Overexpression of human acyl-CoA thioesterase upregulates peroxisome biogenesis

Experimental Cell Research 297 (2004) 127 – 141 www.elsevier.com/locate/yexcr

Overexpression of human acyl-CoA thioesterase upregulates peroxisome biogenesis Mitsuru Ishizuka, a,b Yoshiro Toyama, c Hiroyuki Watanabe, a,1 Yukio Fujiki, d Arata Takeuchi, a,e Sho Yamasaki, a,e Shigeki Yuasa, c,2 Masaru Miyazaki, b Nobuyuki Nakajima, b Shinsuke Taki, a,3 and Takashi Saito a,e,* a

Department of Molecular Genetics, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan b Department of General Surgery, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan c Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan d Department of Biology, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan e Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa 230-0045, Japan Received 19 August 2002, revised version received 10 February 2004 Available online 15 April 2004

Abstract The biological functions of human acyl-CoA thioesterase III (ACTEIII/PTE-1), initially identified as an HIV-1 Nef binding protein, have remained unclear. We report herein that the stable overexpression of ACTEIII/PTE-1 in human and murine T-cell lines resulted in an increase in both peroxisome number and lipid droplet formation in a manner dependent on the amount of the protein. Peroxisome proliferation was evidenced by immunofluorescence staining for catalase, a peroxisome marker protein, as well as by direct peroxisome enumeration on electron micrographs. Consistently, the amount of catalase was elevated as the amount of ACTEIII/PTE-1 was increased. ACTEIII/PTE-1 mutants with reduced enzymatic activity or with the defect in peroxisome localization did not induce peroxisome proliferation, indicating that peroxisome proliferation was mediated by metabolites generated by ACTEIII/PTE-1 within peroxisomes. Finally, thymocytes isolated from a T-cell-specific ACTEIII/PTE-1 transgenic mouse as well as human and murine cell lines of lymphoid and non-lymphoid origins exhibited a similar proliferation of peroxisomes. Thus, ACTEIII/PTE-1 may be involved in the metabolic regulation of peroxisome proliferation. D 2004 Elsevier Inc. All rights reserved. Keywords: Acyl-CoA thioesterase III; Catalase; Human immunodeficiency virus-1; Lipid droplet; Nef; Peroxisome; T cell; Transfection; Transgenic mouse

Introduction The biosynthesis and maintenance of cellular organelles should be tightly controlled to maintain cell function. Many hereditary disorders are said to be associated with

* Corresponding author. Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology (RCAI), 1-7-22 Suehirocho, Tsurumi-ku, Yokohoma 230-0045, Japan. Fax: +81-45-503-7036. E-mail address: [email protected] (T. Saito). 1 Present address: Department of Psychiatry, Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan. 2 Present address: Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan. 3 Present address: Division of Immunology, Department of Immunology and Infectious Diseases, Graduate School of Medicine, Shinshu University, Matsumoto, Nagano 390-8621, Japan. 0014-4827/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.02.029

alterations in organelle morphology and number [1,2]. A number of neurological disorders [3], including Zellweger syndrome [4], neonatal adrenoleukodystrophy, and infantile Refsum disease [5], have been shown to be associated with defects in peroxisome biogenesis and are classified as peroxisome biogenesis disorders (PBDs). Peroxisomes, present in essentially all eukaryotic cells, play important roles in such metabolic pathways as the generation of H2O2, the h-oxidation of fatty acids and the biosynthesis of ether lipids [6]. Peroxisomes are believed to be generated from pre-existing peroxisomes by the incorporation of various peroxisomal proteins [7,8]. However, the mechanism involved in peroxisome biogenesis has not been completely elucidated so far [9]. Genetic studies of fibroblasts isolated from PBD patients and mutant CHO cells have led to the identification of more than 15 complementation groups [10 – 13]. At present, although not all the

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genes responsible for those defects have been cloned, much insight has been obtained regarding the molecular mechanisms by which the products of those genes function in the process of peroxisome biogenesis, including matrix protein incorporation and membrane assembly [14]. Nevertheless, it is obvious that much more effort is necessary to completely understand the molecular mechanism involved in peroxisome biosynthesis. Recently, genetic alterations affecting h-oxidation in peroxisomes were found to influence peroxisomal abundance by two research groups [15,16]. However, those two groups were inconsistent with regard to how h-oxidation regulates peroxisome biosynthesis. Mice lacking acyl-CoA oxidase (AOX) exhibited excessive peroxisomal proliferation in the liver [17], whereas fibroblast cell lines defective in the same enzyme or 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase were shown to contain less peroxisomes [15]. Although it is not clear at present what caused the apparent discrepancy, the reports raised the possibility that the number of peroxisomes is under metabolic control. Recently, two human acyl-CoA thioesterases compartmentalized in peroxisomes were identified as PTE-1 and-2 [18,19], which hydrolyze acyl-CoAs into free fatty acids and SH-CoA. We cloned PTE-1 independently as an HIV-1 Nef binding molecule using a yeast two-hybrid system from a cDNA library established from the human T-cell line Jurkat [20]. As the Nef binding protein exhibited 42% amino acid sequence homology to Escherichia coli thioesterase II [21 –23] and 85% homology to murine PTE-2, we named it acyl-CoA thioesterase III (ACTEIII/PTE-1; [20]). Uniquely, ACTEIII/PTE-1, but not its E. coli homologue thioesterase II, has a serine –lysine– leucine motif termed peroxisome targeting signal 1 (PTS-1) in its C terminus, which is the basis for its presence in peroxisomes [18]. This characteristic is unique and is not found in other thioesterases present in cytosol [19], mitochondria [24 – 26], and lysosomes [27,28]. However, the physiological functions of ACTEIII/PTE-1 are not known to date, and it is surmised that ACTEIII/PTE-1 plays some role in fatty acid oxidation in peroxisomes. In this regard, the observation that compounds known to stimulate peroxisomal proliferation induce the upregulation of ACTEIII/PTE-1 expression may indicate that ACTEIII/PTE-1 plays a role in the metabolic regulation of peroxisome biogenesis. In fact, there was an observation that the transient expression of the enzyme in a cultured cell line reduced the abundance of peroxisomes [15]. It has been shown that the enzymatic activity of ACTEIII/ PTE-1 is enhanced upon binding to Nef protein [20,29] and the association of Nef protein with ACTEIII/PTE-1 is essential for the down modulation of surface CD4 and class I HLA molecules by Nef protein [29]. Interestingly, a fraction of cellular Nef proteins have been found to colocalize with ACTEIII/PTE-1 in peroxisomes [30]. Although the mechanism of the Nef-mediated enhancement of

