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Controls Muc1 Transcription in Trophoblasts
The Jackson Laboratory, Bar Harbor, Maine,1 ,2 Howard Hughes Medical Institute and The Salk Institute, La Jolla, California,3 Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, Arizona4
Received 30 June 2004/ Returned for modification 26 July 2004/ Accepted 20 September 2004
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ABSTRACT |
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(PPAR) is essential for placental development. Here, we show that the mucin gene Muc1 is a PPAR target, whose expression is lost in PPAR null placentas. During differentiation of trophoblast stem cells, PPAR is strongly induced, and Muc1 expression is upregulated by the PPAR agonist rosiglitazone. Muc1 promoter is activated strongly and specifically by liganded PPAR but not PPAR
or PPAR
. A PPAR binding site (DR1) in the proximal Muc1 promoter acts as a basal silencer in the absence of PPAR, and its cooperation with a composite upstream enhancer element is both necessary and sufficient for PPAR-dependent induction of Muc1. In the placenta, MUC1 protein is localized exclusively to the apical surface of the labyrinthine trophoblast around maternal blood sinuses, resembling its luminal localization on secretory epithelia. Last, variably penetrant maternal blood sinus dilation in Muc1-deficient placentas suggests that Muc1 regulation by PPAR contributes to normal placental development but also that the essential functions of PPAR in the organ are mediated by other targets. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PPAR) is an orphan nuclear receptor with diverse biological activities of prime clinical importance (20). It heterodimerizes with RXR to regulate transcription of target genes through response elements (PPREs) comprised of direct repeats of a core motif spaced by 1 bp (underlined) (AGGTCA N AGGTCA; DR1) (15). PPAR is the molecular target for the thiazolidinedione class of insulin sensitizers, which are widely prescribed for the treatment of type II diabetes (9). It is also a key regulator of adipocyte differentiation and regulates genes mediating lipid homeostasis pathways in adipocytes and macrophages (4, 6, 30). In addition, PPAR has been implicated as a differentiation factor and a potential anti-oncogenic target in breast and colon cancer (27).
PPAR deficiency results in death by the 10th day of gestation (E10.0) (3). At this developmental stage, PPAR is expressed abundantly and exclusively in the placenta, and rescue of PPAR null embryos to term by tetraploid chimeras shows that its essential functions are confined to the trophoblast (3). During placentation, structures transducing either maternal or fetal blood interdigitate to form a labyrinthine network of vessels. Histological studies reveal that PPAR null placentas fail to form this vascular labyrinth (3). Fetal blood circulates in the placenta in endothelium-lined vessels that adhere intimately to the trophoblast. In PPAR-deficient placentas, the tight interface between the trophoblast and the fetal endothelium is severely disrupted, and consequently, fetal vessels arrest at the chorionic plate. Once in the placenta, maternal blood leaves the arterial system and bathes the trophoblast through a series of small blood pools, or lacunae, that are lined immediately by the labyrinthine trophoblast (2). These blood pools are dilated and torn in PPAR null placentas, forming an abnormal, continuous blood sinus on the maternal side of the labyrinth, with overt phagocytosis of maternal erythrocytes by junctional zone trophoblasts (3). Normal labyrinthine trophoblast differentiates into a barrier epithelium that separates the maternal and fetal circulations while performing the essential exchange of metabolites between the two (7). This differentiation is critical for vascular remodeling, as demonstrated in various mouse mutants (21). However, the labyrinthine trophoblast of PPAR null placentas fails to undergo typical morphological and cellular changes, such as compaction, syncytium formation, and lipid droplet accumulation (3). Embryos deficient for RXR, alone or in combination with RXRß, exhibit defects that are similar to those seen in PPAR null placentas, demonstrating the functional dependency of PPAR on RXR (22, 31).
