# Cryptochrome and Period Proteins Are Regulated by the CLOCK/BMAL1 Gene: Crosstalk between the PPARs/RXRα-Regulated and CLOCK/BMAL1-Regulated Systems

Authors: Koh-ichi Nakamura; Ikuo Inoue; Seiichiro Takahashi; Tsugikazu Komoda; Shigehiro Katayama
Journal: PPAR Research
 (2008)

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## Abstract

Feeding and the circadian system regulate lipid absorption and metabolism, and the expression of enzymes involved in lipid metabolism is believed to be directly controlled by the clock system. To investigate the interaction between the lipid metabolism system and the circadian system, we analyzed the effect of a CLOCK/BMAL1 heterodimer on the transcriptional regulation of PPAR-controlled genes through PPAR response elements (PPREs). Transcription of acyl-CoA oxidase, cellular retinol binding protein II (CRBPII), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase was altered by CLOCK/BMAL1, and transcriptional activity via PPRE by PPARs/RXRα was enhanced by CLOCK/BMAL1 and/or by PPARs ligand/activators. We also found that CLOCK/BMAL1-mediated transcription of period (PER) and cryptochrome (CRY) was modulated by PPARα/RXRα. These results suggest that there may be crosstalk between the PPARs/RXRα-regulated system and the CLOCK/BMAL1-regulated system.


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## Body

## 1. Introduction

Most living
things, including humans, show a circadian rhythm in their activities. The
molecular mechanism of this circadian rhythm has recently been clarified. The
first discovery was identification of the period (PER) gene inDrosophila [1–3]. Later, the single
minded (SIM) and aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms
were discovered in Drosophila, and
ARNT was identified as a dioxin receptor and as a transcription factor.
Thereafter, the three genes (PER, ARNT, and SIM) were shown to have identical
domains, the so-called PAS domain [4]. In addition, these
transcription factors contain the basic-helix-loop-helix (bHLH)-leucine zipper
domains, and SIM and ARNT form heterodimers [5]. Another
variant of these genes was named the CLOCK gene (CLOCK) by Takahashi et al. [6, 7]. Moreover, Ikeda et al.   [8] cloned the brain
and muscle Arnt-like protein 1 (BMAL1) gene (BMAL1) from brain and
muscle tissue and demonstrated its homology to ARNT, including possession of a
bHLH-PAS domain. Recent studies using the two-hybrid method have shown that
BMAL1 forms a heterodimer with CLOCK. This CLOCK/BMAL1 heterodimer can
bind to the mouse PER gene (mPER)
promoter region, and act as a transcriptional regulator [9]. The base sequence 5′-CACGTG-3′, which is located in the promoter  region, has been named
the E-box. There are three isoforms of the PER gene (PER1,2,3). In
addition, cryptochrome genes (CRY1,2) have been identified [10]. Interacting positive and negative transcriptional-translational
feedback loops have been reported to drive circadian oscillations in both Drosophila and mammals. The
best-characterized feedback loop is in the mouse and involves the regulation of
three mPER1-3 and two mCRY1-2 genes [11].
It is now thought that transcription of mPER and mCRY is driven by
accumulating CLOCK/BMAL1 heterodimers, which in turn bind to consensus E-box
elements (5′-CACGTG-3′) [9].The peroxisome proliferator-activated receptor (PPAR) is a member
of the nuclear receptor superfamily [12]. Three types of
PPARs have been described in  rodents, humans, and amphibians: PPARα, PPARβ
(also called PPARδ), and PPARγ. PPARα is expressed most abundantly in the liver
[13] and is  associated with β-oxidation in the mitochondria
and peroxisomes. Although PPARβ/δ is expressed ubiquitously [14], its functional role remains to be clarified. Activation of PPARα
decreases the fatty acid content of cells through β-oxidation of fatty acids,
whereas PPARγ is a transcription factor expressed selectively in adipose tissue
[15] and seems to be  associated with adipocyte
differentiation. Fenofibric acid is a
synthetic ligand of PPARα, and the thiazolidinedione drugs such as troglitazone
are synthetic ligands of PPARγ. No synthetic ligands of PPARβ/δ are so far used clinically.Steroid hormones such as dexamethasone have been found to regulate
these three PPARs. It is well known that circulating concentrations of some
steroids exhibit circadian variability [16], and recent
observations suggest that feeding cycles are capable of regulating peripheral
clocks independent of light stimulation [17], (Stokkan et al. (2001). A recent study has also shown
that the circadian expression of the PPAR gene (PPAR) is regulated by peripheral oscillators in a
CLOCK-dependent manner [18]. Moreover, we have also shown that
CLOCK and BMAL1 play an important role in lipid homeostasis by
regulating the circadian activation of potential PPAR response
element- (PPRE-) controlled target genes [19]. The
partner of PPAR, RXR, interacts with the CLOCK protein in a
ligand-dependent manner and inhibits CLOCK/BMAL1-dependent activation
via an E-box element [20]. McNamara et al. also recently reported that the retinoid X receptor
(RXR) and retinoic acid receptor (RAR) are capable of interacting
with MOP4 and CLOCK. MOP4 is a transcription factor, and
is also called NPAS2. MOP4 is a
paralog of CLOCK [21, 22] that can  dimerize with BMAL1 and
appears to function in a clockwork mechanism in mouse forebrain. RXR has been shown to bind to PPARs, resulting in formation of
the heterodimer, which then binds to the peroxisome proliferator response
element (PPRE), thereby activating target genes including those coding for
acyl-CoA oxidase (AOX), lipoprotein lipase, acyl-CoA synthase, cellular retinol
binding protein II (CRBPII), and 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) synthase [23, 24].In the
present study, we used AOX, CRBPII, and HMG-CoA synthase promoter reporters to
investigate whether their transcriptional activity is regulated by the
CLOCK/BMAL1 heterodimer. We also investigated whether CLOCK/BMAL1 heterodimers
are capable of modulating the expression of PPAR/RXR-regulated genes through
the endogenous factors regulated by CLOCK/BMAL1.


