From the
A tyrosine phosphatase, i.e. PTPase HA2, was previously
isolated from 3T3-L1 cells and characterized using O-phospho
Tyrosine
When induced to
differentiate into adipocytes, confluent 3T3-L1 preadipocytes undergo
mitotic clonal expansion followed by growth arrest and then coordinate
expression of adipocyte genes. During clonal expansion, expression of
PTPase HA2 increases abruptly and then decreases concomitant with the
transcriptional activation of adipocyte genes. Constitutive expression
of the PTPase by 3T3-L1 preadipocytes using a PTPase HA2 expression
vector prevents adipocyte gene expression and differentiation into
adipocytes. Appropriately timed exposure of transfected preadipocytes
to vanadate (a PTPase inhibitor), just as clonal expansion ceases
restores their capacity to differentiate. Treatment of transfected
preadipocytes with vanadate prior to or during clonal expansion fails
to reverse PTPase HA2-blocked differentiation, whereas treatment of
untransfected preadipocytes during mitotic clonal expansion blocks
differentiation. Vanadate added following clonal expansion has no
effect on differentiation. Thus, a critical tyrosine phosphorylation
event(s) occurs between termination of clonal expansion and initiation
of adipocyte gene expression while a critical tyrosine
dephosphorylation event(s) occurs during clonal expansion.
During the course of studies on insulin-stimulated glucose
uptake by 3T3-L1 adipocytes, we discovered a cellular protein substrate
of the insulin receptor tyrosine
kinase
(1, 2, 3, 4, 5) . This
15-kDa phosphorylated protein, pp15, was subsequently identified as
O-phosphotryosyl
By using
[
Two lines of evidence suggested that protein
tyrosine phosphorylation-dephosphorylation plays a role in preadipocyte
differentiation. First, the protocol used to induce differentiation
includes treatment of preadipocytes with IGF-1 or a non-physiologically
high level of insulin which acts through the IGF-1
receptor
(10) . As the IGF-1 receptor is a mitogenic
ligand-activated tyrosine kinase, it appears that tyrosine
phosphorylation catalyzed by the receptor plays a role in the induction
of differentiation. Second, mitosis which occurs during the clonal
expansion phase of the differentiation program is thought to involve
mitogen-activated protein kinase, an intermediate that undergoes
tyrosine phosphorylation-dephosphorylation during mitogen-stimulated
signal transduction
(11) . It is likely that these mitotic events
and attendant DNA replication allow transacting factors to gain access
to cis elements involved in the transcriptional activation of genes
that control the differentiation program
(12) . As mitotic clonal
expansion ceases, expression of adipocyte-specific genes is initiated
which ultimately gives rise to the terminally differentiated adipocyte
phenotype
(13) .
In view of evidence that protein tyrosine
phosphorylation-dephosphorylation is involved in the induction of
differentiation of preadipocytes into adipocytes and our preliminary
finding
(14) that expression of PTPase HA2 fluctuates during
differentiation, we investigated the effect of constitutive
vector-driven expression of PTPase HA2 on this process. It was found
that 3T3-L1 preadipocytes transfected with a PTPase HA2 expression
vector failed to differentiate and that treatment with vanadate (a
potent PTPase inhibitor) at the appropriate point in the
differentiation program restores their capacity to differentiate.
Sequencing grade trypsin was purchased from Boehringer
Mannheim. All restriction enzymes were from New England Biolabs.
pBCMGneo vector
(15) was from Dr. Karasuyama (Basel,
Switzerland). Sodium vanadate was purchased from Aldrich. Oil-Red-O was
from Matheson Coleman & Bell (Norwood, OH). Antibody directed
against PTPase1B was generously provided by Dr. Jack Dixon (University
of Michigan). Peptide sequencing and oligonucleotide synthesis were
performed in the Johns Hopkins University Protein/Peptide Facility. The
Genebank data search was carried out through the Biomedical
Supercomputing Center, National Cancer Institute, Frederick, MD. Purification of PTPase HA2 and Tryptic Peptide Sequencing-PTPase
HA2 was purified
A computer search for possible sequence similarities of these
peptides (Fig. 1C) to those in the SwissProt protein
data base revealed matches to sequences in different regions of human
PTPase 1B (Fig. 2B)
(30, 31) . The fact
that the first amino acid in three of the four peptide sequences
followed an Arg or Lys in the PTPase 1B sequence verified their tryptic
cleavage sites in the protein (Fig. 2B). A blank in the
first cycle of Edman degradation of peptide 3 is consistent with a Cys
(which is normally destroyed during gas-phase sequencing) following a
Lys in the sequence of human PTPase 1B (Fig. 2B). These
results indicated that PTPase HA2 was a homologue of human PTPase 1B.
Western blot analysis was performed on lysates of the same cell
lines either prior to (in the growth-arrested state) or after having
been subjected to the differentiation protocol for 8 days. As shown in
Fig. 5B, cells harboring the PTPase HA2 expression
vector expressed higher levels of the PTPase than untransfected cells
or cells harboring the insertless vector. Subjecting the cells
(transfected with the PTPase HA2 vector) to the differentiation
protocol had little effect on the expression of the PTPase (results not
shown). It should be noted that there was little difference in the
levels of PTPase protein expressed among the four cell lines
transfected with the PTPase expression vector (Fig. 5B)
despite differences in copy number of the transgene and the much higher
level of exogenous PTPase mRNA expressed by the FS-3 cell line. We are
uncertain as to the cause of this discrepancy. It should be noted that
the level of expression of exogenous PTPase mRNA and of total PTPase
protein by 3T3-L1 cells harboring the expression vector remained high
and constant through the course of differentiation (results not shown).
