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INTRODUCTION |
Preadipocyte factor-1
(pref-1)1 is an epidermal
growth factor (EGF) repeat domain-containing transmembrane protein with
an anti-adipogenic function (1-4). Pref-1 is highly expressed in 3T3-L1 preadipocytes and is totally absent after their differentiation to mature adipocytes. Interfering with this normal repression of
pref-1 during adipogenesis by constitutive expression of
pref-1 in 3T3-L1 preadipocytes or by the addition of soluble pref-1
ectodomain markedly decreases adipocyte conversion (1-4);
pref-1 expression is also abolished during the adipose
conversion of primary rat preadipocytes in cultures, and their
differentiation is inhibited by the pref-1 ectodomain (5). Taken
together, these data, and the detection of soluble pref-1 in
circulation (6), support a functional role for pref-1 in adipocyte
differentiation in vivo. In this regard, pref-1 mRNA
levels were recently shown to be elevated by adipose-specific
expression of SREBP-1c in a transgenic mouse model of congenital
generalized lipodystrophy, a condition characterized by poorly
developed white and brown adipose tissue (7). Pref-1 belongs to that
class of proteins that can act as either transmembrane or soluble
molecules; membrane-associated pref-1 is cleaved at two sites in the
extracellular domain, thereby extending its potential range of function
(4). Therefore, pref-1 is hypothesized to exert its inhibitory function
by mediating cell communication or interaction with an as yet
unidentified receptor protein via its EGF repeats. In addition to
full-length pref-1 (pref-1A), three alternately spliced transcripts
with deletions in the juxtamembrane region are present in 3T3-L1
preadipocytes. Although all four transcripts generate transmembrane
pref-1, two of these undergo processing to a soluble form of pref-1
corresponding to their respective complete ectodomains. The mode of
pref-1 inhibitory function, juxtacrine or paracrine, may therefore
depend on the specific alternate pref-1 transcript expressed. The
presence of EGF-like domains in the pref-1 ectodomain, a protein motif
demonstrated to mediate protein-protein interaction to control cell
growth and differentiation in a variety of biological settings (8-10), suggests that transmembrane and/or soluble pref-1 may function by
interaction of its EGF-like domains with EGF-like domains of the
putative pref-1 receptor present on the cell surface to maintain the
preadipose phenotype.
A combination of the synthetic glucocorticoid dexamethasone and the
phosphodiesterase inhibitor methylisobutylxanthine (MIX), first
employed 20 years ago by Rubin and co-workers (11), is the standard
protocol for in vitro differentiation of 3T3-L1
preadipocytes. Adipocyte differentiation of 3T3-L1 cells is routinely
induced by a 2-day treatment of confluent preadipocytes with 1 µM dexamethasone and 0.5 mM MIX in the
presence of fetal calf serum. 3-5 days after dexamethasone/MIX
removal, the majority of cells attain an adipocyte phenotype. During
the dexamethasone/MIX hormonal induction phase cells briefly express
c-fos, c-jun, and c-myc; decrease
surface area; and undergo postconfluent mitoses, subsequent clonal
expansion, and growth arrest; however no overt adipose conversion
occurs (12, 13). Because the continued presence of dexamethasone/MIX is
not needed for the initial appearance of lipid droplets nor for
maintenance of the adipocyte phenotype, these agents have been
hypothesized to initiate and/or potentiate differentiation signals.
Although the molecular targets of dexamethasone/MIX remain largely
unknown, the basic transcriptional machinery mediating adipocyte
differentiation involves the nuclear hormone receptor PPAR
and the
C/EBP family of transcription factors (14, 15). These transcription
factors, however, are not expressed solely in adipocytes (16-18) and
have additional functions unrelated to lipid metabolism or adipocyte
differentiation (19-23). It is clear that superimposed on their
actions is the ability of cell-extracellular matrix interactions and
the hormone/growth factor microenvironment to modulate adipocyte
differentiation positively or negatively (24, 25). It remains to be
clarified how the dexamethasone/MIX adipogenic agents transduce their
respective differentiation signals at the molecular level. C/EBP
and
C/EBP
are induced approximately 5-10-fold during hormonal treatment
of 3T3-L1 preadipocytes by MIX and dexamethasone, respectively (26,
27), which subsequently leads to PPAR
induction. However, recent
studies argue against C/EBP
as a major effector of dexamethasone
action and indicate that dexamethasone may signal through pathways
unrelated to C/EBP transcription factors (28, 29). The addition of
soluble pref-1 ectodomain to culture media severely inhibits
dexamethasone/MIX induction of differentiation, including inhibition of
C/EBP
and PPAR
expression (30). This supports the hypothesis that
the inhibitory action of pref-1 is exerted early in differentiation and
suggests that adipogenic agents may function in part through down-regulation of this adipo-inhibitory factor.
To determine if the adipogenic effects of dexamethasone/MIX in the
initiation of adipocyte differentiation may be relayed through
down-regulation of the expression of pref-1, we examined the
levels of pref-1 protein and mRNA during exposure of 3T3-L1 preadipocytes to these agents. We determined that pref-1 is
an early target of dexamethasone action and that dexamethasone rapidly decreases pref-1 gene transcription. Furthermore, we found a
strong correlation between the dose response of pref-1
repression by dexamethasone and the effective concentration(s) of
dexamethasone capable of supporting adipocyte differentiation.
Modulation of pref-1 levels by varying dexamethasone/MIX exposure times
indicates that by maximally reducing pref-1 levels we increased the
degree and uniformity of 3T3-L1 adipocyte differentiation. Moreover, lowering of endogenous pref-1 levels in 3T3-L1 preadipocytes, via
antisense pref-1, substitutes in part for dexamethasone treatment in
the promotion of adipogenesis, supporting a model whereby the rapid
decrease of pref-1 gene transcription upon
dexamethasone exposure may be a mechanism whereby glucocorticoids
promote adipocyte differentiation.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
The pref-1 antisense construct,
pAS-pref-1, was prepared by polymerase chain reaction amplification of
the pref-1 coding sequence using the complete pref-1 cDNA sequence
in pcDNA (Invitrogen) as a template. The primers were designed to
add HindIII sites to both ends of the amplified product. The
HindIII-digested polymerase chain reaction fragment was
inserted into HindIII sites of the pcDNA3.1 (Invitrogen)
vector, which includes a neomycin-selectable marker. Orientation was
determined by multiple restriction mapping.
Cell Culture and Transfection--
3T3-L1 preadipocytes were
maintained in Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) containing 10% fetal calf serum. For standard induction of
adipocyte differentiation, confluent 3T3-L1 preadipocytes in fetal calf
serum-containing medium were treated with 0.5 mM MIX and 1 µM dexamethasone. For evaluation of test agents, the
medium was changed 1 day before the experiments, and confluent 3T3-L1
cells were treated for the indicated times and concentrations. For
antisense studies the pAS-pref-1 plasmid construct was transfected via
the calcium phosphate coprecipitation method, and stable clones were
established by selection in 400 µg/ml G418 for 3 weeks. Clones were
expanded and analyzed for the level of endogenous pref-1. For
comparison of differentiation, 1 × 105 cells were
plated in quadruplicate in six-well plates, and 7 days after
differentiation RNA was harvested and subjected to Northern analysis.
