Mrna Turnover in Human T Lymphocytes Associated with CD40

Characterization of RNA Binding Proteins Associated with CD40 Ligand (CD154) mRNA Turnover in Human T Lymphocytes

This information is current as W. F. C. Rigby, M. G. Waugh and B. J. Hamilton of September 29, 2021. J Immunol 1999; 163:4199-4206; ; http://www.jimmunol.org/content/163/8/4199 Downloaded from References This article cites 37 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/163/8/4199.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Characterization of RNA Binding Proteins Associated with CD40 Ligand (CD154) mRNA Turnover in Human T Lymphocytes1

W. F. C. Rigby,2*† M. G. Waugh,* and B. J. Hamilton*

CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3؅ untranslated region Downloaded from 3؅UTR). Two major proteins (p25 and p50) capable of binding the 3؅UTR of CD154 were identiﬁed. The p25 binding activity was) associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3؅UTR of CD154. Rather, their binding was localized to unique sites between nt of the 3؅UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct 811–471 cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these

proteins will help us understand the signals that are necessary for CD154 expression by activated T cells. The Journal of http://www.jimmunol.org/ Immunology, 1999, 163: 4199–4206.

xamination of CD154 deﬁciency states in both humans optimal T cell proliferation and lymphokine production (5–7). A and mice has clearly delineated the central role of the similar lack of correlation between CD154 expression and lym- E interaction of CD154 (CD40 ligand (CD40L)3) with phokine production has been reported with CD28 ligation. In con- CD40 in the development of humoral and cell-mediated immunity trast to its clear-cut effects on stimulation of lymphokine produc- (reviewed in Refs. 1–4). T lymphocyte expression of CD154 is tion (8, 9), conﬂicting data exist on the ability of CD28 ligation to essential for B cell growth/differentiation as well as the formation augment CD154 expression (10, 11). Studies have shown that of germinal centers (reviewed in Ref. 2). Cellular immunity is maximal expression of CD154 requires pharmacologic stimulation by guest on September 29, 2021 equally reliant on the CD154-CD40 interaction, as Ag presentation provided by PMA and calcium ionophores such as ionomycin by dendritic cells and macrophages is profoundly impaired by the (IONO) (5–7). absence of CD154 expression, as is macrophage-mediated killing The induction of CD154 on T lymphocytes can be blocked by of intracellular or extracellular pathogens (3, 4). Given the breadth concurrent treatment with cyclosporine, glucocorticoids, and of the importance of the CD154-CD40 interaction, it is unsurpris- IFN-␥ (5, 11, 12). Cyclosporine treatment has been shown to be ing that CD154 blockade retards the development and progression associated with decreased mRNA accumulation, and its effects are of immune responses in an array of transplantation and autoim- presumed to be transcriptionally mediated, based on the presence mune disease models (2, 4). of NF-AT sites in the promoter region of CD154 (13). Little is Early studies showed that induction of CD154 expression ap- known about the mechanism of action of IFN-␥ or that of stimuli peared different from that in other lymphokine genes. Stimulation of mouse or human T cells by immobilized anti-CD3 elicited very (PMA, IL-12) that enhance CD154 expression under conditions of little (CD4ϩ T cells) or no (CD8ϩ T cells) expression despite optimal anti-CD3 stimulation (14, 15). Other studies indicate pathways that regulate plasma membrane expression of preformed CD154 in tonsillar and synovial T cells, but not in human periph- *Departments of Medicine and Microbiology, Dartmouth Medical School, Lebanon, eral blood T cells (14–16). In contrast to studies of transcriptional † NH 03756; and Veterans Administration Medical Center, White River Junction, VT and posttranslational regulation of this critical molecule, little has 05009 been done to characterize the role of CD154 mRNA turnover in Received for publication March 26, 1999. Accepted for publication August 2, 1999. regulating its expression by these various stimuli. The importance 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 of posttranscriptional regulation in modulating CD154 expression with 18 U.S.C. Section 1734 solely to indicate this fact. is further suggested by its homology to the TNF-␣ family of pro- 1 This work was supported by a Merit Review Award from the Department of Vet- teins (1), because TNF-␣ gene expression is primarily regulated at erans Affairs and the National Institutes of Health (RO1AI34928) and by the Grim- the posttranscriptional level (17). shaw-Gudewicz Foundation. For these reasons, we examined the kinetics of CD154 mRNA 2 Address correspondence and reprint requests to Dr. William F. C. Rigby, Dart- mouth-Hitchcock Medical Center, Lebanon, NH 03756. E-mail address: turnover and found it to be a rapidly degraded mRNA in mitogen- [email protected] activated human peripheral blood T lymphocytes. CD154 mRNA 3 Abbreviations used in this paper: CD40L, CD40 ligand; 3ЈUTR, 3Ј untranslated is not intrinsically labile, because it can be stabilized either acutely region; Act D, actinomycin D; DRB, 5,6-dichloro-1-␤-ribofuranosylbenzimidazole; IONO, ionomycin; AUBP, AU-rich sequence binding proteins; AURE, AU-rich el- or chronically by phorbol ester and calcium ionophore treatment. ement; NEPHGE, nonequilibrium pH gradient electrophoresis. Moreover, we demonstrate two cytosolic RNA binding proteins,