ACTEIII activity is not known in vivo, the functional relationship between ACTEIII/PTE-1 and Nef-induced cellular pathogenesis remains to be determined. In this regard, it is necessary to understand the functions of ACTEIII/PTE-1 in T cells. During the course of our investigation of the role of ACTEIII/PTE-1 in T cells, we expressed human ACTEIII/ PTE-1 cDNA in the human T-cell line Jurkat using a retrovirus-mediated gene transfer method. Unexpectedly, the transfected cell lines exhibited several morphological alterations, such as an increase in peroxisome number and an accumulation of lipid droplets, in a fashion dependent on the amount of ACTEIII/PTE-1. Importantly, an ACTEIII/PTE-1 mutant having reduced enzymatic activity did not exhibit peroxisome proliferation in the Jurkat cell line, indicating that the morphological alteration was mediated by mechanisms dependent on the enzymatic activity of ACTEIII/PTE1. The ACTEIII/PTE-1-mediated peroxisome proliferation was also observed in ACTEIII/PTE-1-transfected cell lines of various origins in addition to human and murine T cell lines. Furthermore, we observed a similar peroxisome proliferation in thymocytes from transgenic mice expressing human ACTEIII/PTE-1 in a T-cell-specific manner. Thus, the effect of ACTEIII/PTE-1 on peroxisome proliferation is not an in vitro artifact caused by artificial culture conditions. Taken together, our study revealed a novel function of ACTEIII/PTE-1 in peroxisome proliferation that is dependent on its amount and activity. The difference between the previous conclusion on the role of ACTEIII/PTE-1 in peroxisome proliferation [15] and our findings will be discussed.

Materials and methods Cells and Abs The human T lymphoma cell line Jurkat (line E6.1), the murine T-cell hybridoma 2B4, the murine B-cell line A20, the murine mast cell line P815, and the murine adrenal cortex cell line Y1 were cultured in RPMI 1640 containing 10% FCS, 100 Ag/ml L-glutamine, 100 units/ml penicillin, 100 Ag/ml streptomycin, and 50 AM 2-mercaptoethanol. A Jurkat clone expressing the ecotropic retrovirus receptor was established by transfection of the ecotropic receptor [31]. NIH3T3, a mouse neuroblastoma cell line; C1300, a kind gift from Dr. K Ando (National Institute of Radiological Science, Chiba, Japan); and a retrovirus packaging cell line, Phoenix, which was kindly provided by Dr. G. P. Nolan (Stanford University); were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, 100 Ag/ml Lglutamine, 100 units/ml penicillin, 100 Ag/ml streptomycin, and 50 AM 2-mercaptoethanol. Donkey anti-mouse Radixin Ab was purchased from Santa Cruz Biotechnology, Inc. Anti-Lck mAb (mol 171) was a kind gift from Dr. Y. Koga (Tokai Univ., Kanagawa, Japan). Rabbit anti-human catalase Ab was prepared as

M. Ishizuka et al. / Experimental Cell Research 297 (2004) 127–141

described [32]. Mouse anti-human ACTEIII/PTE-1 mAb has been newly established in this study by immunizing BALB/c mice with GST-ACTEIII/PTE-1 fusion protein by Dr. H. Kato (Yamasa Corp., Chiba, Japan). This antihACTEIII/PTE-1 mAb can be used for immunoblot and immunoprecipitation (see below). Secondary reagents used were as follows: horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (Upstate Biotechnology, Inc.). Cy3-conjugated antimouse IgG, Cy5-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Jackson Immuno Research (West Grove, PA). Transfections Full-length ACTEIII/PTE-1 cDNAs were subcloned into pMX-IRES-GFP expression vector (pMX-IRES-GFP/ ACTEIII/PTE-1) and transfected into Phoenix cell line using Lipofectamine (Gibco-BRL). The culture medium was replaced with a fresh one 24 h after transfection and supernatants containing recombinant viral particles were collected 24 h later. Virus was enriched 30-fold by centrifugation of collected supernatant at 10,000  g for 16 h at 4jC. 2B4, Eco-Jurkat, A20, P815, NIH3T3, TM4, and Y1 cells (1  105) were infected in 500 Al of medium containing 10 Al of DOTAP (Roche) and 400 Al of condensed viral culture supernatants. Analysis of enzymatic activity of ACTEIII/PTE-1 The enzymatic activity of ACTEIII/PTE-1 was measured by spectrophotometric assay as previously described [20]. Briefly, 2.4 AM recombinant fusion protein of GSTACTEIII/PTE-1 and its mutants or GST-Nef (as control) was incubated with 1 AM acyl-CoA at 37jC in the reaction buffer (50 mM Tris – HCl (pH 7.0), 0.2 mM CaCl2, 1 mM 5,5V-dithiobis(2-nitrobenzoate) (DTNB)) and the free CoASH liberated by the enzymatic activity was detected. Indirect immunofluorescence and confocal microscopy Adherent cells such as NIH3T3 and Y1 cells were grown on cover slips, whereas non-adherent cells such as Jurkat, 2B4, and P815 were fixed in 4 AM paraformaldehyde/PBS for 20 min and washed with PBS twice. Abs used for staining were diluted with PBS containing 0.1 AM saponin and 0.1 mM BSA. Cy3- and Cy5-conjugated Abs were used for the development of the primary antibodies. Digital images of the cells were acquired using a laser scanning microscope (LSM510, ZEISS, Jena, Germany). Immunoblotting Cells were lysed at 4jC for 10 min with RIPA lysis buffer containing 1% NP-40, 0.1% SDS, 280 mM NaCl, 0.2 mM EDTA, 10 mM NaF, 1 mM phenylmethylsul-

