Function of Rara During the Maturation of Neutrophils

Function of RARa during the maturation of neutrophils

Philippe Kastner1 and Susan Chan*,1

1Institut de GeÂneÂtique et de Biologie MoleÂculaire et Cellulaire, CNRS-INSERM-ULP, 1 rue Laurent Fries, BP163, 67404 Illkirch Cedex, C.U. de Strasbourg, France

The retinoic acid receptor a gene is the target of this review (the reader is referred to Chambon, 1996), chromosomal rearrangements in all cases of acute below is a brief summary of the current knowledge promyelocytic leukemia (APL). This recurrent involve- which may be helpful in understanding the role of ment of RARa in the pathogenesis of APL is likely to RAR in the hematopoietic system. re¯ect an important role played by this receptor during the RARa is a nuclear receptor for all-trans (ATRA) dierentiation of immature myeloid cells to neutrophils. and 9-cis (9C-RA) retinoic acid, and belongs to a RARa is a negative regulator of promyelocyte dierentia- family of three closely-related receptors (RARa, b and tion when not complexed with RA, and stimulates this g). RARs are transcriptional regulators which bind to dierentiation when bound to RA. Since RARs are speci®c retinoic acid response elements (RAREs) dispensable for the generation of mature neutrophils, their present in the promoters of their target genes. RARs role thus appears to be to modulatory, rather than do not bind RAREs on their own, but require obligatory, for the control of neutrophil dierentiation. heterodimerization with an RXR (a, b or g) to do so. In vitro, retinoic acid is also a potent inducer of neutrophil RXRs are also nuclear receptors but bind only the 9- cell fate, suggesting that it might play a role in the cis isomer of retinoic acid. Extensive genetic analyses commitment of pluripotent hematopoietic progenitors to both in cell lines and mice have demonstrated that the the neutrophil lineage. Thus, the APL translocations target RAR/RXR heterodimer is the functional unit respon- an important regulator of myeloid cell dierentiation. sible for the transduction of retinoid signals (Kastner et Oncogene (2001) 20, 7178 ± 7185. al., 1997; Chiba et al., 1997; Mascrez et al., 1998). The RAR/RXR complex has two main functions. Keywords: retinoids; granulocyte; dierentiation In the absence of ligand, the heterodimer binds the corepressors SMRT or NCoR, which in turn interacts with histone deacetylase-containing Sin3A complexes, leading to the silencing of target genes (Nagy et al., The RARa gene is the target of chromosomal rearrange- 1997). Binding of ligand provokes the dissociation of ments in all cases of acute promyelocytic leukemia these complexes and promotes the interaction of (APL). These rearrangements lead to the production of RAR/RXR with coactivators that serve to recruit fusion proteins containing a variable N-terminal portion complexes involved in transcriptional activation (for originating from the PML, PLZF, NuMA, nucleophos- review see Minucci and Pelicci, 1999; Glass and min or STAT5b proteins (reviewed in Melnick and Licht, Rosenfeld, 2000). The coactivators target the ligand- 1999), and a ®xed C-terminal portion corresponding to dependent activation function AF-2. How these most of the RARa polypeptide (including the DNA activators function initiating transcription is only binding domain and ligand binding domain). That RARa now becoming clear (Dilworth et al., 2000). Impor- is consistently altered in APL raises the fundamental tantly, the AF-2 functions present in the RAR and question of whether, and how, the rearrangements may RXR moieties can synergize and play a crucial role in perturb a previously unrecognized function of normal mediating retinoid function in vivo, particularly during RARa in controlling the proliferation of promyelocytes development (Mascrez et al., 1998). In addition to and their dierentiation into neutrophils. We will review AF-2, the N-terminal portions of RAR and RXR here the accumulating evidence suggesting that RAR and contain ligand-independent activation functions (AF-1; retinoids may play several roles in the maturation of a Nagpal et al., 1993). With RXRa, genetic studies have hematopoietic stem