EJCB European Journal of Cell Biology 75, 87-96 (1998, February) · © Gustav Fischer Verlag · Jena 87

Tubular peroxisomes in HepG2 cells: Selective induction by growth factors and arachidonic acid

Michael Schradera, Kerstin Krieglsteinb, H. Dariush Fahimi1)a a Division of Medical Cell Biology, Institute for Anatomy and Cell Biology, University of Heidelberg,

Heidelberg/Germany b Division of Neuroanatomy, Institute for Anatomy and Cell Biology, University of Heidelberg, Heidelberg/Germany

This study is dedicated to Dr. M. J. Karnovsky on the occasion of his 701h birthday.

Received August 28, 1997 Accepted October 10, 1997

Peroxisome - nerve growth factor - arachidonic acid We showed recently the plasticity of the peroxisomal compartment in the human hepatoblastoma cell line HepG2 as evidenced by the pres-ence of elongated tubular peroxisomes measuring up to SJU11 next to much smaller spherical or rod-shaped ones (0.1-{).3JU11). Since the occurrence of tubular peroxisomes in a given cell in culture is syn-chronized, with neighboring cells containing either small spherical or elongated tubular peroxisomes, cell counting of immunoftuorescence preparations stained for catalase was used for the quantitative assess-ment of the dynamics of the peroxisomal compartment and the factors regulating this process. Initial studies revealed that the formation of tubular peroxisomes is primarily inftuenced by the cell density as well as by lipid- and protein-factors in fetal calf serum, being independent of an intact microtubular network. Biochemical studies showed that the occurrence of tubular peroxisomes correlated with the expression of the mRNA for 70 kDa peroxisomal membrane protein (PMP70), but not with that of matrix proteins. By cultivation of cells in serum-and protein-free media specific factors were identified which influ-enced the formation of tubular peroxisomes. Among several growth factors tested, nerve growth factor (NGF) was the most potent one inducing tubular peroxisomes and its effect was blocked by K252b, a specific inhibitor of neurotrophin receptor pathway, suggesting the involvement of signal transduction in this process. Furthermore, from several polyunsaturated fatty acids (PUFA) which all induced tubular peroxisomes, the arachidonic acid (AA) was the most potent one. Our observations suggest that tubular peroxisomes are transient structures in the process of rapid expansion of the peroxisomal compartment which are induced either by specific growth factors or by polyunsatu-rated fatty acids both of which are involved in intracellular signaling.

I) Prof. Dr. H. Dariush Fahimi, lnstitut fur Anatomie und Zellbio-logie, Abt. Medizinische Zellbiologie, Universitat Heidelberg, lm Neuenheimer Feld 307, D-69120 Heidelberg/Germany.

Introduction The membrane-bound cell organelles are highly dynamic structures which can exhibit rapid changes of their shape, number and intracellular distribution. Most striking is the occurrence of elongated tubular forms (and networks) which have been described for the ER [54], the Golgi complex [6], lysosomes [53], endosomes [20], mitochondria [22] and perox-isomes [15, 46, 56]. Hitherto, however, little information is available on the mechanism of tubulation and the exact func-tion of tubular organelles. Based on studies with the fungal metabolite Brefeldin A, a certain function for the tubules aris-ing from the Golgi, trans-Golgi network and endosomes is seen in the recycling of membrane and receptors back to the ER or to the plasma membrane [21, 25, 37]. For organelles like mitochondria and peroxisomes, however, the functional importance of tubular forms is presently unknown. Moreover, the molecular mechanisms that regulate the formation of tubular organelles are not known. Brefeldin A-induced mem-brane tubules, for example, require ATP as well as cytosolic and membrane proteins for their formation [10, 38]. Among the cytosolic factors calmodulin is thought to be a membrane tubulation factor [7]. It is further known that most of the tubulation processes are microtubule-dependent [6, 22, 54].

We showed recently [46, 47], that peroxisomes in the well-differentiated human hepatoblastoma cell line HepG2 exhibit several morphologically distinct forms: elongated-tubular ones (up to 5 !J.m), which are commonly found as the sole form in a given cell, and short rod-shaped (0.3 !liD) or small spher-ical (0.1-0.3 !liD) structures. Quantitative analysis of immu-nofluorescence preparations suggests that the spherical perox-isomes arise by fission from the tubular and rod-shaped ones [ 48]. Moreover, the formation of tubular peroxisomes is highly dependent upon the cell density in culture. Tubular perox-isomes are more frequent at lower density when the cells are well separated, and decrease in frequency with higher cell densities [48]. Our data with cytoskeleton-active drugs indi-cate further that microtubules play an important role in deter-

88 M. Schrader, K. Krieglstein, H. D. Fahimi

mining the intracellular distribution of peroxisomes, but are not required for the observed processes of tubulation and fis-sion of peroxisomes in HepG2 cells [48].

In the present study we have investigated the formation of tubular peroxisomes in HepG2 cells further, taking advantage of cultivating the cells in different serum- and protein-free media. This approach enabled us to manipulate the formation of tubular peroxisomes, to enrich them in culture and to identify the specific factors inducing their formation. Among these factors there are different growth factors such as nerve growth factor (NGF) and some polyunsaturated fatty acids (PUFAs), particularly the arachidonic acid (AA) which are involved in intracellular signaling.