Although the list of defects in PPAR null placentas is extensive, no specific target genes have been established for these phenotypes. Here, an effort to identify and characterize transcriptional targets of PPAR revealed that the mucin gene Muc1 is a tightly regulated PPAR target in the placenta and differentiated trophoblast stem cells. This regulation is mediated by the cooperative action of PPAR-binding and nonbinding elements in the proximal part of the Muc1 promoter, whose protein product is confined to the trophoblast layer surrounding the maternal lacunae. This asymmetric distribution is analogous to the previously established localization of MUC1 protein on luminal surfaces of simple secretory epithelia (5) and implicates the maternal lacunae in the placenta as the anatomical analogues of secretory lumens. About half of Muc1 null placentas exhibit dilation of the maternal lacunae, suggesting that Muc1 may participate in this aspect of the PPAR null phenotype. Our data provide new mechanistic insights into PPAR action in trophoblasts, both by implicating it in shared biological regulation of epithelia and trophoblast and by revealing novel combinatorial interactions of PPAR in target regulation.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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+/– (3) or RXR+/– (28) breeder pairs and kept frozen at –80°C. The corresponding genotypes were determined by PCR of yolk sac DNA, as described previously (3), at which stage placentas with similar genotypes were pooled in groups of four, and RNA was extracted with Tri-Reagent. RNA preparations were further purified by treatment with RNase-free DNase and reextraction.
RDA.
Total RNA (1 µg) from either wild-type or PPAR–/– placentas was converted to double-stranded cDNA, using the SMART PCR cDNA synthesis kit (Clontech). This cDNA was amplified through several rounds of long-range PCR, using Advantage Taq polymerase mix (Clontech). The amplified full-length cDNA was digested with DpnII and used to carry out reciprocal representational difference analysis (RDA) essentially as described previously (11), except that amplification of subtracted products was performed by using Advantage Taq polymerase mix and for 13 to 17 amplification cycles only. An additional modification was the supplementation of the subtracted driver cDNA population with Sau3AI-digested PPAR (added to null driver) or lacZ and neo (added to the wild-type [wt] driver) to circumvent differential recloning of these genes. At the end of three rounds of subtraction-amplification, individual bands could be discerned on agarose gels, from which they were isolated and subcloned into pBluescript. Ten plasmid clones from each band were sequenced to determine its predominant composition, and sequences iterated more than once were subjected to BLAST analysis with the National Center for Biotechnology Information database to determine identity as well as being reprobed against RNA from PPAR+/+, PPAR+/–, and PPAR–/– placentas to confirm true differentials.
Trophoblast stem (TS) cell culture. GFP-Trf mouse trophoblast stem cells (29) were cultured on a feeder layer of embryonic fibroblasts in RPMI 1640 medium containing 20% serum, fibroblast growth factor 4 (FGF4; 25 ng/ml; Sigma), and heparin (1 µg/ml), with medium change every other day. Cells were passaged once in the absence of feeder cells in a similar medium supplemented with 70% embryonic fibroblast conditioned medium and then split for the various experiments. Differentiation was accomplished by withdrawing conditioned medium, FGF4, and heparin from the medium. Where appropriate, cultures were supplemented with 1 µM rosiglitazone.
Northern blots, EMSA, transfections, and reporter assays.
Northern blots and an electrophoretic mobility shift assay (EMSA) were carried out as described previously (3, 10). Supershift was performed using concentrated polyclonal -PPAR (H-100) or -RXR (D-20) antibodies (SantaCruz Biotech). Transfections of CV1 cells and reporter assays were carried out with a 48-well format as described previously (9), with some modifications. In short, wells containing 50 to 70% confluent CV1 cells were lipofected with the indicated plasmid combinations, using DOTAP (Avanti Polar Lipids, Inc.). Receptors, reporters, and cytomegalovirus (CMV)-lacZ controls were transfected at 25, 62, and 125 ng/well, respectively. Lipofection medium was replaced 3 to 5 h after transfection with Dulbecco's modified Eagle's medium containing 2% fetal calf serum and the indicated ligand combinations. Cells were extracted 24 to 36 h later and assayed for luciferase and ß-galactosidase activities. Data shown reflect averages and standard deviations for normalized luciferase activity divided by ß-galactosidase activity in triplicate wells from one representative experiment out of at least four repeats with qualitatively similar results.
