Indi an Jou rnal of Experimen tal Biology Vol. 40, August 2002, pp. 874-881

Review Article

Retinoic acid- A player that rules the game of life and death in neutrophils

Kapil Mehta

Division of Cancer Med ici ne, The Univcrsity of Tcxas M. D. Andcrson Cancer Center, 151 S Holcombe Blvd. , Houston, TX.

FAX (7 13) 745-4167; e-mail: kmchta@mdanderson.org

Neutrophil s arc the most prevalent white blood cell s in the circulation. They represent the first line of defense aga inst in vad ing microorgani sms und have been implicated in the pathogenesis of a number of human diseases. In response to vari­ous factors , the pluripotefl[ stem cell s in bone marrow differenti ate into mature neutrophils, enter the blood stream, and di e with in 24 hr via apoptosis. Numerous defects can occur during the process of neutrophil s' differentiation that can mani fest in the form of a variety of clinica l di sorders. Retinoids (Vitamin A and analogues), in general, and all-trans ret inoic acid (tRA), in particular, playa critical role during differentiation of neutrophil s. tRA can directly modulate gene expression via bindi ng to its nuclear receptors, which in turn , can acti vate transcription of genes that are essential for differentiat ion of im­mature cells to neutrophil s. In volvement of rctinoi c acid receptor in pathogenesis of acute promyeloey tic leukemia (APL) , reflects an important role played by thi s receptor in differentiation of immature myeloid cell s to neu trophil s. This review summari zes evidence on in vo lve ment of ret inoie ac id-med iated events in differentiation process of neutrophil s and their subsequcnt apoptosis.

Neutrophils playa major role in host defense mecha­ni sms against microorganisms and in acute infl amma­tion . On an average, about ten million neutrophils are produced and released every minute in the blood stream of an adult human beingl. However, the net number remains constant, suggesting that an equal number of neutrophils must be deleted constantly in order to maintain normal homeostasis. A seri es of events that are orchestered by a variety of factors in the bone marrow lead to the differentiation of primi­tive hematopoietic precursor stem cells into termi­nally differentiated neutrophils2

. Once mature, they enter the blood circulation or the tissues where they functi on as effecter cells in host-defense mechanisms and after relatively short periods they are eliminated from the body through a geneticall y regulated mecha­nism called apoptos is, or programmed cell death 3

.5

. A critical balance between life span of these cell s and their regulated death is important for normal homeo­stasis .

Certain molecu lar flaws in differentiation process can manifest in the form of clinical di sorders. The observations concerning these disorders have led to understanding the basic mechanisms involved in mye-

Abbrcviations uscd : APL-acutc promye loeytic Icukemia; ERGs-early response genes; HDAC-hisotone deaeety lase ; RAR - retinoie acid receptor; RARE - retinoic ac id response element ; RXR-retinoid X receptor; tRA-trans retinoic acid; TGase- transgl utami nase.

lopoiesis. Particularly, studies of acute promyelocytic leukemia CAPL) , a SUbtype of acute myeloid leukemia that is caused by a block in differentiation at the pro­myelocytic stage, have revealed a crucial role for tRA in the process of neutrophil differen tiation 6

. Similarly, the human myeloblast leukemia cell line, HL-60, has provided an excellent in vitro model fo r studying the regulatory mechani sms of myeloid cell di ffe rentia­tion. This cell line can be induced with tRA to differ­entiate toward cells sharing several phenotypes of mature neutrophi Is 7. tRA-differentiated HL-60 cells, like normal neutrophil s, have a limited in vitro life span and undergo spontaneous apoptosis 8

. Thus, HL-60 cells are a good model, not only to investigate the molecular events linked with neutrophils di fferentia­tion, but also to study the apoptotic processes in­volved in programmed cell death . An important fea­ture of neutrophil physiology is their rapid and spon­taneous programming for cell death, a major mecha­ni sm that maintains netrophi I homeostasis in vivo. It is well-known that apoptosis play an important role in clearing neutrophil s from inflamed tissue, which is critica l for limiting the injury to inflamed tissue and subsequent resolution of inflammation . However, the molecu lar mechanisms controlling neutrophil s apop­tosis remain largely unknown . This review summa­rizes the cu rrent information on molecu lar events that lead to tRA-induced differentiation and apoptosis III

myeloid cells.

