-C2 vector (Clontech) between the BglII and HindIII sites downstream of GFP. The GST and GST-HuR constructs were previously described (23). All plasmids were prepared using the plasmid maxiprep kit (Qiagen) according to the manufacturer's instructions.
The fusion proteins were purified as described in (23,24) with the following modifications. The proteins were eluted from the glutathione-agarose beads with three applications of 500 l of glutathione elution buffer (10 mM for the first elution, and 20 mM for the second and third elutions). Proteins were then dialyzed overnight against phosphate-buffered saline at 4°C. The 20 mM glutathione eluates were the most pure (as determined by SDS-PAGE) and were used in all experiments.

Cell Culture, Treatments, and Transfections
Jurkat cells (E6 clone) (American Type Culture Collection (ATCC)) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen) augmented with 10% FBS (Invitrogen). Jurkat cells were stimulated with PHA (Sigma) at 1 g/ml or at 50 ng/ml for the times indicated. RNA stability curves were generated by treatment of cells with actinomycin D (Sigma) at 5 g/ml. Transfections were performed in 12-well plates using 1 g of plasmid DNA and TransPass RV (New England Biolabs) according to the manufacturer's instructions.
HEK 293 cells (ATCC) were maintained in Dulbecco's modified Eagle's medium (Multicell) supplemented with 10% FBS (Sigma). HEK 293 cells were stimulated with 50 ng/ml PMA for the times indicated. Transfections were performed in 10-cm 2 dishes with 8 -16 g of plasmid DNA and Lipofectamine rea-gent (Invitrogen) according to the manufacturer's instructions. Transfections of siRNA were performed with 60 nM duplexes (siHuR or siCtrl)/10-cm 2 cell culture dish, using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instructions (25). HEK 293 cells were transfected at 20% confluence then retransfected with siRNA 24 h later. Knockdown was assessed 52 h after the first transfection.

RNA Immunoprecipitation and RT-PCR
Preparation of mRNA (mRNP) Complexes-Immunoprecipitation and RNA preparation were performed as previously described (26) with several modifications. Briefly, cell extracts were prepared from PHA-stimulated Jurkat cells or PMA-stimulated HEK 293 cells. mRNP lysate (400 l) was precleared with 8 l of protein G-Sepharose, and the precleared lysate was subsequently divided for each immunoprecipitation (IP). IPs were performed using antibodies to HuR (3A2) and IgG1 isotype control antibody (Sigma). Specific messages associated with HuR were defined using RT-PCR.
RT-PCR-RNA was isolated from the immunoprecipitated mRNP complexes using the ChargeSwitch RNA isolation kit (Invitrogen) scaled to 20% of the manufacturer's instructions. Purified RNA was eluted in 30 l of water, and 4 l was reversetranscribed using the Sensiscript reverse transcription kit (Qiagen) according to the manufacturer's protocol in 20 l of final volume. Subsequently, 2 l of cDNA was PCR-amplified with HotStarTaq (Qiagen) using actin, FasL, or GFP cDNA-specific primers (see supplemental Materials and Methods). The sequences of the primers, as well as the PCR conditions, are described in the supplemental Materials and Methods.

Gel Shift
The FasL RNA probes were produced by in vitro transcription as described previously (27). Regions of FasL 3Ј-UTR were PCR-amplified with sense primers containing the T7 promoter in 5Ј (see supplemental Materials and Methods for primer sequences). The purified PCR fragment was used as a template for transcription using [ 32 P]UTP with T7 RNA polymerase (Promega) according to the manufacturer's instructions. The RNA binding assay was performed with purified 300 ng of GST and GST-HuR as described previously (18) except that upon incubation the RNA-protein complex was not treated with RNase T1.

Northern Blotting
Northern blot analysis was performed as described (28). 15 g of RNA was used and was isolated with the RNeasy Plus extraction kit (Qiagen) according to the manufacturer's instructions. After transferring to a Hybond-N membrane (Amersham Biosciences) and UV cross-linking, the blot was hybridized with GFP or 18 S rRNA probes prepared with [ 32 P]dCTP by random priming with Ready-to-go DNA labeling beads (GE Healthcare) according to the manufacturer's instructions. PCR-amplified fragments of GFP and 18 S rRNA were used to generate labeled probes. After hybridization, the membranes were washed and subsequently exposed on BioMax films (Kodak).

