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Laboratoire de Biologie Moléculaire et Cellulaire, Université de Bourgogne, Dijon, France (S.F., S.S., C.G., M.B.); Brain Research Institute (J.B., A.N.) and Institute of Neurology (B.M.), University of Vienna, Vienna, Austria; and Institut National de la Santé et de la Recherche Médicale U488, Le Kremlin-Bicêtre, France (F.C., M.E.E.)
Received September 19, 2002; accepted February 21, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-oxidation
caused by mutations in the ABCD1 (ALD) gene that encodes a
peroxisomal membrane ABC transporter. ABCD2 (ALDR) displays
partial functional redundancy because when overexpressed, it is able to
correct the X-ALD biochemical phenotype. The ABCD2 promoter contains
a putative thyroid hormone-response element conserved in rodents and humans.
In this report, we demonstrate that the element is capable of binding retinoid
X receptor and 3,5,3'-tri-iodothyronine (T3) receptor
(TR) as a heterodimer and mediating T3 responsiveness of
ABCD2 in its promoter context. After a T3 treatment, an
induction of the ABCD2 gene was observed in the liver of normal rats
but not that of TR-/- mice. ABCD2 was not
induced in the brain of the T3-treated rats. However, we report for
the first time that induction of the ABCD2 redundant gene is feasible
in myelin-producing cells (differentiated CG4 oligodendrocytes). The induction
was specific for this cell type because it did not occur in astrocytes.
Furthermore, we observed T3 induction of ABCD2 in human
and mouse ABCD1-deficient fibroblasts, which was correlated with
normalization of the VLCFA -oxidation. Finally, ABCD3
(PMP70), a close homolog of ABCD2, was also induced by
T3 in the liver of control rats, but not that of
TR-/- mice, and in CG4 oligodendrocytes.
). The gene
responsible for X-ALD (ABCD1) belongs to the subfamily D in the ABC
transporter family and encodes a peroxisomal membrane protein called ALDP
(Mosser et al., 1993). The
subfamily D includes the three other genes ABCD2, ABCD3, and
ABCD4 encoding the peroxisomal half-transporters ALDRP (the closest
homolog of ALDP with 63% of identity)
(Lombard-Platet et al., 1996),
PMP70 (Kamijo et al., 1990),
and PMP69 (Holzinger et al.,
1997b), respectively. X-ALD is associated with defective
peroxisomal -oxidation of very-long-chain fatty acids (VLCFA), which
leads to their accumulation in plasma and tissues. It has been postulated that
ALDP, as homodimerized or heterodimerized with one of the three other related
proteins, could provide an entry for VLCFA into the peroxisome.
At present, no completely satisfactory therapy for X-ALD is available
(Moser et al., 2001).
Recently, it has been shown that the drug phenylbutyrate is capable of
normalizing VLCFA levels in fibroblasts from X-ALD patients
(Kemp et al., 1998).
Furthermore, a reduction of the VLCFA excess in plasma and erythrocytes of
X-ALD patients treated with lovastatin has been observed
(Pai et al., 2000). The
studies revealed the induction of the ABCD2 gene expression,
providing a possible mechanism through which the drugs may lower VLCFA levels
in patients with X-ALD (Kemp et al.,
1998; Pai et al.,
2000). Induction of ABCD2 expression and normalization of
VLCFA -oxidation have also been observed in livers of
ABCD1-deficient mice treated with fenofibrate
(Pai et al., 2000). A possible
role for ABCD2 in the clinical course of the disease had already been
suggested because its expression is maximal in the brain and adrenals
(Lombard-Platet et al., 1996).
At the same time, ABCD2 as well as ABCD3 have been shown to
be functionally redundant because their overexpression in X-ALD fibroblasts
allows VLCFA -oxidation to be restored
(Braiterman et al., 1998;
Kemp et al., 1998;
Flavigny et al., 1999;
Netik et al., 1999;
Albet et al., 2001). That
suggests a novel therapeutic strategy for X-ALD because pharmacological
induction of ABCD2 is clearly possible
(Albet et al., 1997;
Kemp et al., 1998;
Pai et al., 2000). At present,
however, the clinical efficacy (improvement in neurological examinations) of
treatment with lovastatin or phenylbutyrate has not been demonstrated
(Pai et al., 2000), and
fibrates seem to be unable to cross the blood-brain barrier
(Berger et al., 1999). Thus,
there is a need to identify new molecules capable of inducing ABCD2
and possibly ABCD3. Our strategy is derived from the capability of
ligand-modulated transcription factors to activate transcription by binding to
DNA response elements. The in silico study of a gene promoter provides
putative response elements, which can then be studied in vitro and in vivo to
define their function.