Histology and immunofluorescence. C57BL/6J Muc1+/– breeder pairs (26) were intercrossed, and pregnancies were timed by monitoring coital plugs. Embryos and placentas were retrieved from pregnant females at the indicated gestational day, and respective genotypes were determined by PCR of DNA from embryonic matter. For histology, placentas were fixed for 24 h in 10% formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin. For immunofluorescence, placentas were fixed overnight in Bouin's solution, washed with running water for 6 h, embedded in paraffin, and sectioned at 5 µm. MUC1 was detected using a diluted hybridoma supernatant containing an Armenian hamster-derived monoclonal antibody against the short cytoplasmic tail (CT2) (S. J. Gendler, unpublished data) and secondary Cy3-conjugated goat anti-Armenian hamster antibody (10 µg/ml; Jackson Immunoresearch). Caveolin-1 was detected by using a polyclonal rabbit antiserum (5 µg/ml; Transduction Labs) and Alexa488-linked goat anti-rabbit antibody (5 µg/ml; Molecular Probes, Inc.). All incubations and washes were carried out in phosphate-buffered saline containing 0.05% Tween 20 and 5% normal goat serum.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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null placentas.
To identify PPAR target genes in trophoblasts, we screened for mRNA species enriched in wt versus PPAR null placentas by using RDA (11). This screen identified the mucin-1 gene, Muc1 (25). Northern blot analysis confirmed that Muc1 is expressed in wt placentas at E9.5 (Fig. 1A, lanes 1 to 2, and 1B, lane 1) and is virtually absent from either PPAR null or RXR null placentas (Fig. 1A, lanes 5 and 6; Fig. 1B, lane 3, respectively). Moreover, placentas heterozygous for either PPAR or RXR express intermediate levels of Muc1 (Fig. 1A, lanes 3 and 4, and 1B, lane 2), suggesting that Muc1 expression is directly proportional to the amount of PPAR-RXR heterodimers in trophoblasts.
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agonist stimulates Muc1 induction in murine trophoblast stem cells.
TS cells proliferate and retain stem cell status as long as they are supplemented with FGF4, heparin, and embryonic fibroblast conditioned medium (29). Once the additives are withdrawn, these cells undergo terminal differentiation, as manifested by the induction of the spongiotrophoblast-specific marker 4311 4 days later (Fig. 2A, lanes 5 and 9). While PPAR is only minimally expressed in proliferating TS cells, it is dramatically induced with the onset of differentiation (Fig. 2B, lanes 1 to 5), recapitulating its association with trophoblast differentiation in the intact placenta (3). Interestingly, rosiglitazone treatment of differentiated TS cells attenuates PPAR expression (Fig. 2B, compare lanes 6 through 9 to lanes 2 through 5), suggesting that PPAR engages in negative autofeedback regulation.
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. In contrast, if TS cells were allowed first to differentiate for 4 days, Muc1 can be induced as early as 4 h after rosiglitazone administration (Fig. 2E, lane 5); Muc1 transcripts continue to accumulate in predifferentiated TS cells for at least 48 h following ligand treatment (Fig. 2E, lanes 6 and 7; also data not shown). These observations suggest that Muc1 responds directly to liganded PPAR in differentiated TS cells and that its failure to express prior to the third day of differentiation likely reflects delayed acquisition of transcriptional competence rather than a slow or indirect response to PPAR.
Robust activation of the Muc1 promoter by PPAR.
The Muc1 promoter was next characterized for response to PPAR by reporter assays with CV1 cells. As shown previously (10), multimerized consensus PPAR response elements (3xDR1) respond readily to rosiglitazone in CV-1 cells in both the absence and presence of cotransfected RXR and PPAR (Fig. 3A). In contrast, the proximal Muc1 promoter (–715 to +33, with +1 denoting the 5' end of Muc1 mRNA) required cotransfection of both PPAR and RXR to induce strong reporter activity (Fig. 3B). Unlike 3xDR1, Muc1 was induced weakly, albeit significantly, by PPAR-RXR even in the absence of an exogenous agonist (three- to fivefold in different experiments), and 1 µM rosiglitazone augmented its activity by an additional three- to fivefold (Fig. 3B). Similar response patterns and magnitudes were exhibited by fragments stretching from the +33 position to as far as –1836 or as near as the –535 position (data not shown). Thus, the proximal 535 bp of the Muc1 promoter contain sequence information that is necessary and sufficient for activation by PPAR. In aggregate, the data presented thus far provide genetic, pharmacological, and transcriptional evidence that Muc1 is a primary PPAR target gene.