Fluorescence-activated Cell Sorting
Cells were counted by using a hemocytometer and adjusted to a concentration of 1 ϫ 10 6 cells/ml. 0.5 ml of cells was washed three times in 1.5 ml of PBS and then resuspended in 0.5 ml of PBS with 2% FBS. Acquisition of data was done on a FACScan (Becton Dickinson), and the analysis of these data was performed with FlowJo software.

Immunofluorescence
Immunofluorescence was performed as described previously (29). Anti-HuR (3A2) was used at a 1:1500 dilution in 1% goat serum/PBS. HuR was detected using a 1:500 diluted Alexa Fluor 488-conjugated goat anti-mouse IgG polyclonal antibody. DAPI staining 1:20,000 was performed after secondary antibody. A Zeiss Axiovision 3.1 microscope was used to observe the cells with a 63 ϫ oil objective, and an Axiocam HR (Zeiss) digital camera was used for immunofluorescence photography.

Quantitative PCR
RNA was isolated for quantitative RT-PCR by using the ChargeSwitch RNA isolation kit (Invitrogen) scaled to 20% of the manufacturer's instructions. RNA was quantitated using the Ribogreen kit (Molecular Probes), and 150 ng was reverse transcribed using the Sensiscript reverse transcription kit (Qiagen). Subsequently, the cDNA was PCR-amplified with primers described in supplemental Materials and Methods, using the Quantitect SYBR Green kit (Qiagen) in a Corbett Rotor Gene real time thermocycler. The Ct value was used to calculate the amount of the cDNA of interest by extrapolation from a standard curve.

