©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of the B-cell Surface Receptor CD40 Induces A20, a Novel Zinc Finger Protein That Inhibits Apoptosis (*)

Vidya Sarma (§) , Zhiwu Lin (§) , Lisa Clark (1), Beth M. Rust , Muneesh Tewari , Randolph J. Noelle (1), Vishva M. Dixit

From the (1) Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109 Department of Microbiology, Dartmouth Medical School, Lebanon, New Hampshire 03756

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD40 activation is critical for B-cell function, leading to activation and expression of cell surface markers, proliferation, immunoglobulin class switching and inhibition of programmed cell death (PCD). Germinal center B-cells, for example, can be prevented from undergoing PCD by CD40 activation. The mechanism by which PCD is inhibited has been an enigma. A potential role for A20, a novel zinc finger protein, in inhibiting B-cell apoptosis was suggested by our previous finding that it is induced by the Epstein-Barr virus LMP-1 gene product, a potent cell death inhibitor. We now show that CD40 activation induces A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, we show that CD40 activation of A20 expression is mediated by inducible binding of NF-B complexes to the A20 promoter and provide evidence for a critical role for Thr (in the CD40 cytoplasmic domain) in activating NF-B.


INTRODUCTION

Programmed cell death (PCD)() is of critical importance not only in the maintenance of tissue homeostasis but also during development and in immune selection. In an attempt to understand the molecular basis of PCD in lymphoid cells, a number of factors have been identified that either directly initiate PCD, such as activation of the Fas receptor (1, 2) , or inhibit cell death, such as activation of the CD40 receptor (3) and expression of the LMP1 gene product of the Epstein-Barr virus (EBV) (4) .

CD40, a member of the tumor necrosis factor (TNF) receptor family, mediates cognate help of T-cells for the survival, growth and differentiation of B-cells. The ligand for CD40 (CD40L) is expressed on activated CD4 T-cells (5) and on binding CD40 expressed on B-cells (6) there ensues a dramatic alteration in B-cell physiology that includes activation, induction of B-cell proliferation, immunoglobulin (Ig) isotype switching and rescue from PCD (7, 8, 9) . The importance of CD40 in B-cell biology has been emphasized by the recent discovery that genetic mutations in the gene for CD40L (10, 11, 12, 13, 14) or defects in CD40 signaling (15) result in an immunodeficiency syndrome characterized by defective Ig class switching and lack of germinal center formation. This is in keeping with previous studies showing that CD40 signaling blocks spontaneous apoptosis of germinal center B-cells and may therefore be instrumental in the selection of B-cells undergoing somatic hypermutation in germinal centers (3) .

We have previously shown that A20, a novel zinc finger protein, is induced by TNF and confers resistance to TNF killing (16) and is induced by the LMP1 gene product of EBV (17) , suggesting a potential role in inhibiting B-cell PCD. In this report we now show that CD40 activation leads to the induction of A20 and that expression of A20 renders B-cell lines resistant to PCD. Additionally, evidence is provided for a critical role for Thr in the CD40 cytoplasmic domain in activating NF-B and the A20 promoter.


MATERIALS AND METHODS

Cell Culture

The EBV-negative cell lines Louckes and BJAB were maintained in RMPI 1640 and 10% fetal bovine serum (HyClone, Logan, UT) supplemented with nonessential amino acids and antibiotics (100 units/ml penicillin, 50 mg/ml streptomycin). The human embryonic kidney cell line was grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and nonessential amino acids plus antibiotics.

CD40 Activation of Louckes and B Cells

Louckes cells were treated for the indicated periods of time (Fig. 1) with anti-CD40 ascites (used at a 1:10,000 dilution of the ascites HG-14, gift from Tom Tedder, Duke University; Ref. 18) or with isotype-matched contol antibody in the presence or absence of cycloheximide (CHX, 10 µg/ml).