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and PPAR.
The distinct biological functions of PPAR, -, and - (14) imply that each must have at least some unique transcriptional targets. However, the promoters of PPAR targets identified so far, such as acyl-coenzyme A oxidase, aP2, lipoprotein lipase, CD36, LXR, and ADRP, are at least in part responsive to more than one PPAR. This pan-specificity is exemplified by the 3xDR1 reporter, which is activated by any of the PPARs in the presence of their corresponding ligands, as shown previously and reiterated here (10) (Fig. 3C). In contrast, the Muc1 promoter is induced solely by PPAR and is entirely refractory to either free or ligand-bound PPAR and PPAR (Fig. 3D). The lack of compensatory activation of the Muc1 promoter by PPAR and PPAR is unique so far among established PPAR targets but is consistent with the cessation of Muc1 expression in PPAR null placentas despite ongoing expression of the two other PPARs (data not shown).
A DR1 element in the proximal Muc1 promoter is a low-affinity PPAR-binding site.
To understand how Muc1 is regulated by PPAR, we first scanned its promoter for potential PPAR response elements (PPREs). A reverse DR1 sequence (5' AGGTGA C AGGTAA 3'; Fig. 4A) was found 
65 bp upstream of the murine Muc1 transcription start site. The orthologous human Muc1 promoter sequence is highly similar (5' AGGTGA C AGGTGA 3') (17). A synthetic oligonucleotide duplex spanning the Muc1 DR1 was bound by a combination of in vitro-translated PPAR and RXR (Fig. 4C, lane 8) but not by RXR or PPAR alone (lane 7; also data not shown). However, combinations of RXR with similar quantities of either PPAR or PPAR (see Fig. 4B) exhibited only residual binding to the same sequence (Fig. 4C, lanes 9 to 12). Reverting the DR1 sequence to a consensus PPRE sequence (5' AGGTCA C AGGTCA 3') within the Muc1 promoter sequence context significantly improved PPAR-RXR binding (Fig. 4C, lane 2) and restored ligand-stimulated binding of PPAR-RXR and PPAR-RXR (lanes 3 to 6). Template competition experiments demonstrated that a 10-fold excess of unlabeled consensus DR1 competed for binding to any of the PPAR-RXR heterodimers as effectively as a 100-fold excess of native Muc1 DR1 (Fig. 4D, compare lanes 5 to 2, 10 to 7, and 15 to 12). Thus, the two-base variation of the Muc1 PPRE compromises its affinity towards all three PPARs equally; its poor interaction with PPAR and PPAR simply reflects their lower inherent DR1 binding potential relative to that of PPAR (Fig. 4C, compare lanes 4 and 6 to lane 2).
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, we assayed a series of reporter constructs, where this element was inactivated by various mutations in the context of the –715
+33 promoter fragment (Fig. 4A). Surprisingly, basal Muc1 promoter activity was increased by either complete deletion of the DR1 or point mutations to either of its halves (Fig. 4E), both of which completely eliminated PPAR binding (Fig. 4F). Although activities of DR1-deficient promoter mutants in the presence of PPAR and rosiglitazone were moderately higher than that of the native Muc1 promoter, the overall effect of PPAR was significantly blunted (Fig. 4E, bottom [fold induction]). These results demonstrate that Muc1 DR1 harbors a basal repressor, and Muc1 expression depends in part on derepression of this element by PPAR. This dependence can be alleviated by eliminating basal repression at the outset.
The human DR1 ortholog (5' AGGTGA C AGGTGA 3') retained robust response to PPAR despite a threefold increase in basal activity (Fig. 4G), demonstrating that Muc1 DR1 is functionally conserved during evolution. In contrast, altering Muc1 DR1 into a consensus PPRE increased basal promoter activity by 50-fold, and while maximal activity in the presence of liganded PPAR was 12-fold higher than that of the native Muc1 promoter, the response differential was blunted from 32x to 7.2x over the basal level (Fig. 4G). Thus, the deviation of Muc1 PPRE from the consensus is critical for basal repression and in turn for tighter dependence of Muc1 on PPAR-mediated derepression, albeit at the expense of maximal expression.
