1 Diseases of Aging Program, Ottawa Health Research Institute at Ottawa
Hospital, University of Ottawa, 725 Parkdale Avenue, Ottawa, Ontario K1Y 4K9,
Canada
2 Biochemical Neuroendocrinology Laboratory, Clinical Research Institute of
Montreal, University of Montreal, Montreal, Quebec H2W 1R7, Canada
* Author for correspondence (e-mail: mmbikay{at}ohri.ca )
Accepted 6 December 2001
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Summary |
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Key words: Gene expression regulation, Serine proteinases, Protease inhibitors, Transcription factors
![]() |
Introduction |
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The determining role of transcriptional regulation in this conversion has
been extensively studied. Two families of transcription factors, the
CCAAT/enhancer-binding proteins (C/EBPs) and the peroxisome
proliferator-activated receptors (PPARs), play particularly important roles as
mediators of adipogenic signals (Gregoire
et al., 1998; Hwang et al.,
1997
). Among members of the C/EBP family, C/EBPß and
C/EBP
regulate the early response to these signals. Their rapid and
transient induction relays the effects of IBMX and Dex, and catalyzes the
transactivation of C/EBP
and PPAR
genes
(Cao et al., 1991
;
Yeh et al., 1995
;
Zhu et al., 1995
). The latter
factors, in turn, activate a variety of adipocyte-specific genes. Transduction
of either factor in multipotent NIH-3T3 fibroblasts has been shown to promote
their conversion into adipocytes (Freytag
et al., 1994
; Tontonoz et al.,
1994
).
Adipocyte differentiation may also be determined by post-translational
modifications of adipogenic proteins. Proteolytic activation of latent
precursors is one such modification. Sterol regulatory element binding protein
1 (SREBP-1), otherwise known as adipocyte determination and differentiation
factor 1 (ADD1), represents an example of an adipogenic protein that needs
activation by proteases. This transcriptional factor is released into the
nucleus from a membrane-bound precursor located in the endoplasmic reticulum
following two successive cleavages at distinct sites, the first by a
pyrolysin-like convertase called site-1 protease
(Sakai et al., 1998) or SKI-1
(Seidah et al., 1999
), the
second by a metalloproteinase known as site-2 protease
(Brown and Goldstein, 1999
).
SREBP-1 regulates genes involved in the biosynthesis of cholesterol and fatty
acids (Brown and Goldstein,
1999
). Its downregulation has been shown to inhibit the
differentiation of 3T3-L1 cells into adipocytes
(Brun et al., 1996
).
Receptors for insulin (InsR) and insulin-like growth factor 1 (IGF-1R) are
other important mediators of adipocyte differentiation
(Accili and Taylor, 1991;
Smith et al., 1988
) that are
activated by limited proteolysis. These receptors are biosynthesized as
inactive precursors and are rendered functional by a single cleavage into two
chains (
and ß) linked by disulfide bonds. Furin, the enzyme
mediating this processing (Bravo et al.,
1994
; Lehmann et al.,
1998
) belongs to a family of serine proteinases known as
proprotein convertases (PCs) (Seidah and
Chretien, 1999
; Zhou et al.,
1999
).
The PC family also includes PACE4, PC1/3, PC2, PC4, PC5/6 and PC7/8. Furin,
PACE4, PC5 and PC7 are widely expressed. PC1 and PC2 are primarily found in
endocrine and neuroendocrine cells. PC4 expression is mostly confined to the
testis. PCs act in the secretory pathways where they cleave precursor proteins
after selected pairs of basic residues. Their substrates include precursors to
hormones and neuropeptides, cell surface receptors, extracellular matrix
components, viral glycoproteins and bacterial toxins
(Seidah and Chretien, 1999;
Zhou et al., 1999
).
