Department of Life Science, College of Science, National Central University, Chung-Li City, Taoyuan, Taiwan
Submitted 23 November 2004 ; accepted in final form 5 January 2005
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ABSTRACT |
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3T3-L1 preadipocyte; mitogen-activated protein kinase; cyclin-dependent kinase
Green tea catechins (GTCs) are polyphenolic flavonoids once called vitamin P (34). Since the discoveries that they have unique chemical structures (Fig. 1) and are major ingredients of unfermented tea (24, 33), they have been found to possess widespread biological functions and health benefits (1, 24, 25, 28, 45). In vivo, GTCs, especially ()-epigallocatechin gallate (EGCG; Fig. 1), lower the incidence of cancers (1, 24, 25, 28, 45), collagen-induced arthritis (16), oxidative stress-induced neurodegenerative diseases (27), and streptozotocin-induced diabetes (38). Also, EGCG can reduce body weight and body fat (18). In support of this antiobese effect of EGCG, other in vivo data have shown that EGCG or EGCG-containing green tea extract reduces food uptake, lipid absorption, and blood triglyceride, cholesterol, and leptin levels as well as stimulating energy expenditure, fat oxidation, HDL levels, and fecal lipid excretion (10, 18, 19, 24). These in vivo observations may be explained by in vitro findings that EGCG and caffeine synergistically with norepinephrine stimulate the thermogenesis of brown adipose tissue (11), that EGCG regulates various enzymes related to lipid anabolism and catabolism, such as acetyl-CoA carboxylase, fatty acid synthase, pancreatic lipase, gastric lipase, and lipoxygenase (24, 48), that EGCG is a potent prooxidant and antioxidant (24, 42, 43), and that EGCG reduced serum- or insulin-induced increases in cell numbers and the triacylglycerol content during a 9-day period of differentiation (19). These in vivo and in vitro observations suggest that green tea EGCG appears to modulate the mitogenic, endocrine, and metabolic functions of fat cells.
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The present study was designed to understand the mechanism of how EGCG acts in reducing the number of 3T3-L1 preadipocytes as they grow. Our specific aim was to investigate whether EGCG-regulated preadipocyte proliferation is dependent on the ERK MAPK and Cdk2 pathways. The mechanistic results of this study may have possible utility in the treatment of obesity using this compound.
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MATERIALS AND METHODS |
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Cell culture. According to a published method (19), 3T3 and 3T3-L1 cells (American Type Culture Collection; Manassas, VA) were grown in DMEM (pH 7.4) containing 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin (GIBCO-BRL) in a humidified atmosphere of 95% air-5% CO2 at 37°C. Medium (10 ml) was replaced every 2 days. Because serum components contain the factors for facilitating 3T3-L1 differentiation from preadipocytes to adipocytes when they are confluent, these cells were subcultured before reaching confluency. Confluent 3T3 were subcultured.
Growth inhibition experiments.
3T3 and 3T3-L1 cells (15,00020,000 cells/cm2) were plated in triplicate wells of a 12-well plate. To determine whether a dose- or growth phase-dependent effect of GTCs on the growth of 3T3-L1 preadipocytes exists, we treated different growth phases (days 1
6 with day 1 being the day of cell inoculum) of cells with epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), or EGCG at various concentrations (0
400 µM) for indicated time periods. After a particular time course of incubation, cells were trypsinized and counted with a hemocytometer using the 0.4% trypan blue exclusion method. To measure cellular proliferation, we modified the method reported by Vollenweider et al. (41) and used a commercially available bromodeoxyuridine (BrdU) ELISA kit (Roche Applied Science; Mannheim, Germany) as follows. 3T3-L1 cells (2,000 cells/well) were plated into a 96-well microplate (tissue culture grade) with 100 µl DMEM supplemented with 10% FBS. After 24 h were allowed for attachment, cells were starved with serum-free DMEM for 36 h, which was then replaced with fresh DMEM containing 10% FBS, the thymidine analog BrdU (10 µM), and each GTC (at the indicated concentrations) for 4 h at 37°C. This allowed BrdU to be incorporated into newly synthesized DNA of dividing cells during their S phase. After incubation, residual cells were washed with 10 mM PBS and then collected by centrifugation at 1,500 rpm for 5 min. Cell pellets were dried at 60°C for 1 h, fixed with 200 µl FixDenat solution/well for 30 min at 15
25°C, probed with mouse anti-BrdU-POD for 1 h, and visualized with the addition of 100 µl 3,3,5,5-tetramethylbenzidine substrate to each well for 5 min for color development. An aliquot of 100 µl of 1 N H2SO4 was added to stop the reaction of each well for the 1-min incubation on a 300-rpm shaker. The absorbance was read at 450 nm using a MRX microtiter plate reader (Dynatech Laboratories; Chantilly, VA). Culture medium alone and cells incubated with anti-BrdU-POD in the absence of BrdU were used as blank controls for nonspecific binding.
MAPK inhibitor treatment.
3T3-L1 preadipocytes, cultured in DMEM containing 10% FBS for 2 days and synchronized with serum-free DMEM for 1 day, were pretreated for 2 h with EGCG (2050 µM), PD98059 (50 µM) (12), and/or U0126 (10 µM) (13). These compounds were dissolved in 100% DMSO (at a final concentration of 0.1%) and then added to fresh DMEM containing 10% FBS during the experiment. After 4 h of incubation, protein amounts of ERK1/2 and phospho-ERK1/2 were measured by Western blot analysis while the number of cells was examined by the trypan blue exclusion method after 48 h of incubation.