FasL mRNA Has a Short Half-life in Activated T Cells-Pre-
vious reports have shown that FasL mRNA is rapidly induced in T lymphocytes upon T cell receptor engagement and mitogen stimulation (1). To define whether this up-regulation was associated with a stabilization of the FasL mRNA, we first assessed its steady-state levels in T cells exposed to various activators. Jurkat T cells were treated with either PHA, a lectin that nonspecifically aggregates cell surface receptors or PMA, a PKC agonist (1,30). We observed a rapid increase in the level of FasL mRNA at 3 h of PHA treatment. In contrast, PMA treatment had a smaller effect on the steady-state levels of FasL mRNA (Fig. 1A). Interestingly, levels of FasL mRNA return to baseline within 6 -12 h of PHA stimulus, indicating that FasL is transiently expressed in response to lectins similarly to other cytokines. In the absence of PHA or PMA, however, the FasL mRNA was hard to detect (Fig. 1A). Next, we determined the half-life of FasL mRNA under these conditions. We performed actinomycin D pulse-chase experiments (31) and determined the half-life of the FasL mRNA using quantitative RT-PCR analysis. Jurkat cells were treated with PHA or PMA for 3 h to induce maximal FasL mRNA expression and then treated with 5 g/ml actinomycin D for different periods of time. We observed that whereas upon PHA treatment the half-life of FasL mRNA in Jurkat cells was slightly over 60 min, upon PMA treatment, the FasL mRNA half-life was Ͼ4 h (Fig. 1B). Of note, because in untreated Jurkat cells the FasL mRNA is difficult to detect, it was not possible to assess its half-life under these conditions. Therefore, the rapid increase in FasL mRNA expression and the difference in its half-life between the two treatments (PHA and PMA) indicate that for this message to be properly expressed, stabilization mechanisms, similar to those reported for other TNF family members, are activated at least for a short period of time.
3Ј-UTR of FasL mRNA Binds to HuR in an ARE-dependent Manner-The stabilization of cytokine mRNAs is usually mediated by U-rich elements such as AREs located in their 3Ј-UTR FIGURE 1. FasL mRNA has a short half-life in activated T cells. A, RNA was isolated from Jurkat cells which were stimulated for 0, 3, 6, 9, and 12 h with 50 ng/ml PMA or 1 g/ml PHA. Fas ligand mRNA expression was determined by quantitative RT-PCR and was normalized to the expression of the 18 S rRNA. All values were determined relative to peak expression at 3 h of PHA treatment and plotted as the percentage Ϯ the S.E. (error bars) of three independent experiments. B, FasL mRNA stability was determined by treatment of Jurkat T cells with PHA or PMA for 3 h followed by 5 g/ml actinomycin D (ActD) treatment for 0, 15, 45, 60, 120, and 240 min. The half-life of FasL mRNA is indicated by the 50% line and corresponds to ϳ60 min upon PHA and Ͼ240 min upon PMA. Fas ligand mRNA was detected by quantitative RT-PCR and was normalized to GAPDH mRNA expression as described above. (12,32). Our initial analysis of the primary sequence of FasL 3Ј-UTR indicated that it contains the typical AUUUA consensus sequence as well as a 300nt U-rich region (supplemental Fig. 1). The mFold prediction software for mRNA folding showed that both TNF␣ and FasL mRNAs could fold and form a highly similar secondary structure ( Fig. 2A). This prediction method has highlighted the existence in the extreme 3Ј-end of the FasL 3Ј-UTR of two conserved U-rich sequences that are similar in structure and localization to the TNF␣ ARE-1 and ARE-2 ( Fig. 2B) (33). These observations suggest that similarly to TNF␣ (18), the expression of FasL mRNA could involve the association of these U-rich sequences (AREs 1 or 2) with the HuR protein. To test this possibility, we first assessed whether HuR can interact with the FasL mRNA in Jurkat T cells. To ensure the expression of FasL mRNA and reduce FasL-induced apoptosis, Jurkat cells were treated for only 2 h with PHA. These cells were then used to perform IP experiments with the anti-HuR antibody (34) followed by RT-PCR analysis. Prior to the IP experiments, the cells were either exposed or not to UV irradiation for 4 min as described (34). We observed that in PHA-treated Jurkat cells, with or without UV treatment, the FasL mRNA specifically associated with HuR compared with an isotype-matched control antibody (Fig. 3A). To map the HuR binding sites in the FasL 3Ј-UTR and to prove that the interaction between HuR and FasL mRNA is direct, the FasL 3Ј-UTR was divided into six 150-nucleotide regions that were used as probes for RNA electromobility shift assay (Fig. 3B). We observed that all regions other than region 2 (R2) bind purified GST-HuR (Fig. 3C). However, regions 5 and 6, which are particularly U-rich (supplemental Fig. 2A), show a better binding to HuR (Fig. 3C, compare lanes 15 and 18 with lanes 3, 6, 9, and  12). We also showed that this interaction is competed away by an excess of the same unlabeled probes (Fig. 3D) but not with an excess of unlabeled R2 probe (supplemental Fig. 3).
