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Short Communications |
(PPAR) and Its Coactivators Steroid Receptor Coactivator-1 and PPAR-Binding Protein PBP in the Brown Fat, Urinary Bladder, Colon, and Breast of the Mouse
From the Department of Pathology, Northwestern University Medical School, Chicago, Illinois
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and act as coactivators to enhance ligand-dependent
transcription. We report here that PPAR, SRC-1, and
PBP are differentially expressed in the brown fat, transitional
epithelium of the urinary bladder, colonic mucosa, and
mammary epithelium of the adult mouse. PPAR and PBP are expressed in
the transitional epithelium of urinary bladder and in brown adipose
tissue, but not SRC-1. In the colonic mucosa, PPAR
expression occurs throughout the villi, whereas the expression
of both SRC-1 and PBP is confined mostly to the crypts. The expression
of both SRC-1 and PBP is prominent in the breast epithelium of
nonpregnant, pregnant, and lactating mice,
whereas PPAR expression appeared prominent during lactation. During
early embryonic development, PPAR, SRC-1,
and PBP are differentially expressed, with only limited cell
types displaying overlapping expression. PPAR and PBP expression
overlapped in the brown fat and urogenital sinus at stage E15.5 of
embryogenesis, whereas SRC-1 expression occurred mostly in
neuroepithelium and cartilage between stages E9.5 and E13.5 of
embryogenesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, PPAR
, and PPAR have
been identified as products of separate genes from Xenopus,
rodents, and humans.8-12
PPARs form heterodimers with
9-cis-retinoic acid receptor (RXR), and this
heterodimer complex binds to peroxisome proliferator response elements
on the target gene promoter as PPAR-RXR heterodimers to initiate
transcriptional activity.13
All three PPAR isotypes are
generally co-expressed in most cell types, but their relative levels of
expression appear to vary from one cell type to the
other.14-16
Recent investigations have revealed that
nuclear receptors also interact with other nuclear proteins designated
as coactivators and co-repressors and form macromolecular complexes
that appear to modulate transcription.17-21
It is highly
feasible that these coactivators and co-repressors may act in an
intricate way to modulate the extent of tissue/cell and
species-specific physiological processes controlled by various nuclear
receptors. In an effort to understand possible tissue- and
species-specific differences in the transcriptional activity of PPAR
isotypes, we initiated studies to identify cofactors that modulate PPAR
transcriptional activity and identified mouse SRC-122
and
PBP23
as PPAR coactivators. Analysis of spatiotemporal
expression patterns of these accessory molecules by in situ
hybridization and immunohistochemical approaches will be necessary to
gain insights into the PPAR-controlled lipid metabolism and adipocyte
differentiation. In this report, we describe the expression patterns of
PPAR and its two activators SRC-1 and PBP in the mouse brown adipose
tissue, urinary bladder, colon, and breast.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male C57BL/6J mice were used in these experiments. Embryos were from timed mating, with stage E0.5 defined as noon of the day when the copulatory plug was observed. Mouse embryos or tissues from the adult mice were fixed in 4% paraformaldehyde for 16 to 20 hours at 4°C and paraffin embedded.
Probes
The sense and antisense riboprobes for in situ hybridization were derived from cDNAs as follows: PPAR (nucleotides 870 to 1390),10 SRC-1 (nucleotides 1 to 530),22 and PBP (nucleotides 1461 to 2217).23
In Situ Hybridization
Sections 5 µm thick were cut under ribonuclease-free
conditions.24
Every 6th sagittal section for the embryos
and every 10th section for the adult tissues were stained with
hematoxylin and eosin. Each plasmid was linearized with appropriate
restriction enzymes in the polylinker to generate sense and antisense
riboprobes in the presence of -S35-labeled UTP, (80
µCi, >1000 Ci/mmol; Amersham Corp., Arlington Heights, IL).
Hybridization, washing, and developing conditions were as described by
Wilkinson and Nieto.25
The sections were exposed in NTB2
emulsion (Eastman Kodak, Rochester, NY) at 4°C for 10 days. Heochst
33258 (Boehringer Mannheim, Mannheim, Germany) was used to counterstain
the nuclei. Visualization and photography were performed using
epifluorescence and dark-field microscopy on a Zeiss Axiophot
microscope or simple dark-field microscopy. Identical conditions were
used for sense and antisense sections.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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was expressed abundantly in all cells
of the transitional epithelium of the urinary bladder (Figure 1
, A and B). PBP expression was also
noted in the urinary bladder, but this expression appeared to be
prominent in the basal cells as compared with the differentiated
umbrella cell layer (Figure 1
, C and F). SRC-1 expression in the
urinary bladder was not appreciable. Transitional urothelium lining the
ureter and renal pelvis also showed intense labeling for PPAR.
