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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2423-2432
Evidence for the Involvement of Both Retinoic Acid Receptor- and
Retinoic X Receptor-Dependent Signaling Pathways in the Induction of
Tissue Transglutaminase and Apoptosis in the Human Myeloma Cell Line
RPMI 8226
By
Bertrand Joseph,
Olga Lefebvre,
Claude Méreau-Richard,
Pierre-Marie Danzé,
Marie-Thérèse Belin-Plancot, and
Pierre Formstecher
From the INSERM U459 «Signaux, Récepteurs et
Différenciation Cellulaire», Faculté de Médecine,
Lille cedex, France.
 |
ABSTRACT |
In this study, we show that both all-trans-retinoic acid
(atRA) and 9-cis-retinoic acid (9-cis-RA) are
potent inducers of tissue transglutaminase (TGase II), an enzyme
involved in apoptosis, at the level of both enzyme activity and mRNA in
the human myeloma cell line RPMI 8226. RPMI 8226 cells were shown to
express mRNAs for all the retinoid receptors subtypes, ie,
RAR , RAR , RAR , RXR, RXR, and RXR. To
identify which of these receptors are involved in regulating TGase II
expression, several receptor-selective synthetic retinoids were used.
Neither CD367, a very potent retinoid that selectively binds and
activates receptors of the RAR family, nor CD2425, an RXR-selective
agonist, induced TGase II when used alone. However, combination of
CD367 and CD2425 resulted in nearly full induction of the enzyme.
Moreover, when used in combination with atRA, CD367 partially
inhibited the atRA-dependent induction of TGase II, whereas
CD2425 enhanced it. The effects of Am 580, CD417, and CD437, three
synthetic retinoids selective for the RARs subtypes RAR, RAR, and
RAR, respectively, were also investigated. None of these compounds
was able to induce TGase II when used alone; however, the combination
of each of them with CD2425 resulted in strong induction of the enzyme
activity, reaching 30% to 50% of the values obtained in the presence
of retinoic acid and suggesting functional redundancy between the RAR
subtypes. Finally, treatment with atRA or the combination of
CD367 and CD2425, but not with CD367 or CD2425 alone, was also shown to
trigger apoptosis in RPMI 8226 cells, with prominent accumulation of
TGase II immunoreactivity in apoptotic cells. Taken together these data
suggest that the induction of TGase II expression and apoptosis in the
RPMI 8226 myeloma cell line required ligand-dependent activation of
both the RAR and RXR receptors.
| |
INTRODUCTION |
TRANSGLUTAMINASES (EC 2.3.2.13, TGase)
are Ca++-dependent enzymes catalyzing the formation of
 (-glutamyl)lysine cross-links between polypeptide
chains.1 Several members of this gene family have been
identified including the type II (tissue)
TGase.2,3 TGase II, the biological functions
of which remain unclear, is an intracellular enzyme found in many cell
types.4 TGase II is implicated in the activation of several
cytokines5,6 and in signal transduction.7
Expression of TGase II has been found associated with apoptosis in
several cell types, and a role for TGase II in this process has been
suggested.8-10
Apoptosis is a genetically controlled process of cell death and is
important for the elimination of cells during morphogenesis, in
embryonic development as well as in many adult
tissues,10,11 and in cancer. Although the function of TGase
II in apoptosis has yet to be elucidated, it has been proposed that
TGase II may cross-link cellular proteins, thereby preventing the
release of intracellular macromolecules.12
all-trans-retinoic acid (atRA) induces TGase II
expression in various cell types.2,13-21 In mouse
macrophages, regulation of TGase II expression by atRA occurs
at the transcriptional level.2 Most of the effects of
atRA on gene expression are mediated by the activation of
nuclear retinoid receptors, RARs and/or RXRs.22 The
RAR and RXR gene family comprises three subtypes named , , and
.22 These subtypes are expressed in a developmental stage- and cell type-specific manner,22 and each may
regulate the expression of different genes. In this study, we examined the retinoid signaling pathways involved in the induction of TGase II
(and apoptosis) in the human myeloma cell line RPMI 8226, using several
retinoid receptor-selective agonists. This cellular model was chosen
because atRA has been proposed for the treatment of multiple
myeloma.
