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Molecular Mechanisms of Insulin-like Growth Factor-I Mediated Regulation of the Steroidogenic Acute Regulatory Protein in Mouse Leydig Cells

Department of Cell Biology and Biochemistry (P.R.M., S.P.C., Y.J., D.M.S.), Texas Tech University Health Sciences Center, Lubbock, Texas 79430; Scott Department of Urology (S.R.K.), Baylor College of Medicine, Houston, Texas 77030; Physiologie et Physiopathologie (R.C.), Université Pierre et Marie Curie, Unité Mixte de Recherche, Centre Nationale de la Recherche Scientifique, 7079 Paris, France; and Institute of Reproductive and Developmental Biology (I.T.H.), Imperial College London, London W12 0NN, United Kingdom

Address all correspondence and requests for reprints to: Douglas M. Stocco, Ph.D., Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430. E-mail: doug.stocco{at}ttmc.ttuhsc.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Growth factors are known to play diverse roles in steroidogenesis, a process regulated by the mitochondrial steroidogenic acute regulatory (StAR) protein. The mechanism of action of one such growth factor, IGF-I, was investigated in mouse Leydig tumor (mLTC-1) cells to determine its potential role in the regulation of StAR expression. mLTC-1 cells treated with IGF-I demonstrated temporal and concentration-dependent increases in StAR expression and steroid synthesis. However, IGF-I had no effect on cytochrome P450 side-chain cleavage or 3ß-hydroxysteroid dehydrogenase protein levels. IGF-I was capable of augmenting N,O'-dibutyrl-cAMP-stimulated steroidogenic responsiveness in these cells. The steroidogenic potential of IGF-I was also confirmed in primary cultures of isolated mouse Leydig cells. IGF-I increased phosphorylation of ERK1/2, an event inhibited by the MAPK/ERK inhibitors, PD98059 and U0126. Interestingly, inhibition of ERK activity enhanced IGF-I-mediated StAR protein expression, but phosphorylation of StAR was undetectable, an observation in contrast to that seen with N,O'-dibutyrl-cAMP signaling. Further studies demonstrated that these events were tightly correlated with the expression of dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 and scavenger receptor class B type 1. Whereas both protein kinase A and protein kinase C signaling were involved in the IGF-I-mediated steroidogenic response, the majority of the effects of IGF-I were found to be mediated by the protein kinase C pathway. Transcriptional activation of the StAR gene by IGF-I was influenced by several transcription factors, its up-regulation being dependent on phosphorylation of the cAMP response element-binding protein (CREB) and the activator protein 1 family member, c-Jun. Conversely, StAR gene transcription was markedly inhibited by expression of nonphosphorylatable CREB (Ser¹³³Ala), dominant negative A-CREB, and dominant negative c-Jun (TAM-67) mutants. Collectively, the present studies identify molecular events in IGF-I signaling that may influence testicular growth, development, and the Leydig cell steroidogenic machinery through autocrine/paracrine regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
LEYDIG CELL STEROIDOGENIC function is predominantly regulated by LH/human choriogonadotropin (hCG) and the influence of multiple signaling pathways (1, 2, 3). The steroidogenic potential of Leydig cells can be modulated not only by LH/hCG but also by peptide and nonpeptide hormones, including growth factors, prostaglandins, and steroids, through endocrine, autocrine, and paracrine regulatory events (2, 4). Growth factors are involved in numerous actions in many different cell types. IGF-I is a growth-promoting peptide, its effects being mediated through a transmembrane tyrosine kinase receptor that is structurally related to the insulin receptor, and modulates many biological functions including steroidogenesis in gonadal cells. However, the mechanism by which IGF-I influences the steroidogenic machinery in Leydig cells remains poorly understood.

The IGF-I system includes the IGF-I ligands (IGF-I and IGF-II), IGF-I receptors, and IGF-I binding proteins (5, 6). Although most of the physiological functions of IGF-I are thought to occur via the IGF-I receptor, six identified IGF-I-binding proteins can bind IGF-I with varying affinities and can influence its activity (5, 6). The binding of IGF-I to its receptor and the subsequent intracellular modifications that occur result in the activation of Ras-Raf-1-MAPKs, a family of serine/threonine kinases involved in regulating a diverse array of cellular and nuclear proteins, including transcription factors (4, 5). The role of IGF-I has been demonstrated in testicular growth and development, control of Leydig cell numbers, and in the onset of steroidogenesis and spermatogenesis (2, 7). Disruption of the gene encoding the IGF-I receptor resulted in developmental delays in reproductive organs, dwarfism, abnormalities in the central nervous system, and infertility in mice (8, 9). In addition, increasing evidence is accumulating that LH/hCG action can also be controlled by various factors including IGF-l (2, 10, 11), suggesting that IGF-I is an important physiological regulator of testicular function.

