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50 Nebigil, C.G. et al. (1995) Agonist-induced desensitization and phosphorylation of human 5-HT1A receptor expressed in Sf9 insect cells. Biochemistry 34, 11954–11962 51 Wu, X. et al. A critical protein kinase C site on the 5-HT1A receptor controlling coupling to N-type calcium channels. J. Physiol. (in press) 52 Lembo, P.M. et al. (1999) Receptor selectivity of the cloned opossum G protein-coupled receptor kinase 2 (GRK2) in intact opossum kidney cells: role in desensitization of endogenous alpha2Cadrenergic but not serotonin 1B receptors. Mol. Endocrinol. 13, 138–147 53 Loisel, T.P. et al. (1999) Activation of the beta(2)-adrenergic receptor-Galpha(s)

complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Galpha(s). J. Biol. Chem. 274, 31014–31019 54 Chen, C.A. and Manning, D.R. (2000) Regulation of galpha i palmitoylation by activation of the 5hydroxytryptamine-1A receptor. J. Biol. Chem. 275, 23516–23522 55 Palczewski, K. et al. (2000) Crystal structure of rhodopsin: a G protein-coupled receptor. Science 289, 739–745 56 Sun, Q.Q. and Dale, N. (1999) G-proteins are involved in 5-HT receptor-mediated modulation of N- and P/Q- but not T-type Ca2+ channels. J. Neurosci. 19, 890–899

57 Ortiz, T.C. et al. (2000) Structural variants of a human 5-HT1a receptor intracellular loop 3 peptide. Pharmacology 60, 195–202 58 Varrault, A. et al. (1994) 5-Hydroxytryptamine1A receptor synthetic peptides. Mechanisms of adenylyl cyclase inhibition. J. Biol. Chem. 269, 16720–16725 59 Lembo, P.M. et al. (1997) A conserved threonine residue in the second intracellular loop of the 5hydroxytryptamine 1A receptor directs signaling specificity. Mol. Pharmacol. 52, 164–171 60 Albert, P.R. et al. (1998) A putative alpha-helical G beta gamma-coupling domain in the second intracellular loop of the 5-HT1A receptor. Ann. New York Acad. Sci. 861, 146–161

Nuclear receptors in cell life and death Lucia Altucci and Hinrich Gronemeyer The balance between cell proliferation and programmed cell death (apoptosis) determines body patterns during animal development and controls compartment sizes, tissue architecture and remodeling. The removal of primordial structures by apoptosis allows the organism to develop sex specifically and to adapt for novel functions at later stages; apoptosis also limits the size of evolving structures. It is a ubiquitous function that is essential for all cells. Although inappropriate regulation or execution of apoptosis leads to disease, such as cancer, there is now evidence for its great therapeutic potential. This would be particularly true if apoptosis could be targeted at defined cell compartments, rather than acting ubiquitously like chemotherapy. Here, we discuss the potential of nuclear receptor ligands, many of which act through their cognate receptors in defined body compartments as modulators of cell life and death, with special emphasis on the molecular pathways by which these receptors affect cell-cycle progression, survival and apoptosis.

Lucia Altucci Dipartimento di Patologia Generale e Oncologia, Seconda Università degli Studi di Napoli, Centro Sperimentale S. ‘Andrea delle Dame‘, Via De Crecchio 7, 80138, Napoli, Italy. Hinrich Gronemeyer* Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC)/CNRS/INSERM/ ULP, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France. *e-mail: [email protected]

NRs (see Glossary), which comprise the receptors for steroid and thyroid hormones, vitamin D, in addition to many ‘orphans’, are members of a large family of ligand-inducible transcription factors that regulate gene-initiated programs at the basis of a plethora of (patho)physiological phenomena. The recent determination of the crystal structures of NR domains has clarified the intra- and intermolecular mechanisms that initiate receptor activation and signal transduction. All NRs are modular proteins harboring one DBD and LBD (Fig. 1). The LBD also comprises the liganddependent AF-2, whereas AF-1 operates autonomously and ligand independently when placed outside the receptor. NRs are transcription factors that: (1) respond directly to a large variety of hormonal and metabolic signals; (2) integrate diverse signaling pathways to targets posttranslationally; and (3) regulate the activities of other major signaling cascades (called signal transduction crosstalk).