Cdk2 plasmid constructs. cDNA encoding wild-type murine Cdk2 (Cdk2+/+) and the dominant negative form of murine Cdk2 (dnCdk2; with a mutation of Asp145 in Cdk2 to Asn145), as described by Heuvel and Harlow (17), was amplified by RT-PCR. Total RNA was isolated from 3T3-L1 preadipocytes with the TRIzol kit, and cDNA was then synthesized from equal amounts of RNA using Moloney murine leukemia virus reverse transcriptase (Invitrogen; Carlsbad, CA). The forward and reverse primers for obtaining Cdk2+/+ cDNA were 5'-ATGGAGAACTTTCAAAAGGTG-3' and 5'-TCAGAGCCGAAGGTGGGGGC-3', respectively. To obtain dnCdk2 cDNA, four primers were needed to introduce a site-specific mutation by overlap exclusion (42), and they were 5'-ATGGAGAACTTTCAAAAGGTG-3', 5'-TCAGAGCCGAAGGTGGGGGC-3', 5'-GTCCAAAGTTTGCCAGCTTG-3', and 5'-CAAGCTGGCAAACTTTGGAC-3'. PCR was performed under the following conditions: an initial denaturing cycle at 94°C for 3 min, followed by 30 cycles of amplification consisting of denaturation at 94°C for 45 s, annealing at 53°C for 30 s, and extension at 72°C for 90 s. A final extension at 72°C for 10 min was added after the last cycle. The PCR product was run on a 1.5% (wt/vol) agarose gel using 40 mM Tris-acetate buffer (pH 8.0) containing 1 mM EDTA and was visualized using 0.5 µg/ml ethidium bromide. The Cdk2+/+ and dnCdk2 products (predicted to be about 900 bp) were cloned to the pTargetT vector (Promega) as described by Sambrook and Russell (36). Before their transfection into the preadipocytes, they were verified with nucleotide sequencing performed at the Institute of Biomedical Sciences, Academia Sinica, Taiwan. The molecular weights of overexpressed Cdk2+/+ and dnCdk2 proteins were verified to be about 34 kDa by Western blot analysis, and their activities were also confirmed to catalyze the phosphorylation of the histone H1 substrate (20).
Cell transfection and overexpression experiments.
We modified the methods reported by Yang et al. (46) to perform our transfection experiments. In some experiments, 3T3-L1 preadipocytes were transiently (24 h) transfected with 15 µg of either the pcDNA3.1 plasmid, pBabe puro vector, pcDNA3.1-MKK6EE (the constitutively active MKK6 mutant designated MKK6EE, a gift from Dr. S. L. Chen), pCMV-MEKK1 (a constitutively active MEKK1-activating JNK kinase, a gift from Dr. S. L. Chen), pBabe puro-MEK1, or pBabe puro-MEK1S217E/S221E (a constitutively active MEK1 mutant designated MEK1EE; a gift from Dr. J. J. Yang). The overexpressed preadipocytes were cotransfected with pSV--galactosidase cDNA whose transfection efficiency was determined by analyzing the
-galactosidase activity. There were insignificant changes in
-galactosidase production from all transfected cells (data not shown). Activities of MEK1, MEKK1, and MKK6 proteins were assessed as measured by Western blot analysis of changes in the amounts of phospho-ERK1/2, phospho-JNK, and phospho-p38, respectively. In other experiments, 3T3-L1 preadipocytes in a 10-cm plate were stably transfected with 15 µg of the pTargetT vector constructed with Cdk2+/+ and dnCdk2 cDNAs. Stable clones were selected with 1 mg/ml of the antibiotic G-418 (BD Biosci Clontech Lab; Palo Alto, CA). Amounts and activities of Cdk2+/+ and dnCdk2 proteins expressed in the stable clones were later determined by immunoblotting and immunoprecipitation, respectively. A 45-µl volume of the TransFast transfection reagent was used during the stable and transient transfections and was comprised of the synthetic cationic lipid (+)-N,N-bis(2-hydroxyethyl)-N-methyl-N-[2,3-di(tetradecanoyloxy)propyl] ammonium iodide and the neutral lipid L-dioleoyl-phosphatidylethanolamine.
Cells transfected with the vehicle or the recombinant plasmid containing MEK1, MEK1EE, or MKK6EE cDNAs were incubated with or without 50 µM EGCG for 48 h. After incubation, the cell number of preadipocytes was examined by trypan blue dye exclusion. Unless otherwise noted, transfected cells with the vehicle, Cdk2+/+ cDNA, and dnCdk2 cDNA were incubated with and without 20100 µM EGCG for the indicated time periods.
Flow cytometric analysis.
Changes in the kinetics of the cell cycle were analyzed by flow cytometry as described by Kokontis et al. (20). 3T3-L1 cells (15,00020,000 cells/cm2) transfected with and without the vehicle, Cdk2+/+, and dnCdk2 plasmids were plated in a 10-cm dish containing 10 ml DMEM supplemented with 10% FBS. One day after inoculation, the medium was replaced with serum-free DMEM to obtain the homogenous cell population. After a 1-day starvation, cells were treated with fresh medium in the presence and absence of EC, ECG, EGC, or EGCG at various concentrations and time periods. The harvested cell pellets were fixed in 70% ethanol (dissolved in 10 mM PBS, pH 7.4) and stored at 20°C until later analysis. Cell pellets were washed with 10 mM cold PBS (pH 7.4), incubated at 37°C for 30 min with 200 µg/ml RNase A (Sigma), and then stained with 4 µg/ml propidium iodide (Sigma) in PBS containing 1% Triton X-100 (Sigma). Cell cycle profiles and distributions were determined by flow cytometric analysis of 104 cells using the CELLQuest program on a FACS Calibur flow cytometer (Becton-Dickinson; San Jose, CA). Clumped cells were excluded from the cell cycle distribution analysis by gating.