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fonyl fluoride, leupeptin (1 Ag/ml), aprotinin (2 Ag/ml), antipain (0.5 Ag/ml), chymostatin (0.5 Ag/ml), pepstatin (1 Ag/ml), and 5 mM iodoacetamide. Cell lysates were subjected to SDS-PAGE and the proteins were transferred to a PVDF membrane (Millipore). The membrane was incubated with primary antibodies and visualized using the ECL detection system (Amersham) with appropriate secondary Abs. RT-PCR Total cellular RNA was extracted by guanidinium –isothiocyanate method, and cDNA was prepared by RT-PCR. Amplification was performed under the following condition: 94jC for 4 min, 22 (for GADPH), 25 (for human acylCoA oxidase, AOX), and 37(for human Peroxisome Proliferator Activated Receptor gamma, PPARg) cycles of 94jC for 15 s, 58jC for 30 s, and 72jC for 1 min. Primers used for AOX were 5V-ACCTACGCCCAGACGGAAAT and 3V-TGCCCACCACAAGCCATC, and for PPARg 5VGCGAGGGCGATCTTGACAG and 3V-GGAGGCCAGCATTGTGTAAA. Establishment and analysis of human ACTEIII/PTE-1-transgenic mice A full-length human ACTEIII/PTE-1 cDNA was cloned into an expression vector containing the proximal lck promoter [33]. The transgenic construct was then injected into the pronuclei of fertilized eggs of C57BL/6 mice. Mice carrying the transgene were screened by PCR using human ACTEIII/PTE-1-specific primers. T cells were purified from either thymus or spleen using MACS (Miltenyi Biotech, Germany) with anti-CD3 mAb. Electron microscopy Adherent cultured cells such as NIH3T3, 293, Y1, and TM4 were fixed with 3% glutaraldehyde and embedded in Epon according to the method of Toyama et al. [34]. Nonadherent cultured cells, such as 2B4, Jurkat, A20, and P815, were fixed by adding glutaraldehyde into the culture medium to a final concentration of 3%. The method was previously established [35]. Part of the thymus from wild-type and transgenic mice was immersed in 3% glutaraldehyde buffered with 10 mM Hepes, and embedded in a conventional manner [34,36]. Ultrathin sections were stained with uranyl acetate and lead citrate and observed on a 1200 EX II (Jeol Co., Ltd., Tokyo). For peroxidase staining, cells were centrifuged, fixed with 1.25% glutaraldehyde and 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4) for 2 h at room temperature, embedded in 1% agar in the same buffer, and fixed again for 2 h. After washing overnight in the same buffer, the cells were incubated for 1 h at 37jC in the medium described by Fahimi [37]: 4.83 ml Tris buffer

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(0.1 M, pH 7.6), 0.17 ml hydrogen peroxide (30% solution) containing 2.5 mg 3,3V-diaminobenzidine (DAB, Dojindo Labs., Kumamoto, Japan). The cells were washed in two changes of Tris buffer, post-fixed with 3% glutar-

aldehyde and 4% paraformaldehyde in 10 mM Hepes buffer, and then processed for conventional electron microscopy. Control specimens were incubated in the medium excluding hydrogen peroxide.

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Fig. 1. Overexpression of ACTEIII/PTE-1 in Jurkat cells results in lipid droplet accumulation and an increase in the number of peroxisomes. (A) Electron micrographs of ACTEIII/PTE-1 transfectants (Tf). Jurkat cells were transfected with pMX-ACTEIII/PTE-1-IRES-GFP or vector alone (MOCK) by retrovirusmediated gene transfer. GFP+ cells were sorted and examined by electron microscopy. Note numerous lipid droplets in cytoplasm (dark vesicles). Bar indicates 5 Am. Bottom: Expression of ACTEIII/PTE-1 in Jurkat Tf. Cell lysates of MOCK Tf and ACTEIII/PTE-1 Tf (1  106 each) were blotted with anti-ACTEIII/ PTE-1 mAb and anti-catalase Ab. Blots for lck are also shown as loading controls. (B) Expression of ACTEIII/PTE-1 in ACTEIII/PTE-1-transfected Jurkat clones. Cell lysates (1  106 each) of Jurkat clones were blotted with anti-ACTEIII/PTE-1 mAb and anti-lck mAb. Densitometric analysis revealed that the amounts ACTEIII/PTE-1 in clones A79, 26, and 106 were approximately 10-, 30-, and 90-fold larger than that of endogenous ACTEIII/PTE-1 in mock transfectant, respectively. Blots for lck are shown as loading controls. (C) Number of lipid droplets in ACTEIII/PTE-1-transfected Jurkat clones. Lipid droplets were counted for 20 cells in electron micrographs of each clone. Histogram analysis revealed the number of cells containing the indicated number of lipid droplets. Cumulative numbers of cells showing the given numbers of lipid droplets are plotted. (D) High-magnification electron micrograph of ACTEIIItransfected Jurkat cell. Arrowhead indicates peroxisomes in ACTEIII/PTE-1 Tf. Bar indicates 5 Am. (E) Number of peroxisomes in ACTEIII/PTE-1-transfected Jurkat clones. The number of peroxisomes was counted for 20 cells in electron micrographs of each clone. Histogram analysis revealed the number of cells containing the indicated number of peroxisomes. Cumulative numbers of cells showing the given numbers of peroxisomes are plotted. (F) High-magnification electron micrograph for peroxidase staining of wild-type (MOCK Tf, the left panel) and ACTEIII/PTE-1-transfected (ACTEIII-Tf, the right two panels) Jurkat cells. Peroxidase in cells was stained as described in Materials and methods. Right panel shows a high-magnification photo for ACTEIII/PTE-1 transfectants. Bar indicates 5 Am. The numbers of peroxisomes analyzed in D and F were assessed in t test and no significant difference was found, indicating that both methods detect peroxisome similarly. (G) Number of peroxisomes in ACTEIII/PTE-1-transfected Jurkat by analyzing peroxidase staining. Number of peroxisomes was counted for about 150 cells according to the method described in Materials and methods. The numbers indicate mean F S.E.M. (H) Immunofluorescence cytostaining of ACTEIII/PTE-1-transfected Jurkat clones. ACTEIII/PTE-1-transfected Jurkat clones A79, 26, 106 and control mock transfectant (MOCK) were stained for ACTEIII/PTE-1 with mouse anti-human ACTEIII/PTE-1 mAb followed by Cy5-labeled secondary Ab, and for catalase with rabbit anti-human catalase Ab followed by Cy3-labeled secondary Ab. Blue spot (Cy5 staining, ACTEIII/PTE-1) and red spot (Cy3 staining, catalase, peroxisome marker protein) clearly merged to form the pink spot. (I) Expression of acyl-CoA oxidase (AOX) in ACTEIII/PTE-1 transfectant. mRNA expression of AOX in MOCK and ACTEIII/PTE-1 transfectants (Tf.) were analyzed by RT-PCR using primers specific for AOX and GADPH (control). A different amount of cDNA (3-fold) was used for each sample. (J) Expression of peroxisome proliferator activated receptor-g (PPARg) in ACTEIII/PTE-1 transfectants. mRNA expression of PPARs in mock (Mock) and ACTEIII transfectants (Tf) was analyzed by RT-PCR using primers specific for PPARg and GADPH (control).