p25 and p50, that specifically interact with the 3Ј untranslated re- lysed by gentle resuspension in 1% Triton X-100 lysis buffer (50 ␮l, 2 ϫ gion (3ЈUTR) of CD154. These proteins appear to be unique and 107 cells) containing 10 mM PIPES (pH 6.8), 100 mM KCl, 2.5 mM MgCl , 300 mM sucrose, 1 mM Pefabloc, and 2 ␮g/ml each of leupeptin distinct based on their M and pI (18–21), and their binding ac- 2 r and pepstatin A before a 3-min incubation followed by a 3-min centrifu- tivity correlates with CD154 mRNA lability. We were also able to gation at 500 ϫ g. The supernatant was aliquoted and stored at Ϫ80°C as localize the site of the interaction of these proteins to a portion of the cytoplasmic fraction. The pellet was gently resuspended in lysis buffer the 3ЈUTR of CD154 that lacks any known cis-acting instability and spun through a 30% sucrose cushion twice. The nuclear pellet was element. Given the differential regulation of lymphokine and gently resuspended with 0.5 vol of nuclei pellet of low salt buffer contain- ␮ ing 10 mM Tris-HCl (pH 7.6), 20 mM KCl, 1.5 mM MgCl2, 0.5 M DTT, CD154 expression (5–7, 10), the demonstration of a novel mRNA 0.2 mM EDTA, 25% glycerol, 2 mM Pefabloc, and 1 ␮g/ml each of leu- instability element(s) would provide a potential mechanism for this peptin and pepstatin A. While vortexing gently, 1.5 vol nuclei pellet of high observation. salt buffer (identical with the low salt buffer except for the presence of 0.5 M KCl) was added dropwise (25). Samples were gently rocked for 30 min before centrifuging at 12,000 ϫ g for 30 min. The supernatant was ali- Materials and Methods quoted and stored at Ϫ80°C as the nuclear fraction. Reagents Polysomes were prepared as described by Brewer and Ross (26). Hu- Actinomycin D (Act D), ␤-ME, PMA, and 5,6-dichloro-1-␤-ribofurano- man PBMC from volunteer donors were homogenized in buffer A (10 mM Tris-HCl (pH 7.60, 1 mM potassium acetate, 1.5 mM magnesium acetate, sylbenzimidazole (DRB) were purchased from Sigma (St. Louis, MO) and ␮ were freshly made up before use. [␣-32P]UTP and CTP (3000 Ci/mmol) 2 mM DTT, 2 g/ml leupeptin and pepstatin A, and 2 mM Pefabloc), and were purchased from NEN. Unlabeled nucleotides, Pefabloc, leupeptin, nuclei were removed by centrifugation. The supernatant was layered over and pepstatin A were purchased from Boehringer Mannheim a 30% sucrose cushion followed by ultracentrifugation at 36,000 rpm for (Indianapolis, IN). 4 h at 4°C. The supernatant was removed as the S130 fraction, and the pellet was resuspended in buffer A and stored in aliquots at Ϫ80°C as the Downloaded from Cell cultures polysome fraction. Human PBMC obtained from volunteer donors by leukapheresis were iso- RNA probes and AUBP assays lated by Ficoll-Hypaque discontinuous gradient centrifugation and cultured at 4 ϫ 106/ml in RPMI 1640 medium (Cellgro) supplemented with 8% The transcription vector containing nt 293–973 (from the stop codon) of Ј heat-inactivated (56°C, 1 h) neonatal bovine serum (Sigma) and 50 ␮g/ml the 3 UTR of human CD154 was provided by Melanie Spriggs. CD154 Ј gentamicin sulfate (United States Biochemical, Cleveland, OH) at 37°C in 3 UTR FL293–973 was generated by linearizing with HinDIII and transcrip- Ј http://www.jimmunol.org/ a humidified atmosphere of 5% CO in air. Cells were stimulated with a tion with T3 RNA polymerase. CD154 3 UTR-H293–811 was generated by 2 Ј concentration of PHA (1 ␮g/ml; Wellcome Reagent, Beckenham, U.K.) linearizing with HphI followed by T3 RNA polymerase. CD154 3 UTR- found to cause maximal stimulation. After overnight culture with PHA, B293–471 was generated by linearizing with BstNI followed by T3 RNA ⌬ Ͼ90% of the cells were CD3 positive. PMA was added to achieve a final polymerase. The 2R1 RNA transcript was generated by EcoRI digestion concentration of 10 ng/ml, while IONO was added to achieve a final con- followed by T7 RNA polymerase transcription (18). ␣ 32 Ͼ 8 ␮ centration of 1 ␮M. - P-labeled mRNAs with sp. act. of 10 cpm/ g RNA were prepared by in vitro transcription in the presence of 50 ␮Ci of [␣-32P]UTP Measurement of mRNA turnover (3000 Ci/mmol) from NEN (Boston, MA), and 0.0125 mM UTP and 2.5 mM ATP, GTP, and CTP from Boehringer Mannheim. RNA probes (8 ϫ In experiments examining mRNA turnover, total cellular RNA was ex- 104 cpm; 3–14 fmol, calculated based on [␣-32P]UTP incorporation) were tracted from cells at the specified times following transcriptional inhibition incubated with 2 ␮g of cytoplasmic extract, 2 ␮g of nucleoplasmic extract, ␮ ␮ ␮ by guest on September 29, 2021 by the addition of DRB (100 M, final concentration) or Act D (5 g/ml). or 0.01 A260 polysomes in 12 mM HEPES (pH 7.9), 15 mM KCl, 0.2 M These concentrations of DRB and Act D were shown to inhibit Ͼ95% of DTT, 0.2 ␮g/ml yeast transfer RNA, and 10% glycerol for 10 min at 30°C. [3H]uridine incorporation by activated T lymphocytes within 5 min, while UV cross-linking was performed at 4°C using a Stratagene UV Stratalinker having no effect on cell recovery or viability during the period of treatment. 1800 (5 min, 3000 microwatts/cm2) followed by RNase digestion (10 U of Total cellular RNA was extracted by acid guanidinium-phenol-chloroform RNase T1 and 20 ␮g of RNase A) for 30 min at 37°C (18). The sample was extraction (22), modified by increasing the 2-ME (Sigma) from 0.1 to 0.7 analyzed under denaturing conditions by 12% SDS-PAGE and dried, and M in the 5 M guanidinium thiocyanate (Fluka Biochemika, Steinheim, autoradiography was performed. In samples analyzed by two-dimensional Germany) denaturing solution. Total cellular RNA was size fractionated by NEPHGE/SDS-PAGE, polysome preparations were first incubated with formaldehyde-agarose gel electrophoresis, blotted onto a Hybond-N nylon radiolabeled RNA, UV cross-linked, and RNase digested as described membrane (Amersham, Arlington Heights, IL) in 20ϫ SSC, and baked above. Extracts were separated in the first dimension with pH 3–10 am- under vacuum at 80°C for 2 h. Filters were prehybridized overnight at 42°C pholines (Bio-Rad, Hercules, CA) at 400 V for 135 min (900 V-h), fol- in 50% formamide, 0.8 M NaCl, 0.1 M PIPES, 0.1% Sarkosyl, 0.1% Ficoll, lowed by second dimension resolution by 12% SDS-PAGE. 0.1% polyvinylpyrrolidone, 0.1% BSA, and salmon sperm DNA (200 ␮g/ ml). Hybridizations were performed at 42°C for 48 h in prehybridization Flow cytometric staining mix containing 10% dextran sulfate and 1 ϫ 106 cpm/ml of cDNA probes ␤ ␣ For detection of CD154 expression on activated T lymphocytes, cells were for CD154, IL-2, human 2-microglobulin, TNF- , HLA-B7, or cyclophi- lin, which had been labeled with [32P]dCTP (3000 Ci/mmol; Amersham,. washed twice in ice-cold PBS with 0.1% BSA and 0.05% sodium azide, Arlington Heights, IL) to a sp. act. of 1–2 ϫ 109 dpm/␮g DNA using a then incubated with purified mouse mAb 24–31 raised against human ␤ CD154 (gift from Randy Noelle), anti-Tac (anti-CD25), or an irrelevant random primer method (23). 2-Microglobulin was used as a loading control because we have found that its mRNA has a longer half-life than actin mouse IgG1 control (27). Cells were then washed in PBS with 0.1% BSA in T lymphocytes (24) (B. J. Hamilton and W. F. C Rigby, unpublished and 0.05% sodium azide, incubated with goat anti-mouse FITC, fixed, and observations). Filters were washed with 0.1ϫ SSC containing 0.02% so- analyzed on a Becton Dickinson FACScan flow cytometer (Mountain dium pyrophosphate and 0.5% Sarkosyl twice at 20°C, then washed with View, CA). Residual dead cells and cell aggregates were excluded from 0.1ϫ SSC containing 0.01% sodium pyrophosphate and 0.5% Sarkosyl analysis by low angle and orthogonal light scatter. four times for 30 min each time at 56°C. Blots were dried and exposed at Ϫ70°C to Kodak XAR film (Eastman Kodak, Rochester, NY) using one Results intensifying screen. Sizes of mRNAs were estimated from the positions of CD154 mRNA is rapidly degraded in mitogen-activated T cells, 28S (4.8 kb) and 18S (2 kb) ribosomal RNA bands present in methylene but is not intrinsically labile blue-stained marker lanes. Changes in mRNA and AU-rich sequence binding protein (AUBP) intensity were measured by densitometry and analysis Previous studies have demonstrated that TNF␣ gene expression is with NIH Image 1.6.1. primarily regulated at the posttranscriptional level (21). Because Preparation of subcellular fractions CD154 belongs to the TNF gene family (1), we examined the rate of CD154 mRNA turnover in PHA-activated PBL by Northern Cytoplasmic preparations were performed as previously described using a method characterized for its lack of contamination by nuclear proteins (18). blotting following DRB treatment (Fig. 1). At 6 and 18 h following Cytoplasmic lysates were prepared by washing the cells twice in ice-cold PHA activation, CD154 mRNA decayed with half-lives of 40 and PBS. All reagents and subsequent steps were used at 4°C. The cells were 45 min, respectively, as calculated by densitometry. Because of The Journal of Immunology 4201