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Growth regulation by NRs: (patho)physiological phenomena

NRs comprise two principal categories: those that stimulate growth and those that interfere negatively with cell proliferation. ERs and ARs are predominantly growth-stimulatory receptors in the major target organs, such as breast and prostate, which is why anti-hormonal therapies are used in the corresponding (hormone-responsive) cancers. By contrast, GRs are mainly antiproliferative because they induce apoptosis in lymphoid cells; thus, GC agonists are useful as anti-leukemia and antiinflammatory agents. Outside the steroid receptor family, RARs, RXRs, VDR and PPARs have pronounced antiproliferative potential, which is usually linked to the capacity to induce differentiation. However, the distinction between proliferative and antiproliferative NRs is rather artificial. Indeed, depending on the cell context, a receptor can exhibit proliferative or antiproliferative activity. Sex steroid hormones and their cognate receptors trigger fundamental physiological processes, particularly in primary and accessory sex organs, by regulating cell life and death. A prototypic example is the hormonal control of the mammary gland. Estradiol stimulates directly the formation of terminal end buds and proliferation of the mammary epithelium, effects that are inhibited by antiestrogens1 and impaired in ERα-deficient female mice2, demonstrating an ERα-mediated pathway of estrogen action. Progesterone might also act as a MITOGEN in breast, triggering lobular-alveolar development during pregnancy. PR-deficient mice fail to form a mammary lobular-alveolar structure upon exposure to estrogen and progesterone3. One of the most clinically important aspects of estrogen is its stimulation of ERα-positive breast

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Glossary AD: activation domain, one or more ADs can constitute an AF. AF-1: activation function 1 in the N-terminal region A/B of nuclear receptors. AF-2: activation function 2 in the LBD of nuclear receptors. Akt: serine/threonine kinase with SH2 and PH domains, activated by inositol (1,4,5) trisphosphate kinase downstream of insulin and other growth factor receptors; Akt phosphorylates glycogen synthase kinase 3 and is involved in stimulation of Ras and control of cell survival. APL: acute promyelocytic leukemia. AR: androgen receptor (systematic name: NR3C4). ATRA: all-trans retinoic acid. Cdk: cyclin-dependent kinase; family of kinases that are only active when they form a complex with cyclins. CD437: 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2naphthalene carboxylic acid; a synthetic ‘atypical’ retinoid. ChIP: chromatin immunoprecipitation; following DNA–protein crosslinking, antibodies against chromatin (e.g. antibodies directed against acetylated histones or transcription factors) constituents are used to enrich by precipitation chromatin fragments containing these antigens and the corresponding DNA sequences are identified and quantitated by the polymerase chain reaction; this novel technique allows one to assess the acetylation status of a gene and the transcription factor loading of its promoter. CKI: Cdk inhibitor; two classes of CKIs are known, the p21CIP1/Waf1 class that includes p27KIP1 and p57KIP2, which inhibit all G1/S Cdks, and the p16INK4 class, which bind and inhibit only Cdk4 and Cdk6. The p21 inhibitor is transcriptionally regulated by the p53 tumour suppressor, is important in the G1 DNAdamage checkpoint, and its expression is associated with terminally differentiating tissues. CNS: central nervous system.

DBD: DNA-binding domain. DHT: dihydrotestosterone. DP: E2F dimerization partner; DP family proteins interact with E2F family proteins to form heterodimers; association of E2F1 and DP1 was shown to lead to cooperative activation of E2F responsive promoters; E2F1–DP1 association is required for stable interaction with pRB, this interaction inhibits transactivation by E2F1–DP1 heterodimers. EGF: epidermal growth factor. ER: estrogen receptor (systematic name: NR3A1 and 2). ERE: estrogen response element. GC: glucocorticoid. GR: glucocorticoid receptor (systematic name: NR3C1). GRE: glucocorticoid response element. HDAC: histone deacetylase. IGF-1: insulin-like growth factor 1. LBD: ligand-binding domain. MEK: MAPK kinase; mitogen-activated protein kinase kinase [also called externally regulated kinase (ERK) kinase]; serine–threonine kinases that are activated when quiescent cells are treated with mitogens, and that therefore potentially transmit the signal for entry into the cell cycle. Mitogen: substance causing re-entry of cells into the cell cycle. κB: nuclear factor-κB; transcription factor; NF-κ originally found to switch on the transcription of genes for the κ class of immunoglobulins in B cells. NMDA: N-methyl-D-aspartic acid; agonist for a class of NMDA receptors found on some vertebrate nerve cells involved in synaptic transmission. NR: nuclear (hormone) receptor. p21: Cdk inhibitor; p21 is capable of binding to both cyclin–Cdk and the proliferating cell nuclear antigen; through tight inhibitory binding to Cdks, p21 inhibits the phosphorylation of pRB by cyclin A–Cdk2, cyclin E–Cdk2, cyclin D1–Cdk4, and cyclin D2–Cdk4 complexes. PCa: prostate cancer.