DR1 sites are established repression targets for members of the COUP-TF family of orphan nuclear receptors (32). However, while COUP-TFs bind to a consensus DR1, none bound to the Muc1 variant (data not shown). Moreover, cotransfection of COUP-TFI and -II did not further repress basal or PPAR-dependent activity of the Muc1 promoter (data not shown), suggesting that repression of Muc1 DR1 is mediated by a different factor. To test whether trophoblasts contain activities that correlate with the basal DR1 repression activity from CV1 cells, electromobility shift assays were carried out with extracts of TS cells at various stages of differentiation. Two major DNA-binding activities were observed (Fig. 4H and I). The first activity interacted readily with consensus PPRE and substantially less with Muc1 DR1. It was identified as endogenous PPAR-RXR heterodimers by mobility that was identical to that of in vitro-translated PPAR-RXR, relative abundance which mirrored PPAR expression during TS cell differentiation (see Fig. 2), and full attenuation by either anti-PPAR or anti-RXR antibodies (Fig. 4I, lanes 5 and 6). Most importantly, the extracts contained an additional DNA-binding activity, which migrated slower than PPAR-RXR heterodimers and was refractory to antibodies against either receptor (Fig. 4H, lanes 1 to 5 and 7 to 11; Fig. 4I, lanes 4 to 9, asterisks). This activity exhibited marked preference towards Muc1 DR1 over the consensus PPRE counterpart, similar to the basal repression pattern in CV1 cells. It peaked at the second day of differentiation, declining by the fourth; a repressor with such a temporal profile would potentially account for the delay in Muc1 expression until later in differentiation despite the earlier induction of PPAR (see Fig. 2). These observations correlate Muc1 DR1-binding activity from TS cells to basal silencing activity in CV1 cells.
Induction of Muc1 by PPAR requires both the DR1 motif and a composite enhancer element.
Although DR1 mutations blunted the response of the Muc1 promoter to PPAR, a considerable response was nevertheless retained, implicating additional elements in coregulating Muc1 with PPAR. This notion was confirmed by a series of successive 5' truncations of the promoter. Fragments extending from as near as –76 or as far as –512 to +33, all containing an intact DR1 element, exhibited a markedly compromised response to PPAR (Fig. 5A; also data not shown). Finer truncation experiments (data not shown) narrowed the critical element to a 56-bp sequence between positions –535 and –480 (56U) (Fig. 5B). Systematic point mutations along the entire length of this element (data not shown), as well as partial truncations (e.g., the –531+33 fragment in Fig. 5A), caused only partial loss of the response to PPAR, suggesting that 56U comprises several additive enhancer modules.
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, respectively (Fig. 5C). However, placing the 56U element upstream of a 108-bp proximal Muc1 promoter fragment that includes the DR1 motif (56U/–108) restored basal silencing and in turn an 70-fold response to ligand-activated PPAR. These analyses indicate that regulation of the Muc1 promoter by PPAR is mediated cooperatively by two distinct elements, a low-affinity PPRE that serves as a basal silencer at –65 and a composite enhancer at –500. Each of these elements is modestly responsive to PPAR on its own, and together they control a robust and specific response to PPAR. Analogous localization of MUC1 in the placenta and luminal epithelia. The MUC1 protein is localized to the apical-luminal surface of simple secretory epithelia, such as the milk ducts of the mammary gland (5, 19), as shown in Fig. 6A. To understand the biological significance of MUC1 expression in the placenta, we sought to determine whether it is localized in this organ to a comparable luminal structure.
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emerges as a potential common regulator of these analogous properties.
Low-penetrance maternal vascular defects in the Muc1 null labyrinth.