Our results provide evidence indicating that the induction of TGase II
gene expression and apoptosis by atRA in RPMI 8226 cells is
mediated through specific retinoid signaling pathways that involve both
RAR(s) and RXR(s).
| |
MATERIALS AND METHODS |
Retinoids.
atRA and 9-cis-retinoic acid (9-cis-RA) were
purchased from Sigma Chemical Co (St Louis, MO); Am580, CD367, CD417,
CD437, and CD2425 were obtained from CIRD Galderma (Sophia Antipolis, France). Retinoids were dissolved in dimethyl sulfoxide at an initial
10 2 mol/L stock concentration and stored at
 20°C in the dark. Dilutions were performed in ethanol. The
final concentration of ethanol in culture was 0.1%.
Cell line and cell treatments.
RPMI 8226 cells (CCL-155 American Type Culture Collection) were
cultured routinely at 37°C, 5% CO2 in complete medium
(RPMI 1640 with Glutamax I, 10% fetal calf serum [FCS] inactivated
by heating, penicillin [100 U/mL], and streptomycin [100 µg/mL]). Viability (higher than 95%) was determined by trypan blue exclusion. TGase induction by retinoids was performed in cells grown in standard medium (RPMI 1640 with Glutamax I, penicillin [100 U/mL],
streptomycin [100 µg/mL], insulin [5 µg/mL], transferrin [5
µg/mL], and sodium selenite [5 ng/mL]) supplemented either with
3% FCS at 7 hours for TGase activity measurement, or with 10% FCS at
48 hours for microscopic studies.
TGase activity measurement.
TGase activity of cell lysates was determined by
Ca++-dependent incorporation of
[3H]putrescine into N,N' dimethylated
casein.23 A total of 107 cells were induced by
retinoid at 5 × 105 cells/mL for 48 hours and lysed
in 150 µL 10 mmol/L Tris-HCl pH 7.5, 1 mmol/L EDTA, 0.5% Triton
X-100. Aliquots of cell extracts (0.2 mg of proteins) were incubated at
30°C in a total volume of 100 µL containing 20 mmol/L Tris-HCl pH
7.5, 5 mmol/L CaCl2, 10 mmol/L mercaptoethanol, 2 mg/mL
N,N' dimethylated casein, 2 µCi [3H]putrescine (30 to
60 Ci/mmol, NEN Life Science, LeBlanc Mesnil, France), and 0.2 mmol/L
putrescine. After 20 minutes, aliquots were spotted on Whatman 3MM
filter paper, fixed and washed in 10% trichloroacetic acid, 5%
trichloroacetic acid, ethanol/acetone (v/v), and acetone. Protein-bound
[3H]putrescine was determined by liquid scintillation
counting. Background value was obtained by substitution of 1 mmol/L
EGTA for CaCl2 in the reaction mixture added to cell
extracts and lysis buffer. Enzyme activity was expressed as picomoles
of [3H]putrescine incorporated into N,N' dimethylated
casein per minute and per milligram of cell lysate protein. Protein
amount was assayed by the Bradford method24 using fraction
V Albumin (Boehringer Mannheim, Mannheim, Germany) as the standard.
Reverse transcriptase-polymerase chain reaction (RT-PCR) and
Northern blot analysis.
Total RNA was isolated from 107 cells using RNAble
(Eurobio, Les Ullis, France) and was used for RT-PCR and Northern blot
analysis.