cancer cell proliferation. Positivity for ERα and a PR is an indication of hormone-dependent growth control4,5 of breast cancers, and experimental data strongly suggest that estrogens have a role in the development and growth of breast cancer6,7. Hence, treatment with antiestrogens is the first line of therapy for estrogenresponsive breast cancer, and antiestrogens, such as tamoxifen, are evaluated for chemoprevention of breast cancer in patients at risk8–12. The uterus is another major estrogen target organ. Proliferation of the glandular epithelium and stroma of the primate endometrium correlates with circulating levels of estrogen and progesterone13–15. Steroid hormone withdrawal results in uterine epithelium apoptosis in several species16. This steroid hormone dependency constitutes a risk for endometrial cancer17,18. This also applies to tamoxifen, which is an ERα antagonist in the breast but a weak agonist in uterine tissues. Androgens also regulate growth of the cognate target organs. The prostate gland is highly responsive to DHT, and androgens are essential for the maintenance of prostate epithelial cell proliferation and differentiation during development; androgen withdrawal by surgical or chemical castration results http://tem.trends.com

PML: putative transcription factor, apparently involved in apoptosis or survival signaling of cells; in APL blasts displaying a t(15;17) chromosomal translocation a PML–RARα fusion protein is formed and is responsible for a differentiation block at the promyelocytic stage. PKA: protein kinase A. PPAR: peroxisome proliferator-activated receptor (systematic name: NR1C1, 2 and 3). PR: progesterone receptor (systematic name: NR3C3). RA: retinoic acid, generic term for ATRA, 9-cis RA and other RAR and/or RXR ligands. RAR: retinoic acid receptor (systematic name: NR1B1, 2 and 3). pRB: retinoblastoma protein. RXR: retinoid X receptor (systematic name: NR2B1, 2 and 3). SMADs: intracellular proteins that mediate signaling from receptors for extracellular TGF-β-related factors. SMADs 1 and 5 are activated (serine/threonine phosphorylated) by bone morphogenetic protein receptors, SMADs 2 and 3 by activin and TGF-β receptors. SMADs activated by occupied receptors form complexes with SMAD4 and move into the nucleus, where they regulate gene expression. SNuRM: selective NR modulator; ligands that act in a cell- or tissue-selective manner, such as ‘boneselective’ estrogens. α/β β: transforming growth factor α/β. TGF-α TH: thyroid hormone. TIF: transcription intermediary factor, also referred to as coactivator (CoA) and corepressor (CoR); TIFs mediate the action of the AFs on NRs to the transcriptional machinery. α: tumour necrosis factor α. TNF-α TR: thyroid hormone receptor (systematic name: NR1A1 and 2). TRAIL: tumor necrosis factor-related apoptosisinducing ligand (also called Apo2L). VDR: vitamin D receptor (systematic name: NR1I1).

in massive apoptosis of the prostatic epithelium and prostate involution19. Because of the essential participation of the AR in the regulation of prostate growth and function, its potential role in the development and progression of PCa has been studied extensively. AR is the target of endocrine therapy for PCa treatment, which aims to reduce the levels of testosterone and DHT. Blockade of the androgensignaling pathway kills PCa cells through induction of programmed cell death20. Several studies have demonstrated the occurrence of AR variants in antiandrogen-resistant PCa (Refs 21–23). GCs have strong growth-regulatory capacities that can be stimulatory or inhibitory, depending on the cell type. The best-recognized and pharmacologically exploited action of GCs is the induction of apoptosis in T cells24–26. In the brain, adrenal steroids inhibit cell proliferation in the dentate gyrus during the early postnatal period and in adulthood. This antiproliferative action of GCs appears to be indirect through an NMDA receptor-dependent excitatory pathway27. However, the collective actions of GCs in the brain are paradoxical in that basal levels are essential for neuronal development, plasticity and survival, whereas stress levels of GCs produce neuronal loss28.