We next assessed the contribution of MUC1 to placental functions downstream of PPAR by histological analysis of Muc1 null placentas. The normal Mendelian distribution and birth size, as well as the full viability of Muc1 null pups (26), provided no prior evidence of defects in Muc1 null placentas. Our analyses confirmed this notion, although detailed histological inspection revealed minor dilations and tears in the maternal lacunae of 50% of Muc1 null placentas between E12.5 and E18.5 (Compare Fig. 7B and D to Fig. 7A and C, respectively). Two out of nineteen Muc1 null placentas, but none of the wild-type placentas, exhibited expansive thrombi (see Fig. 7E), which could represent harsher manifestations of the same defect. Association of these defects with the maternal lacunae is consistent with the localization of MUC1 to layer I of the labyrinth, around these lacunae. Thus, MUC1 may cooperate with additional targets downstream of PPAR to maintain the integrity of maternal blood pools, which are severely torn and hemorrhagic in PPAR null placentas (3). All other PPAR-dependent histopathies, including labyrinthine trophoblast differentiation, fetal vessel permeation, and lipid droplet accumulation, were normal in Muc1 null placentas (data not shown). These last functions are likely regulated by other PPAR target genes.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in embryonic development. Our studies identify the Muc1 gene as a PPAR target in trophoblasts, reveal a novel combinatorial mechanism of gene regulation by PPAR, and implicate PPAR in regulating epithelial functions of trophoblasts. Importantly, Muc1 is the only direct PPAR target so far with no obvious ties to lipid or energy metabolism, demonstrating the versatile, nonredundant functions of this nuclear receptor in different biological systems.
Novel insights into gene regulation by PPAR.
The dependence of Muc1 expression on PPAR is manifested in every regulatory parameter tested. These include the complete shutdown of Muc1 expression in PPAR null placentas, its strong upregulation by an agonist in TS cells, and the robust PPAR-specific response of its promoter. Muc1 thus provides a new, biologically relevant template for mechanistic studies of PPAR-regulated transcription.
Before discussing the details of Muc1 regulation by PPAR, it is important to address the issue of cell type specificity. Because Muc1 is not expressed in all PPAR-expressing tissues, most notoriously adipocytes or macrophages (data not shown), it is clear that its regulation by PPAR in trophoblasts is tissue specific. Therefore, the activation of its promoter in kidney-derived CV1 cells is surprising. However, this activation is robust, specific for PPAR over PPAR and PPAR, and involves complex interactions between two cis regulatory elements, arguing that it is neither coincidental nor promiscuous. Most importantly, a DNA binding activity from TS cells (Fig. 4H and I), whose preference for Muc1 DR1 over consensus PPRE mirrors that of the basal silencing activity in CV1 cells, suggests that components of the Muc1 regulatory network are likely shared between trophoblasts and CV1 cells. In hindsight, the ability of CV1 cells to support the Muc1 response to PPAR may not be as surprising, considering their reported renal epithelial origin (13) and the notion that emerges here of molecular and cellular similarities between trophoblasts and epithelia.
The response of Muc1 to PPAR is an interplay between two cis elements: a direct repeat sequence (DR1) that comprises a variant PPRE and a composite 56-bp-wide enhancer (56U). The 56U element drives robust transcription independently of PPAR, suggesting that it constitutively docks active transcription factors. However, in the basal state, such as in undifferentiated trophoblasts or nontransfected cells, PPAR is absent, and the DR1 element dominantly silences the promoter.
PPAR-RXR heterodimers can bind the variant DR1, albeit with a 10-fold-lower affinity than consensus PPRE. The affinity of RXR-PPAR and RXR-PPAR heterodimers is reduced similarly, suggesting that preferential activation of Muc1 by PPAR is likely not due to deviation from the consensus. Mutating the DR1 motif not only increases basal Muc1 promoter activity but significantly attenuates the response differential, indicating that PPAR regulates derepression of this element. Surprisingly, when canonical PPRE is restored in place of the original DR1, basal repression is lost and the degree of response to PPAR is blunted. Thus, silencing and temporally controlled PPAR-mediated derepression are critical for Muc1 regulation and require modification of the PPRE sequence. However, cell type specificity and expression intensity must be provided elsewhere. We hypothesize that this crucial biological context for Muc1 induction, as well as its remarkably specific response to PPAR, are mediated by the 56U element, where an epithelium-specific enhancer has been previously characterized (1, 16, 23, 24).