The thyroid hormones 3,5,3'-tri-iodothyronine (T3) and
3,5,3',5'-tetra-iodo-L-thyronine (or thyroxine;
T4) play a major role in lipid metabolism and brain maturation
(Bernal and Nunez, 1995). They
stimulate peroxisomal fatty-acid -oxidation
(Just and Hartl, 1983) and
peroxisome biogenesis (Fringes and Reith,
1982). Thyroid hormones modulate gene expression by interacting
with thyroid hormone receptors (TR), which are members of the steroid/thyroid
hormone nuclear receptor superfamily. Two distinct genes (TR
and TR) encode several isoforms, mainly TR1 and
TR1, which have a wide tissue distribution, including liver and brain
(Apriletti et al., 1998). TR
binds to a thyroid hormone response element (TRE) characteristically as a
retinoid X receptor RXR/TR heterodimer. A TRE consists of an imperfect direct
repeat of the consensus hexamer 5'-A(G)GGTCA-3' separated by a
4-base pair spacer (DR+4). Inspection of the ABCD2 promoter sequence
revealed a region conserved in rat, mouse, and human containing a DR+4 motif
(in bold) that differs from the consensus TRE by only 1 base pair when reverse
orientation is considered (rat, —392/—367,
5'-GCAGTTGACCTTATTCGACCTCTCCA-3'; mouse,
—387/—362,
5'-GCAGCTGACCTCATTCGACCTCTCCA-3'; and human,
—402/—377,
5'-GCAGATGGCCTGATTCGACCTCTCCA-3')
(Fourcade et al., 2001).
In the present study, we first demonstrated that the DR+4 motif is a
functional TRE that binds TR1 and mediates T3 activation. We
then investigated the regulation of ABCD2 (and ABCD3)
expression in tissues of rat and cultured murine nervous cells upon
T3 treatment. Finally, we examined whether T3 treatment
could restore -oxidation and VLCFA levels in fibroblasts from
ABCD1-deficient mice and patients with X-ALD and whether the
restoration was correlated with the up-regulation of ABCD2.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-deficient (Ecole Normale
Superieure, Lyon, France) were given chow pellets impregnated with 0.15%
6-propyl-2-thiouracil (Harlan, Gannat, France) for 18 days and injected with
T3 (20 µg i.p. per animal per day) or with 0.9% NaCl solution
(control mice) for the last 3 days.
Cell Culture and VLCFA Analysis. COS-7 cells were grown as described
previously (Fourcade et al.,
2001). C6 rat glioma cells were cultured in a 1:1 mixture of Ham's
F-10 (Invitrogen, Carlsbad, CA) and Dulbecco's modified Eagle's medium
supplemented with 7.5% fetal calf serum. CG4 rat glial cells were propagated
in B104 neuro-blastoma cell-conditioned medium and differentiated to mature
oligodendrocytes by culture in the absence of B104 medium for 3 days before
starting T3 treatment (Louis et
al., 1992). Pure astrocytes were prepared from brain of 18-day-old
Sprague-Dawley rat fetuses (Pallud et al.,
1999). Primary cultures of mixed glial cells were derived from the
brains of newborn rats (Besnard et al.,
1989). Control and ABCD1-deficient human and mouse
fibroblasts were cultured as described previously
(Netik et al., 1999). The
content and the -oxidation rate of VLCFA (24:0) in fibroblasts were
determined as described previously (Netik
et al., 1999).
Northern Blot Analysis. Total RNA was extracted from rat tissues as
described previously (Fourcade et al.,
2001). The kits GenElute Mammalian Total RNA (Sigma) and RNeasy
Mini (QIAGEN, Courtaboeuf, France) were used to prepare total RNA from nervous
cells and fibroblasts, respectively. Membranes containing 20 µg/lane of
total RNA were hybridized with -32P–labeled
ABCD2 and ABCD3 cDNA probes as described previously
(Albet et al., 2001).
