Department of Hygienic Chemistry, College of Pharmacy, Nihon University,
7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
*
Author for correspondence (e-mail:
shimba{at}pha.nihon-u.ac.jp
)
Accepted April 26, 2001
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SUMMARY |
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Key words: Arylhydrocarbon receptor, Adipose differentiation, 3T3-L1 cells
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INTRODUCTION |
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The AhR is postulated to play important roles not only in the regulation of
xenobiotic metabolism but also in the maintenance of homeostatic functions.
Although a physiological ligand for the AhR has yet to be identified, several
reports have shown constitutive activation of the AhR in the absence of
exogenous ligand (Singh et al.,
1996; Chang and Puga,
1998
; Crawford et al.,
1997
). AhR knockout mice
exhibit decreased liver size, hepatic portal fibrosis, decreased constitutive
expression of certain xenobiotic-metabolizing enzymes such as CYP1A2, and
decreased body size over the first 4 weeks of life, relative to their
littermate controls (Fernandes-Salguero et al.,
1995
; Schmidt et al.,
1996
; Mimura et al.,
1997
). Stable transfection of
AhR cDNA into AhR-defective mouse hepatoma cells has shown that the AhR plays
important roles in control of cell cycle progression and differentiation, and
that no exogenous ligands are required for the function (Ma and Whitlock,
1996
). Absence of the AhR
accelerates entry into senescence in fibroblast cells (Alexander et al.,
1998
). The treatment of
cultured embryos with AhR antisense oligonucleotides resulted in a
significantly lower incidence of both blastocyst formation and mean embryo
cell number (Peters and Wiley,
1995
). In cell
differentiation, the AhR is increased during differentiation toward
keratinocytes and monocytes (Wanner et al.,
1995
; Hayashi et al.,
1995
). By contrast, AhR
protein is found to decrease with ongoing adipose differentiation, resulting
in the loss of functional response to xenobiotics (Shimba et al.,
1998
). Consequently, it is
likely that the AhR is involved in some aspects of the developmental and
differentiation processes.
Several lines of evidence suggest that one of the possible roles of the AhR
is negative regulation of adipose differentiation. We and other groups have
demonstrated that TCDD treatment suppresses the conversion of 3T3-L1
fibroblast cells into adipose cells (Philips et al.,
1995; Brodie et al., 1996a;
Brodie et al., 1996b; Shimba et al.,
1998
). A cell clone that lacks
the ability to transport the AhR into the nucleus is resistant to TCDD (Shimba
et al., 1998
). Moreover,
depletion of the AhR during adipogenesis results in the loss of the inhibitory
effects of TCDD (Shimba et al.,
1998
). These results suggest
that the inhibitory effects of TCDD on adipogenesis depend on the AhR. In
vivo, AhR-null mice exhibited fatty metamorphosis in the liver over the first
2 weeks of life (Schmidt et al.,
1996
). Furthermore, the AhR is
not expressed in liver fat storage cells (Riebniger and Schrenk,
1998
). Collectively, AhR
activation can have profound effects on adipose differentiation.
In this study, we have investigated the role of the AhR in adipose differentiation in 3T3-L1 cells. We show that expression of high levels of AhR sense RNA is able to inhibit significantly the accumulation of lipid, as well as the induction of adiposity-specific genes. In contrast, lowering AhR levels in 3T3-L1 cells, using antisense AhR mRNA, induces much greater differentiation. These results strongly suggest that the AhR plays a role in the negative regulation of adipose differentiation in 3T3-L1 cells.
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MATERIALS AND METHODS |
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Stable transfection
To construct the AhR mRNA expression vector, full-length murine AhR cDNA
was subcloned into the mammalian expression vector pRc/CMV2 (Invitrogen),
which contains a selective marker, the neomycin resistance gene. Similarly,
antisense AhR mRNA expression vector was constructed by inserting full-length
AhR cDNA in the antisense orientation. The cells were transfected by using
Lipofectin (Life Technologies) and were allowed to grow in nonselective medium
for 48 hours. The cells were then cultured in medium containing G-418 (450
µg/ml). After 2 to 3 weeks, clones were isolated and expanded individually.
The expression of AhR protein in each clone was analyzed by western
blotting.