Immunoprecipitation. Cdk2 protein was immunoprecipitated according to the method described by Kokontis et al. (20). After experimental treatments, preadipocytes were washed twice in 10 mM PBS and then lysed in 1 ml buffer A [20 mM Tris·HCl (pH 7.6), 1 mM EDTA, 1 mM Na3VO4, 0.2% Triton X-100, and 1 mM PMSF]. The lysate was agitated for 15 min at 4°C and then centrifuged at 14,000 rpm for 10 min to collect the supernatant. The protein content of the supernatant was determined in duplicate by the dye-binding method (6) using a Bio-Rad (Richmond, CA) microplate reader and BSA (Sigma) as a standard. An aliquot of the supernatant (1 mg protein) was preincubated for 1 h at 4°C with Cdk2 antibody or preimmunized normal rabbit serum (NRS; as the control) for 1 h at room temperature or overnight at 4°C. The mixture was incubated with 20 µl protein A-agarose (Santa Cruz Biotechnology) overnight at 4°C. The total amounts of Cdk2, p18, p21, and p27 in the immunoprecipitates were measured by Western blot analysis with each antibody. After normalization to the Cdk2 protein, the amounts of each cyclin-dependent kinase inhibitor (CKI) protein were expressed as a percentage of the control, and changes in their bindings to Cdk2 were indicated. Data obtained from NRS were not shown because of the insignificant changes.
Western blot analysis.
Western immunoblot analysis was performed on supernatant fractions of preadipocytes as described by Kokontis et al. (20). An aliquot of 50 µg of supernatant protein was separated by 12% SDS-PAGE with 2x gel-loading buffer [100 mM Tris·HCl (pH 6.8), 4% SDS, 20% glycerol, 0.2% bromophenol blue, and 10% -mercaptoethanol] and then blotted onto Immobilon-NC transfer membranes (Millipore; Bedford, MA). The immunoblots were blocked for 1 h at room temperature with 10 mM PBS containing 0.1% Tween 20 (PBST) and 5% defatted milk. After the immunoblots were washed with PBST, immunoblot analyses were performed. All primary antibodies (ERK-1, ERK-2, phospho-ERKs, MEK1, p38, phospho-p38, JNK, phospho-JNK,
-actin, Cdc2, Cdk2, p18, p21, p27, cyclin D1, and phospho-histone H1 antisera) were used at a dilution of 1:1,000 (
0.2 µg/ml). Donkey anti-rabbit IgG, donkey anti-mouse IgG, donkey anti-goat IgG, or goat anti-guinea pig IgG conjugated with horseradish peroxidase was used as the secondary antibodies at a dilution of 1:2,000 (
0.2 µg/ml). The immunoblots were visualized using the Western Lightning chemiluminescence reagent plus kit (Perkin-Elmer Life Science; Boston, MA) for 3 min followed by exposure to Fuji film for 2
3 min. Blots were quantified using the FX Pro Plus Molecular Imager (Bio-Rad Laboratories). After normalization to
-actin protein, levels of these intracellular proteins were expressed as a percentage of the control unless otherwise noted.
Cdk2 activity assay. After immunoprecipitation, Cdk2 activity was determined as modified from the method of Kokontis et al. (20). Assays were performed at 37°C for 30 min in a final volume of 25 µl. The final substrate mixture per tube contained 20 mM ATP, 10 µg histone H1, 20 mM Tris·HCl buffer (pH 7.5), 4 mM MgCl2, 0.8 mM EGTA, and Cdk2 immunoprecipitates. The reaction was terminated by the addition of 50 µl SDS-PAGE sample buffer as described above. We removed a 20-µl aliquot of the solution to load onto SDS-PAGE and used anti-phospho-histone H1 as the primary antibody to immunoblot the samples as described above. Changes in the amounts of phospho-histone H1, after normalization to the immunoprecipitated Cdk2 protein, indicated alterations in Cdk2 activity.
Statistical analysis. Data are expressed as means ± SE unless otherwise noted. An unpaired Student's t-test was used to examine differences between the control and EGCG-treated groups. One-way ANOVA followed by the Student-Newman-Keuls multiple-range test was used to examine differences among multiple groups. Differences were considered significant at P < 0.05. All statistical analyses were performed using SigmaStat (Jandel Scientific; Palo Alto, CA).
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RESULTS |
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Differences of EGCG with other specific MEK1 inhibitors.
On the basis of IC50 values of EGCG (2050 µM), PD98059 (50 µM), and U0126 (10 µM) for inhibiting preadipocyte proliferation or reducing MEK1 activity, we tested whether the inhibitory effects of EGCG on preadipocyte growth and MEK1 activity differed from those of other specific inhibitors of the MEK1 protein (Fig. 6). Proliferation of preadipocytes was inhibited by either EGCG, PD98059, or U0126 alone. In addition, their growth was inhibited by a combination of EGCG with either PD98059 or U0126 (Fig. 6A). Total amounts of neither of the proteins, ERK-1 or ERK-2, changed after treatment with each inhibitor (Fig. 6B). Interestingly, EGCG tended to be additive with either PD98059 or U0126 in decreasing MEK1 activity, as indicated by the reduced amounts of phospho-ERK1/2 proteins.