Of all the PCs, furin has the widest variety of proteins among its
substrates. This type 1-membrane-bound enzyme cycles between the TGN and the
surface of all cells (Molloy et al.,
1994). It cleaves its substrates after an R-X-K/R-R (X represents
any amino acid) motif. This motif is found at the processing site of proInsR
and proIGF-1R as well as that of precursors to extracellular matrix components
such as stromelysin-3, fibrillin and membrane type 1 matrix metalloproteinase
(Lönnqvist et al., 1998
;
Santavicca et al., 1996
;
Yana and Weiss, 2000
). Furin
cleavage of these precursors could thus be integral to mitogenic cell
signaling and to plasma membrane remodeling. In this context, furin has been
implicated in the growth and differentiation of gastric surface mucous cells
and of cardiocytes (Konda et al.,
1997
; Sawada et al.,
1997
).
The importance of PCs in adipogenesis has not been examined before. In this report, we describe the regulation of several PC genes during adipocyte differentiation of 3T3-L1 cells. We also show that PC-specific inhibitors block this differentiation at an early stage, confirming the involvement of these proteinases for the process.
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Materials and Methods |
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Establishment of clonal lines of transfectant 3T3-L1 cells
The pcDNA3 expression vector (Invitrogen) and a derivative carrying a
full-length cDNA for the PC inhibitor 1-antitrypsin Portland
(
1-PDX) (Anderson et al.,
1993
; Benjannet et al.,
1997
; Dufour et al.,
1998
; Jean et al.,
1998
; Munzer et al.,
1997
; Tsuji et al.,
1999a
) were transfected into 3T3-L1 preadipocyte cells using the
DOSPER Liposomal Transfection Reagent (Boehringer Mannheim). Independent
G418-resistant clonal cell lines were established from clones picked from
different transfection culture dishes.
Semi-quantitative RT-PCR
Total RNA was extracted using a guanidine isothiocyanate method previously
described (Day et al., 1992).
The RNA was treated with RNase-free DNaseI (Life Technologies) and 2 µg
were reverse-transcribed into cDNA using the SuperScript II Reverse
Transcriptase (Life Technologies). Three microliters of the reverse
transcription reaction were used for PCR amplification of cDNA fragments for
PC1, PC2, PC5, PC7, PACE4, furin, adipsin, PPAR
1 and
2
(collectively called PPAR
),
1-PDX or the ribosomal protein L30
as internal standard. The sequence of the PCR primers is shown in
Table 1. The PCR reaction mixes
contained 0.5 units of rTaq DNA polymerase (Life Technologies), 1x PCR
buffer, 0.2 mM dNTP, 0.5 µM sense and antisense primers for a specific
cDNA, and L30 in a 50 µl volume. PCR was performed with PC or
1-PDX
primers for a total of 32-37 cycles, together with L30 primers for the last 20
cycles. For PPAR
and adipsin semi-quantification, the reaction was
conducted for 25 cycles with their respective primers and L30 primers. The
number of cycles was pre-determined to fall within the linear range of
amplification of each PCR product. Each cycle involved a 94°C/1 minute
denaturation step, a 60°C/1 minute annealing step, and a 72°C/1 minute
polymerization step. The PCR products were electrophoresed on agarose gels,
stained with ethidium bromide, revealed by UV irradiation and analyzed by
densitometry using the National Institutes of Health Image software. The
authenticity of the amplified sequences was verified by restriction enzyme
mapping.
