9 Regulation of Death Receptors

From: Cancer Drug Discovery and Development: Death Receptors in Cancer Therapy Edited by: W. S. El-Deiry © Humana Press Inc., Totowa, NJ

163

9 Regulation of Death Receptors

Udo Kontny, ^MD and Heinrich Kovar, ^P h D

SUMMARY

Apoptotic cell death mediated through activation of death receptors is essential in the regulation of tissue homeostasis in development and differentiation. The expression of the members of the death receptor family is tightly regulated and varies among tissues.

Dysregulation of death receptor expression is implicated in the pathogenesis of various diseases, including cancer, autoimmune disorders, neurodegenerative diseases, and infections. In this chapter we will focus on the stimuli and mechanisms that regulate the expression of death receptors.

TNF RECEPTORS

For tumor necrosis factor (TNF)- _, two different receptors, designated as TNF recep- tor 1 (TNFR1, also known as TNFR p55, CD120a) and TNF receptor 2 (TNFR2, also known as TNFR p75, CD120b) have been described (1). Both receptors have been shown to mediate apoptosis (2). Whereas TNFR1 is constitutively expressed on all nucleated cells, the expression of TNFR2 is primarily restricted to hematopoietic cells (3). The relative ratio of TNFR1 and -R2 varies in different cells. It has been shown that myeloid progenitors downregulate the expression of TNFR1 as they differentiate into mature monocytes (4). TNF receptor expression on the cell surface is regulated by three different mechanisms: (1) regulation of receptor synthesis; (2) shedding of receptors; and (3) internalization of receptors.

The TNF Receptor Promoters

The TNFR1 gene promoter resembles that of housekeeping genes lacking canonical

TATA and CAAT box motifs with multiple start points for transcription (5). Though a

consensus sequence for NF- gB has been described, there is no clear evidence that TNFR1

is regulated by this transcription factor. The promoter of TNFR2 contains consensus

elements for transcription factors involved in T-cell development and activation, such as T-cell factor 1, Ikaros, AP-1, CK-2, interleukin (IL)-6 receptor E, ISRE, GAS, NF-gB, and SP-1 (3,6). The mRNA expression of TNFR2 is upregulated by various cytokines, such as TNF-_, IL-1`, IL-10, and bFGF (7,8). TNFR2 mRNA is downregulated by interferon (IFN)a (8). Following T-cell activation, the expression of TNFR1 is increased, whereas TNFR2 expression is decreased (9).

Regulation of TNF Receptor Expression by Shedding and Internalization Shedding of TNFR has been shown to antagonize the effect of TNF-_ (10,11). How- ever, when soluble TNFR (sTNFR) concentrations are low, they can increase TNF activ- ity by stabilizing the death-inducing factor prolonging its availability to bind to membrane-bound TNFR. Shedding of TNFR1 and -R2 is mediated by the action of a metalloproteinase, probably identical to the TNF-_-converting enzyme (TACE) (12). Its activity is stimulated by hydrogen peroxide and nitric oxide (13,14). Injurious agents are known to induce shedding of TNFR1 from endothelial cells (15). Also, IL-4 has been demonstrated to induce shedding of both TNFR1 and TNFR2 from cultured monocytes (16). The production of sTNFR2 is increased after stimulation of cells with TNF-_, lipopolysaccharide (LPS), or IL-10 (8,17). In contrast, dexamethasone suppresses the release of sTNFR1 and -R2 from human monocytes (18). Internalization of the TNFR1 is observed after stimulating monocytes with LPS or TNF-_ (19).

TNF Receptor Expression and Disease

Increased expression of membrane-bound and soluble TNFR2 is seen in various autoimmune disorders, such as rheumatoid arthritis and Crohn’s disease, and plays a pivotal role in the pathogenesis of these disorders (20,21). Also, increased levels of sTNFR are observed in patients with malignancies including acute myelogenous leu- kaemia, non-Hodgkin’s lymphoma, and breast cancer (22–24).

