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Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes
Biochemical and Biophysical Research Communications 411 (2011) 287–292
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Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes Abu Asad Chowdhury a, Mohammad Salim Hossain a, Mohammad Sharifur Rahman a, Kohji Nishimura b, Mitsuo Jisaka a, Tsutomu Nagaya a, Fumiaki Shono c, Kazushige Yokota a,⇑ a b c
Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514, Japan
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Article history: Received 6 June 2011 Available online 24 June 2011 Keywords: Adipocyte Cyclooxygenase pathway Lipocalin-type prostaglandin D synthase Prostaglandin J2 series Stable transfection Antisense direction
a b s t r a c t Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD2 and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD2 synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE2 was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense LPGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE2 and PGF2a, while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- D 12,14-prostaglandin J2. These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ2 series. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Arachidonate cyclooxygenase (COX) pathway generates prostaglandin D2 (PGD2) with versatile effects, through the coordinated actions of the isoformic enzymes of COX and prostaglandin D synthases (PGDSs) [1]. PGD2 exerts its effects via cell-surface receptors including DP1 and CRTH2 [2,3]. Moreover, PGD2 is non-enzymatically converted to PGJ2 series, such as 15-deoxy- D12,14-prostaglandin J2 (15d-PGJ2) and D 12-PGJ2 [4]. Of these, 15d-PGJ2 is the most potent natural ligand for the nuclear hormone receptor,
Abbreviations: PG, prostaglandin; L-PGDS, lipocalin-type PGD synthase; COX, cyclooxygenase; 15d-PGJ2, 15-deoxy- D 12,14-prostaglandin J2; PPARc, peroxisome proliferator-activated receptor c; G418, geneticin; FBS, fetal bovine serum; RT-PCR, reverse transcriptase-polymerase chain reaction; aP2, adipocyte protein 2; GLUT-4, glucose transporter-4; LPL, lipoprotein lipase; ELISA, enzyme-linked immunosorbent assay. ⇑ Corresponding author. Fax: +81 852 326576. E-mail address: [email protected] (K. Yokota). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.126
peroxisome proliferator-activated receptor c (PPARc) [5,6]. Alternatively, 15d-PGJ2 serves as a negative regulator of prostanoid synthesis and inflammation [7]. White adipose tissue is specialized for the storage and mobilization of fats as fuel molecules. As well, adipocytes serve as signaling cells to secrete bioactive factors like adipocytokines and prostanoids with opposite effects at different life stages. During the differentiation and maturation of adipocytes, PPARc is expressed abundantly and acts as a master regulator of a series of the gene expression leading to adipogenesis [8,9]. Indeed, exogenous PGD2 and PGJ2 series have been demonstrated to promote adipogenesis in PPARc-expressing cultured adipocytes [5,6,10,11]. For the study on the roles of prostanoids in adipocytes and the precursor cells, we have been making use of cultured preadipogenic 3T3-L1 cells [10–13]. Our recent studies have described the contribution of endogenous PGJ2 series to the up-regulation of adipogenesis [10,11]. Earlier, cultured 3T3-L1 cells have been shown to express specifically lipocalin-type PGDS (L-PGDS) during the progress of
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adipogenesis associated with co-expression of PPARc [10,12,14]. Nevertheless, the role of intracellular L-PGDS is still complicated because PGD2 and the related metabolites or derivatives exert other different effects on the function of adipocytes. For example, PGD2 can be metabolized to 9a, 11b-PGF2a, which acts as an anti-adipogenic factor in cultured adipocytes [15]. In addition, 15d-PGJ2 has been implicated in the inhibition of inflammatory response associated with inducible synthesis of COX-2 [7]. Moreover, the action of PGD2 through the cell-surface receptors with different subtypes may not be excluded. Interestingly, active form of L-PGDS has been shown to inhibit the phosphorylation of Akt, a serine/ threonine kinase [16]. The phosphorylated Akt has been previously implicated in spontaneous differentiation into adipocytes in a PGindependent manner [17]. Thus, the functions of L-PGDS in adipocytes at different life stages still remain to be determined. Here, to determine the specific role for L-PGDS in the control of adipogenesis in cultured adipocytes, we attempted to suppress L-PGDS in cultured preadipocytes transfected stably with the mammalian expression vector having the cDNA insert oriented in the antisense direction.
2. Materials and methods 2.1. Materials Authentic prostanoids, arachidonic acid, and rabbit polyclonal antibody for L-PGDS were obtained from Cayman Chemical (Ann Arbor, MI, USA). Geneticin (G418) and Triglyceride E-Test Kit were supplied by Wako (Osaka, Japan). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Solon, OH, USA). SuperFect Transfection Reagent was provided by Qiagen (Valencia, CA, USA). Other materials were obtained as described earlier [12,13,18]. All other chemicals were of reagent or tissue culture grade. 2.2. Cell culture of 3T3-L1 cells for the differentiation and maturation of adipocytes Preadipogenic mouse 3T3-L1 cells were plated at 5 104 cells/ ml in the growth medium and cultured until confluence. The monolayer cells were exposed to the differentiation medium for 45 h and followed by the continued cultures in the maturation medium by changing every 2 days to promote the accumulation of fats during the maturation phase under the established cultured conditions [10–13,19–21]. Exogenous prostanoids and arachidonic acid used for the cell cultures were dissolved in ethanol as a vehicle while A23187 was added to the culture medium after dissolving in dimethyl sulfoxide. The volume of the vehicle was adjusted to 0.2%. 2.3. Construction of L-PGDS expression vector oriented in the antisense direction Total RNA was extracted from brain of BALB/c mouse and used for the amplification of cDNA insert encoding the full-length open reading frame by reverse transcriptase-polymerase chain reaction (RT-PCR) with 50 -ACTTGAATTCCAAATGGCTGCT-30 as a 50 -primer having the EcoRI site and 50 -TGCAAAGCTTGCGTTTACTCTT-30 as a 30 -primer containing the HindIII site. The amplified cDNA fragment was cut doubly with the restriction enzymes, purified, and ligated to the sites of HindIII and EcoRI of the mammalian expression vector, pcDNA3.1(+) with neomycin-resistant gene (Invitrogen, Carlsbad, CA, USA) in the antisense direction. The recombinant DNA was extracted from Escherichia coli DH5a that had been transformed with the ligation product, and subjected to the determination of the DNA sequence as describe before [13,20,21].