Immunoblot analysis
The cells grown in 60 mm dishes were rinsed with ice-cold
phosphate-buffered saline (PBS). The rinsed cells were scraped off the dish,
placed in a microcentrifuge tube, and centrifuged at 5000 g
for 1 minute. The resulting pellets were suspended in the lysis buffer (50 mM
Hepes KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 1 mM DTT,
0.5 mM PMSF, 0.0002% leupeptin and 20% glycerol), vortex-mixed, and rocked at
4°C for 60 minutes. The suspensions were centrifuged for 15 minutes at
10000 g and the resulting supernatants were then frozen until
further analysis. The protein concentration of the extracts was determined
according to the method of Bradford, using bovine serum albumin as standard
(Bradford, 1976). Protein
samples were denatured by heating to 90°C in SDS-reducing buffer and were
resolved by electrophoresis on 10% SDS-polyacrylamide gels. After transfer to
a nitrocellulose membrane, the filters were probed with the antibodies. Color
visualization was performed with secondary antibodies conjugated with alkaline
phosphatase and nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate substrate solution (Promega).
Protein expression was quantified with the use of National Institutes of
Health Image 1.61 software as described previously (Pollenz,
1996
).
Oil red O staining
To judge the states of adipose differentiation by visual inspection,
cultures were fixed with 10% formalin in PBS for 2 hours, rinsed three times
with distilled water and then air-dried. The fixed cells were stained with
0.5% oil red O solution for 1 hour. After staining, the cultures were rinsed
several times with 70% ethanol.
Glycerophosphate dehydrogenase (GPDH) activity
Cells grown in 35 mm culture dishes were rinsed twice with ice-cold PBS,
scraped into 0.2 ml of the extraction buffer (25 mM Tris-HCl (pH 7.5), 1 mM
EDTA), and homogenized with a Teflon pestle drill apparatus. The homogenate
was centrifuged for 10 minutes at 4°C. GPDH activity was assayed in the
supernatant by monitoring the decrease in absorbance at 340 nm of NADH in the
presence of dihydroxyacetone phosphate (Wise and Green,
1979).
Analysis of RNA
Total RNA was extracted from the clones with TRIzol reagent (Life
Technologies) according to the manufacturer's instructions and was analyzed by
northern blotting. Probes corresponding to C/EBPs, PPAR, aP2 and CYP1B1 were
prepared by RT-PCR techniques using Bca PLUS RTase and LA taq with GC buffer
(Takara Biomed, Japan).
In vitro kinase assay
Active p42/p44 MAP kinase was immunoprecipitated in extraction buffer (20
mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% Triton X-100, 2.5
mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM
Na3VO4, 1 µg leupeptin, 1 mM PMSF) from the cells.
Immunoprecipitates were washed twice in extraction buffer followed by two
washes in kinase buffer (25 mM Tris-HCl (pH 7.5), 1 mM glycerophosphate, 0.1
mM Na3VO4, 2 mM DTT, 10 mM MgCl2). Kinase
reaction was incubated in the presence of Elk-1 protein (2 µg) and 200
µM ATP at 30°C for 30 minutes followed by the addition of SDS-reducing
buffer. Samples were run on a 10% SDS-polyacrylamide gels and visualized by
western blotting using anti-phospho Elk-1 antibody.
Flow cytometry
Cells were collected by mild trypsinization and gentle centrifugation and
were fixed in 70% ethanol. The fixed cells were washed twice with PBS and
resuspended in Propidium Iodine (PI) solution (10 mg of DNase-free RNase A and
100 mg of PI per ml of PBS). DNA content in the cells was analyzed with
FACScan (Becton Dickinson).
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RESULTS |
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Isolation of cells overexpressing the AhR or expressing antisense
AhR
To directly assess the participation of the AhR in adipogenesis, 3T3-L1
cells were stably transfected with a vector expressing high levels of
full-length sense AhR mRNA, antisense AhR mRNA or a control vector. After
selection and expansion of stable clones, the level of the AhR protein was
examined by western blotting. On the basis of AhR protein level, two clones
expressing control vector mRNA, three clones expressing AhR sense mRNA and
three clones expressing antisense AhR mRNA were chosen for subsequent studies.