Autoradiograms were quantified by digital imaging, and the relative abundance
of ABCD2 and ABCD3 mRNA was determined by comparison with the mRNA
levels for rat acidic ribosomal phosphoprotein P0 (36B4). The
36B4 probe was a gift from Dr C. Le Jossic-Corcos (University of
Burgundy, Dijon, France).
Semiquantitative PCR. To study ABCD2 and ABCD3
expression in cultured nervous cells, conventional PCR was performed as
described previously (Fourcade et al.,
2001) except that 25 cycles were used for ABCD2.
Amplification of the control 36B4 was carried out using the forward
(F) and reverse (R) primers 5'-AAYGTGGGCTCCAAGCAGATG-3' and
5'-GAGATGTTCAYCATGTTCAGCAG-3', respectively, with 17 cycles and
60°C as the annealing temperature. When ABCD2 expression was
studied in fibroblasts, PCR was conducted using the primers
5'-GAAGCCTCGGACTTTCATCATC-3' (F) and
5'-GTGTAATTATGGGAACATTTTCAC-3' (R) with 32 cycles and 58°C as
the annealing temperature. Gels were quantified by digital imaging, and the
relative abundance of ABCD2 and ABCD3 mRNA was determined by comparison with
the 36B4 mRNA levels.
Real-Time Quantitative PCR. cDNA generated by reverse transcription
from total RNA extracted from fibroblasts was analyzed by quantitative PCR
using the iCycler iQ real-time PCR detection system (Bio-Rad, Hercules, CA).
The primers (nt 1959) 5'-CACAGCGTGCACCTCTAC-3' (F) and (nt 2032)
5'-AGGACATCTTTCCAGTCCA-3' (R) and the TaqMan fluorescent probe (nt
1986) 5'-HEX-CAAAGAGAAGGAGGATGGGATGC-TAMRA-3' were used for
amplification and detection of ABCD2 mRNA. Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA used as the control was analyzed with the primers
(nt 525) 5'-AGGTCATCCATGACAACTTT-3' (F) and (nt 601)
5'-AGTCTTCTGGGTGGCAGT-3' (R) and the probe (nt 562)
5'-FAM-CATGACCACAGTCCATGCCATAMRA-3'. Standard curves for
quantification were obtained using plasmid containing the mouse ABCD2
or GAPDH cDNA (Berger et al.,
1999). For each assay, 1 and 6 ng of reverse-transcribed RNA was
used for the PCR analysis of GAPDH and ABCD2 mRNA, respectively. The
thermocycler was programmed as follows: 95°C for 10 min, and 50 cycles at
95°C for 20 s and 58°C for 50 s.
Electrophoretic Mobility Shift Assay. EMSA was performed as
described previously (Fourcade et al.,
2001), except for the oligonucleotides
5'-AGCTTCAGGGTCATTTCAGGTCCTTGG-3' (F) and
5'-GATCCCAAGGACCTGAAATGACCCTGA-3' (R), which contain
the functional TRE in the long terminal repeat of Moloney murine leukemia
virus (MMLV-TRE). To carry out EMSA with the receptors RXR and TR, proteins
were synthesized in vitro using the transcription-translation–coupled
Reticulocyte Lysate System (Promega, Madison, WI) from the rat RXR-pSG5
(a gift from Dr. S. Green, Zeneca Pharmaceuticals, Cheschire, UK) and human
TR1-pSG5 (provided by Dr. V. Laudet, ENS, Lyon, France) plasmids.
Binding experiments were performed as described above, except that the probe
(30,000 cpm) was incubated with 1 to 4 µl of the unlabeled RXR
and/or TR1 synthesis mixture (or 2 µl of reticulocyte lysate for the
negative control) instead of nuclear extracts.