Since expression of endogenous PTPase HA2 is maximal in 3T3-L1 cells
undergoing mitotic clonal expansion and minimal in growth-arrested
cells (Fig. 3, B and C), it was of interest to
ascertain the effect on cell growth of constitutive vector-driven
expression of the PTPase. As shown in , the doubling times
of the four cell lines harboring the PTPase HA2 expression vector and
control cells were not greatly different (averaging
Constitutive expression of PTPase HA2 had a profound effect on the
capacity of the 3T3-L1 preadipocytes to acquire adipocyte
characteristics, completely suppressing their ability to differentiate
when subjected to the appropriate induction protocol. As illustrated in
Fig. 6
, A and B, cells transfected with the
PTPase HA2 expression vector failed to accumulate significant amounts
cytoplasmic triglyceride as visualized by Oil-Red-O staining.
Nonetheless, cells harboring the PTPase transgene underwent mitotic
clonal expansion typical of differentiating preadipocytes achieving the
same final surface cell density (when subjected to the differentiation
protocol) as control cells that underwent differentiation-associated
clonal expansion (results not shown).
Preliminary experiments showed that exposure of
cells harboring the PTPase HA2 vector to 35 µM sodium
orthovanadate during the entire course of the differentiation program
(day -2 to day 8) inhibited differentiation of control cells and
failed to reverse the blockade of differentiation caused by the PTPase
expression vector. This finding raised the possibility that the time
window of exposure to vanadate might be critical, since upon induction
of differentiation (of wild-type 3T3-L1 cells), the endogenous PTPase
level reaches a maximum during clonal expansion, i.e. on days
1 and 2 (Fig. 3, B and C), and then decreases
as clonal expansion ceases and the coordinate expression of adipocyte
genes begins, i.e. on days 3 and 4 (Fig. 3, B and C). As shown in Fig. 8, exposure of wild-type
and control cells to vanadate on days 3 and 4 did not disrupt
acquisition of the adipocyte phenotype as judged by accumulation of
cytoplasmic triglyceride. Moreover, exposure to vanadate of 3T3-L1
preadipocytes, transfected with the PTPase HA2 expression vector,
partially reversed (in some instances almost completely, e.g. the FS4 line, Fig. 8) both the inhibition of
cytoplasmic triglyceride accumulation (Fig. 8) and the inhibition
of expression of two adipocyte genes, i.e. the C/EBP
The present paper shows that protein tyrosine
phosphorylation-dephosphorylation play an important role at two points
in this differentiation program, i.e. a PTPase-catalyzed
dephosphorylation event(s) which occurs at the time mitotic clonal
expansion is initiated and a tyrosine phosphorylation event(s) which
occurs as preadipocytes complete clonal expansion and begin to
coordinately express adipocyte genes. Several lines of evidence
implicate a phosphotyrosine dephosphorylation event(s) that takes place
at the time of mitotic clonal expansion. At this point in the
differentiation program, expression of PTPase HA2 (an enzyme purified,
characterized, and its cDNA cloned and sequenced in this laboratory;
Fig. 1
and Fig. 2, see also Ref. 7) increases to a maximum
and then falls precipitously as clonal expansion ceases and adipocyte
gene expression occurs (Fig. 3). Suppression of the endogenous
PTPase activity at this stage of the program by exposure of 3T3-L1
preadipocytes to vanadate, a potent PTPase-specific inhibitor, blocks
subsequent differentiation () but has no effect on mitotic
clonal expansion. Exposure of the cells to vanadate either prior to or
following clonal expansion has no effect on differentiation
(). These results indicate that the dephosphorylation
event, critical for differentiation accompanies, but is not involved in
mitotic clonal expansion.
The importance of a tyrosine
phosphorylation event(s) in the differentiation program is indicated by
the finding that constitutive vector-driven expression of PTPase HA2
blocks differentiation (Fig. 6). Thus, 3T3-L1 cells harboring the
PTPase HA2 vector fail to express adipocyte genes, e.g. the
422/aP2 and C/EBP
It will now be necessary to
identify the phosphoproteins that function in the
phosphorylation-dephosphorylation events which regulate differentiation
during and after clonal expansion. Investigations are underway to
identify these proteins.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
We thank Dr. Jack Dixon (University of Michigan) for
providing an anti-PTPase1 antibody and Dr. Wu-Schyong Liu for
assistance in amino acid sequencing of tryptic peptides. We also thank
Natalie Tumminia and Margaret Dailey for expert assistance in preparing
this manuscript.
-422/aP2 protein (a target of the insulin receptor
tyrosine kinase) as substrate. The nucleotide sequence of a PTPase HA2
cDNA showed it to be a homologue of PTPase 1B.
-422(aP2) protein
(4) . It
was found that the phosphoryl group of pp15 turned over rapidly (t < 1 min) and that this turnover was prevented by the trivalent
arsenical, phenylarsine oxide
(PAO)
(
)(1, 2) . Trivalent
arsenicals are known to bind to vicinal or neighboring -SH groups, and
thereby, to block biological activity if such -SH groups are required
for function
(3, 6, 7) . Thus, the enzymatic
activity responsible for turnover of the phosphoryl group of pp15 would
be expected to possess functional neighboring thiol groups.