As shown in Fig. 2A, the cells
transfected with the vector expressing sense AhR mRNA produced more AhR
protein compared with the control vector-transfected cells and untreated
cells, as analyzed by western blotting. In contrast, expression of the
full-length AhR antisense mRNA resulted in a substantial decrease in the
levels of the AhR protein. To confirm the activity of AhR in these clones,
they were treated with 3-MC (1 µM) and the induction of CYP1B1 mRNA was
analyzed. Note that CYP1A1, which is extensively used as the marker for
AhR-mediated cellular response, is not induced in fibroblastic cells,
including 3T3-L1 cells (Gradin,
1999), and therefore CYP1B1
expression was examined in this study. In the sense cells, high levels of
CYP1B1 mRNA were expressed in the presence and absence of 3-MC
(Fig. 2B). By contrast the
antisense cells failed to induce CYP1B1 mRNA, although they expressed a basal
level of CYP1B1 mRNA. These results indicate that the increased levels of AhR
in the sense cells, and the decreased levels of AhR in the antisense cells,
are of sufficient magnitude to affect AhR-mediated signaling activity.
|
The AhR is involved in negative regulation of adipose
differentiation
To evaluate the differentiation potency of the clones, the cells were
cultured to confluence and then treated with a standard
differentiation-induction medium containing DEX, IBMX and insulin. The extent
of differentiation was estimated by adipose staining with oil red O and
measurement of activity of glycerophosphate dehydrogenase (GPDH), a marker
enzyme of adipogenesis. The sense and antisense cell clones, as well as
control cells, showed no signs of lipid accumulation if cultured in the
absence of differentiation-inducing agents (data not shown). When cell clones
were treated with differentiation medium, wild type 3T3-L1 cells and the cells
expressing vector mRNA exhibited a similar degree of lipid accumulation and
induction of GPDH activity (Fig.
3A, middle lane; Fig.
3B). However, the cells overexpressing AhR accumulated a minimum
amount of lipid droplets and very little GPDH activity was induced.
Conversely, the antisense cells showed somewhat more lipid accumulation and
marked induction of GPDH activity compared with the control cells
(Figs 3A,B). These results were
confirmed by measuring the expression of adipocyte-related genes, such as aP2,
CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator activator
receptor (PPAR) 2. As expected, expression of these genes was greatly
induced in the control cells after 6 days of induction
(Fig. 3C). Consistent with the
morphological observations and GPDH activity, a minimum amount of aP2 and
C/EBP
mRNA was induced in the AhR sense cells. Interestingly, the
induction of PPAR
2 was not substantially affected by overexpression of
the AhR. However, enhanced induction of these genes was observed in the
antisense cells (Fig. 3C).
These effects of AhR on the expression of adipocyte-specific genes are similar
to those of TCDD (Liu et al.,
1996
).
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The inhibitory effect of the AhR on adipogenesis is mediated by
p42/p44 MAP kinase
In adipose differentiation, p42/p44 kinase activation is required for
clonal expansion, whereas hyperactivation of p42/p44 MAP kinase cascade
results in inhibition of differentiation (Sale et al.,
1995; Font de Mora et al.,
1997
). As shown in
Fig. 5A, p42/p44 MAP kinase
activity was gradually decreased during adipogenesis in the control cells. By
contrast, p42/p44 MAP kinase activity in the sense cells was maintained
throughout day 5, and, as a result, the activity in the sense cells was higher
than that in the control cells on day 3-5
(Fig. 5A). Western blot
analysis showed that the p42/p44 protein was present in equal amounts through
all the time points (Fig. 5B).
In addition, there was no substantial differences in the p42/p44 protein level
in between the control cells and the sense cells, indicating that the apparent
differences in kinase activity is not simply due to variation in the protein
level (Fig. 5B). To examine
whether the higher activity of p42/p44 MAP kinase observed accounts for the
lower potency of differentiation in the sense cells, cell clones were treated
with PD98059 or U0126, the specific inhibitors of upstream of p42/p44 MAP
kinase. Co-treatment with the conventional differentiation cocktail and
PD98059 or U0126 restores the ability of the sense cells to differentiate
(Fig. 5C). Treatment of the
cells with SB203580, a specific inhibitor of p38 MAP kinase, inhibited
adipogenesis (Fig. 5C). These
results indicate that the hyperactivation of p42/p44 MAP kinase, at least
partly, mediates the inhibitory effect of the AhR on adipose
differentiation.
|
AhR activation inhibits clonal expansion
As shown by the previous result, AhR influences a very early stage in the
differentiation pathway. Thus, we examined the effect of the AhR on clonal
expansion, which is the earliest event in adipogenesis. In a first set of
experiments, wild-type 3T3-L1 cells were treated with 10 nM TCDD and cell
proliferation was determined. Treatment of the cells with TCDD suppressed the
growth of 3T3-L1 cells (Fig.