Plasmids and Cell Transfection. The plasmids DR+4-pGLUC, p2206, and
p748 were described elsewhere (Fourcade et
al., 2001). The p2206
and p748 plasmids, deprived of
the DR+4 motif, were prepared by deletion of the region —748/—312
and —391/—373, respectively. The p2206 plasmid was
constructed by replacing the BglII/HindIII region with the
PCR product used to obtain the p312 construct
(Fourcade et al., 2001). The
p748 plasmid resulted from ligation between
BglII/HindIII double-digested pGL3-Basic and two PCR
fragments. The PCR fragments were amplified from the original promoter
subclone SH-pKS (Fourcade et al.,
2001) using primers that start in the DR+4 motif and contain an
EcoRI site. After ligation, the junction was as follows:
5'-CCAGgaatTCTCCAG-3' (—395/—366), where lowercase
letters represent modified nucleotides from the original sequence allowing the
creation of an EcoRI site (underscored). The constructs were used in
the transient transfection of COS-7 cells as described previously
(Fourcade et al., 2001).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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and TR1.
We first determined whether nuclear proteins could interact with the DR+4
motif present in the rat ABCD2 promoter in EMSA experiments. The
incubation of probe DR+4 or MMLV-TRE (a well-known functional TRE) with
nuclear extracts from rat liver resulted in the formation of two complexes
(Fig. 1A, lanes 2 and 7), which
were reduced by an excess of unlabeled oligonucleotide
(Fig. 1A, lanes 3 and 8).
Cross-competition experiments also showed reduced complexes
(Fig. 1A, lanes 4 and 8). The
unrelated competitor Sp1 did not alter the complexes
(Fig. 1A, lanes 5 and 10). We
further investigated whether the DR+4 motif could bind an RXR/TR heterodimer.
In the presence of RXR and TR1, two retarded complexes migrated
at the same position as the complexes observed with nuclear extracts
(Fig. 1B). The upper complex
probably corresponded with an RXR/TR1 heterodimer
(Fig. 1B, lane 4) because
TR1 alone was sufficient to form the lower complex
(Fig. 1B, lane 3).
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The DR+4 Motif Is a Functional TRE. To determine whether the
DR+4 motif is a functional TRE, COS-7 cells were transfected with DR+4-pGLUC
plasmid (pGLUC contains a -globin promoter upstream of the reporter
gene). When cells were cotransfected with TR1-pSG5 and treated with
T3, a 5-fold increase in luciferase activity was observed
(Fig. 2). As expected, similar
results were obtained using a construct containing the human ABCD2
DR+4 motif (data not shown).
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To confirm that the DR+4 motif functions in its promoter context, we
transfected COS-7 cells with constructs containing fragments of the rat
ABCD2 promoter cloned upstream from the promoterless luciferase gene
(p2206 and p748). Luciferase activity was induced 2.6-fold by using p2206 and
1.6-fold by using p748 in cells cotransfected with TR1-pSG5 and treated
with T3 (Fig. 2).
The induction was completely abolished when transfection was performed with
the cognate plasmids deleted for the DR+4 motif (p2206 and
p748). The proximal part of the ABCD2 promoter in rat, mouse,
and human is strikingly conserved
(Fourcade et al., 2001),
suggesting that the results obtained in rat might be fully applicable to
human.
T3 Induces ABCD2 Expression in the Liver. To
determine whether our in vitro findings have a physiological relevance, we
studied the in vivo effects of T3 on ABCD2 expression in
the liver, a major target of T3
(Feng et al., 2000) and the
only organ in which ABCD2 has so far proved to be inducible
(Albet et al., 1997), and in
the brain, which is the most important organ for an X-ALD therapy.
ABCD3 was also examined because its expression is positively
regulated by T3 (Hartl and
Just, 1987). A preliminary study carried out in adrenalectomized
and castrated rats revealed that a T4 treatment [12.5 µg/100 g
of b.wt./day] for 3 days was sufficient to induce the expression of the
ABCD2 and ABCD3 genes in the liver by 2.6- and 2.0-fold,
respectively, but we did not detect any induction in the brain (data not
shown). Because the ABCD2 and ABCD3 expression in the brain
did not seem to be sensitive to T4, we treated normal rats by
substituting T4 by T3 (the most metabolically active
hormone) and increasing either the duration (7 days) or the dose (100
µg/100 g of b.wt./day). Again, we observed induction of both genes in the
liver, i.e., 1.8- and 3.5-fold increases in the level of ABCD2 mRNA and 1.7-
and 2.5-fold increases in the level of ABCD3 mRNA at the 10- and
100-µgT3 doses, respectively
(Fig. 3A). No change occurred
in the brain (Fig. 3A). Interestingly, thyroidectomized rats exhibited a lowered expression for both
genes in the liver (Fig. 3B),
indicating that T3 plays a role in their basal hepatic expression;
this lowering did not take place in the brain (data not shown).