P]pp15 as authentic substrate to assay
phosphoryl group removing activity, two membrane-associated
PAO-sensitive enzymes, i.e. HA1 and HA2, were purified
10,000-fold from 3T3-L1 adipocytes
(7) . Both HA1 and HA2
possessed the characteristics of protein phosphotyrosine-specific
phosphatases (PTPases) and had molecular masses of
60 and 38 kDa,
respectively. HA2 is expressed by 3T3-L1 preadipocytes and to a lesser
extent by adipocytes, whereas HA1 is expressed only by adipocytes. In
the present paper we show that HA2 possesses extensive amino acid
sequence similarity to PTPase 1B. PTPases are known to be sensitive to
-SH reagents including trivalent arsenicals since a cysteinyl-SH group
(Cys
) is known to function in the catalytic mechanism
(8). The recent three-dimensional x-ray structure of PTPase 1B
(9) shows that the -SH group involved in catalysis is juxtaposed
near another -SH (Cys
) which accounts for the strong
inhibitory action of vicinal-neighboring -SH group reagents, such as
phenylarsine oxide.
20,000-fold from 800 10-cm monolayers of 3T3-L1
preadipocytes following previously described procedures
(7) . The
active PTPase HA2 fractions from the terminal glycerol gradient were
concentrated using Centricon C-30 filters (from Amicon). The
concentrated protein was subjected to SDS-PAGE (10% acrylamide, 16)
(Fig. 1A) and then transferred to a nitrocellulose
membrane in 25 mM Tris, 192 mM glycine, and 20%
methanol, pH 8.3, at 250 mA for 2 h. Protein on the filters was stained
with 0.1% Ponceau-S in 1% acetic acid solution, and the band
corresponding to PTPase HA2 was cut out, destained in 200 mM
NaOH, and the filter segment blocked with 0.5% PVP-40 (Sigma) and 100
mM acetic acid solution for 30 min at 37 °C, followed by
extensive washing in deionized water. After cutting the filter segment
into
1-mm
pieces, trypsin (
1/20 the amount of
PTPase HA2 protein) in 50 ml of 95% 100 mM
NH
HCO
, pH 8.2, and 5% acetonitrile was added
and digestion carried out for 48 h at 37 °C. After centrifugation,
the pellet was washed once with 50 ml of 95% 100 mM
NH
HCO
, pH 8.2 and 5% acetonitrile solution. The
two supernatants were combined and lyophilized three times to remove
all traces of salt. The pellet was dissolved in 100 ml of 0.1%
trifluoroacetic acid and injected into a pre-equilibrated microbore
C-18 reverse-phase HPLC column (Vydac). After washing with 0.06%
trifluoroacetic acid solution (buffer A) at a flow rate of 0.15 ml/min
for 10 min, peptides were eluted with a two-step linear gradient from
100% of Buffer A to 62.5% of Buffer A and 37.5% of 0.052%
trifluoroacetic acid and 80% acetonitrile solution (Buffer B) in 60 min
followed with gradient from 62.5% of Buffer A and 37.5% of buffer B to
25% of Buffer A and 75% of Buffer B in 30 min at 0.15 ml/min flow rate.
The column effluent was monitored at 215 nm, and fractions were
collected manually. Selected fractions were concentrated and subjected
to gas-phase amino acid sequencing using an Applied Biosystems peptide
microsequenator.
Figure 1:
Analysis of tryptic peptides derived
from purified PTPase HA2. A, silver-stained gel after SDS-PAGE
of PTPase HA2 from the terminal glycerol gradient step of purification.
PTPase HA2 from 800 10-cm monolayer 3T3-L1 preadipocytes was carried
through the purification steps described previously (7). An aliquot of
each fraction from the final glycerol (10-30%) gradient
centrifugation step was subjected to 10% SDS-PAGE, and the resulting
gel was silver-stained. PTPase HA2 enzyme activity of each fraction was
assayed using
[P]phospho-Tyr
-422/aP2 protein as
substrate (7); the asterisks designate fractions possessing
phosphatase activity. The molecular mass markers include the following
prestained proteins: myosin (200 kDa), glycogen phosphorylase b (97 kDa), bovine serum albumin (68 kDa), ovalbumin (46 kDa), and
carbonic anhydrase (29 kDa). B, HPLC elution profile of PTPase
HA2 tryptic peptides. After transfer from the gel to a nitrocellulose
filter, the PTPase HA2-containing segments were digested with trypsin
and the resulting peptides applied to a microbore C-18 reverse-phase
column (150
2.1 mm) in 0.1% trifluoroacetic acid, followed by
elution with an acetonitrile gradient (- - - -). Fractions
(200-300 ml) were collected manually based on the
A
(-) to ensure the separation of
individual peaks. Three peptides (designated peaks 1-3)
were sequenced using a gas-phase amino acid sequenator. PVP-40 indicates the point of elution of the reagent used to block
trypsinization of PTPase HA2. C, PTPase HA2 peptide sequences.
Four peptide sequences were obtained from peptide amino acid sequence
analysis of peaks 1-3 (see text for details). The absence of the
first amino acid in peptide 3 indicates an initial blank cycle during
amino acid sequencing.
Reverse-transcribed PCR (RT-PCR)
A pair of peptide
sequences of PTPase HA2, i.e. n-LHQEDNDYINAS-c and
n-FIMGDSSVQDQ-c, were selected as PCR primer sites. A Genebank search
showed identical matches of these peptides to those of PTPase 1B. Based
on this information, the corresponding oligonucleotides, i.e. 5`-GGAATTCGCACCAGGAAGATAATGACTATATCAATGCCAGC-3` and
5`-GGAATTCACTGATCCTGCACTGACGAGTCGCCCATGATG-3` with EcoRI
restriction enzyme sites were synthesized. Reverse-transcription was
carried out with total cellular RNA from day 0 3T3-L1 preadipocytes and
day 5 3T3-L1 adipocytes. The RNA template was heated to 80 °C for 2
min, held at 65 °C for 40 min, and then slowly cooled to 37 °C.