Western Blot Analysis--
Cell monolayers of 3T3-L1
preadipocytes were washed twice with phosphate-buffered saline and
scraped into phosphate-buffered saline containing 2 mM
phenylmethylsulfonyl fluoride. The cell suspension was subjected to
three freeze-thaw cycles, and the crude membrane fraction was recovered
by centrifugation at 13,000 × g for 25 min at 4 °C.
The pellet was dissolved in lysis buffer (20 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Nonidet
P-40, 1 mM EDTA, 2 mM phenylmethylsulfonyl
fluoride) on ice for 30 min, clarified by brief spinning in a
microcentrifuge, and protein content determined (Bio-Rad). The
indicated amount of protein was loaded per lane in SDS-PAGE and
electroblotted onto Immobilon polyvinylidene difluoride membranes
(Millipore) using 10 mM CAPS, 10% methanol transfer
buffer. For immunodetection of proteins, membranes were blocked for
1 h at room temperature in 1 × NET (145 mM NaCl,
5 mM EDTA, 0.25% gelatin, 0.05% Triton X-100 and 50 mM Tris-HCl, 7.4) followed by incubation for 1 h at
room temperature with primary antiserum. For blocking experiments, the
pref-1 antiserum was preincubated with 30 µg of the noted proteins
for 1 h before the addition of the membrane. Detection of the
antigen-antibody complexes was accomplished via goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad), and the peroxidase conjugate was developed with 0.015% H2O2, 16%
methanol, 8.3 mM Tris-HCl, pH 7.4, and 0.05% w/v
4-chloro-1-naphthol. For enhanced chemiluminescence (ECL, Amersham
Pharmacia Biotech) detection, processing was essentially as above
except that 5% bovine serum albumin was used for the blocking step,
and product visualization was according to the manufacturer's instructions.
RNA Preparation and Northern Blot Analysis--
Cell monolayers
were washed twice with phosphate-buffered saline, and total RNA was
prepared by guanidine isothiocyanate/cesium chloride
ultracentrifugation or TRIzol (Life Technologies, Inc.) extraction
methods. RNA was electrophoresed in 1% formaldehyde-agarose gels in
2.2 M formaldehyde, 20 mM MOPS, 1 mM EDTA, stained with ethidium bromide, and transferred to
Hybond N (Amersham Corporation). After UV cross-linking, filters were
incubated at 42 °C for at least 4 h in 50% formamide, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, and 50 µg/ml
herring sperm DNA. After prehybridization, filters were hybridized
under identical conditions to 32P-labeled random primed
cDNA probes for at least 16 h or with strand-specific in
vitro transcribed probes using the MaxiScript kit (Ambion).
Posthybridization washes were for 30 min at room temperature in 1 × SSC, 1% SDS and then in 0.1 × SSC, 0.1% SDS at 65 °C for
1 h. After exposure to x-ray film with intensifying screen at
80 °C, autoradiograms were scanned using an imaging densitometer
(GS670, Bio-Rad) and analyzed with Molecular Analyst (Bio-Rad) or Alpha
Innotech digital imaging software.
Ribonuclease Protection Assays--
A template for the
production of antisense RNA probe was prepared by insertion of a
restriction fragment from bases 132 through 1312 of the pref-1 cDNA
sequence into the EcoRI site of pcDNAI (Invitrogen)
followed by linearization with SmaI at base 970 of the
pref-1 sequence. The SmaI restriction site occurs in the
region that is present in the pref-1A form of the transcript but is
deleted in the alternate forms of the transcript, pref-1B, pref-1C, and pref-1D. Because pref-1C and pref-1D differ at the 5'-end but have the
same 3'-end of their respective deletions, this probe will not
distinguish between these two forms of the transcript. A
32P-labeled antisense riboprobe of 397 base pairs was
generated by use of SP6 RNA polymerase (Promega); this includes 53 base pairs of vector sequence and 342 base pairs of pref-1 cDNA
sequence. The probe was purified by elution from 6% polyacrylamide gel
and employed in a ribonuclease protection assay utilizing the RPAII kit
(Ambion) according to the manufacturer's recommendations. Products
were analyzed on 6% sequencing gels and fragment lengths determined by
comparison with DNA sequencing ladders.
Isolation of Nuclei and Transcription Run-on Assays--
3T3-L1
cells were washed three times with phosphate-buffered saline and
scraped in lysis buffer containing 1% Nonidet P-40, 0.32 M
sucrose, 3 mM MgCl2, 5 mM HEPES, pH
6.9, and 0.5 mM
-mercaptoethanol. Nuclei collected by
centrifugation were washed once by centrifugation in lysis buffer
without Nonidet P-40 and stored in liquid nitrogen in 50 mM
Tris-HCl, pH 7.9, 5 mM MgCl2, 0.5 mM
-mercaptoethanol, and 40% glycerol. Run-on
transcription was carried out at 30 °C for 45 min in a reaction
mixture containing 5 × 107 nuclei and 125 µCi of
[
-32P]UTP in a final volume of 0.5 ml. Labeled RNA was
extracted with phenol and SDS at 65 °C, ethanol-precipitated, and
subjected to a Sephadex G-50 spin column. 5 µg of each plasmid DNA
was denatured by treating with 0.3 N NaOH for 30 min at
4 °C and applied to nitrocellulose filters using a slot-blot
apparatus. Hybridization was carried out with 5 × 107
cpm in 10 mM Tris-HCl, pH 7.4, 0.2% SDS, and 10 mM EDTA at 65 °C for 72 h. The filters were washed
twice in 0.1% SDS, 2 × SSC at 22 °C and in 0.1% SDS,
0.1 × SSC at 60 °C.
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RESULTS |
Dexamethasone, a Key Component of the Adipogenic Mixture, Markedly
Decreases Pref-1 Protein Levels in 3T3-L1 Preadipocytes--
Standard
in vitro differentiation of 3T3-L1 preadipocytes involves
induction of differentiation by treatment of confluent preadipocytes
with dexamethasone/MIX for 48 h in the presence of fetal calf
serum. This results in differentiated adipocytes 3-5 days later. The
exact mechanism whereby dexamethasone/MIX promotes adipocyte
differentiation is not known, and no overt differentiation to the
adipose phenotype occurs during the dexamethasone/MIX treatment phase.
