Articles

Pref-1 (Preadipocyte Factor 1) Activates the MEK/Extracellular Signal-Regulated Kinase Pathway To Inhibit Adipocyte Differentiation

Kyung-Ah Kim, Jung-Hyun Kim, Yuhui Wang, Hei Sook Sul
DOI: 10.1128/MCB.02207-06

ABSTRACT

Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor γ2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.

Preadipocyte factor 1 (Pref-1) is a transmembrane protein having epidermal growth factor (EGF)-like repeats in the extracellular domain, a juxtamembrane region, a single transmembrane domain, and a short cytoplasmic tail. Pref-1 is found in 3T3-L1 preadipocytes but disappears after their conversion into adipocytes (41, 42). Pref-1, therefore, is used as a marker for preadipocytes (38, 49, 54). Adipose conversion can be markedly reduced by interfering with normal repression of Pref-1 during adipocyte differentiation through the constitutive expression of Pref-1 or by the addition of soluble Pref-1 ectodomain in 3T3-L1 cells (39, 42). Conversely, inhibiting Pref-1 by expression of antisense RNA increases the ability of the cells to undergo adipocyte differentiation (40). The importance of Pref-1 has been demonstrated in vivo through the generation of Pref-1 knockout mice and transgenic mice overexpressing Pref-1. Pref-1 knockout mice display growth retardation, skeletal malformation, and accelerated adiposity (26), whereas transgenic mice overexpressing the full ectodomain of Pref-1 in adipose tissue display a decrease in fat mass, reduced expression of adipocyte markers, and lower adipocyte-secreted factors including leptin and adiponectin (22). These in vivo and in vitro studies clearly demonstrate the inhibitory function of Pref-1 in adipogenesis. In this regard, glucocorticoids, which are components of routinely used differentiation agents, enhance adipocyte differentiation partly by down-regulating Pref-1 expression (40). The Pref-1 gene has been shown to be a paternally expressed gene located in a chromosomal region containing six imprinted genes (36, 44, 51). Although the gene(s) responsible for the phenotype is not clear, paternal monoallelic expression of Pref-1 and maternal uniparental disomy have been observed in syntenic chromosomes in various species. Changes in adiposity observed in some of the species may be due to the role of Pref-1 in adipose tissue development. During embryogenesis, Pref-1 is expressed in multiple tissues (12, 19, 42). However, expression of Pref-1 is rapidly abolished in most tissues after birth and becomes restricted to preadipocytes and several neuroendocrine types of cells (10, 14, 20, 42). The Pref-1 expression pattern and the abnormalities detected in Pref-1 knockout and transgenic mice show that Pref-1 may also be involved in embryonic development as well as in adipogenesis.

Growth factors and cytokines are the primary extracellular signaling molecules that control cell growth and differentiation. Growth factors such as platelet-derived growth factor, EGF, fibroblast growth factor, tumor necrosis factor alpha, and transforming growth factor β have been shown to inhibit adipogenesis in vivo and in vitro (13, 16, 29, 34, 37, 43, 48). Conversely, insulin-like growth factor 1 (IGF-1) or a pharmacological concentration of insulin promotes adipocyte differentiation (43). These growth factors and cytokines affect their target cells by binding to specific cell surface receptors and activating intracellular signaling pathways. Although these factors have opposing effects on adipocyte differentiation, they are known to exert their effects through activating mitogen-activated protein kinase (MAPK)-dependent pathway (17). In this regard, there are conflicting reports on the role of the MAPK pathway in adipocyte differentiation. Some have reported a requirement for rapid transient activation of extracellular signal-regulated kinase (ERK) for either clonal expansion (5) or adipocyte differentiation (30, 31), while others have reported inhibition of differentiation by ERK activation (9, 17). Thus, it is likely that ERK activation is tightly and temporally controlled, since under certain conditions and periods ERK activity may be required, while in others ERK activity may impair adipocyte differentiation. Pref-1 shares structural similarity with other EGF-like repeat-containing proteins that can function as soluble or transmembrane proteins. One well-characterized subclass of EGF repeat-containing proteins includes those growth factors that bind and function through EGF receptor, such as EGF, transforming growth factor α, and heparin-binding EGF-like growth factor. These growth factors are made as transmembrane proteins but cleaved to generate mature growth factors (24). Another class of EGF repeat-containing protein includes Notch, which can undergo proteolytic processing and has Delta, Serrate, and Lag-2 proteins as ligands (2). We also found that the membrane form of Pref-1 is proteolytically processed at two sites in the extracellular domain: one located near the fourth EGF repeat and the other in the region proximal to the transmembrane domain, resulting in the 25-kDa small and 50-kDa large soluble forms (39). We found that only the 50-kDa large soluble form is active and sufficient for the inhibition of adipocyte differentiation (25). Furthermore, we have recently shown that tumor necrosis factor alpha converting enzyme is involved in this cleavage (50). Since Pref-1 is cleaved to release an active soluble form and has structural similarity with other EGF-like repeat-containing proteins, we hypothesized that the mode of action of Pref-1 may be similar to those proteins. Thus far, little is known about Pref-1 signaling, its specific receptor, or the mechanism underlying Pref-1 inhibition of adipogenesis.

The aim of the present study was to understand the signaling pathway for Pref-1 and the effect of Pref-1 on adipocyte differentiation. By using mouse embryo fibroblasts (MEF) prepared from Pref-1 null mice that we previously generated, we show, for the first time, that Pref-1 directly increases MEK1/2 and ERK1/2 phosphorylation. During differentiation of wild-type MEF into adipocytes, similar to that of 3T3-L1 cells, there is an initial transient burst of ERK1/2 phosphorylation upon addition of adipogenic agents. In addition, there is a low but significant second increase in ERK phosphorylation peaking at day 2 that parallels the expression levels of Pref-1. This second increase in ERK phosphorylation during MEF differentiation is absent in Pref-1 null MEF, and adipocyte differentiation of Pref-1 null MEF is enhanced compared to that of wild-type MEF. Thus, when the second ERK1/2 phosphorylation found in wild-type MEF is prevented by a specific MEK inhibitor or transient small interfering RNA (siRNA)-mediated ERK1/2 depletion, MEF differentiation is enhanced. Furthermore, Pref-1 treatment restores this second increase in ERK phosphorylation in Pref-1 null MEF, resulting in inhibition of adipocyte differentiation. However, Pref-1 cannot inhibit adipocyte differentiation when ERK1/2 phosphorylation is blocked by MEK inhibitor or ERK siRNA. Overall, these studies show that Pref-1 directly activates the MEK/ERK pathway and that ERK phosphorylation peaking at day 2 is responsible for Pref-1 inhibition of adipogenesis.

MATERIALS AND METHODS

Plasmid construction, protein expression, and purification.The cDNA sequence of the large soluble form of Pref-1, which contains six EGF-like domains and the juxtamembrane region of Pref-1, was ligated in frame to the human Fc region encoding the C-terminal 235 amino acids and then inserted into pcDNA3.1 precut with HindIII and XhoI. The sequence of the construct was verified by dideoxy sequencing. Freestyle 293F cells (Gibco BRL) were grown in 293 expression medium (Invitrogen) and transfected with Pref-1-hFc construct using 293fectin (Invitrogen). Purification of secreted Pref-1-hFc fusion protein from serum-free culture medium was carried out by affinity chromatography using ImmunoPure Plus immobilized protein A (Pierce).