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For quantitation of lipid droplets and peroxisomes, we enumerated both lipid droplets and peroxisomes by counting in a photo of a section of a single cell in electron micrographs for more than 20 cells for each sample. More than 160 cells were counted for peroxidase staining. Only one section was chosen for one cell. The photo was used only for the cells that showed round with the nucleus in the center and equal length of cytoplasm for both sides of the nucleus. Cells that do not fit these criteria were excluded for quantitation.

light microscopy. However, electron microscopy revealed the presence of numerous lipid droplets in a considerable fraction of GFP+ Jurkat cells, which were rarely observed in control cells (Fig. 1A). Limiting dilution culture was carried out to isolate Jurkat clones expressing various amounts of ACTEIII. Of the more than 30 clones thus established, three representative clones, A79, 26, and 106, were chosen for further analysis, expressing roughly 10-, 30-, and 90-fold increases in the amount of ACTEIII, respectively, compared with mock-transfected Jurkat cells as judged from Western blots (Fig. 1B, Table 1). When inspected under an electron microscope (EM), a significant accumulation of lipid droplets was observed only in a clone A106 that exhibited a 90fold increase in the amount of ACTEIII. To quantitatively examine the extent of lipid droplet formation, we enumerated the number of lipid droplets found within a section of a single cell in electron micrographs (one section per cell) for at least 20 cells that clearly show the nucleus and the cytoplasm. The histograms in Fig. 1C show the number of cells plotted against the number of lipid droplets contained within the sections of the cells. There was essentially no difference among clones A79 and A26 and control Jurkat cells (MOCK) as less than one lipid droplet/section was found (summarized in Table 1). In contrast, more than six lipid droplets/section on average were counted in clone A106 sections. The results indicate that lipid droplets were formed when the amount of intracellular ACTEIII/PTE-1 exceeded a certain threshold.

Data Analysis Results are expressed as means F SEM. The value n represents the number of measurements of individual cell samples. Statistical comparisons were done using the Student’s t test with P < 0.05 considered significant.

Results and discussion Accumulation of lipid droplets in Jurkat cells by overexpression of ACTEIII An expression vector for human ACTEIII cDNA was introduced into the human T-cell line Jurkat by retrovirusmediated transfection. This vector (pMX-IRES-GFP/ ACTEIII) carried an internal ribosome entry site (IRES) and cDNA for green fluorescence protein (GFP), 3V of the ACTEIII cDNA, to allow simultaneous expression of ACTEIII and GFP in a cell. Transfected Jurkat cells were then sorted out from bulk population on the basis of GFP expression (GFP+ Jurkat). Western blot analysis showed a roughly 40-fold increase in the amount of ACTEIII protein in the cells (Fig. 1A, Table 1). The cells exhibited normal growth and no morphological changes were apparent by

Peroxisome proliferation but not lipid droplet accumulation is induced in Jurkat clones expressing relatively small amounts of exogenous ACTEIII When examined by high-magnification EM, we noted that in addition to lipid droplet accumulation, the number

Table 1 Expression of ACTEIII and catalase, and increase in the number of peroxisomes and lipid droplets in ACTEIII-transfected cells Parent cell

Transgene

ACTEIII (fold increase)

Catalase (fold increase)

Peroxisome (number/cell)

Lipid droplet (number/cell)

Jurkat

MOCK ACTEIII 79H/A DSKL ACTEIII A79 (clone) ACTEIII A26 (clone) ACTEIII A106 (clone) MOCK ACTEIII MOCK ACTEIII MOCK ACTEIII ACTEIII( ) ACTEIII(+/ ) ACTEIII(+/+)

1.0 45.6 61.5 68.7 7.8 27.6 90.7 1.0 98.2 1.0 55.4 1.0 75.7 1.0 14.0 30.3

1.0 1.6 0.7 1.0 1.1 1.6 2.2 1.0 1.7 1.0 1.2 1.0 1.4 1.0 1.4 1.6

1.8 2.8 0.2 1.3 1.6 4.9 4.3 0.3 1.3 0.2 0.9 0.4 1.6 0.2 1.1 0.8

0.4 F 3.4 F 0.3 F N.D. 1.1 F 0.5 F 6.5 F 0.7 F 2.5 F N.D. N.D. N.D. N.D. N.D. N.D. N.D.

2B4 P815 A20 Thymocyte (ACTEIII-Tg mouse)

F F F F F F F F F F F F F F F F

0.0 24.9 25.9 16.3 1.2 2.7 19.3 0.0 11.8 0.0 23.1 0.0 32.1 0.0 4.7 14.9

F F F F F F F F F F F F F F F F

0.0 0.4 0.1 0.1 0.5 0.1 0.2 0.0 0.4 0.0 0.2 0.0 0.1 0.0 0.2 0.1

N.D.: not detected. Each value represents mean F S.E.M. A79, A26 and A106 were ACTEIII-transfected Jurkat clones. * Statistically significant ( P < 0.01) based on the Student’s t test.