## 2. Materials and Methods

## 2.1. Reagents

Fenofibric acid was provided by Kaken
Pharmaceutical Co. (Tokyo, Japan). We used 10μM fenofibric
acid as a PPARα ligand in our
experiments, a concentration that is below its reported EC50 value
[25].Troglitazone was generously provided by
Sankyo Co. (Tokyo, Japan). The cytotoxicity of the
compounds tested was assessed by the trypan blue dye exclusion method.


## 2.2. DNA Sequencing

DNA sequencing was performed with an
automated sequencer (ABI PRISM 310 Genetic Analyzer; Perkin Elmer, Foster City,
Calif, USA). All
DNA sequences were confirmed by reading both DNA strands.


## 2.3. Western Blotting

An
Amersham ECL kit was used to perform Western blotting to detect specific
immunoreactivity. Briefly, heat-treated 15μg samples of CLOCK/BMAL1 were
subjected to 10% sodium dodecylsulfate polyacrylamide-gel electrophoresis and
transferred to nitrocellulose membranes (Millipore) by semidry blotting.
Molecular weight was determined by using markers (RAINBOW Molecular Weight
Markers, RPN800; Amersham Biosciences, Buckinghamshire, UK).
Antibodies against the proteins coded by the human clock genes PER1, PER2, CRY1, and CRY2 were obtained from Santa Cruz Biotechnology (Calif, USA).Membranes were treated overnight
with TBS-Tween: 5%
dry milk and incubated for 1 hour with 1000-fold diluted goat antibodies to
human PER1, PER2, CRY1,
and CRY2. After washing, the membranes were incubated with
horseradish-peroxidase-conjugated rabbit antigoat monoclonal antibodies.
Antigen detection was performed with a chemiluminescence detection system.


## 2.4. Human PPARα, PPARγ1,
RXRα, CLOCK, and BMAL1 Expression Constructs

DNA fragments encoding full-length human PPARα
(Genebank L-02932), PPARγ1 (Genebank L-40904), and RXRα (Genebank X-52773)
cDNAs were amplified from skeletal muscle cDNA (no.
9514, Takara Shuzo Co., Ltd., Kyoto, Japan) or liver cDNA (no. 9505, Takara Shuzo Co., Ltd.)
and ligated into pCR2.1 (Invitrogen). For PPARα, the 1407 bp Sal I/Not I fragment was subcloned into the Sal I/Not I site of
pCI-neo (Promega), and the vector was named pCI-PPARα. PPARγ1 was subcloned
into the Sal I/Not I site of the vector, and the vector was named pCI-PPARγ1. RXRα
was also subcloned into the Xho I/Not I site of the same vector, and the
vector was named pCI-RXRα.Human full-length cDNAs for CLOCK and BMAL1 were generated by the
method reported previously [26]. The cDNA fragments for CLOCK and
BMAL1 were subcloned into the Sal I/Not I site of pcDNA3 (Invitrogen, Calif, USA), and the
vectors were named pc-CLOCK and pc-BMAL1, respectively.