|
Western blot analysis
Cells were washed with PBS and scraped off dishes in 1 ml of PBS. They were
sedimented by centrifugation, lysed by the addition of 100 µl of SDS-gel
buffer, and heated to 100°C for 5 minutes
(Cao et al., 1991). Extracted
proteins were fractionated by 12% SDS-PAGE and transferred to Immobilon-P
membranes (Millipore). The membranes were incubated with an anti-C/EBPß
antibody (Santa Cruz Biotechnology) and immunoreactive proteins were revealed
by chemiluminescence (Amersham). To equalize protein loading, a preliminary
protein gel was stained with Coomassie brilliant blue for a visual estimation
of the quantity of proteins in the cellular extracts. Nuclear extracts were
prepared as described by Dent and Latchman
(Dent and Latchman, 1993
) from
cells treated for 24 hours with the adipogenesis-inducing agents. Aliquots
equivalent to 50 µg of proteins were treated or not with 50 units of calf
intestine alkaline phosphatase (CIAP) (Boehringer) for 1 hour at 37°C;
they were then diluted in SDS-gel buffer, boiled for 5 minutes and analyzed by
Western blotting for C/EBPß proteins.
To examine proIGF-1R proteolytic processing, we used transfected control
and 1-PDX preadipocytes as well as normal preadipocytes treated at
confluency with 0 or 100 µM dec-RVKR-CMK for 48 hours. Whole-cell extracts
in 60 mM Tris-HCl buffer, pH 7.5/1% SDS were prepared; aliquots corresponding
to 100 µg of proteins were fractionated by 8% SDS-PAGE and analyzed by
immunoblotting as described above using an antibody against the ß subunit
of IGF-1R (Santa Cruz Biotechnology).
IRS-1 phosphorylation was examined by sequential probing of a blot carrying
50 µg of proteins from control and 1-PDX cells, stimulated or not
with insulin (10 µg/ml). Probing was conducted subsequently with an
anti-IRS-1 antibody and, after membrane stripping using Re-Blot Plus-Mild
(Chemicon), with an anti-phosphotyrosine antibody. Both antibodies were
obtained from Santa Cruz Biotechnology.
Electrophoretic mobility shift assay (EMSA)
A 5'-[32P]-labeled double-stranded C/EBP consensus
oligonucleotide (5'-GAT CGA TTG CGC AAT C-3')
(Osada et al., 1996) was used
as probe. The binding mixture contained 10 µg of nuclear extract, 2 µg
of poly dI-dC, 20 mM Hepes, pH 7.9, 4% Ficoll, 0.5 mM DTT, 1 mM
MgCl2, 50 mM KCl and 20,000 cpm of labeled probe. It was incubated
for 40 minutes at 4°C. In competition assays, the mixture was supplemented
with a 100-fold molar excess of unlabeled oligonucleotide prior to adding the
labeled probe. In supershift assays, nuclear extracts were incubated for 90
minutes with 1 µg of an antibody directed against the C-terminus of
C/EBPß prior to adding the labeled probe. The binding mixtures were
electrophoresed at 100 V at room temperature in a 6% nondenaturing
polyacrylamide gel with a 45 mM Tris-borate/1 mM EDTA buffer. The gel was then
dried and subjected to autoradiography.
Immunohistochemistry
Confluent preadipocytes were induced to differentiate for 24 hours; they
were then fixed with 50% ethanol/1% H2O2 and incubated
for 90 minutes with the anti-C/EBPß antibody (diluted 1:250). The
immunoreaction was amplified using a horseradish peroxidase-based Tyramine
Signal Amplification (TSA) Plus DNP kit (NEN Life Science Products), and
revealed using the chromogenic substrate 3,3' diaminobenzidine.
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Results |
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For comparison, we also examined the relative levels of these transcripts
in epididymal white adipose tissue (WAT) from C57B1/6 mice. Compared with
3T3-L1 adipocytes, WAT contained more PACE4 transcripts, less PC7 transcripts
and markedly less furin transcripts (Fig.
1A). These differences may be due to the fact that, unlike 3T3-L1
adipocytes, the WAT is made of a heterogeneous populations of cells
(Smas and Sul, 1995) that may
have distinct patterns of PC transcripts.