cell to a mature neutrophil. shown that the AF-1 function participates in mediating RAR/RXR activity in vivo (Mascrez et al., 2001). The AF-1 function of RARa and RARg, which Molecular aspects of RAR function involves the phosphorylation of a conserved serine residue, also appears to be important for RA- Although a detailed analysis of the molecular under- mediated dierentiation of F9 embryonic carcinoma standing of RAR function is not within the scope of cells (Taneja et al., 1997). Each RAR encodes for at least two major isoforms (RARa1 and RARa2 in the case of RARa, Leroy et *Correspondence: P Kaster and S Chan; al., 1991) which dier in their N-terminal A region. E-mail: [email protected] These A regions contribute to the AF-1 function RARa and neutrophil differentiation P Kastner and S Chan 7179 (Nagpal et al., 1992) and may therefore confer distinct neutrophil development may take place in the transcriptional properties to each RAR isoform. absence of any RAR, as granulopoiesis appears to occur normally in RARa7/7RARg7/7 and RARa17/7RARg7/7 double-de®cient fetuses (Kastner RAR and RXR knock-out mice et al., 2001; Labrecque et al., 1998). In vitro, morphologically normal neutrophils can be generated Mice de®cient for each RAR gene have been produced. from the fetal liver cells of RARa7/7RARg7/7 mutants RARa7/7 mice are viable and exhibit few obvious (but apparently not from the bone marrow cells of defects, aside from abnormal spermatogenesis (Lufkin RARa17/7RARg7/7 fetuses, see below). As RARb is et al., 1993). RARb7/7 and RARg7/7 mice also have not expressed in myeloid cells, the neutrophils in these relatively few defects (Lohnes et al., 1993; Ghyselinck mutant animals do not express any RAR. It is et al., 1997). Animals bearing the targeted disruption of presently unknown if RAR function is dispensable single isoforms have also been generated, all of which, during adult granulopoiesis; the answer to this question with the exception of RARg17/7 mice, appear normal will come from the study of mice whose hematopoietic (Lohnes et al., 1993; Lufkin et al., 1993; Subbarayan et system has been reconstituted with RARa7/7RARg7/7 al., 1997). In contrast to the single mutants, double double-mutant fetal liver cells. homozygote animals exhibit a much larger array of Despite the lack of neutrophil defects in RARa7/7 developmental defects, in most cases leading to mice, RARa appears to mediate some of the eects of lethality, either in utero (the majority of exogenous retinoids on myeloid cells (Kastner et al., RARa7/7RARg7/7 mutants die during fetal develop- 2001). (i) RARa7/7 bone marrow cells produce normal ment) or shortly after birth (RARa7/7/RARb7/7 and numbers of colonies when cultured in the presence of RARb7/7RARg7/7 mutants; Lohnes et al., 1994; 9C-RA (with Epo, I1-3, IL-6 and SCF) while the Mendelsohn et al., 1994). Together, the defects in number of WT colonies is reduced by twofold. (ii) In these mutants recapitulate the complete spectrum of these same assays, the addition of 9C-RA also does not the fetal vitamin A de®ciency syndrome established by induce the expected reduction in colony size. (iii) Warkany and coworkers more than half a century ago Exogenous RA does not enhance granulocyte dier- (Kastner et al., 1995 for review and refs). However, entiation in RARa7/7 mice, both in vitro and in vivo. none of the RAR double mutants show the early These observations therefore suggest that the retinoid embryonic defects that occur when retinoic acid signal is largely transduced through RARa in myeloid synthesis is prevented, such as that seen after the cells. Hence, the presence of a normal neutrophil inactivation of the crucial retinaldehyde dehydrogenase population in RARa7/7 mice further suggests that RALDH2 gene (Niederreither et al., 1999). These retinoic acid is not essential for neutrophil dierentia- results suggest that there is a large degree of functional tion in adult mice. redundancy during development. All three RXRs have also been knocked-out. RXRa7/7 mice die during fetal development of cardiac A role of RA in the commitment of hematopoietic dysfunction (Kastner et al., 1994; Sucov et al., 1994), progenitors