2.4. Stable transfection and cloning of transfectants Parent 3T3-L1 cells were transfected with the mammalian expression vector, pcDNA3.1(+) having the cDNA insert of mouse L-PGDS oriented in the antisense direction or the vector only as a control using the SuperFect Transfection Reagent according to the manufacture’s instructions. The cultured cells were grown in the growth medium supplemented with G418 as described previously [20,21]. The growing cells were employed for the cloning of single cells. The isolated cloned cells were further propagated and subjected to the gene expression analysis of the mRNA and protein levels of L-PGDS as described earlier [10,12]. 2.5. Analysis of expression levels of mRNA and protein Total RNA was extracted from cultured 3T3-L1 cells at the indicated stages of adipocytes as described before [22]. For the specific detection of the expressed genes, the resulting total RNA was used for the analysis by RT-PCR as reported previously [10,20,21]. The amplification of the target gene was conducted using pairs of 50 and 30 -primers specific for each of L-PGDS, adipocyte protein 2 (aP2), leptin, PPARc, glucose transporter-4 (GLUT-4), adiponectin, lipoprotein lipase (LPL), and b-actin as listed in our previous reports [10,12,13,20,21]. The amplified DNA fragments were separated by 1.5% agarose electrophoresis and the DNA sequences were confirmed as described previously [13,20,21]. Cultured 3T3L1 cells were cultured to confluence in the growth phase. The resulting cells were harvested for the analysis of protein expression levels of L-PGDS by Western blot analysis with a rabbit polyclonal antibody for mouse L-PGDS according to our previous method [13,20]. 2.6. Enzyme-linked immunosorbent assay (ELISA) for prostanoids The culture medium was collected and applied to our immobilized ELISA specific for PGD2 [23], PGE2 [13,18,20,21], and D12-PGJ2 [11] as reported previously. For the assay, the conjugate of each PG and bovine c-globulin was used as an immobilized antigen in 96well EISA plate. The immobilized antigen was allowed to react competitively with a specific antibody for each prostanoid species in the presence of standards or samples to be tested. The resulting immunocomplex was detected spectrophotometrically as described earlier [18]. 2.7. Other methods The storage of triacylglycerols after the maturation phase of adipocytes was measured using Triglyceride E-Test Kit as described before [12,13]. Cultured cells were harvested and used for the determination of the cellular proteins after the precipitation of proteins with cold trichloroacetic acid [24]. The accumulation of lipid droplets in adipocytes was monitored by staining with Oil Red O [12,25]. All of the quantified data were represented as the mean ± S.E.M. of three or more experiments. Student’s t test was used for evaluating statistical significance, and the difference was considered to be significant when p < 0.05. 3. Results 3.1. Preparation of cloned stable transfectants with L-PGDS oriented in the antisense direction Cultured 3T3-L1 cells were transfected stably with L-PGDS oriented in the antisense direction. Finally, we isolated several
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Fig. 1. Gene expression of L-PGDS in parent cells and the transfectants. (A) Transcript levels of L-PGDS. Untransfected 3T3-L1 cells and the transfectants with expression vector only or the vector with antisense L-PGDS were planted at 5 104 cells/ml in a 60-mm Petri dish containing 4 ml of the growth medium and grown to confluence. Total RNA was extracted from the confluent cells and subjected to the analysis of the transcript levels of L-PGDS, neomycin-resistant gene in the vector, and b-action as a control. (B) Western blot analysis of L-PGDS protein levels in cultured parent cells and the transfectants. The cell lysates (20 lg in each lane) were analyzed by Western blot analysis using a polyclonal antibody specific for mouse L-PGDS. Labels: parent, untransfected cells; vector, cloned transfectants with expression vector only; R-5 and R-13, cloned transfectants with L-PGDS in the antisense direction.
Fig. 2. Biosynthesis of prostanoids by cultured transfectants with antisense L-PGDS. Parent cells and the transfectants were plated at 5 104 cells/ml in a 35-mm Petri dish containing 2 ml of the growth medium and grown to confluence. The resulting cultured cells were incubated for 2 h with 10 lM arachidonic acid (A) or 10 lM A23187 (B). The culture medium was collected for the quantification of PGD2 by ELISA. Alternatively, the synthesis of PGE2 was determined by ELISA after the confluent cultured cells were incubated for 30 min with 10 lM arachidonic acid (C). Moreover, the monolayer cells were allowed to undergo the differentiation and maturation. The culture medium was collected after 6 days of the maturation phase and subjected to the determination of D 12-PGJ2 by ELISA (D). ⁄p < 0.05 compared with the transfectants with vector only along with vehicle. #p < 0.05 compared with the transfectants with vector only in the presence of arachidonic acid or A23187.
independent clones of the transfectants. Of these, attempts were made to characterize cloned transfectants R-5 and R-13 as representative ones. The neomycin-resistant gene in the vector was expressed in the cloned transfectants with the vector only or that with antisense L-PGDS (Fig. 1A), whereas it was not detectable in untransfected parent cells. As expected, the stable transfection of L-PGDS oriented in the antisense direction significantly suppressed the transcript levels (Fig. 1A) and the protein levels (Fig. 1B) of LPGDS in the cloned transfectants. By contrast, the transfection of cultured preadipocytes with the vector only had almost no influence on the expression levels of L-PGDS.