Preparation of primary MEF and induction of adipocyte differentiation.Primary MEF were isolated from embryos of wild-type and Pref-1 null mice at 13.5 days post coitum. Embryos removed and separated from maternal tissues and yolk sacs were finely minced, digested with 0.25% trypsin-1 mM EDTA for 30 min at 37°C, and centrifuged for 5 min at 1,000 × g (45). The pellet was resuspended in culture medium before plating. Cells were cultured at 37°C in high-glucose Dulbecco's modified Eagle's medium (DMEM; Gibco BRL) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS; Omega) and 100 U/ml penicillin/streptomycin (Gibco BRL). All differentiation experiments were carried out using cells at passage 2. Upon reaching confluence, the cells were split into six-well plates and cultured to confluence. Two days later, the medium was replaced with the standard differentiation induction medium containing 0.5 mM methylisobutylxanthine (MIX), 1 μM dexamethasone (DEX), 10 μg/ml insulin, 10 μM troglitazone, and 10% (vol/vol) FBS. The cells were treated with differentiation agents for 4 days, and the medium was renewed every other day. For preparation of protein for Western blot analysis and total RNA for reverse transcription (RT)-PCR and real-time RT-PCR, cells were harvested before and after induction of differentiation. Oil red O staining was performed at days 0 and 8. Briefly, cells were washed twice with phosphate-buffered saline (PBS) and fixed with 10% formalin in PBS for 1 h. They were washed three times with water and stained with Oil red O (6 parts of 0.6% Oil red O dye in isopropanol and 4 parts of water) for 1 h. Excess stain was removed by washing with water, and the stained cells were dried. Spectrophotometric quantification of the stain was performed by dissolving the stained oil droplets in 100% isopropanol for 10 min. Optical density was then measured at 500 nm. In thymidine incorporation assays, 1 μCi/ml of [3H]thymidine was added to the cultured cells, and the incorporation into DNA was determined with a scintillation counter 2 days after induction of differentiation.

Transfection of small interfering RNAs.siRNAs targeting ERK1/2 (Santa Cruz) were employed. MEF, when 90% confluent, were transfected with 20 μM concentrations of either ERK1/2 siRNAs or control siRNA (Santa Cruz). Fresh medium containing 10% FBS was added 6 h posttransfection. Two days later, medium was replaced with the standard differentiation induction medium containing 0.5 mM MIX, 1 μM DEX, 10 μg/ml insulin, 10 μM troglitazone, and 10% (vol/vol) FBS.

Western blot analysis.MEF before and during adipocyte differentiation were prepared for Western blot analysis by rinsing twice with PBS and scraping cells in Western lysis buffer containing 1 mM Na3VO4, 25 mM NaF, 50 mM Tris-HCl, pH 8, 1 mM EGTA, 150 mM NaCl, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholic acid, and 0.1% protease inhibitors. Cell lysates were sonicated on ice and centrifuged for 15 min at 14,000 × g at 4°C. The protein concentration of each supernatant was determined by the Bradford method (Bio-Rad). Cell lysates were fractioned by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad). After blocking with milk, the membranes were incubated with one of the following: anti-Pref-1, anti-phosphorylated [Thr(P)202/Tyr(P)204] ERK1/2 (Santa Cruz), anti-ERK1/2 (Upstate), anti-phosphorylated [Thr(P)180/Tyr(P)182] p38 MAPK (Cell Signaling), anti-p38 MAPK (Cell Signaling), anti-phosphorylated [Thr(P)183/Tyr(P)185] JNK (Cell Signaling), anti-JNK (Cell Signaling), anti-phosphorylated [Ser(P)217/221] MEK 1/2 (Cell Signaling), or anti-MEK 1/2 antibodies (Cell Signaling). The membranes were then treated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (Bio-Rad). Signals were visualized by enhanced chemiluminescence (PerkinElmer).

RT-PCR.Total RNA was isolated using Trizol reagent (Gibco BRL). Reverse transcription was performed with 2 μg of total RNA, and resultant cDNA populations were amplified by semiquantitative PCR for Pref-1, CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ2 (PPARγ2), fatty acid synthase (FAS), and adipocyte fatty acid-binding protein (aFABP). These were then compared with expression levels of the control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression levels. The primer pairs used in RT-PCR were Pref-1 (5′-GACCCACCCTGTGACCCC-3′ and 5′-CAGGCAGCTCGTGCACCCC-3′), C/EBPα (5′-TGGACAAGAACAGCAACGAG-3′ and 5′-AATCTCCTAGTCCTGGCTTG-3′), PPARγ2 (5′-ACTGCCTATGAGCTCTTCAC-3′ and 5′-CAATCGGATGGTTCTTCGGA-3′), FAS (5′-TGCTCCCAGCTGCAGGC-3′ and 5′-GCCCGGTAGCTCTGGGTGTA-3′), aFABP (5′-ATGAGTACTACATGGCTA-3′ and 5′-CAATGGGGGCCAGATCAT-3′), and GAPDH (5′-CATCACCATCTTCCAGGAGCG-3′ and 5′-TGACCTTGCCCACAGCCTTG-3′). The reaction conditions were as follows: denaturation at 94°C for 30 s; annealing at 50°C (aFABP), 56°C (GAPDH, FAS, and PPARγ2), or 60°C (Pref-1 and C/EBPα) for 30 s; and extension at 72°C for 30 s. Varying cycles of PCR were carried out to determine linear ranges of PCR products. PCR products were electrophoresed on a 2% agarose gel and visualized with ethidium bromide staining.

Real-time RT-PCR was performed with an ABI PRISM 7900 sequence detection system (PE Applied Biosystems) to quantify the relative mRNA levels of gene markers with GAPDH as the endogenous control. cDNAs for C/EBPα or Pref-1 were amplified in real time using TaqMan gene expression assays consisting of a 20× mix of premade unlabeled PCR primers and TaqMan MGB probe labeled with FAM dye (PE Applied Biosystems). For PPARγ2, primers and MGB probe were designed using primer express software version 2.0. The forward primer was 5′-ACTCTGGGAGATTCTCCTGTTGAC-3′, the MGB probe was 5′-CAGAGCATGGTGCCTT-3′, and the reverse primer was 5′-TGCTCATAGGCAGTGCATCAG-3′. Each sample was run in triplicate in a 25-μl reaction mixture using TaqMan Universal PCR master mix according to the manufacturer's instructions. Thermal cycling was initiated with an initial denaturation at 50°C for 2 min and 95°C for 10 min. After this initial step, 40 cycles of PCR (95°C for 15 s; 60°C for 1 min) were performed. Statistical analysis of the quantitative real-time PCR was obtained using the (2−ΔΔCt) method, which calculates the relative changes in gene expression of the target normalized to an endogenous reference (GAPDH) and relative to a calibrator that serves as the control group.