6A). Similarly, overexpression of the AhR slowed cell
proliferation, whereas lowering of AhR protein levels stimulated cell growth
(Fig. 6B). Cell cycle analysis
revealed that, after 18 hours of stimulation with differentiation medium, most
of the sense cells remained in G0/G1 phase, whereas half of the control cells
had entered S phase (Fig. 7).
In antisense cells, somewhat enhanced entry into S phase was observed relative
to that seen in the control cells (Fig.
7).
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Overexpression of the AhR inhibits the Rb protein phosphorylation and
the down-regulation of p107
The retinoblastoma protein (pRB) plays key role in regulating clonal
expansion associated with adipogenesis (Chen et al.,
1996). Also, a direct
interaction of pRB with the AhR has been reported (Ge and Elferink,
1998
). Therefore, to explore
the mechanism by which the AhR inhibits clonal expansion, we analyzed
phosphorylation state in pRB. Immunoblot of pRB in
Fig. 8A showed that the AhR had
no effect on pRB phosphorylation on day 0 (quiescent cells). Upon induction of
differentiation, pRB in control cells and antisense cells was almost entirely
hyperphosphorylated on day 1 (Fig.
8A). This pRB phosphorylation pattern observed is similar to that
in previous report (Shao and Lazar,
1997
). The degree of pRB
phosphorylation in the antisense cells was slightly higher than that in the
control cells. By contrast, the sense cells exhibited minimal degree of pRB
phosphorylation (Fig. 8A).
These data suggest that the delay of cell cycle shown in
Fig. 7 is, at least partly, due
to the lower phosphorylation state of pRB caused by the AhR.
|
In addition to pRB, the related member p107 and p130 have been shown to
play a critical role in differentiation and proliferation (Richon et al.,
1997). Upon stimulation of
differentiation, the expression of p107 was increased on day 1 and was
entirely diminished on day 4 (Fig.
8B). The activated AhR had little effect on the transient increase
of the p107 expression on day 1 but stabilized the expression of p107
throughout day 4 (Fig. 8B). By
contrast, the expression of p130 was not affected by the AhR activation
(Fig. 8C).
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DISCUSSION |
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It is well recognized that PPAR2 plays pivotal roles in adipogenesis
(Tontonz et al., 1994
). It is
expressed in small amounts in preadipocytes, and its synthesis is increased
during the process of adipogenesis. In this study, the AhR-overexpressing
cells exhibited similar level of PPAR
2 expression compared with control
cells, despite their reduced ability for morphological differentiation. Two
possible mechanisms can explain this discrepancy between the normal level of
expression of PPAR
2 mRNA and the lower potency for morphological
differentiation in the AhR-overexpressing cells. First, the AhR may inhibit
the production of endogenous ligand for PPAR
2. This view is compatible
with the results in Fig. 4
showing that treatment with exogenous PPAR
2 ligands, such as
troglitazone, ciglitazone and indomethacin, restored the ability of the
AhR-overexpressing cells to differentiate. The second possibility is that the
AhR signaling pathway may inhibit PPAR
2 activity. TCDD activates
COUP-TF, thereby antagonizing the activity of PPAR
2 (Brodie et al.,
1996
,Brodie et al.,
1996
). We have shown in this
study that the activated-AhR stimulates p42/p44 MAP kinase activity
(Fig. 5A). Treatment of the
cells with PD98059 or U0126 overcame the inhibitory effects of the AhR on
adipogenesis (Fig. 5C). These
results suggest that p42/p44 MAP kinase activity mediates negative regulatory
action of the AhR on adipogenesis. A report by Hu et al. has revealed that the
phosphorylation of Ser112 in PPAR
2 by MAP kinase results in the loss of
transcriptional activity (Hu et al.,
1996
). Collectively, a
possible mechanism is that the AhR inactivates PPAR
2 via stimulation of
MAP kinase. We have also shown that the activation of the AhR affects on the
expression of p107, pRB-related protein
(Fig. 8B). The expression of
p107 is transiently upregulated in clonal expansion stage and is downregulated
after clonal expansion (Fig.
