©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Blockade of Preadipocyte Differentiation by Protein-tyrosine Phosphatase HA2 Is Reversed by Vanadate (*)

Kan Liao (§) , M. Daniel Lane (¶)

From the (1) Department of Biological Chemistry, The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A tyrosine phosphatase, i.e. PTPase HA2, was previously isolated from 3T3-L1 cells and characterized using O-phospho Tyrosine-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.

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.


INTRODUCTION

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-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.

By using [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.

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.


EXPERIMENTAL PROCEDURES

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 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 NHHCO, 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 NHHCO, 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-NaHPO4 solution (0.28 M NaCl, 50 mM HEPES, pH 7.12, 1.5 mM NaHPO4) 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.

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.


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.

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 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).

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).


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).

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 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.

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 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).

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.

  
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



FOOTNOTES

*
This work was supported by a research grant from the National Institutes of Health, NIDDK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L40595.

§
Present address: Dept. of Biology, University of Science and Technology of China, Hefei, Anhui 230026, People's Republic of China.

To whom correspondence should be addressed: Dept. of Biological Chemistry, The Johns Hopkins University, School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: 410-955-3554; Fax: 410-955-0903.

The abbreviations used are: PAO, phenylarsine oxide; PTPase, protein tyrosine phosphatase; C/EBP, CCAAT/enhancer binding protein; 422/aP2, adipocyte fatty acid binding protein; RT-PCR, reverse-transcribed polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; MIX, 3-isobutyl-1-methylxanthine; DEX, dexamethasone; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; IGF, insulin-like growth factor.


ACKNOWLEDGEMENTS

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.


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