A potential point at which these inducing agents exert their effects
may involve down-regulation of the adipo-inhibitory factor pref-1. To
begin to address if these agents affect pref-1 protein levels in 3T3-L1
preadipocytes, antiserum was prepared against a TrpE/pref-1 fusion
protein. Fig. 1A demonstrates
that in Western analysis of crude 3T3-L1 preadipocyte membrane
preparations, the pref-1 antibody detects at least seven discrete
pref-1 protein bands of 45-60 kDa. We have reported that this
heterogeneity likely arises from multiple pref-1 transcripts and
glycosylation of pref-1 protein (2, 4, 30). The specificity of the
signal is demonstrated by the fact that it is competed by preincubation
of the pref-1 antiserum with bacterially produced TrpE/pref-1 protein
but not with TrpE protein alone, and no signal is detected by normal
rabbit serum nor with an antiserum raised against an unrelated TrpE
fusion protein. In addition, no signal resulted when the pref-1
antibody was used in Western analysis of crude membrane fractions
prepared from mouse L cells, a cell line that does not express pref-1
(data not shown).

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Fig. 1.
Effect of differentiation-inducing agents on
pref-1 protein levels in 3T3-L1 preadipocytes. A,
specificity of pref-1 antibody. 50 µg of protein was fractionated per
lane of 10% SDS-PAGE and subjected to Western analysis using indicated
primary antisera (pref-1; N, normal sera; UR,
unrelated) and 30 µg of the indicated competitor proteins, and
products were visualized by color detection as described under
"Experimental Procedures." The bracket indicates pref-1
protein species. Markers in kDa are indicated. B, Western
analysis of control untreated 3T3-L1 cells or those cells treated for
48 h with 1 µM dexamethasone (DEX) and
0.5 mM MIX. C, Western analysis of control
untreated 3T3-L1 cells or cells treated with either 0.5 mM
MIX or 1 µM dexamethasone for 48 h. For panels
B and C, 15 µg of crude membrane extracts of 3T3-L1
preadipocytes was subjected to SDS-PAGE and enhanced chemiluminescence
for Western blot analysis utilizing pref-1 primary antibody. The pref-1
protein bands are indicated by brackets, and markers in kDa
are indicated. Before SDS-PAGE for Western analysis, the protein
content of samples was verified by SDS-PAGE and Coomassie Blue staining
of parallel gels. Three separate sets of experiments were conducted
with essentially identical results, and representative data are
shown.
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Western analysis of pref-1 protein levels after the standard 2-day
incubation of confluent 3T3-L1 preadipocytes with 1 µM dexamethasone and 0.5 mM MIX in combination or individually
is shown in Fig. 1, B and C. Cells treated with
dexamethasone/MIX for 48 h had a drastic reduction in all forms of
the pref-1 protein compared with untreated control cells (Fig.
1B, bracket). No differential effect was noted;
dexamethasone/MIX decreased all forms of the protein in a manner
proportional to their initial levels in control cells. The degree of
reduction in pref-1 protein by dexamethasone was comparable to that of
dexamethasone/MIX, indicating that the decreased pref-1 protein content
was primarily attributable to the effects of dexamethasone (Fig.
1C, bracket). We observed previously that in
pref-1-transfected COS cells, the pref-1 protein half-life is less than
7 h (4). The decrease in pref-1 protein content we observe with
dexamethasone treatment of 3T3-L1 preadipocytes is consistent with this
relatively short half-life. The pattern of pref-1 protein species, with
the disappearance of the higher molecular mass proteins in MIX-treated
cells, resembles that for pref-1 protein in 3T3-L1 cells when
N-linked glycosylation is blocked by tunicamycin (4),
indicating that MIX could possibly alter the glycosylation profile and
subsequently influence protein half-life.
Dexamethasone Decreases All Forms of the Pref-1
Transcript--
Given the striking decrease in pref-1 protein levels
by dexamethasone, we determined if this reflected changes in the pref-1 mRNA level by conducting northern analyses of pref-1 mRNA after a 48-h incubation of confluent 3T3-L1 preadipocytes with 1 µM dexamethasone, 0.5 mM MIX, or 1 µM dexamethasone and 0.5 mM MIX in
combination. Dexamethasone and dexamethasone/MIX treatment affect
pref-1 mRNA levels most dramatically. As shown in Fig. 2A, pref-1 mRNA levels in
dexamethasone-treated cells decreased to approximately 20% of that of
nontreated controls. Furthermore, the reduction of pref-1 mRNA by
dexamethasone/MIX in combination is equal to that observed with
dexamethasone alone. Decreased pref-1 mRNA content by dexamethasone
and dexamethasone/MIX in combination paralleled the effects we observed
at the protein level and suggests that dexamethasone is the primary
agent down-regulating pref-1 levels when 3T3-L1 cells are subjected to
the standard dexamethasone/MIX differentiation protocol. Although not
as pronounced as the effects of dexamethasone, MIX decreased pref-1
mRNA levels somewhat. However, as shown in Fig. 1C, MIX
treatment resulted in a decrease in only the larger species of the
pref-1 protein. Therefore, the Western and Northern analyses we present
indicate that pref-1 is reduced drastically by the dexamethasone
component of the adipogenic mixture and that this occurs during the
dexamethasone/MIX hormonal induction phase, a time period when cells
are presumably receiving differentiation signals. Boney et
al. (31) have reported that in 3T3-L1 preadipocytes, although
neither dexamethasone nor MIX had an effect, decreased pref-1 mRNA
levels were noted only with the complete differentiation mixture of
dexamethasone/MIX plus insulin. These studies probably reflect gene
expression during adipocyte differentiation but not the immediate
effect of glucocorticoids on pref-1 expression.

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Fig. 2.
Down-regulation of pref-1 mRNA by
dexamethasone. Confluent 3T3-L1 preadipocytes were cultured for
48 h in the presence or absence of the indicated agents: 1 µM dexamethasone and 0.5 mM MIX, 1 µM dexamethasone (DEX), 0.5 mM
MIX, or control (no additions to medium). A, Northern
analysis. 5 µg of total RNA was hybridized on Northern blots with
32P-labeled pref-1 cDNA probe. 28 S and 18 S
ribosomal RNA from ethidium bromide-stained gels are shown.
B, ribonuclease protection assay. 5 µg of total RNA
harvested from 3T3-L1 preadipocytes cultured for 48 h in the
presence of 1 µM dexamethasone and 0.5 mM
MIX, 1 µM dexamethasone, or control nontreated
preadipocytes was subjected to RNase protection assay using a pref-1
probe that distinguishes the indicated form of the transcript.
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Northern analysis shows dexamethasone-mediated down-regulation of a
pref-1 mRNA of 1.7 kilobases in 3T3-L1 preadipocytes. This signal,
however, reflects the sum of at least four pref-1 transcripts that we
have identified previously by reverse transcriptase-polymerase chain
reaction and through the isolation and sequence analysis of multiple
independent pref-1 cDNA clones (2). These arise by in-frame
deletions of up to 225 base pairs of the pref-1 juxtamembrane region.