Materials and methods Cell culture HepG2 cells [1] were obtained from the American 'JYpe Culture Collection (ATCC, Rockville, MD/USA). The cells were routinely cul-tured in minimal Eagle's medium (a-MEM) containing 2gfl sodium bicarbonate, 2mM glutamine, 100 U/ml penicillin, 100~-tg/ml strepto-mycin and 10 % fetal calf serum (FCS) (all from Biochrom KG, Berlin/ Germany) at 37°C in a humidified atmosphere of 5% C02 and 95% air. For experiments the cells were washed with serum/protein-free medium and detached by incubating with a trypsin/EDTA solution (0.125 %/0.05% (w/v); Biochrom KG). The cells were harvested with serum/protein-free medium containing 1 mg/ml soybean trypsin inhib-itor (Serva, Heidelberg/Germany) and centrifuged at 70g for 5 minutes (Beckman TJ-6). The cell pellet was then resuspended in the appropri-ate serum/protein-free medium (see below) or in DMEMIN1 contain-ing the supplements indicated below. Cells were plated at a defined density of 2 x 105 cells/ml on glass coverslips in 35 mm culture dishes for immunofluorescence or in 100mm culture dishes (Greiner GmbH, Frickenhausen/Germany) for RNA and membrane preparations. The cells were routinely prepared for immunofluorescence after 24 hours when the tubular peroxisomes reached their maximum.

PC12 (rat pheochromocytoma cell line) and 3T3 mouse fibroblasts were obtained from Dr. K. Unsicker (Institute for Anatomy and Cell Biology, Division of Neuroanatomy, Heidelberg/Germany). PC12 cells were routinely cultured in RPMI medium (RPMI 1640; 10 % horse serum; 5% FCS; 50 ~-tgfml penicillin; 50 ~-tg/ml streptomycin; 100 ~-tgfml neomycin; 2mM glutamine (ail from Gibco, Grand Island, NY/ USA)), whereas 3T3 cells were cultured in a-MEM/10% FCS as described above.

Serum-free/protein-free culture media The following serum- or protein-free culture media were tested: DMEM/N1 (Dulbecco's modified Eagle's medium (DMEM, Gibco); N1: 0.25% BSA; 6.25 x 10·8 M transferrin; 8.3 x 10·7 M insulin; 3 x 10·8 M selenium; 2 x 10·8 M progesterone; 1 x 104 M putrescine (all from Sigma Immunochemicals, Munich/Germany); 50 ~-tgfml penicil-lin; 50 11g/ml streptomycin; 100 ~-tgfml neomycin; according to [4]), PFEK-1 (Biochrom KG; protein-free), Ultraculture (Biowhittaker, Walkersville, MD/USA; 3mg total protein/ml); Hybri Max S2897, a serum- and protein-free hybridoma medium (Sigma).

Pretreatment of fetal calf serum For inactivation of serum proteins FCS was heat-inactivated ( + 80 oc for 45 minutes) or protease-treated (1 mg/ml trypsin, 37°C, 24 hours, stop reaction with 1 mglml soybean trypsin inhibitor (Serva)). In another set of experiments serum proteins were precipitated with increasing concentrations of ammonium sulfate (30% - 80% ), the precipitates were dialyzed against PBS, pH 7.4 and used as supple-ments (see below). Serum lipids were extracted with chloroform/ methanol, air dried and resuspended in 0.5% BSA in PBS, pH 7.4.

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Supplementation studies For further supplementation studies the serum-free DMEM/N1 medium was used. To this medium were then added various growth factors and fatty acids. Growth factors added: Epidermal growth factor (EGF, mouse, sub-maxillary gland; 5-10 ng/ml), saliva (human, M. Schrader; 10% ), hepa-tocyte growth factor (HGF, human, Boehringer Mannheim GmbH, Mannheirn/Germany; 10ng/ml), fibroblast growth factor (bFGF, human, recombinant from E. coli; 10ng/ml), transforming growth fac-tor~ (TGF ~.human, recombinant from E. coli; 1-5ng!ml), ciliary neurotrophic factor (CNTF, rat, recombinant from E. coli; 5nglml), nerve growth factor (NGF, mouse, submaxillary gland, Boehringer Mannheim; 10nglml), brain derived neurotrophic factor (BDNF, human, recombinant from E. coli; 25ng/ml), neurotrophic factor-3 (NT-3, human, recombinant from E. coli; 10ng/ml), (all from IC Che-mikalien, Ismaning/Germany). Fatty acids added: Arachidonic acid (AA, Sigma; 1-50~-tM), arachi-donic acid methyl ester (AAME, Sigma; 25 1-tM), linolenic acid (LA, Sigma; 25-501-tM), palmitic acid (PA, Sigma; 12.5-50~-tM). The fatty acids were dissolved in ethanol at 100 mg/ml stock solutions.

Treatment with K252b To study the effect of NGF further HepG2 cells were pretreated 4 hours after seeding with the protein kinase inhibitor K252b (1 11M -2 ~-tM; for 1 hour) from Norcardiopsis sp. (Kamiya Biomedical Com-pany, Thousand Oaks, CAIUSA). After pretreatment, NGF (10ng/ml) or FCS (10%) were added and cells were prepared for inununofluores-cence after 24 hours.