Statistical analysis.The data are expressed as the means ± standard errors of the means. Statistical analysis was performed using Student's t test for comparison between two groups and one-way analysis of variance with Dunnett's post hoc test for multiple comparisons.

RESULTS

Pref-1 activates ERK1/2 but not p38 MAPK or JNK.Pref-1 contains two proteolytic processing sites in the extracellular domain (39). Upon cleavage, Pref-1 produces a 50-kDa soluble fragment corresponding to the full ectodomain in addition to a smaller soluble fragment of 25 kDa. We have shown that only the large 50-kDa soluble form of Pref-1, but not the transmembrane form or the 25-kDa soluble form, inhibits adipocyte differentiation (25). To analyze the biological function of Pref-1, we prepared recombinant Pref-1 protein which contains the 50-kDa large soluble form of Pref-1 fused to human immunoglobulin G(γ) heavy chain Fc region at the C terminus. Since Pref-1 has been known to function as a dimer, fusion to human Fc (hFc) would enhance its dimerization and bioactivity (20, 28). 293F cells were transfected with the Pref-1-hFc construct, and the secreted Pref-1-hFc fusion protein was purified to near homogeneity from serum-free culture medium by affinity chromatography. Purified Pref-1-hFc migrated as a molecular mass of 85 kDa, which corresponds to the 50-kDa soluble fragment and 35-kDa hFc, as shown by staining with Coomassie blue and by Western blot analysis (Fig. 1A).

FIG. 1.
FIG. 1.

Pref-1 increases phosphorylation of ERK1/2, but not p38 MAPK or JNK, in a time- and dose-dependent manner. (A) Purification of recombinant Pref-1-hFc. Conditioned media and eluted fractions from the protein A column were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by Coomassie staining or by Western blot analysis with antibodies against hFc fragment. Lanes: 1, conditioned media; 2 and 3, eluted fractions from the protein A column. At 80% confluence, primary MEF from Pref-1 null mice were maintained in DMEM with 0.1% FBS for 4 h and then treated with purified Pref-1-hFc or hFc in serum-free media. Cell lysates were harvested at the indicated times after the addition of 50 nM Pref-1-hFc or hFc (B) or 10 min after the addition of the indicated doses of Pref-1-hFc or hFc (C and D) and then subjected to Western blot analysis using antibodies against phosphorylated ERK1/2, total ERK1/2, phosphorylated p38 MAPK, total p38 MAPK, phosphorylated JNK, or total JNK as indicated. Essentially the same results were obtained in three independent experiments.

To investigate the direct effect of Pref-1 on MAPK activation, we employed Pref-1 null MEF isolated from Pref-1 knockout mice that we previously generated (26). These cells were treated with Pref-1 in serum-free media, and the extent of phosphorylation of ERK1/2 [Thr(202)/Tyr(204)] was examined. ERK1/2 phosphorylation increased in a time-dependent manner when the MEF were incubated with 50 nM Pref-1, whereas the abundance of ERK1/2 was not affected by Pref-1 treatment (Fig. 1B). The earliest increase in ERK1/2 phosphorylation was observed 1 min after Pref-1 addition, and the phosphorylation was further increased up to 10 min and then decreased back to basal level 60 min after Pref-1 addition (Fig. 1B). When we treated Pref-1 null MEF with hFc alone as a control, ERK1/2 phosphorylation was not changed (Fig. 1B). The increase in ERK1/2 phosphorylation by Pref-1 was also dose dependent when the MEF were treated for 10 min with various concentrations of Pref-1. ERK1/2 phosphorylation in Pref-1 null MEF increased with Pref-1 treatment up to 50 nM Pref-1 when added to Pref-1 null MEF, whereas hFc did not change ERK1/2 phosphorylation at any concentrations (Fig. 1C). Pref-1 at concentrations higher than 50 nM did not further increase ERK1/2 phosphorylation (data not shown). Conversely, phosphorylation of p38 MAPK and JNK was not affected by Pref-1 treatment (Fig. 1D). These results indicate, for the first time, that Pref-1 increases ERK1/2 phosphorylation and that this increase in phosphorylation is specific for ERK1/2.

Transient increase in ERK1/2 phosphorylation during adipocyte differentiation peaking at day 2 is absent in Pref-1 null MEF.Next, we examined whether the ERK1/2 activation by Pref-1 that we observed is responsible for the biological function of Pref-1, i.e., inhibition of adipocyte differentiation. Pref-1 null MEF is an ideal system since, unlike 3T3-L1 cells, these cells do not have high endogenous Pref-1 but yet undergo adipocyte differentiation. We prepared primary MEF from wild-type and Pref-1 null mice that we previously generated and induced the cells to differentiate into adipocytes by treatment for 4 days with differentiation inducers that included DEX, MIX, and insulin, used routinely as adipogenic agents, as well as troglitazone. As previously reported, we found that troglitazone was necessary for uniform and extensive differentiation of MEF into adipocytes (1). The cells were harvested at various time points before, during, and after this treatment. First, mRNA and protein levels of Pref-1 were examined daily during MEF differentiation by RT-PCR and Western blot analysis, respectively. As shown in Fig. 2A, Pref-1 mRNA levels in wild-type MEF were transiently increased up to day 2 of differentiation and then decreased down to basal levels. Similarly, Pref-1 protein levels were increased transiently at day 2, which subsequently decreased during differentiation. As we have shown previously, Pref-1 was detected as multiple bands due to glycosylation and alternate splicing. We next examined ERK1/2 activation during differentiation of MEF into adipocytes. A rapid but short-lived ERK activation during the initial hour of differentiation has been reported in 3T3-L1 cells (30, 31). Similar to 3T3-L1 cells, addition of differentiation agents induced a rapid but short-lived burst of ERK1/2 phosphorylation peaking at 15 min which subsided to basal levels by 2 h poststimulation (Fig. 2B). In addition, there was a second, low but significant increase in ERK1/2 phosphorylation peaking at day 2 coinciding with Pref-1 levels (Fig. 2A). This second increase in ERK phosphorylation was described for 3T3-L1 cells also but has not been investigated. Pref-1 null MEF, similar to wild-type MEF, also showed the first burst of ERK1/2 phosphorylation. In Pref-1 null MEF, however, we could not detect the second transient ERK1/2 phosphorylation that we observed during differentiation of wild-type MEF into adipocytes. This suggests that the second ERK1/2 phosphorylation we observed in wild-type MEF is due to the presence of Pref-1 because it is absent in Pref-1 null MEF. Therefore, in the subsequent studies, we have focused on the transient increase in ERK1/2 phosphorylation at day 2 to investigate the effect of Pref-1 on the MEK/ERK signaling pathway during MEF differentiation into adipocytes.

FIG. 2.