FIGURE 1. CD154 mRNA is a labile mRNA. Total cellular RNA from FIGURE 2. CD154 mRNA is acutely stabilized by PMA and IONO human PBL activated with PHA for 6 or 18 h followed by treatment with treatment. Total cellular RNA was analyzed by Northern blotting, as de- 100 ␮M DRB for the indicated times was extracted and analyzed by North- scribed, from PHA (20-h)-activated human PBL treated with Act D and ern blotting as described in Materials and Methods. The blot was sequen- solvent control, PMA (10 ng/ml ﬁnal), or IONO (1 ␮M) for the indicated tially probed with 32P-labeled CD154 or ␤ -microglobulin (B M) cDNA 2 2 times. The blot was sequentially hybridized with the indicated 32P-labeled probes and analyzed by autoradiography. cDNA probes. Similar results were seen in two other experiments. Downloaded from this rapid rate of turnover, we compared the rate of CD154 mRNA decay relative to that of IL-2 and TNF␣ mRNA. Following RNA CD154 mRNA. The stabilization of CD154 mRNA by either PMA polymerase II inhibition, CD154 mRNA levels declined rapidly in or IONO lasted longer than that observed with TNF-␣ and IL-2 PHA-activated (20 h) PBL (Fig. 2). The rate of decline was equiv- mRNA, suggesting different kinetics of stabilization. Moreover, in alent to that observed for IL-2 mRNA in human PBL, which has contrast to that found with IL-3 mRNA in a mast cell line (31),