MEHTA: RETI OIC ACID IN NEUTROPHIL DIFFERENTIATIO 875

tRA as transcription regulators As pointed out earlier, tRA plays an important role

in neutrophil differentiation pathway by regulating the cell growth, differentiation and cell death functions. The ability of retinoids to influence these complex biological processes resides in their abi lity to modu­late the ex pression of multiple genes (Tab le I). Some of these genes are expressed immediately following treatment with tRA, while the expression of other genes is delayed . Net effect of tRA-induced gene ex­pression is the inhibition of cell growth and induction of differentiation and apoptosis. tRA-induced expres­sion of target genes is mediated through nuclear re­ceptors that belong to a superfam ily of li gand­inducible transcription factors including teroid, vita­min 0 , thyroid hormone, and peroxisome proliferator­act i vated receptor. Spec i fically, retinoids can bind and ac tivate two major types of nuclear receptors, the reti­noic acid receptors (RARs) and retinoid X receptors (RXRs). Both, RARs and RXRs consist of three sub­types (a-, ~- and y-) that are encoded by separate genes and have been discussed in greater detail in several recent reviews9

- " .

Each of RAR and RXR SUbtypes contains six dis­tinct domains referred to as, A through F, and each of these domains has di verse functions (Fig. I A). A and

A B c

B domains are located at the amino terminal and con­tain isoform specific, ligand-independent transactiva­tion functions , AF- I. The 0 A-binding domain (do­main C) is highly conserved among different retinoid receptor isoforms and binds to specific upstream nu­cleotide sequences (referred to as retinoic acid re­sponse element or RARE), located in the promoter region of the target genes. (Fig. I B). Ligands (reti­no ids) bind to the ligand-binding domain (LBO) or E domain at C-terminus of the receptors that contain sequences involved in dimerization of the receptors, ligand dependent transactivation (AF-2), and translo­cation to the nucleus . Functions for 0 and F domains have not been clearly defined . RARs and RXRs main ly act as heterodimers to affect gene transcription after binding to RAREs in the promoter region of tar­get genes (Fig. I B). RARs can be transcriptionall y activated by binding to either tRA or 9-cis-RA, how­ever RXRs can be activated only by 9-cis-RA (Fig. IC) . Upon ligand binding, activated nuclear receptors that bind to RAREs found in the upstream sequences (promoters) of RA responsive genes and lead to tran­scription of the target genes.

Complex diversity and pleiotropic effects in tRA signaling pathway is provided not only due to ex is­tence of multiple isoforms of RA receptors but also as

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Fig. I-Structure and functions of retinoid receptors. (A)-Schematic representation of retinoid receptor protein depicting various func­tional domains. (B)-A molecular model for retinoid ac ti on. The liganded RAR form s heterodimer with RXR, binds to specific regu la­tory sequences (RARE) in the promoter region of a targel gene A. Transactivation of gene A is a primary event of retinoid acti on. In an another scenario, the product of gene A can itself activate the transcription of a second gene B. Transactivation of gene B represents a secondary act ion of retinoid since its transcription requi res Ihe sy nthesis of protein A. This cascade of gene events leads 10 secondary and terti ary events that eventually produce a phenotype that is characteristic of relinoid action. (C)- Natural isoforms or retinoid receptors (RARs and RXRs) and thcir physiological li gands are li sted .

876 IND IAN J EXP 1310L, AUGUST 2002

a result of combinations of RAR-RXR heterod imers and presence of different ligands. IRA treatment, for

example, can induce express ion of RARa and RAR~ genes, suggesting that tR A can modulate its own re­ceptors, in add ition to differentiation-related genes. A va i labi Ii ty of reti noid I igands to its cognate recep­tors can be regu lated by the level of certain non­receptor proteins, such as cy top lasmi c retinoic acid binding proteins (C RABP) and heat shock proteins. Formation of RXRJRAR heterod imer is required for high-affinity binding to RAREs that is critica l for subsequent retinoid-induced transcripti on of target

Table 1- Modu lation of gene ex pression by tRA during neut ro­phil different iation

Target gene

IlIdllced expressioll

CD38

C/EB Pa CI P/W AF/p2 1 Transglutaminase CDllb Myeloperox idase TRA IL PU.I RAR~ STATs

DOlVllreg ll la led expressioll

c-myc bcl2

RARE in the Function promoter

Yes

Yes Yes Yes Yes Yes ? Yes Yes Yes

? ?

