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Endocrinology, doi:10.1210/en.2007-1580
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Endocrinology Vol. 149, No. 7 3743-3752
Copyright © 2008 by The Endocrine Society

New Enzymes Involved in the Mechanism of Action of Epidermal Growth Factor in a Clonal Strain of Leydig Tumor Cells

Rocío Castilla, Mariana Gadaleta, Ana Fernanda Castillo, Alejandra Duarte, Isabel Neuman, Cristina Paz, Fabiana Cornejo Maciel and Ernesto J. Podestá

Instituto de Investigaciones Moleculares de Enfermedades Hormonales, Neurodegenerativas y Oncológicas, Department of Biochemistry, School of Medicine, University of Buenos Aires, C1121ABG Buenos Aires, Argentina

Address all correspondence and requests for reprints to: Ernesto J. Podestá, Instituto de Investigaciones Moleculares de Enfermedades Hormonales, Neurodegenerativas y Oncológicas, Department of Biochemistry, School of Medicine, University of Buenos Aires, Paraguay 2155, 5th, C1121ABG Buenos Aires, Argentina. E-mail: biohrdc{at}fmed.uba.ar.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The studies presented herein were designed to investigate the effect of mouse epidermal growth factor (mEGF) on arachidonic acid (AA) release in a clonal strain of cultured murine Leydig cells (designed MA-10). In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the steroidogenic acute regulatory (StAR) protein. However, the mechanism by which mEGF releases AA in these cells is not totally elucidated. We show that mEGF produces an increment in the mitochondrial AA content in a short-term incubation (30 min). This AA is released by the action of a mitochondrial acyl-CoA thioesterase (Acot2), as demonstrated in experiments in which Acot2 was down or overexpressed. This AA in turn regulates the StAR protein expression, indirect evidence of its metabolism to lipoxygenated products. We also show that mEGF induces the expression (mRNA and protein) of Acot2 and an acyl-CoA synthetase that provides the substrate, arachidonyl-CoA, to Acot2. This effect is also observed in another steroidogenic cell line, the adrenocortical Y1 cells. Taken together, our results show that: 1) mEGF can induce the generation of AA in a specific compartment of the cells, i.e. the mitochondria; 2) mEGF can up-regulate acyl-CoA synthetase and Acot2 mRNA and protein levels; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ARACHIDONIC ACID (AA) is an essential polyunsaturated fatty acid that plays a pivotal role in cell signaling and proliferation. It can act as a signaling messenger itself or through its metabolites produced mainly by lipoxygenases (LOX, which includes 5-LOX, 12 LOX, and 15 LOX) and cycloxygenases enzymes (1, 2, 3).

We postulated that the generation and export of AA into the mitochondria in steroidogenic cells is the way to lead the fatty acid along the lipoxygenase pathway, thus rendering a specific way to differentiate the generation of lipoxygenase products from cyclooxygenase products to induce the expression of the steroidogenic acute regulatory (StAR) protein and steroidogenesis (4). These events are triggered by steroidogenic hormones and carried out by the concerted action of two enzymes: the acyl-coenzyme A (CoA) synthetase (ACS4) and the mitochondrial acyl-CoA thioesterase (Acot2) (4, 5).

Acot2 was first identified as a 43-kDa phosphoprotein by its capacity to increase mitochondrial steroidogenesis in a cell-free assay (6). The protein was then purified to homogeneity (6). Further cloning and sequencing of its cDNA revealed that it belongs to a thioesterase family with long-chain acyl-CoA thioesterase activity (7), which includes four isoforms with different subcellular localization and a high degree of homology (8, 9). In particular, Acot2 was shown by immunoelectron microscopy to associate with the matrix face of mitochondrial cristae (8). In accordance with the role of Acot2 in steroidogenesis, we detected the protein and its mRNA in all steroidogenic tissues including placenta and brain (7). It is also expressed in heart and liver among other tissues (10, 11).

To date, the induction of Acot2 has been described in liver, triggered by peroxisome proliferator action and fasting state (12), and in diabetic rat heart mitochondria (13). We demonstrated previously that in steroidogenic cells, Acot2 activity is hormonally regulated by phosphorylation and substrate availability (7).

Although it is known that acyl-CoA thioesterases are a group of enzymes that catalyze the hydrolysis of acyl-CoA to the nonesterified fatty acid and CoA (14), only very recently has it been demonstrated that a member of this family participates in a new mechanism for generation and export of fatty acids into the mitochondria. Moreover, we and others have demonstrated that the regulation of mitochondrial ATP synthesis is an important regulator of AA export from mitochondria (15, 16).

ACS4 has been described as an AA-preferring acyl-CoA synthetase that is mainly expressed in steroidogenic tissues such as adrenal cortex, luteal and stromal cells of the ovary, and Leydig cells of the testis (17). We have shown previously that steroidogenic hormones and their second messenger, cAMP, increase ACS4 protein levels in a time- and concentration-dependent way. This enzyme appears rapidly in response to the trophic hormone, with the highest increase after 30 min (18). In steroidogenic cells, the AA released into the mitochondria is determined by the action of Acot2 (19). The availability of its substrate, arachidonyl-CoA (AA-CoA) is, in turn, determined by the hormone-induced increase of ACS4 levels (18). Cho et al. (20) showed that ACS4 expression in a murine adrenocortical tumor cell line, Y1, is also increased by ACTH and suppressed by glucocorticoids.

