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J. Biol. Chem., Vol. 279, Issue 22, 22803-22808, May 28, 2004
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From the Molecular Endocrinology Center, William Harvey Research Institute, Bart's and the London, Queen Mary University of London, London, EC1A 7BE, United Kingdom
Received for publication, February 20, 2004
| ABSTRACT |
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(PPAR
) and its heterodimeric partner retinoid X receptor
(RXR
) in adipocyte nuclear extracts. Co-transfection of PPAR
2/RXR
with the pMC2-R(112/+105)GL3 reporter resulted in transcriptional activation in preadipocytes, and this response required an intact PPRE. Mutation of the PPRE to prevent PPAR
/RXR
binding resulted in a complete abrogation of the pMC2-R(112/+105)GL3 reporter activity in day 3 differentiated 3T3-L1 cells, demonstrating a key role played by this site in regulating MC2-R expression in the murine adipocyte. These data highlight a novel mechanism for mc2-r transcription, which may have significance in both adrenal and extra-adrenal sites of expression. | INTRODUCTION |
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Early studies using the murine 3T3-L1 cell line, a widely used model of adipogenesis, demonstrated the appearance of high affinity ACTH binding sites following treatment of growth-arrested cells with a mixture of adipogenic agents including insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (6). Subsequent characterization of the melanocortin receptors expressed by 3T3-L1 adipocytes revealed the presence of both the MC2 and MC5-R. However, a pharmacological analysis showed that the actions of ACTH were mediated solely through the MC2-R in this cell line (2). ACTH exerts a potent lipolytic effect on the murine adipocyte via the cAMP pathway (7, 8) and has more recently been shown to suppress the expression and secretion of the appetite regulating hormone leptin in differentiated 3T3-L1 cells (9). MC2-R expression may therefore be important in providing feedback between the hypothalamo-pituitary-adrenal axis and peripheral leptin production by adipose tissue.
Studies to date on the transcriptional regulation of the murine and human mc2-r have focused solely on expression in adrenal-derived cell lines such as murine Y1 and human H295R cells (1013). Such studies have demonstrated a role for the transcription factor steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor that is widely expressed by steroidogenic tissues and that is important for the expression of key regulators of steroidogenesis in both the adrenal and gonadal systems (14). However, although heterologous expression of SF-1 can induce mc2-r promoter activity in non-expressing, non-adrenal cell lines such as JEG3 and L cells (10, 15), MC2-R is not expressed by all of the SF-1-positive tissues and, therefore, SF-1 is not sufficient for mc2-r gene expression. In contrast, 3T3-L1 adipocytes have been shown not to express SF-1 (16). In the absence of SF-1, mc2-r gene expression must be regulated via an alternative transcriptional mechanism in this cell line and the elucidation of such a mechanism would greatly improve our understanding of the tissue-specific expression of this receptor.
The following study investigates the transcriptional activation of the mc2-r during 3T3-L1 differentiation. We show that murine mc2-r promoter constructs are activated in adipocytes and that the region of activation maps to a peroxisome proliferator response element (PPRE)-like sequence in the proximal promoter. This is a binding site for the nuclear hormone receptor peroxisome proliferator-activated receptor
(PPAR
), a key regulator of adipogenesis that has been shown to directly regulate the expression of genes such as adipocyte P2 (17) and lipoprotein lipase (18). We show that deletion or mutation of this sequence renders mc2-r promoter constructs uninducible during differentiation into adipocytes or by co-transfection with expression vectors for PPAR
2 and its heterodimeric partner retinoid X receptor
(RXR
).
| EXPERIMENTAL PROCEDURES |
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(pCMX-hRXR
) (19) and PPAR
2 (pCMX-mPPAR
2) (20) were the kind gifts of Prof. R. Evans (Salk Institute, San Diego, CA) and Prof. M. Lazar (University of Pennsylvania, Philadelphia, PA, respectively. Site-directed mutagenesis was performed using the QuikChange protocol (Stratagene). Cell Culture3T3-L1 preadipocytes (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium, 10% fetal calf serum (Sigma) at 37 °C with 5% CO2 and differentiated by treating 2-day post-confluent cells (day 0) with medium containing 0.5 mM 3-isobutyl-1-methyl-xanthine, 0.25 µM dexamethasone, and 1 µg/ml insulin for 2 days. On day 2, medium was replaced with insulin only containing medium (1 µg/ml) for a further 48 h before returning the cells to normal cell culture conditions (day 4).