Each of these transcripts generates a transmembrane protein that
undergoes processing to release soluble products into culture media,
with the nature of these soluble forms dependent on the type of pref-1
transcript expressed: pref-1A and pref-1B release large soluble
products that apparently correspond to their complete ectodomain,
whereas forms pref-1C and pref-1D do not shed their full ectodomain
(4). The soluble pref-1 ectodomain has been demonstrated to inhibit
adipocyte differentiation. Because Northern analysis does not
distinguish among these multiple transcripts, the possibility existed
that the remaining pref-1 mRNA we detect after a 48-h dexamethasone
treatment represents a form of the transcript not subjected to
dexamethasone regulation. We therefore addressed whether the
dexamethasone/MIX differentiation mixture, or dexamethasone alone,
differentially regulates the various pref-1 transcripts. This could in
turn govern the production and release of the soluble pref-1 ectodomain
during the adipose conversion of 3T3-L1 preadipocytes. Ribonuclease
protection assays were conducted with a probe that distinguishes those
transcripts giving rise to the soluble full ectodomain, pref-1A and
pref-1B, from those that do not, pref-1C and pref-1D. Results shown in
Fig. 2B indicate that the longest transcript (pref-1A) is
the most abundant form in 3T3-L1 preadipocytes and that together
pref-1C and pref-1D constitute approximately 10% of the pref-1
mRNA species, and pref-1B 10%. Comparison of the signals for
pref-1A and pref-1B versus those of pref-1C and pref-1D
indicates that there are no differential effects of dexamethasone on
the individual forms of the pref-1 transcript; all four forms are
down-regulated by dexamethasone to the same extent we observed by
Northern analysis. In addition, the use of reverse
transcriptase-polymerase chain reaction to examine the levels of each
of these four major forms of pref-1 transcript in dexamethasone-treated
and control 3T3-L1 preadipocytes showed no differences in the relative
amounts of the various pref-1 transcripts (data not shown). Therefore
dexamethasone has equal inhibitory effects on those forms of pref-1
which shed the full ectodomain and those that do not undergo such processing.
Kinetics of Pref-1 mRNA Down-regulation by
Dexamethasone--
To examine the time course of dexamethasone
down-regulation of pref-1 mRNA during the critical hormonal
induction phase of adipocyte differentiation, RNA was prepared from
3T3-L1 preadipocytes at confluence (time 0) and after culture in the
presence of 1 µM dexamethasone for 3, 6, 12, 24, and
48 h and from untreated control cells at 48 h postconfluence.
The Northern analysis shown in Fig.
3A indicates that a decrease
in pref-1 mRNA is first noted at 6 h, and it decreases through
the final time point of 48 h. At this time we observed a decrease
to approximately 25% of control levels. To illustrate that
glucocorticoid down-regulation of pref-1 mRNA was the result of direct
effects of dexamethasone and not the consequence of adipocyte
differentiation, we assayed the mRNA levels of an early
differentiation marker, PPAR
, and a terminal differentiation marker,
stearoyl-CoA desaturase 1 (SCD1); we did not detect SCD1 mRNA and
observed no increase in PPAR
mRNA levels over the 48-h period
examined.

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Fig. 3.
Kinetics of dexamethasone-mediated decrease
in pref-1 mRNA in 3T3-L1 preadipocytes. A, time
course of pref-1 decrease. RNA was prepared from confluent 3T3-L1
preadipocytes treated with 1 µM dexamethasone for 3, 6, 12, 24, and 48 h and untreated control cells at confluence (time
0) and at 48 h postconfluence (48 hc). Upper
panel, Northern analysis of 5 µg of total RNA with
32P-labeled pref-1 cDNA probe. Ethidium bromide-stained
gel with 28 S and 18 S ribosomal RNA is shown directly below.
Lower panel, Northern analysis was conducted on 20 µg of
total RNA with 32P-labeled PPAR or SCD1 cDNA probes.
The rightmost lane contains total RNA from differentiated
3T3-L1 adipocytes (Ad) as a positive control for signal
detection. B, determination of pref-1 half-life by
actinomycin D treatment. Confluent 3T3-L1 preadipocytes were untreated
( ) or treated (+) with 10 µg/ml actinomycin D. RNA was harvested at
0, 15, 24, and 48 h, and 10 µg of total RNA was subjected to
Northern blot analysis with 32P-labeled pref-1 cDNA
probe. C, graphical comparison of pref-1 mRNA half-time
and time course of dexamethasone down-regulation. The autoradiograms
shown in panels A and B were used for
densitometric scanning. Values were corrected against the intensity of
ethidium bromide staining of ribosomal RNA. Levels of pref-1 mRNA
in the presence of actinomycin D are indicated by solid
boxes. The hollow boxes indicate values for the pref-1
mRNA levels in the presence of dexamethasone for the indicated
times, and the solid circle indicates the value of the
pref-1 decrease after a 48-h treatment with dexamethasone and
actinomycin D in combination.
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To address whether the time course of the decrease of pref-1 mRNA
levels was caused by slow action of glucocorticoids or was attributable
solely to the half-life of pref-1 mRNA, 3T3-L1 preadipocytes were
treated with actinomycin D to block RNA polymerase
II-dependent gene transcription. Total RNA was isolated at
0 h and after 15, 24, and 48 h of exposure of confluent
preadipocytes to 10 µg/ml actinomycin D or from control untreated
cells at 48 h. Northern analysis in Fig. 3B indicates
that the pref-1 mRNA level decreased steadily throughout the time
period analyzed, whereas the level in control cells did not change up
to 48 h. Graphical representation of this data determines a
t1/2 of approximately 24 h for the pref-1 mRNA (Fig. 3C, solid boxes). The time course
of pref-1 mRNA down-regulation during dexamethasone treatment,
derived from the Northern blot in Fig. 3A, is represented as
hollow boxes in the graph in Fig. 3C. Comparison
of the level of pref-1 mRNA over time in actinomycin D and
dexamethasone-treated cultures indicates a very similar rate of pref-1
mRNA decrease. A 50% decrease in the pref-1 mRNA level
occurred after 24 h of dexamethasone exposure, the same time
required for a 50% reduction in the level of pref-1 mRNA during
actinomycin D treatment. Treatment with actinomycin D and dexamethasone
in combination resulted in a decrease in pref-1 mRNA level to 26%
of controls by 48 h (Fig. 3C, solid circle), the same decrease noted with each of these agents alone. These data
indicate that the dexamethasone-mediated decrease of pref-1 mRNA is
most likely solely transcriptional, with dexamethasone acting to
attenuate transcription of the pref-1 gene rapidly and markedly.
Dexamethasone-mediated Repression of Pref-1 Gene
Transcription--
Actinomycin D treatment determines that the pref-1
mRNA half-life is approximately 24 h. This is the same time
required for a 50% decrease in pref-1 mRNA during
dexamethasone exposure and strongly suggests that dexamethasone
acts to repress transcription of the pref-1 gene. To obtain
direct evidence that glucocorticoid down-regulation of
pref-1 is at the transcriptional level, nuclear run-on
assays were conducted using nuclei isolated from dexamethasone-treated and control untreated 3T3-L1 preadipocytes. Fig.