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Overexpression of Cdk2+/+ with EGCG-inhibited growth of preadipocytes.
In a further demonstration of whether Cdk2 protein is required for the effect of EGCG on 3T3-L1 preadipocytes, we stably cloned preadipocytes that overexpressed Cdk2+/+ and examined whether overexpression of Cdk2+/+ could prevent EGCG-induced growth inhibition of preadipocytes after treatment with 20100 µM EGCG for 5 days (Fig. 11). As indicated by the increased cell number, Cdk2+/+-transfected preadipocytes grew more rapidly than vehicle-transfected cells during the 5-day incubation. The growth of the former cells, but not the latter cells, was not changed by EGCG (Fig. 11A). However, overexpression of dnCdk2 with a mutation of Asp145 in Cdk2 to Asn145 slowed down growth of preadipocytes after a 5-day incubation no matter the presence and absence of EGCG (data not shown). When examined at 24 and 48 h of EGCG treatment, EGCG's alteration of the four different phases of the cell cycle (Fig. 11B), EGCG's reduction of levels (Fig. 11C) and activity (Fig. 11D) of Cdk2 protein and the EGCG-altered binding of Cdk2 to p18, p21, and p27 (Fig. 11E) were still observed in vehicle-transfected preadipocytes but not in Cdk2+/+-transfected preadipocytes.
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DISCUSSION |
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The observed decrease in the number of preadipocytes by EGCG could be attributable to its inhibition of cell mitogenesis. This is supported by decreased BrdU incorporation, a measure of DNA replication, and by increased G0/G1 growth arrest of the cell cycle when preadipocytes were incubated with EGCG. However, the observed decreases in the cell number by high doses (100400 µM) of EGCG could also be explained by its induction of cell apoptosis. This is evident by the fact that such high doses of EGCG reduced the cell viability of preadipocytes by 15
30%, induced the appearance of DNA fragmentation (data not shown), and increased the activity of the caspase-3 protein (data not shown), an apoptotic enzyme. Taken together, green tea EGCG may act at different concentrations in regulating mitogenesis and apoptosis of 3T3-L1 preadipocytes. This contention is similar to the reported dose-dependent effect of EGCG on neuroblastoma cells (22, 2627, 42). However, the possibility still remains that EGCG acts at 10
400 µM to induce antimitogenesis and apoptosis of 3T3-L1 preadipocytes via oxidative stress as reported for hepatoma (42) and neuroblastoma cells (22, 26, 43).
Antimitogenic effect of EGCG on preadipocytes depends on ERK pathway. To our knowledge, the MAPK family is an essential part of the signal transduction machinery in signal transmissions from cell surface receptors and environmental stimulation, and it contains three major MAPK subfamilies: ERK, p38, and JNK (4, 31). They have been proposed to serve as signal elements in several types of cells through which EGCG may regulate cell growth (1, 3, 8, 2425, 35, 45) and found to modulate the mitogenic and adipogenic signalings of IGF-I in 3T3-L1 preadipocytes (5). We observed herein that acute (4 h) exposure to EGCG induced a decrease in phosphorylated ERK1/2 in 3T3-L1 preadipocytes but did not alter the total levels of MEK1, ERK-1, ERK-2, p38, phospho-p38, JNK, or phospho-JNK. This suggests that EGCG acts on a specific type of MAPK, especially in the ERK MAPK family. This contention is also partially supported by the fact that chronic (24 or 48 h) exposure to EGCG induced a decrease in the phosphorylated ERK1/2 of preadipocytes, although it did not alter total levels of MEK1 or ERK1/2 proteins. In addition, transient amplification of phospho-ERK1/2 content by transfecting MEK1 cDNA or its active mutant cDNA to 3T3-L1 preadipocytes prevented EGCG-induced decreases in their cell number. The total levels of MEK1 protein in vehicle-, MEK1-, or MEK1EE-transfected preadipocytes were not affected by any of the EGCG treatments. In contrast, overexpression of either MKK6EE, a constitutively active mutant of MKK6 to activate p38 MAPK kinase, or MEKK1 (data not shown), a MEKK1 construct favoring the activation of JNK MAPK kinase, did not prevent EGCG-induced decreases in the number of preadipocytes. Taken together, these findings demonstrate that a suppressive effect of EGCG on preadipocyte proliferation is likely mediated via ERK MAPK-dependent and p38 MAPK- and JNK MAPK-independent pathways and confirms that the ERK MAPK subfamily is important in preadipocyte proliferation, as reported by Boney et al. (5).