ERK1/2 phosphorylation and differentiation of wild-type and Pref-1 null MEF into adipocytes. (A) mRNA (upper) and protein (lower) levels of Pref-1 during differentiation of wild-type MEF were measured by RT-PCR and Western blot analysis using antibody against Pref-1, respectively. (B) MEF from wild-type (WT) or Pref-1 null (KO) mice were induced to differentiate by the treatment with DEX, MIX, insulin, and troglitazone. Total lysates were prepared from cells harvested before (day 0) or at the indicated times after exposure to the differentiation agents and were subjected to Western blot analysis using antibodies specific for phosphorylated ERK1/2 or total ERK1/2 as indicated. (C) Cell proliferation during MEF differentiation into adipocytes. (a) Determination of cell number. MEF from wild-type or Pref-1 null mice or 3T3-L1 cells were seeded in 12-well plates, and at 2 days postconfluence, cells were maintained in normal media (open bars) or differentiation media (solid bars). The cell number was determined using a hemocytometer after trypsinization in triplicates before (day 0) and 2 days after induction of differentiation. (b) [3H]thymidine incorporation at 2 days postconfluence of MEF from wild-type or Pref-1 null mice or 3T3-L1 cells in normal media (open bars), or differentiation media (solid bars) in the presence of 1 μCi/ml of [3H]thymidine. [3H]thymidine incorporated into the cells was determined using a scintillation counter. (D) Oil red O staining of MEF before and 8 days after exposure to the differentiation agents and spectrophotometric quantification of lipid stain at day 8. (E) Semiquantitative RT-PCR analysis of adipocyte marker levels in nondifferentiated and differentiated MEF (left panel) and real-time RT-PCR analysis for C/EBPα and PPARγ2 at day 8 (right panel). Statistical analysis of the real-time RT-PCR was carried out using the (2−ΔΔCt) method, which calculates the relative changes in mRNA levels normalized to an endogenous reference (GAPDH) relative to a calibrator (WT) that serves as the control group and was expressed as fold change. Essentially the same results were obtained in 5 independent experiments using different RNA preparations from MEF. ** P < 0.01. OD 500, optical density at 500 nm.

Studies indicate that established preadipocyte cell lines such as 3T3-L1 and 3T3-F442A cells can undergo several rounds of cell division (mitotic clonal expansion) before undergoing adipocyte differentiation. However, it is not clear whether clonal expansion is an obligatory step in adipocyte differentiation. Pref-1 null MEF still show rapid transient bursts but lack only the second increase in ERK phosphorylation at day 2 during MEF differentiation. However, since the MAPK/ERK pathway is intimately associated with cell growth, we investigated whether cell proliferation is affected in Pref-1 null cells during adipocyte differentiation. We assessed cell proliferation by counting the cells as well as by measuring DNA synthesis by [3H]thymidine incorporation after confluence. Incubation with differentiation inducers did not affect either the cell number (Fig. 2C, a) or incorporation of [3H]thymidine into DNA (Fig. 2C, b) in both wild-type and Pref-1 null MEF, whereas in 3T3-L1 cells, cell number as well as [3H]thymidine incorporation increased almost twofold at 2 days after induction of differentiation. These data show that (i) the mitotic clonal expansion that occurs during 3T3-L1 adipocyte differentiation may not occur in MEF under the culture conditions we employed and (ii) Pref-1-mediated ERK activation at day 2 during differentiation is not related to clonal expansion.

Since the inhibitory function of Pref-1 in adipogenesis has been well documented (39-42), we measured the levels of MEF differentiation to investigate the effect of Pref-1-mediated ERK1/2 phosphorylation on MEF differentiation into adipocytes. At 8 days after exposure to the inducers of differentiation, approximately 30% of MEF from wild-type mice were differentiated into adipocytes, as judged by cell morphology and Oil red O staining (Fig. 2D). In contrast, Pref-1 null MEF showed enhanced differentiation with sustained lipid accumulation with about 70% of the cells acquiring morphological characteristics of mature adipocytes. Quantification of oil red O staining at day 8 showed that the degree of adipose conversion increased approximately threefold in Pref-1 null MEF compared to wild-type MEF (Fig. 2D). We also examined the effect of Pref-1 on MEF differentiation by measuring expression levels of adipocyte markers. First, using mRNAs prepared from wild-type or Pref-1 null MEF before and after differentiation into adipocytes, we performed RT-PCR analysis for C/EBPα and PPARγ2, two key transcription factors for adipogenesis, as well as FAS and aFABP, representative late adipocyte markers. Real-time RT-PCR analysis indicated a >3-fold-higher expression of C/EBPα and PPARγ2 in Pref-1 null MEF than in wild-type MEF. Wild-type MEF, therefore, exhibited lower levels of adipocyte markers than Pref-1 null MEF, reflecting the inhibitory effect of Pref-1 on MEF differentiation into adipocytes (Fig. 2E). Overall, these data suggest that Pref-1 may cause ERK1/2 phosphorylation peaking at day 2 of differentiation and that, in the absence of this ERK phosphorylation, MEF differentiation into adipocytes is enhanced.

Inactivation or depletion of ERK1/2 at day 2 during MEF differentiation enhances adipogenesis.Pref-1 null MEF with enhanced adipocyte differentiation do not display the low but significant phosphorylation of ERK1/2 peaking at day 2 that is observed in wild-type MEF. We therefore tested whether prevention of this second peak of phosphorylation in wild-type MEF can enhance the degree of differentiation into adipocytes. We incubated wild-type MEF with 50 μM PD98059, a pharmacological inhibitor of MAPK/ERK kinase (MEK) that is an upstream kinase for ERK1/2. Since Pref-1 increases ERK1/2 phosphorylation in wild-type MEF 2 days after the induction of differentiation (Fig. 2B), we treated cells for 2 days with PD98059, beginning 2 h after induction of differentiation. Delayed treatment until 2 h after the induction of differentiation would prevent any interference with the initial rapid burst in ERK1/2 activation that occurs during the first hour of treatment with adipogenic agents. PD98059 treatment reduced or prevented the increase in ERK1/2 phosphorylation that was detected at day 2 to day 3 compared to untreated MEF (Fig. 3A). Furthermore, treatment with PD98059 increased the degree of MEF differentiation into adipocytes, as monitored by changes in cell morphology and increased Oil red O staining of lipid droplets (Fig. 3B and C). RT-PCR analysis for C/EBPα, PPARγ2, FAS, and aFABP showed higher mRNA levels of these adipocyte markers in wild-type MEF treated with PD98059 (Fig. 3D). Real-time RT-PCR analysis for C/EBPα and PPARγ2 showed that wild-type MEF treated with PD98059 exhibited >3-fold-higher mRNA levels than untreated MEF. These data suggest that prevention of ERK1/2 activation at day 2 to day 3 during differentiation enhanced the degree of MEF differentiation into adipocytes.

FIG. 3.
FIG. 3.

