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Originally published In Press as doi:10.1074/jbc.M701549200 on July 3, 2007

J. Biol. Chem., Vol. 282, Issue 35, 25445-25452, August 31, 2007
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Role of MAPK Phosphatase-1 in the Induction of Monocyte Chemoattractant Protein-1 during the Course of Adipocyte Hypertrophy*Formula

Ayaka Ito{ddagger}§1, Takayoshi Suganami{ddagger}, Yoshihiro Miyamoto, Yasunao Yoshimasa, Motohiro Takeya||, Yasutomi Kamei{ddagger}, and Yoshihiro Ogawa{ddagger}§2

From the {ddagger}Department of Molecular Medicine and Metabolism and the §Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstitution of Tooth and Bone, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan, the Department of Medicine, Division of Atherosclerosis and Diabetes, National Cardiovascular Center Hospital, 5-7-1 Fujishirodai, Suita, Osaka 560-0005, Japan, and the ||Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

Received for publication, February 21, 2007 , and in revised form, June 14, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Monocyte chemoattractant protein-1 (MCP-1), an important chemokine whose expression is increased during the course of obesity, plays a role in macrophage infiltration into obese adipose tissue. This study was designed to elucidate the role of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) in the induction of MCP-1 during the course of adipocyte hypertrophy. We examined the time course of MKP-1 and MCP-1 mRNA expression and extracellular signal-regulated kinase (ERK) phosphorylation in the adipose tissue from mice rendered mildly obese by a short term high fat diet. We also studied the role of MKP-1 in the induction of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. MCP-1 mRNA expression was increased, followed by ERK activation and down-regulation of MKP-1, an inducible dual specificity phosphatase to inactivate ERK, in the adipose tissue at the early stage of obesity induced by a short term high fat diet, when macrophages are not infiltrated. Down-regulation of MKP-1 preceded ERK activation and increased production of MCP-1 in 3T3-L1 adipocytes in vitro during the course of adipocyte hypertrophy. Adenovirus-mediated restoration of MKP-1 in hypertrophied 3T3-L1 adipocytes reduced the otherwise increased ERK phosphorylation, thereby leading to the significant reduction of MCP-1 mRNA expression. This study provides evidence that the down-regulation of MKP-1 is critical for increased production of MCP-1 during the course of adipocyte hypertrophy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence has accumulated suggesting that obesity is a state of chronic, low grade inflammation; it may represent a potential mechanism whereby obesity leads to the metabolic derangements (1–3). Previous studies demonstrated that the adipose tissue is markedly infiltrated by macrophages in several models of rodent obesities and human obese subjects (4, 5). Using an in vitro co-culture system composed of adipocytes and macrophages, we have provided evidence that a paracrine loop involving saturated free fatty acids (FFAs) and tumor necrosis factor-{alpha} (TNF{alpha}) derived from adipocytes and macrophages, respectively, establishes a vicious cycle that aggravates the inflammatory changes; i.e. marked up-regulation of pro-inflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1)3 and TNF{alpha} and down-regulation of anti-inflammatory adiponectin (6, 7). These findings have led us to speculate that macrophages, when infiltrated, may participate in the inflammatory pathways that are activated in obese adipose tissue.

A previous study with bone marrow transplantation demonstrated that most macrophages in the adipose tissue are derived from the bone marrow (4). In this regard, adipose tissue expression of MCP-1, a major chemokine implicated in the control of monocyte recruitment to the site of inflammation, is increased during the progression of obesity (8, 9) and is roughly correlated with macrophage markers in the adipose tissue (5, 10). These findings suggest that increased production of MCP-1 may be an initial event at the early stage of obesity so as to accumulate macrophages in the adipose tissue. Recently, Kanda et al. and Kamei et al. (11, 12) have independently reported that MCP-1 plays a role in the recruitment of macrophages into obese adipose tissue. It is, therefore, important to know the molecular mechanism for increased production of MCP-1 at the early stage of obesity. Recent studies have demonstrated that multiple intracellular signaling pathways are activated in adipocytes during the course of adipocyte hypertrophy in vitro and in obese adipose tissue in vivo. However, how the inflammatory pathways are activated in adipocytes at the early stage of obesity is still poorly understood.

Mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun NH2-terminal kinase (JNK) are activated in a variety of cellular processes (13). Once activated by the upstream kinases, e.g. MAPK/ERK kinase (MEK), MAPKs are rapidly inactivated by a family of protein phosphatases such as MAPK phosphatase-1 (MKP-1), an inducible dual specificity phosphatase (14, 15). Sakaue et al. showed previously that MKP-1 plays an essential role in 3T3-L1 adipocyte differentiation through ERK down-regulation (16). On the other hand, Bost et al. (17) reported that mice lacking ERK1 (ERK1-/- mice) are protected from high fat diet-induced obesity and insulin resistance. These findings, taken together, suggest that the MAPK pathways play an important role in the adipocyte proliferation and differentiation in vitro and in vivo (18).

