Regulation of CD154 (CD40 Ligand) mRNA Stability During T Cell Activation Gregory S. Ford, Bryan Barnhart, Scott Shone and Lori R. Covey This information is current as of September 25, 2021. J Immunol 1999; 162:4037-4044; ; http://www.jimmunol.org/content/162/7/4037 Downloaded from References This article cites 53 articles, 28 of which you can access for free at: http://www.jimmunol.org/content/162/7/4037.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 25, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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. Regulation of CD154 (CD40 Ligand) mRNA Stability During T Cell Activation1 Gregory S. Ford, Bryan Barnhart, Scott Shone, and Lori R. Covey2 The CD154 protein (CD40 ligand), which is critical to the regulation of both humoral and cellular immune responses, is expressed transiently on the surface of activated CD41 T cells. To determine whether control of mRNA stability contributes to the highly regulated expression of CD154 during T cell activation, CD41 T cells were isolated from human peripheral blood and stimulated for various lengths of time with plate-bound anti-CD3 mAb. At early times after anti-CD3 activation, the CD154 message was found to be very unstable, however, the stability measurably increased after 24–48 h of activation. Similar analyses of TNF-a and c-myc mRNA decay throughout a time course of T cell activation revealed patterns of regulation that were distinct from CD154. Similar to the effect on TNF-a mRNA, stimulation of T cells with PMA 1 ionomycin greatly increased the stability of CD154 message. However, CD154 message stability was only modestly increased in T cells coactivated with anti-CD3 and anti-CD28 at Downloaded from 5 h and not increased by costimulation at 24 h. Finally, an analysis of both mRNA and surface protein expression over a time course of T cell activation with anti-CD3 revealed a rapid induction of expression early after activation. This induction was followed by a more gradual decrease in expression over the next 48 h. Together, these data support a role for posttranscriptional regulation in the control and overall expression of CD154 in activated T cells. The Journal of Immunology, 1999, 162: 4037–4044. ignaling between Th cells and APCs during a specific im- surface expression on activated T cells by increasing receptor-me- http://www.jimmunol.org/ mune response initially involves TCR recognition of an diated endocytosis (12). But, decreased surface expression through S antigenic peptide associated with class II MHC molecules CD40 contact does not correlate with a reduction in the steady- expressed on the surface of the APC. One important consequence state level of CD154 mRNA (15). Also, costimulation through of this interaction is the up-regulation of activation-specific effec- CD28 appears to augment the expression of CD154, which is ac- tor molecules, including the ligand for CD40, CD154, on the sur- companied by an increase in CD154 mRNA (11). However, the face of the CD41 T cells. The interaction between CD154 and its absolute requirement for costimulation on the induction of CD154 receptor CD40, expressed primarily on the surface of B cells and expression is debatable (16–18). Together, these results point to- other APCs, has been shown to be critical for the development of ward CD154 expression being controlled by multiple factors. both humoral and cellular immune responses (reviewed in Refs. These factors may be contingent on the process by which T cells by guest on September 25, 2021 1–3). Although the functional analysis of CD154 has been exten- become activated and the extent to which the cell has received sive, little is known about the mechanisms controlling CD154 ex- additional signals through costimulatory and/or adhesion mole- pression in activated T cells. Regulating the overall expression of cules. Also, the time after activation is an important variable in the CD154 is critical for restricting T helper activity since CD40 is expression of both CD154 mRNA and protein. constitutively expressed on the surface of both Ag-selected and Posttranscriptional control of messenger RNA stability is a par- nonselected B cells. Unrestricted CD154 expression could poten- ticularly important component of the regulation of many human tially lead to the activation of nonantigen-specific B cells and re- genes, such as cytokines and proto-oncogenes, that are expressed sult in autoimmunity (4, 5). in response to specific cellular signals. Rapid changes in overall Studies examining the kinetics of