In steroidogenic cells, once generated and exported, mitochondrial AA exerts its biological action through its lipoxygenated metabolites on the induction of StAR protein, relevant for steroid synthesis (21).

The mouse Leydig tumor line MA-10 retain many of the differentiated functions of normal Leydig cells and display a well-characterized response to the LH and chorionic gonadotropin, with increased levels of intracellular cAMP and secretion of progesterone (22). In the MA-10 cells, steroidogenesis also can be regulated by the mouse epidermal growth factor (mEGF) through a mechanism that does not include an elevation of the cAMP (23).

The epidermal growth factor is a small polypeptide whose main action is the stimulation of cell multiplication, and it acts as positive and negative modulator of differentiated functions in certain cultured cells (24, 25, 26). It is well known that the epidermal growth factor promotes cell growth in different types of cells as a consequence of the AA release (27, 28).

In MA-10 cells, mEGF promotes AA release and metabolism to lipoxygenated products to induce the StAR protein expression (29, 30). mEGF increases the intracellular concentration of AA. Exogenously added AA and mEGF display similar effects on differentiated functions of MA-10 cells as well as sensitivities to inhibitors of AA metabolism to lipoxygenase products (29). In addition, certain lipoxygenase metabolites of AA stimulate MA-10 cell steroidogenesis (29, 31).

The mechanism by which epidermal growth factor (EGF) releases AA in steroidogenic cells was partly described by Majercik and Puett (29) using phospholipase A2 inhibitors, suggesting that this enzyme may be involved in this process. Besides, we already demonstrated in MA10 cells that the enzymes involved in cAMP-stimulated mitochondrial AA release are ACS4 and Acot2. Thus, this mechanism also could be involved in EGF-mediated AA release. Therefore, the aim of this work was to study the participation of ACS4 and Acot2 on EGF-triggered AA release.

The studies presented herein show that mEGF is capable of increasing the levels of intramitochondrial AA. This effect is controlled by the regulation of the expression of the enzymes, Acot2 and ACS4.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Waymouth MB752/1 cell culture media, acrylamide, bis-acrylamide, AA, agarose, formaldehyde, mEGF, BSA, and fatty acid-free BSA were purchased from Sigma Chemical Co. (St. Louis, MO). Ham-F10 cell culture medium, sera, antibiotics, trypsin-EDTA, TriZol reagent, and Lipofectamine 2000 were from Life Technologies, Inc. (Gaithersburg, MD); plastic flasks and dishes were provided by Corning-Costar (Corning, NY). Polyclonal antibodies against Acot2 and ACS4 were previously obtained in our laboratory (5, 18). Anti-β-tubulin monoclonal antibody was bought from Upstate (Lake Placid, NY). Antimitochondrial 39-kDa subunit of the reduced form of the nicotinamide adenine dinucleotide (NADH)-cytochrome c reductase (complex I) was purchased from Molecular Probes, Inc. (Eugene, OR). Electrophoresis supplies, polyvinylidendifluoride membrane, and secondary antibody (horseradish peroxidase conjugated goat antibody) were bought from Bio-Rad Laboratories Inc. (Hercules, CA). An enhanced chemiluminescence kit and Hybond N+ membrane were provided by Amersham Pharmacia Biotech (Buckinghamshire, UK). All other chemicals were commercial products of the highest grade available.

Rat interstitial cells and cell lines
Interstitial cells were obtained from rat testis following a standard and already used procedure (32). Leydig cells were purified from the interstitial cell preparation by a discontinuous Percoll density gradient following an already described procedure (33). Two million purified Leydig cells were treated for the times indicated in the corresponding figure with 10 ng/ml EGF in a final volume of 1 ml at 37 C under carbogen atmosphere.

The MA-10 cell line is a clonal strain of mouse Leydig tumor cells (22). MA-10 cells were generously provided by Mario Ascoli (University of Iowa, College of Medicine, Iowa City, IA) and were handled as originally described (22). The growth medium consisted of Waymouth MB752/1 containing 1.1 g/liter NaHCO3, 20 mM HEPES, 50 µg/ml gentamicin, and 15% horse serum.

Murine Y1 adrenocortical tumor cells, generously provided by Bernard Shimmer (University of Toronto, Toronto, Canada) were maintained in Ham-F10 medium, supplemented with 12.5% heat-inactivated horse serum and 2.5% heat-inactivated fetal bovine serum, 1.2 g/liter NaHCO3, 200 IU/ml penicillin, and 200 µg/ml streptomycin sulfate (34).

Flasks and multiwell plates were maintained at 36 C in a humidified atmosphere containing 5% CO2. mEGF stimulation was performed in culture medium containing 0.1% BSA.

Western blot
Total or mitochondrial proteins (20 µg) were separated on 12% SDS-PAGE and electrotransferred to poly(vinylidene difluoride) membranes (Bio-Rad Laboratories) as described previously (4).