Activation and inhibition of protein kinase C (PKC) To examine the!fole of PKC in the formation of tubular peroxisomes in HepG2 cells cultured under serum-free conditions, PKC was activated by the phorbol ester tetradecanoylphorbol acetate (TPA; 200 ng/ml) or by 1-oleolyl-2-acetyl-sn-glycerol (OAG; 0.2-10 ~-tM). In another set of experiments PKC was inhibited by bisindolylmaleirnide I (BIM; 0.01-1~-tM), and the formation of tubular peroxisomes was induced by the addition of AA (25 1-tM). Cells were also treated with the protein phosphatase inhibitor okadaic acid (1-5 !lhl) prior to the addition of AA (25~-tM). All stock solutions (20~-tM) were prepared in DMSO.

Treatment with peroxisome proliferators HepG2 cells were either cultured in serum-free DMEM/N1 or in a-MEM/10 % FCS up to 5 days in the presence of the following peroxi-some proliferators: eicosatetraynoic acid [23] (ETYA; 10-25 ~-tM; Cay-man Chemical Company, Ann Arbor/MI, USA), bezafibrate [13] (104 -10"6 M), BM 17.0249 [29] (10-4-10-6 M) to study their effect on the formation and disappearance of tubular peroxisomes. The latter two drugs were a gift of Boehringer Mannheim.

Treatment with Brefeldin A HepG2 cells were cultured in a-MEM/10% FCS and were treated 3-4 hours after seeding with Brefeldin A (5-10 ~-tgfml from a 1 mg/ml stock solution in ethanol; for 0.5, 1 and 3 hours; Santa Cruz Biotechnology, Inc., Santa Cruz, CAIUSA) to study its effect on tubule formation, and separately after 24 hours of cultivation to examine its influence upon the disappearance of tubular forms.

Treatment with mlcrotubule-depolymerlzing drugs HepG2 cells were seeded in DMEM/N1 and after 4 hours of cultiva-tion microtubules were depolymerized by addition of nocodazole (15~-tM) or colcemid (500 nM) (Sigma). After 3 hours of drug treat-ment, 10 % FCS was added to stimulate the formation of tubular per-oxisomes. The cells were prepared for inununofluorescence after 24 hours of cultivation.

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Indirect immunofluorescence and quantitative evaluation of peroxisomes HepG2 cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Thton X-100 as described [46]. The following antibodies were used: polyclonal rabbit anti-guinea pig catalase [3], monoclonal mouse anti-~-tubulin (Sigma), dichlorotriazinylaminofluorescein (DTAF)-conjugated goat anti-rabbit IgG (Dianova, Hamburg/Ger-many), rhodamine isothiocyanate (RITC)-labeled goat anti-mouse IgG (Southern Biotechnology Associates, Inc., Birmingham!UK). Samples were examined in a Zeiss Axiophot and photographed on Kodak TMY film. For quantitative evaluations 100 cells per each prep-aration were examined under the fluorescence microscope and cate-gorized as cells with tubular, spherical or rod-shaped peroxisomes [48). The data were analyzed statistically in comparison with the appropri-ate controls using student's t-test.

Membrane preparation HepG2, PC12 and 3T3 cells grown to confluency were rinsed in PBS, detached with a rubber policeman, collected in PBS and centrifuged

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Tubular peroxisomes 89

at 70g, 4°C for 5 minutes (Beckman TJ-6). Cells were homogenized in ice-cold 0.2M Na2C03-buffer containing 1mM PMSF, 1mM E-aminocaproic acid, 0.2mM DTT, 5mM benzamidine, and 10~-tg/ml leupeptin with a Potter S homogenizer (1000 rpm; Braun, Melsungen/ Germany) and were centrifuged at 125000g, 4 °C for 45 minutes in a Beckman L5-65B ultracentrifuge (SW 50 rotor). The resulting mem-brane pellet was resuspended in TVBE-buffer (1 mM NaHC03, 1 mM EDTA, 0.1% ethanol, 0.1% Thton X-100), divided into aliquots, fro-zen in liquid nitrogen, and stored at -80 °C.

Western bloHing Equal amounts of protein (as indicated in Results) were applied to 7.5% polyacrylamide gels (8.5 X 5 X 0.1 em) which were used for SDS-PAGE, followed by electrotransfer to nitrocellulose sheets at 120mA by semidry blotting. A polyclonal monospecific antibody to tyrosine kinase (Trk)-receptors A, B, C (Trk (C-14):sc-11; Santa Cruz Biotech-nology) was employed for immunocomplexing followed by horse rad-ish peroxidase (HRP)-goat anti-rabbit IgG. Immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) technique (Amersham, Braunschweig/Germany).

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Fig. 1. HepG2 cells stained with an antibody to catalase, a peroxisomal marker enzyme, shown by indirect immunofluorescence. Cultivation in a-MEM/10 % FCS induces the formation of elongated tubular peroxisomes (a). In contrast, cultivation in serum-free DMEM/N1 medium leads to the forma-tion of only spherical or rod-shaped per-oxisomes (b). N: nucleus; Bar: 10 llffi·

90 M. Schroder, K. Krieglstein, H. D. Fohimi

Isolation of RNA and preparation of digoxigenin-labeled cRNA probes Total cellular RNA from HepG2 cells cultivated for different time intervals and in different media was extracted by means of guanidine thiocyanate/phenol/chloroform using the Roti-Quick isolation kit (Roth GmbH, Karlsruhe/Germany).

Digoxigenin-labeled cRNA probes were prepared by using a Dig-RNA Labeling Kit (Boehringer Mannheim) as described recently [45]. Briefly, the subcloned plasmids (pGEM-7Zf(-)) containing a full-length eDNA of rat liver catalase, acyl CoA-oxidase and PMP70 were cleaved at the opposite side of the RNA-polymerase promoter used later for in vitro transcription. After phenol/chloroform extraction the linearized plasmids were applied for preparation of cRNA fragments according to the manufacturer's instructions (Boehringer Mannheim).