The reverse-transcription reaction contained 5 µg of heat-treated
RNA, 50 mM KCl, 10 mM Tris-HCl, pH 8.4, 4 mM
MgCl, 1 mM dNTPs, 5 mM primer 2 as
initial primer for reverse-transcription, 20 units of placental RNase
inhibitor, and 50 units of murine reverse transcriptase in 20 ml. After
incubating at 37 °C for 30 min, the reaction mixture was heated to
95 °C for 5 min to inactivate the reverse transcriptase. Ten µl
of the RT reaction mixture were used for DNA amplification in 50 ml
containing 50 mM KCl, 10 mM Tris-HCl, pH 8.4, 1.5
mM MgCl
, 200 mM dNTPs, 1 mM each
primer, and 2.5 units of Taq DNA polymerase. The PCR reaction
was carried out for 30 cycles (94 °C for 30 s, 50 °C for 1.5
min, and 72 °C for 2 min), and half of the product was analyzed by
electrophoresis in 1% agarose gel. The DNA band (
694 bp) was
isolated by glass wool filtration and then reamplified. The reamplified
band was isolated by electrophoresis, digested with EcoRI,
inserted into the pBluescript KS(-) vector (Stratagene) and
sequenced (17). The nucleotide sequences of the RT-PCR products of both
the day 0 and day 5 DNA samples were identical.
Isolation of cDNA Clones
The RT-PCR fragment was
labeled with [-
P]dATP by the random-priming
method
(18) and used as probe to screen 1.2 million phage
plaques of a day 5 mouse 3T3-L1 cell cDNA library
(19) in
[lamda]ZAP (Stratagene) using low stringency conditions
(i.e. hybridization in 20% formamide, 4
SSC (standard
saline citrate), 1
Denhardt's solution, 1% SDS, 50 mg/ml
yeast tRNA, 0.5 mg/ml sodium pyrophosphate, and 50 mM sodium
phosphate, pH 7.0, at 42 °C overnight and washing with 2
SSC and 0.1% SDS 2
30 min at 42 °C). Ten positive clones
were identified and purified. The inserts of these clones were
characterized, and a full-length cDNA clone was constructed.
Isolation and Analysis of RNA
Total RNA was
isolated from proliferating 3T3-L1 cells, confluent cells, 2-day
post-confluent cells, and cells at various differentiation stages by
the guanidine isothiocyanate method
(20) . For Northern blot
analysis, 20 mg of total RNA was denatured with glyoxal and dimethyl
sulfoxide and resolved by electrophoresis on 1% agarose gels as
described
(21, 22) . After transfer to Hybond-N membrane
(Amersham Corp.) and UV cross-linking, the RNA was stained with
methlene blue to locate 28 S and 18 S rRNAs and verify equal loading.
The blot was then hybridized with DNA probes in 50% formamide, 4
SSC, 1
Denhardt's solution, 50 mM sodium
phosphate, pH 7.0, 1% SDS, 50 µg/ml yeast tRNA, and 0.5 mg/ml
sodium pyrophosphate at 42 °C overnight and washed with 1
SSC and 0.1% SDS twice at 50 °C and 0.1
SSC and 0.1% SDS
twice at 65 °C.
Isolation and Analysis of DNA
Chromosomal DNA from
one 10-cm dish of confluent 3T3-L1 preadipocytes was isolated by the
method of Wigler et al.(23) . DNA samples (20 µg
each) were double-digested overnight with XhoI and
BamHI and resolved by electrophoresis on 0.9% agarose gel.
After denaturation, neutralization, and transfer to Hybond-N
membranes
(24) , hybridization was carried out as described for
Northern blotting (above). The copy number of the PTPase HA2 transgene
in chromosomal DNA was quantitated by densitometry normalized to the
signal a known quantity of plasmid PTPase HA2 DNA insert.
Extraction and Analysis of Protein
Total cellular
protein was extracted from proliferating 3T3-L1 cells and cells at each
different stage of the differentiation program. Each 10-cm monolayer of
cultured cells was washed twice with ice-cold phosphate-buffered saline
(PBS), pH 7.5. The cells were then lysed directly on the plate with
boiling 1 Laemmli SDS sample buffer containing 20 mM
dithiolthretol
(16) . The cellular lysate was boiled for 5 min
and protein determined using the BCA protein method (Pierce). Protein
(100 µg) was subjected to 10% SDS-PAGE and transferred to
Immobilon-P membranes (Millipore). After Ponceau-S staining, to ensure
equal loading of protein, membranes were incubated with primary
antibody to the targeted protein, followed with horseradish
peroxidase-conjugated secondary antibody. The immunoreactive protein
was visualized by ECL (enhanced chemiluminescence, Amersham).
Plasmid Construction and Stable
Transfections
Full-length PTPase HA2 cDNA, excised from
pBluescript with EcoRI, was blunt end-inserted into the
XhoI site of the pBCMGneo expression vector
(15) . The
sense insert orientation plasmid, pBCMGFS, was identified by
restriction analysis and used to transfect 3T3-L1 cells. For
transfection, 3 10
low passage 3T3-L1 preadipocytes
were plated onto 10-cm culture dishes the day before and transfected
with 20 µg of pBCMGFS or pBCMGneo control plasmid DNA by the
calcium phosphate precipitation method
(25) . Briefly, 0.5 ml of
DNA-CaCl
(0.25 M) solution was added dropwise to
0.5 ml NaCl-HEPES-Na
HPO4 solution (0.28 M NaCl, 50
mM HEPES, pH 7.12, 1.5 mM Na
HPO4) to form
the DNA-calcium phosphate precipitate. After standing for 30 min, the
mixture was added directly to culture medium of the cell monolayers.