THE CD95 RECEPTOR

The human CD95 antigen (also known as APO-1, Fas) is constitutively expressed in a wide range of hematopoietic and nonhematopoietic cells, and with particular abun- dance in the thymus, liver, and kidney (25).

The CD95 Promoter

The human CD95 gene is a single-copy gene comprising nine exons and eight introns (26,27). The 5' flanking sequence is GC rich (61%), with a high number of CpG dinucle- otides between –590 and –1, and lacks conventional TATA and CAAT boxes (28), properties characteristic of housekeeping genes (29). The promoter sequence contains consensus binding sites for the transcription factors Sp1, AP-1, AP-2, Ets, GF-1, EBP20, c-myb, CREB, GAF, NF-gB, NF-AT, NF-Y, and NF-IL6 (26,27,30). Rudert et al. iden- tified a silencer region between nucleotide positions –1035 and –1008, and a strong enhancer region between –1007 and –964 (31). The transcription factors YB-1, Pur_, and Pur` were shown to bind to the silencer region and produce varying levels of transcrip- tional repression (32). An enhancer element containing recognition motifs for GA-bind- ing protein (GABP) and AP-1 resides between nucleotide positions –862 and –682 (33).

Regulation of CD95 expression has been described for a wide range of stimuli.

Upregulation of CD95 in T-Cell Activation

When T-cells get activated, CD95 mRNA is upregulated and cells gradually become sensitive to CD95L-mediated apoptosis (34). In a first step, signaling through the TCR/

CD3 complex activates the src family tyrosine kinases p56 lck and p59lyn, and the protein-tyrosine kinases ZAP-70 and Syk (35). These kinases trigger three major path- ways: (1) phospholipase Ca1  Ca2+  calcineurin  NF-AT; (2) phosphatidylinositol (PI) 3-kinase  protein kinase (Akt)  S6 kinase; and (3) the Ras/Rac  mitogen- activated protein (MAP) kinase pathway. The MAP kinase pathways consist of three parallel signaling cascades, including the ERK1/2 MAP kinase cascade, the JNK/SAPK cascade, and the p38 MAP kinase cascade. Several targets of MAP kinase have been identified, including c-Jun and c-Fos, components of the transcription factor AP-1. Bind- ing of both AP-1 and GABP to the upstream enhancer element has been shown to be required for initiating CD95 transcription after T-cell activation (33). In addition, a composite binding site for Sp1 and NF-gB transcription factors at positions –295 to –286 is critical for T-cell activation-driven upregulation of CD95 (36).

Regulation of CD95 in Viral Infections

Infection with influenza virus or HIV augments the production of CD95 at the mRNA level (30,37). This may be due to a virus-stimulated increase of NF-IL6 (nuclear factor for interleukin 6 expression) binding to the 5' end of the human CD95 gene, containing eight copies of the NF-IL6 binding motif. NF-IL6 activation is likely to involve posttrans- lational modification, since no increase in NF-IL6 abundancy was observed after viral infection.

CD95 Expression After Genotoxic Stress

Chemotherapeutic drugs and irradiation stimulate upregulation of CD95 in many cell types (38). The increase in CD95 mRNA has been shown to be p53-dependent by various groups (39–42). After exposure to DNA-damaging agents, p53 protein gets stabilized, resulting in elevated steady-state levels (43). Wild-type p53 protein governs cellular activity by regulating downstream genes that control various cellular pathways, includ- ing cell-cycle arrest and apoptosis (44,45). A p53-responsive element is present in the first intron of the CD95 gene. Three additional putative p53 binding elements are con- tained 5' to the gene (40). When the p53-binding intronic region was placed in conjunc- tion with the CD95 promoter in a reporter plasmid, transcriptional activity was strongly induced by wild-type p53. Interestingly, in contrast to bax, another p53-regulated proapoptotic gene, the human CD95 p53-response element is still activated by the dis- criminatory p53 mutants Pro-175 and Ala-143 (46). Different genotoxic treatments cause different phosphorylations of p53. Whereas both irradiation and the chemotherapeutic drug ara-C resulted in p53-induction and apoptosis in the human leukemia cell line BV173, only irradiation was capable of inducing p53-dependent CD95 expression (41).