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Fig. 1. (a) Schematic of the structural and functional organization of NRs. The evolutionary conserved regions C (DBD) and E (LBD) are indicated as boxes and a black line represents the divergent regions A/B, D and F. Two transcription AFs have been described in several NRs, a constitutively active (if taken out of the context of the receptor) AF-1 in region A/B and a ligand-inducible AF-2 in region E. Within these AFs, ADs have been defined. (b) Estrogen receptor DBD complex on a cognate DNA response element. (c) Agonist-induced changes of the LBD, allowing binding of coactivators (the bound coactivator-binding peptide is shown). Figures 1b,c are three-dimensional views derived from the corresponding crystal structures. Abbreviations: See Glossary.

TH initiates apoptosis and leads to tail regression in amphibians during metamorphosis29. In mammals, TH affects brain development, and a role of TR in oligodendroglial and neuronal differentiation and cell death has been proposed30. However, there are several unresolved issues when the effects of congenital hypothyroidism and the TH resistance syndrome on CNS (dys)function are compared with results obtained with TR-knockout and mutant TR-knockin genetic studies in mice31. Retinoid receptors [a family of three RAR and three RXR isotypes] and their cognate ligands, such as vitamin A-derived retinoic acids, are involved in the regulation of embryonic development, cell proliferation, differentiation and apoptosis32–34. A block in embryonic RA synthesis leads to early embryonic death caused by multiple defects, such as lack of axial orientation, incomplete neural tube

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closure, reduction of the trunk region, lack of the heart looping and chamber morphogenesis35. The role of RA signaling in the control of cell death during embryogenesis is apparent from the persistence of interdigital webbing in mice lacking certain combinations of RAR-encoding genes36. The potency of retinoids in modulating cell growth and differentiation has been the basis of their use as anticancer drugs. In animal models, they (and the RXR-selective retinoids) can interfere with the growth of several types of cancer, including melanoma, breast, bladder, and prostate cancers, squamous cell carcinomas of head, neck and skin and neuroblastoma37–39. In humans, efficient treatment of precancerous oral lesions and prevention of second primary tumors of head and neck squamous carcinomas has been reported40,41, and clinical chemoprevention trials with retinoids, rexinoids and atypical retinoids are under way42–44. The prototype for cancer differentiation therapy with retinoids is APL. Treatment with RA associated with chemotherapy is now the first line therapy for these patients, with a survival rate of more than 75% five years after diagnosis45,46. APL is caused by a t(15;17) chromosomal translocation that generates a PML–RARα fusion protein, which is believed to repress signaling by physiological concentrations of RA. Pharmacological doses of RA restore signaling. The above-described studies have provided a wealth of evidence for the major impact of NR signaling on the control of cell growth in virtually all phases of life and in multiple cell types, including cancer cells. Molecular basis and therapeutic perspective of the growth-regulatory potential of NRs

Cell growth is the consequence of the relative importance of the signaling pathways that control cell proliferation, death and survival. NRs can interfere positively or negatively with each of these events, or even simultaneously with two or more, in some cases in a temporal manner. There is some understanding of the molecular mechanisms by which NRs regulate the programs controlling growth; that is, cell cycle progression and arrest, cell survival and apoptosis. Estrogen and progesterone receptors

It is well established that the growth-stimulatory effect of estrogens, mediated through their cognate receptors (ERα and/or ERβ), result, at least in part, from the induction of cell cycle progression (Fig. 2). One particular cell cycle regulatory factor, cyclin D1, is linked to the estrogen-dependent growth response of target tissues. Female mice lacking cyclin D1 were defective in pregnancy-associated mammary tissue proliferation47,48, whereas overexpression of the gene encoding cyclin D1 (Ccnd1) in the mammary gland of MMTV-D1 transgenic mice resulted in abnormal cell proliferation, including hyperplasia and the development of mammary adenocarcinomas49.

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Fig. 2. Scheme illustrating cell-cycle regulation by certain nuclear receptors. The cell cycle phases G0, G1, S, G2 and M are depicted in (a), together with a schematic illustration of the corresponding levels of the various Cdk–cyclin complexes. Some steroid receptors (ER, AR and PR) stimulate expression of the gene that encodes cyclin D1, which interacts with and activates Cdk4. The activated cyclin–Cdk complex phosphorylates pRB, which dissociates from the DP–E2F complex, thus allowing transcription of cell cycle regulatory genes. In an opposite regulatory mode, vitamin D3 and retinoic acids can induce expression of the CKI p21, which blocks Cdk activity, resulting in G1 arrest of treated cells, such as U937. Abbreviations: See Glossary.