For PCR amplification, pairs of primers were selected using the cDNA
sequences of human TGase II from umbilical vein endothelial cells,
human -actin, human RAR, RAR, RAR, and human RXR, RXR, RXR (Table 1). TGase II primers
are located in the coding region of two contiguous exons.25
The reverse transcriptase reaction was performed in a final volume of
20 µL containing 1 µg total RNA (denatured at 68°C for 5 minutes), 20 U RNAsin Ribonuclease inhibitor (Promega,
Charbonnieres, France), 5 µmol/L pd(N)6
(Pharmacia Biotech, Uppsala, Sweden), 0.5 mmol/L dNTPs, and 200 U
Moloney Murine Leukemia Virus Reverse Transcriptase (Promega) in RT
buffer. An aliquot (5 µL) of the RT reaction product was mixed with
20 µL PCR buffer containing 1 U Taq DNA polymerase (Promega), 0.1 mmol/L dNTPs, and 100 ng of each primer. PCR was performed in a
minicycler (MJ Research, Watertown, MA) as follows: 95°C for 5 minutes, then n (15 to 40) cycles consisting of 1 minute at 95°C, 1 minute at 54°C, and 1 minute at 72°C. After the last cycle, incubation at 72°C was prolonged for 10 minutes. TGase II, RARs, RXRs, and -actin PCR reactions were performed separately. PCR products (15 µL) were analyzed on a 2% agarose gel in
TAE buffer (40 mmol/L Tris, 40 mmol/L sodium acetate, 1 mmol/L EDTA, pH 8.4) using a DNA ladder 100-bp marker and a PCR product
from human TGase II cDNA26 (kindly provided by Dr V. Gentile, Naples, Italy) as controls. Samples were analyzed
by Southern blot using as a probe internal primers for amplified
sequence (Table 1). Primers were labeled with 20 µCi
[32P] adenosine triphosphate (ATP; 3,000 Ci/mmol
Dupont-NEN) using 5 to 10 U/µL T4 polynucleotide kinase (Promega).
Northern blot analysis was performed using 8 µg total RNA and cDNA
probes for human TGase II, RAR, RAR, RAR, and RXR, and for
mouse RXR, RXR kindly provided by Dr V. Gentile, Pr P. Chambon (LGME, Strasbourg, France), and Drs R.M. Evans and D.J.
Mangelsdorf (The Salk Institute, La Jolla, CA), respectively. cDNAs
were labeled with 20 µCi [32P]ATP (800 Ci/mmol)
using a multilabeling kit (Boehringer Mannheim).
Autoradiograms were quantified by densitometric analysis with
a rapid electrophoresis apparatus (Helena
Laboratory, Beaumont, TX). Values were normalized using -actin as
external standard.
Morphological studies.
At the indicated times, 15 × 104 cells were taken to
make cytospin preparations. Slides were stored at 20°C. Cell
morphology was observed after staining with May-Grünwald-Giemsa
(MGG). DNA staining with Hoechst 33258 (Bis [benzidine]) was
performed as described by Galli and Fratelli.27 In situ DNA
fragmentation was measured by using the to TUNEL method based on 3'OH
end labeling of DNA breaks with deoxyuridine terminal deoxynucleotidyl
transferase.28 Immunofluorescence detection29
of TGase II was performed by using a mouse monoclonal antibody (IgG)
raised against purified guinea pig TGase II30 and kindly
provided by Dr P.J. Birckbichler (Ardmore, OK). A fluorescein conjugated anti-mouse IgG (Biosys, Compiègne, France) was used for detection by fluorescence microscopy.
| |
RESULTS |
Time course and dose-response induction by atRA of TGase II
activity.
TGase II activity was undetectable in untreated RPMI 8226 cells.
atRA treatment resulted in a time and dose-dependent induction of the enzyme (Fig 1). This induction was
easily detected after 2 days of incubation and increased linearly until
4 days (Fig 1A). Interestingly 9-cis-RA, which activates both
RARs and RXRs, appeared more potent than atRA in dose-response
experiments (Fig 1B).
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| Fig 1.