Biochemical, immunocytochemical, and ligand-binding studies have identified the presence of IGF-I receptors in human (12), porcine (13), rat (14), and mouse (15) Leydig cells. Treatment with IGF-I has been shown to potentiate steroidogenic responsiveness to LH/hCG or cAMP analogs in primary cultures of different Leydig cells (10, 16, 17, 18). It has recently been demonstrated that IGF-I null mice have decreased levels of serum testosterone and steroidogenic acute regulatory (StAR) protein (19), and thus, an investigation on the direct effects of IGF-I on StAR expression seems warranted. StAR is synthesized as a 37-kDa precursor mitochondrial phosphoprotein that is converted to a 30-kDa mature isoform after uptake and processing by the mitochondria (20, 21, 22). StAR has been demonstrated to play an essential role in regulating steroid biosynthesis by mediating the transfer of cholesterol from the outer to the inner mitochondrial membrane where it is converted to pregnenolone (22, 23, 24, 25). The precise role of StAR in the mechanism of cholesterol transfer to the inner mitochondrial membrane is unknown, but the preponderance of evidence indicates that the 37-kDa isoform acts on the outer membrane to effect cholesterol transfer (26, 27). An opposing view indicated that the 30-kDa phosphorylated isoform acting on the inner mitochondrial membrane resulted in the majority of cholesterol transfer (28).

The compelling evidence documenting the critical role of StAR in the regulation of steroidogenesis has been obtained from both basic and clinical studies (23, 29, 30). Transcriptional and/or translational inhibition of StAR expression results in a dramatic decrease in steroid biosynthesis whereas approximately 10–15% of steroid synthesis appears to be mediated through StAR-independent mechanisms (31, 32, 33, 34). Whereas the expression of the StAR protein is predominantly regulated by cAMP-dependent mechanisms, several factors and signaling pathways have been demonstrated to play permissive roles (reviewed in Refs.35 and 36). In addition to the cAMP/protein kinase A (PKA) pathway, the protein kinase C (PKC) pathway was demonstrated to be involved in regulating StAR expression and steroidogenesis in mouse Leydig (37) and adrenal (38, 39) cells. Also, transcriptional regulation of the StAR gene is mediated by several transcription factors (35, 40), including the cAMP response element (CRE)-binding protein (CREB)/CRE modulator, and the activator protein 1 (AP-1) family members Fos and Jun (41, 42, 43, 44, 45). Additional studies have also demonstrated the involvement of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1) and SR-BI (scavenger receptor, class B, type 1) in the regulation of StAR expression and steroidogenesis (46, 47, 48, 49, 50). The present findings demonstrate that the mechanism of action of IGF-I in the regulation of StAR expression and steroid synthesis in mouse Leydig cells involves multiple signaling pathways.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Role of IGF-I on StAR, Cytochrome P450 Side-Chain Cleavage Enzyme (P450scc), 3ß-Hydroxysteroid Dehydrogenase (3ß-HSD), Steroid, and cAMP Levels
Growth factors are known to have diverse roles in regulating StAR expression and steroidogenesis in gonadal cells. Mouse Leydig tumor (mLTC-1) cells treated with 0–500 ng/ml IGF-I demonstrated dose-dependent increases in StAR protein, StAR mRNA, and progesterone synthesis (Fig. 1). IGF-I demonstrated 2.7 ± 0.3- and 3.3 ± 0.4-fold increases in StAR protein (Fig. 1, A and D) and StAR mRNA (Fig. 1, B and D) levels over untreated cells, respectively. Accumulation of progesterone in the media was slightly lower when compared with StAR expression, being 2.0 ± 0.3-fold over basal in response to IGF-I. In contrast, IGF-I did not affect P450scc protein expression (Fig. 1, C and D).

View larger version (51K): [in this window] [in a new window]   Fig. 1. Role of IGF-I on StAR Protein, StAR mRNA, and P450scc Protein Expression, and Progesterone Synthesis in mLTC-1 Cells Cells were treated with 0–500 ng/ml IGF-I for 6 h and subjected to isolation of mitochondria (for immunoblotting) and total RNA (for RT-PCR) to determine StAR and P450scc protein, and StAR mRNA expression, respectively. Representative immunoblots and autoradiogram illustrate the dose-response pattern of StAR protein (panel A), StAR mRNA (panel B), and P450scc protein (panel C) expression. Accumulation of progesterone in the media was determined by RIA (panel D). Integrated optical density values of each band were quantified (normalized with the corresponding L19 bands in case of RT-PCR) and presented in terms of fold response (panel D). Data represent the mean ± SE of three to four independent experiments.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Temporal Response Pattern of IGF-I on StAR, P450scc, and 3ß-HSD Protein Expression and Intracellular cAMP Levels in mLTC-1 Cells Cells were incubated 100 ng/ml with IGF-I for 0–36 h and subjected to immunoblotting with specific antibodies (panels A–C), as described in Materials and Methods. Cells were also treated without or with 100 ng/ml IGF-I for 0–6 h in the presence of 0.5 mM 3-isobutyl-1-methylxanthine, and the levels of intracellular cAMP were determined (panel D, inset) by RIA (± SE, n = 3). Representative immunoblots illustrate expression of StAR (panel A), P450scc (panel B), and 3ß-HSD (panel C) at indicated times. Integrated optical density values of each immunospecific band were quantified and presented in terms of fold response (panel D), which represent the mean ± SE of three independent experiments.