PC inhibitors block adipocyte differentiation
To assess the importance of these PCs for adipogenic conversion, we
produced clonal lines of 3T3-L1 transfectants carrying either the pcDNA3
vector (control cell lines) or a pcDNA3/1-PDX for expression of the
PC-specific inhibitor
1-PDX (
1-PDX cell lines). Because 3T3-L1
cells are notorious for spontaneously giving rise to differentiation-resistant
cells, 3 control and 16
1-PDX transfectant cell lines were established.
These cell lines were induced to differentiate and stained with Oil Red O. A
typical staining before and after adipogenic treatment is illustrated in
Fig. 2A. All 3 control cell
lines stained very strongly with Oil Red O, an indication that they
accumulated substantial amounts of cytoplasmic triglycerides. By contrast, 14
out of the 16 cell lines derived from transfection with the
1-PDX
expression vector exhibited noticeably reduced staining with Oil Red O
(Fig. 2A), indicative of low
triglyceride content. By light microscopy, only 2-5% of cells in these lines
contained lipid vesicles typical of fully differentiated adipocytes (not
shown). Transcripts for
1-PDX were detectable by RT-PCR in the 14
lines. They were absent in control lines. A representative analysis of the PCR
product is shown in Fig. 2B.
All subsequent experiments involving transfected cells were conducted with the
3 control cell lines and 3 randomly selected
1-PDX-positive cell lines.
To ascertain that the effect of
1-PDX is specific, we also used
recombinant
1-PDX, which has been shown to efficiently inhibit cellular
PC activities when added to cell culture medium
(Jean et al., 1998
). As
control, we used the
1-antitrypsin (
1-AT) protein, from which
1-PDX was derived but which is not a PC inhibitor
(Anderson et al., 1993
) As
expected,
1-PDX added in medium at 8 µM blocked adipocyte
differentiation whereas
1-AT had no effect at this concentration
(Fig. 3A).
|
|
To further confirm the importance of PCs for adipocyte differentiation, we
incubated untransfected post-confluent 3T3-L1 cells, for 24 hours before and
for the 48 hours of adipogenic stimulation, in medium containing non-cytotoxic
amounts (20-100 µM) of dec-RVKR-CMK, an irreversible inhibitor of PCs
(Angliker, 1995;
Munzer et al., 1997
;
Santavicca et al., 1996
;
Tsuji et al., 1999a
). Then we
assessed their adipogenic response by Oil Red O staining. Incubation with the
inhibitor reduced the staining in a concentration-dependent manner
(Fig. 3B). Maximum inhibition
was reached at 80 µM dec-RVKR-CMK. The general cellular morphology after
adipogenic treatment is shown in Fig.
3C. Adipocyte conversion was extensive in the absence of the
inhibitor. It was blocked in its presence, partially at 20 µM and
completely at 100 µM. The timing of incubation with the inhibitor was
critical as addition of the latter any time after the two-day induction period
failed to block differentiation (data not shown). Thus, like
1-PDX,
this synthetic inhibitor was able to block the adipocyte conversion of 3T3-L1
cells, reinforcing the view that PCs are crucial for adipogenesis.
Altered expression of adipsin, PPAR and C/EBPß in
1-PDX-expressing cells
To further characterize the phenotype of the 1-PDX-transduced cells,
we examined by semi-quantitative RT-PCR the relative levels of PPAR
and
adipsin. The former is an early marker of adipogenic conversion and the latter
a late marker of the adipocyte phenotype. Representative results are
illustrated in Fig. 4.
Expectedly, the levels of PPAR
and adipsin mRNA transcripts markedly
increased in control cells after adipogenic induction. By contrast, in
1-PDX-expressing cells, a similar treatment produced only a slight and
belated induction in the level of adipsin transcripts and none at all for
PPAR
transcripts.
|
The gene for PPAR is one of those activated by C/EBPß in the
cascade of transcriptional events leading to the adipogenic conversion of
3T3-L1 cells (Zhu et al.,
1995
). We therefore examined how C/EBPß expression was
affected by
1-PDX transduction. By western blot analysis, total
extracts from control and
1-PDX-expressing cells contained comparable
levels of the LIP (liver-enriched inhibitory protein) and LAP (liver-enriched
activating protein) C/EBPß isoforms
(Fig. 5A). These levels
transiently increased during the 2-day treatment with inducing agents, as
expected (Cao et al., 1991
).