Here we show that MCP-1 mRNA expression is increased, which is followed by ERK activation and MKP-1 down-regulation in the adipose tissue from mice rendered mildly obese by a short term high fat diet, when macrophages are not infiltrated. We also demonstrate that ERK activation through MKP-1 down-regulation is involved in increased production of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. This study provides evidence that MKP-1 down-regulation is critical for the inflammatory changes in hypertrophied adipocytes at the early stage of obesity, thereby suggesting that MKP-1 activation may offer a novel therapeutic strategy to treat or reduce the inflammatory changes in adipocytes during the progression of obesity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Rabbit polyclonal antibodies against ERK, phospho-ERK, p38 MAPK, phospho-p38 MAPK, MEK1/2, phospho-MEK1/2, MEK inhibitors PD98059 and U0126, and a p38MAPK inhibitor SB203580 were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against JNK, phospho-JNK, and MKP-1 and a mouse monoclonal antibody against Lamin A/C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were purchased from Sigma or Nacalai Tesque (Kyoto, Japan).

Animal Studies—Four-week-old male C57BL/6J mice were purchased from Charles River Laboratories Japan (Tokyo, Japan). The animals were housed in a temperature-, humidity-, and light-controlled room (12-h light and 12-h dark cycle) and allowed free access to water and chow. Five-week-old mice were fed either the standard chow (Oriental MF, 362 kcal/100 g, 5.4% energy as fat; Oriental Yeast, Tokyo, Japan) or high fat diet (D12492 [GenBank] , 524 kcal/100 g, 60% energy as fat; Research Diets, New Brunswick, NJ) for 15 weeks. They were fasted for 1 h (12:00–13:00) and sacrificed to harvest the epididymal adipose tissue before (n = 10) and 2 weeks (n = 10), 4 weeks (n = 12), 6 weeks (n = 11), 8 weeks (n = 6), and 15 weeks (n = 4) after the experiments. All animal experiments were conducted according to the guidelines of Tokyo Medical and Dental University Committee on Animal Research (No. 0060026).

Histological Analysis—The epididymal WAT was fixed with neutral-buffered formalin and embedded in paraffin. Sections were stained with hematoxylin and eosin and studied under x200 magnification to measure the adipocyte area using Win Roof software (Mitani Corporation, Tokyo, Japan) (19). Immunohistochemical study was carried out using 5-µm thick paraffin-embedded sections for macrophage marker F4/80 as previously described (20, 21).

Cell Culture—3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were maintained as described (6, 7). Differentiation of 3T3-L1 preadipocytes to adipocytes was described elsewhere (6, 7). Cells at day 8 and day 21 after the induction of differentiation were used as non-hypertrophied and hypertrophied adipocytes, respectively (6). Accumulation of triglyceride in adipocytes was detected by oil red O staining (19).

Measurement of Triglyceride Content—Triglyceride content in 3T3-L1 adipocytes was measured as previously reported (22). In brief, 3T3-L1 adipocytes in 35-mm dish were harvested, and cellular lipid was extracted by chloroform-methanol (2:1). After evaporation, precipitation was dissolved in isopropyl alcohol. Triglyceride content was measured using a colorimetric assay kit (triglyceride E-test Wako, Wako Pure Chemicals, Osaka, Japan) according to the manufacturer's instructions.

Quantitative Real-time PCR—Quantitative real-time PCR was performed with an ABI Prism 7000 Sequence Detection System using PCR Master Mix reagent kit (Applied Biosystems, Foster City, CA) as described (6, 19). Primers used were described in supplemental Table S1. Levels of mRNAs were normalized to those of housekeeping gene 36B4 mRNA.

ELISA—The MCP-1, IL-6, and adiponectin levels in culture supernatants were determined by the commercially available ELISA kits (MCP-1 and IL-6, R&D systems, Minneapolis, MN; adiponectin, Otsuka Pharmaceutical, Tokyo, Japan).