The physiological relevance of the effects of IGF-I on StAR expression and steroidogenesis was further evaluated in isolated mouse Leydig cells. As determined by RT-PCR, treatment with 100 ng/ml IGF-I for 6 h resulted in a 2.2 ± 0.3-fold increase in StAR mRNA expression over untreated cells (Fig. 3A). The cAMP analog, N,O'-dibutyrl-cAMP [(Bu)₂cAMP], an inducer of StAR expression through the PKA pathway, demonstrated a 3.8 ± 0.5-fold increase in StAR mRNA expression over basal at a concentration of 0.5 mM. Accumulation of testosterone in the media was elevated to 166 ± 9 and 214 ± 12% by IGF-I and (Bu)₂cAMP, respectively, when compared with control (Fig. 3B), further confirming the involvement of IGF-I in the steroidogenic response.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Effect of IGF-I on StAR mRNA Expression and Testosterone Production in Isolated Mouse Leydig Cells Dispersed adult mouse Leydig cells were treated without (Control) or with 100 ng/ml IGF-I and 0.5 mM (Bu)2cAMP for 6 h, and total RNA was isolated to determine StAR mRNA expression. A representative autoradiogram shows expression of StAR mRNA by RT-PCR using 2 µg of total RNA (panel A). Integrated optical density values of each band were quantified and normalized to the intensity of the corresponding L19 bands (panel B). Accumulation of testosterone in the media is seen in panel B. Data represent the mean ± SE of three independent experiments. Letters above the bars indicate that these groups differ significantly at P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Effects of IGF-I, Insulin, (Bu)2cAMP, or their Combination on StAR mRNA Expression and Steroid Synthesis mLTC-1 cells were treated without (Control) or with 100 ng/ml IGF-I, 1000 ng/ml insulin, 0.5 mM (Bu)2cAMP, 5 µg/ml cycloheximide (CHX), or in combination for 6 h as indicated (panels A–C). Total RNA was extracted for RT-PCR analysis, and a representative autoradiogram demonstrates the effects of these agents on StAR mRNA expression (panel A). Integrated optical density values of each band were normalized to the corresponding L19 bands. Progesterone (panel B) and testosterone (panel C) levels in the media of the same treatments were determined (±SE, n = 3–5). Letters above the bars indicate that these groups differ significantly at least at P < 0.05.

View larger version (32K): [in this window] [in a new window]   Fig. 5. Effects of IGF-I, (Bu)2cAMP, or Their Combination on StAR Promoter Responsiveness Panel A, mLTC-1 cells were transfected with the StAR promoter/luciferase segments (–966–1, –151/–1, and –96/–1 bp) in the presence of pRL-SV40 (Renilla luciferase for determining transfection efficiency), as described in Materials and Methods. PGL3-basic vector (pGL3) was used as a control. Panel B, mLTC-1 cells were also transfected with empty vector (pcDNA3.1), SF-1, C/EBPß, SREBP-1, GATA-4, and Sp1 expression plasmids in the presence of the –151/–1 bp StAR segment. pRL-SV40 was used in this transfection protocol. After 36 h of transfection, cells were treated without (Basal) or with 100 ng/ml IGF-I, 0.5 mM (Bu)2cAMP, or their combination for 6 h, and luciferase activity in the cell lysates was determined as relative light units (RLU). Data represent the mean ± SE of three to four independent experiments.

Role of the PKA and PKC Pathways in IGF-I- and (Bu)₂cAMP-Responsive StAR Expression
To understand signaling events in the IGF-I-mediated steroidogenic response, the roles of the PKA and PKC pathways were assessed in mLTC-1 cells (Fig. 6). Cells treated with 100 ng/ml IGF-I and 0.05 and 0.5 mM (Bu)₂cAMP for 6 h demonstrated approximately 2.7-, 3.8-, and 8.6-fold increases in StAR protein levels in comparison with control, respectively (Fig. 6A). (Bu)₂cAMP (0.05 and 0.5 mM)-induced StAR protein expression was further augmented by 6.3- and 11.4-fold when cells were treated in combination with IGF-I. Inhibition of PKA activity with H-89 (25 µM) significantly decreased (P < 0.05) basal and IGF-I-stimulated StAR expression, suggesting the involvement of endogenous cAMP in IGF-I-mediated steroidogenic responses because the latter had no effect on cAMP levels. Under similar experimental paradigms, progesterone levels were approximately 2-, 60-, and 276-fold higher over basal, with IGF-I and 0.05 and 0.5 mM (Bu)₂cAMP, respectively (data not shown), and, when used in combination, they followed the pattern of StAR expression. These results prompted us to evaluate the phosphorylation status of StAR protein. Phosphorylation of StAR is required for its full activity (54). StAR phosphorylation (P-StAR) was undetectable in control, IGF-I-, H-89-, and IGF-I plus H-89-treated cells but was dose responsive with (Bu)₂cAMP (Fig. 6A). Whereas no P-StAR was detected in response to IGF-I, it augmented (Bu)₂cAMP-induced P-StAR by 1.7- and 1.4-fold over the response seen with 0.05 and 0.5 mM (Bu)₂cAMP, a process strongly inhibited by H-89.