TransfectionsCell lines stably harboring the pMC2-R(1805/+105)GL3 reporter or the empty vector pGL3 were created by calcium phosphate precipitation transfection. Ten micrograms of each plasmid were co-transfected with 0.5 µg of pcDNA3.1 (Invitrogen), which confers resistance to G418S (Geneticin, Invitrogen). Cells were selected and maintained in medium containing 500 µg/ml G418S 24 h after transfection, and the resulting colonies were pooled to produce polyclonal cell cultures. Transient transfections were performed by lipofection using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Cationic lipid was combined in a 2:1 ratio with 2 µg of plasmid DNA and then incubated with the cells under serum-free conditions for 1 h. An equal volume of Dulbecco's modified Eagle's medium, 20% fetal calf serum was then added to the cells, which were subsequently harvested for luciferase assay after 24 h. When differentiating cells were transfected, insulin was included in the media for the 24 h after transfection. All of the transient transfections for luciferase reporter assays included 200 ng of the pRL-CMV Renilla control vector (Promega). For transfections with expression vectors for PPAR
and RXR
, the ratio of reporter, expression vector was kept constant by adding the pCMX empty vector.
Reporter AssaysLuciferase was measured using the Dual Luciferase reporter assay (Promega). Cell lysates were prepared according to the manufacturer's instructions, and luciferase activity was measured using a BioOrbit 1253 luminometer (LabTech International, Sussex, United Kingdom). Reporter activity for transient transfections was calculated by normalizing the reporter luciferase value with that of the Renilla control vector. The activity of the pMC2-R(1805/+105)GL3 reporter in stable cell lines was calculated by normalizing the luciferase values to those of the corresponding pGL3-containing cells.
cAMP AssayCells grown in triplicate wells of a 6-well plate were washed twice with Dulbecco's modified Eagle's medium and then stimulated for 30 min in the presence of 1 mM 3-isobutyl-1-methyl-xanthine with or without ACTH (108 M). The cells were then scraped on ice before being boiled for 5 min. After centrifugation, the supernatant was assayed for cAMP using a competitive binding assay (21). The response of differentiating 3T3-L1 cells to ACTH was calculated for each time point by dividing stimulated cAMP values by unstimulated values.
Reverse Transcriptase (RT) PCRCytoplasmic RNA was harvested from 3T3-L1 cells grown in 6-well plates using the RNeasy miniprep kit (Qiagen) according to the manufacturer's guidelines. 2 µg of RNA was then DNase-treated at 37 °C for 15 min prior to RT. RT was performed at 37 °C for 1 h using Moloney murine leukemia virus-RT and random hexamers (Promega). PCRs were then performed using the cDNA equivalent of 50 ng of cytoplasmic RNA. The following primer sequences (Sigma) were used: MC2-R (forward, 5'-GAGCTGAAGCCAGCAAGC-3', and reverse, 5'-GGATCTGGCTTAGAAGGG-3'); SF-1, (forward, 5'-ATGGAAATGCATCGAATCC-3', and reverse, 5'-AATGCTTGTTGTTCTGGAC-3'); stearoyl-CoA desaturase-1 (SCD-1) (forward, 5'-ACATGCTCCAAGAGATCTC-3', and reverse, 5'-GAGCCTTGTAAGTTCTGTG-3'); glyceraldehyde-3-phosphate dehydrogenase (forward, 5'-TGCACCACCAACTGCTTAG-3', and reverse, 5'-GGATGCAGGGATGATGTTC-3'); and PPAR
2 (forward, 5'-GAGATTCTCCTGTTGACCC-3', and reverse, 5'-AGCTTCAATCGGATGGTTC-3').
Electrophoretic Mobility Shift Assay (EMSA)Probes were created by filling in the 5' overhangs of the annealed oligonucleotides with Klenow DNA polymerase using a mixture of dATP, dGTP, dTTP, and [
-32P]dCTP. Nuclear extracts were prepared by Nonidet P-40-mediated cytoplasmic lysis (22), and EMSA was performed using 10 µg of extract per reaction as described previously (23) using 1 µg of poly(dI·dC)·poly(dI·dC) as competitor (Amersham Biosciences). If antibody was included in the reaction, the initial incubation was extended to 1 h on ice in the presence of 2 µl of rabbit pre-immune serum, PPAR
antibody (Santa Cruz Biotechnology), or RXR
antibody (a kind gift of Prof. R. Evans, Salk Institute, San Diego, CA). Complexes were electrophoresed on a 5% native acrylamide gel, dried, and visualized by autoradiography. Oligonucleotide sequences (Sigma) used were (overhang in lowercase): PPRE wild type upper strand, 5'-gatcTCCCCTTTGGCCTCTCT-3', and PPRE mutant upper strand, 5'-gatcTCCCGATAGGCCTCTCT-3'.