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might be an early step in the transition from benign lesions or hyperplasias to breast carcinoma. Ccnd1 expression is induced by estrogen in hormoneresponsive breast cancer cells and in normal rat uterus after hormonal treatment52,53. Because no classic ERE has been detected in the Ccnd1 promoter, it has been proposed that ER activates Ccnd1 transcription indirectly through heterologous response element(s)52,54. The physical recruitment of ERα to the Ccnd1 promoter in breast cancer cells after estradiol stimulation was demonstrated by ChIP analysis55, confirming a direct link between ERα and cyclin D1 regulation. In addition to the effect of estradiol on Ccnd1 expression, cyclin D1 can interact directly with ERα in a ligand-independent manner and recruit p160 coactivators, thus stimulating the transcription activation potential of ER (Refs 56,57). The physiological significance of this observation is currently unclear. In addition to the ERα–cyclin D1 link, ERα–growth factor crosstalk might contribute to estradiol-dependent end-bud proliferation of mouse mammary gland in vivo2. With the use of implant techniques, it was shown that EGF, which is itself at least partially regulated by estradiol, mediates its mitogenic effect through ERα, whereas blocking EGF action abrogated the estradiol-dependent stimulation of end-bud development58. However, these hormonal effects are apparently not cell autonomous. Tissue recombination experiments with ERα-deficient mice indicated that the hormonal regulation of epithelial proliferation is a paracrine event mediated by receptor-positive stromal cells59. In T47D breast cancer cells, progestins induce a biphasic change in the rate of cell cycle progression, consisting of an initial transient acceleration through G1 phase and an increase in the S phase fraction, followed by G1 arrest. These two distinct, opposing effects could account for the observations that both agonists60 and antagonists61 inhibit the proliferation of PR-positive cells. Antagonist action is accompanied by induction of the CDK inhibitor, CKI p21WAF1, at increased cyclin D1 and cyclin E levels and decreased kinase activity61. Agonist-induced growth inhibition correlates with decreased synthesis of cyclins D1, D3 and E, and increased association of the CKI p27KIP1 with Cdk4 complexes60. Estrogen-stimulated proliferation of uterine tissues, which, in adults, is limited to the epithelial compartment, is ablated by disruption of the gene encoding ERα, thus establishing the crucial role of ERα in the growth response. Members of the EGF family appear to mediate estrogen-induced mitogenesis in the uterus. Experiments demonstrated an upregulation of the levels of uterine EGF and its receptor, TGF-α and IGF-1. Compelling evidence is accumulating for extensive crosstalk between the EGF and ligand-independent ERα signaling pathways, and studies with ERα antagonists and ER-deficient mice showed that the

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Fig. 3. Simplified schematic of retinoid and rexinoid signaling pathways that affect NB4 cell maturation, survival and apoptosis. The rexinoid pathway (a), activated by rexinoid (i.e. RXRselective) agonists and mediated by RXR heterodimers (RXR–‘X’) with an unknown heterodimerization partner or RXR homodimers (RXR–RXR), leads, by default, to immediate apoptosis of immature NB4 cells. This pathway is not affected by RARα antagonists. Several signaling options can rescue NB4 cells from rexinoid-induced apoptosis, including RARα and PKA agonists (both of which activate pathways that lead to cell maturation), and as yet uncharacterized serum factors that induce survival. The second default signaling pathway (b) is dependent on RARα agonists, abrogated by RARα antagonists, and leads to cell maturation followed by postmaturation apoptosis. The receptor species involved have not been unequivocally determined and could involve RXR–RARα or RXR–PML–RARα heterodimers or oligomers of PML–RARα [(PML–RARα)x]. For details, see Benoit et al.88, from which this scheme was modified, with permission. Abbreviations: See Glossary.

mitogenic effect of EGF is mediated by ERα (Refs 2,62). As for breast epithelium, tissue recombination experiments demonstrated that stromal ER is required for the mitogenic effect of estrogen on uterine epithelium63. Uterine endometrial apoptosis in ovariectomized pseudopregnant rabbits can be prevented by progestin administration, demonstrating the antiapoptotic effect of the PR in this system64. The protective effect of progestins appears to correlate with a switch of predominant expression from the pro- (Bcl-xS) to the antiapoptotic (Bcl-xL) forms of the Bcl-2 family member, Bcl-x (Ref. 65). In support of an implication of the Bcl-2 family in balancing endometrial cell life and death, the levels of BAX are modest in proliferative human endometrium and increase dramatically in the secretory phase, whereas BCL-2 shows the opposite66. The discovery of a second ER (ERβ)67 led to several studies that attempted to distinguish the roles of the two ERs in the growth-stimulatory effect, particularly on breast cancer. Knockout experiments in mice suggest that ERα, but not ERβ, supports breast development68,69. ERβ, the levels of which decrease during carcinogenesis, could have a protective effect http://tem.trends.com

against the mitogenic action of estrogens70. It has also been suggested that ERβ might affect prostatic growth71. However, little is known about the molecular programs that might be responsible for the proposed growth-regulatory capacity of ERβ. This matter is also complicated by the observation that the two ERs can form heterodimers72,73.