Immunoblot Assay—Nuclear and cytosolic extracts were prepared by using the Nuclear/Cytosol fractionation kit (Bio-Vision, Mountain View, CA). Separation of nuclear and cytosolic proteins was confirmed by immunoblots with {alpha}-tubulin and lamin A/C antibodies, respectively. Whole cell lysates were prepared using buffer containing 50 mmol/liter HEPES (pH7.5), 150 mmol/liter NaCl, 100 mmol/liter sodium fluoride, 1 mmol/liter EGTA, 1 mmol/liter EDTA, 1% Triton X-100, 2 mmol/liter sodium vanadate, 2 mmol/liter phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Immunoblot assay was performed as described (6). Samples (10–20 µg protein/lane) were separated by 12.5% SDS-PAGE and electrophoretically transferred onto polyvinylidene difluoride filter membrane (PolyScreen; PerkinElmer, Wellesley, MA). After membranes were incubated with primary antibodies for 1 h at room temperature, immunoblots were developed with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-Sciences, Piscataway, NJ) and a chemiluminescence kit (GE Healthcare Bio-Sciences). The signals were detected with LAS3000 (Fuji Photo Film, Tokyo, Japan).

Generation of 3T3-L1 Adipocytes Stably Expressing Coxsackie-Adenovirus Receptor (CAR)—A mouse CAR-expressing plasmid pcDNA3-CAR (23) was kindly provided by Dr. Hiroyuki Mizuguchi (National Institute of Biomedical Innovation, Osaka, Japan). The CAR retroviral expression vector (pMRX-CAR) was constructed by ligating the full-length CAR cDNA into the EcoR1 site of pMRX vector (24) and transfected into Plat-E packaging cells (25) using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. Viral supernatants were harvested from 24 to 48 h after transfection and applied to 3T3-L1 adipocytes in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 5 µg/ml of polybrene (Nacalai Tesque) in a final volume of 5 ml. The stable CAR-expressing 3T3-L1 adipocytes (CAR-3T3-L1 adipocytes) were obtained by 2 µg/ml of puromycin (Nacalai Tesque) selection.

Adenovirus-mediated Expression of MKP-1—The adenoviral vector expressing mouse MKP-1 (Ad-MKP-1) (26), kindly provided by Dr. Jeffery D. Molkentin (University of Cincinnati, Cincinnati, OH), was prepared using HEK293 cells and purified by VIRAPREP adenovirus purification kit (Virapur, LLC, San Diego, CA) as previously described (27). The GFP adenovirus (Ad-GFP; Clontech Laboratories, Palo Alto, CA) was used as a control. The CAR-3T3-L1 adipocytes at day 5 and day 18 after the induction of differentiation were transfected with Ad-MKP-1, incubated for 3 days, and harvested to be used for quantitative real-time PCR and immunoblot assay.

Statistical Analysis—Data are shown as means ± S.E. Statistical analysis was performed using the Student's t test and analysis of variance followed by Scheffe's test. p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
MCP-1 mRNA Expression in the Adipose Tissue from Mice with Diet-induced Obesity—Body weight was increased significantly in mice fed high fat diet for 2 weeks relative to those fed standard diet (p < 0.01) (Fig. 1A). The mice fed high fat diet weighed ~20% more than those fed standard diet for 15 weeks (29.7 ± 0.3 g versus 34.8 ± 1.9 g, p < 0.05). The weight of epididymal white adipose tissue (WAT) was significantly increased in mice fed high-fat diet for 2 weeks relative to those fed standard diet (0.26 ± 0.01 g versus 0.49 ± 0.04 g, p < 0.01). Histological examination revealed appreciable increase in adipocyte cell size in mice fed a high fat diet during the initial 2 weeks, which reached up to ~4-fold larger than that in mice fed standard diet after 15 weeks (Fig. 1B). There were no appreciable infiltration of macrophages in the adipose tissue up to 8 weeks after the experiment, after which interstitial cells stained with F4/80, a marker of activated macrophages, appeared in mice fed high fat diet (Fig. 1C). Correspondingly, F4/80 mRNA expression was also increased in the epididymal WAT in mice fed high fat diet for 15 weeks relative to those fed standard diet (Fig. 1D, left). In mice fed high fat diet, MCP-1 mRNA expression was increased as early as 4 weeks and gradually increased up to 15 weeks after the experiment (Fig. 1D, right). These observations indicate that MCP-1 mRNA expression is increased prior to macrophage infiltration at the early stage of obesity.


Figure 1
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  		FIGURE 1.
Time course of adipocyte hypertrophy and macrophage infiltration in mice with diet-induced obesity. Five-week-old male C57BL/6J mice were fed either SD or HFD for 15 weeks. A, time course of body weight. Open circle, SD; closed circle, HFD. B, time course of adipocyte area. C, macrophage marker F4/80 immunostaining of the epididymal WAT in diet-induced obese mice. Original magnification, x200. Scale bars, 100 µm. D, time courses of F4/80 and MCP-1 mRNA expression. Data in B and D are expressed as the ratio of changes in mice fed HFD to those in mice fed SD. *, p < 0.05; **, p < 0.01 versus SD, n = 4–12 at each time point.