Induction of TGase II activity by retinoic acid in RPMI
8226 cells. Cells were treated with atRA or
9-cis-RA, and TGase II activity was assayed by
[3H]putrescine incorporation into N,N' dimethylated
casein. (A)Time course of the induction of TGase II activity by
107 mol/L atRA. (B) Dose-response curves after
48 hours of induction with various concentration of atRA or
9-cis-RA.
|
|
mRNA TGase II induction by atRA in RPMI 8226 cells.
TGase II induction was also detected at the mRNA level by using a
RT-PCR assay (Fig 2). Whereas the expected
301-bp amplification product could not be detected before the 29th
cycle of PCR in untreated cells, it was clearly observed after only 22 cycles in atRA-treated cells and increased dramatically after
29 and 36 cycles. The 301-bp band specificity was controlled using PCR performed with a bona fide cloned human TGase II cDNA26 and BamH1 cleavage, which yielded the expected digestion pattern
(data not shown). Actin levels were not altered upon atRA
treatment. Taken together these results showed that the
atRA-dependent induction of TGase II in RPMI 8226 cells occurs
at a pretranslational stage.
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| Fig 2.
Induction of TGase II mRNA in RPMI 8226 cells. Cells were
treated or not for 7 hours with 107 mol/L atRA
in defined medium. Total RNA (1 µg) was submitted to RT and PCR (for
22, 28, 34, or 40 cycles), fractionated on 2% agarose gel, transferred
to a Hybond N+ membrane, and hybridized to
32P-labeled oligonucleotidic probes for human TGase II and
actin. (For primers and probes see Materials and Methods).
|
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Expression of RARs and RXRs in RPMI 8226 cell line.
The induction of TGase II by atRA may be directly or indirectly
mediated by nuclear retinoid receptors. To determine which retinoid
receptors could be involved in this regulation, the expression of RARs
and RXRs in RPMI 8226 cells was investigated. Untreated cells expressed
RAR, RAR, RAR, RXR, RXR, and RXR mRNAs at various
levels (Fig 3). The size of the transcripts
was checked by Northern Blot and found to be correct (data not shown).
Treatment of RPMI 8226 cells for 7 hours with 107 M
atRA resulted in an upregulation of RAR and RAR as shown by the sensitive RT-PCR assay used (Fig 3).
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| Fig 3.
Retinoid-receptor profile of RPMI 8226 cell line. Cells
were treated or not for 7 hours with 107 mol/L
atRA in defined medium. Total RNA (1 µg) was submitted to RT
and PCR (for 15, 20, 25, 30, or 35 cycles), fractionated on 2% agarose
gel, transferred to a Hybond N+ membrane, and hybridized
to 32P-labeled oligonucleotidic probes for human RAR,
RAR, RAR, RXR, RXR, RXR, or -actin. (For primers and
probes see Materials and Methods).
|
|
Effects of receptor-selective retinoids.
To evaluate the respective involvement of RARs and RXRs in the
induction of TGase II, we used several retinoids that bind selectively
to and activate specific RARs or RXRs (Fig
4).
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| Fig 4.
Structures of the natural and synthetic
retinoids used. Structures of atRA,
9-cis-RA,4-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl]-benzoic acid (CD367),
(E)-2-[2-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl]-4-thiophenecarboxylic acid (CD2425 or AGN191701),
[4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carboxamido]benzoic acid (Am580),
6-(3-tert-butyl-4-methoxyphenyl)-2-naphthoic acid (CD417), and
6-[3-adamantyl-4-hydroxyphenyl]-2-naphtalene carboxylic acid (CD437).
The selectivity of each compound is indicated according to previously
published dissociation constant and EC50 data.31,32,43 CD2425 does not transactivate RARs, but
transactivates RXRs (EC50 = 54 nmol/L). It binds to RARs
with a very weak affinity (kd = 10,000, 1470, and 712 nmol/L for
RAR, RAR, and RAR, respectively; U. Reichert, personal
communication, March 1997). No RXR binding data have been
published for this compound.