View larger version (48K): [in this window] [in a new window]   Fig. 6. Role of the PKA and PKC Pathways in IGF-I and (Bu)2cAMP-Mediated StAR Expression in mLTC-1 Cells Cells were treated for 15 min with either vehicle, PKA, or PKC inhibitors, and divided into two groups to assess the involvement of the PKA (panel A) and PKC (panel B) pathways in IGF-I- and (Bu)2cAMP-mediated StAR protein expression. The role of the PKA and PKC pathways on StAR mRNA expression using similar experimental conditions was determined. Cells were incubated without or with 100 ng/ml IGF-I, 0.05 and 0.5 mM (Bu)2cAMP, 10 nM PMA, 20 µM GFX, and 25 µM H-89, or a combination of them for 6 h, as indicated (panels A–C). Cells were subjected to mitochondrial isolation for determining StAR protein and P-StAR by immunoblotting, and total RNA for determining StAR mRNA expression by RT-PCR analysis. Representative immunoblots and autoradiogram show expression of StAR and P-StAR using 25 µg of mitochondrial protein (panels A and B), and StAR mRNA expression using 2 µg of total RNA (panel C). Data are representative of three independent experiments. kD, Kilodaltons.

IGF-I responsive StAR mRNA expression demonstrated qualitatively similar results in association with the PKA and PKC signaling pathways (Fig. 6C). mLTC-1 cells treated with 100 ng/ml IGF-I, 10 nM PMA, and 0.5 mM (Bu)₂cAMP demonstrated 3.2-, 8.1-, and 10.4-fold increases in StAR mRNA expression over basal. Whereas IGF-I did not show noticeable effects on StAR mRNA expression in combination with PMA, IGF-I further augmented (Bu)₂cAMP-induced StAR expression by 15.7-fold when compared with controls. Inhibition of the PKA and PKC pathways, by 25 µM H-89 and 20 µM GFX, respectively, markedly decreased IGF-I plus (Bu)₂cAMP- and IGF-I plus PMA-mediated StAR mRNA expression. Because the steroidogenic potential of IGF-I was drastically affected by inhibiting PKC and it had no additional effect in combination with PMA, these results indicate the involvement of PKC signaling in IGF-I responsiveness.

To elucidate a role for the PKC pathway in IGF-I-mediated steroidogenesis, StAR expression in response to PMA was determined and compared with IGF-I. Cells treated with 10 nM PMA showed a significant increase (P < 0.01) in StAR protein expression by 2 h and a maximum response (6.9-fold) by 6 h and thereafter declined slowly with time (Fig. 7). Using similar experimental paradigms, qualitatively similar results were obtained with StAR mRNA expression. In fact, the expression of StAR in response to IGF-I was very similar to that seen with PMA (compare Figs. 2A and 7A). Inhibition of PKC with 20 µM GFX markedly decreased PMA-stimulated steroidogenic responsiveness. Notably, addition of 5 µg/ml cycloheximide to PMA-treated cells attenuated (P < 0.01) both StAR expression and progesterone synthesis (Fig. 7B), an observation similar to IGF-I responsiveness (Fig. 4, A–C). These findings demonstrate clearly that the PKC signaling pathway plays an indispensable role in IGF-I-mediated steroidogenic responses.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Temporal Response of StAR Expression and its Dependence on Ongoing Protein Synthesis to PMA in mLTC-1 Cells Cells were stimulated with 10 nM PMA for 0–36 h and subjected to immunoblotting (using 30 µg of mitochondrial protein) and RT-PCR (using 2 µg of total RNA) analysis for StAR expression (panel A), as described in the legend of Fig. 1. Cells were also treated without (Control) or with 10 nM PMA for 6 h in the absence or presence of 5 µg/ml cycloheximide (CHX), and StAR expression and progesterone accumulation in the media were then determined (panel B). Representative immunoblots and autoradiograms illustrate PMA-stimulated StAR protein and StAR mRNA expression (panels A and B). IOD values of each band were quantified (normalized with the corresponding L19 bands in the case of RT-PCR), and the levels of StAR protein and StAR mRNA are expressed as the maximum activity in terms of fold response (panel A). In both panels, data represent the mean ± SE of three to four independent experiments. Letters above the bars indicate that these groups differ significantly at P < 0.05.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Specific Involvement ERK1/2 Signaling in IGF-I Responsiveness in mLTC-1 Cells Cells were serum starved for 6 h and then treated with 100 ng/ml IGF-I for 0–30 min and ERK1/2 phosphorylation (P-ERK1/2) and total ERK1/2 in 25 µg total protein were determined by immunoblotting. Representative immunoblots illustrate expression of P-ERK1/2 and ERK1/2 in response to IGF-I (panel A). Integral optical density (IOD) values presented for P-ERK1 (p44), P-ERK2 (p42), and ERK1/2 were compiled from four independent experiments (panel A). Serum-starved cells were incubated for 3 min with 0–100 ng/ml of IGF-I. Representative immunoblots illustrate the dose-response pattern of P-ERK1/2 and ERK1/2 to IGF-I using 20 µg of total cellular protein (panel B). Panel C, Cells were pretreated with either vehicle or the MEK inhibitors, PD98059 (PD, 50 µM) and U0 (12 µM) for 15 min, and then stimulated for 3 min without (Control) or with 100 ng/ml IGF-I, or a combination of them as indicated. Cellular protein (22 µg) was analyzed by immunoblotting for P-ERK1/2 and ERK1/2. Data are representative of four independent experiments.