 
Dysregulation of Adipocytokine Production during the Course of Adipocyte Hypertrophy—To explore the molecular mechanisms underlying adipocyte hypertrophy, we cultured 3T3-L1 adipocytes up to 21 days after the induction of differentiation; they exhibited a gradual increase in lipid accumulation from day 8 to day 21 during the course of adipocyte hypertrophy as revealed by oil-red O staining (Fig. 2A) and triglyceride content (Fig. 2B). In this study, insulin-induced glucose uptake was preserved up to day 21 (supplemental Fig. S1).

Quantitative real-time PCR analysis revealed that MCP-1 mRNA expression was significantly increased up to day 21, ~6-fold higher than that in 3T3-L1 adipocytes (day 8) (p < 0.01), in parallel with increased cell size and lipid accumulation (Fig. 2C). Expression of IL-6 mRNA was also increased during the course of adipocyte hypertrophy. The IL-6 mRNA levels in 3T3-L1 adipocytes (day 21) were ~5-fold higher than those in 3T3-L1 adipocytes (day 8) (p < 0.01). By contrast, adiponectin mRNA expression showed significant reduction (up to 30%) during the course of adipocyte hypertrophy (p < 0.01). The MCP-1, IL-6, and adiponectin concentrations in the culture media were roughly parallel to their respective mRNA levels (Fig. 2D). The expression patterns of adipocytokines in hypertrophied 3T3-L1 adipocytes (day 21) were similar to those found in obese adipose tissue. We also confirmed that mRNA expression patterns of adipogenesis-related markers such as peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), adipocyte fatty acid-binding protein (aP2), fatty-acid transport protein 1 (FATP1), and CCAAT/enhancer-binding protein {alpha} (C/EBP{alpha}) in hypertrophied 3T3-L1 adipocytes were consistent with those in obese adipose tissue (supplemental Fig. S2). In this study, we used 3T3-L1 adipocytes cultured for 8 and 21 days after differentiation as non-hypertrophied (day 8) and hypertrophied (day 21) adipocytes, respectively.


Figure 2
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  		FIGURE 2.
Changes in adipocytokine expression during the course of adipocyte hypertrophy in vitro. A, morphological changes of 3T3-L1 adipocytes during the course of adipocyte hypertrophy (day 8-day 21) as revealed by oil-red O staining. Original magnification, x200. Scale bars, 100 µm. B, triglyceride accumulation in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. C and D, changes in adipocytokine mRNA expression (C) and secretion (D) in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. **, p < 0.01 versus day 8. n = 4.

 
Activation of MAPK Pathways during the Course of Adipocyte Hypertrophy—To explore the role of MAPK activation in the dysregulation of MCP-1 production during the course of adipocyte hypertrophy, we examined phosphorylation of ERK, p38 MAPK, and JNK in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. Immunoblot analysis revealed that phosphorylation of ERK and p38 MAPK is increased in hypertrophied adipocytes relative to non-hypertrophied adipocytes (Fig. 3A). In this study, there was no significant induction of phosphorylation of JNK during the course of adipocyte hypertrophy (Fig. 3A). Treatment of hypertrophied adipocytes with MEK inhibitors, PD98059 and U0126, for 24 h significantly reduced MCP-1 mRNA levels (Fig. 3B left, p < 0.01) and secretion in the culture media (Fig. 3B right, p < 0.01). Moreover, the effect of the MEK inhibitors on MCP-1 mRNA expression was observed as early as 6 h after the treatment (Fig. 3C). Furthermore, phosphorylation of ERK was increased in the nuclear fraction rather than in the cytosolic fraction from hypertrophied adipocytes (Fig. 3D, p < 0.01). We also confirmed that phosphorylation of MEK is increased in hypertrophied adipocytes (Fig. 3E, p < 0.01). On the other hand, no such inhibitory effect was observed when treated with a p38 MAPK inhibitor, SB203580 (Fig. 3, B and C). These observations suggest that increased mRNA expression and secretion of MCP-1 in hypertrophied adipocytes are due at least in part to MEK-ERK activation.