Thus, the susceptibility of CD95 to p53-mediated upregulation is dependent on the genotoxic signal.

Regulation of CD95 by Biological Response Modifiers

TNF-_ induces CD95 expression by activating NF-gB. This upregulation has been

shown to be dependent on the RelA subunit of NF-gB (47).

Binding of IFN- a to its receptor leads to the activation of tyrosine kinases of the Janus family (JAK). Activated JAKs then phosphorylate subunit 1 of the IFN-a receptor (IFNaR1), which subsequently serves as a docking site for signaling and transactivation (STAT) proteins, followed by their phosphorylation. The STAT proteins, mainly STAT1, form homo- or heterodimers, translocate to the nucleus, and regulate gene transcription by binding to gamma activating sequences (GAS elements) in the promoter of IFN-a regulated genes (48). IFN-a has been shown to upregulate CD95 in cells from various tumors, including gliomas, Ewing’s sarcoma, colon cancer, and in normal epithelial cells (49–52). The dependency of IFN-a- induced CD95 upregulation on STAT1 was demon- strated in the colon cancer cell line HT29. No increase in CD95 was observed in STAT1- deficient cells, but transfection of STAT1 restored CD95 responsiveness to IFN-a in these cells (53).

IL-12 has been shown to directly upregulate surface expression of CD95 in the human osteosarcoma cell line SAOS and the human breast cancer cell lines MDB-MB-231 (54).

There is preliminary evidence that this increase in CD95 expression involves the NF-gB pathway (54). Stimulation of CD95 may add to the antitumor activity of IL12 in several preclinical animal tumor models (55).

In lung cancer cells, the synthetic retinoid CD437 induces increased CD95 expression, which is dependent on wild-type p53 (56).

EXPRESSION OF DEATH RECEPTOR DR3

Activation of death receptor (DR)3 is capable of inducing both NF-gB and apoptosis (57). The DR3 gene locus is located on human chromosome band 1p36.2-p36.3 (58). This gene locus is frequently deleted in neuroblastoma and other neuroectodermal tumors. In medulloblastoma/PNET, expression of DR3 is significantly associated with survival (59). The DR3 promoter and its regulation have not yet been described.

THE TRAIL RECEPTORS DR4 AND DR5

TNF-related apoptosis-inducing ligand (TRAIL, also known as APO2L) interacts spe- cifically with five different receptors. Death receptors DR4 (also known as TRAIL-R1) and DR5 (also known as TRAIL-R2, TRICK2, and KILLER) are type I transmembrane pro- teins, which contain an intracellular death domain (60). Binding of TRAIL to receptors DR4 and DR5 leads to apoptosis via recruitment of adaptor proteins such as Fas-associ- ated death domain (FADD), resulting in activation of the caspase system. Decoy receptor DcR1 (also known as TRAIL-R3 or TRID), a glycosylphosph-atidylinositol-linked pro- tein, DcR2 (also known as TRAIL-R4 or TRUNDD), and osteoprotegerin contain either no cytoplasmic death domain or a truncated death domain. Therefore binding of TRAIL to these receptors does not result in apoptosis.

Regulation of DR4 and DR5 T

DR4 P

The 5' flanking region of the DR4 gene has been recently characterized (61). Three

putative binding sites for AP-1 were identified, with the site located at –350/–344 being

tested and shown to be functionally active. TPA, a strong inducer of AP-1, enhanced the

binding of this DR4 AP-1 site to nuclear extracts and increased transcription of DR4.