Membranes were then incubated with 5% fat-free powdered milk in 500 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.5% Tween 20 for 60 min at room temperature with gentle shaking. The membranes were then rinsed twice in 500 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 0.5% Tween 20 and incubated overnight with the appropriate dilutions of primary antibody at 4 C: 1:4,000 rabbit polyclonal anti-Acot2, 1:1,500 rabbit polyclonal anti-ACS4, 1:5,000 mouse monoclonal anti-β-tubulin, and 1:20,000 mouse monoclonal antimitochondrial 39-kDa subunit of the NADH-cytochrome c reductase (complex I). Bound antibodies were developed by incubation with secondary antibody (1:5,000 goat antirabbit and 1:10,000 goat antimouse horseradish peroxidase conjugated) and detected by chemiluminescence.

Isolation of mitochondria
Isolation of mitochondria was done as described (35). Briefly, MA-10 cell cultures were washed with PBS, scraped in 10 mM Tris-HCl (pH 7.4), 250 mM sucrose, 0.1 mM EDTA, 10 µM leupeptin, 1 µM pepstatin A, and 1 mM EGTA (buffer A), homogenized with a Pellet pestle motor homogenizer (Kimble Kontes, Vineland, NJ), and centrifuged at 600 x g for 15 min. The supernatant obtained was centrifuged at 10,000 x g for 15 min and rendered a mitochondrial pellet that was washed once with buffer A and resuspended in 10 mM Tris-HC (pH 7.4), 10 µM leupeptin, 1 µM pepstatin A, and 1 mM EGTA.

[1-14C]AA incorporation in MA-10 cells
MA-10 cells were labeled after a previously described methodology (36), with minor modifications. [1-14C]AA (NEN Life Science Products, Boston, MA; specific activity 53 mCi/mmol) was added to the cultures (1 µCi/ml per well; 1 well = 2 x 106 cells) in serum-free Waymouth MB752/1 containing 0.5% fatty acid-free BSA. After 5 h of incubation at 37 C in a humidified atmosphere containing 5% CO2, the cells were incubated for 30 min in the presence or in the absence of 10 ng/ml mEGF.

After these treatments, the cells were washed with serum-free Waymouth medium containing 0.5% fatty acid-free BSA. Mitochondrial pellets were obtained and resuspended as described above and were then sonicated. Protein concentration was measured and lipids were extracted from equal amounts of mitochondrial proteins (500 µg) from each treatment having previously added 500 ng of unlabeled AA.

Lipid extraction was performed twice with ethyl acetate (six volumes per one volume of mitochondrial fraction). The organic phase was then collected and dried under nitrogen at 25 C and analyzed by two successive thin-layer chromatographies on silica gel. Radioactive spots were developed using a Storm phosphorimager (Amersham Biosciences, Stockholm, Sweden) after 1 wk of exposition, and the spot intensities were analyzed using ImageQuant 5.2 software (Amersham Biosciences).

RNA extraction and semiquantitative RT-PCR
Total RNA from the different treatment groups was extracted using TriZol reagent following the manufacturer’s instructions (Life Technologies, Inc.-BRL, Grand Island, NY).

The reverse transcription and PCR analyses were made using 2 or 4 µg of total RNA for cell lines or purified Leydig cells, respectively. The cDNAs generated were further amplified by PCR under optimized conditions using the primer pairs listed below.

The primers used for isolation and amplification of the Acot2 (amplicon size 846 bp) were: sense primer, 5'-AATGGTGGCCTCGTCTTTCGC-3' and antisense primer, 5'-ATAGCAAGGCCAAGTTCACCC-3' (8). For amplification of ACS4 (amplicon size 419 bp), the sense primer, 5'-AATGGTGGCCTCGTCTTTCGC-3', and the antisense primer, 5'-ATAGCAAGGCCAAGTTCACCC-3', were used. For amplification of mouse StAR cDNA (amplicon size 1224 bp), the sense primer, 5'-GGACCTTGAAAGGCTCAGGAAGAACAACCC-3', and the antisense primer, 5'-GGATTAGTAGGGAAGTCGGCACAATGATGG-3' (37), were used. Primers specific for a 405-bp segment of L19 ribosomal protein were used as housekeeping gene (38). For the comparison of the amount of amplified Acot2, ACS4, and StAR produced from different RNA samples, the amplified L19 product of each sample was used as an internal standard, using the sense primer, 5'-GAAATCGCCAATGCCAACTC-3', and the antisense primer, 5'-TCTTAGACCTGCGAGCCTCA-3' (39).

The reaction conditions were one cycle of 94 C for 5 min, followed by 23 cycles for ACS4; 25 for Acot2; 28 for StAR; or 23 for L19 of 94 C for 30 sec, 56 C for 30 sec, and 76 C for 90 sec. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification. PCRs for Acot2 amplification included 5% dimethylsulfoxide.

PCR products were resolved on a 1.5% (wt/vol) agarose gel containing 0.5 µg/ml of ethidium bromide to determine the molecular sizes of the ACS4, Acot2, StAR, and L19 amplicons. The gel images were acquired with the GelPro analyzer (IPS, North Reading, MA). The levels of the ACS4, Acot2, StAR, and L19 mRNA were quantitated using a computer-assisted image analyzer (ImageQuant 5.2) and the PCR results for each sample were normalized by L19 mRNA as an internal control.

