CD40: A Growing Cytoplasmic Tale

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Science's STKE  15 Jun 2004:
Vol. 2004, Issue 237, pp. pe25
DOI: 10.1126/stke.2372004pe25


CD40, a member of the tumor necrosis factor (TNF) receptor family that is expressed on B cells, monocytes, dendritic cells, endothelial cells, and epithelial cells, as well as on B cell lymphomas and carcinomas, activates multiple signaling pathways. In B cells, the response to CD40 is complex and depends on the maturation status of the cell. It is well established that CD40 can promote cell survival through up-regulation of the expression of genes encoding antiapoptotic proteins. However, a new role for CD40 signaling is being recognized in promoting progression through the cell cycle. The roles of the phosphoinositide 3-kinase, mitogen-activated protein kinase, and nuclear factor κB pathways in mediating CD40 stimulation of the cell cycle are described.

CD40 is a member of the tumor necrosis factor (TNF) receptor family that is expressed on B cells, monocytes, dendritic cells, endothelial cells, and epithelial cells, as well as on B cell lymphomas and carcinomas (1). Development of the acquired immune response is dependent on bidirectional signaling mediated by CD40 and its ligand CD154, which is expressed primarily on activated CD4+ T cells (1, 2). Specifically, CD40 signaling is essential for activation and proliferation of B cells by T cell–dependent antigens, germinal center formation, immunoglobulin (Ig) class switching, affinity maturation of B lymphocytes, and the development of plasma and memory B cells. Hyper-IgM syndrome, an X-linked immunodeficiency resulting from mutation of CD154, illustrates this well: Patients with this syndrome exhibit reduced ability to generate T cell–dependent antibody responses; lack circulating IgG, IgA, and IgE; and are unable to generate memory B cells. In addition, CD40 has recently been shown to be an important regulator of the production of inflammatory mediators, cell survival, and prolonged antigen presentation by dendritic cells (2). Interest in the molecular mechanisms of CD40 signaling has generally focused on CD40-mediated regulation of apoptosis. This is because CD40 signaling can rescue autoreactive B cells from clonal deletion by apoptosis; consistent with this, enforced and prolonged expression of CD154 can result in the production of autoantibodies (3, 4). Moreover, whereas CD40 signals lead to survival in normal B cells, in lymphoma both inhibition and induction of apoptosis have been reported, depending on the maturation state of the B cell [reviewed in (1)]. Thus, delineation of the key survival signals involved has obvious potential for the development of novel cancer and autoimmunity therapies. In addition, there has been an upsurge in interest in the signaling mechanisms underlying the emerging role for CD40 in promoting cell cycle progression of B cells, not least because these studies have identified potential novel targets for intervention in B cell malignancies.

CD40 is a 45-kD transmembrane glycoprotein that exists as a monomer, and the binding of CD154 causes clustering of CD40 in lipid raft domains at "synapses" between interacting cells in order to propagate intracellular signals (5, 6). CD40 lacks intrinsic catalytic activity, but the cytoplasmic domain of CD40 has two binding sites for TRAF (TNF receptor–associated factor) proteins (7). TRAFs have several functional domains, including a zinc finger domain and a RING finger domain, both of which may mediate DNA binding, as well as a TRAF domain that enables protein-protein interactions. TRAFs 1, 2, 3, 5, and 6 have been found in association with CD40, and these adaptors couple CD40 to the phosphoinositide 3-kinase (PI3K), phospholipase Cγ (PLC-γ), mitogen-activated protein kinase (MAPK-ERK, p38, and JNK), and nuclear factor κB (NF-κB) signaling pathways [F1; reviewed in (1, 8)].