F F F F F F F F F F F F F F F F

0.4 0.8 0.1* 0.4 0.3 0.6* 0.7* 0.1 0.3* 0.1 0.2* 0.1 0.3* 0.1 0.3* 0.2*

0.2 1.1* 0.1 0.3 0.3 1.0* 0.2 0.8*

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of peroxisomes, which are electron-dense, monolayer-surrounded microbodies in the cytoplasm, was increased in ACTEIII transfectants (Fig. 1D). The relative number of peroxisomes was determined using the method similar to lipid droplet, that is, counting the peroxisome number in the section (one section per cell) for more than 20 cells (Fig. 1E and Table 1 for summary). A statistically significant increase ( P < 0.01 as determined by the Student’s t test) was observed for clone A106 (average peroxisome number per section: 4.3 F 0.7) in addition to the GFP+ Jurkat population (2.8 F 0.8), compared with control cells (1.8 F 0.4). In contrast, no peroxisome proliferation was observed in clone A79 despite its ACTEIII/PTE-1 expression of more than 10-fold compared to control cells. Interestingly, clone A26 that did not show lipid droplet accumulation exhibited peroxisome proliferation to an extent similar to that observed for A106 (4.9 F 0.6 peroxisomes per section; Fig. 1E), in spite of the fact that A26 expressed 3-fold less ACTEIII/PTE-1 than A106 (Fig. 1B, Table 1). We also confirmed that the electron-dense microbodies counted were peroxisomes by staining for peroxidase using the substrate DAB, that is exclusively localized in peroxisomes. The increase of peroxisome in transfectants was confirmed (Figs. 1E and 1F), and the number of peroxisomes was correlated in two different quantitation protocols. To confirm peroxisome proliferation in clones A26 and A106, immunofluorescence staining for catalase, a peroxisomal marker protein, was carried out. Consistent with the relative peroxisome numbers enumerated above, there were much more vesicles positive for catalase in clones A26 and A106 than in control cells and clone A79 (Fig. 1H). It was noted that some catalase-fluorescence-positive vesicles in clones A26 and A106 were clearly larger than those in control cells. Such large vesicles seemed to represent amalgamated images of several peroxisomes located close to each other because the average size of peroxisomes as observed in electron micrographs was not significantly different among the cells examined (data not shown). The total amounts of catalase were also increased by 1.6- and 2.2-fold in clones A26 and 106, respectively, compared to that in control cells (Table 1). Together with the enumeration on electron micrographs and catalase immunofluorescence, the biochemical results indicate that the overexpression of ACTEIII/PTE-1 in the human T-cell line Jurkat resulted in the upregulation of peroxisome proliferation. We confirmed peroxisome proliferation furthermore by measuring the enhanced expression of peroxisomal acyl-CoA oxidase (AOX), which is a well-known PPARa target gene and a marker for peroxidase proliferation. Indeed, RT-PCR analysis revealed that ACTEIII/PTE-1 transfectants express several fold higher level of AOX than mock-transfected Jurkat cells (Fig. 1I). Because peroxisome proliferator-activated receptors (PPAR) regulate AOX expression and fatty acid metabolism, the expression of PPARs (a, g, and y) was

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also analyzed by RT-PCR. PPAR a, g, and y were all detected in Jurkat cells. While there was no significant difference in the expression of PPARa and y between mock and ACTEIII/ PTE-1 transfectants (data not shown), the expression of PPARg was clearly increased in ACTEIII/PTE-1 transfectants (Fig. 1J). These results suggest that ACTEIII/PTE-1 may induce peroxisome proliferation through augmentation/ activation of PPARg. Peroxisome proliferation and lipid droplet accumulation in other types of cells expressing human ACTEIII/PTE-1 Murine T-cell hybridoma 2B4 was also transfected with the same human ACTEIII/PTE-1. The transfected 2B4 cells expressed human ACTEIII/PTE-1 at a level approximately 100 times higher than that of control transfectants (Fig. 2A and Table 1), well above the amounts required for peroxisome proliferation as well as lipid droplet formation. In fact, the transfected 2B4 cells showed both peroxisome proliferation (Fig. 2C) and lipid droplet formation (Fig. 2D) as well as an increase in the amount of catalase (Figs. 2A and 2B and Table 1). These results indicate that human ACTEIII/ PTE-1 can function in murine cells regardless of species, and suggest that the downstream machinery leading to peroxisome proliferation and lipid droplet accumulation triggered by ACTEIII/PTE-1 overexpression is conserved between mouse and human. In agreement with this suggestion, other murine cell lines, A20, a B-cell lymphoma line (Fig. 2) and P815, a mastcytoma line (data not shown), exhibited similar peroxisome proliferation and elevation in the amount of catalase upon forced expression of human ACTEIII/PTE-1 (Figs. 2A – 2D). In addition, we observed increases in the amount of catalase and the number of peroxisomes in a human adrenal cortex-derived cell line, Y1, a mouse fibroblast cell line, NIH3T3 (Fig. 3), a human sertoli cell originated cell line, TM4, and a human fibroblast cell line, 293 (data not shown). The increase in the amount of catalase in NIH3T3, Y1, 293, and TM4 was 1.9 F 0.3, 1.7 F 0.2, 1.2 F 0.1, and 1.2 F 0.1, respectively, and indicated that the effect of ACTEIII/PTE-1 is not cell-type-specific. It is therefore likely that a metabolic pathway(s) common to all these cell types mediates the peroxisome proliferating effects of ACTEIII/PTE-1. In these cells, lipid droplet formation was not observed probably because the expression of ACTEIII protein was not sufficiently high to induce the lipid droplet formation. Enzymatic activity of ACTEIII/PTE-1 is required for the induction of peroxisome proliferation In human ACTEIII/PTE-1, the amino acid residues in the region corresponding to the active center of the E. coli counterpart thioesterase II including amino acids (VHSVHS) [22] are highly conserved. Thus, although the active center of human ACTEIII/PTE-1 was not formally identi-

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fied, it was assumed that the conserved histidine residue corresponding to the same amino acid residue that was shown to be essential for E. coli enzymatic activity [21,22,38] was also critical for the enzymatic activity of

human ACTEIII/PTE-1. On the basis of this assumption, an ACTEIII/PTE-1 mutant was created by replacing the histidine residue at position 79 with alanine (79H/A). The acylCoA hydrolyzing activity of the mutant enzyme 79H/A