RNA quantitation by Northern- and dot blot RNase protection assay For Northern blots identical amounts of total RNA (30 f.tg) were sub-jected to denaturing agarose gel electrophoresis followed by overnight capillary blotting (20 x SSC). For quantitative analysis of mRNA, a novel dot blot RNase protection assay was used [59]. Briefly, 1-2 f.tg RNA were directly applied in a constant volume of 1 f.tl onto a nylon membrane (Qiagen, Chatsworth, CA/USA), hybridized with the appropriate denatured cRNA probes and incubated with RNase A (1 f.tg/ml, Boehringer Mannheim). Digoxigenin-labeled RNA hybrids were detected by the ECL technique using an anti-digoxigenin anti-body conjugated to alkaline phosphatase (Boehringer Mannheim). Signals obtained were quantitated by densitometry using a Chromo-scan 3 scanner (Joyce-Loeb! Ltd., Gateshead/UK). The 28S rRNA band was used to normalize the amount of loaded RNA.

Results

Influence of different cell culture media on the formation of tubular peroxisomes Approximately 40-50% of HepG2 cells grown for 24 hours under our routine culture conditions ( a-MEM with 10% FCS) at a density of 2 x 105 cells/ml contained tubular peroxisomes

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::E ll. ~ » 0 :r: Fig. 2. Influence of different serum- and protein-free cell culture media on the formation of tubular peroxisomes in HepG2 cells. Cells were seeded at a density of 2 x 105 cellslml, incubated for 24 hours and prepared for immunofluorescence to determine the frequency of cells containing tubular peroxisomes. The protein-free (PFEK-1, Hybri Max S2892) or serum-free (DMEM/N1) culture media inhibit the formation of tubular forms, while the addition of FCS (10%) to these media induces their formation up to the control values ( a-MEM/10% FCS). Cultivation in the serum-free "Ultraculture" medium (Ucult) induces significant formation of tubular peroxisomes.

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DMEMIN1

FCS (10%)- -l Saliva (10%)- -I

NGF·

bFGF

TGF]3

HGF

BDNF

NT-3

CNIF

EGF

Insulin

0 10 20 30 40 50

% tubular peroxisomes

Fig. 3. Effect of different growth factors on the formation of tubular peroxisomes in HepG2 cells. Cells were seeded in DMEM/N1 supple-mented with different growth factors (for abbreviations and concentra-tions see Material and methods). Note that amongst the growth factors the nerve growth factor NGF (lOng/ml) shows the strongest induction of tubular peroxisomes.

(Fig. 1a) with the remaining cells containing mostly spherical and rod-shaped organelles [46]. The influence of different cul-ture media on the formation of tubular peroxisomes is shown in Fig. 2. Whereas in protein-free (PFEK-1, Hybri Max S2897) or serum-free media (DMEM/N1) the tubular peroxisomes were almost absent and most cells contained spherical or rod-shaped forms (Fig. 1b), the addition of 10% FCS to those media restored the tubular forms to a level comparable to that in routine cultures (Fig. 2). Higher concentrations of FCS (20%) did not increase the frequency of tubular forms. Cul-tivation in "Ultraculture" medium, a commercial preparation, the exact composition of which is kept as a trade-secret, led on the other hand to a massive increase in tubular peroxisomes (approximately 70% ). It should be noted that HepG2 cells cultured in the described serum- or protein-free media exhib-ited comparable morphology and numerical density as in the cells cultivated routinely in the presence of FCS. The cells remained attached and were well-spread tolerating the serum-free conditions for several weeks.

Both protein and lipid components in FCS induce tubular peroxisomes Ammonium sulfate fractionation of serum revealed that the most potent serum proteins inducing tubular peroxisomes in 28% of the cells were precipitated with 40% ammonium sul-fate. Interestingly, inactivation of serum proteins by heat ( + 80 °C) or by protease treatment (trypsin) did not complete-ly destroy the ability of FCS to induce tubular peroxisomes (not shown) indicating the involvement of non-proteinaceous factors in tubule formation. To probe the effect of lipids on the tubulation of peroxisomes, serum lipids were extracted with chloroform/methanol. Addition of those lipids to HepG2 cells seeded in DMEM/N1 led to a concentration-dependent increase of tubular peroxisomes (not shown).

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Growth factors and particularly NGF induce the formation of tubular peroxisomes Taking advantage of the well-known presence of growth fac-tors in saliva [28] our initial observation was made with HepG2 cells cultivated in DMEM/N1 to which 10% human saliva was added. Indeed, this addition led to the formation of tubular peroxisomes to an extent comparable to the effect of 10% FCS (Fig. 3). In further experiments defined growth fac-tors were added to HepG2 cells seeded in DMEM/Nl. The DMEMIN1 with 10% FCS (maximum of tubular peroxi-somes) and DMEMIN1 without additional supplements (mini-mum of tubular peroxisomes) served as positive and negative controls. The influence of different growth factors on the formation of tubular peroxisomes in HepG2 cells is summa-rized in Fig. 3. Addition of EGF, insulin or CNTF to serum-free DMEMIN1-medium did not induce tubular peroxisomes. HGF, TGF~, bFGF, and the neurotrophins BDNF and NT-3 showed only a slight effect with approximately 10% of all cells containing tubular peroxisomes. The strongest induction was obtained with NGF stimulating the formation of tubular per-oxisomes in 21.5% of all cells. This is almost half of the maxi-mal value induced by 10% FCS ( 45 % tubular forms) under comparable cell density conditions.