Figure 1: CD40 activation induces A20 transcript (A and B). RNA was isolated from the EBV-negative Louckes B-cell line or from primary B-cells following exposure to agonist CD40 monoclonal or isotype-matched control antibody in the absence or presence of CHX. A20 expression was determined by Northern blot analysis. RNA isolated from human umbilical vein endothelial cells treated with TNF, and CHX was run as a positive control for A20 expression. The bottom part of each panel is a photograph of ethidium bromide stained 18S rRNA to indicate equivalency of loading.



Mononuclear cells were isolated from the peripheral blood of healthy volunteers and monocytes removed by adherence as described in Ref. 19. B-cells were separated from T-cells by rosetting with 2-aminoethylisothiouronium bromide-treated sheep red blood cells again as described in Ref. 19. Isolated B-cells were either treated with monoclonal anti-CD40 ascites (at a dilution of 1:10,000) or with isotype-matched control antibody for 4 h prior to RNA extraction.

Northern Blot

Total RNA was isolated using a previously described guanidine hydrochloride procedure (20) . Ten µg of RNA was resolved on a 1% agarose-formaldehyde gel, blotted onto nitrocellulose, and hybridized to a P-labeled A20 cDNA probe using hybridization and washing conditions as described previously (20) .

Plasmids

The A20 expression construct was obtained by cloning the entire coding region of A20 into the pZEMneo vector (21) which also contained a neoR cassette allowing for selection in G418.

The construction and characterization of the native and mutant CD40 expression vectors has been described previously (22) .

The A20 promoter reporter plasmid A20PstCAT, which contains nucleotides -233 to +12 of the wild type A20 promoter inserted upstream of the chloramphenicol acetyltransferase (CAT) gene, has been previously described (23, 24) . The A20dmCAT construct is identical except both B sites within this region of the A20 promoter have been inactivated by site-directed mutagenesis (23, 23) .

Transfection and CAT Assay

Louckes and BJAB cells (5 10) were transfected by electroporation (200 V, 960 microfarads) and after 2 days in culture, clonal lines were obtained by selection in medium containing 3.5 mg/ml G418 (Geneticin, Life Technologies, Inc.).

293 cell transient transfections were performed as per published procedures using 5 µg of plasmid/2 10 cells (25) . Transfected cells were treated for 10 h with a 1:10,000 dilution of the HG-14 anti-CD40 ascites or isotype-matched control antibody 36 h post-transfection. Cells were harvested, extracts prepared, and CAT enzyme activity assayed according to standard protocols (23, 24) . CAT activity was quantitated using a Betascope 603 Blot analyzer (Betagen, MA).

Metabolic Labeling and Immunoprecipitation

A20 expression in the stably transfected cell lines was assessed by metabolic labeling with [S]methionine and cysteine (100 µCi/ml) followed by immunoprecipitation as described previously (17) .

Flow Cytometry Analysis

Vector control and CD40 transfected cells were washed once in phosphate-buffered saline containing 1% bovine serum albumin, blocked with 10% normal goat serum for 20 min, and then incubated with a 1:500 dilution of the HG-14 anti-CD40 ascites or isotype-matched control antibody for 30 min at 4 °C. After washing twice in phosphate-buffered saline, cells were incubated with a 1:50 dilution of fluorescein-conjugated goat anti-mouse IgG (Cappel) for 20 min. at 4 °C, washed twice and analyzed by flow cytometry on a FACScan (Becton-Dickinson).

Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)

Nuclear extracts used in EMSAs were prepared as described previously (23, 24) , from 293 cells expressing either the native or mutant (CD40A) receptor. Cells were treated with a 1:10,000 dilution of the HG-14 anti-CD40 ascites prior to extract preparation. EMSA was performed as described by us in previous publications (23, 24) . Briefly, a 43-base pair double-stranded oligonucleotide containing residues -74 to -32 of the A20 promoter (A20B) was fill-in labeled using Klenow fragment. Binding reactions containing 10 µg of nuclear protein extract, 1 µg of poly(dIdC)-(dIdC), 0.1 ng of P-end-labeled A20B probe, and binding buffer (23, 24) were incubated 15 min at room temperature. For competition studies, 10 µg of unlabeled double-stranded oligonucleotide was added to the binding reaction. The sequences of these oligonucleotides have been published previously (23, 24) . Products were resolved on a 4% polyacrylamide gel and analyzed by autoradiography.

Apoptosis Assay

Apoptosis was assessed by the use of the fluorescent DNA-staining dye acridine orange to reveal nuclear morphology essentially as described in Refs. 4 and 26.


RESULTS AND DISCUSSION

CD40 Activation Induces A20 Expression

The effect of CD40 activation on A20 expression was examined by Northern blot analysis. The activation of the CD40 receptor, which abrogates B-cell apoptosis, resulted in the induction of A20 both in EBV-negative B-cell lines, Louckes (Fig. 1A) and BJAB (data not shown) and in primary B-cells (Fig. 1B). The induction of A20 transcript was rapid, occurring within an hour, and in the presence of CHX, there was a superinduction of the transcript. This finding is consistent with A20 being an immediate early or primary response gene whose induction is independent of intervening protein synthesis.

A20 Expression Protects B-cells from Apoptosis

To determine whether or not A20 influenced B-cell survival, the EBV-negative human B lymphoma cell lines BJAB and Louckes were transfected with an A20 expression construct or vector alone as a control. EBV-negative B-cell lines are sensitive to apoptosis following transfer into medium containing a sub-optimal concentration (0.1%) of fetal calf serum (FCS) and have been used extensively to characterize factors that modulate B-cell apoptosis (4) . An additional advantage of these cells is that entry into apoptosis is easily recognized by staining cellular DNA with acridine orange and assessing nuclear fragmentation by fluorescence microscopy (Fig. 2A). As shown in Fig. 2(B and C), four independent A20 transfectants on a BJAB background and three on a Louckes background expressed A20 protein as confirmed by metabolic labeling and immunoprecipitation. These A20 transfectants displayed significant, albeit partial, resistance to PCD when compared to an equal number of vector control transfectants.


Figure 2: A20 expression confers resistance to apoptosis. A, acridine orange staining of vector control transfected Louckes cells maintained in 10% FCS (leftpanel) or 0.1% FCS (rightpanel) for 5 days. Nuclear condensation characteristic of apoptosis is clearly evident. B and C, BJAB (B) and Louckes (C) clonal cell lines transfected with an A20 expression construct and the corresponding vector control transfectants (VB and VL) were transferred to 0.1% FCS containing medium and 6 days later scored for apoptotic morphological changes following acridine orange staining. A minimum of 100 cells were counted from triplicate cultures and values shown represent averages ± standard deviations. The bottompanels of B and C show A20expression in transfected and vector control lines as assessed by metabolic labeling and immunoprecipitation.



Partial resistance to PCD in B-cells is also provided by Bcl-2 (27) ; however, contrary to an earlier report, more recent work by the same authors now indicates that Bcl-2 is not CD40-inducible (28) . Nevertheless, it is entirely feasible that the heightened state of resistance to PCD conferred by CD40 activation is due to a synergistic interaction between A20 and a Bcl-2-like protein. In fact, we have recently found that Bcl-x, a related anti-apoptotic gene, is CD40-inducible.()