View larger version (50K): [in this window] [in a new window]   Fig. 9. Role of the ERK1/2 Pathway in IGF-I- and (Bu)2cAMP-Mediated StAR Protein Levels and Progesterone Synthesis, and Their Relevance to DAX-1 and SR-BI Expression mLTC-1 cells were pretreated with either vehicle or the MEK inhibitors PD98059 (PD, 50 µM) and U0 (12 µM) for 15 min, and then were divided into two groups. One group was incubated without (Control) or with 100 ng/ml IGF-I in the absence or presence of PD or U0 for 6 h (panel A). The other group was treated with 0.5 mM (Bu)2cAMP in the presence or absence of PD and U0, or in combination with IGF-I, (Bu)2cAMP, PD, and U0 for 6 h, as indicated (panel B). Representative immunoblots illustrate StAR protein expression and StAR phosphorylation (P-StAR) using 25 µg of mitochondrial protein. Accumulation of progesterone in the media of these treatment groups was determined and expressed as ng/mg of protein (panels A and B). Similarly, mLTC-1 cells were treated for 6 h without (Control) or with IGF-I, (Bu)2cAMP, PD, U0, or their combination as indicated (panels C and D). Total RNA was isolated from different treatment groups and subjected to the expression of StAR, DAX-1, and L19 mRNAs (panel C), and SR-BI and L19 mRNAs (panel D) by RT-PCR analysis using 2.5 µg of RNA. Integrated optical density (IOD) values of StAR, DAX-1, and SR-BI bands were quantified and normalized with the corresponding L19 bands and presented (panels C and D). Note the different scales on both sides (panel C). Data represent the mean ± SE of three to four independent experiments. Letters above the bars indicate that these groups differ significantly at least at P < 0.05.

To further understand the decrease in steroid synthesis associated with MEK inhibition, we investigated the expression levels of the scavenger receptor class B type 1 (SR-B1), the lipoprotein receptor involved in providing steroidogenic cells with cholesterol for steroid biosynthesis. Treatment of 100 ng/ml IGF-I demonstrated a 1.8 ± 0.3-fold increase in SR-B1 mRNA expression over basal (Fig. 9D). In contrast, the expression of SR-BI mRNA was 6.2 ± 0.6-fold in response to 0.5 mM (Bu)₂cAMP when compared with controls. It can be clearly seen that both 50 µM PD and 12 µM U0 were capable of decreasing (P < 0.05) IGF-I and (Bu)₂cAMP-stimulated SR-B1 mRNA expression. These inhibitors also affect basal SR-B1 expression. Thus, the decrease in steroid synthesis in response to PD and U0 could be a result of a decrease in cholesterol availability to the mitochondria as a result of an inhibition in SR-B1 expression.

CREB Phosphorylation and Transcriptional Activation of CREB by IGF-I
The role of posttranslational modification of a CREB/CRE modulator family member in the regulation of StAR expression and steroidogenesis has been demonstrated (41, 43). Thus, it was essential to determine whether IGF-I plays a role in CREB phosphorylation. As depicted in Fig. 10A, phosphorylation of CREB (P-CREB) at Ser133 was increased by 3.6 ± 0.7-fold over basal at 30 min in response to 100 ng/ml IGF-I and began to decrease thereafter with time. No changes were observed in the levels of CREB protein during this 180-min period. Also, the phosphoactivating-CREB antibody (Ab) detected an additional band for phosphorylated transcription factor 1 (P-ATF-1, Ser63), as both CREB and ATF-1 are 100% homologous for consensus phosphorylation sequences (60). Indeed, P-ATF-1 demonstrated an expression pattern similar to that of P-CREB. Additionally, 0–500 ng/ml IGF-I increased P-CREB and P-ATF-1 in a dose-response manner without affecting the total amount of CREB protein (Fig. 10B).