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We first analyzed effects of CD367 and CD2425, two retinoids highly
selective for the RAR and the RXR receptors families, respectively.
CD367 binds to and activates RAR, RAR, and RAR at nanomolar
concentrations,31 whereas CD2425 (or AGN 191701) is a RXR
panagonist.32 RPMI 8226 cells were treated for 48 hours with these retinoids at concentrations ranging from
109 to 106 mol/L, either alone or
in combination with 107 mol/L atRA. This
concentration of atRA, yielding only suboptimal (about 30%)
TGase II induction, was chosen to detect any positive or negative
modulation of the response by the synthetic retinoid used. Results are
presented in Fig 5. When used alone, and at every concentration tested, the RAR-selective retinoid CD367 did not
cause any increase in the TGase II activity (Fig 5A). Moreover, it
appeared able to partially inhibit the atRA-dependent TGase II
induction. When used alone the RXR-selective compound, CD2425 appeared
also unable to induce TGase II activity (Fig 5B). However, CD2425 in
combination with atRA strikingly enhanced the atRA
response in a dose-dependent manner, the maximum being observed at
107 mol/L CD2425 (threefold enhancement when
compared with 107 mol/L atRA alone). At
higher CD2425 concentrations (106 mol/L) combination
with 107 mol/L atRA appeared less efficient.
Finally, as shown in Fig 5C, combination of both RAR and RXR-selective
agonists resulted in synergistic induction of TGase II, reaching 80%
of the value obtained with 107 mol/L atRA.
Taken together these observations suggested that activation of both RAR
and RXR is required for induction of TGase II.
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| Fig 5.
Effects of RAR and RXR-receptor-selective retinoids or
both on TGase II activity in RPMI 8226 cells. Cells were treated for 48 hours with 107 mol/L atRA, varying doses of
CD367 (A), CD2425 (B), and their combination (C), in defined medium for
7 hours, and then in 3% FCS-supplemented medium.
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The next step in our studies was to determine if one of the RAR
subtypes, ie, RAR, , or , was selectively involved in the control of TGase II expression. RPMI 8226 cells were treated as before
in the presence of either Am 580, CD417, or CD437, three synthetic
retinoids that are RAR, , or selective, respectively, when
used at nanomolar concentrations.33,34 As shown in
Fig 6, RAR subtype selective retinoids used
alone were inactive. However, when used at 109 mol/L
concentration in combination with CD2425, each of them induced TGase II
activity to an extent varying from 25% to 45% of the level obtained
after treatment with 107 mol/L atRA.
Interestingly, no further induction was observed when the RAR subtype
ligands were used at higher, nonselective concentrations. Thus, the
various RAR subtypes that are all expressed in RPMI 8226 displayed
functional redundancy with respect to the synergistic induction of
TGase II in the presence of an RXR-selective ligand.
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| Fig 6.
Effect of RAR-, -, and -receptor-selective
retinoids in combination with a RXR-receptor-selective retinoid on
TGase II activity in RPMI 8226 cells. Cells were treated for 48 hours
with either 107 mol/L atRA or 107
mol/L CD2425 (RXR selective) and various doses of Am580 (RAR selective), CD417 (RAR selective), or CD437 (RAR selective) in
defined medium for 7 hours, and then in 3% FCS-supplemented medium.
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Correlation between the induction of TGase II and apoptosis in RPMI
8226 cells.
Several studies have suggested a role for TGase II in
apoptosis.8-10 To examine whether the atRA-induced
TGase II expression in RPMI 8226 cells was correlated with the
induction of apoptosis, we performed both indirect immunofluorescence
detection of TGase II protein (Fig 7, left
panel) and MGG staining (Fig 7, right panel) of cells within 6 days of
addition of different retinoids to the culture . Apoptosis was
quantified by counting apoptotic cells after MGG and Hoechst 33258 staining. Count of TUNEL-positive cells gave similar results
(Fig 8).