to the neutrophil lineage? but RXRb7/7 and RXRg7/7 animals are viable and mostly normal (Kastner et al., 1996; Krezel et al., RA has the capacity to drive pluripotent hematopoietic 1996). The production of compound RXR/RAR progenitors into the granulocyte lineage, as seen in mutants (Kastner et al., 1994, 1997) has shown that assays using the multipotent cell lines FDCPmix A4 there is a strong synergy between RXRa and RAR (Tsai and Collins, 1993) and EML (a line that mutations, as RXRa/RAR double mutants recapitulate overexpresses a dominant-negative RARa;Tsaiet al., most of the defects observed in RAR-only double 1994). RA can also suppress the formation of erythroid mutants. Recent work using spatio-temporally con- and macrophage colonies, and induce a concomitant trolled mutagenesis of RXRa has also demonstrated an increase in the number of neutrophil colonies, from important role for this receptor in several adult tissues, primary human progenitors (Gratas et al., 1993; van including skin, liver and adipose tissue (Imai et al., Bockstaele et al., 1993; Labbaye et al., 1994; Tocci et 2001a,b; Li et al., 2000). RXRb and RXRg appear to al., 1996). Importantly, Tocci et al. (1996) have shown be more limited in their function than RXRa and, to that this eect directly results from a cell-fate date, have been implicated in spermatogenesis (Kastner conversion by RA. In their experiments, single et al., 1996) and dopamine signaling in the striatum CD34+ fetal liver cells were plated in methylcellulose- (Krezel et al., 1998). containing medium in the presence of Epo, IL3 and SCF; 2 days later, individual daughter cells were replated in a similar medium in the presence or absence RARs are dispensable for granulopoiesis of RA. Invariably, progenitors that normally gave rise to erythroid or macrophage colonies in the absence of RARa-de®cient mice have normal numbers of neu- RA dierentiated into granulocytes in the presence of trophils in their peripheral blood and hematopoietic all-trans RA. Similarly, Paul et al. (1995) showed that organs (Kastner et al., 2001), demonstrating that RARa cells from the eosinophil precursor cell line is not required for neutrophil dierentiation. In fact, AML14.3D10 are redirected to become neutrophils

Oncogene RARa and neutrophil differentiation P Kastner and S Chan 7180 by RA. These results indicate that RA can reprogram ganded RARa can antagonize dierentiation. Over- hematopoietic progenitors already engaged in a non- expression of WT RARa1 in immature hematopoietic granulocyte lineage to the neutrophil fate. cells readily blocks cytokine-induced neutrophil dier- It is unclear whether RA acts endogenously as a entiation at the promyelocyte stage in the absence of speci®cation factor for the neutrophil lineage. To date, exogenous RA (Onodera et al., 1995; Du et al., 1999). no evidence for such a role has been described. In The inhibition observed here is reminiscent of the contrast, several observations suggest otherwise: (i) block seen when a C-terminally-truncated `dominant- vitamin A-de®ciency leads to an increased number of negative' RARa (RARa403) lacking the ligand-depen- neutrophils in mice (Kuwata et al., 2000; Kastner et al., dent AF-2 function is overexpressed in immature 2001), an observation incompatible with a major role myeloid cells. Collins and coworkers have shown that for retinoids in specifying neutrophil fate; (ii) similar overexpression of RARa403 can immortalize hemato- numbers of granulocyte, macrophage and mixed poietic progenitors at the promyelocyte stage (leading granulocyte/macrophage colonies are grown from to the generation of the MPRO cell line; Tsai et al., progenitors of normal or VAD mice (Kuwata et al., 1994) and at the multipotent progenitor stage (leading 2000), (iii) competitive reconstitution experiments with to the generation of the EML cell line; Tsai et al., 50 : 50 mixes of RARa7/7 and WT bone marrow cells 1994). The interpretation of these results has generally do not reveal a disadvantage of RARa7/7 cells in pointed to a role for RA in neutrophil dierentiation. contributing to the neutrophil lineage (our unpublished However, it is equally possible that