However, when nuclear extracts from these transfectant lines were analyzed by
C/EBPß-specific EMSA, the complexes observed with nuclear extracts from
1-PDX-expressing cells were of lesser intensity and of faster
electrophoretic mobility than those observed with nuclear extracts from
control cells (Fig. 5B, lane 1
versus lane 4). In both cases, the oligonucleotide electrophoretic shift was
specifically due to C/EBPß binding since the complexes could be retarded
in the presence of an anti-C/EBPß antibody
(Fig. 5B, lanes 2,5) and
abrogated in the presence of unlabeled competing oligonucleotide
(Fig. 5B, lanes 3,6). These
results suggested that, compared with control cells,
1-PDX-expressing
cells contained less or less active C/EBPß in their nucleus. We conducted
a comparative western blot analysis of the nuclear extracts to verify these
possibilities. Nuclear extracts from control cells contained two LAP
immunoreactive bands of nearly equal intensities; those from
1-PDX-expressing cells contained noticeably less of both isoforms, and
even less of the slower-migrating one (Fig.
5C, lanes 1,2). The latter represented a phosphorylated form of
the faster-migrating isoform, as shown by its disappearance in CIAP-treated
nuclear extracts (Fig. 5C,
lanes 3,4).
|
Reduced translocation of C/EBPß into the nucleus of stimulated
1-PDX-expressing cells was further confirmed by immunohistochemistry.
The results are shown in Fig.
5D. After a 24-hour stimulation with the adipogenic agents,
C/EBPß immunoreactivity in control cells was concentrated in the nucleus
(Fig. 5Db), it was mostly
perinuclear in the
1-PDX-expressing cells
(Fig. 5Dd). The specificity of
the immunoreaction was ascertained by omitting the anti-C/EBPß antibody
in the protocol (Fig.
5Da,c).
PC inhibitors block the processing of prolGF-1 receptors and mitotic
clonal expansion
The observed blockage by PC inhibitors of the transcriptional cascade
leading to adipocyte differentiation was most probably due to a failure in the
signaling pathways normally induced by adipogenic treatment. The insulin and
IGF-1 signaling pathway is one that is likely to be affected by the inhibitor
since the latter may reduce activation of proIGF-1R by furin
(Lehmann et al., 1998). To
verify that the proIGF-1R processing is abolished by PC inhibition, 3T3-L1
confluent preadipocytes were treated or not with dec-RVKR-CMK and analyzed for
proIGF-1R processing by immunoblotting. The results are shown in
Fig. 6A. In the absence of the
inhibitor, there was complete proIGF-1R processing since only the mature
IGF-1R ß chain was detected (Fig.
6A, lane 1). In dec-RVKR-CMK-treated cells, by contrast, proIGF-1R
was much more abundant than the mature IGF-1R ß chain
(Fig. 6A, lane 2). We observed
the same block of proIGF-1R processing in the
1-PDX-expressing cells
compared with control cells (Fig.
6A, lanes 3,4). This observation confirms the role of PCs in
proIGF-1R processing in 3T3-L1 cells. It also suggests that the block of
adipocyte differentiation may be due to inhibition of proIGF-1R
processing.
|
To confirm that the IGF-1R pathway was affected, we analyzed IRS-1
phosphorylation in control and 1-PDX cells
(Fig. 6B). This analysis was
conducted by sequential probing of a blot carrying proteins from control and
1-PDX cells, stimulated or not with insulin. The insulin concentration
used was high enough to activate type 1 IGF-1R. The first probing conducted
with an anti-IRS-1 antibody revealed an IRS-1 band in both control and
1-PDX cells. The second probing conducted with an anti-phosphotyrosine
antibody revealed a band overlapping that of IRS-1 only in insulin-stimulated
control cells.