Յ ␣ http://www.jimmunol.org/ been well characterized for its lability (t1/2, 30 min) with acti- PMA appeared more potent than IONO in stabilizing TNF- and vation by either anti-CD3 or PHA (28, 29). IL-2 mRNA. It is clear that the high level of CD154 surface ex- Following RNA polymerase inhibition, TNF-␣ mRNA levels pression induced by activation with concurrent PMA and IONO is declined more rapidly than IL-2 and CD154. In addition, the dis- associated with stabilization of CD154 mRNA (32, 33). Our find- appearance of TNF-␣ mRNA was accompanied by a loss of size ings now indicate that the change in CD154 mRNA stability con- heterogeneity, consistent with poly(A) deadenylation occurring be- ferred by either PMA or IONO treatment can occur very rapidly in fore degradation of the mRNA (30). In contrast, IL-2 and CD154 activated cells, even in the absence of mRNA synthesis. mRNA declined without a clear transition or trend to homogeneity, Ј suggesting that their degradation does not pass through a specific Identification of CD154 3 UTR binding proteins by guest on September 29, 2021 deadenylation step. Thus, CD154 mRNA turnover appears to be The turnover rate of many mRNA is regulated by 3ЈUTR cis- quantitatively more rapid and qualitatively distinct from that of acting elements whose activity is transduced through their inter- TNF-␣. action with specific trans-acting factors (19, 21, 34). We therefore This experiment additionally demonstrates that CD154 mRNA examined cytosolic extracts from resting and activated peripheral is unstable, but can be stabilized by pharmacologic stimuli (PMA, blood T cells for the presence of proteins that could bind to in vitro IONO) concurrent with RNA polymerase II inhibition, as has been transcribed RNA corresponding to the 3ЈUTR of CD154. To spe- reported with cytokine mRNA (31) Using this same approach, we cifically identify the proteins that bound the RNA, UV cross-link- demonstrated that addition of PMA or IONO immediately after ing was used to establish a covalent bond between the nucleotides Act D treatment resulted in rapid stabilization of lymphokine and in direct contact with the protein. Exhaustive digestion with RNase

FIGURE 3. Diagrams of RNA probes. A diagram of the 3ЈUTR of CD154 with nucleotide number from the stop codon is shown followed by the three RNA probes used for the binding assays. Also shown is the sequence for the ⌬2R1 probe, used as a cold competitor of RNA binding in Fig. 7. 4202 REGULATION OF CD40 LIGAND (CD154) mRNA TURNOVER

FIGURE 4. PMA and IONO modulate binding of a 25-kDa protein to CD154 3ЈUTR. A, Cytoplasmic and nuclear extracts from resting and PHA- activated (12 h) PBL were analyzed for binding to in vitro transcribed [32P]CD154-FL RNA by SDS-PAGE and autoradiography as described in Materials and Methods. Arrows denote p50, p43, p36, and p25 binding activities in cytoplasmic and nuclear extracts. B, In a separate experiment, PHA-activated (12 h) lymphocytes were treated with 100 ␮M DRB for 15, 30, and 60 min plus DMSO control, PMA (10 ng/ml), 1 ␮M IONO, or PMA and IONO combined. The cytoplasmic extracts were analyzed for binding activity to in vitro transcribed [32P]CD154-FL RNA. The lane marked R shows the binding activity in lymphocytes cultured for 12 h in the absence of PHA activation. Downloaded from

is performed, leaving only the nucleotide that is covalently bound to the protein. Following SDS-PAGE, if the protein-associated nucleotide is radiolabeled, autoradiography will identify the relevant protein expressing this binding activity. For this set of studies, we 32