the dominant- results). Nonetheless, the potent activity of RA in negative RARa described above functions like WT inducing neutrophil commitment in vitro remains RARa, although the activity of the former cannot be intriguing, and further research is required to assess alleviated by the low levels of retinoids present in the its possible relevance in vivo. culture medium. In addition, ex vivo neutrophil dierentiation occurs faster for BM cells derived from RARa7/7 compared to WT mice (Kastner et al., 2001). RARa is a bidrectional regulator of neutrophil Thus, loss of RARa removes an activity that maintains dierentiation myeloid cells at an immature stage. Collectively, these observations indicate that RARa is a negative RA has also been shown to promote the dierentiation regulator of granulocyte dierentiation. Of special of normal, as well as leukemic, immature myeloid cells signi®cance, treatment of WT mice with a saturating already engaged in the neutrophil lineage. This was dose of ATRA leads to a decrease in the size of the ®rst demonstrated for the HL60 promyelocytic cell line most immature (Mac-1+Gr-17/lo) and an increase in (Breitman et al., 1980) and later extended to primary that of the intermediate (Mac-1+Gr-1lo/+) granulocyte promyelocytes (Gratas et al., 1993). Moreover, the populations (Kastner et al., 2001), suggesting that not addition of a RAR antagonist (BMS493) to cultures of all of the RAR molecules are liganded under normal bone marrow cells in the presence of G-CSF and SCF circumstances in vivo, and are therefore free to act as results in a slowdown of neutrophil dierentiation negative regulators. (Kastner et al., 2001), suggesting that serum retinoids Thus, RARa is never a neutral bystander during normally play a role in the positive control of this neutrophil dierentiation: it inhibits dierentiation dierentiation. The role of endogenous retinoids in when not bound to retinoic acid and promotes controlling neutrophil dierentiation in vivo has been dierentiation when bound to RA. It is therefore clear more elusive. The best evidence has come from data why ligand de®ciency (as in VAD) or the use of RAR looking at the short-term eects of BMS493 in vivo antagonists is not functionally equivalent to lack of the which, when systemically administered to mice for 3 RAR itself. In the former, only the ligand-dependent days, leads to an accumulation of immature granulo- dierentiating activity is abolished while the blocking cytes in the bone marrow, as assessed by Mac-1 and activity is maintained and perhaps even enhanced. This Gr-1 expression, and cell morphology (Kastner et al., model predicts that a functional RAR is required for 2001). A similar increase in the proportion of immature the full eects of vitamin A de®ciency on dierentia- neutrophils was seen in vitamin A-de®cient mice tion, a hypothesis that we are currently testing by (Kastner et al., 2001). Even though VAD is long to generating VAD RARa7/7 mice. Interestingly, a establish, often leading to secondary phenotypes, the similar scenario has been observed with thyroid similarity between the eects of VAD and BMS493 hormone receptor b (TRb). Mice de®cient for TRb administration strongly suggests that retinoids are do not exhibit signs of hypothyroidism, while animals involved in controlling the dierentiation of neutrophil carrying a point mutation speci®cally abbrogating the under physiological steady state settings. ability of TRb to bind thyroid hormone do (Hashimo- If indeed RA plays an important role in controlling to et al., 2001). Together, these results show that neutrophil dierentiation, it might seem surprising, nuclear receptor knock-outs may not always be the even contradictory, that this dierentiation occurs best models for studying the role of the cognate ligand, rather normally in RARa7/7 mice. This paradox may especially when the ligand functions in part to relieve be explained by the observation that unliganded RARa the repression exerted by the unliganded receptor. is an active mediator of the eects of RA de®ciency. Improved mutations that knock-out the speci®c ability Several experiments have demonstrated that unli- to carry out ligand-dependent and -independent