In cultured prostate tumor cells, androgens stimulate the expression of cell cycle genes Cdk2 and Cdk4 and repress the expression of the gene encoding CKI p16INK4a, resulting in increased Cdk activities74. In view of the biphasic, dose-dependent influence on the proliferation of LNCaP cells – low concentrations of androgen stimulate, whereas high concentrations inhibit proliferation and induce strong expression of differentiation markers – it has been proposed that the effect on Cdks represents the low concentration response, which leads to increased pRB phosphorylation, E2F-1 protein levels and E2F activity, and increased production of the E2F target gene products, E2F-1 and cyclin A. At high androgen concentrations, the pRB is largely hypophosphorylated, resulting in low E2F activity and low concentrations of mRNA encoding E2F-1 and cyclin A, and a marked increase of p27KIP1 protein levels75. Two other growth-signaling options have been reported for the AR. Migliaccio et al.76 found that a ligand-induced ternary AR–ER–SRC complex activates the Src–Raf-1–Erk-2 pathway and stimulates PCa cell proliferation, whereas Reutens et al.77 proposed a crosstalk between cyclin D1 and AR, which implies that cyclin D1 binding to the AR might repress ligand-dependent AR activity by directly competing for P/CAF binding. The breast cancer susceptibility gene product BRCA1 has also been implicated in the transcription activation potential of AR (Ref. 78). However, the contribution of these various signaling options to androgen action in (patho)physiological growth of the prostate remains elusive. Although the growth-stimulatory effect of androgens appears to result from effects on cyclin–Cdk function, their absence leads to apoptosis induced by distinct signaling paradigms. In mice, castration leads to apoptotic cell death in the reproductive tract, which is characterized by downregulation of Bcl-2 production and subsequent increased synthesis of the death ligand receptor, Fas. This regression of the reproductive tract is Fas mediated, because it is absent in mice lacking functional Fas (Ref. 79). Castration reduces Cdk2 and Cdk4 expression in rat ventral prostate, and normal levels can be restored with androgen. Although castration has little, if any, effect on the synthesis of CKI, androgen treatment results in the virtual disappearance of p21 (p27 exhibits a transient initial increase) and both Cdk2 and Cdk4 protein levels increase80.

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Retinoic acid receptors (RARα, -β, -γ and RXRα, -β, -γ)

The vitamin A-derived RA regulates essential processes during embryogenesis and cell proliferation and differentiation in the adult34,35. These highly pleiotropic effects result from the combinatorial action of their six receptors (RARα, -β and -γ, and RXRα, -β and -γ), which form RAR–RXR heterodimers, and act as ligand-inducible transcription regulatory factors. RXRs can also form homodimers, in addition to heterodimers with various other NRs (e.g. VDR, TR, PPAR and orphan receptors), thereby modulating multiple signaling pathways (reviewed in Refs 81–83). Although the cancer chemotherapeutic and chemopreventive activity of retinoids is well established, only recently have studies on the molecular basis of APL and the role of retinoids and rexinoids on APL cell growth and differentiation, in addition to studies on the action of atypical retinoids, provided clues about the molecular players involved in growth control. APL is caused by the formation of a PML–RARα fusion protein as the consequence of a translocation involving chromosomes 15 and 17. This protein is believed to act as a dominant–negative factor that represses target gene induction by physiological concentrations of ATRA; this is the result of an increased efficiency of interaction with corepressor complexes (also termed histone deacetylase complex or HDAC) of the fusion protein relative to RAR. Pharmacological doses of ATRA dissociate HDACs from PML–RARα and restore ligand signaling, thus leading to promyelocyte maturation and apoptosis45,84,85. Recently, Altucci et al.86 identified (some of) the growth regulatory pathways by which ATRA triggers cell life and death decisions during, and subsequent to, differentiation along the granulocyte lineage in promyelocytic NB4 cells and the blasts of APL patients. ATRA initially induces a plethora of early and late survival programs. Synthesis of the anti-apoptogenic Bcl-2 family member Bcl-2a1 (also known as Bfl-1 or A1) is detectable after 2 h and increases nearly 200-fold, followed by increased synthesis of the apoptosis inhibitory proteins cIAP-2 and NAIP. In addition, TNF-α signaling switches from the apoptosis to the survival mode, involving NF-ΚB activation, which, in turn, boosts expression of its target gene, Bcl2a1. It is not yet clear whether these antiapoptotic programs are required for, or depend on, promyelocyte maturation. ATRA induction of the death ligand, TRAIL (also called Apo2L) finally causes postmaturation apoptosis of APL blasts86. In addition to the above-described postmaturation apoptosis, a RAR-independent signaling pathway has been described that involves crosstalk between RXR and PKA agonists87. Moreover, a rexinoid-dependent default death pathway that triggers apoptosis of immature promyelocytic cells in the absence of survival factors has been detected recently88. Rexinoid-induced apoptosis displayed all the features http://tem.trends.com