## 2.5. Luciferase Reporter Assay

Transient transfections
for the reporter assay were performed with Tfx-50 reagent (Promega) by
following the protocol recommended by the manufacturer. 293T human kidney cells
(293T cells), CV-1 monkey kidney cells (CV-1 cells), NIH3T3 cells, and COS-1
cells were used because of their high transfection efficiency.All luciferase reporter
assays were performed using
charcoal-dextran-treated medium (no. SH30068.03; HyClone, Utah, USA). When
medium is treated with charcoal, bioactive factors, such as testosterone,
thyroxine, triiodothyronine, vitamin A, 1,25-dihydroxy-vitamin D, and 17β-estradiol,
are adsorbed by the charcoal, and the concentration of all these hormones is
greatly reduced (as stated in the manufacturer’s protocol). Since all these
hormones act through nuclear receptors such as PPAR, all
luciferase-reporter assays were performed using
charcoal-dextran-treated medium.We used AOX, CRBPII, and HMG-CoA synthase promoter regions [24] ligated into the  Kpn I/Nco I sites of pGL3-Basic (Promega)
upstream of the luciferase gene, and called these constructs pAOX-Luc,
pCRBPII-Luc, and pHMG-Luc, respectively.
Typically, genes
(3 μg) were inserted into 1×106 cells in 100-mm dishes for pAOX-Luc,
pCRBPII-Luc, or pHMG-Luc, and
for pCI-PPARα,
pCI-PPARγ1, pCI-RXRα, pcDNA-CLOCK, or pcDNA-BMAL1. To measure transcription
rates induced by PPARs/RXRα and CLOCK/BMAL1 via the E-box, we used the promoter
region of mPER1 cloned
into the Kpn I/Nco I sites of pGL3-Basic upstream of the luciferase gene, a
construct named pPER-Luc. There are three E-box elements (5′-CACGTG-3′) in the
promoter region (about 2,000 base pairs) of mPER1 [26].pPPRE-Luc, pCRBPII-Luc,
pHMG-Luc or pPER-Luc, together with pRL-TK, in the presence or absence of
pCI-PPARα, pCI-PPARγ1, pCI-RXRα, pcDNA-CLOCK, or pcDNA-BMAL1 vectors, were cotransfected
into cells which were grown in 24-well plates. The total amount of DNA
transfected (0.6 μg) was normalized with a carrier DNA (pCI-neo or pcDNA3).
After 24 hours, cells were stimulated with the PPARs ligand/activator for
another 24 hours. Finally, luciferase activity for pAOX-Luc, pCRBPII-Luc,
pHMG-Luc, or pPER-Luc was normalized to Renilla luciferase activity. All these activities were measured according to the
manufacturer's instructions (Promega).


## 2.6. Transient Transfection/Cotransfection

pCI-PPARα (3 μg), pCI-PPARγ1 (3 μg), pCI-RXRα (3 μg), pcDNA-CLOCK (3 μg), or pcDNA-BMAL1 (3 μg) were transiently transfected
into 1×106 cells in 100 mm dishes by using Tfx-50 Reagent
(Promega) according to the manufacturer’s instructions. After 24 hours, the cells
were stimulated with a test compound for 24 hours and were then analyzed by
Western blot assay.