The ERK-dependent effect of EGCG observed in 3T3-L1 preadipocytes is also strengthened by our findings that two specific inhibitors of Erk MAPK, PD98059 (12) and U0126 (13), alone inhibit cell growth and MEK1 activity, as shown with reduced phospho-ERK1/2 (5, 40), and that either of them used to treat preadipocytes within the IC50 range speeds up EGCG-induced reduction in the amounts of phosphorylated ERK1/2 and, to a lesser extent, in the number of cells. It appears that EGCG works differently from PD98059 and U0126 in reducing levels of phosphorylated ERK1/2 proteins. Whereas PD98059 prevents MEK1 activation by Raf (12), U0126 directly protects ERK from being phosphorylated by MEK1 (13). In cell-free systems, the inhibition of EGCG on MAPK activity is competitive with the myelin basic protein substrate and is noncompetitive with ATP (47). In contrast, the activities of certain protein phosphatases are stimulated by 15% by 1050 µM EGCG (47). In cultured cells, phosphorylation of ERK1/2 can be regulated by a variety of factors, including growth factors, G protein-coupled receptors, tyrosine kinase receptors, and Raf and MEK1 kinases (4, 31). We measured the amounts of phospho-ERK1/2 protein after 3T3-L1 preadipocytes were treated with either 1 nM IGF-I or 10 nM IGF-II in the presence and absence of 50 µM EGCG. EGCG did significantly prevent the increase in phosphorylated ERK1/2 by either IGF-I or IGF-II (Fig. 7) and concomitantly reduced IGF-I receptor activity, as indicated by a decrease in the phosphotyrosine-IGF-I receptor and an association of the IGF-II receptor with Gi
-2 protein (unpublished observations). Alternatively, it is possible that EGCG induces a decrease in phosphorylated ERK1/2 from preadipocytes via reducing the phosphorylation of MEK1 and the association of Raf with MEK1 as reported for H-Ras-transformed cells (9). However, confirming this requires more thorough studies.
Antimitogenic effect of EGCG on preadipocytes depends on Cdk2 pathway.
Cdks are key regulators of the cell cycle in vertebrate cells. They are related to the effects of EGCG in modulating cell mitogenesis and growth arrest of most cancer cells (1, 2325) and can serve as the main controller of mitogenesis and mitotic clonal expansion of preadipocytes (40). We observed herein that doses of 20100 µM EGCG decreased Cdk2 activity at 4, 24, and 48 h and reduced its protein levels at 48 h but not at 4 and 24 h. Also, EGCG dose dependently induced G1 growth arrest at 24 and 48 h after treatment. In addition, increased Cdk2 activity via the transfection of Cdk2+/+ cDNA to preadipocytes prevented EGCG-induced decreases in their Cdk2 activity and cell number and EGCG-induced increases of G1 arrest, whereas decreased Cdk2 activity via the transfection of dnCdk2 cDNA to preadipocytes slowed down the 5-day growth of preadipocytes and increased their G1 growth arrest (data not shown). These observations suggest that the effect of EGCG of inducing preadipocyte antimitogenesis and growth arrest is dependent on a Cdk2 pathway and requires inactivation of the Cdk2 protein. Because cyclin D1 is a G1 cyclin associated with Cdk4 and Cdk6 proteins, which favor cell cycle arrest at the G1 checkpoint (4), decreased cyclin D1 protein expression by EGCG for 24
48 h suggests the possibility of Cdk4- and Cdk6-related effects of EGCG on preadipocyte growth arrest. However, we observed herein that 20 µM EGCG for 48 h did not affect the total levels of Cdc2 protein and that 100 µM EGCG for 48 h reduced the levels of Cdc2 protein less than that of Cdk2. Accordingly, EGCG appears to act on a specific type of Cdks in preadipocytes, but further studies are required to illustrate this contention. Because Cdc2 takes over as the predominant Cdk activity in the early G2/M transition of the cell cycle (4), the observed decrease in Cdc2 protein expression by 100 µM EGCG suggests the possibility of the action of EGCG on the G2/M phase of preadipocytes.
Regulation of Cdk2 activity of in vivo cultured cells occurs at multiple levels, involving the synthesis of subunits and the association of inhibitory proteins such as p21 and p27 (29, 37). On these bases, a decrease in the Cdk2 activity of 3T3-L1 preadipocytes induced by 4 h of EGCG treatment may have resulted from the observed increase in the association of Cdk2 with p21 and p27 by EGCG. However, the short-term effect of EGCG in reducing Cdk2 activity should be unrelated to the availabilities of p21, p27, and Cdk2 because EGCG did not alter their protein levels in this period. However, increased levels of p21 and p27, but not of p18 or Cdk2, observed with the 24-h EGCG treatment may be responsible for the increased association of Cdk2 with p21 and p27, but not p18, by EGCG, thereby leading to low Cdk2 activity and a subsequent rise in the percentage of G0/G1 arrest. In addition, the decreased levels of Cdk2 protein and increased levels of p21 and p27, but not p18, observed with the 48-h EGCG treatment may explain the EGCG-induced increase in the association of Cdk2 with p21 and p27, but not p18, thereby resulting in decreases in Cdk2 activity and increases in the percentage of G0/G1 arrest. These results suggest that EGCG may act on a particular type of preadipocyte in the CKI family to reduce Cdk2 activity. It would be of interest if other types of CKIs, such as p53 (4), were also found to be involved in the action of EGCG on preadipocyte growth arrest as reported in cervical cells (35). As the sequestration of p21 and p27 is mediated via the induction of cyclin D1 and cyclin D2 protein synthesis rates (32), the 24- and 48-h decreases in cyclin D1 protein expression of preadipocytes by EGCG may also explain the EGCG-induced increase in the association of Cdk2 with p21 and p27. Further studies to determine whether EGCG affects the association of cyclin D1 with these CKIs would help clarify this notion.