3.2. Biosynthesis of PGD2, PGE2, and D12-PGJ2 by cloned transfectants stably expressing antisense L-PGDS To assess the intracellular activity of L-PGDS in the stable transfectants or the related cells, the confluent cells during the growth phase were incubated with 10 lM exogenous free arachidonic acid (Fig. 2A) or 10 lM calcium ionophore A23187 (Fig. 2B). In all cases, the biosynthesis of PGD2 was appreciably attenuated in the transfectants with antisense L-PGDS. However, no significant difference was observed between the parent cells and the transfectants with the vector only. All of tested parent cells and the transfectants did
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Fig. 3. Storage of fats in cultured transfectants with L-PGDS in the antisense direction. Parent cells and the transfectants were plated and grown to confluence as described in Fig. 2. The monolayer cells were allowed to undergo the differentiation and maturation. The amount of triacylglycerols was quantified after 6 days and 12 days of the maturation phase (A). ⁄p < 0.05 compared with the transfectants with vector only after 6 days of the maturation phase. #p < 0.05 compared with the transfectants with vector only after 12 days of the maturation phase. Furthermore, the cultured cells were viewed by phase-contrast microscopy and differential-interference microscopy after Oil Red O staining at 200 magnification in addition to the observation of macroscopic views (B). Alternatively, the confluent cells were maintained in the growth medium for another 8 days by changing the same culture medium for the evaluation of spontaneous adipogenesis (C). The resulting cells were subjected to the determination of the amount of triacylglycerols. ⁄p < 0.05 compared with the transfectants with vector only. The cultured cells under the same conditions as the panel C were observed as microscopic views or macroscopic views as described in the panel B (D).
not show significant changes in the ability to stimulate the synthesis of PGE2 from exogenous free arachidonic acid (Fig. 2C). We have recently described the increased ability to generate endogenous PGJ2 series during the maturation phase [10,11]. Here, endogenous synthesis of D12-PGJ2 was measured after 6 days of the maturation phase (Fig. 2D). Both cloned transfectants with antisense L-PGDS synthesized endogenous D 12-PGJ2 at significantly lower levels compared to those of control cells. 3.3. Fat storage in stable transfectants with antisense L-PGDS during the maturation phase We then examined whether the reduction in the expression levels of L-PGDS could influence the storage of fats during the maturation phase. The storage of fats after 6 and 12 days was appreciably higher in the transfectants with antisense L-PGDS than that in the control cells (Fig. 3A). Similarly, the enhanced storage of fats was also confirmed by the observation of microscopic and macroscopic views of cultured adipocytes during the maturation phase (Fig. 3B). Alternatively, we noticed the spontaneous
adipogenesis of our confluent transfectants with antisense L-PGDS after the maintenance for total 8 days in the growth medium without further hormonal stimulations for undergoing the differentiation and maturation phases (Fig. 3C and D). These combined results clearly indicate that the sustained suppression of L-PGDS levels induces the up-regulation of adipogenesis. 3.4. Gene expression of adipocyte-specific markers in stable transfectants with antisense L-PGDS during the maturation phase To define whether the elevation of the fat storage in the stable transfectants with antisense L-PGDS could be due to the positive regulation of adipogenesis program, the transcript levels of adipocyte-specific markers in the transfectants were analyzed during the maturation phase (Fig. 4A). The transfectants with antisense L-PGDS expressed aP2, leptin, and GLUT-4 as typical adipogenesis markers at higher levels than other control cells although the expression levels of PPARc, LPL, and adiponectin remained unchanged. These findings overall support the positive regulation of adipogenesis program in the transfectants with antisense L-PGDS.
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Fig. 4. Gene expression levels of adipocyte-specific markers in cultured transfectants. Parent cells and the transfectants were plated and grown to confluence as described in Fig. 1. The monolayer cells were allowed to undergo the differentiation and maturation. Total RNA was extracted from cultured cells after 12 days of the maturation phase and applied to the analysis of transcript levels of aP2, leptin, PPARc, GLUT-4, adiponectin, LPL, and b-actin as a control (A). Alternatively, parent cells and the transfectants were plated and grown to confluence as described in Fig. 2. The monolayer cells were allowed to undergo the differentiation and maturation. During the maturation phase, the cultured cells were treated with either vehicle, PGE2 or PGF2a, or 15d-PGJ2 at 1 lM for total 6 days by replacing every 2 days with the fresh maturation medium containing the corresponding prostanoid species (B). After the treatments, the cells were harvested for the determination of the amount of triacylglycerols. ⁄p < 0.05 compared with the transfectants treated with vehicle. The macroscopic views of cultured cells under the same conditions as the panel B were observed after staining with Oil Red O (C).
3.5. Response to exogenous anti-adipogenic or pro-adipogenic prostanoids in stable transfectants with antisense L-PGDS during the maturation phase We investigated determine whether the cloned stable transfectants with antisense L-PGDS could respond to exogenous prostanoids. The cultured cells were treated with either of 1 lM PGE2 or PGF2a as anti-adipogenic prostanoids or 1 lM 15d-PGJ2 as a pro-adiogenic prostanoid during the maturation phase and then subjected to the quantification of the accumulated fats (Fig. 4B) and the observation of macroscopic views of cultured cells (Fig. 4C). Both PGE2 and PGF2a appreciably suppressed the storage fats in the transfectants with antisense L-PGDS. On the other hand, 15d-PGJ2 increased the fat storage additionally at a significant level. The results indicate that our cloned stable transfectants with antisense L-PGDS can respond to exogenous anti-adipogenic and pro-adipogenic prostanoids in a manner similar to the untransfected parent cells as described earlier [10,13]. 4. Discussion The function of PGD2 in adipocytes and the precursor cells is complicated because of its enzymatic or non-enzymatic conversion to other types of species exerting their effects differently on adipogenesis in adipocytes. To manipulate the intracellular levels of L-PGDS involved in the biosynthesis of PGD2 and the related PGJ2 derivatives, we undertook the approach to suppress the expression of L-PGDS by the sustained expression of antisense RNA for L-PGDS in cultured 3T3-L1 preadipocytes. The continuously expressed antisense RNA has been shown to interfere specifically the gene expression of the desired target [26]. The present study confirmed the appreciable suppression of the transcript and protein levels of L-PGDS in cultured preadipocytes transfected stably with L-PGDS
in the antisense direction. Additionally we provided the evidence for the substantial decrease in the synthesis of PGD2 in the cloned transfectants with antisense L-PGDS. Furthermore, the levels of D 12 -PGJ2 derived from PGD2 after 6 days of the maturation phase were much lower in the stable transfectants with antisense L-PGDS than the control cells. By contrast, the generation of PGE2 was not significantly influenced in the transfectants with antisense L-PGDS. The combined results clearly provided the evidence that our transfection method inhibited selectively the synthesis of PGD2 and the related PGJ2 series without affecting other pathways including upstream COX isoforms and other PGE synthases. The current study demonstrated that our cloned transfectants with antisense L-PGDS were able to enhance more greatly the storage of fats during the maturation phase. The accelerated accumulation of fats was accompanied by the up-regulated gene expression of typical adipogenesis markers, such as aP2, leptin, and GLUT-4, which are known to be under the control of PPARc [27]. These findings support the notion that the stimulation of fat storage in the transfectants is closely linked with the promotion of a series of adipogenesis program during the life stages of adipocytes. Additionally, we found the spontaneous adipogenesis in confluent monolayers of the cloned transfectants with antisense L-PGDS without undergoing the differentiation and maturation phases, providing another clear evidence for the up-regulation of adipogenesis. Moreover, our study confirmed that the transfectants with antisense L-PGDS responded to anti-adipogenic PGE2 and PGF2a and pro-adipogenic 15d-PGJ2 during the maturation phase as normally as that of the parent cells as described earlier [10,13]. The present results appear to be consistent with an earlier study where L-PGDS-knockout mice showed accelerated accumulation of triacylglycerols in hypertrophic adipocytes [16]. By contrast, a previous study has reported the reduced adipogenesis in cultured adipocyes using the siRNA interference of L-PGDS [28].