CD154 expression have mRNA levels can be more easily achieved when both transcription shown maximal levels of surface expression 6–24 h after T cell rate and message stability are altered (reviewed in Ref. 19). The activation with various stimuli. The amount, as well as the kinetics rapid and transient expression of CD154 during T cell activation of CD154 expression over a time course of activation, appear to suggest that a layer of control may be at the posttranscriptional depend on the type of activation stimuli and on costimulatory in- level. teractions provided by B cells or APCs (6–15). For example, T cell Several observations suggest that CD154 may be regulated at contact with CD40-expressing cells clearly down-regulates CD154 the level of message stability. First, the CD154 gene shares con- siderable sequence homology with its family member TNF-a, which has been shown to be regulated in part by control of mes- Division of Life Sciences, Rutgers, The State University of New Jersey, Piscataway, sage stability (20–22). Second, the CD154 message, like TNF-a, NJ 08854 contains multiple copies of the AUUUA sequence in its 39 un- Received for publication June 30, 1998. Accepted for publication December 28, 1998. translated region (39-UTR)3. This element has been identified as a The costs of publication of this article were defrayed in part by the payment of page sequence motif that affects the message stability of many mRNAs charges. This article must therefore be hereby marked advertisement in accordance (23–25). The mRNA stability of several cytokines is correlated with 18 U.S.C. Section 1734 solely to indicate this fact. 1 with RNA-binding activities that selectively bind to AUUUA mul- This work was supported in part by National Institutes of Health Grant AI37081 (to 9 L.R.C.) and by grants from the Charles and Joanna Busch Memorial Fund and the timers in the 3 -UTR of these cytokine messages during T cell Nicholas C. Palchuk Research Grant, Rutgers, The State University of New Jersey. activation (21, 26). 2 Address correspondence and reprint requests to Dr. Lori R. Covey, Division of Life Sciences, Nelson Biological Laboratories, Rutgers, The State University of New Jer- sey, 604 Allison Road, Piscataway, NJ 08854. E-mail address: covey@biology. 3 Abbreviations used in this paper: UTR, untranslated region; ARE, AU-rich ele- rutgers.edu ments; ion, ionomycin; Act D, actinomycin D. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 4038 POSTTRANSCRIPTIONAL REGULATION OF CD154 mRNA More recent studies have directly demonstrated functional rela- RNA isolation and Northern blot analysis tionships between AU-rich element (ARE)-binding trans-acting Total RNA was isolated from 5 3 106 to 1 3 107 cells using the Trizol factors and the control of mRNA stability. For example, Hel-N1, reagent (Life Technologies, Gaithersburg, MD). RNA (6–15 mg) were run a protein isolated on the basis of homology to the drosophila em- in 1.2% agarose/formaldehyde gels and transferred to supported nitrocel- bryonic lethal abnormal vision (ELAV) gene, appears to stabilize lulose. Membranes were hybridized to random-primed 32P-labeled probes overnight at 42°C and then washed three times at 68°C for 15 min with 23 the glucose transporter (GLUT-1) mRNA when overexpressed in 1 9 SSC 0.5% SDS. After hybridization with the 28S rRNA probe, mem- 3T3 cells by binding to AU-rich 3 -UTR sequences (27). Another branes were washed three times at 68°C for 15 min with 13 SSC 1 0.5% member of the ELAV-like family, HuR, binds in vitro to ARE SDS, followed by a single wash for1hat68°C with 0.13 SSC 1 0.1% found in several mRNAs like IL-3 and c-fos (28). Interestingly, SDS. Membranes were exposed to XAR-film or scanned using the Storm this protein has in vitro RNA-binding specificity to different AU- PhosphorImager system (Molecular Dynamics, Sunnyvale, CA) for signal quantification. rich sequences that correlates with the ability of those sequences to direct in vivo mRNA decay (28, 29). Stabilizing or destabilizing RNA-binding factors that recognize AU-rich 39-UTR sequences DNA and RNA probes could regulate CD154 message stability during T cell activation. The human CD154 cDNA, isolated from the D1.1 Jurkat T cell line (7, 30) To investigate whether mRNA stability contributes to the over- was used as a probe on Northern blots. For the analysis of TNF-a expres- all regulation of CD154 expression, we have examined CD154 sion, a 1.1-kb human TNF-a cDNA was excised from the pE4 plasmid mRNA stability under different conditions of T cell activation.