After 4 h at 37 °C in the CO
incubator, cells were
shocked with 10% dimethyl sulfoxide-PBS for 3 min and then cultured for
24 h in Dulbecco's modified Eagle's medium (DMEM)
containing 10% calf serum. G418 (350 µg/ml) was added for
selection, and resistant foci were isolated and cultured.
Cell Culture and Differentiation of 3T3-L1
Preadipocytes
The 3T3-L1 preadipocytes were cultured in DMEM
with 10% calf serum and allowed to achieve confluence (designated as
day -2). Differentiation was induced by adding 1 µg/ml
insulin, 1 mM dexamethasone (DEX), and 0.5 mM
3-isobutyl-1-methylxanthine (MIX) to 2-day post-confluent cells
(designated as day 0) in DMEM with 10% fetal bovine
serum
(26, 27) . After 48 h (i.e. on day 2), the
medium was replaced with DMEM containing 10% fetal bovine serum and 1
µg/ml insulin, and the cells were then fed every other day with
DMEM containing 10% fetal bovine serum. By day 3, expression of
adipocyte mRNAs (e.g. the 422/aP2 message) occurs and by day 4
small cytoplasmic triglyceride droplets are evident. By day 8-9,
the cells are fully differentiated
(28) . In vanadate reversal
experiments, the cell differentiation was induced in the usual manner
as above. After 48 h, 35 µM sodium vanadate was added to
the normal feeding medium. Vanadate treatment lasted for 2 days with a
second addition of 35 µM sodium vanadate made after the
first day.
Oil-Red-O Staining
3T3-L1 adipocytes were washed
with PBS and fixed for 2 min with 3.7% formaldehyde in PBS. A 0.5%
Oil-Red-O isopropanol solution was diluted with 1.5 volume of water,
filtered, and added to the fixed adipocyte monolayers for 1 h. Cells
were then washed and stained triglyceride droplets visualized and/or
photographed (29).
RESULTS
Amino Acid Sequencing and cDNA Cloning of PTPase
HA2
PTPase HA2 was purified 10,000-fold to near homogeneity
from 800 10-cm monolayers of 3T3-L1 preadipocytes by procedures
described previously (7). As a final step in the purification the
protein was subjected to SDS-PAGE (Fig. 1A) and then
transferred to a nitrocellulose membrane. The segment of the membrane
containing PTPase HA2 was cut out and digested with trypsin, and the
tryptic peptides were subjected to HPLC on a microbore C-18
reverse-phase column. The elution profile revealed several major peaks,
three of which, i.e. peaks 1, 2 and 3 in
Fig. 1B, were subjected to gas-phase amino acid
microsequencing. Peaks 1 and 3 were each found to be comprised of two
distinct peptides (peptides 3 and 4 in peak 1 and peptides 1 and 2 in
peak 3, see Fig. 1C). Owing to the large differences
(
3-fold) in the amounts of the two peptides in peaks 1 and 3 at
each cycle of Edman degradation, the amino acid sequences of both
peptides could be readily deduced. The ordering of amino acids in
peptides 1 and 2 was verified by the sequence of the peptide in peak 2
which contained the first 5 amino acids in the sequence of peptide 2.
Figure 2:
Nucleotide and deduced amino acid
sequences of PTPase HA2 cDNA. A, nucleotide and deduced amino
acid sequences of a PTPase HA2 cDNA cloned from a mouse 3T3-L1 cell
cDNA library. The deduced amino acid sequence is shown under the
nucleotide sequence using the standard single-letter code.
Underlined amino acids identify the four peptides obtained by
amino acid sequence analysis of PTPase HA2-derived tryptic peptides and
the bold lettering identifies amino acids in the PTPase active
center. An asterisk identifies the terminal codon. The
polyadenylation signal AATAAA sequence was not found in the cDNA.
Nevertheless, the cDNA is close in size to the transcript shown on the
Northern blot analysis in Fig. 5. B, comparison of the amino
acid sequence of mouse (3T3-L1 cell) PTPase HA2 with that of human
PTPase 1B. Vertical dashes indicate amino acid identity; the
two phosphatases possess 83% amino acid
identity.
Oligonucleotide primers were prepared which corresponded to the
amino acid sequences of peptide 1 (5` 3`) and peptide 2 (3`
5`) taking into consideration the nucleotide sequences at
comparable sites in PTPase 1B cDNAs found in the GenEMBL data base.
RT-PCR using these primers and RNA isolated from confluent 3T3-L1
preadipocytes or day 5 3T3-L1 adipocytes gave rise to the same
694-bp fragment whose identity was verified by sequencing. This
694-bp RT-PCR fragment was then used as probe to screen a day 5 3T3-L1
cell
Zap cDNA library (19). Ten positive clones were isolated and
shown by sequencing to represent the same mRNA. As shown in
Fig. 2A the full-length 1969-bp cDNA had an open reading
frame encoding 432 amino acids with a calculated molecular mass of 49.5
kDa. The apparent discrepancy between the molecular mass derived from
the cDNA and that of purified PTPase HA2 (38 kDa by SDS-PAGE) suggests
that the purified protein may have been post-translationally modified
or undergone proteolysis during purification. The amino acid sequence
of PTPase HA2 shares 83% identity with that of human PTPase 1B
(Fig. 2B). The full-length cDNA constructed from two
overlapping cDNA clones corresponds to a 1.9-kb mRNA which is in good
agreement with the size of an mRNA detected by Northern blot analysis
(Fig. 5A) of RNA isolated from 3T3-L1 cells. In addition
to the 1.9-kb transcript, a transcript similar in size to 28 S rRNA
(
4.8 kb) was detected using the PTPase cDNA as probe. The amount
of this transcript, however, varied in different blots with no
consistent pattern of expression; thus, it is not clear whether this
RNA represents an intermediate in mRNA processing or a nonspecific
hybridizing RNA.