Plasmid transfection
MA-10 cells were transiently transfected. One day before transfection, MA-10 cells (5 x 105 cells/well) were grown up to 80% confluence onto 24-well plates. Transfection was performed with either 1 µg of pRc-CMVi plasmid containing Acot2 sense and Acot2 antisense cDNA (4) or the empty vector as control in Opti-MEM medium and 2 µl Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the instructions of the manufacturer. Cells were placed into normal culture medium 6 h after transfection and grown for a further 48 h. The cells were afterward used as described in the respective figures. Transfection efficiency was approximately 30% as estimated by counting fluorescent cells transfected with pRc/CMVi plasmid containing the enhanced form of green fluorescent protein.

Protein quantification and statistical analysis
Protein was determined by the method of Bradford (40) using BSA as a standard. Statistical analysis was performed by Student’s t test or ANOVA followed by the Student-Newman-Kuels test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of mEGF on ACS4 expression in MA-10 cells
We have shown previously that steroidogenic hormones and their second-messenger increase ACS4 protein levels in a time- and concentration-dependent way. The AA release into the mitochondria is regulated by the hormone-induced increase of ACS4 levels, which in turn increases the availability of mitochondrial Acot2 substrate. ACS4 appears rapidly in response to the trophic hormone with the highest increase after 30 min (18).

On that basis, we analyzed the effect of mEGF on ACS4 expression.

The dose-response pattern of ACS4 protein expression after mEGF treatment was studied (Fig. 1AGo). The MA-10 cells stimulated for 30 min by increasing doses of mEGF (0–100 ng/ml) showed a dose-dependent increase in the levels of ACS4 protein. The rise in ACS4 protein levels was significant (P < 0.01) at 1 ng/ml, with maximum increase –2-fold, compared with the control, obtained with 10 ng/ml.


Figure 1
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  		FIG. 1. Dose- and temporal-response pattern of ACS4 expression in MA-10 cells in response to mEGF. Cells were treated with increasing doses of mEGF (0–100 ng/ml) for 30 min (A) or with mEGF (10 ng/ml) at indicated times (B and C). A and B, Cellular proteins were subjected to SDS-PAGE, and Western blot analysis was performed using sequentially anti-ACS4 and anti-β-tubulin antibodies. Specific bands were detected by enhanced chemiluminescence. Both panels show a representative Western blot and the quantitative representation of three independent experiments. The integrated OD of ACS4 protein levels were quantified for each band and normalized with the corresponding β-tubulin signal. Results are expressed as means ± SD. **, P < 0.01; ***, P < 0.001 vs. nontreated cells. C, Total RNA was extracted from different treatment groups and subjected to RT-PCR analysis of ACS4 mRNA expression. The RT-PCR products were resolved in ethidium bromide-stained agarose gels (1.5%). Images of the gels were acquired and a representative image is shown. The integrated OD of ACS4 mRNA expression was quantified for each band and normalized with the corresponding L19 mRNA bands. Data represent the means ± SD of three independent experiments. ***, P < 0.001 vs. nontreated cells.

 
We then analyzed the time kinetics ACS4 protein expression after mEGF treatment by Western blot (Fig. 1BGo). Cells stimulated by mEGF (10 ng/ml) showed a biphasic ACS4 protein expression pattern. The magnitude of the response was significant (P < 0.001) at 15 min and had a maximum at 1 h. At 4 h it reached values below those of control. Afterward the expression level increased again, with approximately 2-fold at 5 h.

To provide additional insights in the steps involved in up-regulation of ACS4 protein concentration, we investigated whether treatment with mEGF had any effect on ACS4 mRNA levels (Fig. 1CGo).

After mEGF (10 ng/ml) stimulation, the ACS4 mRNA levels rose gradually up to 2 h when they showed a maximum 2-fold increase over nonstimulated cells. Then the mRNA expression decreased to reach levels similar to the control at 4 h; afterward a new increment was noticeable at 5 and 6 h of mEGF treatment.

Effect of mEGF on Acot2 expression in MA-10 cells
Steroidogenic hormones regulate Acot2 by substrate availability and phosphorylation. Nevertheless, when we analyzed the Acot2 expression in mEGF-treated cells, we observed an increment in its expression. As was done for ACS4, we analyzed by Western blot the time-response of mitochondrial Acot2 protein levels after mEGF treatment (Fig. 2AGo). Cells stimulated with mEGF (10 ng/ml) showed a biphasic Acot2 protein expression pattern with an increment significant (P < 0.001) at 15 min, a maximum at 30 min, and a second peak at 6 h (5.3- and 8.5-fold increase in the level of Acot2, respectively).