generated a [ P]UTP-radiolabeled RNA corresponding to the por- http://www.jimmunol.org/ tion (680 nt) of the CD154 3ЈUTR that is highly conserved (ϳ70%) across human and mouse species (Fig. 3). Incubation of the [32P]UTP-radiolabeled CD154 RNA with cytoplasmic and nuclear extracts from control and PHA-activated peripheral blood T cells demonstrated a cytoplasmic protein of 25 kDa as the major 3ЈUTR binding activity (Fig. 4A). A 50-kDa CD154 binding activity was also observed, also only in cytoplasmic extracts. The binding activity of each protein was present in resting T cells and

was unaffected by PHA activation. Nuclear extracts demonstrated by guest on September 29, 2021 a different pattern of RNA binding proteins, with 55-, 43-, and 36-kDa proteins being observed. Because the addition of PMA or IONO to mitogen-activated T cells acutely stabilized CD154 mRNA, we examined the effects of these agents on CD154 3ЈUTR binding proteins in the context of RNA polymerase II inhibition (Fig. 4B). With PMA and/or IONO treatment, p25 binding activity was clearly reduced 15 and 30 min after their addition. After 60 min of DRB treatment, the relative difference in p25 binding between DMSO controls and PMA/ IONO-treated cells was no longer apparent, because cytosolic p25 binding declined with DRB treatment in the DMSO controls. This decline in p25 binding activity with DRB treatment was variably seen in controls (see below). There was no clear correlation between p50 binding activity and modulation by PMA/IONO. How- ever, broadening of the p50 binding activity was observed, suggesting either posttranslational modiﬁcation of the p50 or

recruitment of other RNA binding proteins of similar Mr.Inad- dition to the p25 and p50, minor p43 and p36 binding activities were observed, each of which increased with DRB treatment. In FIGURE 5. PMA/IONO activation of PBL modulates binding of a 25- particular, the p36 binding activity appeared to be increased by the kDa protein to CD154 3ЈUTR. Upper panel, Cytoplasmic extracts from acute addition of PMA and/or IONO. resting PBL as well as those activated for 16 h with PHA or PMA/IONO Prior studies have demonstrated that significant CD154 expres- followed by 100 ␮M DRB treatment for the specified times were analyzed sion requires activation with PMA/IONO (5–7) and is associated 32 for binding to in vitro transcribed [ P]CD154-FL RNA by SDS-PAGE and with stabilization of CD154 mRNA (32, 33) (B. J. Hamilton and autoradiography. Arrows denote p50, p43, p36, and p25 binding activity. W. F. C. Rigby, unpublished observations). Therefore, we com- The lane marked R shows the binding activity in lymphocytes cultured for 16 h in the absence of PHA activation. Lower panel, Densitometric anal- pared PHA and PMA/IONO activations of PBL for their effects on Ј ysis of p50 and 25 binding as a function of PHA and PMA/IONO activa- CD154 3 UTR binding proteins in the context of RNA polymerase tion for 16 h (0 m DRB) relative to resting levels as well as DRB treatment II inhibition with DRB (Fig. 5). As seen previously, cytosols from for the specified times. Open symbols indicate PHA activation; black sym- resting and PHA-activated PBL exhibited comparable levels of bols represent PMA/IONO activation. p25 binding. Levels of p25 binding activity remained constant for The Journal of Immunology 4203

p25 and p50 CD154 3ЈUTR binding proteins are polysomal Several studies have indicated that polysomal loading is a requisite step in rapid mRNA turnover (33, 35). Given the observed lability of CD154 mRNA, we examined the polysomes of resting and activated human T lymphocytes for the presence of CD154 3ЈUTR binding proteins. These studies showed that p25 binding activity is present on polysomes and is unaffected by PHA activation (Fig. 6A). With DRB treatment, polysomal p25 binding activity increased and was maintained for 2 h. In contrast, concurrent treatment with IONO reduced polysomal p25 binding activity. Low levels of polysomal p50, p43, and p36 binding activity were observed in this polysome preparation and appeared to be slightly increased by IONO treatment, but were only evident in DRB- treated cells. In other experiments, only the p25 binding activity was detectable on polysomes. Postpolysomal supernatants (S130) expressed very weak binding activity (p43 and p90), without clear patterns of modulation by IONO. Based on these ﬁndings, we con- Ј clude that p25 is a polysome-associated CD154 3ЈUTR binding

FIGURE 6. p25 and p50 CD154 3 UTR binding proteins are polyso- Downloaded from Ј mally associated. A, PHA-activated (16 h) lymphocytes were treated with protein whose binding activity correlates with CD154 3 UTR 100 ␮M DRB plus DMSO (Ϫ lanes) or PMA/IONO (ϩlanes) for 60 and mRNA lability. In contrast, a clear correlation among the other