View larger version (42K): [in this window] [in a new window]   Fig. 10. Time- and Dose-Response Pattern of CREB Protein Expression and Its Transcriptional Activation by IGF-I Panel A, mLTC-1 cells were treated with 100 ng/ml IGF-I for 0–180 min. Total cellular protein (30 µg) was electrophoresed and immunoblotted, and representative immunoblots show CREB phosphorylation (P-CREB), ATF-1 phosphorylation (P-ATF-1), and CREB in response to IGF-I. Cells were also incubated with 0–500 ng/ml of IGF-I for 30 min, and 28 µg of total protein was analyzed for P-CREB, P-ATF-1, and CREB by immunoblotting (panel B). Results are representative of three independent experiments. Panel C, mLTC-1 cells were transfected with empty vector (pcDNA3.1), wild-type CREB (WT-CREB), CREB-M1 (nonphosphorylatable CREB), and A-CREB (dominant-negative CREB), in the presence of the –151/–1 StAR promoter segment. Cells were cotransfected with pRL-SV40 for normalization of transfection efficiency. After 36 h of transfection, cells were treated without (Basal) or with 100 ng/ml IGF-I, 0.5 mM (Bu)2cAMP, or a combination of them for an additional 6 h, and luciferase activity in the cell lysates was determined and expressed as reporter response [RLU (relative light units)]. Data represent the mean ± SE of four independent experiments.

IGF-I and c-Jun Responsiveness and Their Relevance to StAR Gene Transcription
Because c-Jun can up-regulate StAR gene transcription (42, 44), we assessed its role in IGF-I responsiveness. mLTC-1 cells transfected with the pathway-specific transactivator plasmid, pFA2-c-Jun, in the presence of pFR-Luc demonstrated a dose-dependent increase in luciferase activity, a response enhanced maximally by 2.7 ± 0.4-fold over basal with 100 ng/ml IGF-I (Fig. 11A). Cells cotransfected with pFA2-c-Jun in the presence of a dominant-negative form of c-Jun (TAM-67) abrogated the c-Jun-mediated response to IGF-I.

View larger version (26K): [in this window] [in a new window]   Fig. 11. Involvement of c-Jun in IGF-I Responsiveness and StAR Gene Transcription mLTC-1 cells were transfected with the c-Jun transactivator plasmid (pFA2-c-Jun) in the presence of pFR-luc reporter plasmid (panel A), as described in Materials and Methods. The pRL-SV40 was included in the transfection protocol to normalize transfection efficiency. The specificity of c-Jun responsiveness was assessed using TAM-67 (a dominant-negative c-Jun). Panel B, mLTC-1 cells treated with 100 ng/ml IGF-I for 0–120 min and 23 µg of total cellular protein were used for determining c-Jun phosphorylation (P-c-Jun) and c-Jun by immunoblotting. Representative immunoblots show P-c-Jun, Jun D phosphorylation (P-Jun D), and c-Jun in response to IGF-I. Data are representative of three independent experiments. Panel C, Utilizing the –151/–1 StAR promoter segment, mLTC-1 cells were transfected either with empty vector (pcDNA3.1), wild-type c-Jun (c-Jun), TAM-67, or a combination of them, in the presence of pRL-SV40. After 36 h of transfection, cells were treated without (Basal) or with varying (0–100 ng/ml, panel A) or fixed (100 ng/ml, panel C) doses of IGF-I, and luciferase activity in the cell lysates was determined. Data represent the mean ± SE of four independent experiments. RLU, Relative light units.

The relevance of c-Jun phosphorylation in response to IGF-I was evaluated at the level of StAR gene transcription utilizing the –151/–1 StAR reporter segment (Fig. 11C). mLTC-1 cells transfected with the c-Jun expression plasmid demonstrated an additional 2.2 ± 0.4-fold increase in luciferase activity over the response seen with 0.5 mM (Bu)₂cAMP in mock-transfected cells. In spite of a lack of IGF-I response, it further enhanced (Bu)₂cAMP-induced (2 fold) StAR reporter activity mediated by c-Jun. The activation of c-Jun-mediated StAR reporter activity by (Bu)₂cAMP was markedly inhibited by TAM-67, and IGF-I failed to modulate (Bu)₂cAMP-stimulated responsiveness. Taken together, these findings demonstrate that IGF-I-responsive CREB and c-Jun signaling play essential roles in regulating StAR transcription, especially the synergy between IGF-I and (Bu)₂cAMP, in mouse Leydig cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Steroidogenic responsiveness of gonadal cells to gonadotropins can be modulated by circulating peptides or locally produced factors secreted by cells within respective tissues. Basic and clinical studies implicate the role of growth factors in regulating testicular function, including spermatogenesis, and these factors can interact with LH/hCG in potentiating their action on Leydig cells. Steroidogenesis in Leydig cells involves a complex interaction of a diversity of hormones and multiple signaling pathways (2, 3, 31, 61). Steroid hormone biosynthesis is initiated upon translocation of cholesterol from the outer to the inner mitochondrial membrane, the site of the P450scc enzyme, a process mediated by the StAR protein (23, 24, 25). The present studies extend our knowledge of the manner in which IGF-I acts to drive StAR expression and the steroidogenic machinery in mouse Leydig cells.