Fig. 2. Murine T-cell hybridoma and B-cell line show increases in catalase expression and the number of peroxisomes upon ACTEIII/PTE-1 overexpression. (A) Expression of transfected human ACTEIII/PTE-1 in murine T-cell hybridoma 2B4 and murine B-cell line A20. 2B4 and A20 were transfected with pMXIRES-GFP/ACTEIII/PTE-1 (ACTEIII) or the control vector (MOCK) and GFP+ cells were sorted out. Cell lysates (1  106 each) prepared from transfectants were blotted with anti-ACTEIII/PTE-1 mAb and anti-catalase Ab. Blots for lck are included as loading controls. (B) Immunofluorescence cytostaining for ACTEIII/PTE-1 and catalase in transfectants. The distribution of ACTEIII/PTE-1 and catalase was analyzed by confocal laser microscopy in ACTEIII/PTE-1 transfectants (ACTEIII) and control mock transfectants (MOCK) of 2B4 and A20 cells. ACTEIII/PTE-1 was stained with anti-human ACTEIII/PTE-1 mAb followed by Cy5-labeled secondary Ab, and catalase was stained with anti-human catalase Ab followed by Cy3-labeled secondary Ab. Blue spot (Cy5 staining, ACTEIII) and red spot (Cy3 staining, catalase) clearly merged to form the pink spot. (C) Number of peroxisomes in ACTEIII/PTE-1-transfected 2B4 and A20 cells. The number of peroxisomes was counted for 20 cells in electron micrographs of MOCK and ACTEIII/PTE-1 transfectants. Note that the number of peroxisomes was larger in ACTEIII/PTE-1 Tf than in MOCK, similar to ACTEIII/PTE-1-transfected Jurkat. (D) Number of lipid droplets in ACTEIII/PTE-1transfected 2B4 and A20 cells. The number of lipid droplets was counted for 20 cells in electron micrographs of MOCK and ACTEIII/Tf Note that A20 did not induce any lipid droplet while the number of peroxisome was increased.

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Fig. 2 (continued).

expressed as a glutathione S-transferase (GST) fusion protein was less than 20% of that of the GST wild-type enzyme (Fig. 4A). This indicates that although the histidine residue might not be the active center itself, it played an important role in the enzymatic activity. The mutant ACTEIII/PTE-1 was expressed in Jurkat cells as described above. The amount of mutant 79H/A was roughly 60-fold larger than that of endogenous ACTEIII/PTE-1 in the transfectants as estimated by Western blotting (Fig. 4B, Table 1). Electron micrographs showed that the number of lipid droplets was not elevated and the average number of peroxisomes/ section was significantly reduced in 79H/A-transfected Jurkat cells compared with mock transfectants (Fig. 4C and Table 1), with the amount of catalase being reduced accordingly (Fig. 4D). As the mutant 79H/A expression level in 79H/A-transfected cells was lower than that of clone A106 that exhibited lipid droplet formation, we could not conclude if the reduction in enzyme activity affected the ability of ACTEIII/PTE-1 to induce lipid droplet formation. However, as the expression levels of the mutant 79H/A in these cell lines were much higher than those in clone A26, it became clear that the enzymatic activity of ACTEIII/PTE-1 was indispensable for peroxisome proliferation. Localization of ACTEIII/PTE-1 in peroxisomes is necessary for inducing peroxisome proliferation ACTEIII/PTE-1 has the peroxisome targeting signal 1 (PTS-1) at its C terminus and in fact, a majority of cytoplasmic ACTEIII/PTE-1 are found within peroxisomes [18]. The results of our immunofluorescence staining for

ACTEIII/PTE-1 and catalase (Fig. 1F) agree well with previous observations. However, in the case of clone A106 in which peroxisome proliferation occurred due to ACTEIII/PTE-1 overexpression, significant fraction of ACTEIII/PTE-1-positive staining did not overlap with catalase-positive staining, thereby suggesting that the amounts of ACTEIII/PTE-1 and catalase within each peroxisome are different and raising the possibility that ACTEIII/PTE-1 may function in trans in increasing the number of peroxisomes, possibly through its products such as fatty acids or by degrading acyl-CoA. Alternatively, we cannot exclude the possibility that ACTEIII/PTE-1 misrouted to other compartment due to the overexpression. Nevertheless, the PTS-1 motif seemed necessary for peroxisome proliferation induced by ACTEIII/PTE-1 because DSKL, a truncated ACTEIII/PTE-1 mutant lacking the PTS-1 signal (Figs. 4B –4E), did not induce peroxisome proliferation in Jurkat cells (Figs. 4D and 4E and Table 1). These results suggest that ACTEIII/PTE-1 present in peroxisomes but not in cytoplasm modified the metabolic processes occurring in the peroxisomes that lead to peroxisome proliferation. In this regard, we noted that Jurkat cells expressing ACTEIII/ PTE-1 with reduced enzymatic activity (79H/A) exhibited a slight but significant reduction in peroxisome number (Fig. 4F, Table 1). We infer that the occupancy of a ‘‘niche’’ within peroxisomes by such an incompetent enzyme resulted in insufficient peroxisome biogenesis. Again, however, the amount of the truncated mutant was less than the amount of ACTEIII/PTE-1 protein in A106 (Fig. 4B), making it impossible to examine if the PTS-1 signal is indeed necessary for lipid droplet formation.

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Fig. 3. ACTEIII/PTE-1 overexpression leads to an increase in catalase expression and peroxisome proliferation in non-lymphoid cells. Immunofluorescence staining of ACTEIII/PTE-1-transfected NIH3T3 and Y1 cells. NIH3T3 and an adrenal cortex cell line, Y1, were transfected with pMX-IRES-GFP/ACTEIII/ PTE-1 (ACTEIII) or the control vector (MOCK). GFP+ cells were sorted out and stained for ACTEIII/PTE-1 with anti-human ACTEIII/PTE-1 mAb followed by Cy5-labeled secondary Ab, and for catalase with anti-human catalase Ab followed by Cy3-labeled secondary Ab. ACTEIII transfectants exhibited increases in the expression of catalase and ACTEIII.