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T3 Does Not Induce ABCD2 and ABCD3
Expression in TR—/— Mice. To
confirm the involvement of TR in the T3 induction of ABCD2
observed in the liver, we treated wild-type and
TR-/- mice with T3 and examined the gene
expression in the liver. All of the mice were pretreated with
6-propyl-2-thiouracil because T3 and T4 are markedly
increased in mice lacking TR
(Gauthier et al., 1999). After
18 days of pretreatment, the serum levels of free T4 and
T3 were 24.4 and 7.9 pmol/l in the TR-/-
mice (not injected with T3), respectively, indicating that the
levels of thyroid hormones were normalized. The serum levels of free
T4 (3.8 pmol/l) and T3 (1.4 pmol/l) in the pretreated
wild-type mice (not injected with T3) were similar to those
observed in thyroidectomized animals.
Figure 4 shows that
T3 induction of ABCD2 (x2.0) and ABCD3
(x2.8) occurred in the liver of wild-type mice as expected but not in
the TR-/- mice. In the animals not injected with
T3, the levels of ABCD2 and ABCD3 were higher in
the TR-/- mice than in the wild-type mice
(Fig. 4), as a result of the
differences in the serum levels of T3 and T4 through pleiotropic
effects of the thyroid hormones.
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ABCD2 and ABCD3 Are Up-Regulated by T3 in CG4 Oligodendrocytes but not in Astrocytes. We observed no induction of ABCD2 and ABCD3 by T3 in the whole brain. However, such an analysis might not detect induction restricted to only one type of nervous cell. We therefore performed analyses by Northern blotting and semiquantitative PCR on cell lines and primary cultures of rat glial cells. We treated oligodendrocyte-differentiated CG4 cells with 125 or 500 nm T3 for 3 days. The ABCD2 mRNA level was enhanced by 2.3- and 4.8-fold, and the ABCD3 mRNA level by 1.2- and 2.6-fold, after the dose of T3 (Fig. 5A). When CG4 cells were exposed to 100 nm T3 for different times (2 to 10 days), the induction of ABCD2 and ABCD3 was maintained for all the period of treatment (Fig. 5B). On the other hand, we observed no induction for both genes in C6 cells treated with 100 nm T3 for 3 days (Fig. 5A) or shorter times (6, 24, and 48 h) (data not shown). When mixed primary cultures of oligodendrocytes (approximately 40%) and astrocytes were treated with 100 nm T3 for 3 days, the induction of ABCD2 was still visible although low [x1.24 ± 0.04 as mean ± S.E. (n = 11) from three independent cell preparations; several cultures were conducted from each cell preparation and analyzed by RT-PCR in duplicate] (Fig. 5C). Indeed, no change in ABCD2 and ABCD3 expression occurred in primary cultures of pure astrocytes treated with 0.1 or 1 µM T3 for 3 days (Fig. 5C). The results indicate for the first time that the induction of ABCD2 and ABCD3 in cells of the central nervous system is possible. Furthermore, they suggest that the induction can occur in an oligodendrocytic cell type, the target for X-ALD therapy.
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T3 Induction of the ABCD2 Gene Is Correlated with
Normalization of the X-ALD Biochemical Phenotype. Overexpression of
ABCD2 in fibroblasts from ABCD1-deficient mice or from X-ALD
patients is known to restore -oxidation of VLCFA and to reduce their
intracellular level (Kemp et al.,
1998; Flavigny et al.,
1999; Netik et al.,
1999; Albet et al.,
2001). We thus treated such fibroblasts with T3 to
induce ABCD2 gene expression and to examine the effects of this
induction on -oxidation of VLCFA. We first investigated the dependence
of the ABCD2 mRNA expression on the T3 dose (10 to 100 nm) and the
duration (2 to 10 days) of treatment in ABCD1-deficient mouse
fibroblasts using quantitative PCR. Although large differences between
individual experiments probably obscured statistically significant differences
between treatments, we observed a T3 dose-dependent increase in the
amount of ABCD2 mRNA in cells T3-treated for 2 days
(Fig. 6A). However, the
ABCD2 induction seemed to be transitory, because after treatment with
100 nm T3 for 10 days, the ABCD2 mRNA level was close to the level
measured in untreated cells (Fig.