Figure 5:
Constitutive expression of PTPase HA2 in
pBCMGFS-transfected cells. A, Northern blot of total cellular
RNA isolated from untransfected 3T3-L1 cells (3T3-L1) and
3T3-L1 cells transfected with the control pBCMGneo vector
(control) or with the PTPase HA2 expression vector, pBCMGFS,
transfected cells (FS-1-4) during cell division
(D) or in the growth-arrested confluent state (C).
Twenty µg of RNA were analyzed and probed with
P-labeled PTPase HA2 cDNA probe. Exogenous PTPase
indicates the transcript derived from the transgene and endogenous
indicates the transcript from the cellular PTPase HA2 gene. B,
Western analysis of PTPase HA2 extracted from the cell lines described
above on day 0 (i.e. 2-day post-confluent cells) and 8 days
after the induction of differentiation. Extracts containing 100 µg
of protein were subjected to SDS-PAGE (10% acrylamide) and Western blot
analysis with anti-PTPase antibody. PTPase indicates the
40 kDa
protein band corresponding to PTPase HA2.
Expression of PTPase HA2 during Differentiation of 3T3-L1
Preadipocytes
When exposed to the appropriate complement of
external inducers, 3T3-L1 preadipocytes differentiate into adipocytes.
To ascertain whether PTPase HA2 is differentially expressed during this
process, total cellular RNA and protein extracted from cells at various
stages of differentiation were analyzed. Northern blot analysis
revealed that expression of PTPase HA2 mRNA is regulated during
differentiation, the level of expression being highest during mitotic
clonal expansion and lowest during growth arrest
(Fig. 3B). Thus, following induction of differentiation,
the expression of PTPase HA2 message and protein increases dramatically
(day 0 to day 2, Fig. 3, B and C) concomitant
with clonal expansion, and then falls as the cells undergo growth
arrest and begin to coordinately express adipocyte genes, including the
C/EBP, insulin receptor, 422/aP2, SCD1, GLUT4, as well as others
genes
(28, 32, 33, 34, 35, 36, 37, 38) that give rise to adipocyte characteristics.
Figure 3:
Temporal relationship between
differentiation-induced mitotic clonal expansion of 3T3-L1 cells and
expression of PTPase HA2 mRNA and protein. A, change in cell
number during differentiation. Differentiation was induced by exposure
of growth-arrested (2-day post-confluent) 3T3-L1 preadipocytes
(designated as day 0) to medium containing MIX, DEX, and
insulin for 2 days (days 1 and 2 during which clonal expansion occurs),
followed by medium with insulin for 2 additional days (days 3 and 4) as
described under ``Experimental Procedures.'' As clonal
expansion ceases on day 3, the expression of adipocyte genes and
the accumulation of cytoplasmic triglyceride begin. Days are numbered
beginning on the day differentiation is induced with MDI (MDI refers to
MIX, DEX, and insulin treatment). In B and C
expression of PTPase HA2 mRNA and protein, respectively, are shown.
Total cellular RNA and protein were extracted on the days indicated
during the differentiation program. Twenty µg of RNA were subjected
to Northern blot analysis using full-length PTPase HA2 cDNA as probe;
100 µg of protein were subjected to SDS-PAGE after which Western
immunoblot analysis was performed with antibody directed against rat
PTPase 1 which cross-reacts with PTPase HA2.
Effect of Constitutive Vector-driven Expression of PTPase
HA2 on the Preadipocyte Differentiation Program
To determine
whether the inappropriate timing of expression of PTPase HA2 might
alter the course of the differentiation program, the effect of
constitutive expression of the phosphatase on the program was
investigated. A bovine papilloma virus-based PTPase HA2 expression
vector, pBCMGFS, was constructed (Fig. 4A) in which the
full-length PTPase HA2 cDNA including the 5`-untranslated region was
inserted in the sense orientation just 3` of a human cytomegalovirus
gene promoter
(15, 35) . In addition to a neomycin
resistance gene, this vector contains bovine papilloma virus sequences
that allow extrachromosomal replication at high copy number, as well as
integration into the cellular genome. The PTPase expression vector and
the parental vector lacking an insert (pBCMGneo) were separately
transfected into 3T3-L1 preadipocytes. Selection with G418 generated
several PTPase positive and control cell lines. Quantitative Southern
blot analysis indicated copy numbers of 2, 1, 8, and 1, respectively,
for four representative cell lines, i.e. pBCMGFS-1, -2, -3,
and -4 (Fig. 4B), that were used for the studies
described below.