Figure 2
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  		FIG. 2. Temporal response pattern of mEGF-stimulated Acot2 expression level in MA-10 cells. Cells were stimulated with mEGF (10 ng/ml) for the indicated times (0–6 h). A, Mitochondrial proteins were subjected to SDS-PAGE, and Western blot analysis was performed using sequentially anti-Acot2 and antimitochondrial 39-kDa subunit of the NADH-cytochrome c reductase (comp I) antibodies. Specific bands were detected by enhanced chemiluminescence. A representative Western blot and the quantitative representation of three independent experiments are shown. The integrated OD of Acot2 protein levels were quantified for each band and normalized with the corresponding complex I signal. Results are expressed as means ± SD. **, P < 0.01; ***, P < 0.001 vs. nontreated cells. B, Total RNA was extracted from different treatment groups and subjected to RT-PCR analysis of Acot2 mRNA expression. The RT-PCR products were resolved in ethidium bromide-stained agarose gels (1.5%). Images of the gels were acquired and a representative image is shown. The integrated OD of Acot2 mRNA expression was quantified for each band and normalized with corresponding L19 mRNA bands. Data represent the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. nontreated cells.

 
We studied whether the increment in Acot2 protein levels shown above was also accompanied by an increment in the Acot2 mRNA (Fig. 2BGo). Indeed, cells stimulated with mEGF (10 ng/ml) also showed a biphasic pattern of Acot2 mRNA expression with two peaks, at 30 min and 5 h (1.7- and 2.4-fold increase in the level of Acot2 mRNA over nonstimulated cells, respectively). The magnitude of the response was significant (P < 0.01) at 15 min.

Effect of mEGF on ACS4 and Acot2 expression in Y1 cells
To evaluate whether mEGF can induce ACS4 and Acot2 in a different system in which the hormone-stimulated AA release into mitochondria was also described, we tested the effect of mEGF in adrenocortical cells using Y1 cell line. This cell line showed a dose-dependent increase in the levels of ACS4 protein, with the maximal increase obtained with 10 ng/ml (data not shown).

We then performed the kinetic studies (0 at 3 h) of both ACS4 and Acot2 protein expression after mEGF treatment by Western blot (Fig. 3Go, A and C). Cells stimulated with mEGF (10 ng/ml) showed an increment in the expression of both enzymes with a maximum at 30 min (2.9- and 2.0-fold ACS4 and Acot2, respectively, compared with nonstimulated cells) and decreased until 3 h.


Figure 3
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  		FIG. 3. Effect of mEGF on ACS4 and Acot2 expression in adrenal Y1 cells. Cells were stimulated by mEGF (10 ng/ml) for the indicated times (0–3 h). A and C, Cellular (A) or mitochondrial (C) proteins were subjected to SDS-PAGE, and Western blot analysis was performed using sequentially anti-ACS4 and anti-β-tubulin (A) or sequentially anti-Acot2 and antimitochondrial 39-kDa subunit of the NADH-cytochrome c reductase (comp I) antibodies. Specific bands were detected by enhanced chemiluminescence. Both panels show a representative Western blot and the quantitative representation of three independent experiments. The integrated OD of ACS4 or Acot2 protein levels were quantified for each band and normalized with the corresponding β-tubulin or complex I signal, respectively. Results are expressed as means ± SD. **, P < 0.01; ***, P < 0.001 vs. nontreated cells. B and D, Total RNA was extracted from different treatment groups and subjected to RT-PCR analysis of ACS4 and Acot2 mRNA expression. The RT-PCR products were resolved in ethidium bromide-stained agarose gels (1.5%). Images of the gels were acquired and a representative image is shown. The integrated OD of ACS4 or Acot2 mRNA expression was quantified for each band and normalized with corresponding L19 bands. Data represent the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. nontreated cells.

 
The time response analysis of ACS4 and Acot2 mRNA expression was also performed and shown in Fig. 3Go, B and D.

Cells stimulated by 10 ng/ml mEGF showed a significant increase (P < 0.01) in ACS4 mRNA levels at 0.5 h with a maximum at 1 h (2.5-fold increase, compared with nonstimulated cells). At 30 min, the Acot2 mRNA showed a little but significant (P < 0.05) increase over nonstimulated cells. At longer times the expression decreased.

Effect of mEGF on mitochondrial-free AA content
As mentioned, in MA-10 cells, ACS4 and Acot2 are involved in the compartmentalized release (i.e. mitochondria) of AA stimulated through a cAMP-mediated pathway (4, 19). Because mEGF is another factor known to enhance the AA release (27, 29) and that this growth factor regulates the expression of these two enzymes in these steroidogenic cells, we studied whether mEGF stimulates the mitochondrial AA release.

For this purpose MA-10 mouse Leydig tumor cells were labeled with [1-14C]AA for 5 h after which they were incubated in the presence or absence of mEGF 10 ng/ml for 30 min. As shown in Fig. 4AGo, mEGF treatment increased AA content into the mitochondria (1.5-fold), compared with control cells.