During the embryonic development, PPAR expression appeared prominent
in the urogenital sinus at E15.5 of embryogenesis (Figure 1
, D and E).
Brown adipose tissue expressed PPAR abundantly at stage E15.5 of
embryogenesis along with PBP (Figure 1
, G to I), and this level of
expression was also maintained in the adult brown fat (not shown).
SRC-1 expression was not detected either in the embryonic or adult
brown fat.
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, SRC-1, and PBP displayed a limited
extent of overlapping expression (Figure 1
, J to O). PPAR expression
appeared dynamic throughout the colonic mucosa, with intense labeling
of all cells lining the villi (Figure 1
, J and M). In contrast, the
expression of both PBP (Figure 1
, K and N) and SRC-1 (Figure 1
, L and
O) appeared restricted almost exclusively to cells lining the crypts.
Another site of differential expression of PPAR and its two
coactivators was the mammary gland, in which PBP and SRC-1 transcripts
were detectable in the virgin, pregnant, and lactating mouse mammary
epithelium (Figure 2
, A to C). In the
mammary gland of virgin mice, few ducts and acinar elements were
present among the white adipose tissue, and these revealed the SRC-1
and PBP transcripts, but very little or no expression of PPAR
(Figure 2A
and data not shown). During pregnancy, there was a
remarkable proliferation of ductular and acinar structures, and these
showed a prominent expression of both PBP and SRC-1 (Figure 2
, B and
C). Lactating mammary epithelium also expressed both coactivators, and
during this phase a modest level of expression of PPAR was observed
(not shown).
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. This specific expression continued in
stage E13.5 embryo (Figure 2F)
with primordia of the developing
vertebrae, femur head, and ribs showing high SRC-1 expression.
Interestingly, olfactory epithelium also displayed high SRC-1
expression. The sense controls for E9.5 and E13.5 (Figure 2
, E and G,
respectively) showed no significant signal. We were unable to detect
any significant expression of PPAR at these E9.5 and E13.5
embryogenic developmental time points (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is an important member of the nuclear receptor
superfamily.7,9,10
It has been shown to regulate adipocyte
differentiation and thus has been implicated as a key protein that may
be involved in regulating biological responses, such as thermogenesis
and fat metabolism.7
Evidence also suggests that ligands
for PPAR are highly effective in reducing levels of inflammatory
cytokines and other mediators of inflammatory responses generated by
activated macrophages.26,27
Furthermore, recent studies
also demonstrate that PPAR and its heterodimeric partner act
synergistically in the conversion of monocytes into foam
cells.28,29
In the present study, we determined the mRNA
expression patterns of PPAR, SRC-1, and PBP in several mouse tissues
by in situ hybridization to gain additional insights into
the possible functional role of PPAR at biological sites other than
the adipose and lymphoid tissues. In addition, we are also interested
in whether, at such biological sites, the PPAR expression is
coincident with the two coactivators, ie, SRC-1 and PBP. This
information is essential in understanding which coactivators are
biologically relevant in mediating PPAR signaling. Our results
demonstrate that, in the urinary bladder and brown fat, SRC-1 is not
co-expressed with PPAR in the adult and during embryogenesis,
whereas PBP is spatiotemporally co-expressed with PPAR in these two
tissues (Figure 1
, A to I, and data not shown), thus suggesting that
SRC-1 is perhaps not biologically relevant in PPAR signaling at
these sites. In addition, the expression of PPAR and PBP in brown
adipose tissue and urothelium during embryogenesis implies a possible
role for these proteins in the differentiation of brown adipose tissue
and transitional epithelium of the urinary bladder. Another important
possibility is that PPAR, which is activated by
15-deoxy-
12,14
prostaglandin J2 which is
present abundantly in urine, may serve as a regulator of inflammatory
response in the urinary bladder. Because chronic inflammation appears
to predispose the transitional epithelium of the urinary bladder mucosa
to neoplastic conversion, it is reasonable to speculate that PPAR,
by virtue of its ability to inhibit inflammatory response and induce
differentiation, may be important in the pathogenesis of bladder
cancer.