The Cdk2 activity of in vivo cultured cells is also regulated at phosphorylation-dephosphorylation levels (29, 37). In rat aortic smooth muscle cells, increased Cdk2 activity by endothelin is mediated via either the activation of Erk and Cdc25A (a phosphatase) or the inactivation of WEE1 (an inhibitory kinase) but is prevented by the inactivation of ERK and the activation of WEE1 (7). Whereas Cdk2 activity is inactivated through phosphorylation at Tyr15 by WEE1, it is restored through dephosphorylation at Tyr15 by Cdc25A (7). Activities of WEE1 and Cdc25A are, respectively, inactivated and activated through phosphorylation by the activation of ERK, and vice versa (7). Accordingly, the observed EGCG reduction in Cdk2 activity in 3T3-L1 preadipocytes may be mediated by the inactivation of ERK. This explanation is supported by observations that decreased amounts of phospho-ERK1/2 proteins by EGCG occur in parallel to the reduced activity, but not protein levels, of Cdk2 by EGCG over a 48-h period. Because Cdk2 activity in in vivo cells is also regulated by the association of stimulatory proteins, such as cyclin E (4, 29), more investigations are needed to clarify whether the production of cyclin E protein and its association with Cdk2 are altered by EGCG and thereafter cause decreases in Cdk2 activity. However, an interesting observation not shown here is that the cyclin D1 protein is able to associate with Cdk2, and such an association can be altered with EGCG treatment (unpublished observations). This suggests that EGCG may result in altered cyclin-Cdk2 protein complexes in 3T3-L1 preadipocytes as reported in mouse liver cells (14).
Catechin-specific effect. GTCs have numerous biological activities that can possibly provide various health benefits (1, 2425, 28, 45). In most cases, but not all, gallated catechins, especially EGCG, are more active than other catechins. This contention is supported by our findings in 3T3-L1 preadipocytes that at the same dose and duration of treatment, EGCG was generally more effective than EC, ECG, and EGC in changing the number of cells, the amount of incorporated BrdU, percentages of the four phases of the cell cycle, activities of MEK1 and Cdk2, and levels of Cdk2, cyclin D1, and CKIs. The observed catechin-specific effects of green tea suggest that EGCG may act differently from EC, EGC, and ECG in regulating preadipocyte growth. According to the nature of the unique structures of these catechins tested (Fig. 1) (24, 33), EGCG contains the largest number of hydroxyl groups on its three aromatic rings among the tea catechins, and these hydroxyl groups may be important for hydrogen bonding. Also, EGCG has both gallyl and galloyl groups, which have some conformational flexibility, that may also be important for interactions with other molecules. Further exploration of the chemical basis of the antimitogenic activity on preadipocytes by EGCG is needed to understand differences in the mechanism of EGCG's action compared with those of EC, EGC, and ECG on these processes.
Growth phase-dependent effect. The effects of green tea EGCG in reducing the number of 3T3-L1 preadipocytes are dependent on the latent, proliferative, and plateau phases of preadipocyte growth. This is evidenced by our observations that the IC50 values of EGCG for reducing the number of day 1-6 cells differed during the 72-h treatment. This suggests that latent, log-phase, and confluent preadipocytes have different sensitivities to this tea catechin. This is similar to the studied growth phase-dependent effect of IGF-I on the activation of MAPK protein phosphorylation in 3T3-L1 preadipocytes, particularly proliferating cells (5). A possible explanation is that various endogenous properties of the three phases of cells occur, thereby leading to EGCG's signal proceeding in different phases of cells. The findings that EGCG decreased the amounts of phospho-ERKs in day 3 cells, but not in day 1 or 5 cells, and that the basal amounts of phospho-ERKs in day 3 cells were the largest among these stages of cells (Fig. 4C) actually support this contention. Determining whether the number and affinity of the EGCG receptor (39) and its associated signal proteins exhibit different presences in the latent, proliferative, and confluent phases of preadipocytes should help elucidate their distinct sensitivities to EGCG.
Differential effects of EGCG on 3T3 and 3T3-L1 cells. Mechanistic studies of green tea EGCG have reported its cell type-dependent manner (1, 24, 25, 45). This may be explained by the fact that the sensitivity of different normal, transformed, and cancer cell lines to green tea EGCG varies (24), although such differences may be due to the cell culture techniques and assay methods employed. In this study, we used the same experimental culture conditions and assay methods to look at whether a cell type-specific effect of EGCG occurs. We observed that the IC50 value of EGCG for reducing the cell number was lower in 3T3-L1 cells than in 3T3 fibroblast and human KB oral cancer cells (data not shown), supporting the existence of different sensitivities of these cell lines to EGCG, as similarly reviewed by Liao et al. (24). Such differences between 3T3 fibroblasts and 3T3-L1 preadipocytes induced by EGCG may be explained by observations that the decrease in phosphorylated ERK1/2 of the former cells in response to 48-h EGCG treatment was much less than that in the latter cells (Fig. 9) and that levels of Cdc2 and Cdk2 proteins were, respectively, decreased and increased by EGCG in the former cells, whereas Cdc2 levels in the latter cells were decreased much less by EGCG than were Cdk2 levels (Fig. 10). These observations suggest that EGCG may work differently in the two cell lines in modulating mitogenesis via altering different phases of their cell cycles and/or the extent of apoptosis and that the Cdk2 transducer may play different roles with EGCG in the two cell lines. It should be noted that in certain cells, Cdks are activated during apoptosis (49), although it is unknown whether the increase in Cdk2 protein levels in 3T3 fibroblasts by EGCG is related to the mechanism through which EGCG induces a decrease in the cell number. Other different characteristics between 3T3 fibroblasts and 3T3-L1 preadipocytes have been reported, although the latter cells are subcloned from the former cells (15). It would be worthwhile to explore whether any of them are responsible for the differential effects of EGCG on 3T3 and 3T3-L1 cells.