A20 Induction upon CD40 Activation Is Mediated by B Sites in the A20 Promoter

The exact mechanism by which CD40 transduces signals is unknown. CD40 does possess a short cytoplasmic tail (63 amino acid residues) and mutagenesis studies indicate that Thr in the cytoplasmic domain is essential for signal transduction (29) . Since CD40 possesses no intrinsic signaling capacity (e.g. kinase activity), signal transduction is likely to be mediated by associated molecules (22) . Regardless of the exact mechanism, it is now evident that CD40 activation induces the transcription factor NF-B. NF-B consists of a variety of homo- and heterodimeric complexes homologous to the proto-oncogene c-rel with the ability to differentially regulate transcription through recognition of variant B elements. Given that LMP1 induction of A20 is mediated through the inducible binding of NF-B to B sites within the A20 promoter (23, 24) , we asked whether CD40 activation similarly induces A20. To address this question and to determine the exact role of Thr in CD40 signaling, two stably transfected cell lines were established that expressed either native CD40 or mutant CD40 in which the Thr was converted to Ala (CD40A). CD40 surface expression in the stably transfected 293 cells was confirmed by FACS analysis using HG-14, an agonist CD40 monoclonal antibody (18) (Fig. 3A). Transient transfections were performed using these cell lines to determine first, if CD40 induction of A20 was mediated by B elements within the A20 promoter and second, if Thr was essential for A20 induction. Transfection of the wild type A20 promoter CAT reporter construct, A20PstCAT, led to a significant induction of CAT activity on cross-linking surface expressed CD40 with the agonist monoclonal antibody (Fig. 3B). In contrast, no significant difference was seen on transfection with A20dmCAT in which the two B elements were mutated, confirming the importance of these B elements in conferring CD40 responsiveness to the A20 promoter. Additionally, no significant induction of the native A20PstCAT construct was observed on cross-linking mutant CD40 (CD40A), indicating that Thr was critical for engagement of the B signal transduction pathway. Electrophoretic mobility shift assays (EMSA) were performed to confirm that CD40 activation induced the binding of NF-B proteins to B sites within the A20 promoter. Nuclear extracts prepared from cells expressing native CD40, in the absence or presence of agonist CD40 antibody, were incubated with a double-stranded oligonucleotide probe containing both A20 B sequences. An electrophoretically retarded complex was clearly induced on CD40 activation (Fig. 3C) and was effectively competed by a 50-fold molar excess of double-stranded oligonucleotides containing the A20 or HIV B elements, but not by an unrelated IL-2 octamer binding sequence. In contrast, no electrophoretically retarded complex was evident in nuclear extracts prepared from cells expressing mutant CD40 (CD40A) in the presence or absence of agonist CD40 antibody. Taken together, these findings indicate that CD40 activation of A20 expression is mediated by the inducible binding of NF-B complexes to the A20 promoter and provide evidence for a critical role for Thr in activating NF-B.


Figure 3: Thr is required for CD40-mediated activation of A20 promoter CAT expression. A, 293 cells were stably transfected with either a native or a mutant CD40 (Thr Ala; designated CD40A) expression construct and surface expression confirmed by FACS analysis using an agonist CD40 monoclonal (HG-14) or isotype-matched control antibody (C-Ab). B, representative cell lines expressing either native or mutant CD40 (CD40A) were transiently transfected with the indicated A20 promoter CAT constructs and CAT activity measured following exposure to agonist or isotype-matched control antibody. C, activation of native CD40 induces binding of NF-kB to the A20 promoter. Nuclear extracts were prepared from cells expressing either the native or mutant CD40 receptor following stimulation with agonist monoclonal (+) or isotype-matched control antibody (-), and electrophoretic mobility assays performed as described previously. For competition studies, 10 µg of unlabeled double stranded-oligonucleotide was added to the binding reaction. The specific DNA-protein complex is indicated by the arrow.



In summary, these studies demonstrate that A20 significantly protects B-cell lines from PCD and suggests that this may be an important mechanism utilized by physiological inhibitors of PCD like CD40 and viral inhibitors of PCD such as the LMP-1 gene product of EBV. Finally, these data are the first to demonstrate that A20 inhibits PCD induced by factors other than TNF, in this case serum deprivation, and thereby indicates a broader role for this novel zinc finger protein in inhibiting PCD, especially in B-cells. A cautionary note, however, is that, unlike Bcl-2 and related family members, A20 does not appear to be a general antidote against programmed cell death as certain forms of death, such as glucocorticoid-induced apoptosis that are potently inhibited by Bcl-2, are not inhibited by A20.()


FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA 61348 (to V. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
The two first authors contributed equally to this paper.