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Thus, it is sill ambiguous about the role of L-PGDS in adipogenesis due to the complex actions of PGD2 and the related species at different stages of adipocytes. Previous studies have described that 15d-PGJ2 is the most potent natural ligand to activate PPARc as a master regulator of adipogenesis [5,6]. Furthermore, exogenous 15d-PGJ2 and D 12-PGJ2 have been shown to be effective in the stimulation of adipogenesis in cultured adipocytes during the maturation phase [10,11]. As well, cultured adipocytes at the later maturation phase are capable of producing endogenous PGJ2 series [10,11]. Hence, we initially hypothesized that stable transfection of cultured preadipocytes with antisense L-PGDS should suppress the adipogenesis program leading to the storage of fats by interfering the endogenous synthesis of those pro-adipogenic prostanoids. Nevertheless, our cloned preadipocytes transfected with antisense L-PGDS exhibited adipogenesis at higher levels. Although the exact mechanism remains obscure, a possible contribution to it might involve the reduced synthesis of 9a, 11b-PGF2a, known as an anti-adipogenic prostanoid [15]. This compound can be formed from the enzymatic reduction of PGD2 in some tissues [29]. Alternatively, PGD2 itself is known to bind to its specific receptors termed the DP1 and CRTH2 receptors [2,3], and to increase the intracellular levels of cAMP [2]. This second messenger might potentially transmit the anti-adipogenic signal to the untransfected parent cells at some life stages of adipocytes by analogy with the EP4 receptor of anti-adipogenic PGE2 [30]. As a separate mechanism independent of endogenous prostanoids, spontaneous differentiation of cultured 3T3-L1 preadipocytes has been reported to occur without standard hormonal stimulation while expressing the active phosphorylated form of Akt kinase [17]. Currently, we also found the spontaneous differentiation of our cloned transfectants with antisense L-PGDS. Recently, L-PGDS has been described to inhibit the Akt phosphorylation in other cells [16]. Therefore, the reduced expression of L-PGDS in our transfectants with antisense L-PGDS may account partly for the up-regulation of adipogenesis through the activation of Akt kinase. Further extensive studies remain to be done to define the detailed mechanisms. In conclusion, we established cloned preadipocytes transfected permanently with L-PGDS in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited an appreciable increase in the fat storage in adipocytes during the maturation phase. These findings provided the unique features of the stable transfectants with antisense L-PGDS, which are useful for further studies to dissect the molecular and cellular mechanisms underlying the effects of L-PGDS through PG-dependent or PG-independent manners. References [1] O. Hayaishi, Sleep-wake regulation by prostaglandins D2 and E2, J. Biol. Chem. 263 (1988) 14593–14596. [2] Y. Boie, N. Sawyer, D.M. Slipetz, et al., Molecular cloning and characterization of the human prostanoid DP receptor, J. Biol. Chem. 270 (1995) 18910–18916. [3] H. Hirai, K. Tanaka, O. Yoshie, et al., Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seventransmembrane receptor CRTH2, J. Exp. Med. 193 (2001) 255–261. [4] T. Shibata, M. Kondo, T. Osawa, et al., 15-Deoxy- D 12,14-prostaglandin J2: a prostaglandin D2 metabolite generated during inflammatory processes, J. Biol. Chem. 277 (2002) 10459–10466. [5] B.M. Forman, P. Tontonoz, J. Chen, et al., 15-Deoxy- D 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARc, Cell 83 (1995) 803–812. [6] S.A. Kliewer, J.M. Lenhard, T.M. Willson, et al., A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor c and promotes adipocyte differentiation, Cell 83 (1995) 813–819.