Statistical AnalysisANOVA was performed where appropriate followed by Student's t test. p values <0.05 were considered significant.
| RESULTS |
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The second aspect of MC2-R expression to be measured was promoter activity. 3T3-L1 preadipocytes were stably transfected with the pMC2-R(1805/+105)GL3 reporter. These cells were differentiated, and the luciferase activity at each time point was compared with that of cells expressing the empty vector pGL3. Promoter activity was inferred from these two measurements, and the results show a 2-fold rise in promoter activity in the immediate 24 h following hormonal induction (Fig. 1B). The mc2-r promoter was activated maximally on day 3, and beyond this time point, the activity was found to diminish. A 24-h time lag was observed between changes in the MC2-R promoter and detectable changes in the response to ACTH stimulation. For example, the onset of a functional response to ACTH on day 2 was preceded by promoter activation on day 1, and the peak response to ACTH on day 4 was preceded by peak promoter activity on day 3. In subsequent experiments in which reporter activity was used as a measure of promoter activity in adipocytes, the day 3 time point was chosen as the optimum time to harvest cell lysates following transient transfection.
The transcriptional activation of the mc2-r was also inferred by RT-PCR analysis of RNA harvested from differentiating 3T3-L1 cells using intron-spanning primers (Fig. 1C). Consistent with the observed increase in promoter activation, MC2-R message was detectable 24 h after hormonal induction and this appearance preceded the up-regulation of the adipocyte marker SCD-1 (24) by 72 h. MC2-R transcriptional activation therefore precedes the attainment of terminal characteristics of differentiation and the accumulation of lipid, which was not apparent until day 3 post-induction as measured by Oil-Red O staining.2 RT-PCR was also used to measure SF-1 expression in 3T3-L1 cells. The results confirmed the absence of mRNA for this factor throughout the time course of differentiation, and this result prompted a search for an alternative positive acting factor(s) driving the basal activity of the promoter in adipocytes.
A 59-bp Region of the Proximal Promoter Enhances mc2-r Transcription in the AdipocyteIn an attempt to characterize regions of the mc2-r promoter that confer enhanced activity in the adipocyte, a 5' deletion series was transiently transfected into both undifferentiated and differentiated cells and the activities of the constructs were compared (Fig. 2A). Consistent with the results obtained using stable cells, the activity of the full-length pMC2-R(1805/+105)GL3 reporter was significantly higher in day 3 adipocytes compared with undifferentiated cells and similar significantly enhanced levels of luciferase activity were observed for all of the deletion constructs between 1805 and 112. This suggested that each of these constructs contained within their sequence an adipocyte-specific enhancer. Further deletion of the promoter between 112 and 53 effectively abolished this enhanced activity, indicating that the enhancer sequence(s) resided between these two markers. When this 59-bp region of sequence was analyzed using MatInspector (25), a putative PPRE was identified between 95 and 83 relative to the transcription start site (Fig. 2B). The 13-bp sequence shared a 77% similarity with a consensus direct repeat-1 site to which PPAR
is known to bind as a heterodimer with RXR
(26). PPAR
2 is the adipocyte-specific PPAR
isoform, and it is up-regulated during 3T3-L1 differentiation (27). RT-PCR was used to compare the mRNA profile of PPAR
2 with that of the MC2-R in a time course of differentiation of 3T3-L1 cells. Both mRNAs were up-regulated within the first 24 h following hormonal induction, and therefore, an increase in PPAR
2 levels might explain the activation of the mc2-r (Fig. 2C). This coincidence of PPAR
2/MC2-R expression together with the presence of a PPRE in the regulatory region of the mc2-r promoter highlighted PPAR
2 as a potential candidate regulator of MC2-R expression.