Blocking ERK1/2 activation peaking at day 2 by MEK inhibitor, PD98059, increases MEF differentiation into adipocytes. (A) At 2 days postconfluence, MEF from wild-type mice were induced to differentiate by exposure (day 0) to DEX, MIX, insulin, and troglitazone. PD98059 (50 μM) was added 2 h after the addition of the differentiation agents, and the cells were treated for 2 days. Cell were harvested at the indicated times and total lysates were subjected to Western blot analysis using antibodies specific to phosphorylated ERK1/2 or total ERK1/2. (B) Cells under microscope (left panel) and oil red O staining for lipid accumulation (middle and right panels) at day 8 of differentiation. (C) Spectrophotometric quantification of lipid stain at day 8 of differentiation was shown. OD 500, optical density at 500 nm. (D) Total RNA was extracted for semiquantitative RT-PCR of adipocyte markers (left panel) and real-time RT-PCR analysis of C/EBPα and PPARγ2 (right panel) at day 8. Statistical analysis of the real-time RT-PCR was obtained as described for Fig. 2. Essentially the same results were obtained from three independent experiments. **, P < 0.01.

To rule out any drug-related pleiotropic effects, wild-type MEF were transfected with siRNA specifically targeted for ERK1/2 and then induced to differentiate into adipocytes. As shown in Fig. 4A, treatment with adipogenic agents caused a rapid and transient burst of ERK1/2 phosphorylation peaking at 15 to 30 min in both ERK1/2 siRNA-transfected and control siRNA-transfected cells. However, transfection with siRNA targeted for ERK1/2, but not with control siRNA, reduced the abundance and phosphorylation of ERK1/2, as observed during the first 2 days after induction of differentiation. MEF transfected with ERK1/2 siRNA and exposed to differentiation inducers for 4 days showed enhanced differentiation into adipocytes, as measured by changes in cell morphology and Oil red O staining (Fig. 4B). In agreement with the accumulation of lipid droplets, quantification of Oil red O staining at day 8 showed about 1.5-fold-higher lipid staining in ERK1/2 siRNA-transfected cells than in control siRNA-transfected cells (Fig. 4C). At 8 days postinduction, wild-type MEF transfected with ERK1/2 siRNA also expressed higher mRNA levels of adipocyte markers than did cells transfected with control siRNA measured by RT-PCR (Fig. 4D). The C/EBPα and PPARγ2 mRNA levels measured by real-time RT-PCR were >4-fold higher in wild-type MEF transfected with ERK1/2 siRNA than in cells transfected with control siRNA. These complementary data obtained from using MEK inhibitor to prevent ERK1/2 phosphorylation and siRNA-mediated ERK1/2 depletion both indicate that the increase in ERK1/2 phosphorylation 2 days postinduction of differentiation is not required for MEF differentiation into adipocytes but has inhibitory function in adipocyte differentiation. These data also suggest that the endogenous Pref-1 present in these wild-type MEF may have contributed to ERK1/2 phosphorylation, resulting in the lower degree of adipocyte differentiation in wild-type MEF than in Pref-1 null MEF.

FIG. 4.
FIG. 4.

ERK1/2 siRNA-mediated deletion of ERK1/2 at day 2 enhances adipocyte differentiation of MEF. (A) MEF at 90% confluence were transfected with ERK1/2 siRNA or control siRNA. Fresh medium containing FBS was added 6 h posttransfection, and the cells were incubated for an additional 2 days. Culture media were then replaced with the differentiation induction media. Total lysates from cells harvested at the indicated times after the addition of the differentiation agents were subjected to Western blot analysis using antibodies specific to phosphorylated ERK1/2 or total ERK1/2. (B) At day 8, cells were viewed microscopically (left panel) and stained with Oil red O for lipid accumulation (middle and right panels). (C) Spectrophotometric quantification of the lipid stain at day 8 of differentiation was shown. OD 500, optical density at 500 nm. (D) Total RNA was used for semiquantitative RT-PCR of adipocyte markers (left panel) and real-time RT-PCR analysis of C/EBPα and PPARγ2 (right panel) at day 8. Statistical analysis of the real-time RT-PCR was obtained as described for Fig. 2. Essentially the same results were obtained from three independent experiments. *, P < 0.05; **, P < 0.01.

Pref-1 treatment restores ERK1/2 phosphorylation at day 2 in inhibiting differentiation of Pref-1 null MEF into adipocytes.Thus far, we have shown that Pref-1 inhibition of adipocyte differentiation correlates with the Pref-1-mediated phosphorylation of ERK1/2 peaking at day 2 in wild-type MEF that is absent in Pref-1 null MEF. In light of this observation, we questioned whether the treatment of Pref-1 null MEF with Pref-1 can restore ERK1/2 phosphorylation that we observed in the wild type at day 2 during differentiation. We prepared Pref-1 null MEF and incubated them with 100 nM Pref-1 or hFc for the first 4 days in addition to differentiation inducers. Figure 5A shows that the treatment of Pref-1 null MEF with Pref-1 accompanied a clearly detectable increase in phosphorylation of ERK1/2 at day 1 that was sustained until day 3, while there was no change in ERK1/2 phosphorylation in untreated Pref-1 null MEF (control) or in cells treated with hFc. Furthermore, Pref-1 treatment decreased adipocyte differentiation of Pref-1 null MEF to 30%, as judged by cell morphology and Oil red O staining, compared with 70% observed in control or hFc-treated cells (Fig. 5B). When we treated Pref-1 null MEF with hFc alone, MEF differentiation into adipocytes was not changed. Similarly, quantification of Oil red O staining at day 8 showed an about 60% decrease in lipid accumulation in Pref-1 null MEF treated with Pref-1 compared to untreated Pref-1 null MEF or cells treated with hFc (Fig. 5C). In agreement with our finding on lipid accumulation, the induction of mRNA expression of adipocyte differentiation markers including C/EBPα, PPARγ2, FAS, and aFABP after 8 days of differentiation was also reduced in Pref-1 null MEF treated with Pref-1. Conversely, in the controls, hFc treatment did not change levels of these adipocyte differentiation markers, supporting Pref-1 inhibition of MEF differentiation into adipocytes (Fig. 5D). Real-time RT-PCR analysis for C/EBPα and PPARγ2 confirmed the inhibitory effect of Pref-1 on MEF differentiation into adipocytes. Our results suggest that Pref-1 treatment restores ERK1/2 phosphorylation at 2 days postdifferentiation in Pref-1 null MEF and inhibits MEF differentiation into adipocytes. We also examined the time course of C/EBPα and PPARγ2 induction during adipocyte differentiation by real-time RT-PCR. The C/EBPα mRNA levels in wild-type MEF substantially increased up to day 3 but did not increase further during the 8-day differentiation period (Fig. 5E). Pref-1 null MEF and Pref-1 null MEF treated with hFc had an approximately twofold-higher level of C/EBPα mRNA than that observed in wild-type MEF, which further increased during differentiation up to day 8. Pref-1 treatment in Pref-1 null MEF decreased C/EBPα mRNA levels down to wild-type levels. PPARγ2 mRNA levels in wild-type MEF were barely detectable and were not substantially increased in the first few days after differentiation but were observed to increase at day 8. Conversely, Pref-1 null MEF and Pref-1 null MEF treated with hFc showed a marked increase in PPARγ2 mRNA levels during the first 3 days that further increased until day 8 (threefold-higher level than in wild-type MEF). Pref-1 null MEF treated with Pref-1 did not show a significant increase in PPARγ2 mRNA levels during the first 3 days, although they had somewhat higher levels at day 8 which were still lower than those in wild-type MEF. These results show that although both C/EBPα and PPARγ2 levels were affected by Pref-1, PPARγ2 expression is more profoundly suppressed by Pref-1 in MEF cells during adipocyte differentiation. The addition of exogenous Pref-1 in Pref-1 null MEF caused inhibition of adipocyte differentiation. Indeed, this inhibition was even more pronounced than the effect found in wild-type MEF. This is likely because the level achieved by adding exogenous Pref-1 in Pref-1 null MEF was higher than the endogenous Pref-1 level in wild-type MEF. This was reflected by sustained ERK activation observed upon Pref-1 treatment in Pref-1 null MEF (Fig. 5A). Overall, these results indicate that Pref-1 treatment can restore ERK1/2 phosphorylation at day 2 during differentiation of Pref-1 null MEF, resulting in inhibition of differentiation primarily by preventing induction of PPARγ2 expression.