Figure 4
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  		FIG. 4. Effect of mEGF on mitochondrial-free AA content. MA-10 cells were labeled for 5 h at 37 C with [1-14C] AA (1 µCi/ml) per 2 x 106 cells in serum-free media containing 0.5% fatty acid-free BSA. Then cells were incubated in the presence or absence of 10 ng/ml mEGF for 30 min. After washing the cells, mitochondrial fraction was obtained as described in Materials and Methods. The mitochondria were sonicated and lipids were extracted with ethyl acetate. The organic phase was collected and dried under nitrogen. The dried extracts were dissolved in chloroform-methanol [9:1 (vol/vol)] and analyzed by thin-layer chromatography on silica gel plates. A, Representative autoradiography showing [1-14C]AA spots from mitochondrial fractions and the corresponding quantification of three representative experiments. Bars denote levels (in arbitrary units) of [1-14C]AA in mitochondria. Results are expressed as the means ± SD from three independent experiments. *, P < 0.05 vs. control. MA-10 cells were transfected with the empty pRc-CMVi (mock) or the pRc-CMVi containing Acot2 antisense cDNA (Acot2as) or the full-sense Acot2 cDNA (Acot2s), as described in Materials and Methods. After 48 h, MA-10-transfected cells were labeled and stimulated as described above. Mitochondrial proteins were subjected to SDS-PAGE, and Western blot analysis was performed using sequentially anti-Acot2 and antimitochondrial 39-kDa subunit of the NADH- cytochrome c reductase (comp I) antibodies (B), and intramitochondrial AA was determined as described above (C). B, Western blot representative of three independent experiments. C, Representative autoradiography showing [1-14C]AA spots in mitochondrial fractions. Autoradiography spots quantification by densitometry. Bars denote levels (in arbitrary units) of [1-14C]AA in mitochondria. Results are expressed as the means ± SD from three independent experiments. a, P < 0.01 vs. control mock-transfected cells; b, P < 0.01 vs. mEGF-stimulated mock-transfected cells; c, NS vs. control Acot2as-transfected cells; d, P < 0.001 vs. mEGF-stimulated mock-transfected cells; e, P < 0.001 vs. control Acot2s-transfected cells.

 
We have also shown that in the hormone stimulated mechanism of AA release, Acot2 is the enzyme that releases AA into the mitochondria. To study the role played by Acot2 in the intramitochondrial release of AA by mEGF, we performed experiments aimed at silencing the expression of Acot2 or at overexpressing the protein in MA-10 cells. For this purpose MA-10 cells were transiently transfected with pRc-CMVi plasmid containing either the antisense or the full-sense Acot2 cDNA. The transfected cells were stimulated by mEGF (10 ng/ml) for 30 min and AA levels were analyzed.

The effectiveness of cDNA transfection was analyzed by determination of the intramitochondrial levels of Acot2 protein by Western blot. As shown in Fig. 4BGo, the Acot2 expression was diminished in cells transfected with pRc-CMVi plasmid containing antisense Acot2 cDNA, and it was incremented in cells transfected with full-sense Acot2 cDNA both compared with the cells transfected with the empty plasmid and mEGF stimulated (Fig. 4BGo).

As was seen previously in Fig. 4AGo, mitochondria from cells treated with mEGF had an increment in the AA levels (1.6-fold) (Fig. 4CGo). This AA increase did not take place in cells containing low levels of Acot2. On the other hand, cells with higher levels of Acot2 expression showed an increment in AA content (3.4-fold), compared with the AA levels of mitochondria from cells transfected with the empty plasmid. There is no significant difference among the AA content under nonstimulated conditions, disregarding the transfected plasmid.

Effect of Acot2-knockdown on StAR expression
As mentioned above, at least one of the biological actions of AA under cAMP or mEGF stimulation in MA-10 cells is the regulation of StAR protein expression (31, 41, 42). Thus, we tested whether the mitochondrial AA released by mEGF is involved in StAR expression by semiquantitative RT-PCR.

StAR mRNA levels were increased 1.7-fold, compared with nonstimulated cells in mock-transfected cells stimulated by mEGF for 6 h (Fig. 5Go). Transfection of the cells with Acot2 antisense plasmid impaired mEGF action on StAR mRNA. Considering there is only 30% efficiency in cell transfection, the effect is not completely abolished in these cells, and there is still an increase in StAR mRNA under mEGF treatment. The effect of mEGF on StAR mRNA levels was strongly reduced in cells transfected with the plasmid containing Acot2 antisense, probably due to the lower mitochondrial-free AA content in these cells.


Figure 5
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  		FIG. 5. Effect of Acot2 knockdown on StAR expression in MA-10 cells. Mock-transfected (mock) or Acot2 antisense transfected (Acot2as) MA-10 cells were incubated in the presence or absence of mEGF (10 ng/ml) for 6 h, and total RNA was extracted from different treatment groups and subjected to RT-PCR analysis of StAR mRNA expression. The RT-PCR products were resolved in ethidium bromide-stained agarose gels (1.5%). Images of the gels were acquired and a representative image is shown. The levels of StAR mRNA are presented as the integrated OD of StAR mRNA expression and normalized with the corresponding L19 mRNA bands. Data represent the means ± SD of three independent experiments. a, P < 0.05 vs. control mock-transfected cells; b, P < 0.05 vs. mEGF-stimulated mock transfected cells.

 
Effect of mEGF on ACS4 and Acot2 expression in primary Leydig cell cultures
Although MA-10 cells are very well accepted as steroidogenic model, to extrapolate the results obtained above to physiological conditions, we choose to analyze the effect of mEGF on the expression of these two key enzymes involved in AA intramitochondrial release in primary Leydig cell cultures, which better reflect cellular physiology than cell lines. Thus, the kinetics of ACS4 and Acot2 mRNA and protein expression was determined in freshly isolated and purified rat Leydig cells under mEGF treatment. The results are shown in Fig. 6Go. ACS4 (Fig. 6AGo) showed a peak of mRNA expression at 3 h (2.9-fold, compared with the control), whereas Acot2 (Fig. 6BGo) had a significant increment at 3 h (2-fold) and a peak at 5 h (4.2-fold, compared with the control). Regarding protein expression, EGF showed a biphasic effect on both enzymes, with a rapid increase of ACS4 and Acot2 protein levels at 15 min and a second peak at 3 h (Fig. 6CGo).