## 2.7. Deletion Models for the CRBPII-Luc Plasmid (See Figure1), pAOX-Luc Plasmid, and pHMG-Luc Plasmid

The CRBPII gene promoter (5′-gtgtcccactctgctgtcacAGGTCAcAGGTCAcAGGTCAcAGTTCAttttcctgtctctgtc-3′, from −670 to +63)
contains four (5′-AG(G/T)TCA-3′) repeat sequences [23]. We determined whether or
not the action of CLOCK and BMAL1 on the CRBPII promoter was dependent on the
number of AGGTCA sequences. Deletions were introduced into the CRBPII-Luc plasmid (from −670
to +63) by the PCR method or with the Exo/Mung Bean Deletion kit (Stratagene,
Calif, USA) to generate the following reporter constructs: del-0-CRBPII-Luc (5′-cctgtctctgtc-3′, from −599 to +63), del-1-CRBPII-Luc (5′-cAGGTCAttttcctgtctctgtc-3′,
from −610 to +63), del-2-CRBPII-Luc (5′-cAGGTCAcAGGTCA ttttcctgtctctgtc-3′,
from −617 to +63), del-3-CRBPII-Luc (5′-cAGGTCAcAGGTCAcAGGTCAttttcctgtctctgtc-3′, from −624 to
+63),
and del-4-CRBPII-Luc (5′-c AGGTCAcAGGTCAcAGGTCAcAGGTCAttttcctgtctctgtc-3′, from −631 to +63). The
del-0-CRBPII-Luc plasmid contained no (5′-AG(G/T)TCA-3′) repeat sequences; the
del-1-CRBPII-Luc plasmid contained one (5′-AG(G/T)TCA-3′) repeat sequence; the
del-2-CRBPII-Luc plasmid contained two (5′-AG(G/T)TCA-3′) repeat sequences; the
del-3-CRBPII-Luc plasmid contained three (5′-AG(G/T)TCA-3′) repeat sequences,
and the del-4-CRBPII-Luc plasmid contained four (5′-AG(G/T)TCA-3′) repeat
sequences.Figure 1
Delations of PPAR response elements (5′-AG(G/T)TCA-3′) from the promoter region of the cellular retinol binding protein II (CRBPII)-Luc plasmid.Deletions were also introduced into the
pAOX-Luc plasmid or pHMG-Luc plasmid to generate reporter constructs by the
same methods as for the deletions of the CRBPII-Luc plasmid.


## 2.8. Statistical Analysis

Four independent experiments were performed,
and each experiment was performed in triplicate. Parametric data are expressed
as the mean ± standard deviation (SD). Differences between groups were
evaluated by Scheffé's F test. Differences were
analyzed by repeated-measures ANOVA.