One means of achieving transcriptional cooperativity between the 56U and DR1 elements is for a constitutive, tissue-specific 56U-bound transcription complex of factors and cofactors to tether PPAR-RXR heterodimers to the Muc1 promoter. Ligands increase the interaction of PPAR with various coactivators and could accordingly enhance cooperativity by recruiting PPAR to an integral coactivator component of the 56U-bound complex. Such model would explain the puzzling ability of PPAR and rosiglitazone to directly activate the 56U element (see Fig. 5C, 56U/45). Tethering through 56U should in turn greatly facilitate interaction of the PPAR-RXR heterodimer with its low-affinity DR1 target, which would further cement an active transcription complex on the Muc1 promoter. This model envisions PPAR agonists as mediators of cooperativity between promoter elements, and hence in the control of transcriptional context and specificity, beyond their known role as transcriptional pacemakers.
The combinatorial complexity of Muc1 regulation by PPAR has not been documented previously with other PPAR-regulated promoters. Future studies should reveal whether this form of regulation is unique to Muc1 or whether it has simply been overlooked heretofore.
PPAR, MUC1, and the analogies between trophoblast and epithelia.
The identification of Muc1, a classical marker of luminal epithelia, as a PPAR target in trophoblasts suggests an analogy between the placenta and prototypic luminal epithelia. In the placenta, MUC1 is confined exclusively to the apical surface of the labyrinth, surrounding the lacunae that conduct maternal blood. This pattern reiterates the luminal localization of MUC1 in prototypic glandular epithelia (5). Although glandular lumens contain gland secretions, and the placental "lumens" conduct blood, the analogous distribution of MUC1 in both highlights their architectural similarities and suggests that they share additional properties. The analogy extends to the induction of Muc1 expression upon differentiation of the mammary gland during pregnancy and lactation (19), which resembles its induction during trophoblast differentiation. PPAR is expressed abundantly in the mammary epithelium and other luminal epithelia (12, 18), and it is therefore plausible that its role in these tissues may resemble its placental function.
Muc1 deficiency is not lethal, and does not cause classical manifestations of placental defects, such as intrauterine growth retardation. Approximately 50% of Muc1-deficient placentas exhibit mild structural anomalies in the maternal lacunae, which could reflect a partial role of Muc1 down-regulation in the overt dilation and breakage seen in the lacunae of PPAR null placentas (3). It is equally possible that the major function of Muc1 downstream of PPAR is nonstructural. For example, the MUC1 protein may primarily function to protect the placenta against other genetic or maternally borne insults, such as bacterial pathogens (8). The incomplete penetrance of the phenotype may reflect the variable extent or frequency of these putative insults. The full function of Muc1 has yet to be revealed, but regardless, its elaborate regulation by PPAR suggests that it is a functionally important target rather than a coincidental one. At the same time, the mild Muc1 null phenotype implies that additional targets transduce the essential developmental signals of PPAR in the placenta.
This study finds that the expression of PPAR is tightly regulated in TS cells. In undifferentiated TS cells, PPAR expression is minimal and is confined to rare cells that have differentiated spontaneously (immunofluorescence data [not shown]). Within hours of FGF4 and conditioned medium deprivation, PPAR expression is induced dramatically in the vast majority of cells in the culture, suggesting that it is an early determinant in the differentiation of all trophoblast lineages. These observations align with the importance of PPAR for trophoblast differentiation and placental development in the whole animal (3). The rapid induction of PPAR in differentiating TS cells is reminiscent of its early induction during adipogenesis in vitro and in vivo (30). In contrast, Muc1 is expressed neither in nascent nor in mature adipocytes, whereas adipogenic PPAR target genes, such as aP2, CD36, LXR and lipoprotein lipase, are either absent from the placenta or impervious to the status of PPAR in the organ (data not shown). These differences suggest that while PPAR is intimately involved in early differentiation of both trophoblasts and adipocytes, its mechanisms of action and downstream targets are distinct in each cell type.
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ACKNOWLEDGMENTS |
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R.M.E. is an investigator of the Howard Hughes Medical Institute at The Salk Institute. This work was supported in part by NIH HD044103 to Y.B. and by CORE CA34196 to The Jackson Laboratory.
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FOOTNOTES |
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DR-1 are the mutations introduced experimentally into the sequence in the context of either oligonucleotide probes or full promoter constructs. Equal quantities of in vitro-translated PPARs (B) were mixed with RXR