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Sustained expression of lipocalin-type prostaglandin D synthase in the antisense direction positively regulates adipogenesis in cloned cultured preadipocytes Abu Asad Chowdhury a, Mohammad Salim Hossain a, Mohammad Sharifur Rahman a, Kohji Nishimura b, Mitsuo Jisaka a, Tsutomu Nagaya a, Fumiaki Shono c, Kazushige Yokota a,⇑ a b c
Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima 770-8514, Japan
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Article history: Received 6 June 2011 Available online 24 June 2011 Keywords: Adipocyte Cyclooxygenase pathway Lipocalin-type prostaglandin D synthase Prostaglandin J2 series Stable transfection Antisense direction
a b s t r a c t Adipocytes express preferentially lipocalin-type prostaglandin (PG)D synthase (L-PGDS) that is responsible for the biosynthesis of PGD2 and other related prostanoids with pro-adipogenic or anti-adipogenic effects. To evaluate the role of L-PGDS in cultured adipocytes and the precursor cells, we attempted to interfere the intracellular expression of L-PGDS in cultured 3T3-L1 preadipocytes by stable transfection with a mammalian expression vector having the full-length cDNA of L-PGDS oriented in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited the reduction in the transcript and protein levels of L-PGDS, resulting in the significant inhibition of the PGD2 synthesis from exogenous and endogenous arachidonic acid. By contrast, the synthesis of PGE2 was not influenced appreciably, indicating no interfering effects on cyclooxygenases and PGE synthases. The stable transfection with antisense L-PGDS induced markedly the stimulation of fat storage in cultured adipocytes during the maturation phase. In addition, the spontaneous accumulation of fats occurred in the transfectants with antisense L-PGDS without undergoing the stimulation with inducing factors. The gene expression studies revealed the enhanced expression of adipocyte-specific markers in the transfectants with antisense LPGDS, indicating the up-regulation of adipogenesis program. The stimulated adipogenesis was significantly reversed by anti-adipogenic prostanoids including PGE2 and PGF2a, while the storage of fats was additionally enhanced by pro-adipogenic 15-deoxy- D 12,14-prostaglandin J2. These results suggest that the stably reduced expression levels of L-PGDS regulates positively adipogenesis program in a cellular mechanism independent of pro-adipogenic action of PGJ2 series. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Arachidonate cyclooxygenase (COX) pathway generates prostaglandin D2 (PGD2) with versatile effects, through the coordinated actions of the isoformic enzymes of COX and prostaglandin D synthases (PGDSs) [1]. PGD2 exerts its effects via cell-surface receptors including DP1 and CRTH2 [2,3]. Moreover, PGD2 is non-enzymatically converted to PGJ2 series, such as 15-deoxy- D12,14-prostaglandin J2 (15d-PGJ2) and D 12-PGJ2 [4]. Of these, 15d-PGJ2 is the most potent natural ligand for the nuclear hormone receptor,
Abbreviations: PG, prostaglandin; L-PGDS, lipocalin-type PGD synthase; COX, cyclooxygenase; 15d-PGJ2, 15-deoxy- D 12,14-prostaglandin J2; PPARc, peroxisome proliferator-activated receptor c; G418, geneticin; FBS, fetal bovine serum; RT-PCR, reverse transcriptase-polymerase chain reaction; aP2, adipocyte protein 2; GLUT-4, glucose transporter-4; LPL, lipoprotein lipase; ELISA, enzyme-linked immunosorbent assay. ⇑ Corresponding author. Fax: +81 852 326576. E-mail address: [email protected] (K. Yokota). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.06.126
peroxisome proliferator-activated receptor c (PPARc) [5,6]. Alternatively, 15d-PGJ2 serves as a negative regulator of prostanoid synthesis and inflammation [7]. White adipose tissue is specialized for the storage and mobilization of fats as fuel molecules. As well, adipocytes serve as signaling cells to secrete bioactive factors like adipocytokines and prostanoids with opposite effects at different life stages. During the differentiation and maturation of adipocytes, PPARc is expressed abundantly and acts as a master regulator of a series of the gene expression leading to adipogenesis [8,9]. Indeed, exogenous PGD2 and PGJ2 series have been demonstrated to promote adipogenesis in PPARc-expressing cultured adipocytes [5,6,10,11]. For the study on the roles of prostanoids in adipocytes and the precursor cells, we have been making use of cultured preadipogenic 3T3-L1 cells [10–13]. Our recent studies have described the contribution of endogenous PGJ2 series to the up-regulation of adipogenesis [10,11]. Earlier, cultured 3T3-L1 cells have been shown to express specifically lipocalin-type PGDS (L-PGDS) during the progress of
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adipogenesis associated with co-expression of PPARc [10,12,14]. Nevertheless, the role of intracellular L-PGDS is still complicated because PGD2 and the related metabolites or derivatives exert other different effects on the function of adipocytes. For example, PGD2 can be metabolized to 9a, 11b-PGF2a, which acts as an anti-adipogenic factor in cultured adipocytes [15]. In addition, 15d-PGJ2 has been implicated in the inhibition of inflammatory response associated with inducible synthesis of COX-2 [7]. Moreover, the action of PGD2 through the cell-surface receptors with different subtypes may not be excluded. Interestingly, active form of L-PGDS has been shown to inhibit the phosphorylation of Akt, a serine/ threonine kinase [16]. The phosphorylated Akt has been previously implicated in spontaneous differentiation into adipocytes in a PGindependent manner [17]. Thus, the functions of L-PGDS in adipocytes at different life stages still remain to be determined. Here, to determine the specific role for L-PGDS in the control of adipogenesis in cultured adipocytes, we attempted to suppress L-PGDS in cultured preadipocytes transfected stably with the mammalian expression vector having the cDNA insert oriented in the antisense direction.
2. Materials and methods 2.1. Materials Authentic prostanoids, arachidonic acid, and rabbit polyclonal antibody for L-PGDS were obtained from Cayman Chemical (Ann Arbor, MI, USA). Geneticin (G418) and Triglyceride E-Test Kit were supplied by Wako (Osaka, Japan). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Solon, OH, USA). SuperFect Transfection Reagent was provided by Qiagen (Valencia, CA, USA). Other materials were obtained as described earlier [12,13,18]. All other chemicals were of reagent or tissue culture grade. 2.2. Cell culture of 3T3-L1 cells for the differentiation and maturation of adipocytes Preadipogenic mouse 3T3-L1 cells were plated at 5 104 cells/ ml in the growth medium and cultured until confluence. The monolayer cells were exposed to the differentiation medium for 45 h and followed by the continued cultures in the maturation medium by changing every 2 days to promote the accumulation of fats during the maturation phase under the established cultured conditions [10–13,19–21]. Exogenous prostanoids and arachidonic acid used for the cell cultures were dissolved in ethanol as a vehicle while A23187 was added to the culture medium after dissolving in dimethyl sulfoxide. The volume of the vehicle was adjusted to 0.2%. 2.3. Construction of L-PGDS expression vector oriented in the antisense direction Total RNA was extracted from brain of BALB/c mouse and used for the amplification of cDNA insert encoding the full-length open reading frame by reverse transcriptase-polymerase chain reaction (RT-PCR) with 50 -ACTTGAATTCCAAATGGCTGCT-30 as a 50 -primer having the EcoRI site and 50 -TGCAAAGCTTGCGTTTACTCTT-30 as a 30 -primer containing the HindIII site. The amplified cDNA fragment was cut doubly with the restriction enzymes, purified, and ligated to the sites of HindIII and EcoRI of the mammalian expression vector, pcDNA3.1(+) with neomycin-resistant gene (Invitrogen, Carlsbad, CA, USA) in the antisense direction. The recombinant DNA was extracted from Escherichia coli DH5a that had been transformed with the ligation product, and subjected to the determination of the DNA sequence as describe before [13,20,21].