Figure 6
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  		FIG. 6. Temporal response pattern of mEGF-stimulated ACS4 and Acot2 expression level in primary Leydig cell cultures. Cells were stimulated with mEGF (10 ng/ml) for the indicated times (0–6 h). Total RNA was extracted from different treatment groups and subjected to RT-PCR analysis of ACS4 (A) and Acot2 (B) mRNA expression. The RT-PCR products were resolved in ethidium bromide-stained agarose gels (1.5%). Images of the gels were acquired and a representative image is shown. The integrated OD of ACS4 and Acot2 mRNA expression was quantified for each band and normalized with corresponding L19 mRNA bands. Data represent the means ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001 vs. nontreated cells. C, Cellular (upper Western blot) or mitochondrial (lower Western blot) proteins were subjected to SDS-PAGE, and Western blot analysis was performed using sequentially anti-ACS4 and anti-β-tubulin or sequentially anti-Acot2 and antimitochondrial 39-kDa subunit of the NADH-cytochrome c reductase (comp I) antibodies. Specific bands were detected by enhanced chemiluminescence. Both Western blots are representative of two independent experiments, each of them performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The main three conclusions of the present work are: 1) mEGF can induce the generation of AA in a specific compartment of steroidogenic cells, i.e. the mitochondria; 2) mEGF can up-regulate ACS4 and Acot2 mRNA and protein levels, which are involved in the generation of intramitochondrial AA; and 3) mEGF-stimulated intramitochondrial AA release leads to StAR protein induction, which is an indirect evidence of its metabolism to lipoxygenated products.

AA is released by mEGF in a compartmentalized fashion using the same effective concentrations of the growth factor previously described in total homogenates (29). Moreover, we demonstrated for the first time that a growth factor can regulate the expression of two enzymes, ACS4 and Acot2, involved in lipid metabolism in two related cell lines and in primary Leydig cell cultures. The necessity of protein synthesis in the effect of mEGF on StAR expression is in line with previous results indicating that the action of the growth factor on StAR mRNA levels is abolished by treatment of steroidogenic cells with a protein synthesis inhibitor such as cycloheximide (30).

Regarding Acot2 expression, mEGF enhances both mRNA and protein levels in a parallel time-dependent fashion. However, mEGF treatment increases ACS4 protein levels at shorter times than the mRNA levels (15 min vs. 1 h). We have already described, under short-time cell stimulation, that ACS4 expression requires only protein synthesis (18). In the present work, it seems that EGF has an effect similar to cAMP, initially increasing ACS4 protein levels, without modification of mRNA.

Detailed kinetics reveals that mEGF-induced ACS4 and Acot2 expression is biphasic. Regarding mRNA, the biphasic expression cannot be seen in freshly isolated and purified Leydig cells. However, although it is not statistically significant, there is a slight increase of ACS4 mRNA at short times. On the contrary, the effect of mEGF is biphasic for both ACS4 and Acot2 when protein levels are evaluated in both MA-10 and purified Leydig cells. Other authors demonstrated a similar pattern in other systems (43, 44). They concluded that the growth factor differentially regulates two distinct signaling pathways to render the same final effect at different time frames. Given that we did not analyze the signal transduction pathways involved in the effects observed in MA-10 cells, more studies should be performed to sustain this hypothesis.

The involvement of Acot2 in the generation of AA in the mitochondria by mEGF is supported by the results obtained in the experiments in which AA released into the mitochondria decreased when Acot2 expression was suppressed, and it increased when Acot2 was overexpressed. The generation of the fatty acid in the mitochondria by induction of the Acot2 expression is different from the mechanism used by cAMP in steroidogenic cells. cAMP does not change the expression of Acot2 but regulates its activity by protein phosphorylation and substrate availability (18). To this date, the induction of Acot2 has also been described in liver, triggered by peroxisome proliferator action and fasting state (12), and in heart mitochondria of diabetic rats (16). To our knowledge, our results provide the first evidence that mEGF regulates the expression of Acot2 and ACS4, both enzymes involved in AA release.

Because mEGF increases the expression of ACS4, we cannot rule out that this growth factor could also regulate the activity of Acot2 by substrate availability. This phenomenon is observed under steroidogenic hormone action, a condition in which ACS4 is the rate-limiting enzyme in the induction of intramitochondrial AA release (18). Given that Acot2 has consensus sites of phosphorylation by protein kinase C and that this kinase is involved in the mEGF signal transduction pathway (45), we cannot discard that mEGF might also regulate Acot2 phosphorylation state.