4 shows a high level of pref-1
gene transcription in 3T3-L1 preadipocytes (control). In contrast,
dexamethasone-treated cells have a marked decrease in pref-1
transcription to approximately 20% of that in control cells,
indicating negative transcriptional regulation of the pref-1 gene by glucocorticoids. Although nuclear run on assays showed marked
effects of dexamethasone on pref-1 transcription, there were
no appreciable differences in the transcription of actin or
glyceraldehyde phosphate dehydrogenase in dexamethasone-treated and
control 3T3-L1 cells, nor did we detect any increase in PPAR
(data
not shown). These data indicate that not only is the dexamethasone effect specific for pref-1 gene transcription, but the
decrease in pref-1 gene transcription is not the result of
adipocyte differentiation.

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Fig. 4.
Dexamethasone-mediated transcriptional
down-regulation of the pref-1 gene in 3T3-L1
preadipocytes. Nuclei from 3T3-L1 preadipocytes untreated
(Control) or treated with 1 µM
dexamethasone (DEX) were prepared and used for run-on
transcription assays as described under "Experimental Procedures."
The [32P]UTP-labeled transcripts were hybridized to
indicated plasmid DNA fixed on nitrocellulose filters: glyceraldehyde
phosphate dehydrogenase (GAPDH), pBluescript
(Vector), pref-1, and actin.
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The Concentration Dependence of Dexamethasone-mediated Pref-1
mRNA Down-regulation Correlates with Dexamethasone Efficacy in
Adipocyte Differentiation--
In the standard dexamethasone/MIX
differentiation conditions for 3T3-L1 preadipocytes, dexamethasone is
employed at 1 µM. To determine the dose response of
pref-1 mRNA to dexamethasone, 3T3-L1 cells were incubated for
48 h with dexamethasone concentrations from 1 nM to 1 µM. Northern analysis shown in Fig.
5A was quantified and is
presented graphically in Fig. 5B. At 1 nM
dexamethasone, the lowest concentration employed, pref-1 mRNA
levels were the same as those of nontreated control cells.
Concentrations of 10 nM dexamethasone and higher decreased
pref-1 mRNA to approximately 20% of that in untreated controls,
identifying from 1 nM to 10 nM as the critical
range for dexamethasone-mediated down-regulation of pref-1 mRNA.
Within this range, dexamethasone concentrations of 3, 5, and 7 nM decreased pref-1 mRNA levels to 85, 62, and 33% of
controls, respectively. Compared with levels in differentiated adipocytes, we observed low but detectable levels of PPAR
mRNA in 3T3-L1 preadipocytes; dexamethasone had no effect on PPAR
mRNA over the range of concentrations tested. The lack of an
induction of PPAR
mRNA and the absence of SCD1 mRNA (Fig.
5A, lower panel) indicate that the decrease of
pref-1 mRNA we observe is not the result of adipocyte conversion
but rather the direct effect of dexamethasone on pref-1 mRNA levels
during the critical 48-h hormonal induction phase.

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Fig. 5.
Dose response of dexamethasone
down-regulation of pref-1 mRNA. A, Northern
analysis. Upper panel, 5 µg of total RNA from 3T3-L1 cells
treated for 48 h with the indicated concentrations of
dexamethasone (DEX) was subjected to Northern analysis using
32P-labeled pref-1 cDNA probe. Concentrations employed
were: no dexamethasone addition (lane 1), 1 nM (lane 2), 3 nM (lane
3), 5 mM (lane 4), 7 nM
(lane 5), 10 nM (lane 6), 100 nM (lane 7), and 1 µM (lane
8). The 28 S and 18 S ribosomal RNA on the ethidium
bromide-stained gel is shown below. Lower panel, 20 µg of
total RNA from 3T3-L1 cells treated for 48 h with the indicated
concentrations of dexamethasone was subjected to Northern analysis
using PPAR or SCD1 32P-labeled cDNA probes. Doses
employed were: no dexamethasone addition (lane 1), 10 nM (lane 2), 100 nM (lane
3), 10 µM (lane 4), 100 µM
(lane 5). The rightmost lane contains 20 µg of
total RNA from differentiated 3T3-L1 adipocytes (Ad).
B, dexamethasone dose-response plot. The autoradiogram shown
in panel A was subjected to scanning densitometry, and
values were corrected for variations in the amount of RNA loaded per
lane.
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Our results indicate that pref-1 mRNA is markedly down-regulated by
the 1 µM dexamethasone concentration employed in the
standard protocol for 3T3-L1 differentiation. Furthermore, pref-1
mRNA was decreased to a similar extent at 100 nM and
slightly less at 10 nM. We hypothesized that if
pref-1 down-regulation by dexamethasone is a mechanism
whereby glucocorticoids promote adipogenesis, then this should also be
reflected by the effectiveness of these dexamethasone concentrations in
supporting adipogenesis. To test the effects of reduced dexamethasone
concentrations, confluent preadipocytes were initiated to differentiate
by a 48-h exposure to a combination of 0.5 mM MIX and 1 µM dexamethasone or at reduced dexamethasone concentrations of 100 pM, 1 nM, 3 nM, 5 nM, 7 nM, 10 nM,
and 100 nM in the presence of 0.5 mM MIX. 5 days after the removal of agents, the extent of adipocyte
differentiation was determined by expression of mRNAs for the
adipocyte markers PPAR
, SCD1, and aFABP, and is shown in Fig.
6A; these data are plotted in Fig. 6B. Cultures treated with 0.5 mM MIX alone
showed nearly no adipose conversion (Fig. 6A, lane
1). Minimal, if any, adipocyte differentiation occurs, as judged
by morphology (data not shown) and expression of adipocyte markers,
with dexamethasone concentrations of 1 nM and lower. A low
but detectable increase in expression of adipocyte markers is observed
at 3 nM dexamethasone which increases markedly by 10 nM dexamethasone to, on average, levels of 75% of the
maximal, 1 µM, level. Taken with the dose response of
pref-1 mRNA to dexamethasone shown in Fig. 5, A and
B, with the critical range identified to be between 1 and 10 nM, these data not only reveal that dexamethasone
concentrations 100 times lower than the standard 1 µM can
significantly decrease pref-1 mRNA levels and support adipocyte
differentiation, but they also indicate a strong correlation between
dexamethasone-mediated down-regulation of pref-1 and the
degree of differentiation of 3T3-L1 preadipocytes in response to
dexamethasone concentration.

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Fig. 6.