Our current data demonstrate that IGF-I is capable of increasing expression of StAR protein, but not StAR phosphorylation, and steroid synthesis in mouse Leydig cells. The stimulatory effect of IGF-I on StAR expression and steroid synthesis was dependent on ongoing protein synthesis, and these responses were not associated with an alteration of intracellular cAMP (34, 62). The steroidogenic potency of IGF-I was evident both in mLTC-1 and in freshly isolated mouse Leydig cells and was qualitatively similar to that of insulin, providing evidence that the two hormones are probably acting through similar mechanisms. The action of IGF-I on steroidogenesis in mLTC-1 cells was specific and was mediated through its own receptors because we demonstrated that they possess high-affinity IGF-I binding sites [dissociation constant (K_d) 0.68 nM] with a capacity of 172 fmol/mg protein as determined by Scatchard analysis (data not shown). Consistent with this finding, IGF-I receptors have previously been identified in Leydig cells of several species (12, 13, 14, 15). Alterations in DNA synthesis were not observed with longer periods of IGF-I treatment, indicating that IGF-I may act as a differentiation factor rather than a mitogenic factor. Hence, the influence of the latter can be ruled out as a cause of the increased steroidogenic response in adult mouse Leydig cells (16).

IGF-I and insulin are also capable of potentiating (Bu)₂cAMP-mediated StAR expression and steroid synthesis in mouse Leydig cells, an observation in agreement with previous findings (16, 18, 63). No significant effect of IGF-I was observed on either P450scc or 3ß-HSD protein levels in mLTC-1 cells. Likewise, IGF-I had no effect on P450scc mRNA expression, although it increases hCG-stimulated P450scc levels in primary cultures of rat Leydig cells (64, 65). An elevation of P450scc and 17-hydroxylase mRNA levels in porcine Leydig cells has also been reported (10). The lack of an IGF-I/insulin response on basal and (Bu)₂cAMP-mediated progesterone synthesis in MA-10 cells might be due to the absence of functional receptors for these ligands (66). MA-10 and mLTC-1 are Leydig cells cloned from mouse tumors. The latter cell line secretes significant amounts of testosterone together with constitutively higher levels of progesterone, StAR, P450scc, and 3ß-HSD and thus more closely resembles primary Leydig cells (present data and Refs.51, 52 , and 67). Therefore, the steroidogenic potential of IGF-I is dependent upon the relative abundance of receptors and receptor-ligand interactions, the endocrine status of the donor, species specificity, and the short- or long-term experimental conditions.

Mapping of the mouse StAR promoter demonstrated the importance of the first 150 nucleotides from the transcription start site, which contains multiple recognition motifs for sequence-specific transcription factors, in transcriptional regulation of the StAR gene (35, 40, 42, 45, 53, 68, 69, 70). In the present study, no noticeable effect of IGF-I on StAR promoter activity was found up to 1 kb of the 5'-flanking region, suggesting that element(s) responsive to IGF-I may lie upstream of this segment or in intronic sequences of the gene (71, 72). However, IGF-I potentiated the response of (Bu)₂cAMP in StAR promoter responsiveness with the segments that responded to (Bu)₂cAMP. Additionally, we observed that several transcription factors were involved in regulating transcription of the StAR gene mediated by IGF-I, including SF-1, C/EBPß, GATA-4, CREB, and c-Jun. Consistent with this, the regulation of porcine StAR promoter activity by IGF-I has been shown to occur in conjunction with GATA-4 and C/EBPß (73). However, it also remains possible that IGF-I can modulate signaling pathways downstream of cAMP/PKA that are involved in the steroidogenic response.

Whereas the cAMP/PKA pathway is undoubtedly the major signaling cascade regulating StAR expression and steroidogenesis, several other pathways are involved in these processes as well (35, 36). Earlier and present findings demonstrate that a low level of cAMP is sufficient for expression of the StAR protein (34, 74), most notably phosphorylated StAR, and increasing steroid synthesis. Whereas intracellular cAMP was not affected by IGF-I, the PKA inhibitor H-89 decreased basal and IGF-I-mediated StAR expression, demonstrating that endogenous cAMP plays an essential role in IGF-I responsiveness. Previous studies have demonstrated an indispensable requirement for phosphorylation of StAR to attain its full biological activity (54, 75). Two putative conserved PKA phosphorylation sites (Ser56/57 and Ser194/195, in murine and human StAR, respectively) were identified and characterized, and the predominant involvement of the latter site in enhancing the activity of the StAR protein and steroidogenesis was illustrated (54). Utilizing a specific Ab against phosphorylated Ser194 in the StAR protein, no P-StAR was detected in response to IGF-I. On the other hand, (Bu)₂cAMP greatly increased P-StAR, a response further augmented by IGF-I, demonstrating a critical role for phosphorylation in obtaining maximal steroid synthesis (54, 75, 76). Importantly, the IGF-I response was found to be dependent on PKC signaling, as inhibition of PKC by GFX markedly decreased StAR expression and steroidogenesis. In addition, PMA increased StAR protein expression but not P-StAR and only slightly elevated steroid production, a situation similar to that seen with IGF-I. Both IGF-I and PMA demonstrated a qualitatively parallel pattern in StAR expression that was dependent on ongoing protein synthesis. Whereas IGF-I potentiated the action of (Bu)₂cAMP, it had no additional effects in combination with PMA. This observation indicates that the action of IGF-I in steroidogenesis is predominantly mediated through the PKC pathway in mouse Leydig cells.