Differenti ation

Different iation Cell cyc le inhib itor Apoptosis Adhesion Pro-innam matory Apoptos is DifTerenti at ion Differen tiati on Transcripti on factors

Cel l grow th Apoptosis

DR5-RlIRE REPRESSED

genes. M ost RAREs have been identified in the regu­latory regions of genes whose transcripti on is induced by retino ids (T ab le I ). These cognate response ele­ments consist of direct repeats (DR) of 2 core motifs in the seq uence AGGTCA(X)nAGGTCA. The most common spac ing observed in RAREs is S bp (DRS); however, RAR Es containing these or similar motifs separated by I (DR 1) or 2 bp (DR2) arc also common.

Curren t view on tRA-med iated regulation of target gene ex press ion is that the unliganded RA R-RXR heterodimer constit utes a hi gh affin i ty receptor and can bind to the RARE in upstream seq uence of the target gene. Nevertheless, in the absence of li gand or in the presence of some antagon ists, the receptor's target gene stays in repressed form. This is due to the recruitment of histone deacetylase (HDAC) conta in ­ing complexes that are tethered th rough corepressors, such as the silencing mediator for retinoid and thyroid hormone receptors (SMRT) or the nuc lear receptor co-repressor (NCoR) to a non-liganded RAR-RXR dimmer (Fig. 2A). Thi s process results in histone deacetyl ation, chromatin compaction and silencing of the target-gene. Current understandi ng of RAR­mediated gene transactivation is illustrated in Figure 2B. The binding of RAR ligand (e.g. tRA) destabi ­li zes the corepressor binding and induces all os teric changes in the ligand-b inding domain. Thi s results in the release of repressor-protein complex and subse­quent recruitment of transcriptional coact ivator com­plexes (containing CREB-binding protein [CBP]/p300 or other co-activators) that con tain or recruit histone

TnANSACTlVATIO:\

Fig. 2-Schemati c representation of the basal repression and retinoid- induced transcripti onal regul at ion of the target gene(s). (A)-Nucleosomes consist of DNA (bl ack line) wrapped around hi stone octo illers (black circles). The binding of unli gated RAR/RXR heterod illler to RAR E (DRS-RAR E) in promoter region of the target gene, results in the recruitment of a transcrip ti onal repressor com­plex that compri ses N-CoR, mSin3a, SMRT, and a hi stone deacety lase (HDAC- I). l-mAC-1 ac ti vity helps to suppress the tr:mscri ptiOllJl ac ti vity. (B) - Addition of tR A results in a conformati onal change in RARlRXR heterodimer that leads to release of repressor complex and recru itment of a transcriptional-acti vator complex that has histone acetyltransferase (HAT) ac ti vity. Hi stone acetyla tion (Ac) of DNA rel axes chromatin and creates a permi ssive state for promoter activation.

MEHTA : RETINO IC ACID I NEUTROPHIL DIFFERE T IATION 877

acetyltransferase (H AT)12-1.!. Thus, binding of li gand to RARJRXR dimers converts it from a transcripti ona l repressor (Fig. 2A) to a transcripti onal activator (Fig. 2B) . Evidently, modulation of transcriptional ac ti vit y in vo lves close links between acety lation/deacety lation and stabi 1 iza ti on/destabil izat ion of repress i ve chromo­somes.