FIG. 5.
FIG. 5.

Pref-1 increases phosphorylation of ERK1/2 and inhibits adipocyte differentiation in Pref-1 null MEF. (A) MEF from Pref-1 null mice at 2 days postconfluence (day 0) were induced to differentiate by treatment with DEX, MIX, insulin, and troglitazone in the presence (100 nM) or absence of Pref-1-hFc or hFc for 4 days. Total lysates from cells harvested at the indicated times were subjected to Western blot analysis using antibodies to detect phosphorylated ERK1/2 or total ERK1/2. (B) Cell morphology (left panel) and Oil red O staining for lipid accumulation (middle and right panels) at day 8. (C) Spectrophotometric quantification of lipid stain at day 8 was shown. OD 500, optical density at 500 nm. (D) Total RNA was extracted and used for semiquantitative RT-PCR of adipocyte markers (upper panel) and real-time RT-PCR analysis of C/EBPα and PPARγ2 (lower panel) at day 8. Statistical analysis of the real-time RT-PCR was obtained as described for Fig. 2. **, P < 0.01. (E) Real-time RT-PCR analysis of C/EBPα and PPARγ2 during MEF differentiation. ⧫, wild-type MEF; ▪, Pref-1 null MEF; ▴, Pref-1 null MEF treated with hFc; •, Pref-1 null MEF treated with Pref-1-hFc.

Pref-1 inhibition of MEF differentiation is through ERK1/2 phosphorylation peaking at day 2.Next, to further determine if Pref-1 inhibition is due to the ERK1/2 phosphorylation at day 2 during differentiation, we treated Pref-1 null MEF with both PD98059 and Pref-1 for the first 4 days of differentiation. Compared to 70% differentiation observed in Pref-1 null MEF without any treatment (Fig. 3B and Fig. 6A), Pref-1 treatment alone decreased adipocyte differentiation of these cells to 30%, as judged by cell morphology and Oil red O staining (Fig. 6A). Unlike wild-type MEF that showed enhanced differentiation upon treatment (Fig. 4), PD98059 treatment alone in Pref-1 null MEF had no effect on differentiation. Furthermore, when we treated Pref-1 null MEF with Pref-1 together with PD98059, the degree of adipocyte differentiation was not affected by Pref-1 treatment, as judged by morphology and Oil red O staining. Similarly, mRNA levels of adipocyte markers, including C/EBPα, PPARγ2, FAS, and aFABP, clearly showed that Pref-1 treatment in Pref-1 null MEF decreased adipocyte differentiation in the absence of PD98059 (Fig. 6B). However, Pref-1 did not inhibit adipocyte differentiation in the presence of PD98059. We also employed siRNA targeted for ERK1/2. Unlike effects observed in wild-type MEF, transfection of Pref-1 null MEF with ERK1/2 siRNA had no effect on MEF differentiation, as judged by morphology and Oil red O staining (Fig. 6C) as well as adipocyte marker expression levels (Fig. 6D). As predicted, Pref-1 treatment caused inhibition of adipocyte differentiation of Pref-1 null MEF transfected with control siRNA. In contrast, Pref-1 treatment did not inhibit differentiation when cells were transfected with siRNA for ERK1/2. These data provide clear evidence that ERK1/2 activation is required for Pref-1-mediated inhibition of adipogenesis and that the inhibitory effect of Pref-1 on MEF differentiation is through ERK1/2 phosphorylation.

FIG. 6.
FIG. 6.

ERK1/2 phosphorylation is required for Pref-1-mediated inhibition of MEF differentiation. (A) MEF from Pref-1 null mice at 2 days postconfluence were induced to differentiate by exposure to the differentiation media containing DEX, MIX, insulin, and troglitazone in the presence of Pref-1 alone or with PD98059 for 4 days or PD98059 alone for 2 days. At day 8, cells were viewed microscopically (left panel) and stained with Oil red O for lipid accumulation (right panel). (B) Total RNA was extracted and used for semiquantitative RT-PCR of adipocyte markers (upper panel) and real-time RT-PCR analysis of C/EBPα and PPARγ2 (lower panel) at day 8. Statistical analysis of the quantitative real-time PCR was obtained as described for Fig. 2. (C) MEF at 90% confluence were transfected with ERK1/2 siRNA or control siRNA. Fresh medium containing 10% FBS was added 6 h posttransfection, and the cells were incubated for an additional 2 days. Media were then replaced with the standard differentiation induction media. At day 8, cells were viewed microscopically (left panel) and stained with Oil red O for lipid accumulation (right panel). (D) Total RNA was extracted and used for semiquantitative RT-PCR of adipocyte markers (upper panel) and real-time RT-PCR analysis of C/EBPα and PPARγ2 (lower panel) at day 8. Statistical analysis of the quantitative real-time PCR was obtained as described for Fig. 2. Essentially the same results were obtained from three independent experiments. **, P < 0.01 versus control group. +, present; −, absent.

Pref-1-mediated ERK1/2 phosphorylation is through activation of MEK1/2.Consistent with other growth factors, we demonstrated that Pref-1 increases ERK1/2 phosphorylation in a dose- and time-dependent manner. We have also shown that Pref-1 inhibits adipocyte differentiation by inducing a low but significant increase in ERK1/2 phosphorylation peaking at day 2 during adipocyte differentiation. The phosphorylation of ERK1/2 is known to be regulated by the upstream kinase MEK, a dual-specificity serine/threonine kinase. To further elucidate the mechanism by which Pref-1 activates ERK1/2, we investigated the effect of Pref-1 on MEK activation. As shown in Fig. 7A, MEK1/2 phosphorylation increased in a time-dependent manner when the Pref-1 null MEF were incubated with 50 nM of Pref-1, whereas the abundance of MEK1/2 was not affected by Pref-1 treatment. The earliest increase in MEK1/2 phosphorylation was observed 1 min after Pref-1 addition, and the phosphorylation was further increased up to 5 min, which is somewhat earlier than the ERK1/2 phosphorylation we observed upon Pref-1 treatment. When we treated Pref-1 null MEF with hFc alone as a control, MEK1/2 phosphorylation was not changed. The increase in MEK1/2 activation by Pref-1 was concentration-dependent, since treatment for 10 min with 50 nM Pref-1 had a higher effect on MEK1/2 phosphorylation than treatment with 10 nM Pref-1 (Fig. 7B). Overall, these studies clearly show that Pref-1, soluble inhibitor of adipocyte differentiation, activated the MEK/ERK signaling pathway.