The effect of CD40 ligation on apoptosis can be explained by the increased transcription of genes encoding antiapoptotic members of the Bcl-2 family, such as Bcl-xL, A1, and A20, which act to promote the survival of B cells (914). However, it is well established that the induction of antiapoptotic genes, encoding such proteins as Bcl-xL, does not promote cell cycle progression in the absence of CD40 signaling (1114). Indeed, it has recently been shown that the survival-promoting effects of Bcl-2 and Bcl-xL correlate with enhanced G0/G1 arrest and cell cycle delay, with the resulting lower metabolic state possibly contributing to the prevention of cell death (15). Nonetheless, although immature WEHI-231 B cells that overexpress Bcl-xL still undergo antigen receptor [also known as B cell receptor (BCR)]–mediated growth arrest, overexpression of the transcription factor E2F can overcome this arrest in the cell cycle (16). (This is presumably because E2F regulates many of the genes required for the G1-to-S phase transition, such as those encoding DNA polymerase-α, thymidine synthetase, and cyclins D3, E, and A.) These results suggest that CD40 regulates the survival and proliferation of immature B cells through distinct pathways. Indeed, mutagenesis studies have shown that distinct regions of CD40 are required to stimulate the activity of regulators of cell cycle progression and expression of the gene encoding Bcl-xL (12). Thus, independent from Bcl-XL up-regulation, CD40 signaling also results in decreased expression of the gene encoding the cyclin-dependent kinase (CDK) inhibitor p27kip-1 and increased expression and activation of the cyclin D–dependent kinases Cdk4 and Cdk6, which are essential for progression through the G1 phase of the cell cycle (12, 14). These kinases phosphorylate the retinoblastoma (Rb) pocket domain protein, resulting in release and activation of the E2F transcription factor (17). Interestingly, although cell cycle arrest induced by ectopic expression of p27kip-1 does not alter levels of Bcl-xL or A1, CD40-mediated cell survival is partially reduced, which suggests that cell cycle progression may ultimately be required for CD40-mediated survival of B cells (14). The CD40-driven stimulation of cyclin D and Cdk4 mRNA and protein abundance and the corresponding decrease in expression of p27kip-1, which are required for quiescent B cells to enter G1 phase, are dependent on activation of caspase-6 and suppression of apoptosis and caspase-3 activity (18). Inhibitor of apoptosis, c-IAP—which can bind, inhibit, ubiquitinate, and promote the proteasomal degradation of caspase-3, but not caspase-6 (18)—can be found in association with CD40-coupled TRAFs (19), and this may be one mechanism for the differential regulation of caspase activity. Thus, early recruitment of IAP may explain how CD40 signaling can inhibit apoptotic executioner caspase activity while stimulating the activity of caspases required for cell cycle entry and progression.

CD40 coupling to NF-κB signaling plays key roles in both cell survival and proliferation. Indeed, studies investigating the link among CD40, NF-κB, and aggressive neoplastic cell growth in non-Hodgkin’s lymphoma found that CD40 was anchored in lipid rafts, leading to constitutive activation of NF-κB and stimulation of CD154 gene expression, which in turn results in continual autonomous CD40-mediated stimulation of survival and proliferation (5). Although NF-κB stimulates the expression of genes encoding such target proteins as Bcl-XL, which appear solely to be associated with cell survival (20), NF-κB also stimulates the proto-oncogene pim-1, which encodes a kinase whose abundance correlates with increased survival and proliferation of B lymphocytes (21).

Similarly, prolonged CD40 coupling to NF-κB signaling results in sustained increases in c-Myc mRNA and protein levels (22, 23), which are associated with rescue from BCR-driven immature B cell growth arrest and apoptosis (20). By contrast, BCR-mediated growth arrest and apoptosis of immature B cells correlates with a transient (<1 hour) increase in c-Myc mRNA and protein, followed by a rapid fall below basal levels that is associated with increased abundance of the tumor suppressor protein p53 and the CDK inhibitors p21waf and p27kip. The molecular events linking sustained c-Myc activity to cell cycle progression have not been fully delineated, but it is clear that c-Myc transcriptional activity stimulates the gene encoding CUL1, a member of the Cullin family of scaffolding proteins that form Skp1–Cullin–F-box (SCF) complexes (24). SCF complexes are the ubiquitin ligases that promote the degradation of negative regulatory proteins, such as p21waf and p27kip, to allow G1-to-S phase progression. In addition, because SCF-type ubiquitin ligases can also ubiquitinate and hence target IκB (the negative regulator of NF-κB) for degradation, stimulation of c-Myc is likely to produce a positive feedback amplification loop of CD40-proliferative signaling. CD40 signaling also acts to suppress the expression of the gene encoding CTCF, which is a transcription factor that represses c-myc expression, thus allowing sustained c-myc expression and cell cycle progression (25). Indeed, CTCF was reported to be a key negative regulator of B cell proliferation (25). In addition to its repression of c-myc, CTCF induces expression of the gene encoding p19Arf, which sequesters the p53 inhibitor MDM2, resulting in activation of p53 and stimulation of the genes for p21waf (a mediator of growth arrest) and, ultimately, Bax (a mediator of apoptosis) (25).