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| Fig 8.
Quantification of apoptosis. Cells were incubated for 6 days in the absence of retinoids or in the presence of
107 mol/L atRA, 107 mol/L CD367,
107 mol/L CD2425, or the combination of both
107 mol/L CD367 and 107 mol/L CD2425.
Count of apoptotic cells was performed on cytospin preparations after
MGG coloration, Hoechst 33258 staining, or TUNEL assay. In each case
the percentage of apoptotic cells was determined in duplicate counts of
400 cells. Mean values ±SD from six independent experiments are
shown.
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As shown in Fig 7A, after treatment of RPMI 8226 cells with
107 mol/L CD367 or 107 mol/L
CD2425 alone, neither apoptotic cell death (right panel) nor TGase II
(left panel) were induced. The apoptotic index in the CD367- or
CD2425-treated cultures was not different from that in control (Fig 8).
Thus, mere activation of endogenous RARs by a RAR-selective retinoid
such as CD367 or of endogenous RXRs by a RXR-selective retinoid such as
CD2425 is insufficient to induce apoptosis in RPMI 8226 cells.
Interestingly, treatment with 107 mol/L
atRA, the natural panagonist ligand, resulted in both TGase II
induction (Fig 7B, left panel) and significant apoptosis. As shown in
Fig 7B (right panel), numerous cells displayed the characteristics of a
classical apoptotic phenotype with condensed nuclei, extensive
cytoplasmic blebs, marked decrease in cellular volume, and cellular
fragmentation. The apoptotic index of atRA-treated cells was
approximately 12% (Fig 8). Finally, after 6 days of culture in the
presence of both 107 mol/L CD367 and
107 mol/L CD2425, we observed the same morphological
evidence of apoptosis as in atRA-treated cells (Fig 7C, right
panel). The apoptotic index was in the 18% range (Fig 8). After
atRA treatment TGase II appeared induced in cells undergoing
apoptosis and accumulated in apoptotic bodies (Fig 7C, left panel).
Taken together, our results showed that the induction of TGase II and
apoptosis by retinoids in RPMI 8226 cells is linked and that the
activation of both RARs and RXRs is required for the induction of
apoptosis. Interestingly, the same requirement was observed for the
inhibition of cell proliferation elicited by retinoids in RPMI 8226 cells (Fig 9).
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| Fig 9.
RPMI 8226 cells growth in the presence of various natural
and synthetic retinoids. Cell proliferation was evaluated by direct counting (Trypan blue assay).
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| |
DISCUSSION |
In this study we showed that retinoic acid induces TGase II expression
in a dose-dependent manner at both the mRNA and the enzyme activity
levels in RPMI 8226 cells. A retinoic acid-dependent expression of the
protein was also shown in intact cells by indirect immunofluorescence
assay. In RPMI 8226 cells, there is no detectable basal expression of
TGase II, a situation also observed in other cellular models, including
HL-60 human leukemia cells, a model extensively used for the study of
the mechanisms controlling TGase II expression.15
Conversely, in some other cell types and tissues, significant basal
expression of TGase II has also been reported.35-38 In most
cellular models, however and whatever the basal level, retinoic acid
induced TGase II, with only few exceptions,35,39 probably
related to some poorly understood postreceptor resistance.