MKP-1 Down-regulation during the Course of Adipocyte Hypertrophy—We next examined expression of members of the MKP family during the course of adipocyte hypertrophy. Interestingly, we detected substantial amounts of MKP-1 mRNA and protein in non-hypertrophied adipocytes, which are markedly down-regulated in hypertrophied adipocytes (Fig. 4, A and B, p < 0.05). There were no obvious changes in MKP-2 and MKP-3 mRNA levels during the course of adipocyte hypertrophy (Fig. 4A). We also observed that MKP-1 mRNA expression is significantly down-regulated in the adipose tissue from mice fed high fat diet for 2- and 4-weeks relative to those fed standard diet (Fig. 4C, p < 0.05). In addition, phosphorylation of ERK was significantly increased in the adipose tissue from mice that received 4-, 6-, 8-, and 15-week high fat diet relative to those fed standard diet (Fig. 4D, p < 0.05). These observations, taken together, suggest that MKP-1 is down-regulated in hypertrophied adipocytes, which is accompanied by ERK activation in vivo.


Figure 3
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  		FIGURE 3.
Role of MAP kinases in MCP-1 mRNA expression in hypertrophied adipocytes. A, phosphorylation of MAP kinases during the course of adipocyte hypertrophy. Representative immunoblots of ERK, p38 MAPK and JNK quantification of phosphorylation levels. *, p < 0.05; **, p < 0.01 versus day 8. n = 4. B, effect of 24-h-treatment with MAP kinase inhibitors on MCP-1 mRNA expression (left) and secretion (right) in hypertrophied 3T3-L1 adipocytes (day 21). PD, PD98059, 20 µmol/liter; U, U0126, 10 µmol/liter; SB, SB203580, 10 µmol/liter. **, p < 0.01 versus vehicle treated day 21. n = 6. N.S., not significant. C, effect of 6-h-treatment with MAPK inhibitors on MCP-1 mRNA expression in hypertrophied 3T3-L1 adipocytes (day 21). D, phosphorylation of ERK in the cytosolic and nuclear fractions from non-hypertrophied (day 8) and hypertrophied (day 21) 3T3-L1 adipocytes. Representative immunoblots of ERK and quantification of phosphorylation levels. E, phosphorylation of MEK during the course of adipocyte hypertrophy. Representative immunoblots of MEK and quantification of phosphorylation levels. **, p < 0.01 versus day 8. n = 4–6.

 
Generation of CAR-3T3-L1 Adipocytes—Because MKP-1 down-regulation may be responsible for the induction of MCP-1 during the course of adipocyte hypertrophy, we next examined the effect of MKP-1 restoration on ERK activity and MCP-1 mRNA expression in hypertrophied adipocytes. Because hypertrophied 3T3-L1 adipocytes are difficult to transfect with plasmid- or even virally encoded genes, we generated CAR-3T3-L1 adipocytes as described under "Experimental Procedures." There was no obvious difference in lipid accumulation between CAR-3T3-L1 and 3T3-L1 adipocytes without retroviral infection (control 3T3-L1 adipocytes) (Fig. 5, A and B) during the course of adipocyte differentiation and hypertrophy. We also confirmed no appreciable difference in mRNA expression of adipogenesis-related markers and adipocytokines between the cells (supplemental Fig. S3). Similar to control 3T3-L1 adipocytes, CAR-3T3-L1 adipocytes exhibited the up-regulation of MCP-1 during the course of adipocyte hypertrophy (Fig. 5C). The transfection efficiency was markedly increased in CAR-3T3-L1 adipocytes relative to control 3T3-L1 adipocytes as judged by Ad-GFP infection (Fig. 5D).


Figure 4
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  		FIGURE 4.
Changes in MKP-1 expression during the course of adipocyte hypertrophy in vitro and in vivo. A, changes in mRNA expression of MKP family during the course of adipocyte hypertrophy. n = 6. B, changes in MKP-1 protein levels in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. Representative immunoblots of MKP-1 and quantification of protein levels. n = 4. *, p < 0.05; **, p < 0.01 versus day 8. C and D, time course of MKP-1 mRNA expression (C) and ERK phosphorylation (D) levels in mice with diet-induced obesity. Representative immunoblots of ERK and quantification of phosphorylation levels. Data in C and D are expressed as the ratio of changes in mice fed HFD to those in mice fed SD. *, p < 0.05; **, p < 0.01 versus SD. n = 4–12 at each time point.

 


Figure 5
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  		FIGURE 5.
Generation of 3T3-L1 adipocytes stably expressing CAR (CAR-3T3-L1 adipocytes). A and B, lipid accumulation of CAR-3T3-L1 adipocytes and control 3T3-L1 adipocytes (ct) during the course of adipocyte differentiation and hypertrophy as revealed by oil-red O staining (A) and triglyceride content (B). C, changes in MCP-1 mRNA expression in CAR-3T3-L1 adipocytes. D, efficiency of adenovirus-mediated gene transfer in CAR-3T3-L1 adipocytes using Ad-GFP. PC, phase contrast view; GFP, GFP fluorescence view. Original magnification, x200. Scale bars, 100 µm. **, p < 0.01 versus day 8. n = 4.