An important finding of the current study is the differential
expression of PPAR, SRC-1, and PBP in the colonic mucosa. The
expression of all three molecules appeared abundant in the cells lining
the colonic crypts. PPAR expression appeared robust in the
differentiated enterocytes of the colon, whereas the expression of the
two coactivators is confined mostly to the cells within the crypts of
these villi (Figure 1
, J to O). It is possible that the
co-expression of both coactivators in cells lining the crypts may be
synergistically regulating the PPAR activity in determining a
differentiation commitment of the immature enterocytes. Thus, PPAR
may play a basal function in the undifferentiated enterocytes but
possibly requires additional coactivators for its optimal functioning
in mature intestinal epithelium. The relative paucity of SRC-1 and PBP
in the differentiated epithelium lining the intestinal villi suggests
the presence of additional coactivators of PPAR in the mature
epithelium of the villi. It will be of interest to determine whether
PGC-1,30
a recently discovered PPAR coactivator that is
cold-inducible in brown adipose tissue, is also expressed in the
differentiated epithelium of the intestinal villi along with PPAR.
Strong RXR expression has been reported in the intestinal
mucosa,31
which appears similar to PPAR distribution
described in the present study, suggesting a biological role for these
two heterodimerization partners in this cell type.
On a similar note, both PBP and SRC-1 were also identified in the
ductular epithelium of the nonlactating and pregnant breast tissue, in
which PPAR was only found at low levels (Figure 2
, A to C, and data
not shown). Nonetheless, during lactation, PPAR expression in the
breast appeared prominent. From these findings, it is tempting to
speculate that PBP and SRC-1 regulate ductular proliferation but
require PPAR for differentiation. Recently, SRC-1-null mice were
shown to have defects in ductular branching of the breast
epithelium.32
SRC-1, and to some extent PBP, modulate the
activity of other members of the steroid receptor family besides
PPAR. Thus, an understanding of their biological role is essential,
especially during early development, because embryogenesis is under the
tight control of different nuclear receptors.
We have previously shown that PBP is ubiquitously expressed in several
mouse tissues as early as stage E9.5 of mouse gestation. Because PBP
and SRC-1 both interact with several common nuclear receptors, we also
investigated the developmental regulation of SRC-1 in the present
study. Unlike PBP, SRC-1 expression appeared to be limited to neural
tissue and the somites in the stage E9.5 embryo, thus suggesting that
SRC-1 may be important in the differentiation of somites into cartilage
and muscle. In this regard, its expression is coincident to
RXR.31
We were unable to detect significant expression
of PPAR in the somites. It will be of interest to ascertain whether
PPAR and - isotypes are co-expressed with SRC-1. If this is true,
then it is tempting to speculate that RXR and PPAR and PPAR
along with SRC-1 are the relevant players in regulating target genes in
the somites. SRC-1 is also a member of the basic
helix-loop-helix-PAS family of transcription
factors,33
and to date, its putative heterodimerization
partner has not been identified. Thus, our results regarding its
expression pattern allude to putative biological sites for this
purpose.
Detailed analysis of the relative levels of co-expression of the
three PPAR isotypes in a variety of tissues will be necessary in view
of the increasing functional implications of these three receptors. It
has been reported that in the rat, PPAR and PPAR are co-expressed
in the same cell types in the intestinal mucosa.15
In the
rabbit urinary tract, PPARß and PPAR are co-expressed in the
transitional epithelium.16
A limitation of our study is
that it does not provide information regarding the protein expression
pattern of these nuclear proteins. As appropriate reagents become
available, such characterization would be valuable. Nevertheless, mRNA
expression profiles are an important step toward beginning to
understand the biology of transcription factors and co-factors.
Although the PPAR probe we used for in situ hybridization
in this study was not designed to distinguish between PPAR1 from
PPAR2,10
these results provide important preliminary
understanding of the differential expression of PPAR and its two
coactivators. Our study focused on selected tissues that have either
been shown to be biologically regulated by PPAR (eg, fat) or that
demonstrated novel expression profiles for these transcription factors.
The knowledge of the interplay between PPARs and accessory proteins
becomes important in understanding the control of certain biological
processes, especially during development, differentiation, and
carcinogenesis. These results confirm and extend some of the previous
findings on the expression of PPAR14-16
and provide
novel expression profiles for its coactivators, SRC-1 and PBP.
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Footnotes |
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Supported by National Institutes of Health Grant GM-23750 (to JKR) and a Department of Veterans Affairs Merit Review Grant (to AVY and MSR).
Accepted for publication May 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) gene: alternative promoter use and different splicing yield two PPAR isoforms. Proc Natl Acad Sci USA 1995, 92:4318-4323, -ß, and - in the adult rat. Endocrinology 1996, 137:354-366[Abstract]
. Gene Exp 1996, 6:185-195
agonists inhibit production of monocyte inflammatory cytokines. Nature 1998, 391:82-86[Medline]
promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 1998, 93:241-252[Medline]
. Cell 1998, 93:229-240[Medline]
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