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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2000 as doi:10.1096/fj.00-0363fje. |
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Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain
2Correspondence: Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain. E-mail: gombau{at}porthos.bio.ub.es
SPECIFIC AIMS
The expression of the uncoupling protein-3 (UCP-3) gene in skeletal muscle is under a strict transcriptional regulation. However, the molecular mechanisms controlling the human UCP-3 gene promoter in relation to muscle cell-specific expression and hormonal regulation have not been established. Here we report that the UCP-3 gene is substantially expressed in muscle cells only when differentiated and that UCP-3 promoter activity is largely dependent on MyoD. A novel regulatory pathway for UCP-3 gene expression is established by the identification of retinoic acid (RA) as a major inducer of UCP-3 gene transcription and an RA-responsive element is identified in the proximal region of the human UCP-3 gene promoter.
PRINCIPAL FINDINGS
1. Retinoic acid induces UCP-3 mRNA expression in differentiated
C2C12 and L6E9 muscle cells
Northern blot analysis revealed that neither C2C12 nor L6E9 (mouse
and rat muscle cell lines) expressed detectable levels of UCP-3 mRNA in
the myoblastic stage. Nevertheless, when cells were differentiated and
acquired a myotube phenotype, low but detectable levels of UCP-3 mRNA
were present. When the cells were nondifferentiated, treatment with
all-trans RA had no effect on UCP-3 expression.
Nevertheless, treatment of myotubes with all-trans RA
induced UCP-3 mRNA expression by 8- to 10-fold over basal levels in
both cell types. When 9-cis RA was added to 4, day
differentiated L6E9 cells induction of UCP-3 mRNA expression was about
50% of the induction observed with all-trans RA.
2. Retinoic acid activates the expression of the human UCP-3 gene
promoter in L6E9 cells MyoD is required for the basal promoter activity
and RA responsiveness
We have cloned and sequenced a 3 kb fragment of the human
UCP-3 promoter and this was fused to luciferase reporter gene. The
promoter–reporter constructs -2903hUCP3-Luc and -1588hUCP3-Luc were
used in transient transfection studies in L6E9 cells. Results with both
constructs showed very low basal luciferase activity. To test the
hypothesis that a myogenic factor could be involved in UCP-3 promoter
regulation, we cotransfected the -2903hUCP3-Luc and -1588hUCP3-Luc
with the MyoD expression plasmid, and a 7-fold and 10-fold induction of
the activity over the basal values were obtained, respectively
(Fig. 1
).
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To analyze the role of retinoids in UCP-3 promoter activity,
cotransfection experiments with expression vectors for RAR and RXR were
performed. Results showed that when -1588hUCP3-Luc was transfected in
the absence of cotransfected MyoD, no significant differences over the
basal level were obtained either by cotransfection with RAR
or
RXR alone or together or by the addition of their agonists,
all-trans RA or 9-cis RA. When MyoD was
cotransfected, RA caused a significant induction of the promoter.
Furthermore, when a RAR expression vector was cotransfected,
induction by all-trans RA was much higher (Fig. 1)
. In
contrast, exposure to 9-cis RA in the presence of
cotransfected RXR had no significant effect on UCP-3 promoter
activity. Experiments performed with both types of receptor together
showed a higher induction due to cotransfection, which was
significantly increased by exposure to all-trans RA.
3. Deletion and point mutation analysis of the human UCP-3 promoter
reveals an RA-responsive element in the proximal promoter region
To search for regulatory elements responsible for the effects of
RA on the hUCP3 promoter, a set of deletion mutants was created. A
major deletion that contained only the proximal 5' region of the gene,
-165hUCP3-Luc, retained the same basal activity as the -1588hUCP3-Luc
and was activated eightfold by MyoD. All-trans RA in the
presence of cotransfected MyoD and RAR induced 30-fold the activity
of -165hUCP3-Luc. An additional deletion mutant, -61hUCP3-Luc, still
responded to MyoD but no induction was observed after RA treatment,
indicating that elements for RA responsiveness are present in the human
UCP-3 promoter region between -165 and -61 and are distinct to those
mediating MyoD-dependent activity. Point mutations in a proximal
DR1-like element (see Fig. 2C
) generated on the -165hUCP3-Luc or in the -1588hUCP3-Luc
revealed that none of these mutations affect the ability of MyoD to
activate the expression of the gene. In the presence of MyoD and
RAR, mut1UCP3-Luc lost around 80% of the induction by
all-trans RA when compared to -165hUCP3-Luc, whereas
mut2UCP3-Luc lost around 75%. Mutation of both sites
in the same construct (mut1/mut2hUCP3-Luc)
abolished the activation of the promoter by RA, demonstrating the
involvement of these sites in the regulation of hUCP3 promoter by
all-trans RA.