 
Effect of MKP-1 Restoration in Hypertrophied Adipocytes—Infection with Ad-MKP-1 significantly increased MKP-1 mRNA expression in hypertrophied CAR-3T3-L1 adipocytes (Fig. 6A, left). We confirmed that MKP-1 protein levels in hypertrophied CAR-3T3-L1 adipocytes infected with Ad-MKP-1 are roughly comparable to those found in non-hypertrophied CAR-3T3-L1 adipocytes (Fig. 6A, right). In this setting, MKP-1 restoration in hypertrophied CAR-3T3-L1 adipocytes resulted in a marked reduction of ERK phosphorylation (Fig. 6B, p < 0.05) and MCP-1 mRNA expression (Fig. 6C, p < 0.01), which is roughly comparable to those found in non-hypertrophied CAR-3T3-L1 adipocytes. Interestingly, adiponectin mRNA expression was significantly increased and IL-6 mRNA expression tended to be reduced in hypertrophied CAR-3T3-L1 adipocytes with Ad-MKP-1 infection (supplemental Fig. S4), suggesting the anti-inflammatory effect of MKP-1 in hypertrophied adipocytes. Of note, there was no obvious difference in mRNA expression of adipogenesis-related markers by Ad-MKP-1 infection (supplemental Fig. S4). We also confirmed that Ad-MKP-1 infection does not affect lipid accumulation and insulin-induced glucose uptake in hypertrophied CAR-3T3-L1 (supplemental Fig. S5). These observations, taken together, indicate that restoration of MKP-1 does not affect adipocyte differentiation and hypertrophy but improves the overall inflammatory changes in hypertrophied adipocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies showed that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that the inflammatory changes induced by the cross-talk between adipocytes and macrophages are critical for the pathophysiology of obesity and thus the metabolic syndrome (4, 5). The molecular mechanisms underlying the recruitment of macrophages into obese adipose tissue have not fully been elucidated, but there is considerable evidence suggesting the involvement of MCP-1, which is increased during the course of obesity (8, 9). It is, therefore, important to know how MCP-1 is increased in hypertrophied adipocytes at the early stage of obesity, when macrophages are not infiltrated. This study was designed to elucidate the signaling pathway that mediates increased production of MCP-1 at the early stage of obesity.


Figure 6
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  		FIGURE 6.
Effect of MKP-1 restoration on ERK phosphorylation and MCP-1 mRNA expression in hypertrophied adipocytes. A, adenovirus-mediated restoration of MKP-1 in hypertrophied CAR-3T3-L1 adipocytes (day 21). Changes in MKP-1 mRNA (left) and protein (right) expression. Representative immunoblots of MKP-1 and quantification of protein levels. B and C, effect of MKP-1 restoration on ERK phosphorylation (B) and MCP-1 mRNA expression (C). *, p < 0.05; **, p < 0.01 versus Ad-GFP, (day 21). n = 6.

 
In this study, we found that expression of MCP-1 mRNA is increased in the adipose tissue from mice rendered mildly obese by a short term high fat diet. Histologically, there is marked increase in adipocyte cell size with no obvious macrophage infiltration, suggesting that MCP-1 mRNA expression is increased in the adipose tissue prior to macrophage accumulation in vivo. Two recent studies with transgenic mice overexpressing MCP-1 in the adipose tissue and/or MCP-1-deficient mice showed that MCP-1 plays a role in the recruitment of macrophages into obese adipose tissue (11, 12). Furthermore, Weisberg et al. (28) reported the attenuation of macrophage content and inflammatory changes in the adipose tissue from mice lacking C-C motif chemokine receptor-2, a major receptor for MCP-1, during a long term high fat diet. Together with recent evidence that MCP-1 mRNA expression and secretion in primary cultured adipocytes from obese subjects are positively correlated with their cell size (29), our data herein support the concept that increased production of MCP-1 in hypertrophied adipocytes at the early stage of obesity contributes to increased infiltration of macrophages into the adipose tissue at the late stage of obesity.