6A). The pattern of ABCD2 expression was similar when
semiquantitative RT-PCR was used (data not shown). In ABCD1-deficient
mouse fibroblasts exposed to 100 nm T3 for 2 days, the rate of
C24:0 -oxidation increased by 4.3-fold and thus reached a higher level
than in untreated WT fibroblasts (Fig.
6B). C24:0 -oxidation returned to its initial rate after 6
days of T3 treatment. With a delay of a few days, the transitory
effect of T3 on VLCFA -oxidation affected the C26:0 cell
level. Indeed, we observed an approximately 45% decrease in the C26:0 content
in cells treated with T3 for 4 or 6 days
(Fig. 6C). The effect
disappeared in fibroblasts treated for 10 days. We obtained similar results
with X-ALD human fibroblasts (Fig.
6C). The present data suggest that normalization of the X-ALD
biochemical phenotype by T3 results from up-regulation of the
ABCD2 gene expression.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-oxidation (Kemp et al.,
1998; Flavigny et al.,
1999; Netik et al.,
1999; Albet et al.,
2001). In the present study, we were interested in identifying
novel molecules that could up-regulate ABCD2 expression to provide
the basis for pharmacological treatment for patients with X-ALD. By a
molecular analysis of the ABCD2 promoter in rat, mouse, and human, we
found a conserved DR+4 motif fitting the TRE consensus sequence
(Fourcade et al., 2001). In
the present study, we demonstrated that the rat or human ABCD2 DR+4
was able to mediate T3 up-regulation of the reporter luciferase
gene in transient cell transfection experiments. The T3 induction
obtained with the plasmids containing the proximal rat ABCD2 promoter
was moderate, as already seen for other promoters transfected in COS-7 cells
(Simonides et al., 1996).
Moreover, it has been reported that the cotransfection of an RXR
expression plasmid enhances gene induction by T3
(Simonides et al., 1996). This
finding, as well as our demonstration that the ABCD2 DR+4 can bind
RXR/TR1 in vitro, suggests that the DR+4 motif might mediate the
9-cis-retinoic acid induction of ABCD2 observed in an
embryonal carcinoma human cell line
(Troffer-Charlier et al.,
1998). Indeed, the 1.0-kb proximal region of the mouse or human
ABCD2 promoter, which is also highly conserved in rat, has been shown
to be sufficient to promote the 9-cis-retinoic acid activation of a
reporter gene cotransfected with RXR
(Pujol et al., 2000). Thus,
retinoic acid would be likely to boost T3 induction of
ABCD2.
The effects of T3 on ABCD2 expression were then studied
in vivo. We observed an increase in the levels of ABCD2 mRNA in the liver but
not in the brain of the hyperthyroid rats. The T3 induction of
ABCD2 in the liver requires the presence of TR. Moreover,
ABCD2 expression was decreased in the liver but not in the brain of
the thyroidectomized rats. Thus, thyroid hormone seems to be necessary to
maintain the steady-state level of ABCD2 expression, at least in the
liver. Thyroid status might be involved in the clinical variability of X-ALD
because physiological changes in the regulation of the ABCD2
redundant gene may be beneficial (induction) or not (no induction) for the
biochemical status of patients. Our results demonstrate that alteration of the
thyroid status could be used to modify the expression of
T3-sensitive redundant genes. The doses of T3 used in
our experiments may seem unacceptable for humans because of potential side
effects. However, the possibility of selectively targeting the TR
receptor with T3 analogs, such as GC-1, might reduce some
deleterious effects of thyroid hormones
(Baxter et al., 2001).