Thymocytes isolated from transgenic mice for human ACTEIII/PTE-1 exhibit peroxisome proliferation A transgenic construct for human ACTEIII/PTE-1 cDNA with the proximal lck promoter was prepared to establish mice expressing high levels of human ACTEIII/PTE-1 in a T-cell-specific manner. The transgenic mice expressed hu-

man ACTEIII/PTE-1 protein in thymus and spleen (Fig. 5A). There was a notable difference in the amount of human ACTEIII/PTE-1 protein between thymocytes and spleen cells, perhaps due to the higher transcriptional activity of the lck promoter in immature T cells than in mature ones [39 – 41]. The overexpression of ACTEIII/PTE-1 in thymocytes induced an increase in the expression of catalase,

Fig. 4. Function of two mutants of ACTEIII/PTE-1 lacking either enzymatic activity (79H/A) or peroxisome localization (DSKL). (A) Reduced enzymatic activity of the 79H/A ACTEIII/PTE-1 mutant. Spectrophotometric assay was performed using recombinant proteins of wild type and mutant (79H/A) of ACTEIII/PTE-1. Recombinant Nef protein was used as control. Asterisk indicates statistically significant difference ( P < 0.01) based on the Student’s t test. (B) Expression of ACTEIII/PTE-1 and catalase in mutant ACTEIII/PTE-1 transfectants. Cell lysates from Jurkat transfectants expressing wild-type (ACTEIII) and mutant (79H/A and DSKL) ACTEIII/PTE-1 and clone A106 as well as control cells (MOCK) were blotted with anti-ACTEIII/PTE-1 mAb and anticatalase Ab. Note that the amounts of catalase are significantly small in 79/H/A. Blots for lck are included as loading controls. Bottom: Quantitative evaluation of catalase expression in each cell line is shown as the catalase/Lck ratio. Asterisk indicates statistically significant difference ( P < 0.05) based on the Student’s t test. (C) Numbers of peroxisomes and lipid droplets in mutant ACTEIII/PTE-1 transfectants. Both numbers were counted for 20 cells in electron micrographs of cells of MOCK Tf and 79H/A or DSKL Tf. Histogram analysis revealed the number of cells containing the indicated numbers of peroxisomes and lipid droplets. Note that the number of peroxisomes was less in 79H/A than in MOCK, whereas the number of lipid droplets was not changed. (D) Immunofluorescence staining of Jurkat expressing mutant ACTEIII/PTE-1. Expressions of ACTEIII/PTE-1 and catalase were analyzed by confocal laser microscopy in mutant ACTEIII/PTE-1 transfectants (79H/A and DSKL) and control mock transfectant (MOCK) of Jurkat. ACTEIII/PTE-1 was stained with anti-ACTEIII/PTE-1 mAb followed by Cy5-labeled secondary Ab, and catalase was stained with anti-catalase Ab followed by Cy3-labeled secondary Ab. (E) Quantitative measurement of the numbers of peroxidases and lipid droplets in various Jurkat transfectants expressing wild-type and mutant ACTEIII/PTE-1. The numbers of peroxisomes (upper panel) and lipid droplets (lower panel) were counted for 20 cells in electron micrographs of cells of ACTEIII/PTE-1expressing Jurkat clones (A79, A26, A106) and transfectants expressing mutant ACTEIII/PTE-1 (79H/A and DSKL). Histogram analysis revealed the numbers of peroxisomes and lipid droplets (mean F SD) in each transfectant.

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Fig. 5. Thymocytes from human ACTEIII/PTE-1-Tg mice exhibit peroxisome proliferation. (A) Expression of transgenic human ACTEIII/PTE-1 in thymus and spleen of Tg mice. Cell lysates (2  106 each) prepared from thymocytes and splenocytes from ACTEIII/PTE-1-Tg mice (Tg(+)) and normal littermate (Tg( )) were blotted for ACTEIII/PTE-1 and Lck. (B) Increase in catalase expression in thymocytes of ACTEIII/PTE-1-Tg mice. Cell lysates of thymocytes from mice carrying ACTEIII/PTE-1 homozygous (Tg(++)) or heterozygous (Tg(+/ ))transgene, and non-transgenic control mice (Tg( )) were blotted for catalase and lck. Bottom: Quantitative increase in catalase expression as shown by the catalase/Lck ratio. (C) Electron micrographs of thymocytes from ACTEIII/PTE-1-Tg mice (Tg(+)) and littermate control mice (Tg( )). Arrowheads indicate peroxisomes. Bars indicate 5 Am. (D) Peroxisome proliferation in thymocytes from ACTEIII/PTE-1-Tg mice. The number of peroxisomes was counted for 70 – 100 cells in electron micrographs of thymocytes from ACTEIII/ PTE-1-Tg mice (ACTEIII-Tg(+)) and control wild-type mice (WT/ACTEIII-Tg( )). Histograms represent the number of cells containing the indicated number of peroxisomes per cell. The number represents mean F S.E.M. Note that ACTEIII/PTE-1-Tg mice homozygous for transgene exhibited almost the same results as Tg mice with heterozygous transgene shown as ACTEIII-Tg (data not shown).

particularly in homozygous Tg mice (Fig. 5B). We observed that thymocytes isolated from the transgenic mice contained significantly larger amounts of both catalase and peroxisomes than those from nontransgenic littermates under EM analysis (Figs. 5C and 5D), even though the amount of human ACTEIII/PTE-1 expressed in those cells was several times smaller than that in Jurkat clone A106 (Table 1). However, spleen cells did not show such a peroxisome proliferation and lipid droplet accumulation was not evident in either thymocytes or spleen cells in the transgenic animals (Fig. 5C and data not shown). We infer that the expression level of human ACTEIII/PTE-1 in these cells was too low to induce lipid droplet accumulation and that the expressed protein level in thymocytes was slightly above the lower limit to induce peroxisome proliferation (Table 1). Nevertheless, it is clear that the overexpression of human

ACTEIII/PTE-1 in vivo in mice causes peroxisome proliferation. Thus, peroxisome proliferation observed in murine and human cell lines was not an artifact peculiar to in vitro cultured cell lines. Mechanism of lipid droplet formation upon ACTEIII/PTE-1 overexpression Although ACTEIII/PTE-1 was first identified as an HIV1 Nef binding protein by a yeast two-hybrid system [20], its physiological functions remain unclear. In this regard, lipid droplet formation and peroxisome proliferation as revealed by this study are the first ACTEIII/PTE-1 functions to be demonstrated even though those alterations were seen when the enzymes were overexpressed. Although lipid droplet accumulation rarely occurs in normal cells, there was a case