Early following differentiation induction, 3T3-L1 preadipocytes undergo
2 rounds of mitotic clonal expansion as they express the early adipogenic
genes (Tang and Lane, 1999
).
It was recently demonstrated that mitotic clonal expansion was induced only by
insulin and not by IBMX or Dex (Qiu et
al., 2001
). We therefore examined whether this event was altered
in
1-PDX cells. To do so, we induced post-confluent control and
1-PDX cells and, three days later, we determined their number. For
control cells, the number increased almost fourfold, whereas it did not
significantly change in
1-PDX cells
(Fig. 6C), indicating that
1-PDX expression affected the mitotic clonal expansion step.
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Discussion |
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To assess the importance of PCs in the adipocyte differentiation of 3T3-L1
cells, we produced transfectant cell lines expressing the PC inhibitor
1-PDX. The majority (14/16) of these cell lines failed to convert into
adipocytes when subjected to an adipogenic treatment. The two lines that were
able to convert were not characterized. They may have derived either from
spontaneously G418-resistant cells, from cells that had incorporated a
fragment of the expression vector containing the resistance gene but not the
1-PDX transgene, or from cells that had incorporated the full vector
but in a transcription-repressing genomic environment. Endogenous expression
of
1-PDX did not cause any change in cell growth or in the levels of
apoptosis markers (not shown). The fact that addition of recombinant
1-PDX or the synthetic PC inhibitor dec-RVKR-CMK to the culture medium
of a native population of 3T3-L1 cells induced a similar differentiation block
is a strong indication that the observations made with
1-PDX
transfectant lines were due to the action of this inhibitor and not caused by
some clonal variation among 3T3-L1 cells. Other peptidyl CMKs such as
N-
-p-Tosyl-L-Phe-CMK and H-Glu-Gly-Arg-CMK had no inhibiting effect on
adipocyte differentiation when added in the medium (not shown).
The differentiation block was associated with absence of PPAR
induction. These factors regulate a variety of genes for proteins involved in
lipid metabolism (Tontonoz et al.,
1994
). PPAR gene activation is mediated by members of the C/EBP
(Wu et al., 1996
) and SREBP
(Fajas et al., 1999
) families.
The PPAR
gene promoter carries two functional C/EBP binding sites
(Zhu et al., 1995
) and two
E-boxes (Fajas et al., 1999
).
Adipogenic stimuli induce as much C/EBPß in
1-PDX-expressing cells
as in control cells. However, the amount of C/EBPß LAP isoform,
particularly its phosphorylated isoform, is dramatically reduced in the
nucleus. Moreover, nuclear C/EBPß from
1-PDX-transduced cells,
together with a consensus C/EBP oligonucleotide, forms a complex of faster
electrophoretic mobility on EMSA. The abnormal EMSA and western blot banding
patterns of nuclear C/EBPß from
1-PDX-expressing cells may be due
to inefficient post-translational modification of the factor. Thus, nearly
complete failure of C/EBPß translocation into the nucleus and abnormal
interactions with its binding sites in the PPAR
promoter may explain
why this gene is not activated by adipogenic signals in
1-PDX-expressing cells. C/EBPß is an early response factor in the
adipogenic signaling pathways as indicated by its rapid increase following
adipogenic stimulation. This regulation is reportedly mediated by cAMP in
response to IBMX (Yeh et al.,
1995
). It has been reported that C/EBPß can be phosphorylated
at multiple sites and that, depending on the phosphorylated site, its
DNA-binding activity either increases or decreases
(Trautwein et al., 1993
;
Trautwein et al., 1994
;
Piwien-Pilipuk et al., 2001
).