2.4. Stable transfection and cloning of transfectants Parent 3T3-L1 cells were transfected with the mammalian expression vector, pcDNA3.1(+) having the cDNA insert of mouse L-PGDS oriented in the antisense direction or the vector only as a control using the SuperFect Transfection Reagent according to the manufacture’s instructions. The cultured cells were grown in the growth medium supplemented with G418 as described previously [20,21]. The growing cells were employed for the cloning of single cells. The isolated cloned cells were further propagated and subjected to the gene expression analysis of the mRNA and protein levels of L-PGDS as described earlier [10,12]. 2.5. Analysis of expression levels of mRNA and protein Total RNA was extracted from cultured 3T3-L1 cells at the indicated stages of adipocytes as described before [22]. For the specific detection of the expressed genes, the resulting total RNA was used for the analysis by RT-PCR as reported previously [10,20,21]. The amplification of the target gene was conducted using pairs of 50 and 30 -primers specific for each of L-PGDS, adipocyte protein 2 (aP2), leptin, PPARc, glucose transporter-4 (GLUT-4), adiponectin, lipoprotein lipase (LPL), and b-actin as listed in our previous reports [10,12,13,20,21]. The amplified DNA fragments were separated by 1.5% agarose electrophoresis and the DNA sequences were confirmed as described previously [13,20,21]. Cultured 3T3L1 cells were cultured to confluence in the growth phase. The resulting cells were harvested for the analysis of protein expression levels of L-PGDS by Western blot analysis with a rabbit polyclonal antibody for mouse L-PGDS according to our previous method [13,20]. 2.6. Enzyme-linked immunosorbent assay (ELISA) for prostanoids The culture medium was collected and applied to our immobilized ELISA specific for PGD2 [23], PGE2 [13,18,20,21], and D12-PGJ2 [11] as reported previously. For the assay, the conjugate of each PG and bovine c-globulin was used as an immobilized antigen in 96well EISA plate. The immobilized antigen was allowed to react competitively with a specific antibody for each prostanoid species in the presence of standards or samples to be tested. The resulting immunocomplex was detected spectrophotometrically as described earlier [18]. 2.7. Other methods The storage of triacylglycerols after the maturation phase of adipocytes was measured using Triglyceride E-Test Kit as described before [12,13]. Cultured cells were harvested and used for the determination of the cellular proteins after the precipitation of proteins with cold trichloroacetic acid [24]. The accumulation of lipid droplets in adipocytes was monitored by staining with Oil Red O [12,25]. All of the quantified data were represented as the mean ± S.E.M. of three or more experiments. Student’s t test was used for evaluating statistical significance, and the difference was considered to be significant when p < 0.05. 3. Results 3.1. Preparation of cloned stable transfectants with L-PGDS oriented in the antisense direction Cultured 3T3-L1 cells were transfected stably with L-PGDS oriented in the antisense direction. Finally, we isolated several
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Fig. 1. Gene expression of L-PGDS in parent cells and the transfectants. (A) Transcript levels of L-PGDS. Untransfected 3T3-L1 cells and the transfectants with expression vector only or the vector with antisense L-PGDS were planted at 5 104 cells/ml in a 60-mm Petri dish containing 4 ml of the growth medium and grown to confluence. Total RNA was extracted from the confluent cells and subjected to the analysis of the transcript levels of L-PGDS, neomycin-resistant gene in the vector, and b-action as a control. (B) Western blot analysis of L-PGDS protein levels in cultured parent cells and the transfectants. The cell lysates (20 lg in each lane) were analyzed by Western blot analysis using a polyclonal antibody specific for mouse L-PGDS. Labels: parent, untransfected cells; vector, cloned transfectants with expression vector only; R-5 and R-13, cloned transfectants with L-PGDS in the antisense direction.
Fig. 2. Biosynthesis of prostanoids by cultured transfectants with antisense L-PGDS. Parent cells and the transfectants were plated at 5 104 cells/ml in a 35-mm Petri dish containing 2 ml of the growth medium and grown to confluence. The resulting cultured cells were incubated for 2 h with 10 lM arachidonic acid (A) or 10 lM A23187 (B). The culture medium was collected for the quantification of PGD2 by ELISA. Alternatively, the synthesis of PGE2 was determined by ELISA after the confluent cultured cells were incubated for 30 min with 10 lM arachidonic acid (C). Moreover, the monolayer cells were allowed to undergo the differentiation and maturation. The culture medium was collected after 6 days of the maturation phase and subjected to the determination of D 12-PGJ2 by ELISA (D). ⁄p < 0.05 compared with the transfectants with vector only along with vehicle. #p < 0.05 compared with the transfectants with vector only in the presence of arachidonic acid or A23187.
independent clones of the transfectants. Of these, attempts were made to characterize cloned transfectants R-5 and R-13 as representative ones. The neomycin-resistant gene in the vector was expressed in the cloned transfectants with the vector only or that with antisense L-PGDS (Fig. 1A), whereas it was not detectable in untransfected parent cells. As expected, the stable transfection of L-PGDS oriented in the antisense direction significantly suppressed the transcript levels (Fig. 1A) and the protein levels (Fig. 1B) of LPGDS in the cloned transfectants. By contrast, the transfection of cultured preadipocytes with the vector only had almost no influence on the expression levels of L-PGDS.