RPMI 8226 cells express all the retinoic acid receptors mRNA,
suggesting that all the RARs and RXRs proteins are also expressed in
these cells, which could therefore constitute a very attractive model
for the elucidation of their respective role in the control of various
target genes by retinoic acid. A more limited pattern of expression of
the retinoic acid receptors has been reported in HL-60 and SPOC-1
cells, two models recently used for the study of the control of
expression of TGase II by retinoids. HL-60 expressed only RAR,
RXR, and RXR, whereas SPOC-1 cells expressed only RAR, RAR,
and RXR.32,40
RAR subtype (, , or ) and RXR-specific retinoids are extremely
useful ligands that, when used at appropriate selective concentrations,
allow the investigation of the respective contribution of the various
RARs and RXRs in the molecular control of a given retinoic
acid-induced response.34,41 Our results showed that in
RPMI 8226 cells, TGase II induction required the activation of both the
RAR and the RXR pathways. Our report is the first trying to elucidate
the molecular mechanism of the control of TGase II by retinoic acid and
its correlation to apoptosis in human myeloma cells. However, this
regulation has been already investigated in other human cells by using
the same kind of approach. In the HL-60 promyelocytic leukemia cell
line, TGase II induction was observed in the presence of both RAR/RXR
panagonists and RXR-selective ligands,42-45 implying that
the activation of RXR alone is able to elicit the response. The
implication of the RAR pathway is less clear, because conflicting
results have been reported using TTNPB, an RAR-selective ligand, in two
different HL-60 subclones. In the HL-60 cdm-1 clone, TTNPB was
inactive,42 whereas in the HL-60 CCL240 clone it induced
TGase II activity at the same level as retinoic acid- and RXR-specific
ligands.45 These discrepancies are probably related to
differences in the cellular context of the two subclones.
Interestingly, the HL-60 cdm-1 line is a differentiation-resistant line.32 In NB4 cells, a human promyelocytic leukemia cell
line expressing both the wild-type RAR and the PML-RAR fusion
protein (resulting from the reciprocal chromosomal translocation
t(15:17) that is the molecular hallmark of acute promyelocytic
leukemia, RAR- and RAR-selective retinoids were able to induce TGase
II, whereas RXR-selective retinoids were inactive.39
Moreover, an RAR antagonist completely inhibited the expression of
TGase II. Thus, in these cells expression of TGase II appeared to be
controlled by RARs rather than by RXR, and a peculiar role for
PML-RAR in the regulation has also been reported. Interestingly, the
specific involvement of RAR in the control of TGase II expression
has been also documented in rat and mouse fibroblasts and rat
tracheobronchial cells by using both RAR- and RXR-selective
retinoids40,46 and modulation of RAR level by
transfecting sense and antisense RAR-expression vectors.47 RXR-selective ligands displayed neither activity when used alone nor synergy with RAR-selective ligands.40
Taken together these data indicate that, whereas retinoic acid is able
to induce TGase II in a large variety of tissues and species, the
molecular mechanism by which it exerts this control differs strikingly
and involves different retinoid-receptor subtypes according to the cell
model used. The pattern of expression of the various RARs and RXRs in a
given cell, the promoter context of the TGase II gene, and more
generally the cellular context are probably responsible for the
diversity observed. Recently, the mouse TGase II promoter has been
cloned and its inducibility by retinoids has been studied in CV-1 cells
transfected with various RARs and RXRs.48 In the CV-1 cells
context this promoter was shown to be activated by both RAR and RXR
pathways, a result different from the RAR preference observed in mouse
and rat cells.
We observed that activation of both the RAR and RXR pathways is
required to obtain a synergistic induction of TGase II. Such a synergy
has been already reported for other retinoic acid-induced responses.34 A potentiation of the effect of a
RAR-selective ligand on HL-60 cell differentiation by RXR-selective
ligands has been also reported.49 Such synergic and
potentiating effects probably involve the activation of RAR-RXR
heterodimers and a cooperation between the ligand-dependent activation
functions of both partners.34 No retinoid response element
has been identified on the partial sequence of the human TGase II
promoter published so far,50 and the existence of such
elements in this promoter remains to be documented. Our results suggest
that a subtle equilibrium between the respective activation levels of
RARs and RXRs has to be reached to obtain maximum synergy (see the
opposite effects of the combination of CD2425 v CD367 with
atRA in Fig 5A and B). The observed enhancing effect of CD2425,
when added to 107 mol/L atRA, could be
explained by a better activation of RXR, because atRA activates
mainly RARs; its ability to activate RXRs also in living cells probably
results from partial conversion into 9-cis-RA. Interestingly,
9-cis-RA, which activates both RARs and RXRs, appeared more
efficient than atRA for the induction of TGase II (Fig 1B).