Oncogene RARa and neutrophil differentiation P Kastner and S Chan 7181 functions are required to understand the physiological crucial and unique role, which cannot be substituted roles of these receptors. for by RARg, during the parietal dierentiation produced by the combined action of RA and cAMP (Taneja et al., 1997). Furthermore, RARa and RARg Functional speci®city for RARa in granulopoiesis? are also markedly dierent in their ability to induce transcription of certain target genes in F9 cells (Chiba Although both RARa and RARg are expressed in et al., 1997). granulocytes (Labrecque et al., 1998), our data suggest that RARa7/7 cells lack both the negative and positive RAR-dependent activities in this lineage. RARa7/7 Do RARa1 and RARa2 play unique roles during cells are resistant to RA and to the RAR antagonist granulocyte dierentiation? BMS493. RARa therefore seems to be the major RAR operating during granulocyte dierentiation. Is this due Both RARa1 and RARa2 are expressed in cells of the to a functional speci®city of this particular receptor or neutrophil lineage (Labrecque et al., 1998; A Zelent, does it merely re¯ect its relative abundance in myeloid personal communication), although it is unclear what cells? their speci®c roles are in these cells. One clue may lie in There is evidence that RARa is functionally speci®c. the con¯icting data produced from our laboratory Du et al. (1999) have shown, using retroviral delivery (Kastner et al., 2001) and those of Labrecque et al. of various RARs into immature hematopoietic pro- (1998). We reported that myeloid progenitors from genitors (i.e. lineage-depleted bone marrow cells from RARa7/7 adult bone marrow or RARa7/7RARg7/7 5-¯uorouracil-treated mice), that overexpression of fetal liver are not blocked in their dierentiation (and RARa, but not RARb or RARg, inhibits granulocyte in fact dierentiate with a faster kinetics than WT dierentiation of transduced cells cultured in the cells). In contrast, Labrecque et al. (1998) showed that presence of IL-3, IL-6, GM-CSF and SCF. In addition, granulocyte progenitors from RARa17/7RARg7/7 only RARa is able to induce proliferation of primary 18.5 d.p.c. fetal bone marrow are mostly blocked at myeloid progenitors (as measured by the potential to the myelocyte/metamyelocyte stage of dierentiation form colonies after three passages in methylcellulose when cultured with SCF, IL3 and Epo; further culture). These observations show that only unliganded dierentiation was induced by the addition of RA. RARa can inhibit granulocyte dierentiation. Since RARa2 is presumably still expressed in RARa-speci®city appears to extend to retinoic acid- RARa17/7RARg7/7 myeloid cells, RARa1 and RARa2 mediated eects as well. Mehta and coworkers (Drach might perform distinct functions in promyelocytes. The et al., 1994; Mehta et al., 1997) studied RA-dependent results from Labrecque et al. (1998) suggest that RARa2 transcription of the CD38 gene in RAR-transduced is a more potent inhibitor of dierentiation than HL60R cells (which are resistant to retinoids due to the RARa1, but that RARa1 may be better at mediating expression of a truncated dominant-negative RARa); RA-induced dierentiation. It might therefore be only transfected RARa, not RARb or g, was able to interesting to investigate whether the two RARa rescue RA-induced CD38 transcription. In addition, isoforms dierentially regulate dierentiation and Du et al. (1999) have shown that immature bone proliferation in the presence and absence of ligand. marrow progenitors transduced with high amounts of RARa and RARg (but not RARb) are highly sensitive to the antiproliferative activity of RA. Together, these Apoptosis observations indicate that RARa is functionally the best RAR to mediate both the ligand-dependent and - Retinoids have been implicated in the apoptotic independent activities of RARs during neutrophil process of myeloid cell lines like HL60 (Ueno et al., maturation. 