Retinoid receptors in neutJ'ophil differentiation and apoptosis

In hematopoietic cells, apoptosis appears to be an essential component of terminal differentiati on. Under steady-state condit ions, large production of neutro­phil s is counterbalanced by their eliminati on in the ti ssues without eliciting an inflammatory response . Aging neutrophil s typi ca ll y die by apoptosis and are subsequently cleared by loca l macrophages. Many cytok ines, such as GM-CSF, G-CSF, interleukin -3, and interleukin-S, can increase the circul atin g time of mature granulocytes from 8 to 48 hr, probabl y by de­lay ing their apoptosisl 5

. D espite many such observa­ti ons, little information is ava ilab le on the bio logical signals th at tri gger the process of apoptos is in normal neut rophil s.

It is apparen t that retinoid-induced differenti ati on and apoptosis in myeloid ce ll s are receptor-med iated

and receptor-selective events. Using human myeloid leukemia (HL-60) cell s th at expressed speci fic sub­types of retinoid receptor (RAR or RXR), we demon­

strated th at act i va ti on of RARo: was sufficient to in­duce terminal differenti ation of HL-60 cell s into neu­

trophil s, while ligand activati on of RXRo: induced DNA fragmentati on and morphologica l changes, I .. f . 16 S' '1 I N { 17 Claractenstic 0 apoptos ls . Iml ar y, agy el n .

have used receptor-spec ifi c synthet ic retinoids th at preferentially ac tivate either RARs or RXRs to dem­onstrate that li gand acti vation of RARs is necessary to induce differentiation , whereas ac ti vation of RXRs is essential fo r the inducti on of apoptos is in HL-60 cell s. Yet in another study, Ueno el at.

18, by using a RAR­

selec ti ve antagon ist th at prevents reti noid-i nduced RARs-dependent transact ivation of target genes , have obtained simil ar resu lts. One major caveat of using syntheti c retinoids in these studi es is that these com­pou nds can be metabolized or isomeri zed inside the ce ll and thu s may ex hibit altered receptor spec i ficity. [n contrast, we have utili zed retrov iral vector­

med iated gene transduction to introduce RAR o: or

RXRo: genes into HL-60R cell s, which harbor a mu­

taled non-funct ional RARo: genel9. We have observed

that 9-cis RA treatmen t of RXR-o: transduced cells primarily lead to apoptos is of HL-60 cell s, while tR A

A 11':-'1 tRA II<A ·intilh:l'd Jilll'n'lIli:ltioll i~ bkx'k(.'u dll l' to it dlllllinant Ill' l!:tl i \ 1..'lIll1latilHi il l 1<. A Kit g~ lle

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Fig. 3-Retinoid-induced differentiation and apoptos is of myeloid leukemia HL-60 cells. (A)- HL-60R cells harbour a fun cti onal mu­tati on in RARa gene that results in a non-functional RARa protein and renders the cell s resistant to tRA. (B) - Retrov irally- transduced expression of fun clional RARa in HL-60R cell s restores the ability of these cell s to differentiate in response to tRA trealment. (C)-Transduction of RXRa cDNA , renders HL-60R cell s hi ghl y sensili ve to RXR-se lecti ve li gands (e.g ., 9-cis RA) and induce massive apoptosis in these cell s without morphologica l differentiation. (D) - RARa nuclear receptors in wild-lype HL-60 ce lls when bind to and are act ivated by appropriate li gand (e .g. tRA) result in granul ocy ti c differenti ati on of the cells. The differentiation process is assoc iated with induction of several new genes, includ ing RXRa receptors. It is conceivable Ihat tRA is isomeri zed to 9-cis RA ill Silll, which can then bind and activate RXRs, lead ing to onset of apoptos is in differenti ated HL-60 ce ll s.

878 INDIAN J EXP BIOL, AUGUST 2002

treatment of RA Rcx-transduced cell s primarily in­duces granulocytic differentiation (Fig. 3) 16. These studi es, using differen t experimental approaches, reach similar conc lusions and support the involve­ment of RAR and RXR in the regulati on of differen­ti ation and apoptosis, respecti vely, in HL-60 cell s.