120 min. Polysome (0.005 A260) and S130 fraction (cell equivalent) were RNA binding proteins, polysomal association, and CD154 mRNA analyzed for binding activity to the [32P]CD154 3ЈUTR-FL RNA. The lane stability was not evident because of their variable presence in poly- marked R shows the binding activity in resting lymphocytes. B, Two-di- somal preparations. mensional NEPHGE/SDS-PAGE was performed on a polysomes (0.2 The rapid modulation of p25 binding activity in the context of A260) prepared from PHA-activated (12 h)/DRB (2 h)-treated cells incu- RNA polymerase II inhibition is consistent with posttranslational http://www.jimmunol.org/ Ј bated and UV cross-linked to CD154 3 UTR-FL RNA. regulation, perhaps by phosphorylation. Two-dimensional NEPHGE analysis of the polysomal p25 binding to the CD154 3ЈUTR reveals three closely related binding proteins, with pI val- 90 min following DRB treatment, while p50 binding activity in- ues of ϳ7 (Fig. 6B). The central isoform demonstrated the most creased (Fig. 5). PMA/IONO activation resulted in loss of p25 binding activity. Due to the absence of specific antisera, we cannot binding activity at all time points examined. In contrast, p50 bind- unambiguously determine whether these isoforms represent the ing activity was increased relative to PHA activation before and same protein, although it seems likely, given the specificity of the after DRB treatment. PMA/IONO activation followed by DRB observed binding. In the polysome fraction analyzed for this ex- by guest on September 29, 2021 treatment resulted in marked induction of p36 binding activity rel- periment, we saw no other CD154 binding activity, so we were ative to that seen with PHA activation, while lesser effects were unable to conclude whether p50 or other CD154 3ЈUTR binding seen for the p43. As described above, the p43 and p36 proteins proteins yielded similar profiles. were minimally evident, if at all, without DRB treatment. These data demonstrate that both chronic or acute treatment with PMA/ Ј IONO induces changes in CD154 3ЈUTR binding proteins. In par- Localization of p25/p50 binding to nt 471–811 3 UTR of ticular, these data suggest a relationship between p25 binding ac- CD154 tivity and CD154 mRNA turnover, but do not exclude possible To determine the site(s) at which p25 and p50 interact with the roles of the other RNA binding proteins. 3ЈUTR of CD154, we examined the abilities of various cold RNA

FIGURE 7. p25 and p50 bind speciﬁcally to CD154 3ЈUTR RNA. Cytoplasmic extract (2 ␮g) from PHA-activated (12 h) lymphocytes was analyzed for binding to [32P]CD154 3ЈUTR-FL RNA in the presence of a 0-, 10-, 30-, or 100-fold excess of cold competitor RNAs CD154 3ЈUTR-B, -H, and FL, and ⌬2R1 (see Fig. 3 for probes). Arrows indicate p50 and p25 binding activities. 4204 REGULATION OF CD40 LIGAND (CD154) mRNA TURNOVER

Table I. Mitogen activation can induce CD25, but not CD154 expressiona

Anti- IgG1 Control CD154 Anti-CD25

Treatment % MFI % MFI % MFI

PHA (4 h) 5 28 11 15 23 15 PMA/IONO (4 h) 10 11 72 70 19 14 PHA (18 h) 4 32 17 15 77 52 PMA/IONO (18 h) 8 29 79 66 80 30

a Flow cytometry data from a representative experiment is shown as % positive cells with the mean ﬂuorescent intensity (MFI) of the positive cells on a log scale also shown. Note that increase in % CD154 positive cells with PHA is associated with MFI lower than that seen with irrelevant control. This is in contrast to anti-CD25 expression with PHA activation for 18 h, which is equal to that seen with PMA/IONO treatment.