1998; Monczak et al., 1997). They may also control the The apparent high degree of speci®city in the rate of granulocyte apoptosis in vivo; Kuwata et al. granulocyte lineage stands in sharp contrast to the (2000) have shown that there is a clear reduction in the large functional redundancy observed from extensive rate of apoptosis of granulocytes from vitamin A- genetic studies of RAR function during development, de®cient mice. However, as apoptosis is the endpoint where most RAR activities require the combined of neutrophil maturation, the retinoid-triggered apop- removal of two receptors to be abbrogated (reviewed totic response might just correspond to one facet of the in Kastner et al., 1995). These observations have dierentiation program controlled by retinoids. Some suggested that RARs may perform broadly similar studies, using myeloid cell lines, have shown, however, functions and can readily substitute for each other, that the dierentiation and apoptotic responses elicited provided their expression levels are adequate. A similar by RA can be separated. In the leukemic cell line PLB- redundancy has been demonstrated in F9 embryonic 985, Monczak et al. (1997) showed that RA provokes carcinoma cells (Rochette-Egly and Chambon, 2001 for apoptosis without triggering dierentiation, and that review). Interestingly, some functional speci®city is activation of both RAR and RXR was required. observed in this system as well. Although RARg (the Likewise, activation of RXR leads to apoptosis in the most abundantly expressed RAR in F9 cells) is absence of dierentiation in HL60R cells (Mehta et al., required for all retinoid functions, RARa plays a 1996). Interestingly, these studies have also pointed to

Oncogene RARa and neutrophil differentiation P Kastner and S Chan 7182 a critical role for RXR activation, in agreement with CAAT/enhancer binding proteins (C/EBPs) data from Boehm et al. (1995) who reported that RXR-selective agonists trigger apoptosis in human Perhaps one of the most promising candidates is myeloid cell lines. C/EBPe. C/EBPe transcription is rapidly induced by At the mechanistic level, it was recently shown that retinoic acid in the absence of protein synthesis in RA induction of TRAIL, a cell surface ligand for HL60 and NB4 cells (Chih et al., 1997; Liu et al., death receptors, is reponsible for the RA-induced 2000), and a functional RARE has been identi®ed in apoptosis of NB4 and PLB-985 cells (Altucci et al., the C/EBPe promoter (Park et al., 1999). C/EBPe is 2001). As TRAIL induction is a rather late event therefore likely to be a direct RAR target gene in occurring only after 48 h of RA stimulation, it is granulocytes. In addition, C/EBPe is a critical regulator unlikely that TRAIL is directly induced by the RAR/ of terminal granulocyte dierentiation (Yamanaka et RXR complex. Curiously, earlier events following RA al., 1997). However, C/EBPe is expressed normally in stimulation are the induction of several anti-apoptotic RARa7/7 mutants (our unpublished results), a fact pathways, including the strong induction of the BCL2 which is not too surprising given the normal neutrophil homolog BCL2A1, and induction of NFkB activity dierentiation that occurs in these mice. It is possible (Altucci et al., 2001; Liu et al., 2000). Thus, even that C/EBPe transcription is repressed by unliganded though the endpoint will be death, retinoids may RARa, an interesting possiblity that deserves further modulate survival and death of myeloid cells in a investigation. Since C/EBPe is required at a rather late temporally complex manner. stage in the dierentiation (around the metamyelocyte stage), this target gene cannot account for the block at the promyelocyte stage that results from overexpressed Stem cell maintenance RARa. Interestingly, another member of the CAAT/ enhancer binding protein family, GADD153 (also Purton et al. (1999, 2000) have recently shown that RA named CHOP) has been found to be induced by RA is potent in maintaining the `stem cell-ness' of murine in a cycloheximide-insensitive manner in NB4 cells (Liu Lin7c-kit+Sca-1+ cells (a population highly enriched et al., 2000). These results point to genes encoding C/ for hematopoietic stem cells (HSC)) in vitro, mainly by EBP factors as important RAR target genes during blocking their maturation. Cells cultured in the granulocyte dierentiation. presence of ATRA