To whom correspondence should be addressed: Dept. of Pathology, University of Michigan Medical School, 1301 Catherine St., Ann Arbor, MI 48109-0602. Tel: 313-747-0264; Fax: 313-764-4308; E-mail: vishva_dixit@med.umich.edu.

The abbreviations used are: PCD, programmed cell death; EBV, Epstein-Barr virus; LMP1, latent membrane protein 1; TNF, tumor necrosis factor; FCS, fetal calf serum; EMSA, electrophoretic mobility shift assay; CHX, cycloheximide; CAT, chloramphenicol acetyltransferase.

V. Sarma, G. Nunez, and V. M. Dixit, manuscript in preparation.

A. Opipari, H. M. Hu, and V. M. Dixit, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Ian Jones for help with the preparation and Suzanne Suchard for critical reading of the manuscript.


REFERENCES
  1. Trauth, B. C., Klas, C., Peters, A. M., Matzku, S., Moller, P., Falk, W., Debatin, K. M., and Krammer, P. H.(1989) Science 245, 301-305 [Medline] [Order article via Infotrieve]
  2. Itoh, N., Yonehara, S., Ishii, A., Yonehara, M., Mizushima, S., Sameshima, M., Hase, A., Seto, Y., and Nagata, S.(1991) Cell 66, 233-243 [Medline] [Order article via Infotrieve]
  3. Tsubata, T., Wu, J., and Honjo, T.(1993) Nature 364, 645-648 [CrossRef][Medline] [Order article via Infotrieve]
  4. Henderson, S., Rowe, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E., and Rickinson, A.(1991) Cell 65, 1107-1115 [Medline] [Order article via Infotrieve]
  5. Armitage, R. J., Fanslow, W. C., Strockbine, L., Sato, T. A., Clifford, K. N., Macduff, B. M., Anderson, D. M., Gimpel, S. D., Davis-Smith, T., Maliszewski, C. R., Clark, E. A., Smith, C. A., Grabstein, K. H., Cosman D., and Spriggs, M. K.(1992) Nature 357, 80-82 [CrossRef][Medline] [Order article via Infotrieve]
  6. Stamenkovic, I., Clark, E. A., and Seed, B.(1989) EMBO J. 8, 1403-1410 [Abstract]
  7. Banchereeau, J., dePaoli, P., Valle, A., Garcia, E., and Rousset, F. (1991) Science 251, 70-72 [Medline] [Order article via Infotrieve]
  8. Liu, Y. J., Joshua, D. E., Williams, G. T., Smith, C. A., Gordon, J., and Maclennan, I. C.(1989) Nature 342, 929-931 [CrossRef][Medline] [Order article via Infotrieve]
  9. Zhang, K., Clark, E. A., and Saxon, A.(1991) J. Immunol. 146, 1836-1842 [Abstract/Free Full Text]
  10. Allen, R. C., Armitage, R. J., Conley, M. E., Rosenblatt, H., Jenkins, N. A., Copeland, N. G., Bedell, M. A., Edelhoff, S., Disteche, C. M., Simoneaux, D. K., Fanslow, W. C., Belmont, J., and Spriggs, M. K. (1993) Science 259, 990-993 [Medline] [Order article via Infotrieve]
  11. Korthauer, U., Graf, D., Mages, H. W., Briere, F., Padayachee, M., Malcolm, S., Ugazio, A. G., Notarangelo, L. D., Levinsky, R. J., Kroczek, R. A.(1993) Nature 361, 539-541 [CrossRef][Medline] [Order article via Infotrieve]