## 3. Results and Discussion

To determine whether there is crosstalk between the PPRE-mediated
transcription system and the E-box-mediated transcription system, we first
measured transcriptional activation of the target genes by PPARs/RXRα and
CLOCK/BMAL1 via PPRE. All the
experiments were performed with human kidney 293T cells and CV-1 monkey kidney
cells. Because introduction of pAOX-Luc and pCRBPII-Luc into human kidney 293T
cells, and of pHMG-Luc into CV-1 monkey kidney cells, was particularly
efficient, only these results are presented.The effect ofCLOCK/BMAL1 (see Figure 2) on transcriptional activity of AOX (pAOX-Luc) was determined by a reporter gene assay in 293T cells.
PPARα/RXRα-mediated transcription of the AOX gene via PPRE in 293T cells was
enhanced in charcoal-treated medium by cotransfection with the CLOCK/BMAL1
expression constructs (0∼30 ng). Unexpectedly,
the enhanced PPARs/RXRα-mediated transcription of the AOX gene was
significantly decreased by cotransfection of higher amounts (30∼90 ng) of the CLOCK/BMAL1 expression constructs in
charcoal-treated medium (see Figure 2). When luciferase-reporter
assays were performed using
medium not treated with charcoal-dextran, the results were the exact opposite. Thus, different
results are obtained depending on whether the sample is treated or is not treated with charcoal-dextran (data not
shown). There are indications that serum stimulation can regulate the
transcription of clock genes [27], and the levels of
factors regulating such genes may be altered in culture media when samples are
treated with charcoal-dextran. Shirai  et
al.  [28]  have reported that PPARα is involved in circadian clock control
independently of the  suprachiasmatic nucleus (SCN), which is the central clock in
mammals. Therefore, it is possible that
a PPAR ligand acts on the expression of CLOCK/BMAL1, and that consistent
results depend on the complete removal of this ligand from the serum used.Dose-dependent alteration by theCLOCK/BMAL1 gene (0–220 ng) of
the transcriptional activity of PPARα/RXRα
mediated through PPRE (5′-AGGTCA-3′) elements located in the promoter of acyl-CoA oxidase (AOX) in human kidney 293T
cells in charcoal-treated medium. Alteration by the CLOCK/BMAL1 gene alone (a), plus the PPARα/RXRα gene (50 ng) (b), and plus
the PPARα ligand/activator fenofibric acid (10 μM) (c) in activity of transcription by PPARα/RXRα through PPRE (5′-AGGTCA-3′) elements located in the promoter of AOX. Luciferase activity for pAOX-Luc was
normalized to the luciferase activity of pRL-TK. Experiments were performed in
triplicate, and four independent experiments were performed. All data are means
± SD.
(a)(b)(c)The retinoid receptors RXR and RAR
are capable of interacting with MOP4 and CLOCK [20]. The diminished
PPARs/RXRα-mediated transcription caused by cotransfection of larger amounts of
the CLOCK/BMAL1 heterodimer (30∼90 ng) may be
attributable to binding between CLOCK and RXRα. However, binding between CLOCK
and RXRα does not appear to occur after cotransfection of smaller amounts of
the CLOCK/BMAL1 heterodimer (0∼30 ng), indicating that
there may be an optimal ratio of CLOCK/BMAL1 to PPARα/RXRα. The CLOCK, BMAL1, PPARα, and RXRα gene
products form heterodimers via the bHLH domain to act as transcription factors.
As mentioned above, McNamara et al. have reported that the partner RXRα gene product for PPARα can bind to CLOCK, resulting in decreased
transcriptional activity of PPARα
[20]. Previously, we have found that RXRα  can bind to CLOCK and, further,
that PPARα can bind to CLOCK. In addition, PPARα can bind to BMAL1, and RXRα
can bind to CLOCK. The binding affinities of these interactions are different (Inoue I: Crosstalk between PPARα/RXRα
gene and clock gene (BMAL1/CLOCK). 35th, Japan Atherosclerosis Society, Kyoto
, September, 2003). The
relative transcriptional activities of the various heterodimers of CLOCK,
BMAL1, PPARα, and RXRα remain to be determined.We performed a Western blot analysis of 293T cells transfected
with various amounts ofCLOCK/BMAL1 (0∼100 ng). The increase in CRY1 and PER1 protein levels in the
nucleus peaked at the 30 ng dose of CLOCK/BMAL1 (see Figure 3). Transcription of the mPER and mCRY
genes is now thought to be induced by accumulating CLOCK/BMAL1 heterodimers,
which, in turn, bind to consensus E-box elements. A consensus E-box element is
present in the promoter of the PER  and CRY genes. That is, the CLOCK/BMAL1 heterodimer binds to the E-box,
5′-CACGTG-3′, located in the promoter regions of the PER and
CRY genes, such that the PER and CRY genes can be
regulated by the CLOCK/BMAL1 heterodimer. The PER and CRY gene products have
been shown to inhibit the binding of CLOCK/BMAL1 to consensus E-box elements of the PER and CRY genes, resulting in a