for 14 days retain their short- and long-term capacity to repopulate lethally-irradiated c-myc recipients, while those cultured without RA do not. Moreover, adding an RAR antagonist to the cultures As c-myc expression is down-regulated by RA in arrests all repopulating activity, its primary eect being NB4 cells in a cycloheximide-independent fashion to induce maturation, as the number of more mature (Liu et al., 2000), it may therefore be a primary CFU-S cells increased in these cultures. Studies by RAR target gene in early myeloid cells. Interestingly, Zauli et al. (1995) have suggested that RA also acts the activities mediated by RARa is reminiscent of eciently to promote stem cell survival. Using puri®ed that exerted by the Myc/Mad system. A switch from CD34+ human cord blood cells, these authors showed the binding of the Myc/Max to the Mad/Max that ATRA (at both low (10710 M) or high (1076 M) heterodimer on target genes has been shown to be doses) can prevent apoptosis induced by serum or critical in a cell's decision to proliferate or dier- growth factor starvation. It is currently unknown entiate, particularly in the myeloid lineage (Ayer and which RARs are involved in these processes. Interest- Eisenman, 1993; Hurlin et al., 1995; Cultraro et al., ingly, the data presented by the studies suggest that 1997; Foley et al., 1998). In Mad17/7 mice, RA inhibits maturation in early pluripotent stem cells granulocyte dierentiation proceeds with slower ki- in a manner opposite to that observed at later stages netics than in WT animals, opposite to that seen in where it acts to promote dierentiation and apoptosis. RARa7/7 mutants (Foley et al., 1998). A direct role for An important question is whether retinoic acid plays a c-myc in mediating RA function has been shown in F9 physiological role in controlling HSC maturation; cells where c-myc transcription is down-regulated by quanti®cation of these cells in RAR knock-out mice, RA. This down-regulation appears to be essential for as well as in VAD mice, will be required to assess the dierentiation, as inhibition of c-myc expression by role of retinoid signaling in the maintenance of the antisense oligonucleotides results in the same eect HSC pool in vivo. (Griep and Westphal, 1988).

p21 Target genes and mechanisms The cyclin-dependent kinase inhibitor p21 (WAF/ Understanding how RARa controls granulopoiesis at CIP1) is a well-studied target gene of p53 and is the molecular level will require identi®cation of critical important in mediating cell cycle arrest. In the target genes regulated both by the unliganded and monocyte cell line U937, p21 is induced by vitamin liganded receptor. Several candidate target genes have D3; this induction is essential for vitamin D3-induced been identi®ed. dierentiation (Liu et al., 1996b). p21 is probably

Oncogene RARa and neutrophil differentiation P Kastner and S Chan 7183 also a direct target gene of RA, and is induced by overcoming the dierentiation block exerted by these retinoids in U937 (Liu et al., 1996a), HL60 (Jiang et proteins. Second, the fusion proteins are usually al., 1994) and NB4 cells (Liu et al., 2000). In NB4 expressed at higher levels than WT RARa in APL cells, p21 induction does not require protein synthesis cells (Kastner et al., 1992; Pandol® et al., 1992), a (Liu et al., 2000). Furthermore a functional RARE factor that probably contributes to their dominant has been found in the p21 promoter (Liu et al., activity. Since RARa is the only RAR that can mediate 1996a). Given the proposed role of p21 in vitamin all the ligand-independent and -dependent activities D3-induced monocytic dierentiation, it is tempting during granulocyte dierentiation, it is likely that the to speculate that it might be equally important in APL fusion proteins eectively antagonize dierentia- mediating RA activity during neutrophil dierentiation by amplifying a normal function of RARa as a tion. negative regulator. Understanding how RARa controls granulocyte dierentiation may also help in understanding how Other genes RARs, or related nuclear receptors, control dierentia- Other genes have been identi®ed to be regulated by tion in other cellular