complex pattern of regulation. These data are consistent with the interpretation that the binding of CD154 3ЈUTR by p25 is a de- Downloaded from stabilizing signal. These findings correlate not only with our studies of CD154 mRNA turnover, but also with the inability of PHA to induce CD154 expression at all time points examined (Table I). FIGURE 8. PMA/IONO modulates binding of p25 and p50 to distinct When these same cytosols were examined for their binding to sites in CD154 3ЈUTR RNA. A, Cytoplasmic extracts from PBL activated radiolabeled CD154 3ЈUTR-H (nt 293–811), an interesting result with PMA/IONO for 0, 3, 6, 16, and 24 h were assayed for binding activity 32 32 emerged. Whereas p25 binding activity was markedly depressed, Ј http://www.jimmunol.org/ to [ P]CD154 3 UTR-FL (nt 293–973) RNA (left) and [ P]CD154 p50 binding activity was much less affected by PMA/IONO treat- 3ЈUTR-H (right). B, Cytoplasmic extracts from resting and PHA- 293–811 ment. This finding indicates that p50 is still present in the cytosols, activated lymphocytes were assayed for binding activity to CD154 3ЈUTR- FL and CD154 3ЈUTR-H RNA transcribed in vitro with either [32P]UTP or but its apparent disappearance is due to reduced binding to CD154 Ј [32P]CTP. 3 UTR FL (nt 293–973). This reduction in p50 binding activity induced by PMA/IONO treatment is apparently dependent on nt 811–973, because their deletion in radiolabeled CD154 3ЈUTR-H transcripts to compete for binding to the radiolabeled CD154 tran- (nt 293–811) restores binding to normal levels. A similar, but script. Using cytoplasmic lysates from PHA-activated PBL, we lesser, effect is seen with p25 binding with these different CD154 Ј observed that unlabeled RNA containing nt 293–973 (CD154 3 UTR probes. Based on the differential binding and modulation of by guest on September 29, 2021 3ЈUTR FL) and 293–811 (CD154–3ЈUTR-H) competed equally p25 and p50 binding activity in these and previous experiments, Ј well for protein binding to the radiolabeled CD154 3ЈUTR FL we conclude that the binding of p50 and p25 to the 3 UTR of transcript (Fig. 7). In contrast, cold competition with RNA tran- CD154 occurs independent of each other. scripts containing nt 293–471 was minimal, while no inhibition Moreover, this different binding pattern suggests the possibility was seen with D2R1, which contains four reiterated AUUUA se- that p50 and p25 bind different sites within nt 471–811 of the Ј quences. In other studies, we and others have found that these 3 UTR of CD154. This latter interpretation is supported by exam- reiterated AUUUA sequences are efficiently bound by heteroge- ining p25 and p50 binding activity with in vitro transcribed CD154 Ј Ј 3 UTR-FL293–973 or CD154 3 UTR-H293–811 radiolabeled with ei- neous nuclear ribonuclear protein A1 and elav-like proteins (18– 32 21). We therefore conclude from these studies that the interaction ther [ P]UTP or CTP (Fig. 7B). The p25 binding activity could be Ј UV cross-linked to each radiolabeled nucleotide in either tran- of the p25 and p50 with the CD154 3 UTR can be localized to nt 32 471–811. The lack of effect of unlabeled D2R1 on p25 and p50 script, but not if labeled with [ P]ATP (data not shown). Because binding to CD154 3ЈUTR and the absence of an AUUUA sequence UV-mediated transfer of radiolabel to protein occurs with a direct between nt 471–811 argues strongly against these proteins interaction between the protein and the radiolabeled nucleotide, representing AUBP. these data indicate that the p25 directly contacts both cytidines and uridines when it binds the 3ЈUTR of CD154. This is in contrast to p25 and p50 bind to distinct sites in the 3ЈUTR of CD154, and the p50, which is labeled by UV cross-linking to a greater degree PMA/IONO activation modulates their binding activity by the UTP-labeled transcript. These data indicate that p25 and p50 bind at distinct sites found between nt 471–811 of CD154 Our studies indicated that p25 binding activity could be modulated 3ЈUTR. acutely by PMA/IONO treatment and directly correlated with CD154 mRNA instability. CD154 mRNA was labile at all time points tested with PHA activation, while the converse was true Discussion when PMA/IONO was the activating stimulus (32, 33) (B. J. Ham- Previous studies have demonstrated that induction of significant ilton and W. F. C. Rigby, data not shown). This relationship be- surface expression of CD154 on human peripheral blood T lym- tween p25 and p50 binding and CD154 mRNA turnover was there- phocytes requires concurrent stimulation with PMA/IONO (5–11). fore examined in the context of PMA/IONO activation over time Anti-CD3 or mitogenic stimulation induces little or no CD154 ex- (Fig. 8A, left panel). Following PMA/IONO activation, p25 bind- pression, in contrast to their ability to activate proliferation or lym- ing to [32P]UTP-labeled CD154 3ЈUTR FL (nt 293–973) was de- phokine production (5–7). This differential in CD154 protein expressed at all time points tested (up to 24 h). In contrast, p50 pression correlates with the mRNA lability that occurs with PHA binding activity was regulated in a more complex manner, being activation. Since submission of this manuscript, nearly identical depressed at 3 and 6 h, then increasing at 16–24 h, suggesting a rates of CD154 mRNA turnover at these time points were reported The Journal of Immunology 4205 with anti-CD3 stimulation of human PBL (33). In addition, we indicate that p25 and p50 independently interact at different sites in demonstrate that CD154 mRNA can be acutely stabilized by either this region, suggesting the presence of two cis-acting elements PMA or IONO treatment even in the presence of concurrent RNA present in this region. Because no AUUUA sequence is found polymerase II inhibition. The rapidity of these effects as well as between nt 471–811, these data suggest that p25 and p50 binding their presence in the absence of transcription are consistent with do not bind the AU-rich elements that play roles in cytokine the hypothesis that CD154 mRNA turnover is regulated by specific mRNA turnover. This conclusion is supported by the inability of proteins whose function can be modulated at a posttranslational AURE-containing RNA to block their binding to CD154 3ЈUTR. event such as phosphorylation. Third, cross-linking studies indicate that the p25 directly interacts Previous work has indicated that the turnover and translation of with both cytidines and uridines, further evidence that it does not Ј labile mRNA are conferred by 3 UTR cis-acting elements, such as bind AURE. Moreover, the Mr and pI of p25 and p50 are different the AU-rich elements (AURE) (35–37). These AURE act as bind- from those of the various AUBP that have been identified (18–21). ing sites for specific RNA binding proteins, which, in turn, mod- Our attempt to identify and characterize these proteins is under- ulate mRNA turnover (19, 21). Based on these studies, we exam- way, so as to permit their functional characterization. ined T lymphocyte cytosols for the presence of proteins capable of Based on our data, we advance the following model of CD154 specifically binding the 3ЈUTR of CD154 mRNA. These studies mRNA turnover. In normal human peripheral blood T lympho- yielded at least four cytosolic proteins capable of binding the cytes, CD154 mRNA lability is maintained by constitutively ex- 3ЈUTR of CD154, of which the major proteins were p25 and p50, pressed polysomal 3ЈUTR RNA binding proteins, p25 and p50, while minor proteins of 36 and 40 kDa were observed. whose binding activity is unaffected by the strong proliferative