Next, we mapped regions 5 and 6 more precisely, to determine the number of HuR binding sites in these regions. Regions 5 and 6 of the FasL 3Ј-UTR were further subdivided into six 50-nucleotide subregions (Fig. 4A). RNA electromobility shift assay experiments as described above showed strong interactions between HuR and some of the subregions, notably subregions 5.2, 5.3, 6.1, 6.2, and 6.3 (Fig. 4B). Therefore, our results indicate that there are at least four distinct U-rich HuR binding sites in regions 5 and 6 in addition to the weaker binding sites in regions 1, 3, and 4 (Fig. 3C). Furthermore, because the binding of HuR to these fragments can be competed by the same unlabeled probes (Fig. 4C), these interactions seem to be specific. Together, these observations argue that HuR binds directly to the 3Ј-UTR of FasL in an ARE-dependent manner.
HuR Protein Is Required for Expression of an mRNA Containing FasL 3Ј-UTR-The data described above suggest that ARE sequences could collaborate with HuR to ensure the rapid expression of FasL mRNA during T cell activation. Hence, we assessed whether the expression of FasL mRNA depends on HuR in cells treated with activators such as PMA or PHA. Ideally, we would have liked to test this possibility in the context of the full-length FasL message by following its expression in the presence or absence of HuR. However, expressing the fulllength FasL mRNA caused massive cell death in different cell lines, including Jurkat cells regardless of stimulus (data not shown). Therefore, we used GFP reporter constructs in which we fused the FasL 3Ј-UTR to the GFP coding sequence. The entire FasL 3Ј-UTR was included in the reporter construct because any or all of the HuR binding sites described in Figs. 3 and 4 could mediate regulatory effects. Surprisingly, flow cytometry experiments showed that the level of expressed GFP was reduced by Ͼ55% in untreated Jurkat cells transfected with GFP-FasL 3Ј-UTR compared with GFP alone (Fig. 5A). This is probably due to other RNA-binding proteins which, in the absence of any stimulus, bind the FasL 3Ј-UTR in trans and promote its rapid decay. To eliminate the possibility that poor transfection efficiency in Jurkat cells led to the evaluation of a selected population, we transfected HEK 293 cells with the same constructs and obtained similar results by flow cytometry (Fig. 5B). In agreement with GFP protein levels, Northern blot analysis showed that in the absence of any treatment, the amount of GFP-FasL 3Ј-UTR mRNA in HEK 293 cells was significantly decreased compared with the GFP control (Fig. 5, C  and D). These experiments suggest that in the absence of extracellular stimulus, the FasL 3Ј-UTR mediates the rapid decay of the GFP reporter mRNA.
Due to the effects of PMA or PHA treatment on FasL expression, we investigated whether these actions were recapitulated on the expression of the GFP-conjugated FasL 3Ј-UTR. The GFP-FasL 3Ј-UTR or GFP plasmids were transfected into HEK 293 cells which were then treated or not with PMA. It is well established that HEK 293 cells activate the PKC pathway in  OCTOBER 8, 2010 • VOLUME 285 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 31133 response to PMA but not to PHA (35). Additionally, our stability experiments presented in Fig. 1B clearly showed that PMA has a much stronger stabilizing effect on the half-life of the FasL mRNA than PHA. Therefore, to mimic the effect seen in activated Jurkat cells, we decided to treat the HEK 293 cells with PMA for 4 h. Using flow cytometric analysis we observed that in untreated cells the expression of GFP protein encoded from the GFP-FasL 3Ј-UTR mRNA was reduced by ϳ40%, whereas in PMA-treated cells this expression was reestablished to almost normal level (Fig.  6A). Using Northern blot analysis we observed an increase in the expression of both GFP and GFP-FasL 3Ј-UTR mRNA after 2-8 h of PMA stimulation (Fig. 6B). Consistent with the protein results (Fig.  6A) and the RNA results presented in 5C, the levels of GFP mRNA are much higher than the levels of GFP-FasL 3Ј-UTR mRNA in untreated cells (Fig. 6B, compare lanes 1 and  6). Of note, as seen in Jurkat cells for FasL mRNA (Fig. 1), in the absence of any treatment, sometimes the GFP-FasL 3Ј-UTR mRNA is hard to detect (Fig. 6B, lane 6).