It is already known that the AA release and its metabolism to lipoxygenated products are part of the mechanism by which mEGF acts on steroidogenic cells to induce the StAR protein. The mechanism by which EGF releases AA in steroidogenic cells was partly described by Majercik and Puett (29) using phospholipase A2 inhibitors, suggesting that this enzyme may be involved in this process. This conclusion was taken on the basis that there was an inhibition of AA release when the cells were treated with bromophenacyl bromide and quinacrine, both compounds originally described as inhibitors of phospholipase A2. We cannot discard that mEGF may also release AA in this cell type using the activation of the phospholipase A2 pathway. However, it is worth mentioning that to our knowledge there is no evidence of phospholipase A2 activation in steroidogenic cells by hormone or mEGF. Moreover, bromophenacyl bromide and quinacrine, generally used to inhibit the phospholipase A2 activity, are potent inhibitors of Acot2 (46), and thus, they are not useful to identify the enzymes involved in the process. The inhibitors of the phospholipase A2 act on Acot2 activity due to the existence of a serine lipase motif in the catalytic domain of the thioesterase (47). It will be of interest to determine whether mEGF and cAMP provide free AA from different sources, e.g. phospholipids or cholesterol esters to the ACS4 enzyme.

Majercik and Puett (29) also mentioned that mEGF- stimulated AA release itself is difficult to demonstrate. They explained this difficulty with the fact that AA release itself would be transient, very rapid, discrete, and localized, resulting in a specific hormone-induced signal below the limits of detection. Thus, they suggested that a sensitive assay should be used to study AA release.

This concept is very important because AA accumulation is very different between the organelles. For example, the nucleus in steroidogenic cells is an organelle that accumulates very high levels of free AA (19). Therefore, the hormone-dependent release of free AA determined in whole cells may be difficult to detect because the release of AA occurs in a specific compartment of the cells as shown in this study.

It is known that mEGF produces less that 10% of steroid synthesis in MA-10 cells, compared with cAMP-stimulated steroid production. However, mEGF has a striking synergistic effect on steroidogenesis when the MA-10 cells are stimulated with a combination of both agents (23). Although the mechanism responsible for this phenomenon has not been described in detail previously or in the present study, the fact that both mEGF and cAMP share the same mechanism to induce AA release and that mEGF increases the expression of Acot2 and cAMP does not may contribute to the synergistic effect. The expression of both enzymes, ACS4 and Acot2, is increased by mEGF in a time-dependent and biphasic fashion, being both peaks within the short-term period (up to 8 h) of mEGF action in which mEGF activates steroid biosynthesis without affecting cAMP levels (23). There is strong evidence that AA and lipoxygenase products are involved in a generalized mechanism of regulating steroidogenic function working in the regulation of StAR expression. In MA-10 Leydig cells it has been shown that the inhibition of the lipoxygenase activity results in a decrease in mEGF and AA-stimulated progesterone production (29). In addition, a stimulatory effect of 12-hydroxyeicosatetraenoic acid (HETE), 15 HETE, and 15-HPETE on MA-10 cell steroidogenesis has also been demonstrated (29). In this paper we show that intramitochondrial AA release is at least one of the intermediary events involved in StAR expression because the decrease in Acot2 and intramitochondrial AA results in a decrease of StAR expression.

Most of the components described in this steroidogenesis activating process, EGF, ACS4, AA and AA lipoxygenated metabolites, are also implicated in carcinogenesis (27, 28, 48, 49). Several lines of evidence indicate that the level of free AA in cells determine apoptosis or proliferation (50). In the gas trointestinal system and liver, ACS4 is expressed at very low levels. However, there is strong evidence that the ACS4 pathway may be very important in colon and hepatocellular carcinoma (51, 52). In both cases, there is an overexpression of ACS4, which prevents AA-induced apoptosis. Moreover, a decrease in the expression of ACS4 in hepatocellular carcinoma cells leads to the inhibition of cell proliferation. ACS4 has been described to be overexpressed in the transition from normal to preneoplastic mammary tissue (52).

The overexpression of ACS4 promotes cell proliferation by increasing the levels of AA-CoA esters. However, the mechanism by which AA-CoA esters may work is still uncertain. Although the mitogenic effect of mEGF is not observed in MA-10 cells, this growth factor is known as a mitogenic factor. Our present report may indicate that AA-CoA esters produced by induction of ACS4 could affect cell proliferation by generation of mitochondrial AA and metabolism to lipoxygenated products.

In summary, in the present work, we presented evidence that mEGF action on AA release is produced by an increment in the expression of ACS4 and Acot2 to release AA in a specific compartment of the cell, i.e. the mitochondria. This mechanism is, at least in part, responsible for the induction of StAR protein.


   Footnotes
 
This work was supported in part by National Research Council (Consejo Nacional de Investigaciones Cientificas y Técnicas), University of Buenos Aires, and National Agency of Scientific and Technological Promotion.

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2008

Abbreviations: AA, Arachidonic acid; AA-CoA, arachidonyl-CoA; Acot2, acyl-CoA thioesterase; ACS4, acyl-CoA synthetase; CoA, coenzyme A; EGF, epidermal growth factor; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; mEGF, mouse epidermal growth factor; NADH, reduced form of the nicotinamide adenine dinucleotide; StAR, steroidogenic acute regulatory.

Received November 19, 2007.

Accepted for publication March 21, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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