Dexamethasone-dependent decrease
of pref-1 mRNA correlates with dexamethasone efficacy in adipocyte
differentiation. A, Northern analysis. Confluent 3T3-L1
preadipocytes were treated with 0.5 mM MIX alone
(lane 1) or in combination with dexamethasone
(DEX) concentrations of 100 pM (lane
2), 1 nM (lane 3), 3 nM
(lane 4), 5 nM (lane 5), 7 nM (lane 6), 10 nM (lane
7), 100 nM (lane 8), or 1 µM
(lane 9). After 48-h treatment cells were allowed to
differentiate for 5 days and were then subjected to Northern analysis
and hybridization with PPAR , SCD1, and aFABP probes. Ethidium
bromide staining of 18 S rRNA is shown. B, dose-response
plot. The autoradiogram shown in panel A was quantitated by
scanning densitometry, and values were corrected against 18 S rRNA and
plotted as a percent of the 1 µM dexamethasone treatment
level (lane 9, panel A) for each marker mRNA
(solid triangle, PPAR ; hollow triangle, aFABP;
solid circle, SCD1).
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Time-dependent Dexamethasone Down-regulation of Pref-1
mRNA Predicts the Degree of Subsequent Adipocyte
Differentiation--
Our data show that during the dexamethasone/MIX
adipogenic induction phase, pref-1 mRNA levels and protein are
markedly reduced. This is consistent with attenuation of
pref-1 gene transcription by dexamethasone determined by
transcription run-on assay. We identify dexamethasone to be the primary
component responsible for pref-1 down-regulation during the
early, hormonal induction phase of the adipocyte differentiation
process. Furthermore, the observed correlation between the
concentration of dexamethasone which down-regulates pref-1 mRNA and
that which supports adipocyte differentiation is consistent with the
concept that a possible mechanism whereby dexamethasone exerts its
adipogenic effects is through down-regulation of pref-1. To
address this further we next determined if there is a relationship
between the extent of the decrease in pref-1 mRNA at the end of the
dexamethasone/MIX treatment phase and the degree of subsequent terminal
differentiation to adipocytes. To this end, we modulated pref-1 levels
in 3T3-L1 preadipocytes by varying the dexamethasone exposure times.
Because dexamethasone alone is not sufficient for differentiation of
these cells, the effects of prolonged dexamethasone exposure time were, by necessity, determined in the context of the dexamethasone/MIX differentiation protocol. The standard dexamethasone/MIX protocol involves a 48-h treatment, at which time we have shown the pref-1 mRNA has been reduced by approximately 80%.
To address the effects of intermediate or prolonged dexamethasone
exposure times on differentiation, 3T3-L1 cells were treated with
dexamethasone/MIX for 7, 24, 48, and 96 h. Fig.
7A shows the level of pref-1
mRNA present at each of these dexamethasone/MIX treatment times.
The 24-h half-life we determined for pref-1 mRNA predicts that at
96 h pref-1 mRNA levels will be reduced to approximately 6%
of initial levels. After the indicated dexamethasone/MIX exposure periods, cultures were shifted to normal growth medium and continued until 6 days after the onset of dexamethasone/MIX exposure. At this
time, several days after dexamethasone/MIX had been removed from growth
medium, cells were judged for degree of differentiation by
morphological criteria including cell shape and lipid accumulation and
by the expression of the adipocyte marker mRNAs PPAR
and SCD1(Fig.
7B). Cells exposed to dexamethasone/MIX for 7 h, a time at which a decrease in pref-1 mRNA levels is not readily apparent, showed minimal if any morphological and molecular indications of
differentiation at 6 days after the initiation of differentiation. In
comparison, lowering pref-1 mRNA levels via longer dexamethasone treatment periods increased the extent of adipocyte differentiation. The most dramatic morphological differences are apparent between 48 and
96 h, and this is reflected by the Northern analysis shown in Fig.
7B. Maximal reduction of pref-1 mRNA by dexamethasone, the 96-h treatment period, resulted in virtually 100% of the cells attaining typical adipocyte morphology with marked lipid accumulation. SCD1 levels rise dramatically with increasing dexamethasone/MIX exposure times and correlate well with overall morphological
differentiation. PPAR
levels, apart from the 7-h treatment, are not
as indicative of adipocyte differentiation because similar PPAR
mRNA levels are detected as the result of the 24-, 48-, and 96-h
dexamethasone/MIX treatment times. Given that dexamethasone may have
multiple effects in adipocyte differentiation, it would be premature to
conclude that the effects of dexamethasone in adipocyte differentiation are mediated solely through its ability to dramatically reduce pref-1
levels. These data, however, clearly indicate that dramatically lowering pref-1 levels, via prolonged exposure to dexamethasone/MIX, greatly optimizes differentiation.

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Fig. 7.
Decreased pref-1 mRNA by prolonged
exposure to dexamethasone/MIX enhances adipocyte differentiation.
A, Northern analysis of pref-1 mRNA. RNA was prepared
from 3T3-L1 preadipocytes (0 h) or after exposure to the standard
dexamethasone (DEX)/MIX treatment for 7, 24, 48, and 96 h and subjected to Northern analysis with a 32P-labeled
pref-1 cDNA probe. The lower panel shows 28 S and 18 S
ribosomal RNA on the ethidium bromide-stained gel. B,
correlation of dexamethasone-mediated pref-1 decrease with extent of
subsequent adipocyte differentiation. 3T3-L1 preadipocytes were exposed
to dexamethasone/MIX for the times indicated below, after which agents
were removed. Differentiation was continued through 6 days after the
onset of the dexamethasone/MIX treatment. Upper panel,
photomicrography of representative fields of live cells after the
indicated dexamethasone/MIX exposure periods. Lower panel,
Northern analysis with 32P-labeled cDNA probes for
SCD1, PPAR , and ethidium bromide-stained 28 S and 18 S ribosomal
RNA. Dexamethasone/MIX exposure times were: 7 h (lane
1), 24 h (lane 2), 48 h (lane 3),
and 96 h (lane 4).
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Decreasing Pref-1 Levels by Transfection of Antisense Pref-1 in
3T3-L1 Cells Attenuates Dexamethasone Dependence of Adipocyte
Differentiation--
Our results indicate that dexamethasone, a key
component of the adipogenic differentiation mixture, down-regulates
pref-1 mRNA during the hormonal treatment phase. Furthermore, the
time course of the decrease in pref-1 mRNA and the transcription run-on assays reveal that transcriptional suppression of the pref-1
gene by dexamethasone occurs immediately upon the addition of
dexamethasone. This decrease in pref-1 mRNA is therefore one
of the earliest responses known to date to a component of the
adipogenic differentiation mixture. Our studies herein indicate that we
are able to affect the extent of subsequent adipocyte differentiation
by modulating pref-1 mRNA levels by the dexamethasone
concentration or treatment time employed. In these studies, however,
although we showed that the extent to which dexamethasone lowered
pref-1 mRNA levels correlated with adipocyte differentiation,
effects of changes in dexamethasone concentration or treatment time,
other than those on pref-1, cannot be excluded.