HuR Posttranscriptionally Regulates FasL mRNA
HuR has previously been reported to translocate to the cytoplasm to stabilize target mRNAs in a PKC-dependent manner (36). In agreement with these findings, we observed a rapid translocation of HuR from the nucleus to the cytoplasm in HEK 293 cells that became visible after 4 h of PMA treatment (Fig. 6C). Therefore, we tested whether the interaction of GFP-FasL 3Ј-UTR with HuR is dependent on PKC activation. Cells transfected with this reporter gene were treated for various periods of time with PMA and then used to perform an IP experiment with the anti-HuR antibody which was followed by RT-PCR analysis. The data showed that GFP-FasL 3Ј-UTR associated with HuR only after 4 -8 h of PMA treatment (Fig. 6D). Next, we tested whether HuR is required for PMAmediated expression of GFP-FasL 3Ј-UTR mRNA. We depleted HuR expression in HEK 293 cells containing GFP-FasL 3Ј-UTR or GFP control using specific siRNA duplexes (37) in the presence or absence of PMA. We were able to deplete HuR expression by Ͼ55% (Fig. 6E). Our experiments showed that in these HuRdepleted cells, PMA treatment did not affect GFP expression; however, it failed to induce the expression of GFP-FasL 3Ј-UTR mRNA (Fig. 6, F and G). It is interesting to note that the steady-

. Fas ligand mRNA associates with HuR via U-rich sequences in the 3-UTR.
A, Jurkat cells stimulated with 1 g/ml PHA for 2 h were either exposed to UV for 4 min (34) or not. These cells were then used to prepare total cell extracts. HuR was immunoprecipitated from Jurkats with the anti-HuR (3A2) antibody ( lanes  2 and 4). A mouse IgG1 antibody was used as an isotype-matched specificity control (lanes 1 and 3).  (lanes 6 and  14), 0.1ϫ (lanes 7 and 15), 1 ϫ (lanes 8 and 16), 10ϫ (lanes 9 and 17), and 100ϫ (lanes 10 and 18) amounts of unlabeled probe. C and D, representative gel shift blots of two independent experiments are shown. state levels of GFP mRNA alone seem also to depend on HuR expression in the presence or absence of PMA treatment (Fig.  6F, compare lanes 5 and 6 with lanes 9 and 10). These observations indicate that HuR is indeed required for the increased expression of GFP-FasL 3Ј-UTR mRNA in PMA activated HEK 293 cells.