8B; Richon et al.,
1997
). Studies using
p107-deficient fibroblast cells revealed that p107 suppresses PPAR
2
activity in adipose differentiation (Classon et al.,
2000
). Thus, the downregulation
of p107 is essential for activation of PPAR
(Classon et al.,
2000
). However, as we presented
in Fig. 8B, the activation of
the AhR stabilized the expression of p107, but not that of p130
(Fig. 8B). Therefore, the
stabilization of p107 expression by the AhR may result in lower PPAR
2
activity. Taken together, these results strongly suggest that PPAR
2
activation pathway is the target for the AhR. Further studies on the crosstalk
between the PPAR
2 activation pathway and the AhR signaling pathway may
reveal new aspects of adipogenesis-related signal transduction.
Recent reports showed a direct interaction of the AhR with pRB (Ge and
Elferink, 1998; Puga et al.,
2000
). In addition, the AhR
associates preferentially with the hypophosphorylated, active form of pRB (Ge
and Elferink, 1998
). The roles
of pRB in adipogenesis have been amply demonstrated. It has been shown that
the ability of SV 40 large T antigen to block adipogenesis is dependent on its
ability to sequester pRB (Higgins et al.,
1996
). In addition, it has
been demonstrated that fibroblast cells from RB-deficient mouse embryos are
unable to undergo adipose conversion, and ectopic expression of RB enables
RB-/- fibroblasts to differentiate (Chen et al.,
1996
). Furthermore, pRB has
been shown to physically interact with C/EBPs, to promote the binding of
C/EBPß to DNA response element and to increase its transactivation
capacity (Chen et al., 1996
).
The interaction between the AhR and pRB may affects on these roles of pRB in
adipogenesis. Given the known activity of pRB as a cell cycle regulator, one
of the roles of pRB in adipogenesis is regulation of clonal expansion (Classon
et al., 2000
). Clonal expansion
has generally been regarded as a prerequisite event for adipose
differentiation. Inhibition of clonal expansion by treatment with drugs such
as rapamycin or TNF
results in failure of the subsequent
differentiation process in 3T3-L1 cells (Yeh et al.,
1995
; Lyle et al.,
1998
). In this study we
present data showing that activation of the AhR inhibits clonal expansion in
adipose differentiation (Fig.
6). Cell cycle analysis revealed that the AhR-overexpressing cells
lag in G0/G1 phase, with subsequent decreased entry into the S phase
(Fig. 7). We also found that
overexpression of the AhR inhibits the phosphorylation of pRB, whereas
underexpression of the AhR stimulates pRB phosphorylation
(Fig. 8A). The importance of
hyperphosphorylation of pRB for the commitment of cells to undergo adipose
conversion has been described previously (Shao and Lazar,
1997
). Collectively, these
results suggest that the represses of pRB phosphorylation by the AhR results
in delay of clonal expansion and subsequent progress of differentiation
program. In addition to the roles in clonal expansion, as described above, pRB
increases the transactivation capacity of C/EBPß via a direct
interaction. This tempts us to speculate that the association of the AhR with
pRB limits the binding capacity of pRB to C/EBPß. In such a scenario, the
activity of C/EBPß could be suppressed. C/EBPß is known to
positively regulate the expression of C/EBP
. By contrast, the AhR
suppresses the induction of C/EBP
(Fig. 3C; Phillip et al.,
1996). Therefore, suppression of the C/EBP
expression and subsequent
lower morphological differentiation in the AhR-activated cells could be due to
less interaction of pRB with C/EBPß. However, this model has to be proven
in the future study.