Figure 4:
Construction of a PTPase HA2 expression
vector and Southern blot analysis of genomic DNA from untransfected
cells and cells transfected with the pBCMGFS PTPase HA2 expression
vector. A, the PTPase HA2 cDNA is shown in which the open
boxes represent the 5`- and 3`-untranslated regions, and the
hatched box represents the coding region. Full-length PTPase
was excised from pBlue PTPHA2 with EcoRI, filled in, and
blunt-end inserted into the blunted XhoI site of the 14.85-kb
pBCMGneo vector in the sense orientation 3` of the human
cytomegalovirus giving rise to pBCMGFS. B, genomic DNA
isolated from wild-type 3T3-L1 cells (3T3-L1) and 3T3-L1 cells
transfected with the pBCMGneo (control) and pBCMGFS
(FS-1-4) vectors were from one 10-cm dish of confluent
cells. Twenty µg of each DNA sample were digested with
XhoI and BamHI restriction enzymes which cut at the
XhoI site in the PTPase HA2 cDNA and at the BamHI
site in the vector to generate a 1.6-kb fragment. After resolving
the DNA on 0.9% agarose gels, Southern blots were hybridized with the
P-labeled
1-kb PTPase HA2 cDNA 3` end fragment
obtained by digesting pBluePTPHA2 with XhoI which cuts in the
XhoI site in the PTPase HA2 and in the vector just 5` to the
T3 promoter. Standards of pBCMGFS vector were used for quantitation of
the copy number of the transgene in the transfected cell
lines.
Northern analysis verified that the
pBCMGFS-transfected cell lines exhibited a high level of expression of
the exogenous PTPase HA2 mRNA in both the proliferating and
growth-arrested confluent states (Fig. 5A). Expression
(albeit at a lower level than the exogenous PTPase message), of the
endogenous PTPase HA2 message occurred in proliferating, but not
growth-arrested preadipocytes (Fig. 5A). The FS-3 cell
line, which possessed the highest copy number of the transgene,
exhibited the highest level of expression of exogenous PTPase HA2
message (which as indicated below did not lead to a comparable high
level of expression of the protein). 3T3-L1 cells that either had not
been transfected or had been transfected with the insertless control
vector (pBCMGneo) expressed low levels of endogenous PTPase message in
the proliferating state and lower levels in the growth-arrested state.
15.5 h), cells
expressing the PTPase transgene exhibiting slightly longer doubling
times. However, cells expressing the transgene achieved a 30-40%
greater cell density at confluence than control cells ().
Nevertheless, such cells exhibited density-dependent growth inhibition
and were unable to proliferate in soft agar (results not shown).
Figure 6:
Effect of constitutive expression of
PTPase HA2 on the accumulation of cytoplasmic triglyceride by 3T3-L1
preadipocytes subjected to the differentiation protocol. A untransfected and transfected (either with the control pBCMGneo or
PTPase HA2 pBCMGFS expression vector) 3T3-L1 preadipocytes were plated
at 0.8 10
cells/6-cm dish. Two days after reaching
confluence (designated as day 0) cells were subjected to the
differentiation protocol as described under ``Experimental
Procedures.'' Eight days after the induction of differentiation
cell monolayers were fixed, and cytoplasmic triglyceride was stained
with Oil-Red-O. Control refers to cells transfected with
pBCMGneo (without the PTPase insert); FS-mix refers to pooled
foci of cells transfected with the PTPase HA2 vector, pBCMGFS; and
FS-1-6 refers to individual clonal cell lines harboring
the pBCMGFS PTPase HA2 expression vector. B, light micrographs
are of typical fields (at higher magnification) of Oil-Red-O-stained
untransfected 3T3-L1 cells (right panel) or 3T3-L1 cells
harboring the PTPase HA2 pBCMGFS expression vector (FS-1)
(left panel) 8 days after being subjected to the
differentiation protocol.
To verify that constitutive
expression of the PTPase HA2 transgene also suppresses the expression
of genes which give rise to the adipocyte phenotype, RNA isolated from
cells exposed to the differentiation protocol was subjected to Northern
analysis using probes for C/EBP and 422/aP2 mRNAs. It should be
noted that expression of both the C/EBP
and 422/aP2 genes is
normally activated when 3T3-L1 preadipocytes are induced to
differentiate. Moreover, C/EBP
is known to be a pleiotropic
transcriptional activator of a group of adipocyte genes, including the
422/aP2 gene, during differentiation
(33) and has been shown to
be required and sufficient for the induction of preadipocyte
differentiation
(35, 39) . As shown in Fig. 7,
expression of the C/EBP
and 422/aP2 messages was virtually
abolished in cell lines that constitutively express exogenous PTPase
HA2. The low level of expression of the 422/aP2 message in FS-4 cells
(which harbor the PTPase HA2 expression vector) was apparently due to
the fact that a small fraction of these cells underwent differentiation
(see Fig. 6A). Nevertheless, all cell lines transfected
with the PTPase HA2 expression vector (including several others not
shown) exhibited suppressed (often completely) expression of the
C/EBP
and 422/aP2 genes. The fact that inappropriate expression of
PTPase HA2 inhibited acquisition of adipocyte characteristics suggested
that phosphorylation of a protein(s) on tyrosine is required for
terminal differentiation.
Figure 7:
Effect of constitutive expression of
PTPase HA2 on the expression of adipocyte genes by 3T3-L1 preadipocytes
subjected to the differentiation protocol. Untransfected and
transfected (with the control pBCMGneo or PTPase HA2 pBCMGFS expression
vector) 3T3-L1 preadipocytes were cultured and subjected to the
differentiation protocol as described under ``Experimental
Procedures.'' Total cellular RNA, isolated from the cells on the
days indicated after initiating differentiation on day 0, was subjected
to Northern blot analysis using C/EBP and 422/aP2 cDNA probes
(35). The results of four transfected cell lines
(FS-1-4) are shown. D designates preconfluent
dividing cells and C cells at point of reaching confluence
(i.e. day -2).