reduction in the transcriptional activity of these genes [9, 29, 30]. From our results, DNA binding of the CLOCK/BMAL1
heterodimer can be regulated
by the protein products of the PER and CRY genes, suggesting that the PER and CRY gene products
can alter the transcriptional activity of the AOX, CRBPII, and HMG-CoA synthase
genes via their PPRE. Further research is necessary to
confirm this suggestion. Oishi et
al. [18] have reported that CLOCK is involved in the transactivation of PPARα. There is the possibility that
increased protein expression of PPARα caused by the CLOCK gene acts directly on
the PPRE. The increases in endogenous PER1 and CRY1 protein levels
induced by exogenous CLOCK/BMAL1 may decrease the PPRE-mediated
transcriptional activity of AOX. Another report suggests that CRY is
capable of acting directly on CLOCK/BMAL1, which would mean that CRY is capable
of regulating the gene expression of CLOCK/BMAL1 [31]. Our
results suggest that the promoter transcriptional activity of pCRBPII could be
modulated by the CLOCK and BMAL1 genes within the dose range 0–100 ng. However,
the change in pCRBPII promoter transcriptional activity induced by the CLOCK
and BMAL1 genes also showed the same tendency over the dose range 100–220 ng
(data not shown), suggesting a similar mechanism of action.Figure 3
Western blotting analysis demonstrating changes in
Period 1 (PER1), Period 2 (PER2), Cryptochrome 1 (CRY1), and Cryptochrome 2
(CRY2) protein levels in the cytosol and nucleus by theCLOCK/BMAL1 gene (0–100 ng). Experiments were performed in
triplicate, and four independent experiments were performed. A typical set of
data is shown.The pAOX-Luc construct contains six
(5′-AGGTCA-3′) repeat sequences, and when all six (5′-AGGTCA-3′) repeat
sequences were deleted, no change in transcriptional activity of the AOX
promoter was observed in 293T cells transfected with the same amounts of
CLOCK/BMAL1 in charcoal-treated medium (see Figure 4).Dose-dependent
changes in activity of PPARα/RXRα-mediated
transcription via the acyl-CoA oxidase (AOX) deletion
promoter by the CLOCK/BMAL1 gene (0–90 ng) in
human kidney 293T cells. Alterations in PPRE-mediated transcription of AOX by the CLOCK/BMAL1 gene
alone (a), plus the PPARα/RXRα gene
(50 ng) (b), and plus the PPARα ligand/activator fenofibric acid (10 μM) (c).
The luciferase activity of del-pAOX-Luc was normalized to the luciferase
activity of pRL-TK. The del-AOX-Luc plasmid contained no repeat sequences
(5′-AGGTCA-3′). Experiments were performed in triplicate, and four independent
experiments were performed. All data are means ± SD.
(a)(b)(c)As stated above, the CRBPII promoter
also contains four (5′-AG(G/T)TCA-3′) repeat sequences, and when all four
(5′-AG(G/T)TCA-3′) repeat sequences were deleted, no change in transcriptional
activity of the CRBPII promoter was observed in 293T cells transfected with the
same amounts of CLOCK/BMAL1 in charcoal-treated medium (see Figure 5). When the
CRBPII promoter containing one repeat sequence (5′-cAGTTCAt-3′), two repeat sequences (5′-cAGGTCAcAGTTCAt-3′), or three repeat sequences (5′-cAGGTCAcAGGTCAcAGTTCAt-3′) was transfected, the change in transcriptional activity of the CRBPII promoter
gradually increased in 293T cells transfected with the same amounts of
CLOCK/BMAL1. When transfected with promoter containing four repeat sequences (5′-cAGGTCAcAGGTCAcAGGTCAcAGTTCAt-3′), the expected change
in transcriptional activity of the CRBPII promoter was observed in 293T cells
transfected with the same amounts of CLOCK/BMAL1 (see Figure 5).Figure 5
Dose-dependent changes in activity of the cellular retinol
binding protein II (CRBPII) promoter in human kidney 293T cells induced by theCLOCK/BMAL1 gene (0–140 ng) plus the PPARα/RXRα gene
(50 ng). The del-0-CRBPII-Luc plasmid contained no repeat sequences (5′-AG(G/T)TCA-3′);
the del-1-CRBPII-Luc plasmid contained one repeat sequence (5′-AG(G/T)TCA-3′);
the del-2-CRBPII-Luc plasmid contained two repeat sequences (5′-AG(G/T)TCA-3′);
the del-3-CRBPII-Luc plasmid contained three repeat sequences
(5′-AG(G/T)TCA-3′), and the del-4-CRBPII-Luc plasmid contained four repeat
sequences (5′-AG(G/T)TCA-3′). The luciferase activity of all pCRBPII-Luc
plasmids was normalized to the luciferase activity of pRL-TK. Experiments were
performed in triplicate, and four independent experiments were performed. All
data are means ± SD.A similar tendency was observed in CV-1 cells transfected with the
promoter of the HMG-CoA synthase gene (see Figure6(a)),
which contains direct (5′-AAAAACTGGGCCAaAGGTCT-3′) repeat sequences [24]. When these direct (5′-AAAAACTGGGCCAaAGGTCT-3′) repeat sequences were deleted from the promoter of the HMG-CoA synthase gene, no change in transcriptional activity of the HMG-CoA synthase promoter was observed in CV-1 cells transfected with the same