To address specifically if dexamethasone-mediated down-regulation of
the anti-adipogenic factor pref-1 is a mechanism whereby glucocorticoids promote adipocyte differentiation we employed antisense
pref-1. This thereby allows a reduction in the level of endogenous
pref-1 outside of its modulation by dexamethasone. We theorized that if
a major functional role of dexamethasone in promoting adipocyte
differentiation was via its reduction of pref-1 levels, then reducing
these levels independent of dexamethasone treatment by transfection of
the antisense pref-1 sequence would substitute for dexamethasone
treatment in the promotion of adipocyte differentiation. We transfected
3T3-L1 preadipocytes with a pref-1 antisense expression construct.
After selection and expansion of stable clones, approximately 40 clonal
lines of 3T3-L1 preadipocytes were screened for a reduction in
endogenous pref-1 mRNA level via Northern analysis, and two that
had markedly reduced pref-1 mRNA levels were identified. One of
these antisense clones was further studied. Endogenous pref-1 mRNA
levels of this antisense clone by Northern analysis are shown in Fig.
8A, along with a control clone
that had pref-1 mRNA levels similar to those before transfection.
At confluence, antisense and control cells were treated with 0.5 mM MIX alone (no dexamethasone) or in combination with 2 nM, 10 nM, or 1 µM dexamethasone.
In addition to the standard 1 µM dexamethasone, we chose
to test the differentiation capacity of these cells in response to
these lower dexamethasone concentrations. This was based on our studies
in Fig. 6, showing that although it was not as effective as 1 µM dexamethasone, 10 nM dexamethasone was
highly effective in supporting adipocyte differentiation, but 2 nM was not. After subjecting antisense and control cells to
the indicated differentiation conditions, the extent of adipocyte conversion was determined by Northern analysis for a panel of adipocyte
differentiation markers, fatty acid synthase, SCD1, PPAR
, and aFABP
(Fig. 8B), and signals were quantitated and corrected for
RNA loading. Whereas the antisense cells showed a similar level of
expression of these markers at 2 nM, 10 nM, or
1 µM dexamethasone, control cells exhibited the same
dexamethasone dose responsiveness in their conversion to adipocytes as
was shown in Fig. 6. SCD1 expression was 70% at 10 nM and
33% at 2 nM compared with SCD1 mRNA levels at 1 µM dexamethasone. A similar dexamethasone dose dependence
was noted with PPAR
, fatty acid synthase, and aFABP mRNA levels.
In the absence of dexamethasone, but in the presence of 0.5 mM MIX, although control cultures showed no expression of
adipocyte marker mRNAs such as SCD1 and aFABP, the antisense clone
showed low but significant SCD1 and aFABP mRNA levels (Fig. 8C). Because of the low overall degree of adipose conversion
in these cells, the autoradiogram was subjected to prolonged exposure and therefore should not be compared directly with that in Fig. 8B. These data therefore indicate that in the presence of
0.5 mM MIX, the same degree of differentiation of antisense
cells occurs in response to 2 nM, 10 nM, and 1 µM dexamethasone, and low but detectable differentiation
occurs in the absence of dexamethasone. Furthermore, of the initial 40 clones isolated, the two clones with reduced pref-1 mRNA showed
enhanced adipocyte conversion and none of those that did not show
decreased pref-1 mRNA levels showed increased differentiation at
lowered dexamethasone concentration (data not shown). Fig.
8D shows photomicrographs of live antisense cells after
differentiation in the noted dexamethasone concentrations. Adipose
conversion is indicated by rounded morphology and lipid accumulation.
At 2 nM, 10 nM, and 1 µM
dexamethasone, nearly complete adipose conversion of cells was
observed; even without added dexamethasone approximately 5-10% of
cells differentiated to adipocytes. Taken together, these data support
a model whereby a major function of dexamethasone in the
adipoconversion process is through its ability to effectively reduce
pref-1 transcription.

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Fig. 8.
Antisense pref-1 promotes adipocyte
differentiation and results in adipocyte differentiation at reduced
dexamethasone concentrations. A, decrease of endogenous
pref-1 mRNA in antisense 3T3-L1 cells. Northern analysis for pref-1
mRNA was conducted on 5 µg of total RNA from either control or
antisense cells. The ethidium bromide staining of 18 S rRNA is shown
below. B, adipocyte differentiation of control and antisense
cells. Control and antisense cells were subjected to differentiation
with 0.5 mM MIX and the indicated concentrations of
dexamethasone (DEX): 2 nM (lane 1),
10 nM (lane 2), 1 µM (lane
3); total RNA was harvested 7 days later. Northern blots were
hybridized to random primed probes for fatty acid synthase
(FAS), SCD1, PPAR , and aFABP. The ethidium bromide
staining of the 18 S rRNA is shown below. C, Northern
analysis of cells analyzed as in panel B after treatment
with 0.5 mM MIX alone. D, photomicrographs of
antisense cells treated with the indicated concentrations of
dexamethasone in the presence of 0.5 mM MIX.
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DISCUSSION |
Studies on adipocyte differentiation indicate that signals to
differentiate are likely combinatorial and involve growth arrest, proper extracellular matrix environment, transcriptional effectors, and
factors in fetal calf serum which may inhibit or potentiate responses
of known modulators (24, 25). For 3T3-L1 preadipocytes, signals for the
initiation of differentiation are provided by the combined exposure to
the hormonal inducing agents dexamethasone and MIX. Two-dimensional gel
electrophoresis indicates that levels of more than 100 as yet
uncharacterized proteins change during the first 5 h of initiation
of differentiation (32). Because pref-1 is an inhibitor of adipogenesis
and its down-regulation is integral to 3T3-L1 adipocyte
differentiation, we hypothesized that pref-1 levels may be repressed by
the differentiation-inducing agents dexamethasone/MIX. We present
findings here that the promotion of adipogenesis by dexamethasone is
mediated in part by its repression of pref-1. Our
observations are the first direct link between a well established agent
that induces adipocyte differentiation and pref-1 regulation. We have
demonstrated that pref-1 protein and mRNA levels are decreased
dramatically by dexamethasone/MIX and that dexamethasone is the primary
agent acting to decrease pref-1 expression. Although the
transcriptional activation of genes during terminal adipocyte
differentiation has received considerable study, pref-1 is
one of a few genes identified to be repressed during the
dexamethasone/MIX initiation phase. The similar time course of decrease
in pref-1 mRNA levels in both dexamethasone-treated and actinomycin
D-treated 3T3-L1 preadipocytes indicates that dexamethasone acts to
attenuate pref-1 gene transcription immediately; we find by
transcription run-on assays that dexamethasone acts to repress
pref-1 directly. To our knowledge, this immediate
down-regulation of pref-1 gene expression is the earliest
response to the dexamethasone component of the differentiation mixture.