Different combinations of the described growth factors with NGF did not lead to an increase of the half-maximal effect of NGF alone. In contrast, combinations with EGF or TGF~ seemed to have an inhibitory effect (not shown).

The effect of NGF on the formation of tubular peroxisomes is mediated by Trk-receptor signaling To examine whether the effect of NGF on the formation of tubular peroxisomes is mediated by a specific neurotrophin

DMEMINI

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% tubular peroxisomes Fig. 4. Inhibition of NGF-stimulated Trk-receptor signaling with the specific inhibitor K252b. HepG2 cells were seeded in DMEM/Nl con-taining the supplements indicated in the Figure and prepared for immunofluorescence after 24 hours of incubation. Note that pretreat-ment of HepG2 cells with 1 or 2 1-1M K252b inhibits the stimulating effect of NGF (10 nglml) on the formation of tubular peroxisomes. This inhibitory effect of K252b was revoked by the addition of 10 % FCS which induced tubular forms.

Tubular peroxisomes 91

receptor (Trk A, B, C), HepG2 cells were pretreated with a specific inhibitor (K252b) of the tyrosin-kinase-mediated sig-nal transduction pathway of the Trk-receptor. K252b shows its optimal inhibitory effect at 1 !J.M, whereas it is non-inhibitory at concentrations less than 1 11M and toxic at concentrations above 2 !J.M after prolonged incubation times. Pretreatment with 1 11M and 211M K252b inhibited completely the stimulat-ing effect of NGF on the formation of tubular peroxisomes (Fig. 4). However, the addition of 10% FCS after the inhibi-tion of the NGF-receptor cascade by K252b induced the maxi-mal formation of tubules indicating that additional factors other than NGF in serum must be involved. Treatment with 2 11M K252b before addition of 10 % FCS reduced the induc-tion of tubular peroxisomes. This could be due to a toxic effect of K252b after prolonged incubation times. These observa-tions suggest that HepG2 cells might possess a NGF-specific, Trk-like receptor.

To verify this issue further, membrane fractions of HepG2 cells and control cells (PC12, 3T3) were prepared and used for immunoblots with an antibody to Trk-receptors A, B, C (Fig. 5). PC12 cells which express Trk A show a typical band with a molecular mass of approximately 140 kDa, whereas 3T3 cells which do not express a neurotrophin receptor are com-pletely negative. HepG2 cells, however, show a distinct immu-noreactive band with a molecular mass of approximately 90 kDa suggesting the presence of a putative NGF-receptor.

Polyunsaturated fatty acids in general and arachidonic acid in particular induce the formation of tubular peroxisomes After the preliminary observations with extracted serum lipids, defined fatty acids were added to DMEMIN1 medium to study their effect on the formation of tubular peroxisomes in HepG2 cells (Fig. 6). Interestingly, only the polyunsatu-rated fatty acids tested (AA, AAME, LA) were found to induce the formation of tubular peroxisomes, whereas PA had no effect. Among the polyunsaturated fatty acids, AA (C20:4w6) showed the most prominent effect, which in the

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buffer from PC12 cells (lane a), HepG2 (lane b) and 3T3 fibroblasts (lane c) incubated for visualization ofTrk-receptors A, B, C. 15 1-1g pro-tein was applied per lane. Notice that the putative Irk-receptor in HepG2 cells (lane b) has a smaller molecular weight than in PC12 cells (lane a) and that 3T3 fibroblasts are completely negative (lane c).

92 M. Schrader, K. Krieglstein, H. D. Fahimi

DMEM/Nl

FCS(I0%)-

50 jiM AA-

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% tubular peroxisomes Fig. 6. Effect of fatty acids on the formation of tubular peroxisomes in HepG2 cells. Cells were seeded in DMEM/Nl supplemented with different concentrations of fatty acids {AA- arachidonic acid; AAME - arachidonic acid methyl ester; LA - linolenic acid; PA - palmitic acid) as described in Material and methods. Note the prominent effect of arachidonic acid (AA), whereas palmitic acid {PA) shows no effect.

range between 10-25 !J.M was comparable to the effect of 10 % FCS and at 50 !J.M was even stronger. The stimulatory effects of AAME and LA (C18:3w3) were less pronounced.

Manipulation of PKC has no effed on the formation of tubular peroxisomes Experiments with activators and inhibitors of PKC revealed that neither the activators TPA or OAG nor the PKC inhibitor BIM had any stimulatory or inhibitory effects on the forma-tion of tubular peroxisomes in HepG2 cells. After inhibition of PKC by BIM tubular peroxisomes could be induced under serum-free conditions by the addition of AA up to control val-ues (not shown). This was the same after treatment with the phosphatase inhibitor okadaic acid (not shown).

Peroxisome proliferators do not induce tubular peroxisomes in HepG2 cells HepG2 cells cultured in serum-free DMEMIN1 or in a-MEM/ 10 % FCS were treated with different concentrations of a variety of peroxisome proliferators (ETYA, bezafibrate, BM 17.00249). Under serum-free conditions none of the prolifera-tors tested was able to induce tubular peroxisomes (not shown). Furthermore, treatment with peroxisome prolifera-tors in the presence of serum (a-MEM/10% FCS) had abso-lutely no stimulatory or inhibitory effect on the process of tubulation or fission of peroxisomes (not shown).