Thyroid hormone plays an essential role in the developing brain
(Bernal and Nunez, 1995;
Rodriguez-Pena, 1999). In the
brain of the young rat, postnatal days 8 to 30 correspond to the period of
most extensive oligodendrocyte maturation and myelination. T3
concentration in the brain reaches a peak approximately 2 weeks after birth,
which correlates with an increase in TR1 expression and in the activity
of type II iodothyronine deiodinase, the enzyme responsible for the conversion
of T4 to T3 in the brain. Expression of myelin
protein-encoding genes in the rodent brain, including the
myelin-basic-protein gene in which the presence of a TRE has been
demonstrated (Pombo et al.,
1999) and of genes encoding the peroxisomal -oxidation
enzymes, reaches its highest point during the postnatal period. ABCD2
expression, which progressively increases after birth and reaches a maximum
level at approximately days 15 to 21 in the brain of rat
(Albet et al., 2001) and mouse
(Berger et al., 1999), seems to
match the local T3 bioavailability. Furthermore, the population of
microperoxisomes reaches a peak at the period of myelin formation
(Adamo et al., 1986). All of
these findings, and our observation that ABCD2 is
T3-inducible in CG4 oligodendrocytes, suggest that during brain
development, ALDRP is involved in the transport of high amounts of a lipid
required for myelination through T3 induction of ABCD2
expression. This lipid might be docosahexaenoic acid (C22:6 n-3) or
its precursor C24:6 n-3 (Su et
al., 2001). Docosahexaenoic acid accumulates specifically in the
brain during development (Martinez,
1992). Recently, docosahexaenoic acid has been presented as a
ligand for RXR and thus could participate with T3 in ABCD2
induction (de Urquiza et al.,
2000).
An important question is whether ABCD2 is inducible in the adult
brain. Once brain development ends, ABCD2 expression remains at a
high level in human (Holzinger et al.,
1997a) and rodents (Berger et
al., 1999; Albet et al.,
2001), perhaps through a T3-independent mechanism,
which may explain why ABCD2 expression levels do not vary in the
brain of T3-treated or hypothyroid rats. Indeed, Strait et al.
(1997) observed a rapid
increase in myelin-basic-protein expression upon T3
treatment during the first 3 days of differentiation of O-2A oligodendrocytes.
However, the levels of myelin-basic-protein mRNA were not different any longer
in O-2A cells cultured for 10 days in the presence or absence of
T3, indicating that the terminal expression levels were maintained
independently of T3 in differentiated oligodendrocytes. In
contrast, we observed no increase in the ABCD2 mRNA levels in the absence of
T3 during the culture of differentiated CG4 oligodendrocytes. Our
findings do not support the hypothesis that the high levels of ABCD2
expression in the adult brain may be independent of T3. On the
other hand, although ABCD2 is expressed in astrocytes as well as in
oligodendrocytes of adult mouse brain
(Troffer-Charlier et al.,
1998), we observed T3 induction of ABCD2 only
in CG4 oligodendrocytes and not in astrocytes. This suggests that
ABCD2 induction could be restricted to only a single cell type in
adult brain, providing an explanation for the absence of detectable variation
in ABCD2 expression observed during an analysis of whole-brain mRNA
from T3-treated and hypothyroid rats. Induction may also occur in
one or a few specific brain regions. The subependymal zone and hippocampus of
the adult rodent and human brain are known to contain multipotential stem
cells, which are capable of de novo generation of neurons and glia.
Interestingly, when exposed to T3, the multipotential stem cells
generate clones composed entirely of cells with oligodendrocyte morphology
(Johe et al., 1996). A study
of the regulation of ABCD2 expression in stem cells expanded from
neurogenic regions should be of great interest because the stem cells can
rapidly generate myelin-forming cells.
Despite only 38% homology with ALDP, the half-transporter PMP70 can also
partially substitute for ALDP because VLCFA -oxidation is restored in
X-ALD fibroblasts transfected with ABCD3 cDNA
(Braiterman et al., 1998;
Kemp et al., 1998). Thus,
ABCD3 could become a target gene in the same way as ABCD2
for pharmacological therapy of X-ALD. We observed T3 up-regulation
of ABCD3 expression in rat liver as well as in CG-4 cells. Computer
analysis of the mouse and human ABCD3 promoters (0.4 and 3.3 kb,
respectively) did not reveal the existence of a putative TRE
(Gärtner et al., 1998).