Although inhibition of each of the PI3K, ERK, JNK, or p38 signaling cascades appears to have little effect on CD40-mediated B cell survival, abrogation of any of these pathways profoundly suppresses CD40-mediated proliferative responses (20). Some recent studies have provided clues to the key roles of these pathways in CD40 coupling to cell cycle progression. For example, coupling of CD40 to p38 MAPK and PI3K may promote cell cycle progression by decreasing the expression of genes encoding Ndr1, Rb1, Rb2, BTG-2, and SPA-1, the expression of which results in growth arrest (20). Ndr1 is a novel differentiation-related gene involved in growth arrest and terminal differentiation (26); Rb1 and Rb2 are members of the Rb pocket protein family of tumor suppressors (27); BTG-2 is encoded by the p53-inducible gene PC3, a member of the PC3/BTG/Tob gene family involved in G1-to-S phase arrest (28); and SPA-1 (sparse) is a Rap-1 guanosine triphosphatase (GTPase) activating protein that inhibits mitogen-induced cell cycle progression (29). Similarly, CD40 induction of caspase-6–like activity results in the cleavage of special AT-rich sequence-binding protein-1 (SATB1) (18), which is a transcriptional repressor that plays a role in maintaining cells in a quiescent state. Moreover, inhibition of PI3K signaling by BCR coupling to 3′-inositol phosphatase in immature B cells also results in the increased expression of the gene encoding CTCF, decreased expression of c-myc, and growth arrest (25). Thus, the abortive BCR-to-PI3K signaling observed in immature B cells provides a rationale for the transient c-Myc signal observed relative to that obtained after mitogenic stimulation of CD40 or, indeed, by the BCR in mature cells. CD40 signaling therefore acts not only to induce genes required for cell cycle progression, but also to decrease the expression of genes for transcriptional repressors that actively suppress proliferation.

Finally, coupling of CD40 to the ERK and PI3K pathways is also likely to promote cell cycle progression through stabilization of c-Myc activity (25, 30, 31). ERK phosphorylates newly synthesized c-Myc protein on Ser62, resulting in stabilization. By contrast, glycogen synthase kinase–3 (GSK-3) phosphorylates c-Myc at Thr58, which promotes dephosphorylation of Ser62 by protein phosphatase 2A (PP2A) and targets c-Myc for degradation through the ubiquitin-proteasome pathway (30). PP2A has also been implicated in the induction of the expression of the gene for CTCF (25), perhaps suggesting a dual-pronged mechanism of PP2A-mediated c-Myc down-regulation. Because GSK-3 activity is negatively regulated by PI3K-dependent Akt-mediated phosphorylation of GSK-3, CD40 coupling to ERK and PI3K would promote c-Myc stabilization and cell cycle progression. We have recently shown CD40-mediated rescue from BCR-mediated growth arrest to be dependent on cycling activation of ERK throughout the cell cycle (13, 32), which presumably results in c-Myc stabilization. This requirement for cycling ERK may therefore be a consequence of CD40-mediated increase in c-Myc abundance being due to modulation of protein stabilization, rather than (or in addition to) transcriptional activation. Indeed, such a phosphorylation-dephosphorylation regulatory mechanism would allow CD40-mediated amplification of c-Myc protein levels to be flexible, rapid, and transient, preventing deregulation of cell cycle control. Perhaps consistent with this idea, a large number of c-myc alleles amplified in Burkitt’s lymphoma carry a mutation at Thr58 (30, 31). Furthermore, the ERK signaling pathway is aberrantly active in Hodgkin’s disease (33). Moreover, inhibition of such ERK signaling results in G2-to-M cell cycle arrest, which suggests that blocking ERK activation and consequent c-Myc stabilization may have therapeutic potential (33).


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Fig. 1.

Signaling mechanisms underlying coupling of CD40 to cell cycle progression. Oligomerization of CD40 in lipid raft domains recruits TRAFs to couple to the PI3K, PLC-γ, MAPK (ERK, p38, and JNK), and NF-κB signaling pathways. NF-κB activation and decreased expression of the gene encoding the transcriptional repressor CTCF results in sustained induction of c-Myc expression and abundance and in decreased activity of cell cycle inhibitors. In addition, CD40 signaling is coupled to the caspase-6–dependent reduction in p27kip-1 levels, induction of the genes encoding cyclin D and Cdk4, and activation of Cdk4 and 6, resulting in hyperphosphorylation of Rb, release of E2F and DNA synthesis, cell cycle progression, and proliferation. SCF, Skp1–Cullin–F-box complex

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