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in which treatment with azide thymidine (AZT), an antiretroviral drug, resulted in lipid accumulation in T cells [42]. AZT is known to cause mitochondrial myopathy characterized by defects in the enzymes of the respiratory chain, which are associated with the decreased h-oxidation of fatty acids [6,43 –46]. This suggests that the perturbation of lipid metabolism underlies lipid droplet formation. Although the ability of human ACTEIII/PTE-1 to hydrolyze acyl-CoA has been shown only in in vitro cell-free systems, a yeast peroxisomal thioesterase highly similar to human ACTEIII/ PTE-1 was shown to be essential for growth, suggesting that peroxisomal thioesterase is critically involved in fatty acid oxidation in peroxisomes in vivo [18]. We speculate that lipid droplet accumulation is a consequence of an increase in the amounts of free fatty acids generated by the catalysis by ACTEIII/PTE-1. It is unlikely that the free fatty acids themselves form lipid droplets within the cytoplasm. One possible mechanism is that the increase in the amounts of free fatty acids leads to the formation of insoluble triglycerides that accumulate as lipid droplets within the cell. An examination of the chemical composition of the lipid droplets is necessary to clarify the mechanism by which ACTEIII/PTE-1 induces lipid droplet formation. So far, we have been unsuccessful in establishing Jurkat cell lines expressing a mutant ACTEIII/PTE-1 with reduced enzymatic activity at levels sufficient for inducing lipid droplet formation. Thus, the above-mentioned hypothesis remains and it is necessary to establish high expressor cell lines to examine directly if the enzymatic activity of ACTEIII/PTE1 is involved in lipid droplet formation. We have not observed significant alterations of cell growth/death and functions such as cytokine production in Jurkat clone A106. Thus, the physiological meaning of lipid droplet formation remains to be clarified. In addition, considering that ACTEIII/PTE-1 is active on a variety of acyl-CoA esters including branched chain acyl-CoAs, this thioesterase might regulate intraperoxisomal levels of acyl-CoA/CoASH [47,48]. ACTEIII-induced peroxisome proliferation and its implication in the mechanism of physiological peroxisome proliferation Another effect of ACTEIII/PTE-1 overexpression found in this study is peroxisome proliferation. Interestingly, our analysis of Jurkat clones expressing different amounts of ACTEIII/PTE-1 showed that the amounts required for lipid droplet accumulation are larger than those for peroxisome proliferation (Fig. 1E, Table 1). However, this may merely reflect a difference in sensitivity between these two alterations. These observations suggest that the mechanism involved in peroxisome proliferation is distinct from that in lipid droplet accumulation. Peroxisome proliferators are compounds that induce hypolipidemia by altering the gene expression of various enzymes involved in h-oxidation, such as long chain ACTE

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[26,49,50]. The mechanism of peroxisome proliferator-mediated increase in peroxisome number is not clear. On the basis of our current results, it is therefore tempting to consider that ACTEIII/PTE-1 is a downstream target of the peroxisome proliferator gene activator, leading to peroxisome proliferation. On the other hand, the gene induction by peroxisome proliferators is believed to be mainly mediated by peroxisome proliferator activated receptors (PPARs), which are a class of transcriptional activators [6,51,52]. Interestingly, free fatty acids have been shown to bind to and activate PPARs in terms of gene induction [53,54]. Therefore, ACTEIII/PTE-1 may activate PPARs by generating free fatty acids. Indeed, we observed that PPARg but not PPARa was increased by ACTEIII/PTE-1 overexpression. This is consistent with the findings that mouse PPARa appears to be responsible for peroxisome proliferation, whereas human PPARa was reported to have mutation and they are not functional [57]. PPARg appears active for regulating peroxisome proliferation although the contribution of PPARy cannot be excluded [58]. The activated PPARs may induce gene expression leading to peroxisome proliferation. One of such known genes is AOX, and indeed, we found that ACTEIII/PTE-1 transfectants increase AOX expression, supporting this mechanism. It is possible that these two mechanisms act in parallel or synergistically to accelerate peroxisome biogenesis. Nevertheless, our current study revealed the importance of ACTEIII/PTE-1 in the process of peroxisome proliferation. Our current result that ACTEIII/PTE-1 can increase the number of peroxisomes when overexpressed in the thymus as well as in various cell lines is not consistent with that of a previous study in which a myc-tagged ACTEIII/ PTE-1 induced a decrease in peroxisome number [15]. In that study, the amount of ACTEIII/PTE-1 was not determined. Our study revealed a certain threshold for ACTEIII/PTE-1 for inducing peroxisome proliferation. It is therefore possible that the amount of the myc-tagged enzyme was below the threshold. Moreover, peroxisome abundance was estimated by immunofluorescence microscopy using the anti-myc antibody [15], in contrast to the anti-catalase antibody used in our current study. Because the distribution of myc tag in their study reflected merely that of the ACTEIII/PTE-1 protein itself and not the peroxisomes, it is difficult to determine if the number of peroxisomes was actually altered. This is an important point as the distribution of ACTEIII/PTE-1 protein and that of peroxisomes as represented by catalase immunofluorescence did not overlap (Fig. 1). In addition, we enumerated directly the number of peroxisomes identified morphologically on electron micrographs. Thus, we conclude that ACTEIII/PTE-1 overexpression results in an increase in peroxisome number. Chang et al. [15] predicted that ACTEIII/PTE-1 reduces peroxisome number by inhibiting h-oxidation in peroxisomes via the degradation of the substrate acyl-CoA. However, as we discussed above, the degradation of acyl-CoA generates free

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fatty acids that activate PPARs [55,56] and may result in peroxisome proliferation.

Acknowledgments We thank Dr. H. Kato (Yamasa Corp.) for establishing the anti-ACTEIII/PTE-1 mAb, Dr. C. Shimizu for the transgenic mice, M. Sakuma and R. Shiina for experimental help, and H. Yamaguchi and Y. Kurihara for secretarial assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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