FIG. 7.
FIG. 7.

Pref-1 increases MEK1/2 phosphorylation in a time- and dose-dependent manner. At 80% confluence, Pref-1 null MEF were maintained in DMEM with 0.1% FBS for 4 h and then treated with purified Pref-1-hFc or hFc in serum-free media. Total cellular proteins were harvested at the indicated times after the addition of 50 nM Pref-1-hFc or hFc (A) or 10 min of treatment at indicated doses of Pref-1-hFc or hFc (B) and then subjected to Western blot analysis using antibodies specific for phosphorylated MEK1/2 or total MEK1/2.

DISCUSSION

Despite numerous in vitro as well as in vivo studies demonstrating that preadipocyte secreted factor, Pref-1, inhibits adipocyte differentiation (39-42), the signaling pathway for Pref-1 has not been elucidated. We show, for the first time, that the purified soluble Pref-1 protein directly stimulates MEK/ERK phosphorylation. Pref-1 treatment increases MEK1/2 and ERK1/2 phosphorylation in a time- and dose-dependent manner in Pref-1 null MEF and in serum-free media in the absence of other growth factors. Furthermore, this effect of Pref-1 on the MAPK pathway is specific to ERK1/2 but not p38 MAPK or JNK. In the present study, we employed MEF isolated from Pref-1 null mice that lack endogenous Pref-1. 3T3-L1 cells have significant levels of endogenous Pref-1, limiting their usefulness in studying the Pref-1 signaling pathway. Using Pref-1 null MEF made it possible for us to clearly demonstrate Pref-1 mediated MEK/ERK phosphorylation.

Since Pref-1 is found in preadipocytes but its expression is extinguished during differentiation into adipocytes (42, 53), Pref-1 is now commonly used as a unique preadipocyte marker by researchers (8, 38, 49, 54). In general, down-regulation of Pref-1 during MEF differentiation into adipocytes is similar to that observed during 3T3-L1 differentiation. However, the Pref-1 level is transiently increased in MEF upon treatment with adipogenic inducers. Unlike 3T3-L1 preadipocytes that are already committed to the adipocyte lineage and can be induced to terminally differentiate into adipocytes, MEF are functionally similar to mesenchymal stem cells that have not undergone commitment but can differentiate into various mesenchymal lineages, including osteoblasts and chondrocytes as well as adipocytes (27). It is likely that the adipogenic inducers we employed are capable of inducing MEF commitment into adipocyte lineage as well as terminal differentiation into adipocytes. The transient increase in expression of Pref-1, a preadipocyte marker, in MEF upon treatment with adipogenic agents may reflect commitment of MEF to the preadipocyte lineage. In this regard, we observed a similar expression pattern of Pref-1 during differentiation of C3H10T1/2 cells into adipocytes (data not shown).

We have previously shown that forced expression of Pref-1 or addition of soluble Pref-1 inhibits 3T3-L1 adipocyte differentiation upon treatment with DEX/MIX in the presence of 10% fetal bovine serum, containing significant levels of insulin/IGF-1 (25, 39, 41, 42). However, Zhang et al. reported that forced expression of Pref-1, although inhibiting adipocyte differentiation in the standard conditions, did not inhibit adipocyte differentiation of 3T3-L1 cells in the presence of IGF-1 or high concentrations of insulin (53). These researchers suggested that the Pref-1 effect may be via regulation of IGF-1 receptors, since the overexpression of Pref-1 was accompanied by a reduction in the level of the mature form of the IGF-1 receptor, an impairment of IGF-1 receptor-mediated ERK1/2 activation, and an increased requirement for IGF-1/insulin to induce adipocyte differentiation. Conversely, Ruiz-Hidalgo et al. reported that antisense mediated Pref-1 down-regulation in BALB/c 3T3 cells enhanced IGF-1-dependent activation of ERK1/2 without affecting IGF-1 receptor levels, insulin receptor substrate 1 levels, or insulin receptor substrate 1 phosphorylation in response to IGF-1 (33). However, Ruiz-Hidalgo et al. did not observe any change in ERK activation upon sense-strand expression of Pref-1. Although contradictory explanations for the effect of Pref-1 on IGF-1 receptor levels were proposed, both of these reports suggest that Pref-1 suppresses IGF-1-mediated ERK1/2 activation. In our present study, however, we demonstrate an effect of Pref-1 on ERK1/2 phosphorylation in serum-free media lacking all growth factors, including insulin and IGF-1. Moreover, contrary to the reports by Zhang et al. and Ruiz-Hidalgo et al., Pref-1 increases, not suppresses, ERK1/2 activation. We conclude that Pref-1 activates the MEK/ERK pathway without involving other growth factors, including IGF-1. In addition, we found that the transient increase in ERK1/2 phosphorylation in wild-type MEF at day 2 does not occur in Pref-1 null cells, and Pref-1 treatment of Pref-1 null cells restores this ERK1/2 phosphorylation. Although we observed a rapid and direct activation of MEK/ERK in serum-free conditions, during MEF differentiation, Pref-1 increases ERK1/2 phosphorylation at 2 to 3 days upon Pref-1 treatment in Pref-1 null MEF. The reasons for the apparent differences in time course of ERK1/2 phosphorylation are not clear. It may be that the presence of adipogenic agents affects Pref-1-mediated ERK activation. It is also possible that activation of the yet to be identified Pref-1 receptor, although present in MEF, might be transiently increased after induction of differentiation. Regardless, the reasons for the apparent discrepancy between our results and those of Zhang et al. or Ruiz-Hidalgo et al. are not obvious. Those researchers employed cell lines that are known to express high levels of endogenous Pref-1. Structurally, Pref-1 is most closely related to Delta by the number of EGF repeats and by other conserved amino acid residues. However, unlike those proteins that function by binding to Notch, Pref-1 does not contain the Delta, Serrate, and Lag-2 domain that is conserved in all Notch ligands and shown to be the functional binding domain in Notch ligand interaction (3, 21, 23). In addition, Notch is not known to activate ERK directly, although it may indirectly affect ERK activation by regulating other growth factor receptors or their signaling pathways (11, 45). Our present study suggests that Pref-1 exerts its biological functions independent of Notch receptor or its downstream effects, including expression of hairy and enhancer of split, a basic helix-loop-helix transcription factor (18). In our cell system, we could not detect changes in hairy and enhancer of split expression upon Pref-1 treatment (data not shown).