From a toxicological perspective, it is not clear whether the AhR-dependent
effects of TCDD reflect the enhanced physiological function of the AhR, or the
specific actions of the TCDD-AhR complex, or a combination of both. In this
study, the features of AhR-overexpressing cells were observed in the absence
of exogenous ligand. However, the cells exhibit several similarities with
TCDD-treated cells: (1) inhibition of differentiation; (2) induction of
CYP1B1; (3) alteration of expression level of adipocyte-related genes; and (4)
growth delay. These results indicate that overexpression of the AhR can mimic
the effects of exogenous ligand. Thus, TCDD may induce its toxicological
effects, at least partly, by overactivating the physiological AhR signaling
pathway. If so, the AhR may play several unidentified physiological roles in
homeostasis, because TCDD causes a variety of toxic reactions in living
material (Poland and Knutson,
1982; Safe,
1986
; Landers and Bunce,
1991
; Pohjanvitra and
Tuomisto, 1994
). Understanding
the detailed mechanisms of the toxicological action of TCDD may reveal further
physiological roles of the AhR.
In summary, this study is the first to report that the AhR participates in
the negative regulation of adipose differentiation in the absence of exogenous
ligands. The proposed mechanism by which the AhR inhibits adipose
differentiation is that the AhR inhibits PPAR2 activation pathway by
stimulating p107 expression and/or p42/p44 MAP kinase, and the inhibition of
PPAR
2 signaling pathway results in lower morphological differentiation
in the AhR-activated cells. Furthermore, the AhR inhibits pRB phosphorylation,
resulting in delay of clonal expansion and the subsequent progress of
differentiation program. Dynamic regulation of the AhR is observed in mouse
embryos (Abbott et al., 1994
),
suggesting that the AhR plays a role during development and differentiation.
Therefore, the data presented in this paper will provide opportunities to
carry out studies in order to better understand the physiological role of the
AhR in development and differentiation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Abbott, B. D., Perdew, G. H. and Birnbaum, L. S. (1994) Ah receptor in mouse embryonic palate and the effects of TCDD on receptor expression. Toxicol. Appl. Pharmacol. 126, 16-25.[Medline]
Alexander, D. L., Ganem, L. G., Fernandez-Salguero, P.,
Gonzalez, F. and Jefcoate, C. R. (1998) Aryl-hydrocarbon
receptor is an inhibitory regulator of lipid synthesis and of commitment to
adipogenesis. J. Cell Sci.
111,3311
-3322.
Brodie, A. E., Manning, V. A. and Hu, C. Y. (1996) Inhibitors of preadipocyte differentiation induce COUP-TF binding to a PPAR/RXR binding sequence. Biochem. Biophys. Res. Commun. 228,655 -661.[Medline]
Brodie, A. E., Azarenko, V. A. and Hu, C. Y. (1996) 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibition of fat cell differentiation. Toxicol. Lett. 84, 55-59.[Medline]
Bradford, M. M. (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72,248 -254.[Medline]
Carver, L. A. and Bradfield, C. A. (1997)
Ligand-dependent interaction of the aryl hydrocarbon receptor with a novel
immunophilin homolog in vivo. Biol. Chem.
272,11452
-11456.
Chang, C. and Puga, A. (1998) Constitutive
activation of the aromatic hydrocarbon receptor. Mol. Cell.
Biol. 18,525
-535.
Chen, P. L., Riley, D. J., Chen, Y. and Lee, W. H. (1996) Retinoblastoma protein positively regulates terminal adipocyte differentiation through direct interaction with C/EBPs. Genes Dev. 10,2794 -2804.[Abstract]
Classon, M., Kennedy, B. K., Mulloy, R. and Harlow, E.
(2000) Opposing roles of pRB and p107 in adipocyte
differentiation. Proc. Natl. Acad. Sci. USA
97,10826
-10831.
Crawford, R. B., Holsapple, M. P., Kaminski, N.
(1997) Leukocyte activation induces arylhydrocarbon receptor
up-regulation, DNA binding, and increased Cyp1a1 expression in the absence of
exogenous ligand. Mol. Pharmacol.
52,921
-927.
Denis, M., Cuthill, S., Wikstrom, A. C., Poellinger, L. and Gustafsson, J. K. (1988) Association of the dioxin receptor with Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor. Biochem. Biophys. Res. Commun. 155,801 -807.[Medline]
Fernandes-Salguero, P., Pineau, T., Hilbert, D. M., McPhail, T., Lee, S. S. T., Kimura, S., Nebert, D. W., Rudikoff, S., Ward, J. M. and Gonzalez, F. J. (1995) Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science 268,722 -726.[Medline]
Fernandez-Salguero, P. M., Ward, J. M., Sundberg, J. P. and Gonzalez, F. J. (1996) Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol. Appl. Pharmacol. 140,173 -179.[Medline]
Font de Mora, J., Porras, A., Ahn, N. and Santos, E. (1997) Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation. Mol. Cell. Biol. 17,6068 -6075.[Abstract]
Gradin, K., Toftgard, R., Poellinger, L. and Berghard, A.