We conclude that the antimitogenic effect of EGCG on 3T3-L1 preadipocytes is dependent on the Erk MAPK and Cdk2 pathways and is likely mediated through decreases in their activities (Fig. 14). While shown to be mediated by ERK MAPK, signaling is demonstrated to be largely independent of the p38 MAPK and JNK MAPK pathways. Decreases in Cdk2 activity by EGCG may be due to its effect on this particular member of the CKI family. In general, EGCG is more effective than other structurally related GTCs in changing mitogenic signals. The signaling of EGCG in 3T3-L1 preadipocytes differs from that in 3T3 fibroblasts. Changes in the endogenous signals of preadipocytes induced by EGCG may help increase our understanding of the modulatory mechanism of green tea EGCG on body weight and fat cells (1819, 24). Future studies on discovering the EGCG receptor in fat cells and on characterizing its oxidative stress are needed to elucidate the mechanisms of how EGCG signals reduce the activities of MEK1 and Cdk2 proteins.
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ACKNOWLEDGMENTS |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* P.-F. Hung and B.-T. Wu contributed equally to this work.
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REFERENCES |
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2. Allison DB, Fontaine KR, Manson JE, Stevens J, and Vanitallie TB. Annual deaths attributable to obesity in the United States. JAMA 282: 15301538, 1999.
3. Balasubramanian S, Efimova T, and Eckert RL. Green tea polyphenol stimulates a Ras, MEKK1, MEK3, and p38 cascade to increase activator protein 1 factor-dependent involucrin gene expression in normal human keratinocytes. J Biol Chem 277: 18281836, 2002.
4. Becker WM, Kleinsmith LJ, and Hardin J. The World of the Cell (5th ed.). San Francisco, CA: Pearson Education, 2003.
5. Boney CM, Smith RM, and Gruppuso PA. Modulation of insulin-like growth factor I mitogenic signaling in 3T3-L1 preadipocyte differentiation. Endocrinology 139: 16381644, 1998.
6. Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248254, 1976.[CrossRef][ISI][Medline]
7. Chen S and Gardner DG. Suppression of WEE1 and stimulation of CDC25A correlates with endothelin-dependent proliferation of rat aortic smooth muscle cells. J Biol Chem 279: 1375513763, 2004.
8. Chung JY, Huang C, Meng X, Dong Z, and Yang CS. Inhibition of activator protein 1 activity and cell growth by purified green tea and black tea polyphenols in H-ras-transformed cells: structure-activity relationship and mechanisms involved. Cancer Res 59: 46104617, 1999.
9. Chung JY, Park JO, Phyu H, Dong Z, and Yang CS. Mechanisms of inhibition of the Ras-MAP kinase signaling pathway in 30.7b Ras 12 cells by tea polyphenols ()-epigallocatechin-3-gallate and theaflavin-3,3'-digallate. FASEB J 15: 20222024, 2001.
10. Dulloo AG, Duret C, Rohrer D, Girardier L, Mensi N, Fathi M, Chantre P, and Vandermander J. Efficacy of a green tea extract rich in catechin polyphenols and caffeine in increasing 24-h energy expenditure and fat oxidation in humans. Am J Clin Nutr 70: 10401045, 1999.
11. Dulloo AG, Seydoux J, Girardier L, Chantre P, and Vandermander J. Green tea and thermogenesis: interaction between catechin-polyphenols, caffeine and sympathetic activity. Int J Obes Relat Metab Disord 24: 252258, 2000.[CrossRef][Medline]
12. Engelman JA, Lisanti MP, and Scherer PE. Specific inhibitors of p38 mitogen-activated protein kinase block 3T3-L1 adipogenesis. J Biol Chem 273: 3211132120, 1998.
13. Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, and Trzaskos JM. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem 273: 1862318632, 1998.
14. Gonzales AJ, Goldsworth TL, and Fox TR. Chemical transformation of mouse liver cells results in altered cyclin D-CDK protein complexes. Carcinogenesis 19: 10931102, 1998.[Abstract]
15. Green H and Kehinde O. Sublines of mouse 3T3 cells that accumulate lipid. Cell 1: 113116, 1974.[CrossRef][ISI]
16. Haqqi TM, Anthony DD, Gupta S, Ahmad N, Lee MS, Kumar GK, and Mukhtar H. Prevention of collagen-induced arthritis in mice by a polyphenolic fraction from green tea. Proc Natl Acad Sci USA 96: 45244529, 1999.
17. Heuvel SVD and Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 262: 20502053, 1993.[ISI][Medline]
18. Kao YH, Hiipakka RA, and Liao S. Modulation of endocrine systems and food intake by green tea epigallocatechin gallate. Endocrinology 141: 980987, 2000.
19. Kao YH, Hippakka RA, and Liao S. Modulation of obesity by a green tea catechin. Am J Clin Nutr 72: 12321241, 2000.
20. Kokontis JM, Hay N, and Liao S. Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol Endocrinol 12: 941953, 1998.
21. Kopelman PG. Obesity as a medical problem. Nature 404: 635643, 2000.[ISI][Medline]
22. Levites Y, Amit T, Youdim MBH, and Mandel S. Involvement of protein kinase C activation and cell survival/cell cycle genes in green tea polyphenol ()-epigallocatechin 3-gallate neuroprotective action. J Biol Chem 277: 3057430580, 2002.