systems. An altered thyroid RA in a number of myeloid cell lines. Liu et al. (2000) hormone receptor a (TRa), v-erbA, is an oncogene have come up with a large number of genes which are carried by the avian erythroblastosis virus; it functions regulated by RA, using the combined approaches of to inhibit the dierentiation of erythroid progenitors suppression-subtractive hybridization, dierential dis- (Metz and Graf, 1992 for review). Overexpression of play and microarrays. Other types of studies have also WT TRa inhibits the dierentiation of erythroblasts identi®ed target genes (Matikainen et al., 1996; (Bauer et al., 1998). Thyroid hormone may also be Lawson et al., 1999; for review, see Lawson and implicated in erythroid dierentiation as it has been Berliner, 1999). Interestingly, a large number of genes identi®ed as the serum component required for encode for proteins of regulatory pathways common spontaneous dierentiation of an erythroleukemia cell to all cells. However, since many of them were line (Dinnen et al., 1994). However, TRa7/7 mice identi®ed in cells expressing abnormal RARs (NB4, appear to have normal erythropoiesis (Fraichard et al., MPRO), caution should be exercised with respect to 1997), similar to the normal granulopoiesis seen in their relevance in the control of granulopoiesis by RARa7/7 mice. These observations suggest that TRa endogenous RARs. Furthermore, all of the target might play a similar dual role in the control of genes identi®ed to date are regulated by RA. To our erythroid dierentiation, for which the negative knowledge, no gene has been shown to be repressed regulatory part may be enhanced by v-erbA. PPARg, by the unliganded receptor. In fact, data obtained a nuclear receptor closely related to RAR, may play a from the study of F9 cells de®cient for RAR/RXR similar role in adipocyte dierentiation (for review see have not shown an upregulation of target genes in the Fajas et al., 1998); similarities in this system include a absence of RA (Chiba et al., 1997). Repression by dynamic regulation of the Myc/Max and Mad/Max unliganded RAR may therefore aect only a small complexes (Pulverer et al., 2000) and the involvement fraction of RAR target genes. Nonetheless the of C/EBP proteins (Lane et al., 1999). Finally, identi®cation of these genes is critical to under- oligodendrocyte dierentiation (involving RAR and standing how RARa regulates granulopoiesis. TR, see Rodriguez-Pena, 1999 for review), and monocyte dierentiation (involving VDR) may involve the same mechanisms. Nuclear receptors may thus be Implications for APL and beyond part of a conserved molecular framework adopted by dierent cell types to modulate their dierentiation. The present knowledge of the role of endogenous Unraveling how RARa controls granulocyte dieren- RARs in the control of granulocyte dierentiation may tiation at the molecular level is likely to have a broad shed some light on the function of translocated RARa signi®cance. in APL. There is now solid evidence that the fusion proteins generated in APL possess strong repressive properties in the absence of RA (He et al., 1998; Hong et al., 1997; Lin et al., 1998; Grignani et al., 1998; Guidez et al., 1998), and that these properties are crucial for the leukemic phenotype. Since repression by RARa plays a normal function in granulopoiesis, it appears that the fusion proteins merely enhance this physiological activity. This enhancement may be brought about by two distinct mechanisms. First, the Acknowledgments The work of our laboratory was supported by the fusion proteins are intrinsically more potent at Association pour la Recherche sur le Cancer, the Centre repression than WT RARa, as they are either National pour la Recherche Scienti®que, the Institut completely refractory to (PLZF-RAR), or require National de la SanteÂ et de la Recherche MeÂ dicale, the higher concentrations of (PML-RAR), RA, thus HoÃ pital Universitaire de Strasbourg, the ColleÁ ge de France making physiological levels of RA ineective at and Bristol-Myers Squibb.

Oncogene RARa and neutrophil differentiation P Kastner and S Chan 7184 References

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Oncogene