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of programmed cell death and was inhibited by RXR, but not RAR antagonists. Several types of survival signals could block rexinoid-induced apoptosis: RARα agonists switch the cellular response towards differentiation and induce the production of antiapoptotic factors. Activation of the PKA pathway in the presence of rexinoid agonists induces maturation and blocks immature cell apoptosis. Addition of non-retinoid serum factors also blocks cell death without inducing cell differentiation. These findings supported a model (Fig. 3) according to which rexinoids activate in promyelocytic cells a default death pathway on to which several other signaling paradigms converge. However, the molecular details of rexinoid apoptosis and the anti-apoptogenic survival/differentiation signaling mechanisms have yet to be elucidated. A link between the TRAIL death signaling pathway and so-called atypical retinoids has been observed in several solid cancer models. CD437induced apoptosis in lung, prostate and squamous cell carcinoma cells correlates with the increased production of the TRAIL receptors, DR4 and DR5 and/or Fas (Refs 89–91). Although the molecular details of expression of TRAIL receptors induced by CD437 or the similar compound 4-HPR are under scrutiny, the possibility of drug-dependent stimulation of both a tumor-selective death ligand and its receptors is a challenge to pharmacological drug design. Vitamin D3 receptor

Apart from its action on bone growth and mineralization, 1,25-dihydroxycholecalciferol (vitamin D3), exhibits antiproliferative and differentiation-promoting activities towards several malignant cell types, including breast cancer92, prostate cancer93, colorectal adenoma and carcinoma cells94, melanoma cells95 and myeloid leukemia cells96. Although these data established an antiproliferative activity of vitamin D3 and support its potential usefulness as an anticancer agent, serious side effects such as hypercalcemia and soft tissue calcification prevent the use of vitamin D3 in the treatment of cancer. Therefore, major efforts have been devoted to the development of new dissociated vitamin D3 analogues with high antiproliferative and low calcemic activity. Vitamin D3 is antiproliferative, inhibiting cell cycle progression and/or inducing apoptosis. G1 arrest induced by vitamin D3 has been attributed to several molecular events, such as induction of the Cdk inhibitors p21WAF1 and p27KIP1, inhibition of CDK2 activity, hypophosphorylation of pRB, and suppression of E2F activation93,97–99. In particular, p21 transcription is induced by vitamin D3 in a VDR-dependent manner, and a functional vitamin D3 response element has been identified in the promoter of the gene encoding p21. Interestingly, overexpression of the gene encoding p21WAF1