  12. Fuleihan, R., Ramesh, N., Loh, R., Jabara, H., Rosen, F. S., Chatila, T., Fu, S. M., Stamenkovic, I., and Geha, R. S.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2170-2173 [Abstract]
  13. Inui, S., Kaisho, T., Kikutani, H., Stamenkovic, I., Seed, B., Clark, E. A., and Kishimoto, T.(1990) Eur. J. Immunol. 20, 1747-1753 [Medline] [Order article via Infotrieve]
  14. Aruffo, A., Farrington, M., Hollenbaugh, D., Li, X., Milatovich, A., Nonoyama, S., Bajorath, J., Grosmaire, L. S., Stenkamp, R., and Neubauer, M., Roberts, R. L., Noelle, R. J., Ledbetter, J. A., Francke, U., and Ochs, H. D.(1993) Cell 72, 291-300 [Medline] [Order article via Infotrieve]
  15. Conley, M. E., Larche, M., Bonagura, V. R., Lawton, A. R., III, Buckley, R. H., Fu, S. M., Coustan-Smith, E., Herrod, H. G., Campana, D., and Lawton, A. R.(1994) J. Clin. Invest. 94, 1404-1409 [Medline] [Order article via Infotrieve]
  16. Opipari, A. W., Hu, H.-M., Yabkowitz, R., and Dixit, V. M.(1992) J. Biol. Chem. 267, 12424-12427 [Abstract/Free Full Text]
  17. Laherty, C. D., Hu, H.-M., Opipari, A. W., Wang, F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 24157-24160 [Abstract/Free Full Text]
  18. Kansas, G. S., and Tedder, T. F.(1991) J. Immunol. 147, 4094-4102 [Abstract/Free Full Text]
  19. Coico, R. (ed)(1994) Current Protocols in Immunology, John Wiley & Sons, Inc., New York
  20. Sarma, V., Wolf, F. W., Marks, R. M., Shows, T. B., and Dixit, V. M. (1992) J. Immunol. 148, 3302-3312 [Abstract/Free Full Text]
  21. Uhler, M. D., and McKnight, G. S.(1987) J. Biol. Chem. 262, 15202-15207 [Abstract/Free Full Text]
  22. Hu, H.-M., O'Rourke, K. M., Boguski, M. S., and Dixit, V. M.(1994) J. Biol. Chem. 269, 30069-30072 [Abstract/Free Full Text]
  23. Krikos, A., Laherty, C. D., and Dixit, V. M.(1992) J. Biol. Chem. 267, 17971-17976 [Abstract/Free Full Text]
  24. Laherty, C. D., Perkins, N. D., and Dixit, V. M.(1993) J. Biol. Chem. 268, 5032-5039 [Abstract/Free Full Text]
  25. O'Rourke, K. M., Laherty, C. D., and Dixit, V. M.(1992) J. Biol. Chem. 267, 24921-24924 [Abstract/Free Full Text]
  26. Tewari, M., and Dixit, V. M.(1995) J. Biol. Chem. 270, 3255-3260 [Abstract/Free Full Text]
  27. Liu, Y.-J., Mason, D. Y., Johnson, G. D., Abbot, S., Gregory, C. D., Hardie, D. L., Gordon, J. C., and MacLennan, I. C. M.(1991) Eur. J. Immunol. 21, 1905-1910 [Medline] [Order article via Infotrieve]
  28. Holder, M. J., Wang, H., Milner, A. E., Casamayor, M. A., Armitage, R., Spriggs, M. K., Fanslow, W. C., MacLennan, I. C. M., Gregory, C. D., and Gordon, J.(1993) Eur. J. Immunol. 23, 2368-2371 [Medline] [Order article via Infotrieve]
  29. Berberich, I., Shu, G. L., and Clark, E. A.(1994) J. Immunol. 153, 4357-4366 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.