Moreover, it appears that acquisition of binding activity during mitotic
clonal expansion involves phosphorylation of C/EBPß
(Tang and Lane, 1999
). Our
results do not imply that lack of C/EBPß translocation into the nucleus
is due to an abnormal phosphorylation. They simply suggest that
1-PDX
prevents PC activation of proproteins involved in the signaling pathways
leading to C/EBP activation and nuclear translocation. The identity of these
precursors remains to be determined.
A signaling pathway that is most certainly affected by this inhibitor
involves the InsR/IGF-1R and the mitogen-activated protein kinase pathway
(Boney et al., 1998). Both
receptors are proven furin substrates
(Bravo et al., 1994
;
Lehmann et al., 1998
). Both
are known to be crucial for the proliferation and adipocyte conversion of
3T3-L1 cells (Accili and Taylor,
1991
; Smith et al.,
1988
). In this study, we have shown that
1-PDX as well as a
synthetic inhibitor of PCs can block proIGF-1R proteolytic maturation to
IGF-1R in preadipocyte cells. Unprocessed receptors would not efficiently bind
IGF-1 or insulin and would thus be unable to initiate the downstream signaling
normally observed with their processed forms. Signal transduction by these
peptides leads to phosphorylation of insulin-receptor substrate 1 (IRS-1)
(Myers et al., 1994
). We show
in this study that this phosphorylation does not occur in insulin-stimulated
1-PDX-expressing 3T3-L1 cells. We also show the mitotic clonal
expansion step, which depends on insulin induction
(Qiu et al., 2001
), is
inhibited in
1-PDX cells..
For optimal inhibitory effect, the synthetic inhibitor dec-RVKR-CMK must be added to post-confluent cells 24 hours before and 48 hours during adipogenic treatment. This timing is required presumably to reduce the amounts of pre-existing active PCs and PC-processed adipogenic products, such as IGF-1R, and to prevent activation of those induced during adipogenesis. The differentiation block is not reversed by removal of the inhibitor, suggesting that active PCs are most crucial in the early steps of adipocyte conversion.
Which, among furin, PACE4 and PC7, is the most critical enzyme for the
adipocyte differention of 3T3-L1 cannot be determined solely from the use of
the dec-RVKR-CMK synthetic inhibitor, as the latter inhibits all three
convertases indiscrimately. However, there are several reasons to believe that
furin is the determining enzyme in this process. First, it is the primary
maturation enzyme for several adipocyte signaling molecules including IGF-1R,
InsR and the low density lipoprotein receptor-related protein
(Bravo et al., 1994;
Ko et al., 1998
;
Lehmann et al., 1998
). Second,
its rapid induction early during adipogenic stimulation, in the same time
window as when blockage of differentiation by the synthetic PC inhibitor is
most effective, suggests that it is needed to promote the processing of these
and other precursor proteins. Third, of the major three PCs found in 3T3-L1
preadipocytes, PACE4 is barely detectable at the crucial early steps of
induction and PC7, which is more readily detected in these cells, is poorly
inhibitable by
1-PDX compared with furin
(Benjannet et al., 1997
;
Jean et al., 1998
). Finally,
furin has been implicated in the regulation of growth and differentiation of
other cell types including gastric surface mucous cells and cardiocytes
(Konda et al., 1997
;
Sawada et al., 1997
).
Moreover, it is interesting to note that the furin gene maps in a mouse
chromosome 7 region that has been shown by quantitative trait linkage analysis
to affect adiposity in a dominant fashion
(Taylor and Phillips, 1996
).
As for PACE4 and PC7, their expression pattern would be compatible with yet
unknown roles late in adipocyte differentiation.
To summarize, we have presented evidence that PCs play an important role in the adipocyte differentiation of 3T3-L1 cells. We have shown that blockage of adipose conversion with PC-specific inhibitors is associated with a dramatic reduction of the nuclear translocation of the C/EBPß factor.
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Acknowledgments |
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