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4. A DR1-like region in the human UCP-3 gene binds RAR and RXR from
L6E9 nuclear extracts
To search for the proteins that bind the RA-responsive DR1-like
sequence, we performed electrophoretic mobility shift assays with L6E9
nuclei extracts. We used the UCP3-DR1 oligonucleotide, corresponding to
the -79/-57 region of the UCP-3 gene, as a labeled probe (Fig. 2)
.
Four retarded bands appeared: A1, A2, B, and NS. Competition assays
using the nonlabeled UCP3-DR1 probe revealed that the NS band is
nonspecific and the other bands (A1, A2, and B) were specific. The B
band disappeared when either DR5-RARE1 or DR2-RARE2 was added to the
incubation buffer. An excess of an unlabeled unrelated oligonucleotide
(NS) was used as a negative control and, as expected, no competition
effect was detected (Fig. 2A
). The presence of RAR
antibody in the incubation media prevented the formation of the
retarded band B, and a partial reduction in the intensity of the band B
was also observed with the RXR antibody. In addition, the RXR
antibody prevented the formation of bands A1 and A2 whereas the ETS
antibody had no effect (Fig. 2B
). Therefore, it is concluded
that band B is formed by a complex containing RAR and RXR whereas bands
A contain RXR but not RAR. Competition analysis with an excess of
unlabeled oligonucleotides that carry the mutations assayed in the
functional promoter analysis (UCP3-DR1m1 and
UCP3-DR1m2, Fig. 2C
) were performed to identify
the sites involved in the DNA–protein binding. The unlabeled
UCP3-DR1m1 oligonucleotide competed for proteins that form
the complexes A1 and A2. The UCP3-DR1m2 oligonucleotide
competed for the formation of the RAR-containing complex leading to
band B. The oligonucleotide UCP3-DR1m3 had no competition
effect. Thus, this result strongly indicates that site 1 is required
for binding RAR/RXR complexes whereas site 2 is also involved in the
formation of other RXR binding complexes that do not include
RAR.
CONCLUSIONS
Here we have established that the UCP-3 gene is expressed in
cultured muscle cells only when differentiated. Moreover, RA has been
identified as a powerful stimulator of UCP-3 gene expression, although
expression of the UCP-3 gene was sensitive to RA only when cells were
in the myotube stage. These findings are consistent with the behavior
of the human UCP-3 gene promoter when transfected into myoblasts. Basal
expression of the promoter was extremely low and there was no
sensitivity to RA. However, MyoD conferred to the UCP-3 gene promoter a
substantial expression as well as sensitivity to RA. MyoD is a master
regulator of the differentiation program of muscle cell, and a
requirement of the UCP-3 gene promoter for MyoD may be responsible for
the preferential expression of the gene in differentiated muscle cells
and its skeletal muscle-specifc expression in vivo
(Fig. 3
).
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The finding that RAR enhanced the responsiveness of the UCP-3 gene promoter to all-trans RA whereas RXR and 9-cis RA were much less effective indicates the presence of a RARE in the UCP-3 gene. In most of the RA-responsive genes studied so far, RXR has an auxiliary role of providing the heterodimerization partner for RAR and we observed that cotransfection of RAR plus RXR was maximally effective in inducing the UCP-3 gene promoter.
A major RARE of the human UCP-3 gene is identified in the proximal promoter region, between -71 and -59. It consists of a DR1-like motif containing an AGGTCA sequence separated by one base pair from another imperfect half site. This DR1 element was required for RA responsiveness and it bound a nuclear protein complex from L6E9 muscle cells that contains RAR and RXR. We have observed that the DR1 sequence in the UCP-3 promoter also binds other RXR-containing complexes from cell muscle nuclei. The expression of the UCP-3 gene is sensitive in vivo to several hormonal signals other than RA that also act through nuclear receptors, such as, for instance, fatty acid derivatives (via PPARs) or thyroid hormones (via thyroid hormone receptors). Further research will be needed to establish whether the UCP-3 gene promoter is sensitive to activation by other ligands and whether the DR1 RARE identified here acts as a multihormonal cis-acting element in the UCP-3 gene.
The presence of deletion and point mutation constructs that are responsive to MyoD but unresponsive to RA indicated that the RARE in the UCP-3 gene promoter is distinct and physically separated in the DNA from the cis-acting elements eliciting MyoD action. Mapping the precise site for MyoD action in the UCP-3 gene promoter is beyond the scope of the present study, but it appears that MyoD acts through the minimal promoter region of the UCP-3 gene in the absence of a conventional E-box binding element. If, as described in other muscle-specific genes, MyoD activates gene expression by interaction with the basal transcription machinery, such a mechanism would provide a consistent explanation for the dramatic requirement of UCP-3 promoter activity, both basal or RA-stimulated, for MyoD.
In summary, RA action constitutes a novel pathway of regulation of UCP-3 gene expression that, in addition to MyoD action, may be critical for the differentiation and development-dependent regulation of the UCP-3 gene expression in skeletal muscle.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0363fje. To cite this article, use FASEB J. (September 8, 2000) 10.1096/fj.00-0363fje 
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