Brefeldin A has no effect on tubular peroxisomes HepG2 cells were treated with Brefeldin A (5 and 10 !J.g/ml) which causes the tubulation of the Golgi complex and of the endosomal compartment and prepared for immunofluores-cence after different time intervals. Addition of Brefeldin A

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3-4 hours after seeding did not stimulate the formation of tubular peroxisomes. Moreover, when Brefeldin A was added after 24 hours of cultivation, it did not influence the expected reduction (by fission) of tubular peroxisomes (data not shown).

Tubular peroxisomes can be formed in the complete absence of an intact microtubular network We showed recently that treatment of HepG2 cells with a variety of microtubule-depolymerizing drugs ( colcemid, noco-dazole, vinblastine) leads to an enrichment of tubular peroxi-somes [ 48]. This phenomenon was further analyzed by seeding HepG2 cells in DMEMIN1 which completely supresses the formation of tubular peroxisomes. After complete depolymer-ization of microtubules with colcemid (500 nM) or nocodazole (15 !J.M) the formation of tubular peroxisomes was stimulated by addition of FCS. Indeed, tubular peroxisomes were induced at almost the same rate in the complete absence of microtubules (Fig. 7) confirming the independence of their formation from the microtubular network. Moreover, both rnicrotubule-depolymerizing drugs alone (in the absence of FCS) showed a stimulatory effect on the formation of tubular peroxisomes (Fig. 7).

The formation of tubular peroxisomes correlates with the expression of PMP70 mRNA To investigate whether the transcription of the peroxisomal matrix enzymes catalase, acyl-CoA oxidase and the peroxi-somal membrane protein PMP70 is correlated with the forma-tion of tubular peroxisomes, we monitored the expression of

DMEMINI

FCS (10%)

Nod+FCS-

Colci+FCS

Noci-FCS

Colci-FCS-

0 10 20 30 40 so % tubular peroxisomes

Fig. 7. Influence of the microtubule-depolymerizing drugs nocoda-zole (15 JJ.M) and colcemid (500 nM) on the formation of tubular perox-isomes. Cells were seeded in DMEM/Nl and microtubules were depolymerized after 4 hours of cultivation. Note that tubular peroxi-somes can be induced by addition of FCS (10%) even after complete depolymerization of microtubules with nocodazole {Noc) or colcemid (Cole). Moreover, in the serum-free medium, the destruction of the microtubular system stimulates the formation of tubular peroxisomes.

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those genes. Total RNA was prepared from cells grown in a-MEM/10% FCS for different time intervals (8 hours, 24 hours, 4 days) and from cells grown in DMEM/N1 and "Ultra-culture" medium for 24 hours. Quantitative analysis (Figs. 8a, b, c) revealed a correlation between the expression of PMP70 mRNA and the formation of tubular peroxisomes. PMP70 mRNA level increases after 24 hours of cultivation of HepG2 cells, when the tubular peroxisomes reach their maximum and decreases after 4 days of cultivation, when HepG2 cells con-tain no more tubular peroxisomes. In addition, cultivation in "Ultraculture" medium which is a strong inducer of tubular peroxisomes leads to an increase in the PMP70 mRNA level (Fig. 8a). In contrast, after cultivation in DMEM/Nl which inhibits the formation of tubular peroxisomes, the PMP70 mRNA level remains low. The mRNAs coding for catalase and

a • • • •

A B C D E

b ••• •

c • • • Fig. Sa-c. Dot blot RNase protection assay for mRNAs coding for 70 kDa PMP (PMP70; a), acyl CoA-oxidase (AOX; b) and catalase (CAT; c) in HepG2 cells cultured under different conditions. Total RNA was prepared from cells grown in a-MEM/10% FCS for 8 hours (A), 24 hours (B) and 4 days (C) and from cells grown in DMEM/N1 (D) and in "Ultraculture" medium (E) for 24 hours. The RNA (2!1g/ dot) was directly dotted in a constant volume of 1111 onto a nylon mem-brane and hybridized with Dig-labeled cRNA probes followed by RNase digestion of non-hybridized RNA. Signals obtained were quantified by densitometry and represent relative values for compari-son between the different groups. 28S rRNA was used to normalize the amount of loaded RNA. Note that the relative signal densities for PMP 70 mRNA increase after 24 hours of cultivation in a-MEM/10% FCS, when the tubular peroxisomes reach their maximum and decrease after 4 days of cultivation, when HepG2 cells contain no tubular, but only rod-shaped and spherical peroxisomes. In contrast, the relative signal densities for AOX- and CAT-mRNA do not decrease after 4 days of cultivation, when tubular peroxisomes disappear and thus do not correlate with the frequency of tubular peroxisomes.

Tubular peroxisomes 93

acyl-CoA oxidase were also increased after 24 hours of cultiva-tion in a-MEM/10% FCS and after cultivation in "Ultracul-ture" medium (Fig. 8b, c). In contrast to PMP70 mRNA, how-ever, a decrease after 4 days of cultivation in a-MEM/10% FCS of those mRNAs was not observed. The acyl-CoA oxi-dase mRNA level increased further and catalase mRNA remained constant.