(1999) Repression of dioxin signal transduction in fibroblasts.
J. Biol. Chem. 274,13511
-13518.
Ge, N. and Elferink, C. J. (1998) A direct
interaction between the arylhydrocarbon receptor and retinoblastoma protein.
J. Biol. Chem. 273,22708
-22713.
Hankinson, O. (1995) The arylhydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol.35 , 307-340.[Medline]
Hayashi, S., Okabe-Kando, J., Honma, Y. and Kawajiri, K. (1995) Expression of Ah receptor (TCDD receptor) during human monocytic differentiation. Carcinogenesis 16,1403 -1409.[Abstract]
Higgins, C., Chatterjee, S. and Cherington, V. (1996) The block of adipocyte differentiation by a C-terminally truncated, but not by full-length, simian virus 40 large tumor antigen is dependent on an intact retinoblastoma susceptibility protein family binding domain. J. Virol. 70,745 -752.[Abstract]
Hu, E., Kim, J. B., Sarraf, P. and Spiegelman, B. M.
(1996) Inhibition of adipogenesis through MAP kinase-mediated
phosphorylation of PPAR gamma. Science
274,2100
-2103.
Landers, J. P. and Bunce, N. J. (1991) The Ah receptor and the mechanism of dioxin toxicity. Biochem. J. 276,283 -287
Liu. P. C. C., Phillips, M. A. and Matsumura, F. (1996) Alteration by 2,3,7,8-tetrachlorodibenzo-p-dioxin of CCAAT/enhancer binding protein correlates with suppression of adipocyte differentiation in 3T3-L1 cells. Mol. Pharmacol. 49,989 -997.[Abstract]
Lyle, R. E., Richon, V. M. and McGehee, R. E. (1998) TNF alpha disrupts mitotic clonal expansion and regulation of retinoblastoma proteins p130 and p107 during 3T3-L1 adipocyte differentiation. Biochem. Biophys. Res. Commun. 247,373 -378.[Medline]
Ma, Q. and Whitlock, J. P. (1996) The aromatic hydrocarbon receptor modulates the Hepa 1c1c7 cell cycle and differentiated state independently of dioxin. Mol. Cell. Biol. 16,2144 -2150.[Abstract]
Ma, Q. and Whitlock, J. P. (1997) A novel
cytoplasmic protein that interacts with the Ah receptor, contains
tetratricopeptide repeat motifs, and augments the transcriptional response to
2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Biol.
Chem. 272,8878
-8884.
Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, T.
N., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M. and Fujii-Kuriyama
Y. (1997) Loss of teratogenic response to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the Ah (dioxin)
receptor. Genes Cells 2,645
-654.
Nambu, J. R., Lewis, J. O., Wharton, K. A. and Crews, S. T. (1991) The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development. Cell 67,1157 -1167.[Medline]
Peters, J. M. and Wiley, L. M. (1995) Evidence that murine preimplantation embryos express arylhydrocarbon receptor. Toxicol. Appl. Pharmacol. 134,214 -221.[Medline]
Philips, M., Enan, E., Liu, C. C. and Matsumura, F.
(1995) Inhibition of 3T3-L1 adipose differentiation by
2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Cell
Sci. 108,395
-402.
Pohjanvitra, R. and Tuomisto, J. (1994) Short-term toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals: effects, mechanisms, and animal models. Pharmacol. Rev. 46,483 -549.[Medline]
Poland, A. and Knutson, J. (1982) 2,3,7,8-Tetrachloro-dibenzo-p-dioxin and related aromatic hydrocarbons: examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol. 22,517 -524.[Medline]
Pollenz, R. S. (1996) The arylhydrocarbon receptor but not the arylhydrocarbon receptor nuclear translocator protein is rapidly depleted in hepatic and nonhepatic culture cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Mol. Pharmacol. 49,391 -398.[Abstract]
Predew, G. H. (1988) Association of the Ah
receptor with the 90-kDa heat shock protein. J. Biol.