DISCUSSION
Regulation of cytokine mRNAs by posttranscriptional mechanisms at the level of splicing, subcellular localization, stability, and translational efficiency is requisite for their appropriate expression (38). However, this level of regulation has never been investigated for FasL. In this study, we demonstrate that the expression of the FasL mRNA is regulated posttranscriptionally by a mechanism that involves the HuR protein. The FasL 3Ј-UTR harbors two ARE sequences that are similar in structure and U content to the TNF␣ AREs. Similarly to the TNF␣ message, HuR directly binds to FasL mRNA in an ARE-dependent manner. This binding depends on the activation of PKC-induced pathways that in turn trigger the rapid cytoplasmic accumulation of HuR leading to the expression of FasL mRNA. Therefore, collectively, our data support a model whereby HuR protein plays a key role in the transient expression of FasL mRNA in response to PKC activators. This suggests that HuR could be involved in T lymphocyte activation and selection which directly affects the homeostasis of the lymphoid system and the duration of the immune response.
HuR is typically involved in relocalizing, stabilizing, and modulating the translational efficiency of AREcontaining mRNAs. In the case of TNF␣, there are two AREs in the 3Ј-UTR. Interestingly, these AREs mediate distinct posttranscriptional effects on TNF␣ message (18,33). The first ARE binds HuR and is responsible for LPS-mediated TNF␣ expression. Although normally this region mediates the destabilization and the translational repression of TNF␣ mRNA in macrophages, upon LPS stimulus, this ARE also allows for the stabilization of the TNF␣ message via HuR (18,39). Although it has been suggested that ARE2 modulates TNF␣ mRNA expression and protein abundance (18,39), the molecular mechanisms behind these effects are still unknown. Our observations suggest that although FasL and TNF␣ mRNAs have little sequence similarity in the first ARE, the secondary structure of the hairpin required for the recruitment of AREbinding proteins (33) is conserved in addition to considerable sequence and structural homology in the second ARE (Fig. 2). This indicates that these two TNF superfamily members could be posttranscriptionally regulated by similar mechanisms. Indeed, consistent with TNF␣, our study shows that HuR associates strongly and specifically with two regions of the FasL 3Ј-UTR which span both putative AU-rich regulatory regions, including the fragment (6.2) which contains the AUUUA pentamer. Although this suggests that HuR regulates the half-life of FasL mRNA via association with these two AREs, the fact that HuR also interacts with other U-rich elements in the FasL 3Ј-UTR (Fig. 3) argues that association with multiple regions could also be required for the HuR-mediated activation of FasL mRNA expression. The expression of the TNF␣ mRNA in macrophages (38) may likewise parallel the up-regulation of FasL mRNA expression mediated by HuR driven by mitogenic stimulus that activates posttranscriptional mechanisms such as mRNA relocalization and/or stabilization.
Here, we show that PKC activation correlates with the transient expression of FasL mRNA (Fig. 6). Cell treatments with the PKC agonist PMA lead to the expression of FasL or the GFP-FasL 3Ј-UTR mRNAs in a mechanism that involves their association with HuR in an ARE-dependent manner. This is consistent with previous reports showing that HuR is phosphorylated by PKC␣ at serines 158 and 221 (40). Interestingly, Ser 221 is located within the hinge region of HuR that is known to regulate its nucleocytoplasmic shuttling (29). The activation of PKC has been recently tied to the relocalization of HuR to the cytoplasm and the stabilization of target mRNAs in this subcellular compartment (41). This is concordant with previous findings, showing that T cell activation via PKC pathways triggers the HuR-mediated cytoplasmic translocation of the CD83 mRNA, and this involves the phosphorylation of the HuR protein ligand APRIL (15). Our results raise the possibility that FasL mRNA could be regulated the same way because PKC activation causes HuR to relocalize to the cytoplasm, and this correlates with an increase in its association with a reporter mRNA fused to the FasL 3Ј-UTR (Fig. 6, C  and D). Indeed, protein ligands such as pp32 and APRIL have been shown to regulate the export of HuR and some of its mRNA targets in different cell systems (41). Hence, the PKC-mediated phosphorylation of HuR may potentially provide a mechanism for FasL mRNA not only to be stabilized but also to be rapidly translocated to the translation machinery in the cytoplasm for protein synthesis. Exploring the implication of HuR protein ligands such as pp32 and APRIL in regulating the cellular movement of FasL mRNA in activated T cells could help us better understand how the expression of this message is modulated during normal conditions and during the activation of an immune response.
Although there is currently some controversy toward the functions of FasL in the immune system (1), there is a consensus that the Fas/ FasL signaling pathway is required for immune tolerance and homeostasis as evidenced by natural mouse mutants and autoimmune lymphoproliferative syndrome patients (1). In addition, there have been several studies linking defects in Fas induced apoptosis to autoimmune diseases such as systemic lupus erythematosus. One study in particular links the number of microsatellite repeats in the FasL 3Ј-UTR to systematic lupus erythematosus (42). Interestingly, this microsatellite repeat is located in region 5.2, which we show binds HuR very strongly (Fig. 4). Thus, it is possible that HuR-mediated posttranscriptional regulation of FasL is important for the maintenance of immune tolerance.
Our observations indicate that despite the high level of expression of HuR protein in unstimulated cells, the FasL is rapidly degraded (Fig. 1). It is possible that under these conditions, the FasL mRNA remains in the nucleus where it associates with factors known to promote the AU-rich-mediated mRNA decay pathway (43). There are several well characterized activators of AU-rich-mediated mRNA decay that are members of the CCCH zinc finger protein family, which includes proteins such as tristetraprolin (TTP), butyrate response factor 1 (BRF1), and KH-type splicing regulatory protein (KSRP) (44 -47). It has been shown that under different growth conditions HuR competes for binding to AREs with KSRP and TTP proteins (41). In addition, HuR has been described as a key player in the transfer of some of its target transcripts from processing bodies (a site of mRNA decay) to polysomes (a site of translation) (48). Thus, it is possible that in   lanes 1 and 6) or cells treated with 50 ng/ml PMA for 2, 4, 6, and 8 h (lanes 2-5 and 7-10) were harvested and used for Northern blot analysis with specific radioloabeled DNA probes against GFP mRNA and 18 S as loading control. Representative blots of three independent experiments are shown. C, HEK 293 cells were left untreated (panels 1 and 2) or treated for 4 h with 50 ng/ml PMA (panels 3 and 4), fixed, permeabilized, and stained with 3A2 (anti-HuR) followed by anti-mouse conjugated to Alexa Fluor 488 (panels 1 and 3) and DAPI (panels 2 and 4). A single representative field for each cell treatment of three independent experiments is shown. Scale bars, 20 m. D, RNA IP of extracts from HEK 293 cells stimulated with PMA 50 ng/ml for 1 (lanes 1 and 5), 2 (lanes 2 and  6), 4 (lanes 3 and 7), and 8 (lanes 4 and 8) h with 3A2 (anti-HuR) (lanes 5-8) antibody or mouse IgG1 isotype control (lanes 1-4). Immunoprecipitation was followed by RT-PCR for GFP (upper) and ␤-actin (lower) as a positive control. E and F, HEK 293 cells were mock-, siHuR-, or control siRNA (siCtrl)-transfected at 0 and 24 h. This was followed by GFP or GFP-FasL 3Ј-UTR transfection and PMA treatment for 4 h. E, total cell extracts from HEK 293 cells treated with siRNA and transfected with GFP-FasL 3Ј-UTR and treated or not with PMA as described above were collected and used for Western blotting with the anti-HuR and tubulin antibodies. F, total mRNA was prepared from the same samples described in E or from cells transfected with GFP plasmid and was used for Northern blot analysis with GFP and 18 S rRNA radiolabeled probes. G, levels of GFP mRNA were quantified and standardized as described in Fig. 5D. Levels for each treatment were plotted as the percentage Ϯ S.D. (error bars) of two independent experiments. stimulated T cells, where PKC pathways are activated, binding of HuR to the FasL mRNA is favored, leading to its stabilization and rapid translation. Therefore, assessing the implication of proteins such as TTP, BRF1, and KSRP in regulating FasL mRNA expression before and after stimulation and defining how their function could be counterbalanced by HuR could provide new strategies to control T lymphocyte responses under normal and or pathological conditions. This may lead to the identification of novel therapeutic targets/tools for the treatment of autoimmune diseases.