APL: An in vivo model fo r tRA-induced differentia­tiun- During early 1990s, the elucidat ion of molecu­lar pathways that lead to development of acute pro­myelocytic leukemia (APL) provided a strong para­digm for the connection between ill vitro and in vivo observat ions and supported th e role for tR A in com­mitting hematopoieti c ce ll s to granu locyti c pathway . APL is a subtype of acute myeloid leukemia that is characteri zed by a block in differentiation at the pro­myelocytic stage6

. It is becoming increas ingly ev ident that APL is uniquely sensiti ve to tRA and clinical tri­als indicate th at tRA induces complete rem iss ions by differentiation and even tual eliminat ion of the malig­nant clone. The great maj ority (~99%) of APLs ex­hibit a characteristic t( I 5: 17) chromosome transloca­tion that generates an aberrant chimeri c protein (PML-RAR), fusing a portion of PML gene on chro­mosome IS with RARcx on chromosome 17 (Fig. 4). Several functional domains are altered in PML-RAR

fusion. RARcx is truncated at N-terminus and lacks A region (Fig. 4) which is considered to be essenti al for promoter transact i vat ion. Other domains, including DNA-binding domain , li gand-b inding domain, RXR heterod imerization domai n are intact in PML-RXR. PML gene, on the other hand, is trunca ted at C­termi nus resulting in loss of a serine rich region. Thi s region is considered to be a target for phosphoryla­tion.

PML-RA R fusion protein interferes wi th the physi­ologic functions of PML and RA Rcx proteins and ex­erts dominant nega tive effec t. Consequentl y, expres­sion of PML-RA R fusion protein in APL ce ll s blocks thei r differentiation at promyelocytic tage, lead ing to accumulati on of immature hematopo ietic cells in bone marrow. In agreement to thi s conviction, overexpres­sion of a dominant negative RAR cx has been shown to cause a di rferential block at the pro myelocytic stage20

.

Recently , transgenic mice expressing PML-RARcx have block at the promyelocyti c stage of myeloid maturation in blast cell s implicating important role for PML-RAR cx abnormal receptor protein in leuko-

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Moreover, PML protein is involved in regu lation of cell proliferation and apoptosis . Cell s Jack ing PML

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Fig. 4-Exprcss ion of oncogenic RARa rcceptor protcin in APL cells due to chromosomal translocation . Chromosomal translocation involvcs RARa gcnc on chromosomc 17 and prolllycl ocytic Icukcm ia (PML) gcnc on chromosomc 15. Thc brcak oints may vary in P 11. gcne, howcvcr. it is always locatcd in thc same point in RARa gcne. tRA at physiologic conccntrations ( 1-10 nM) can bind and activate \\ illl-type RARa, btlt not PML-RARa. As a result of thi s, APL cell s, wh ich harbour PML-RARa fusion protein arc blockcd in promyc locy tic stagc of difrcrcllliati on. Howcver, at pharmacological concclllra ti ons (~ I jlM) , tRA can activale thc ru ~ion protcin . causing transactivation of larget gcnes, difTcrcnt i:ll ion of APL ce ll s and rcmissions in APL patients.

MEHTA: RETI OIC ACID IN EUTROPHIL DIFFERENTIATION 879

are resistant to apoptosis by ga mma irrad iati on, grow fas ter and have longer survi val time, whil e cell s over ex press ing PM L undergo apoptosis by the same stimulus12 It has been shown that PML is located in the nucleus of normal cell s in punctared nuclear struc­tures (PODs) or nuclear bod ies associated with nu­clear matrix, however, in PML-RARcx pos itive APL cell s, loca li zation and the normal pattern of nuclear bod ies are di srupted. Overall, the data obtained sug­gest th at di sru ption of PML function may also con­tri bute to APL pathogenesis.

Bas is fo r dramatic efficacy of tRA against APL is the ability of pharmacolog ica l doses of tRA to over­come repress ion of signaling caused by PML-RAR fusion protein . Restoration of signal ing leads to dif­fe renti ati on of APL cell s and then to postmaturati on apoptosis23

. tRA induces differenti ation of immature leukemic blas ts into terminall y differenti ated granulo­cy ti c ce ll s that is associated with clinical remi ss ions (Fig. 4).

tRA-inducible genes in neutrophil differentiation and apoptosis

Modul ation of severa l putat ive target genes during tRA-induced maturat ion of neutrophils suggests that tRA can directly affect nell trophil maturati on by switching on or off certain subset of genes. These genes include early response genes (ERGs) that pos­sess funct ional RARE within their promoter and can be induced in the presence of cyclohexamide. Induc­tion of ERGs req uire tR A- induced heterod imeri za ti on of RAR-RXR whi ch can then bind and acti vate the target promoter in conjuncti on with other transcrip­ti onal factors. Secondary response genes include those genes that ex hibit tRA inducti on but fail to be ex­pressed in the pre ence of cyc lohexamide and do not possess RARE within their promoters (Fig. I B).