Chem. 263,13802
-13805.
Puga, A., Barnes, S. J., Dalton, T. P., Chang, C., Knudsen, E.
S. and Maier, M. A. (2000) Aromatic hydrocarbon receptor
interaction with the retinoblastoma protein potentiates repression of
E2F-dependent transcription and cell cycle arrest. J. Biol.
Chem. 275,2943
-2950.
Reyes, H., Reisz-Porszasz, S. and Hankinson, O. (1992) Identification of the Ah receptor nuclear translocator protein (arnt) as a component of the DNA binding form of the Ah receptor. Science 256,1193 -1195.[Medline]
Richon, V. M., Lyles, R. E. and McGehee, R. E.
(1997) Regulation and expression of retinoblastoma proteins p107
and p130 during 3T3-L1 adipocyte differentiation. J. Biol.
Chem. 272,10117
-10124
Riebniger, D. and Schrenk, D. (1998) Nonresponsiveness to 2,3,7,8-tetrachlorodibenzo-p-dioxin of transforming growth factor ß1 and cyplA1 gene expression in rat liver fat-storing cells. Toxicol. Appl. Pharmacol. 152,251 -260.[Medline]
Safe, S. H. (1986) Comparative toxicology and mechanism of action of polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu. Rev. Pharmacol. Toxicol. 26,371 -399.[Medline]
Sale, E. M., Atkinson, P. G. A. and Sale, G. J. (1995) Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J. 14,674 -684.[Abstract]
Sassone-Corsi, P. (1997) Molecular clocks. PERpetuating the PASt. Nature 389,443 -444.[Medline]
Schmidt, J. and Bradfield, C. A. (1996) Ah receptor signaling pathways. Annu. Rev. Cell Dev. Biol. 12,55 -89.[Medline]
Schmidt, J. V., Su, G. H., Reddy, J. K., Simon, M. C. and
Bradfield, C. A. (1996) Characterization of a murine Ahr null
allele: involvement of the Ah receptor in hepatic growth and development.
Proc. Natl. Acad. Sci. USA
93,6731
-6736.
Shao, D. and Lazar, M. (1997) Peroxisome
proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha,
and cell cycle status regulate the commitment to adipocyte differentiation.
J. Biol. Chem. 272,21473
-21478
Shimba. S, Todoroki, T., Aoyagi, T. and Tezuka, M. (1998) Depletion of the arylhydrocarbon receptor during adipose differentiation in 3T3-L1 cells. Biochem. Biophys. Res. Commun. 249,131 -137.[Medline]
Singh, S. S., Hord, N. G., Predew, G. H. (1996) Characterization of the activated form of the arylhydrocarbon receptor in the nucleus of HeLa cells in the absence of exogenous ligand. Arch. Biochem. Biophys. 329,47 -55.[Medline]
Smenza, G. L. (1998) Hypoxia-inducible factor 1: Master regulator of O2 homeostasis. Curr. Opin. Genet. Dev. 8,588 -594.[Medline]
Sogawa, K. and Fujii-Kuriyama, Y. (1997) Ah receptor, a novel ligand-activated transcription factor. J. Biochem. 122,1075 -1079.[Abstract]
Tontonz, P., Hu, E. and Spiegelman, B. M. (1994) Stimulation of adipogenesis in fibroblasts by PPAR gamma2, a lipid-activated transcription factor. Cell 79,1147 -1156.[Medline]
Wanner, R., Brommer, S., Czarnetzki, B. M. and Rosenbach, T. (1995) The differentiation-related upregulation of aryl hydrocarbon receptor transcript levels is suppressed by retinoic acid. Biochem. Biophys. Res. Commun. 209,706 -711.[Medline]
Wise, L. S. and Green, H. (1979) Participation of one isozyme of cytosolic glycerophosphate dehydrogenase in the adipose conversion of 3T3 cells. J. Biol. Chem. 254,273 -275.[Abstract]
Yeh, W. C., Bierer, B. A. and McKnight, S. L. (1995) Rapamycin inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells. Proc. Natl. Acad. Sci. USA 92,11086 -11090.[Abstract]