amounts of CLOCK/BMAL1 in charcoal-treated medium (see Figure 6(b)).The effect of the CLOCK/BMAL1 heterodimer on PPARα target genes. Dose-dependent changes in activity of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase promoter (a) induced by the CLOCK/BMAL1 gene (0–140 ng) in
the presence and absence of fenofibric acid (10μM) in CV-1 monkey kidney cells
plus the PPARα/RXRα gene (50 ng). When the promoter of the HMG-CoA synthase gene did not contain
direct repeat sequences (del-pHMG-Luc), no change in transcriptional activity of
the HMG-CoA synthase promoter was observed (b). The luciferase activity of pHMG-Luc
was normalized to the luciferase activity of pRL-TK. Experiments were performed
in triplicate, and four independent experiments were performed. All data are
means ± SD.
(a)(b)A similar tendency toward changes in transcriptional activity of
the CRBPII promoter (see Figures7(a), 7(b)) was observed when the PPARγ
ligand/activator troglitazone was added. Transcription of AOX, CRBPII, and
HMG-CoA synthase was increased by CLOCK/BMAL1, and the transcriptional activity
via the PPRE of PPARs/RXRα was enhanced by CLOCK/BMAL1 and/or PPARs
ligand/activator. These results shown in
Figures 6 and 7 clearly indicate that CLOCK/BMAL heterodimer acts as a
positive functional regulator of PPARα and
PPARγ1. However, it is not clear that
whether CLOCK/BMAL heterodimer directly reacts with the promoter regions of target
genes of PPARs,
or bounds to the PPARs /RXRα proteins themselves.The effect of the CLOCK/BMAL1 heterodimer on
PPARγ target genes. Changes in activity of PPARγ1/RXRα-mediated (50 ng) (a) transcription of the cellular retinol binding protein II (CRBPII) promoter by the CLOCK/BMAL1 gene (50 ng) in human kidney
293T cells in the presence and absence of the PPARγ ligand/activator
troglitazone (0–30 μM). The del-0-CRBPII-Luc plasmid contained
no repeat sequences (5′-AG(G/T)TCA-3′). The luciferase activity of pCRBP-Luc
was normalized to the luciferase activity of pRL-TK. Experiments were performed
in triplicate, and four independent experiments were performed. All data are
means ± SD. *P < .05 versus cells not transfected with the CLOCK/BMAL1 gene. Differences were analyzed by repeated-measures ANOVA.
(a)(b)Next, to determine
whether the effect ofCLOCK/BMAL1 on transcription of mPER is
modulated by PPARα/RXRα or PPARγ1/RXRα, we investigated the effect of the CLOCK/BMAL1 gene on transcription of the PER genein 239T cells. Transcription of the mPER1 gene was altered by co-transfection with the
CLOCK/BMAL1 gene (see Figures 8(a), 8(b)); and by cotransfecting
PPARα/RXRα
(see Figure 8(a)) or PPARγ1/RXRα, (see Figure 8(b)) we also found that the PPARα/RXRα
or PPARγ1/RXRα genes modulate transcription of mPER
indicating that PPARα/RXRα and PPARγ1/RXRα function in the negative regulators of
the CLOCK/BMAL1-dependent transcriptions. Recently,
Shirai  et al.  [32] reported that ligands for PPARα and PPARγ do not
significantly affect the transcriptional activity of the mouse PER gene. In our
study, transcriptional activation of the mPER gene by the CLOCK/BMAL1 gene was
altered by transfection of the PPARα/RXRα and PPARγ/RXRα genes compared with that of the control, in
apparent contradistinction to the results of Shirai et al. However, we used
charcoal-treated medium, which would not contain intrinsic ligands for PPARα,
PPARγ, and RXRα making the difference between the two sets of results
difficult to interpret.Inhibition
of CLOCK/BMAL1 byPPARα and PPARγ1. The PPARα/RXRα (a) gene (50 ng) and the PPARγ1/RXRα (b) gene (50 ng) abolished activation of the Period1 (Per1) promoter by the CLOCK/BMAL1 gene (0–50 ng) in human kidney 293T cells in the presence of the PPARα
ligand/activator fenofibric acid (10 μM) (a) and PPARγ ligand/activator troglitazone (10 μM) (b). The luciferase activity of
pPER-Luc was normalized to the luciferase activity of pRL-TK. Experiments were
performed in triplicate, and four independent experiments were performed. All
data are means ± SD. *P < .05 versus cells not transfected with the PPARα/RXRα gene or PPARγ1/RXRα gene. Differences were
analyzed by repeated-measures ANOVA.
(a)(b)We believe
that the change in transcriptional activity of AOX, CRBPII, and HMG-CoA
synthase caused by the presence of the CLOCK/BMAL1 heterodimer may be regulated
via the PPRE (5′-AGGTCA-3′). The PPRE is the site of action not only of PPARα/RXRα but also
of PPARβ/RXRα and PPARδ/RXRα.In conclusion, the results of this study suggest that there is
crosstalk between binding to the PPRE and binding to the E-box by theCLOCK/BMAL1 gene and by the PPARα/RXRα or PPARγ/RXRα genes, and that modulation of the PPRE by the CLOCK/BMAL1 gene affects the activity of
the PPARα/RXRα gene, the PPARγ/RXRα gene, and/or PPARα/γ
ligand/activators.



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*Source: 348610-2008-02-19.xml*