Apart from Pref-1, contradictory observations on the role of the ERK pathway in adipocyte differentiation have been reported. Eliminating ERK1/2 by antisense oligonucleotide strategy or inhibiting ERK1/2 phosphorylation by MEK inhibitors has been reported to prevent insulin-induced differentiation of 3T3-L1 cells into adipocytes (31, 35, 46). In this regard, it has been shown genetically in mice that ERK is necessary for adipogenesis and adiposity in vivo (7, 15, 32) and that MEF and preadipocytes isolated from ERK1 knockout mice have impaired adipogenesis (7). These observations suggest that ERK pathway may be required for or may play a positive role in adipogenesis. In contrast, others have shown that ERK activation inhibits adipocyte differentiation. Activation of the ERK pathway has been shown to mediate inhibition of adipocyte differentiation produced by various growth factors (4, 17). Retinoblastoma protein has also been shown to promote adipogenesis by suppressing ERK1/2 activation (15). To explain these apparently contradictory results of ERK activation on adipogenesis, it has been speculated that activation of ERK might have opposing effects during adipogenesis. In early during adipocyte differentiation, ERK has to be activated for a proliferative step, but later in adipocyte differentiation, ERK needs to be shut off to prevent PPARγ phosphorylation (6). In our present study, analogous to that in 3T3-L1 cells, addition of adipogenic agents induces a rapid but transient burst of ERK1/2 phosphorylation peaking at 15 min, which then subsided to basal levels by 2 h poststimulation. We also detected a second, albeit lower, increase in ERK1/2 phosphorylation peaking at day 2 that corresponded to Pref-1 abundance. Similar to wild-type MEF, Pref-1 null MEF also showed the first burst of ERK1/2 phosphorylation. However, we could not detect the second transient ERK1/2 phosphorylation during Pref-1 null MEF differentiation into adipocytes. This indicates that this second ERK1/2 phosphorylation detected in wild-type MEF, but not in Pref-1 null MEF, is due to the presence of Pref-1. Our study predicts that even though the first rapid burst of ERK activation may be required for adipocyte differentiation, as others have reported, the second peak of ERK phosphorylation actually has an inhibitory effect on adipocyte differentiation. Although the mitotic clonal expansion phase may precede the adipogenic program in 3T3-L1 preadipocytes, whether cell proliferation is an obligatory step during early adipocyte differentiation in general is not known. Tang et al. showed that when MEF were subjected to the same differentiation protocol as in 3T3-L1 preadipocytes, a subset of the MEF underwent mitotic clonal expansion and adipocyte differentiation (46). In our present study, the cell number or DNA synthesis did not increase early during adipocyte differentiation of MEF. These contradictory results regarding mitotic clonal expansion during MEF differentiation may be due to the differences in experimental conditions. We employed troglitazone, an agonist of PPARγ, in addition to the standard differentiation inducers, whereas Tang et al. used DEX, MIX, and insulin, standard adipogenic agents for 3T3-L1 differentiation. As reported by Alexander et al. we found that the standard differentiation medium used for 3T3-L1 adipogenesis was not sufficient to induce high level differentiation of MEF into adipocytes (1). It may be that PPARγ agonist is required for induction and/or acceleration of MEF differentiation into adipocytes. In our present study, we employed MEF freshly isolated after passaging only once before inducing them to differentiate into adipocytes. Because the size of MEF is smaller than that of 3T3-L1 cells, we have used higher number of MEF than 3T3-L1 cells to reach 100% confluence. In these conditions, cell number and [3H]thymidine incorporation did not increase after treatment with adipogenic inducers, suggesting that cell proliferation or DNA synthesis is not a required step for MEF differentiation into adipocytes. More importantly, we can conclude that Pref-1-mediated ERK activation is not related to mitotic clonal expansion.

The present study demonstrates the importance of time dependence in ERK activation in regulating adipocyte differentiation. The rapid and transient burst in ERK activation upon treatment of adipogenic agents has been shown to be critical for adipocyte differentiation. Park et al. and Prusty et al. reported that ERK phosphorylation rapidly and transiently increases within an hour following exposure of DEX/MIX/insulin and that this ERK activation enhances C/EBPβ phosphorylation with subsequent induction of C/EBPα and, therefore, adipogenesis (30, 31). On the other hand, the role of the second ERK activation that occurs later during adipocyte differentiation has not been investigated. Although ERK knockout causes impaired adipogenesis and adiposity (7, 32), these studies by design can only determine the effects of early, but not later, ERK activation, which we propose plays the negative role in adipogenesis. The temporal effect of ERK activation may also explain the perceived discrepancy between our results and those that did not show enhanced MEF differentiation with MEK inhibitor treatment (6, 14). We treated the cells 2 h after the addition of adipogenic agents to avoid affecting the early rapid transient burst in ERK phosphorylation, while others treated during the entire duration of differentiation. Furthermore, we employed troglitazone in addition to the standard adipogenic agents. Consistent with our results, Prusty et al. reported that troglitazone can rescue the inhibition of differentiation by MEK inhibitor that blocks the early transient ERK activation caused by DEX/MIX/insulin treatment (31). Similarly, this may also explain our observation that PD98059-treated Pref-1 null MEF differentiated into adipocytes at a similar degree as cells without treatment. It has been reported that PPARγ activity is negatively regulated by phosphorylation upon growth factor-induced ERK activation (9, 17). It is possible that once PPARγ is induced during 3T3-L1 differentiation, its transcriptional activity is reduced by phosphorylation mediated by ERK activation. Since we found that PPARγ2 mRNA levels, although detectable, are not increased at early stages of differentiation of wild-type MEF, it is likely that even in the presence of troglitazone, Pref-1-mediated MEK/ERK phosphorylation inhibits adipogenesis at a step prior to PPARγ2 phosphorylation. Although we cannot rule out possible PPARγ2 phosphorylation by Pref-1-mediated ERK activation, especially at later time points, it is more likely that Pref-1 acts via suppression of PPARγ2 expression, as shown in Fig. 5E. The mechanism by which Pref-1 mediated ERK phosphorylation regulates PPARγ expression is unknown. It is possible, however, that Pref-1-induced ERK activation may regulate expression of transcription factor(s) that can repress PPARγ expression during adipogenesis. In this regard, it has recently been shown that the ERK/MAPK cascade can regulate levels of GATA-3, a transcription factor found in white adipose tissue and reported to suppress PPARγ expression (47, 52). Further studies aimed at identifying targets of the MEK/ERK pathway as well as the Pref-1 receptor are critical to better understand Pref-1 function and signaling.

In conclusion, we show that (i) Pref-1 activates MEK1/2 and ERK1/2 directly and selectively, (ii) Pref-1 inhibition of adipocyte differentiation requires Pref-1-mediated ERK1/2 phosphorylation, and (iii) Pref-1-mediated inhibition of adipocyte differentiation accompanies prevention of PPARγ2 induction. The precise downstream target(s) of ERK activation mediating Pref-1 inhibition of MEF differentiation remains to be determined.

ACKNOWLEDGMENTS

We thank Weiming Ruan for his help during the early stage of this study in preparing Pref-1-hFc and ERK1/2 phosphorylation. We also thank Mary Ann Williams for her careful review of the manuscript.

This work was supported by National Institutes of Health grants DK50828 and DK68439 (to H.S.S.).

FOOTNOTES

    • Received 25 November 2006.
    • Accepted 21 December 2006.

  • ↵▿ Published ahead of print on 8 January 2006.

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