3.2. Biosynthesis of PGD2, PGE2, and D12-PGJ2 by cloned transfectants stably expressing antisense L-PGDS To assess the intracellular activity of L-PGDS in the stable transfectants or the related cells, the confluent cells during the growth phase were incubated with 10 lM exogenous free arachidonic acid (Fig. 2A) or 10 lM calcium ionophore A23187 (Fig. 2B). In all cases, the biosynthesis of PGD2 was appreciably attenuated in the transfectants with antisense L-PGDS. However, no significant difference was observed between the parent cells and the transfectants with the vector only. All of tested parent cells and the transfectants did
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Fig. 3. Storage of fats in cultured transfectants with L-PGDS in the antisense direction. Parent cells and the transfectants were plated and grown to confluence as described in Fig. 2. The monolayer cells were allowed to undergo the differentiation and maturation. The amount of triacylglycerols was quantified after 6 days and 12 days of the maturation phase (A). ⁄p < 0.05 compared with the transfectants with vector only after 6 days of the maturation phase. #p < 0.05 compared with the transfectants with vector only after 12 days of the maturation phase. Furthermore, the cultured cells were viewed by phase-contrast microscopy and differential-interference microscopy after Oil Red O staining at 200 magnification in addition to the observation of macroscopic views (B). Alternatively, the confluent cells were maintained in the growth medium for another 8 days by changing the same culture medium for the evaluation of spontaneous adipogenesis (C). The resulting cells were subjected to the determination of the amount of triacylglycerols. ⁄p < 0.05 compared with the transfectants with vector only. The cultured cells under the same conditions as the panel C were observed as microscopic views or macroscopic views as described in the panel B (D).
not show significant changes in the ability to stimulate the synthesis of PGE2 from exogenous free arachidonic acid (Fig. 2C). We have recently described the increased ability to generate endogenous PGJ2 series during the maturation phase [10,11]. Here, endogenous synthesis of D12-PGJ2 was measured after 6 days of the maturation phase (Fig. 2D). Both cloned transfectants with antisense L-PGDS synthesized endogenous D 12-PGJ2 at significantly lower levels compared to those of control cells. 3.3. Fat storage in stable transfectants with antisense L-PGDS during the maturation phase We then examined whether the reduction in the expression levels of L-PGDS could influence the storage of fats during the maturation phase. The storage of fats after 6 and 12 days was appreciably higher in the transfectants with antisense L-PGDS than that in the control cells (Fig. 3A). Similarly, the enhanced storage of fats was also confirmed by the observation of microscopic and macroscopic views of cultured adipocytes during the maturation phase (Fig. 3B). Alternatively, we noticed the spontaneous
adipogenesis of our confluent transfectants with antisense L-PGDS after the maintenance for total 8 days in the growth medium without further hormonal stimulations for undergoing the differentiation and maturation phases (Fig. 3C and D). These combined results clearly indicate that the sustained suppression of L-PGDS levels induces the up-regulation of adipogenesis. 3.4. Gene expression of adipocyte-specific markers in stable transfectants with antisense L-PGDS during the maturation phase To define whether the elevation of the fat storage in the stable transfectants with antisense L-PGDS could be due to the positive regulation of adipogenesis program, the transcript levels of adipocyte-specific markers in the transfectants were analyzed during the maturation phase (Fig. 4A). The transfectants with antisense L-PGDS expressed aP2, leptin, and GLUT-4 as typical adipogenesis markers at higher levels than other control cells although the expression levels of PPARc, LPL, and adiponectin remained unchanged. These findings overall support the positive regulation of adipogenesis program in the transfectants with antisense L-PGDS.
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Fig. 4. Gene expression levels of adipocyte-specific markers in cultured transfectants. Parent cells and the transfectants were plated and grown to confluence as described in Fig. 1. The monolayer cells were allowed to undergo the differentiation and maturation. Total RNA was extracted from cultured cells after 12 days of the maturation phase and applied to the analysis of transcript levels of aP2, leptin, PPARc, GLUT-4, adiponectin, LPL, and b-actin as a control (A). Alternatively, parent cells and the transfectants were plated and grown to confluence as described in Fig. 2. The monolayer cells were allowed to undergo the differentiation and maturation. During the maturation phase, the cultured cells were treated with either vehicle, PGE2 or PGF2a, or 15d-PGJ2 at 1 lM for total 6 days by replacing every 2 days with the fresh maturation medium containing the corresponding prostanoid species (B). After the treatments, the cells were harvested for the determination of the amount of triacylglycerols. ⁄p < 0.05 compared with the transfectants treated with vehicle. The macroscopic views of cultured cells under the same conditions as the panel B were observed after staining with Oil Red O (C).