Of these proteins, p25 and p50 were polysomal, consistent with stimulus provided by mitogens such as PHA or anti-CD3. We be- Downloaded from their potential role in CD154 mRNA turnover. Indeed, p25 was the lieve that PHA and anti-CD3 are equivalent, as recent studies re- major binding activity on polysomes, and in some preparations ported nearly identical rates of CD154 mRNA turnover with anti- little p50 was observed. Addition of PMA and/or IONO rapidly CD3 stimulation of human PBL (33). Thus, it is likely that TCR decreased binding activity of the p25 on both polysomes as well as engagement is similarly deﬁcient in modulating p25 and/or p50 in cytoplasmic lysates. Polysomal p25 binding activity inversely binding activity. These proteins appear to bind at unique sites

correlated with acute and chronic stabilization of CD154 mRNA, within the 3ЈUTR and do not apparently interact with AURE. Un- http://www.jimmunol.org/ suggesting its role as a destabilizing trans-acting factor. Two-di- der these conditions, polysomal p25 and/or p50 can bind to specific mensional NEPHGE analysis of polysomal CD154 3ЈUTR binding sites in the CD154 3ЈUTR and facilitate rapid mRNA degradation activity indicates that the p25 consists of three isoforms with minor (32). In the presence of a PMA/IONO signal, these RNA binding differences in charge, suggesting a phosphoprotein. This finding is proteins are phosphorylated and are no longer able to bind CD154 consistent with its identity as a potential target for a kinase trig- 3ЈUTR, resulting in multiple rounds of translation of each CD154 gered by PMA or IONO. Because PHA stimulation does not alter mRNA, yielding enhanced surface expression. It is unlikely that p25 binding in resting lymphocytes, it suggests that the putative CD28 ligation on the T cells provides a signal equivalent to that kinase that phosphorylates p25 is not activated by optimal condi- seen with PMA/IONO, as we and others do not see substantive tions of PHA stimulation, as measured by proliferation and lym- changes in CD154 protein expression, mRNA turnover, or RNA by guest on September 29, 2021 phokine production. Given the minimal induction of CD154 ex- binding protein profile (10, 33) (B. J. Hamilton and W. F. C. pression by either mitogen or anti-CD3 (5–7), a similar lack of Rigby, unpublished observations). Given the unique importance of effect of this pathway of CD154 mRNA turnover seems likely. CD154 in regulating cellular and humoral immunity, understand- In contrast to the modulation of p25 binding activity by signals ing this important pathway of its regulation will have relevance to (PMA/IONO) that stabilize CD154 mRNA, a similar functional developing novel immunomodulatory approaches in the treatment role cannot be inferred from these data for these other CD154 of cancer and autoimmune disease. Indeed, dysregulated expres- 3ЈUTR binding proteins. The other CD154 3ЈUTR (p50, p43, and sion of CD154 in systemic lupus erythematosus has been reported p36) binding proteins did not clearly correlate with the stabiliza- (38), and it is possible that this effect is mediated through changes tion of CD154 mRNA by the acute addition of PMA or IONO to in mRNA stability. mitogen-activated cells. It is possible that these proteins influence CD154 mRNA turnover and translation not by their binding to References specific cis-acting elements, but through the recruitment of other 1. Hollenbaugh, D., H. D. Ochs, R. J. Noelle, J. A. Ledbetter, and A. Aruffo. 1994. proteins that target the mRNA for translation or degradation. In- The role of CD40 and its ligand in the regulation of the immune response. Im- terestingly, the cytoplasmic p43 and p36 binding activities are only munol. Rev. 138:23. seen following RNA polymerase II inhibition, while similarly 2. Foy, T. M., A. Aruffo, J. Bajorath, J. E. Buhlmann, and R. J. Noelle. 1996. Immune regulation by CD40 and its ligand gp39. Annu. Rev. Immunol. 14:591. sized binding proteins are present in the nucleus in untreated cells. 3. Noelle, R. J. 1996. CD40 and its ligand in host defense. Immunity 4:415. These data suggest the hypothesis that the nuclear proteins capable 4. Grewal, I. S., and R. A. Flavell. 1998. CD40 and CD154 in cell-mediated im- Ј munity. Annu Rev. Immunol. 16:111. of binding the CD154 3 UTR are shifted from the nucleus to the 5. Roy, M., T. Waldschmidt, A. Aruffo, J. A. Ledbetter, and R. J. 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Martin, P. J., J. A. Ledbetter, Y. Morishita, C. H. June, P. J. Beatty, and CD154 RNA restored binding. Although these data highlight the J. A. Hansen. 1986. A 44 kDa cell surface homodimer regulates interleukin 2 difficulty in precisely mapping the binding sites of RNA binding production by activated human T lymphocytes. J. Immunol. 136:3282. proteins with short oligoribonucleotides, truncation analysis and 9. Thompson, C. B., T Lindsten, J. A. Ledbetter, S. L. Kunkel, H. A. Young, Ј S. J. Emerson, J. M. Leiden, and C. H. June. 1989. CD28 activation pathway competition studies clearly identifies nt 471–811 in the 3 UTR as regulates the production of multiple T cell-derived lymphokines/cytokines. Proc. essential for p25 and p50 binding activity. Mapping studies also Natl. Acad. Sci. USA 86:1333. 4206 REGULATION OF CD40 LIGAND (CD154) mRNA TURNOVER

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