We find that not only the standard 1 µM concentration but
also 10 nM dexamethasone down-regulates pref-1
markedly while initiating effective adipocyte differentiation. Furthermore, 1 nM dexamethasone was not effective at either
dexamethasone-mediated down-regulation of pref-1, nor
did it support adipocyte differentiation. This indicates a strong
correlation between the dexamethasone concentration effective in
supporting adipocyte differentiation and dexamethasone-mediated
down-regulation of pref-1. In addition, the degree of
reduction of pref-1 mRNA during the dexamethasone/MIX exposure
period correlates with the extent of adipocyte conversion which occurs
during subsequent terminal differentiation. Reducing pref-1 mRNA to
nondetectable levels in preadipocytes, via a longer dexamethasone/MIX
exposure period, greatly accentuates adipocyte differentiation.
Moreover, employing antisense pref-1 to lower endogenous pref-1
mRNA levels partially relieved the dexamethasone requirement for
differentiation, indicating that dexamethasone promotes adipogenesis,
in part, through transcriptional down-regulation of
pref-1.
The observation that the transcription factors C/EBP
and C/EBP
are induced approximately 5-10-fold during hormonal treatment of
3T3-L1 preadipocytes by MIX and dexamethasone, respectively (26, 27),
led to the suggestion that C/EBP
and C/EBP
might relay the
adipogenic effects of dexamethasone/MIX early in adipocyte differentiation by increasing PPAR
levels. Subsequent functional studies by Farmer and co-workers have addressed this hypothesis in
detail (28, 29). Expression of ectopic C/EBP
in NIH 3T3 cells
obviates the MIX requirement for their differentiation. This indicates
that C/EBP
may be a primary effector of MIX action in adipocyte
differentiation. However, differentiation of these cells is enhanced
dramatically by inclusion of dexamethasone, with a clear dexamethasone
dose dependence of PPAR
mRNA observed. To this end it had been
proposed that the dexamethasone-mediated induction of C/EBP
could
lead to the formation of C/EBP
·C/EBP
heterodimers, which were
postulated to be more transcriptionally active than C/EBP
homodimers. To the contrary, however, C/EBP
was found to contribute
only minimally to functional C/EBP complexes during the initial stages
of differentiation of dexamethasone-treated C/EBP
-expressing NIH 3T3
cells (28). Moreover, dexamethasone treatment was necessary for
adipogenic differentiation of NIH 3T3 fibroblasts ectopically
expressing high levels of both C/EBP
and C/EBP
(28), indicating
that dexamethasone must be fulfilling an adipogenic function other than
enhancing C/EBP
levels. Our studies herein clearly show that
dexamethasone does not increase expression of PPAR
mRNA in
3T3-L1 preadipocytes. Based on our data here we propose that
down-regulation of pref-1 may be a function of dexamethasone
in promoting adipogenesis. Additional target genes that relay
dexamethasone action during adipocyte differentiation remain to be clarified.
It is of interest that we identify dexamethasone as the major component
of the dexamethasone/MIX differentiation mixture that mediates the
down-regulation of pref-1 because dexamethasone has been shown to have a physiological role in several other models of
adipocyte differentiation. Mesenchymal stem cells with the potential to
form the mesodermal cell types of adipose tissue, muscle, bone, and
cartilage have a widespread distribution in the connective tissue
compartments of many organs and organ systems (33-35). In
vitro studies show their differentiation to be dependent upon
dexamethasone treatment in a dose- and time-dependent
manner, with indications that the formation of adipocytes is
particularly related to dexamethasone exposure. In RCJ 3.1 cells, a
clonal population derived from 21-day fetal rat calvaria, prolonged
dexamethasone exposure favored maintenance of the adipocyte and muscle
phenotypes (34). For the D1 bone-marrow derived pluripotent mesenchymal cell line, dexamethasone stimulates their differentiation into adipocytes at the expense of osteoblast differentiation
(35).
In contrast to our studies in 3T3-L1 preadipocytes, where treatment
with 1 µM dexamethasone for 2 days drastically
down-regulates pref-1 mRNA levels to 20% of that in nontreated
controls, previous studies on pref-1 in a human neuroblastoma cell line
indicate that dexamethasone increases pref-1 mRNA expression in
these cells (36). In this study, the low but detectable levels of
pref-1 mRNA in neuroblastoma cells increased 8-9-fold upon
exposure to 100 µM dexamethasone for 7 days. It is
unclear whether the week-long dexamethasone exposure affected the
differentiation state of the neuroblastoma cells, which in turn might
alter pref-1 mRNA levels. The opposite responses to dexamethasone
in these two cell types might be attributable to different experimental
conditions; in addition, multiple examples exist for opposite
glucocorticoid effects on the regulation of a given gene, depending on
the cell or tissue type examined. For example, the phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids in the liver and
kidney and repressed in adipose tissue (37). The current general model
for glucocorticoid action is that transcriptional activation involves
binding of the receptor complex to simple glucocorticoid regulatory
elements. On the other hand, repression and more complex regulation
occur via composite glucocorticoid elements and require action of a
glucocorticoid receptor combined with one or more nonreceptor
sequence-specific regulators. In the latter case, regulation is
dependent upon the presence or absence of accessory DNA-binding
proteins in a given cell type. Futher studies are needed to identify
the elements in the pref-1 promoter responsible for
dexamethasone-mediated repression (38).
We do not at present know the cellular pathways that are affected as
the consequence of pref-1 down-regulation via dexamethasone, which in turn may mediate adipocyte differentiation. Indications are
that permanent exit from the cell cycle may mark the irreversible commitment to adipocyte differentiation, and both PPAR
and C/EBP
have been shown to be involved in growth arrest (39, 40). Studies in
3T3-L1 cells suggest that dexamethasone could be responsible for
establishing the postmitotic growth arrest state that is required for
adipocyte differentiation (41). A recent report indicates that tumor
necrosis factor-
treatment of mature adipocytes, which causes a
modulation of their phenotype wherein they decrease lipid content and
take on a fibroblastic appearance, decreases PPAR
levels whereas
pref-1 expression remains permanently abolished (42). This
argues that pref-1 is not involved in the later, lipogenic stages of
terminal differentiation but that its effects are exerted early during
the commitment of preadipocytes to the differentiation program,
i.e. the dexamethasone/MIX treatment period. This is
consistent with our observations here and with our previous findings
that 3T3-L1 cells subjected to the dexamethasone/MIX differentiation
protocol in the presence of soluble pref-1 have markedly reduced
expression of PPAR
. Our data clearly demonstrate that
transcriptional repression of pref-1 is an early action of dexamethasone in 3T3-L1 adipocyte differentiation. The fact that pref-1 transcription is blocked by dexamethasone, a
component of the standard differentiation mixture, indicates that
down-regulation of pref-1 by glucocorticoids may be a
mechanism for promoting adipogenesis. In sum, our data support a model
whereby exposure of 3T3-L1 cells to dexamethasone/MIX provides initial
signals for differentiation and that these include rapid attenuation of pref-1 transcription.