Several members of C/EBP fami ly of transcription factors have been shown to pl aya role in hematopoi­esis. C/EB PE in parti cular, has been implicated to be a tRA target gene during neutrophils maturation. For example, differentiation of APL cell line (NB4) is assoc iated with inducti on of thi s gene as early as I hr after 9-eis RA treatment. Consistent with these find­ings was the observation that C/EBPE promoter con­tains a function al RARE24. Similarly, CD38 th at has been originally described as a c!ifferentiation marker on T and B cells, is rapidly induced in response to tRA treatment of HL-60 cell s and APL cell s and its promoter has a functional RARE25. The prec ise fun c­ti on. of CD38 in neutrophil s differenti ation remains to

be elucidated but it is known to catalyze the synthes is of cyclic ADP-ribose, a metabolite of NAD+ that can induce ca lcium mob ili zati on26. CIP/W AF/p21 is an­other direct target gene of tRA whose promoter con­tains a functional RARE. The p21 protein binds and inhibits the function of a cyclin-dependent protein kinase resulting in growth arrest of the cells in G I phase.

In contrast to CIEBP and p2 J genes, the expression of e- //lye oncogene is down-regulated by tRA in a cyclohexamide independent fashion 27 . In fact , the ac­ti vation mediated by RARcx is remini scent of that ex­erted by Myc/Mad system in ce ll 's decision to proli f­erate or differenti ate. Several other genes have been identified whose ex press ion is modulated by tRA in various myeloid cells. For example, recentl y Liu el 0 1.28 have used the combined approaches of sup­press ion-subtraction hybridization, differential dis­play , and microarray to identi fy everal genes that are induced in response to tRA treatment of APL cell s. In another study, Li an el 01.

29 used the microarray and 2-D protein electrophores is analysis to identi fy severa l known and new genes whose expression is modul ated du ring tRA-induced differentiati on of a murine pro­myelocytic leukemia cell line. Interestingly, in both the studi es a large number of genes that are modul ated by tRA, encoded proteins th at are in volved in cell growth and apoptot ic regul atory pathways. Similar studies on the identi fica tion of critica l genes th at are regul ated by unliganded and li gnaded RARs will be useful to understand the process of granulopo ies is at mo lecul ar level.

As discussed in a previous section, tRA-induced differentiation of neutrophil s is shortlived and the dif­ferenti ated cells are destined to undergo apoptosis within 24 hr. tRA can induce several changes that may render these cell s susceptible to apoptosis. In­creased expression of a protein cross linking enzyme, ti ssue transglutaminase (TGase), fo r example, has been linked to retinoid-induced terminal differentia­tion and arrest of cell growth in several hematopoietic cell s3o

. Hi gh levels of TGase have also been demon­strated in several cell types undergoing apoptotic cell death 31

• Activation of TGase is thought to be respon­sible for formation of intracellular cross-linked pro­tein polymers that constitute the major component in an apoptotic envelope.

Does tRA-induced TGase expression has any role in the execution of apoptosis? Transfection studies have been carried out to directly address this questi on .