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facilitates differentiation of myeloid cells98. Furthermore, levels of the antiapoptotic molecule BCL-2 have been shown to be reduced after vitamin D3 treatment in some cell lines, including human breast cancer cells100, and BCL2 overexpression blocks vitamin D3-induced apoptosis of human prostate cancer cells in culture101. Although these results suggest a possible role for BCL2 downregulation in vitamin D3-induced apoptosis, it is not clear whether reduction of BCL2 expression is sufficient itself to induce apoptosis. In colon cancer cells, vitamin D3-induced apoptosis is not consistently associated with BCL2 downregulation but rather with increased synthesis of pro-apoptotic BAK (Ref. 94). Finally, it has been reported that the sensitivity of MCF-7 cells to vitamin D3-induced apoptosis does not depend on the production of a functional p53 tumor suppressor protein and does not involve the activation of known caspases102. Vitamin D3 induces the caspase-dependent cleavage of MEK, resulting in a loss of MEK synthesis and Erk1/2 signaling. Moreover, Akt signaling was found to be potently inhibited in cells induced to undergo apoptosis by vitamin D3 (Ref. 103). Furthermore, SMAD3, one of the SMAD proteins downstream in the TGF-β signaling pathway, has been reported to act as coactivator for VDR-dependent transactivation104,105, thus establishing crosstalk between TGF-β and vitamin D3 signaling. One important insight into the molecular signaling events by which vitamin D3 exerts an antiproliferation and differentiation-inducing effect is the identification of the CKI p21WAF1 as a transcriptional target of VDR (Fig. 2); note that p21 is also a RAR target gene106. The development of new vitamin D3 analogs that exert a dissociated action on Ca2+ homeostasis and differentiation might yield cancer-therapeutic and, perhaps, cancerpreventive drugs. Glucocorticoid receptor

Acknowledgements The authors thank Michel Lanotte and colleagues for efficient collaboration and exchange of ideas. LA thanks INSERM and the Italian Government (L. R. n. 41, 1999) for support. Work at the IGBMC was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg and BristolMyers Squibb.

GCs have pronounced effects on metabolism, differentiation, proliferation and apoptosis of certain cells. GRs, in addition to some other NRs, regulate such gene programs not only by directly binding to GREs but also through signal transduction crosstalk – for example, by interfering with AP-1 and NF-κB activities107. Although there is evidence that GC-induced peripheral T-cell apoptosis requires the DNA-binding-dependent component of GR action (and does apparently not involve AP1crosstalk)108,109, apart from the possible implication of some GR-regulated factors (MYC, JUN, ΙκΒ, inositol (1,4,5) trisphosphate receptor), little is known about the molecular signaling cascade(s) by which GCs induce lymphoid cell death110. Disappointingly, even a screen of 7074 genes, of which 163 were regulated by GCs, did not reveal a clear death-signaling pathway and led the authors to speculate that GR upregulation, which is a http://tem.trends.com

prerequisite for apoptosis111, leads to a metabolic disequilibrium that causes cell death112. Summary

NRs have a major impact on growth regulation in embryogenesis, organ development and homeostasis and, in particular, life and death decisions in many cell types. They are prime pharmacological targets because, in addition to their regulatory power, their ligands are small and amenable to combinatorial chemistry. Importantly, ligand design can dissociate receptor-associated function, thus allowing specification of a desired pharmacological effect113,114. To exploit the ability of NRs to regulate growth and pave the way towards novel tools for cancer therapy and cancer prevention, the pathways by which the receptors regulate cell proliferation, survival and death need to be identified. Although little is known about these regulatory pathways, some principles can be noted. • The effects of NRs on growth are cell- or tissuespecific and might even be diametrical in different tissues. It is therefore important to generate socalled SNuRMs, which inhibit mainly tissue-selective activities. This can be achieved by considering the cell specificity of the activation functions AF-1 and AF-2, limiting receptor action by choosing weak agonists, partial or neutral antagonists or inverse agonists, exploiting the p160 coactivator selectivity of NRs (Ref. 115; M. Gehin and H. Gronemeyer, unpublished data from TIF2-deficient mice), or by generating dissociated ligands identified in properly engineered screening systems. • Three types of signaling can be discerned that trigger, directly or indirectly, NR effects on growth. (1) The proliferation-stimulatory action of estrogen in the mammary gland is, at least in part, the result of direct transcription activation of cyclin D1, a component of the cell cycle machinery, by the cognate receptor. The importance of this regulation was recently highlighted by the observation that cyclin D1 ablation protects mice against oncogeneinduced breast cancer116. Conversely, the growthinhibitory action of RAs and vitamin D3 in the myeloid compartment is at least in part the result of transcription activation of CKIs, particularly p21. Other components of the cell cycle machinery can also be NR targets, such as Cdks for AR, but these effects are less well defined. (2) Crosstalk with other signaling pathways can be required for survival, as in the case of rexinoid apoptosis88, or the NR might itself be a mediator of growth factor action, as is the case for ER in the uterus. (3) The third option is the apoptotic machinery, best illustrated by the induction of the death ligand TRAIL by RA in APL (Ref. 86). However, TRAIL receptors are also induced by atypical retinoids, and the BCL-2 family of apoptosis regulators can be involved in growth regulation, as in the case of the survival effect of progesterone on endometrium65.

Review

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