Discussion

The observations presented here have revealed the dynamic nature of the peroxisomal compartment in HepG2 cells by generating elongated tubular forms in response to various external and internal signals.

Tubular peroxisomes are not unique to HepG2 cells Whereas in most previous studies on peroxisomes in cultured cells, they were described as spherical particles [17, 19, 33, 39, 44], we reported recently that in freshly seeded HepG2 cells a certain percentage of cells contained elongated tubular peroxi-somes measuring up to 5 f.tm (46, 48]. This initial observation has been confirmed in the meantime by Duclos et al. [11]. It must be emphasized, however, that tubular peroxisomes are not unique to HepG2 cells and indeed were observed by elec-tron microscopy in cardiomyocytes [18], sebaceous and uropy-gial glands [15, 58], regenerating hepatocytes [56], and entero-cytes [41]. Recently, they were also mentioned in cultured Leydig cells [30] and in yeast cells after overexpression of per-oxisomal membrane proteins [12, 35].

Interestingly, all peroxisomes in a given HepG2 cell were synchronized exhibiting either the tubular or alternatively the much smaller spherical or rod-shaped forms, but rarely both forms side by side. The tubular peroxisomes were transient structures which dependent on cell density reached their maxi-mum at 24 hours after seeding and decreased thereafter, disap-pearing almost completely at 72 hours [48]. The frequency of cells with tubular peroxisomes was highly constant, once the cell density and the time of observation (24 hours) were main-tained. This enabled us to devise a quantitative approach based on counting and classification of whole cells containing the different forms of peroxisomes in immunofluorescence preparations in order to study the dynamics of tubular peroxi-somes.

The formation of tubular peroxisomes requires specific serum factors In several serum- and protein-free culture media tested (e.g. DMEM/N1, Hybri-Max), the formation of tubular peroxi-somes was markedly reduced (Fig. 2), demonstrating the importance of specific serum factors in their induction. Only the "Ultraculture" medium, containing unspecified growth factors induced a higher rate of tubular peroxisomes than 10% FCS. Supplementation studies using serum-free media with precipitated, heat-inactivated or protease-treated serum pro-teins and extracted lipids confirmed the importance of both serum-proteins and -lipids. Therefore, the effects of defined growth factors as well as fatty acids were investigated in subse-quent studies.

94 M. Schrader, K. Krieglstein, H. D. Fahimi

The indudion of tubular peroxisomes by NGF is mediated by a Trk-like receptor and tyrosine-kinase signal transdudion pathway Initial observations with saliva, which is known to contain growth factors [28] and with "Ultraculture" medium suggested the involvement of growth factors in the formation of tubular peroxisomes. Supplementation studies with defined growth factors in serum-free medium confirmed this impression revealing NGF as the most potent one while insulin, EGF and HGF showed no or only slight effects (Fig. 3). This NGF-effect on peroxisomes is not completely unexpected since this growth factor induces the expression of catalase, a peroxi-somal marker enzyme, in rat brain [14] and in PC12 cells [43]. More significantly, the stimulating effect of NGF upon tubular peroxisomes was completely inhibited by K252b, a specific inhibitor of Trk-receptor signaling (Fig. 4) suggesting the involvement of tyrosin-kinase signal transduction pathway in mediation of the NGF-effect in HepG2 cells. Since normal adult human liver was reported recently to lack any of the neurotrophin-receptors of the Trk-family [50] we analyzed by Western blotting the membrane fractions of HepG2 cells and found indeed a distinct cross-reactive band with a lower mo-lecular weight than Trk A, the high-affinity receptor for NGF (Fig. 5). The expression of this receptor on HepG2 cells is consistent with its reported detection in a variety of non-neuronal tissues [9, 26, 31] and could be explained by the ana-plastic nature of HepG2 cells.

Arachidonic acid and polyunsaturated fatty acids induce the formation of tubular peroxisomes The initial observation that the frequency of cells with tubular peroxisomes was increased by a chloroform/methanol extract of FCS led to supplementation studies with fatty acids which clearly revealed PUFAs and particularly AA as inducers of tubular peroxisomes (Fig. 6). PUFAs and AA are well-known to increase the transcription of peroxisomal lipid ~-oxidation enzymes and can cause peroxisome proliferation [40] by two different mechanisms. Firstly, by activation of the peroxisome proliferator-activated receptor (PPAR-a) since AA and PUFAs have been shown to be activators of this nuclear recep-tor [23]. Alternatively, the effect of AA could be mediated via the adenylate cyclase and the PKC signaling pathway [2, 32]. Experiments with activators (TPA, OAG) and inhibitors (BIM) of PKC, as well as with peroxisome proliferators (ETYA, bezafibrate, BM 17.0249), however, all showed no effect on tubulation of peroxisomes. The low response of human hepatocytes and HepG2 cells to peroxisome prolifera-tors is well-known [13] and is supposedly due to low-level or inappropriate expression or structural difference of PPAR-a [49].

The observations with several PUFAs such as AA and LA exhibiting half-maximal response, could be possibly due to activation of the postulated "PUFA response factor" [5] in the induction of tubular peroxisomes. This notion is based on the apparently highly specific action of AA at very low concentra-tions (l-lO!J.M) compared to its methyl ester derivative. The direct participation of AA - and not one of its metabolites - is confirmed also by experiments with ETYA, which blocks the cyclooxygenase and lipooxygenase pathways of AA metabo-lism, and which did not affect the induction of tubular peroxi-somes by AA (unpublished observations).