There are multiple intracellular signaling pathways activated in adipocytes during the course of adipocyte hypertrophy. The data of this study demonstrate that 3T3-L1 adipocytes, when cultured alone up to 21 days after differentiation, is capable of up-regulating MCP-1 and IL-6 and down-regulating adiponectin in parallel with increased cell size and lipid accumulation, which are comparable to those in obese adipose tissue. Moreover, mRNA expression patterns of some adipogenesis-related markers are also roughly parallel to those in obese adipose tissue, suggesting that 3T3-L1 adipocytes cultured from day 8 to day 21 serve as the useful in vitro experimental model system to investigate the molecular mechanism for the dysregulation of adipocytokine production during the course of adipocyte hypertrophy.

In this study, we observed that ERK and p38 MAPK are activated in hypertrophied 3T3-L1 adipocytes. Moreover, increased production of MCP-1 is significantly suppressed by MEK inhibitors as early as 6 h after the treatment, but not by a p38 MAPK inhibitor. We also demonstrated that ERK phosphorylation is significantly increased in the nuclear fraction but not in cytosolic fraction obtained from non-hypertrophied (day 8) and hypertrophied (day 21) 3T3-L1 adipocytes, suggesting that ERK activation occurs mostly in the nucleus rather than in the cytoplasm of adipocytes during the course of adipocyte hypertrophy. These observations are consistent with the concept that MKP-1 acts as a negative regulator of MAPKs within the nucleus (30). Furthermore, we observed that phosphorylation of MEK is increased in hypertrophied adipocytes. Together with a recent report that MAPKs are involved in the regulation of MCP-1 in human adipose tissue (31), these observations suggest that increased production of MCP-1 in hypertrophied adipocytes is mediated at least in part thorough MEK-ERK activation. In this regard, using the TRANSFAC (6.0) data base, we also searched for transcriptional factor binding sites in the mouse, rat, and human MCP-1 promoter and found a consensus AP-1 binding site 3–4-kb upstream of the transcriptional start site. Moreover, there are previous reports showing that cytokine-induced MCP-1 is mediated at least in part through the activation of ERK and AP-1 (32, 33). It is, therefore, conceivable that decrease in MKP-1 leads to the activation of ERK and AP-1 transcriptional activity within the nucleus, thereby increasing MCP-1 production during the course of adipocyte hypertrophy.

In this study, we demonstrate for the first time that both MKP-1 mRNA and protein levels are significantly down-regulated during the course of adipocyte hypertrophy in vitro. Moreover, restoration of MKP-1 in hypertrophied adipocytes reduces the otherwise increased ERK phosphorylation and thus MCP-1 mRNA expression. These observations, taken together, suggest that ERK activation through MKP-1 down-regulation is involved in increased production of MCP-1 in 3T3-L1 adipocytes during the course of adipocyte hypertrophy. The above discussion is consistent with the in vivo observation that ERK is activated, which is followed by MKP-1 down-regulation in the adipose tissue at the early stage of obesity, when there is no appreciable macrophage infiltration. Thus, reduced MKP-1 expression may be one of the early events during the progression of obesity in vivo, thereby leading to increased production of MCP-1 through the activation of ERK. In this regard, constitutive activation of ERK as a result of low induction of MKP-1 confers stronger resistance of immortalized cells than that of normal human fibroblasts to a cancer therapy called photodynamic therapy (34, 35). It is also noteworthy that MKP-1 expression is down-regulated in human ovarian cancer cell lines, where its forced re-expression reduces their malignant potential, suggesting the role of MKP-1 in the progression of human ovarian cancer (36). Thus, imbalance between MKP-1 and MEK activities as a result of MKP-1 down-regulation may cause ERK activation, thereby leading to increased production of MCP-1 in hypertrophied adipocytes.

It is also important to know the upstream signaling pathways responsible for MKP-1 down-regulation during the course of adipocyte hypertrophy. Recent studies have suggested the involvement of multiple intracellular signaling pathways in the inflammatory changes in adipocytes in vitro and in obese adipose tissue in vivo. For instance, Özcan et al. (37) reported that obesity is associated with the induction of ER stress predominantly in the adipose tissue and liver and demonstrated that ER stress is a central feature of obesity-related insulin resistance and type 2 diabetes. On the other hand, Furukawa et al. showed that ROS production is increased in parallel with lipid accumulation in 3T3-L1 adipocytes and that oxidative stress induces the dysregulation of adipocytokine production (38). It is, therefore, interesting to investigate the relationship among ER stress induction, ROS production, and MKP-1 activation during the course of adipocyte hypertrophy and/or at the early stage of obesity. Lin et al. (39, 40) demonstrated previously that MKP-1 degradation via the ubiquitin-proteasome pathway is stimulated by ERK, thereby leading to the sustained activation of ERK. Whether MKP-1 is thus down-regulated in hypertrophied adipocytes or not must await further investigations.