23. Liang YC, Lin-Shiau SY, Chen CF, and Lin JK. Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by ()-epigallocatechin-3-gallate. J Cell Biochem 75: 112, 1999.[ISI][Medline]
24. Liao S, Kao YH, and Hiipakka RA. Green tea: biochemical and biological basis for health benefits. Vitam Horm 62: 194, 2001.[CrossRef][ISI][Medline]
25. Lin JK, Liang YC, and Lin-Shiau SY. Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade. Biochem Pharmacol 58: 911915, 1999.[CrossRef][ISI][Medline]
26. Mendel S, Weinreb O, Amit T, and Youdim MB. Cell signaling pathways in the neuroprotective actions of the green tea polyphenol ()-epigallocatechin-3-gallate: implications for neurodegenerative diseases. J Neurochem 88: 15551569, 2004.[CrossRef][ISI][Medline]
27. Mendel S and Youdim MB. Catechin polyphenols: neurodegeneration and neuroprotection in neurodegenerative diseases. Free Radic Biol Med 37: 304317, 2004.[CrossRef][ISI][Medline]
28. Mitscher LA, Jung M, Shankel D, Dou JH, Steele L, and Pillai SP. Chemoprotection: a review of the potential therapeutic antioxidant properties of green tea (Camellia sinensis) and certain of its constituents. Med Res Rev 17: 327365, 1997.[CrossRef][ISI][Medline]
29. Morgan DO. Principles of CDK regulation. Nature 374: 131134, 1995.[CrossRef][ISI][Medline]
30. Niimi T, Kumagai C, Okano M, and Kitagawa Y. Differentiation-dependent expression of laminin-8 (alpha 4 beta 1 gamma 1) mRNAs in mouse 3T3-L1 adipocytes. Matrix Biol 16: 223230, 1997.[CrossRef][ISI][Medline]
31. Pearson G, Robinson F, Gibson TB, Xu BE, Karandikar M, Berman K, and Cobb MH. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocr Rev 22: 153183, 2001.
32. Perez-Roger I, Kim SH, Griffiths B, Sewing A, and Land H. Cyclins D1 and D2 mediate Myc-induced proliferation via sequestration of p27kip1 and p21cip1. EMBO J 18: 53105320, 1999.
33. Roberts EAH. The chemistry of tea fermentation. J Sci Food Agric 3: 193198, 1952.[ISI]
34. Rusznyak S, and Szent-Gyorgyi A. Vitamin P: flavanols as vitamins. Nature 138: 27, 1936.
35. Sah JF, Balasubramanians S, Eckert RL, and Rorke EA. Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. J Biol Chem 279: 1275512762, 2004.
36. Sambrook J and Russell DW. Molecular Cloning: a Laboratory Manual (3th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Lab Press, 2001.
37. Sherr CJ and Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev 9: 11491163, 1995.[CrossRef][ISI][Medline]
38. Song EK, Hur H, and Han MK. Epigallocatechin gallate prevents autoimmune diabetes induced by multiple low doses of streptozotocin in mice. Arch Pharmacol Res (Seoul) 26: 559563, 2003.
39. Tachibana H, Koga K, Fujimura Y, and Yamada K. A receptor for green tea polyphenol EGCG. Nature 11: 380381, 2004.
40. Tang QQ, Otto TC, and Lane MD. Mitotic clonal expansion: a synchronous process required for adipogenesis. Proc Natl Acad Sci USA 100: 4449, 2003.
41. Vollenweider P, Clodi M, Martin SS, Imamura T, Kavanaugh WM, and Olefsky JM. An SH2 domain-containing 5' inositolphosphatase inhibits insulin-induced GLUT4 translocation and growth factor-induced actin filament rearrangement. Mol Cell Biol 19: 10811091, 1999. 13.
42. Waltner-Law ME, Wang XL, Law BK, Hall RK, Nawano M, and Granner DK. Epigallocatechin gallate, a constituent of green tea, represses hepatic glucose production. J Biol Chem 277: 3493334940, 2002.
43. Weinreb O, Mandel S, and Youdim MBH. cDNA gene expression profile homology of antioxidants and their antiapoptotic and proapoptotic activities in human neuroblastoma cells. FASEB J 17: 935937, 2003.
44. Wolf AM and Colditz GA. Current estimates of the economic cost of obesity in the United States. Obesity Res 6: 97106, 1998.[Abstract]
45. Yang CS and Wang ZY. Tea and cancer. J Natl Cancer Inst 85:10381049, 1993.[Abstract]
46. Yang LC, Yang SH, Tai KW, Chou MY, and Yang JJ. MEK inhibition enhances bleomycin A5-induced apoptosis in an oral cancer cell line: signaling mechanisms and therapeutic opportunities. J Oral Pathol Med 33: 3745, 2004.[CrossRef][ISI][Medline]
47. Yasokawa M, Goto N and Hashimoto. Inhibition of mitogen-activated protein kinase by ()-epigallocatechin gallate in vitro. J Biochem Mol Biol Biophys 3: 177181, 1999.
48. Yeh CW, Chen WJ, Chiang CT, Lin-Shiau SY, and Lin JK. Suppression of fatty acid synthase in MCF-7 breast cancer cells by tea and tea polyphenols: a possible mechanism for their hypolipidemic effects. Pharmacogenetics 3: 267276, 2003.
49. Zhou BB, Li H, Yuan J, and Kirschner MW. Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurket cells. Proc Natl Acad Sci USA 95: 67856790, 1998.