There is evidence that DR4 is a DNA-damage-inducible gene that is p53-regulated in some types of cancer cells (62). When HPV E6 was transfected into wild-type p53 lung cancer cells, leading to decreased levels of p53 protein, DR4 induction by DNA-damag- ing agents was suppressed. Conversely, transfection of exogenous wild-type p53 led to the upregulation of endogenous DR4 in cells with mutant p53.

T

DR5 P

As in TNFR, CD95, and DR4 genes, DR5 contains a TATA-less promoter. There are two SP1 sites responsible for basal transcriptional activity. Transient transfection assays with serial 5' deletion mutants identified the minimal promoter region between –198 and –116 (63).

DR5

53

Exposure of various human cancer cell lines to doxorubicin, etoposide, or irradiation led to induction of DR5 in cells with wild-type p53 status, but not in cells with mutant p53 (64). Overexpression of wild-type p53 in p53-deficient cells caused induction of DR5.

A p53 DNA-binding site was identified within intron 1, and mutation of this binding site led to loss of DR5 inducibility in reporter gene assays (65). Interestingly, p53-dependent upregulation of DR5 seems to be restricted to wild-type p53 cells undergoing apoptosis but not cell-cycle arrest when exposed to DNA-damaging agents (66).

R

DR4

DR5 T

NF-gB

Etoposide has been shown to upregulate DR4 and DR5 expression in epithelial cells such as breast cancer and human embryonic kidney cells (67). The induction involves the nuclear factor gB (NF-gB) pathway and can be blocked by expression of kinase-inactive MEK kinase 1 (MEKK1) or dominant-negative inhibitor of NF-gB (IgB). Ravi et al.

demonstrated that TNF-_ increased expression of DR4 and DR5 by inducing degradation of IgB and subsequent activation of NF-gB (68). This upregulation was dependent on the c-Rel subunit of NF-gB. Upregulation of DR5 but not DR4 through NF-gB has been described in various epithelial cell lines after exposure to TRAIL (69). In fact, a NF-gB binding site has been described between +385 and +394 in intron 1 of the DR5 gene (63).

R

DR4

DR5

C

In prostate cancer cells, protein levels of DR4 and DR5 were upregulated up to eight- fold when cells were treated with paclitaxel (70). Interestingly, protein induction was not associated with an increase in mRNA compatible with a posttranscriptional mechanism.

Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 protein expression but not DR4 expression (71). When cells were pretreated with these drugs at clinically achievable concentrations, apoptosis induced by TRAIL was signifi- cantly increased.

R

DR4

DR5

B

R

M

IFN-a was shown to downregulate mRNA and surface expression of DR4 and DR5 in

human fibroblasts (72). This effect was associated with inhibition of NF-gB. In contrast,

in various tumor cell lines, DR5 expression is stimulated by IFN-a (73) independently

from the p53 status, and is delayed in STAT1-null cells. Likewise, the glucocorticoid

dexamethasone has been demonstrated to elevate DR5 mRNA expression by a mecha-

nism unrelated to p53 in cancer cell lines (73). Interestingly, the glucocorticoid pred-

nisone, though also inducing apoptosis, did not induce DR5 transcription.

I

V

I

TRAIL R

E

Human herpes virus 7 (HHV-7) is endemic in the adult population and represents a potential opportunistic agent in immunocompromised individuals. HHV preferentially infects CD4+ T-cells. Infection of T-cells is associated with marked downregulation of DR4, making cells resistant to TRAIL-mediated apoptosis (74). In contrast, infection of human fibroblasts with cytomegalovirus (CMV) leads to increased DR4 and DR5 expres- sion (72). Interestingly, CMV infection activates NF-gB, which in turn has been shown to signal upregulation of DR4 and DR5 (67,75).

E

TRAIL R

C

N

D

In colon cancer, the inducible cyclooxygenase-2 (COX-2) gene, which regulates pros- taglandin biosynthesis, is upregulated (76). When HCT-15 colon cancer cells lacking endogenous COX-2 were transfected with COX-2 cDNA, TRAIL-induced apoptosis was attenuated, accompanied by transcriptional downregulation of DR5 and by increased Bcl-2 expression (77). The nonsteroidal antiinflammatory drug sulindac sulfide inhibited COX-2 enzymatic activity and reversed COX-2-mediated downregulation of DR5 mRNA and protein.