|
/RXR
in Adipocyte Nuclear ExtractsA series of experiments were carried out to test the ability of the 95/83 sequence to bind to endogenous PPAR
/RXR
in 3T3-L1 adipocytes. A radiolabeled double-stranded oligonucleotide probe corresponding to the 95/83 sequence was prepared and used in EMSA with both preadipocyte and day 3 adipocyte nuclear extracts (Fig. 3). No factors binding to this sequence were observed in preadipocyte nuclear extracts, but a DNA-protein complex was seen when adipocyte nuclear extracts were used (lanes 1 and 2). This result was consistent with the hypothesis that the transcription factor regulating the expression of the MC2-R is expressed as the cells begin to differentiate. The binding observed with adipocyte nuclear extract could be abolished by mutating 3 of the 4 core bp (28) within the direct repeat-1 sequence, and this inactivation of the site formed the basis of later mutational analysis (lanes 3 and 4). When in vitro translated RXR
and PPAR
2 were combined with the 95/83 probe, three bands were observed, the largest of which had the same mobility as that formed with day 3 adipocyte nuclear extracts (lane 8). Antibodies to both RXR
and PPAR
were able to supershift the complex formed with adipocyte nuclear extracts (lanes 6 and 7) as well as the upper band formed with in vitro translated protein (lanes 9 and 10), confirming the presence of these two proteins in the gel shift complexes. The lower complex is retarded with the RXR antibody and represents RXR homodimers that are known to bind to a direct repeat-1 site (lane 9) (29, 30).
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/PPAR
2 in Preadipocytes and This Response Maps to the 95 PPREHaving determined that endogenous RXR
and PPAR
in adipocyte nuclear extracts could bind to the 95/83 sequence, the ability of these factors to transactivate the MC2-R promoter was tested. 3T3-L1 preadipocytes were transfected with pMC2-R(112/+105)GL3 and expression constructs for PPAR
2 and RXR
. Consistent with previous studies of PPRE-containing promoters (27, 31), transfection of either PPAR
2 or RXR
expression vectors alone had no effect on the activity of the pMC2-R(112/+105)GL3 reporter in 3T3-L1 preadipocytes. However, when both PPAR
2 and RXR
expression vectors were transfected together with the pMC2-R(112/+105)GL3 reporter, a 9-fold induction of luciferase activity was observed (Fig. 4A). This response to PPAR
2/RXR
was mapped by co-expressing the two factors with a series of fine deletion constructs prepared between 112 and 53, the two markers that had previously been shown to contain the adipocyte-specific enhancer (Fig. 3B). This analysis showed that deletion between 104 and 86, which effectively removes the putative PPRE, resulted in a dramatic reduction in the ability of the promoter to respond to PPAR
2/RXR
. Mutating the 95/83 sequence in the context of the pMC2-R(112/+105)GL3 reporter by introducing, by site-directed mutagenesis, the 3-bp mutation that had previously been shown to abolish PPAR
2/RXR
binding in day 3 differentiated cell extracts (Fig. 3, lane 4) reduced the PPAR
2/RXR
response in preadipocytes to 20% of that of the wild type reporter. These results indicated the requirement of the 95 PPRE in mediating the effects of heterologously expressed PPAR
2/RXR
in undifferentiated 3T3-L1 cells. However, to assess whether or not endogenous PPAR
2/RXR
contribute to the basal activity of the mMC2-R promoter during adipogenesis, the wild-type and 95 PPRE-mutated pMC2-R(112/+105)GL3 reporter constructs were transfected into differentiating 3T3-L1 cells and their activity was compared on day 3 post-induction (Fig. 4D). These data demonstrate the complete abrogation of reporter activity in adipocytes following mutagenesis of the 95 PPRE in the context of the pMC2-R(112/+105)GL3 construct.
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| DISCUSSION |
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Although the up-regulation of MC2-R in murine adipocytes has been documented (33), the precise timing of this event was unknown. We have shown that mc2-r transcription is activated within the first 24 h following hormonal induction, several days prior to the expression of mature adipocyte markers such as SCD-1 (24) and the morphological changes associated with lipid accumulation. The early nature of MC2-R expression raises the possibility that this receptor plays a role in the process of differentiation itself, and the effect of ACTH treatment during adipogenesis is currently being investigated. Also, in addition to its early expression, the transcriptional activation of the mc2-r promoter appears to be transient with peak activity on day 3 and a peak ACTH response on day 4. PPAR
levels remain elevated throughout the time course of differentiation; therefore it is possible that the mc2-r promoter is subject to transcriptional repression after day 3. The mechanism of this down-regulation of promoter activity is the focus of ongoing studies.