During the course of this study, Wu et al. (41) have reported that mice deficient in MKP-1 (MKP-1-/- mice) exhibit enhanced MAPK activity in the adipose tissue, reduced adipocyte cell size relative to wild-type littermates, and resistance to diet-induced obesity as a result of increased lipid metabolism in the liver and oxygen consumption in the skeletal muscle. Using mice with congenital deficiency of MKP-1, however, the authors did not address the role of MKP-1 in adipocytes during the course of adipocyte hypertrophy or at the early stage of obesity. In this regard, Bost et al. (17) reported that ERK1-/- mice have decreased adiposity and fewer adipocytes than wild-type littermates, and are resistant to high fat diet-induced obesity and insulin resistance. In this study, we demonstrated that ERK activation through the down-regulation of MKP-1 plays a role in increased production of MCP-1 in hypertrophied adipocytes during the course of obesity. It is, therefore, tempting to speculate that ERK activation through the down-regulation of MKP-1 plays an important role in the regulation of adipocyte differentiation, adiposity, and high fat diet-induced obesity in vivo. In this study, we also found that restoration of MKP-1 improves the dysregulation of adipocytokine production in hypertrophied adipocytes, which may improve obesity-related insulin resistance via adipocytokine mechanism in vivo. The pathophysiologic role of MKP-1 down-regulation in hypertrophied adipocytes at the early stage of obesity in vivo must await further investigation.

To obtain hypertrophied 3T3-L1 adipocytes whose MKP-1 levels are restored to those of non-hypertrophied 3T3-L1, we tried to produce 3T3-L1 adipocytes stably expressing MKP-1 using the retrovirus vector and observed that they are unable to differentiate into lipid-laden mature adipocytes.4 This is consistent with the concept that ERK should be on and off properly during adipogenesis in vitro (16, 42). Although the adenoviral vector has been widely used for the introduction of exogenous genes in non-proliferating cells, 3T3-L1 adipocytes, particularly when hypertrophied, are transfected with less efficiency because of the scarcity of CAR (43–45). In this study, we generated 3T3-L1 adipocytes stably expressing CAR (or CAR-3T3-L1 adipocytes), which is infected with the adenoviral expression vector with ease, even after being hypertrophied. Importantly, there are no appreciable differences in adipogenesis, lipid accumulation, and adipocytokine expression during the course of adipocyte differentiation and hypertrophy between CAR-3T3-L1 adipocytes and control 3T3-L1 adipocytes. This study has verified the usefulness of CAR-3T3-L1 adipocytes as the unique experimental tool to investigate the molecular basis for adipocyte differentiation and hypertrophy.

In conclusion, this study represents the first demonstration that ERK activation through MKP-1 down-regulation is involved in increased production of MCP-1 in adipocytes at the early stage of obesity. The data of this study suggest that MKP-1 activation may offer a novel therapeutic strategy to reduce the otherwise increased production of MCP-1 in hypertrophied adipocytes at the early stage of obesity and thus macrophage infiltration into the adipose tissue at the late stage of obesity.


   FOOTNOTES
 
* This work was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Ministry of Health, Labor, and Welfare of Japan, and research grants from the Astellas Foundation for Research on Metabolic Disorders, Astellas Foundation for Research on Medicinal Resources and The Ichiro Kanehara Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S5. Back

1 Supported by the Tokyo Biochemical Research Foundation. Back

2 To whom correspondence should be addressed: Dept. of Molecular Medicine and Metabolism, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-5280-8108; Fax: 81-3-5280-8108; E-mail: ogawa.mmm{at}mri.tmd.ac.jp.

3 The abbreviations used are: MCP-1, monocyte chemoattractant protein-1; MAPK, mitogen-activated protein kinase; MKP-1, mitogen-activated protein kinase phosphatase-1; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/ERK kinase; CAR, coxsackie-adenovirus receptor; WAT, white adipose tissue; GFP, green fluorescent protein; ER, endoplasmic reticulum; ROS, reactive oxygen species; SD, standard diet; HFD, high fat diet; IL, interleukin. Back

4 A. Ito, T. Suganami, and Y. Ogawa, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Hiroshi Nishina for critical reading of the manuscript. We thank Dr. Toshio Kitamura for Plat-E, Dr. Hiroyuki Mizuguchi for pcDNA3-CAR, Dr. Shoji Yamaoka for pMRX, and Dr. Jeffery D. Molkentin for MKP-1 adenoviral expression vector. We are also grateful to the members of the Ogawa laboratory for discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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