Toxic bile salts promote liver injury and the development of liver cirrhosis (78). The toxic bile salt glycochenodeoxycholate (GCDC) was shown to induce apoptosis in a hepatocellular carcinoma cell line, which was associated with increased expression and aggregation of DR5 but not DR4 (79).

REGULATION OF DEATH RECEPTOR DR6

The death receptor DR6 has been characterized by Pan et al. (80). DR6 is expressed in most human tissues. In LnCAP prostate cancer cells, TNF-_ induced DR6 mRNA and protein levels (81). This induction could be blocked by inhibiting NF-gB activation.

Increased expression of DR6 has been described in late-stage prostate tumors compared to normal tissue and tumor cell lines derived from less advanced prostate cancers.

DECOY RECEPTORS (DCR)

The TRAIL-binding receptors DcR1 and DcR2, as well as the Fas ligand-binding receptor DcR3, are called decoys because they do not contain an intracellular death domain and protect cells from the cytotoxic effect of the respective death ligand (82).

Promoter Structure of DcR1

The promoter structure of the human DcR1 gene has been investigated by Ruiz et al.

(83). The promoter contains a TATA-consensus box. Deletion analysis identified a mini- mal promoter region within the first 33 nucleotides upstream of the transcription initia- tion site. Transcriptional activity increases with extended size of 5' fragments, suggesting functional binding sites for additional, not yet identified transcription factors that coop- erate with the basal transcription machinery in the regulation of the DcR1 gene.

Regulation of Expression of Decoy Receptors

Sheikh et al. demonstrated that DcR1 is upregulated in p53 wild-type but not p53-

negative cell lines after exposure to X-ray (84). When a human temperature-sensitive p53

was transfected into p53-null H1299 lung carcinoma cells, DcR1 was upregulated upon shift to the permissive temperature. Also, doxorubicin, a p53-inducing drug, was able to activate a DcR1 promoter construct in MCF7 cells (83). However, abrogation of p53 activity by HPV E6 protein through proteasome-dependent degradation did not affect the transcriptional activity of this promoter fragment, indicating that the p53-responsive site must be located outside the promoter region of –502, +42.

Overexpression of cRel, a member of the Rel/NF-gB family of transcription factors leads to upregulation of DcR1 in HeLa cells and to resistance against TRAIL-induced apoptosis (85). Upregulation of DcR1 was also achieved by TNF-_ via activation of endogenous Rel/NF-gB factors.

Maeda et al. demonstrated upregulation of DcR2 and downregulation of DcR3 in human keratinocytes after exposure to ultraviolet B irradiation by a mechanism that remains to be identified (86).

Fresh neuroblastoma cells and various tumor cell lines have been shown not to express DcR1 and DcR2. This lack of decoy receptor expression was demonstrated to be asso- ciated with dense hypermethylation of their promoter regions (87). Treatment with 5-aza- 2'deoxycytidine resulted in partial demethylation and restored mRNA expression of DcR1 and DcR2.

Abundant levels of DcR1 and DcR3 have been observed in tumors of the gastrointes- tinal tract (84,88). It is hypothesized that this overexpression confers growth advantage by inhibiting TRAIL- and CD95-mediated destruction of tumor cells.

CONCLUSION

Although control and fine-tuning of death receptor transcription and activity is com- plex and cell-type specific, several regulatory proteins seem to play key roles. Among them p53 and NFgB are frequently involved in the regulation of several death receptors and may contribute to the cytotoxicity of chemo- and radiotherapeutic agents. Therefore, restoration of function of these regulatory proteins, which is frequently compromised in cancer cells, is a major goal in the development of biology-based new tumor-treatment strategies.

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