Reversal by Vanadate of the PTPase HA2-induced Blockade
of Differentiation
The possibility was considered that exposure
of the FS cell lines to a PTPase inhibitor at an appropriate point in
the differentiation program might reverse the inhibition of
differentiation caused by vector-driven expression of PTPase HA2.
Vanadate was selected because it is a PTPase-specific inhibitor and
because cells can be cultured in its presence without adverse side
effects. Furthermore, previous work in our laboratory
(2) had
shown that the t of the phosphoryl group of pp15
(O-phospho Tyr-422/aP2 protein), a substrate of
PTPase HA2
(4) , was markedly lengthened by exposure of 3T3-L1
cells to vanadate. Experiments to determine the concentration of
vanadate which 3T3-L1 cells could tolerate showed that a level of
<50 µM for a period of 2 weeks had no adverse effects
(results not shown).
and
422/aP2 genes (Fig. 9). In contrast, exposure to vanadate on days
1 and 2 (i.e. during mitotic clonal expansion) inhibited
differentiation of control cells and failed to reverse the
vector-driven PTPase HA2 blockade by cytoplasmic triglyceride
accumulation and adipocyte gene expression (). It should
be noted that the extent to which vanadate led to recovery of
cytoplasmic triglyceride accumulation varied among the different cell
lines, most likely due to differences in the level of expression of
transfected PTPase HA2.
Figure 8:
Reversal by vanadate of the inhibition of
differentiation caused by the constitutive expression of PTPase HA2.
Untransfected 3T3-L1 preadipocytes and preadipocytes transfected with
the control pBCMGneo or the pBCMGFS PTPase HA2 expression vectors
(lines FS-2-5) were subjected to the differentiation protocol.
Following withdrawal of MIX, DEX, and insulin (day 0-2), on day 2
cells were treated with insulin, with or without 35 µM
sodium vanadate for 2 days (a second addition of 35 µM
vanadate was made on day 3), and then were carried in culture until day
8 after which they were then stained with
Oil-Red-O.
Figure 9:
Reversal by vanadate of the inhibition of
expression of C/EBP and 422/aP2 proteins caused by the
constitutive expression of PTPase HA2. Protein extracts of several cell
lines in the experiments described in Fig. 8 were prepared on day 8 and
then subjected to SDS-PAGE and then Western blot analysis using in
A, anti-C/EBPa antibody and in B, anti-422/aP2
antibody (35). The two isoforms of 42 kDa (full-length) and 30 kDa of
C/EBP
(42) were detected. Day 0 3T3-L1 preadipocytes and day 8
3T3-L1 adipocytes served as controls.
Taken together these results suggest that:
1) a protein tyrosine phosphorylation event(s) that occurs on days 3
and/or 4 (the period during which adipocyte gene transcription is
initiated) is necessary for terminal differentiation of preadipocytes,
and 2) a vanadate-inhibitable protein phosphotyrosine dephosphorylation
event(s) which occurs on days 1 and/or 2 (the period during which
mitotic clonal expansion occurs) is necessary for subsequent terminal
differentiation.
DISCUSSION
During differentiation, preadipocytes acquire the full
complement of enzymes and accessory proteins with which to carry out
differentiated adipocyte functions
(12, 40) . Following
commitment to the adipose lineage, quiescent preadipocytes become
susceptible to the combination of external modulators (i.e. IGF-1, glucocorticoid, and an agent that increases the
intracellular level of cAMP) which trigger the sequence of events which
give rise to the adipocyte phenotype
(27) . Upon induction
preadipocytes undergo several required rounds of mitotic clonal
expansion and again become quiescent as the coordinate transcriptional
activation of adipocyte genes is initiated
(28) . Certain
mitogens, e.g. fibroblast growth factor and platelet-derived
growth factor, are capable of stimulating post-confluent mitosis of
preadipocytes, but are incapable of inducing differentiation; thus DNA
replication appears to be necessary, but not sufficient for induction
of differentiation
(40) . It has been suggested that DNA
replication and changes in chromatin structure that accompany mitotic
clonal expansion may increase the accessibility of cis elements to
transacting factors which activate (or derepress) transcription of
genes that give rise to the adipocyte characteristics
(40) . As
clonal expansion ceases the coordinate activation of adipocyte-specific
genes occurs. Several transcription factors, notably C/EBP and
PPAR/FAAR, whose expression is induced at this stage of the
differentiation program, appear to function as pleiotropic regulators
of adipocyte gene transcription
(12) . It seems likely that the
time windows during which these events occur are tightly controlled
ensuring that the differentiation program proceeds in an orderly
manner.
genes (Fig. 7), and also fail to
accumulate cytoplasmic triglyceride (Fig. 6), the hallmark of the
adipocyte. It should be noted that C/EBP
is differentially
expressed
(33, 41) , transcriptionally activates a number
of adipocyte genes including the 422/aP2 gene
(33) , and is not
only required
(35) , but is sufficient to induce differentiation
in the absence of external modulators
(39) . These findings and
the fact that vanadate treatment of cells harboring the PTPase HA2
expression vector reverses the blockade of differentiation
(Fig. 8) only when administered after clonal expansion ceases and
adipocyte gene expression begins indicates that the essential tyrosine
phosphorylation event(s) occurs during this time window. Consistent
with this view, expression of the endogenous PTPase HA2 gene in
wild-type 3T3-L1 cells declines rapidly during this period of the
differentiation program (Fig. 3).
Table:
Doubling time and confluent cell number
of 3T3-L1 cells and pBCMGneo or pBCMGFS-transfected 3T3-L1 cells
Table:
Summary of the effect of vanadate at
different stages of the differentiation program
/EMBL Data Bank with accession number(s) L40595.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.