IND IAN J EXP BIOl. AUGUST 2002

Introducti on of full-length TGase cDNA in fibro­blasts, human neuroblastoma, and l-929 fibroblast cel ls resu lted in an increased spon taneous apoptos is or rendered the ce ll s highly susceptible to vari ous death­inducing stimuli. Cons istent w ith these resul ts was the finding that neurobl as toma cell s, when transfected with a fragment of the human TGase cDNA in an an­ti sense orientation, ex hibited red uced su ceptibility to RA-induced apoptos is. These observa ti ons further support the intent that TGase express ion is an essen­tial component of apoptos is. Several stud ies have re­vea led th at retinoids are genera l and poten t ph ys io­logica l illducers or TGase" . M olecular mechani sms involved in regu lat ion of TGase expression have been ex tensively studi ed. By introducing full-length RXR cD A into tR A-res istant Hl-60R ce ll s, we have ob­served that specific express ion and activatioll of RXR results in a marked increase in TGase express ion l6

.

RXR-induced increase in TGase expression in these ce ll s is assoc iated w ith their increased propensity to undergo spon taneous apoptos is.

Iso lation and characteri za ti on of gui nea pig, hu­man, and mouse TGase gene promoters provide the important tool s for elucidating regulatory mechanisms or TGase express ion. An cdys is of 5' upstream nucleo­tide sequences in TGase gene revea ls th at a functi onal TGase promoter con tains potential responsive ele­ments and binding sites for multiple factors, including glucocorticoid , Il-6, A P-I and AP-2. Functional ana lys is of human TGase promoter revea ls that its core reg ion , which includes a TAT A box, four SP I sites, and four potential NF-I sites wi thin 134 bp up­stream of the translation start site, is sufficient for accompli shing high consti tuti ve transc riptional activ­it i 2

. These invest igators have further shown th at four SPI sites contribute most significan tl y to hi gh basal promoter ac ti vi ty. Interestingly, the sequences corre­sponding to RAREs arc not identified in either guinea pig or human TGase promoters, suggest ing that RA­dependent upregulation of TGase may be mediated outside thi s region. However, the results obtained by

agy el 01 .. 13, reveal th at cis-acting elements neces­

sa ry for directing retinoid-dependent transactivation of mouse TG ase are embedded within the prox imal 3.8 kb DNA that r1anks 5' end of TGase gene. Dele­tion ana lysis of the promoter led to identification of two reg ions criti ca l for retinoid-dependent ac tivati on of TGase. The most upstream one, locatcd 1.7 kb up­strl:<llll of transcript ion start site, is rctinoid response element (mTGRRE I ) since it contains a triplicate reti­noid receptor bind ing motif. Coupled wi th a short

DNA segment approx imately I kb upstream of tran­scripti on start site, mTGRREI med iates full promoter acti vat ion by retinoic ac id.

At the molecular level , it has been recently shown that tR A- induced expression of TRAI L , a ce ll surface ligand for death receptors, is respons ible for its apop­toti c acti v i ty in NB4 cel ls23. Since TRAIL ind uct ion is rather a late event occurring only after 48 hI' of tRA trea tment, it is unlikely that TRAIL is direct ly in­duced by tRA. Interest ingly, earli er events fo llow ing tRA treatmeI1l include induction o f certain an ti­apoptot ic pathways such as, induct ion of a BCl2 homologue, BCl2A I and activat ion of NFKB23

.28

.

These observat ions suggest that even though the end point is death, tRA may modulate survival and death o f myeloid cells in a temporall y complex way.

Tn conclusion , apoptos is, which is a normal fate of many somati c ce ll s, especial I yin the hematopoietic system, may be a ti ghtl y con trolled event by retinoids. Thus, retinoids may regulate the rate at which these ce ll s undergo di fferentiation and apoplos is, depending on the RARs or RXR SUbtype express ion. A prec ise understanding of events leadi ng to these processes is likely to give new insight into normal and abnormal leukocy te survi va l. The experimental evidence dis­cussed in thi s rev iew suggests that ac tivati on of RARs induces the genes linked to cellular differentiation ; while act i va ti on of RXRs induces genes linked to apoptos is. Further understanding of such pathways that selectively control the cell growth and different ia­ti on ['un cti ons, may suggest ways to contro l ce ll sur­vival in diseases ranging from ap last ic anemia to leu­kemia.

Acknowledgement The work in author's laboratory is supported in part

by grants from The Department of Defense and the U.S. Food and Drug Administration (FDR-000923). I apolog ize to the co lleagues whose work cou ld not be cited due to space restri ct ions.

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