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Tubular peroxisomes are induced in the absence of microtubules The interaction of peroxisomes with microtubules was demonstrated recently both in vitro [48] and in vivo [39]. In addition, it was noted that depolymerization of microtubules did not affect the shape of tubular peroxisomes [ 48], although tubular lysosomes and mitochondria are known to collapse and become rounded in the absence of microtubules [22, 53]. This observation together with our finding that Brefeldin A treatment had no effect on the tubulation of peroxisomes indi-cate that the latter process is inherently different from the tubulation of other membrane-bound organelles. Using the serum-free culture medium DMEM/Nl we have now found that the depolymerization of microtubules induced the forma-tion of tubular peroxisomes (Fig. 7), suggesting that the dis-sociation of peroxisomes from microtubules would induce their tubulation. Alternatively, since the depolymerization of microtubules itself is known to activate different protein ki-nases [34, 51] and transcription factors [42], the latter pro-cesses could initiate the tubulation of peroxisomes. Another possibility could be that the destruction of microtubules per-turbs the functions of other cell organelles which in turn would induce the tubulation process. Indeed, a functional cross-talk between peroxisomes and mitochondria has been postulated [52].

Tubular peroxisomes in HepG2 cells represent a transient rapidly growing form, comparable to the 11peroxisome reticulum 11 in rat hepatocytes Thbular peroxisomes similar to those shown here by immuno-fluorescence have been described in rat liver undergoing regeneration after partial hepatectomy [56]. Serial section reconstruction revealed that the tubular segments represented interconnections between bulbous (spherical) regions forming a "reticulum" [56] as originally postulated by Lazarow et al. [27]. More importantly, the "peroxisomal reticulum" was a transient structure being present only at the initial stage of liver regeneration (16-32 hours) and disappeared at later stages [57] and is indeed not found in normal adult rat liver [55]. The similarity of patterns of gene expression in regen-erating rat liver and HepG2 cells seeded at low density - as in this study- has been shown [24].

The temporary nature of tubular peroxisomes and of the "reticulum" in regenerating rat liver implies that they are formed only under "induced" conditions, and that they could represent an enlarged precursor compartment for the forma-tion of new spherical peroxisomes by fission (Fig. 9). Consis-tent with this hypothesis, spherical peroxisomes arranged like "beads on a string" can be observed in HepG2 cells at the time of disappearance of tubular peroxisomes [48]. Under steady state conditions (confluent cultures), however, a different regulatory mechanism consisting of simple division of small rod-shaped particles must be operating which is sufficient to maintain the supply of the organelle (Fig. 9). Similarly, in yeast cells tubular peroxisomes are observed mainly after the induction by oleic acid or after overexpression of the mem-brane protein Pexllp [12, 35, 36].

An important observation of the present study was the con-comitant rise of mRNA for PMP70 with the formation of tubular peroxisomes and its subsequent decrease with reduc-tion of their frequency. In contrast, a decrease of mRNAs encoding for two matrix enzymes catalase and acyl-CoA oxi-dase was not observed (Fig. 8). This is consistent with the

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Morphogenesis of Peroxisomes in HepG2 Cells Comparison of induced and constitutive pathways

cell density growth factors PUFAs MT -destruction

MT

Fig. 9. A hypothetical model for the morphogenesis of peroxisomes in HepG2 cells. Tubular peroxisomes are induced in cultures at low density (induced conditions) from rod-shaped or spherical ones. Their formation is cell density-dependent requiring specific growth factors. In addition, polyunsaturated fatty acids or the destruction of the mi-crotubular network stimulate their formation. The tubular peroxi-somes divide by fission giving rise to spherical peroxisomes. The microtubular network (MT) is required for the uniform distribution of peroxisomes after fission, but is not necessary for the formation and fission of tubular peroxisomes. Under steady state conditions (conflu-ent cultures), cells with tubular forms are absent and peroxisomes are formed by simple division of small spherical or rod-shaped ones (con-stitutive formation).

higher surface to volume ratio in tubular peroxisomes and could imply that they may contain larger amounts of other per-oxisomal membrane proteins such as those involved in biosyn-thesis of ether glycerolipids [16]. On the other hand, the tubu-lar peroxisomes can also accommodate larger amounts of per-oxins, proteins involved in the biogenesis of the organelle, most of which are membrane-bound [8]. In this respect, Pexllp deserves particular attention. This 32 (27) kDa mem-brane protein has been characterized in different yeast strains [12, 35] and it was suggested that it could be involved in the fission process [36]. The elucidation of mechanisms involved in tubulation and fission of peroxisomes in HepG2 cells re-quires the cloning and sequencing of the mammalian homo-logue of Pexllp and similar membrane proteins. The serum-free cell culture system as presented in this study provides an excellent model for the functional analysis of Pexllp and other factors involved in the fission of tubular peroxisomes.

Acknowledgements. The technical assistance of Heribert Mohr, Tina Stroh and Jutta Fey, and the support of Gabriele Kramer, Inge From-mer and Marvin Schanz in counting peroxisomes is gratefully acknowl-edged. The activators and inhibitors of PKC were a generous gift from Dr. M. Radloff (University of Hamburg/Germany). We are also grate-ful to Prof. Dr. K. Unsicker, Dr. Eveline Baumgart and Prof. Dr. Dr. Alfred Yolk! (University of Heidelberg/Germany) for many helpful discussions. - Supported by SFB 352, project C7.

Tubular peroxisomes 95

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