Thus, in the presence of CD2425, the response to atRA was
shifted to a 9-cis-RA-like response with better TGase II
induction. Conversely, in the presence of CD367, the RAR-selective ligand, the balance was shifted toward lower induction (Fig 5A). This
inhibitory effect could result from a disequilibrium between RAR and
RXR activation. The relative level of activation of the two receptor
types need probably to be finely tuned to obtain maximal activation,
and in the presence of CD367 this equilibrium could be disturbed (with
a too-high stimulation of the RAR partner, when compared with RXR). We
previously showed that CD367 induces the formation of RARs homodimers
in vitro51; however, the occurrence of such homodimers is
not documented in vivo. Moreover, recent data obtained in our
laboratory showed that CD367 and atRA do not interact in the
same way with RAR and RAR mutants (B. Lefebvre et
al, submitted; S. Mailfait, in preparation). In fact, CD367, although
considered as a RAR panagonist, displays a variable efficiency
according to the response tested (U. Reichert and S. Michel, CIRD Galderma, personal communication, March 1997). It is
therefore possible that for the specific control of TGase II induction,
CD367 behaves as a partial agonist, displaying also weak antagonist
activity. Another possibility is an interference of CD367 with the
metabolism of atRA and its isomerization into 9-cis-RA.
Whereas the combination of a RAR panagonist (CD367) with a RXR agonist
(CD2425) resulted in a synergic induction of TGase II and apoptosis at
a level close to the one obtained with atRA (but slightly
lower, further supporting the hypothesis of a partial agonist activity
in our model), the combination of the various RAR-subtypes-selective
ligands tested with CD2425 resulted in similar but only partial
synergic induction of TGase II.
These results suggest that full TGase II induction by atRA
probably involved the activation of all the RAR subtypes, ie, RAR, , and present in RPMI 8226 cells, and that these subtypes are partially redundant for this induction. Such a functional redundancy has been already observed in other models.34
Finally, our observation that the expression of TGase II and the
induction of apoptosis are regulated by retinoids in a very similar way
in our cells is in good agreement with previous reports suggesting that
TGase II plays a role in apoptosis.8-10,52,53 With their
complete equipment in RARs and RXRs receptor subtypes, RPMI 8226 cells
afford a very suitable model for further studies of the molecular
control of apoptosis by retinoids in human myeloma.
| |
FOOTNOTES |
Submitted January 2, 1997;
accepted November 11, 1997.
Supported by grants from the Institut National de la Santé et de
la Recherche Médicale (INSERM), the Ligue du Nord Contre le
Cancer, the Association de la Recherche contre le Cancer (ARC), the
CIRD GALDERMA, and Université de Lille II. INSERM U459 belongs to
IFR 22 supported by INSERM, CH et U de Lille, Université de Lille
II, and Centre Oscar Lambret.
Address reprint requests to Pierre Formstecher, INSERM U459 «Signaux,
Récepteurs et Différenciation Cellulaire», Faculté de médecine, 1 place de Verdun 59045 Lille cedex, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Birckbichler for the gift of the monoclonal antibody to
TGase II, Dr V. Gentile for the gift of the TGase II plasmid, and Drs
D.J. Mangelsdorf, P. Chambon, and R.M. Evans for the gift of the RARs
and RXRs plasmids. We are also indebted to B. Masselot, P. Segard, P. Plouvier, and S. Tournay for technical and secretarial assistance. We
thank Drs Uwe Reichert and Serge Michel (CIRD GALDERMA) for providing
RAR- and RXR-selective ligands and for helpful discussion.
| |
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Abstract
Introduction
Abstract
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