3.5. Response to exogenous anti-adipogenic or pro-adipogenic prostanoids in stable transfectants with antisense L-PGDS during the maturation phase We investigated determine whether the cloned stable transfectants with antisense L-PGDS could respond to exogenous prostanoids. The cultured cells were treated with either of 1 lM PGE2 or PGF2a as anti-adipogenic prostanoids or 1 lM 15d-PGJ2 as a pro-adiogenic prostanoid during the maturation phase and then subjected to the quantification of the accumulated fats (Fig. 4B) and the observation of macroscopic views of cultured cells (Fig. 4C). Both PGE2 and PGF2a appreciably suppressed the storage fats in the transfectants with antisense L-PGDS. On the other hand, 15d-PGJ2 increased the fat storage additionally at a significant level. The results indicate that our cloned stable transfectants with antisense L-PGDS can respond to exogenous anti-adipogenic and pro-adipogenic prostanoids in a manner similar to the untransfected parent cells as described earlier [10,13]. 4. Discussion The function of PGD2 in adipocytes and the precursor cells is complicated because of its enzymatic or non-enzymatic conversion to other types of species exerting their effects differently on adipogenesis in adipocytes. To manipulate the intracellular levels of L-PGDS involved in the biosynthesis of PGD2 and the related PGJ2 derivatives, we undertook the approach to suppress the expression of L-PGDS by the sustained expression of antisense RNA for L-PGDS in cultured 3T3-L1 preadipocytes. The continuously expressed antisense RNA has been shown to interfere specifically the gene expression of the desired target [26]. The present study confirmed the appreciable suppression of the transcript and protein levels of L-PGDS in cultured preadipocytes transfected stably with L-PGDS
in the antisense direction. Additionally we provided the evidence for the substantial decrease in the synthesis of PGD2 in the cloned transfectants with antisense L-PGDS. Furthermore, the levels of D 12 -PGJ2 derived from PGD2 after 6 days of the maturation phase were much lower in the stable transfectants with antisense L-PGDS than the control cells. By contrast, the generation of PGE2 was not significantly influenced in the transfectants with antisense L-PGDS. The combined results clearly provided the evidence that our transfection method inhibited selectively the synthesis of PGD2 and the related PGJ2 series without affecting other pathways including upstream COX isoforms and other PGE synthases. The current study demonstrated that our cloned transfectants with antisense L-PGDS were able to enhance more greatly the storage of fats during the maturation phase. The accelerated accumulation of fats was accompanied by the up-regulated gene expression of typical adipogenesis markers, such as aP2, leptin, and GLUT-4, which are known to be under the control of PPARc [27]. These findings support the notion that the stimulation of fat storage in the transfectants is closely linked with the promotion of a series of adipogenesis program during the life stages of adipocytes. Additionally, we found the spontaneous adipogenesis in confluent monolayers of the cloned transfectants with antisense L-PGDS without undergoing the differentiation and maturation phases, providing another clear evidence for the up-regulation of adipogenesis. Moreover, our study confirmed that the transfectants with antisense L-PGDS responded to anti-adipogenic PGE2 and PGF2a and pro-adipogenic 15d-PGJ2 during the maturation phase as normally as that of the parent cells as described earlier [10,13]. The present results appear to be consistent with an earlier study where L-PGDS-knockout mice showed accelerated accumulation of triacylglycerols in hypertrophic adipocytes [16]. By contrast, a previous study has reported the reduced adipogenesis in cultured adipocyes using the siRNA interference of L-PGDS [28].
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Thus, it is sill ambiguous about the role of L-PGDS in adipogenesis due to the complex actions of PGD2 and the related species at different stages of adipocytes. Previous studies have described that 15d-PGJ2 is the most potent natural ligand to activate PPARc as a master regulator of adipogenesis [5,6]. Furthermore, exogenous 15d-PGJ2 and D 12-PGJ2 have been shown to be effective in the stimulation of adipogenesis in cultured adipocytes during the maturation phase [10,11]. As well, cultured adipocytes at the later maturation phase are capable of producing endogenous PGJ2 series [10,11]. Hence, we initially hypothesized that stable transfection of cultured preadipocytes with antisense L-PGDS should suppress the adipogenesis program leading to the storage of fats by interfering the endogenous synthesis of those pro-adipogenic prostanoids. Nevertheless, our cloned preadipocytes transfected with antisense L-PGDS exhibited adipogenesis at higher levels. Although the exact mechanism remains obscure, a possible contribution to it might involve the reduced synthesis of 9a, 11b-PGF2a, known as an anti-adipogenic prostanoid [15]. This compound can be formed from the enzymatic reduction of PGD2 in some tissues [29]. Alternatively, PGD2 itself is known to bind to its specific receptors termed the DP1 and CRTH2 receptors [2,3], and to increase the intracellular levels of cAMP [2]. This second messenger might potentially transmit the anti-adipogenic signal to the untransfected parent cells at some life stages of adipocytes by analogy with the EP4 receptor of anti-adipogenic PGE2 [30]. As a separate mechanism independent of endogenous prostanoids, spontaneous differentiation of cultured 3T3-L1 preadipocytes has been reported to occur without standard hormonal stimulation while expressing the active phosphorylated form of Akt kinase [17]. Currently, we also found the spontaneous differentiation of our cloned transfectants with antisense L-PGDS. Recently, L-PGDS has been described to inhibit the Akt phosphorylation in other cells [16]. Therefore, the reduced expression of L-PGDS in our transfectants with antisense L-PGDS may account partly for the up-regulation of adipogenesis through the activation of Akt kinase. Further extensive studies remain to be done to define the detailed mechanisms. In conclusion, we established cloned preadipocytes transfected permanently with L-PGDS in the antisense direction. The cloned transfectants with antisense L-PGDS exhibited an appreciable increase in the fat storage in adipocytes during the maturation phase. These findings provided the unique features of the stable transfectants with antisense L-PGDS, which are useful for further studies to dissect the molecular and cellular mechanisms underlying the effects of L-PGDS through PG-dependent or PG-independent manners. References [1] O. Hayaishi, Sleep-wake regulation by prostaglandins D2 and E2, J. Biol. Chem. 263 (1988) 14593–14596. [2] Y. Boie, N. Sawyer, D.M. Slipetz, et al., Molecular cloning and characterization of the human prostanoid DP receptor, J. Biol. Chem. 270 (1995) 18910–18916. [3] H. Hirai, K. Tanaka, O. Yoshie, et al., Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seventransmembrane receptor CRTH2, J. Exp. Med. 193 (2001) 255–261. [4] T. Shibata, M. Kondo, T. Osawa, et al., 15-Deoxy- D 12,14-prostaglandin J2: a prostaglandin D2 metabolite generated during inflammatory processes, J. Biol. Chem. 277 (2002) 10459–10466. [5] B.M. Forman, P. Tontonoz, J. Chen, et al., 15-Deoxy- D 12,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPARc, Cell 83 (1995) 803–812. [6] S.A. Kliewer, J.M. Lenhard, T.M. Willson, et al., A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor c and promotes adipocyte differentiation, Cell 83 (1995) 813–819.
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