Systematic analysis of the mc2-r promoter revealed the presence of an adipocyte-specific enhancer element(s) within a 59-bp region of the proximal promoter. In addition to the putative PPRE at 95/83, a potential signal transducer and activator of transcription (STAT) site was also identified within this region between 68 and 60. STATs 1, 3, and 5 have been shown to be induced during adipogenesis (34, 35), and a dominant negative STAT5 construct can down-regulate a number of genes activated during adipogenesis (35). EMSA experiments using a double-stranded oligonucleotide probe corresponding to the 68/60 sequence failed to show binding to either preadipocyte or adipocyte nuclear extracts, and this sequence was therefore unlikely to be important for MC2-R expression in these cells.2 In contrast, the 95/83 PPRE binds PPAR
2/RXR
heterodimers in adipocyte nuclear extracts and mediates PPAR
2/RXR
-induced transcriptional activation. Furthermore, mutation of this sequence renders the promoter inactive in the adipocyte in the presence of endogenous PPAR
/RXR
and thereby demonstrates the important role played by the 95 PPRE in regulating the expression of the MC2-R gene. These data explain the ability of the MC2-R to be expressed in the adipocyte in the absence of SF-1.
The question as to whether or not human adipocytes express the MC2-R has not been satisfactorily resolved. Early studies looked for a lipolytic response to ACTH equivalent to that of the murine adipocyte in both human and primate tissues (36, 37) and concluded that such a response was absent. Such studies, together with a number of attempts to document the tissue distribution of melanocortin receptor mRNAs (36, 38), have concluded that the MC2-R is not present in human adipocytes, and this lack of expression might be explained by the absence of an equivalent PPRE-like sequence in the proximal promoter of the human mc2-r. However, an analysis of the distal human promoter sequence revealed the presence of at least three putative PPRE-like elements,2 and recent work using both human subcutaneous adipose tissue and human embryonic stem cell line capable of undergoing adipogenesis demonstrated the expression of mc2-r mRNA in the human adipocyte (39). Interestingly, the cell line used in this study is analogous to the 3T3-L1 cell line, being of embryonic mesenchymal origin. The expression of the MC2-R in human adipocytes may therefore be developmentally regulated in adipose tissue as it is in the testes, where expression has been shown to be restricted to fetal Leydig cells and down-regulated in the adult (3).
It is interesting to speculate as to the role of PPAR
in the regulation of mc2-r in tissues other than the adipocyte. For example, PPAR
has been shown to be abundantly expressed in the adrenal gland (40) and this may explain the ability of MC2-R expression to be maintained in SF-1-haploinsufficient mice in which adrenal MC2-R expression is higher in mutant mice than in their wild type siblings (41, 42), suggesting the existence of compensatory mechanisms. PPAR
expression has also been demonstrated in the human pituitary gland where thiazolidinedione PPAR
agonists are a novel therapeutic target for ACTH-secreting adenomas (43, 44). The MC2-R is also expressed in the pituitary gland and has been proposed to function in maintaining a negative feedback loop controlling ACTH secretion (45, 46). Our results suggest a possible mechanism by which thiazolidinediones might exert their anti-tumorigenic effects upon pituitary adenomas through activation of MC2-R expression. Increased MC2-R expression would serve to enhance the proposed negative feedback of ACTH on cell growth that has been previously demonstrated in adrenocortical tumor cells (47, 48).
In conclusion, these data elucidate a novel mechanism of transcriptional regulation for the murine mc2-r and suggest potentially exciting areas of further study in the adrenal and extra-adrenal sites of expression such as the pituitary gland.
| FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 44-20-7601-7444; Fax: 44-20-7601-8468; E-mail: p.j.king{at}qmul.ac.uk.
1 The abbreviations used are: MC2-R, melanocortin 2 receptor; ACTH, adrenocorticotropic hormone; MC5-R, melanocortin 5 receptor; SF-1, steroidogenic factor-1; PPRE, peroxisome proliferator-response element; m, murine; h, human; RXR, retinoid X receptor; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; RT, reverse transcriptase; SCD-1, stearoyl-CoA desaturase-1; STAT, signal transducer and activator of transcription. ![]()
2 L. A. Noon, A. J. L. Clark, and P. J. King, unpublished results. ![]()
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