The T3 induction levels observed for ABCD3 in the present
study were lower than those for ABCD2. However, the higher content of
PMP70 in the peroxisomal membrane in comparison with ALDRP could compensate
for a relatively low induction level in the context of partial functional
redundancy.
Pharmacological therapy for genetic disease is aimed at up-regulating
redundant genes to compensate for a biochemical defect. Even if the assumption
that VLCFA excess in brain triggers the inflammatory response associated with
progressive demyelination in X-ALD is still in debate, a decrease in the VLCFA
content in brain may be beneficial for patients. In ABCD1-deficient
fibroblasts, we observed a transitory decrease in C26:0 accumulation
correlated with an increase in ABCD2 expression, suggesting that the
restoration of the VLCFA -oxidation resulted from up-regulation of
ABCD2. Several T3 regulation levels are known to play an
important role in maintaining the intracerebral T3 content that is
relatively constant during changes in thyroid status. Thus, increased
T3 concentrations result in depleted TR and type II iodothyronine
deiodinase levels and in enhanced activities of type I and III deiodinases,
the two enzymes that inactivate T3
(Ortiz-Caro et al., 1987;
Kohrle, 1999). Similar
T3 regulation may occur in fibroblasts and may explain the
transitory effects of T3 treatment on VLCFA -oxidation and
ABCD2 expression. Such regulation might be different in
oligodendrocytes, because the induction of ABCD2 was maintained in
T3-treated CG4 cells for 10 days.
In conclusion, we demonstrated that ABCD2 is a T3-responsive gene both in rodent and human cells through a classic TRE and that T3 treatment can induce ABCD2 expression and correct transiently the VLCFA accumulation in X-ALD fibroblasts. Furthermore, we observed that ABCD2 in CG4 oligodendrocytes is responsive to T3, although ABCD2 up-regulation was not found in the whole brain, unlike the liver, of the rat upon T3 treatment. Further studies using ABCD1-deficient mice will be required to evaluate the efficacy of T3 (or T3 analogs) treatment on ABCD2 mRNA and ALDRP protein levels and VLCFA content in the brain.
|
Acknowledgements |
|---|
-/- mice, and Hugo Moser, Sonja
Forss-Petter, and Pamela Talalay for critical reading of the manuscript. |
Footnotes |
|---|
ABBREVIATIONS: X-ALD, X-linked adrenoleukodystrophy; VLCFA, very-long-chain fatty acids; DR+4, direct repeat with a 4-base pair spacer; RXR, retinoid X receptor; T4, 3,5,3',5'-tetra-iodo-L-thyronine (thyroxine); T3, 3,5,3'-tri-iodothyronine; TR, thyroid hormone receptor; TRE, thyroid hormone response element; PCR, polymerase chain reaction; F, forward; R, reverse; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electrophoretic mobility shift assay; MMLV-TRE, Moloney murine leukemia virus thyroid hormone response element; RT-PCR, reverse transcriptase-polymerase chain reaction; WT, wild type; kb, kilobase.
Address correspondence to: Dr. Maurice Bugaut, LBMC, Faculté des Sciences Gabriel, 6 Bd Gabriel, 21000 Dijon, France. E-mail: mbugaut{at}u-bourgogne.fr
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) or cotransfected with
TR
) or both (
); the control cells
were neither treated nor cotransfected (
). Luciferase activity was
expressed as the x-fold induction in relation to untreated pGLUC-transfected
cells. Data represent the mean ± S.E. of three to five independent
experiments performed in triplicate wells. Statistically significant
differences (Student's t test) from controls are indicated by *,
P < 0.01; **, P < 0.001. NS, not significant.
,
control; 
, T3-treated CG4 cells. Assays were conducted in
duplicate, and data represent the means of two independent experiments. C,
semiquantitative RT-PCR analysis of ABCD2 and ABCD3 mRNA from primary
astrocytes (Astro) and from mixed primary cultures of astrocytes and
oligodendrocytes (AO) treated with 100 nM T3 for 3 days.
), and 100 nM
T3 (