The MAPK/ERK signaling cascade has been demonstrated to regulate StAR expression and steroidogenesis; however, conflicting reports are found in different steroidogenic cells (34, 55, 56, 57, 58, 77, 78, 79). For example, inhibition of MEK activity with PD and U0 has been shown to be associated with stimulation (59, 77), inhibition (34, 56, 57), or no effect (78, 79, 80) on the steroidogenic response. Our data demonstrate that activation of ERK1/2 phosphorylation in response to IGF-I was diminished by PD and U0 without affecting ERK1/2 protein synthesis. However, MEK inhibition increases basal and IGF-I-mediated StAR expression, but decreases progesterone synthesis. This seeming contradiction can be explained by previous findings and could be associated with stimulus specificity, as inhibition of MEK was demonstrated to enhance basal and LH-induced StAR expression but attenuate steroid synthesis both in primary and in mouse Leydig tumor cells (55). In the present study, it was also demonstrated that MEK inhibitors were capable of decreasing (Bu)₂cAMP-responsive StAR expression and steroid synthesis, an observation in agreement with previous studies (34, 56, 57). In a recent study, PD was demonstrated to have no effect on (Bu)₂cAMP-stimulated StAR expression in MA-10 cells (81). In granulosa cells, inhibition of MEK has been shown to be associated with increases in basal and LH/FSH-mediated StAR expression and steroid biosynthesis (59, 77). Furthermore, whereas PD markedly attenuated IGF-I-induced progesterone production, no apparent effect of this inhibitor was found in insulin-stimulated human granulosa cells (78). These findings demonstrate a complex role for the MAPK/ERK cascade in the regulation of the steroidogenic response that appears to be dependent on receptor-effector coupling and is tissue and stimulus specific. Thus, it is plausible that activation of the ERK1/2 cascade with IGF-I and (Bu)₂cAMP involves discrete signaling events and indicates the importance of other factor(s) in the steroidogenic response. Exploration of IGF-I- and (Bu)₂cAMP-responsive StAR expression and steroid synthesis in association with MEK inhibition demonstrated that induction and/or attenuation of StAR expression was inversely correlated with DAX-1 (48, 59). In agreement with this, we recently demonstrated the involvement of DAX-1 in the regulation of steroidogenesis and StAR expression in mouse MA-10 and rat R2C Leydig tumor cells (50). PD and U0 decreased the expression of SR-BI, possibly inhibiting the availability of mitochondrial cholesterol and resulting in an attenuation of steroid synthesis. Studies are currently underway to determine whether other factors, in addition to DAX-1 and SR-BI, might influence the MEK-associated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

The binding of IGF-I to its receptor in the plasma membrane results in receptor autophosphorylation, phosphorylation of intracellular proteins, receptor clustering, and differentiation of target cells (4, 5, 6). Our data show that IGF-I induces phosphorylation of the transcription factor CREB at Ser133. This posttranslational modification of CREB, leading to an increase in StAR gene transcription, was markedly diminished by a nonphosphorylatable CREB (Ser¹³³Ala) and a dominant-negative A-CREB (41, 43). IGF-I was capable of increasing phosphorylation of the AP-1 family member, c-Jun, and the importance of this phenomenon in StAR’s transcriptional regulation was demonstrated using a dominant-negative TAM-67 that drastically affected c-Jun responsiveness. It is interesting to note that inhibition of CREB and c-Jun responsiveness abrogated the synergy between IGF-I and (Bu)₂cAMP on StAR gene expression. The role of IGF-I in CREB phosphorylation and its relevance in transcription has been previously shown in PC12 neuronal cells (82). In earlier studies we demonstrated that Fos and Jun could bind to the CRE2 site in the StAR promoter and were involved in StAR gene regulation (44). Moreover, peptide-mapping studies demonstrate that MAPK-related proteins can interact with AP-1 transcription factors, especially c-Jun (83). Thus, the cooperation and/or interaction of different signaling pathways may represent cross-talk, a well-known event in signal transduction, which appears to link IGF-I responsive StAR expression and steroidogenesis in Leydig cells.

Taken together, these findings demonstrate the involvement of multiple IGF-I signaling events associated with the regulation of the steroidogenic machinery in mouse Leydig cells. The lack of StAR phosphorylation in response to IGF-I indicates the importance of phosphorylation at Ser194 in obtaining maximal activity of the StAR protein in steroid synthesis. The steroidogenic potential of IGF-I (or related growth factors) in modulating cAMP-induced StAR transcription mediated by different transcription factors, especially by CREB and c-Jun, undoubtedly involves a multitude of processes, many of which remain to be determined.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Plasmids, Transfections, and Reporter Assays
The 5'-flanking regions (–966/–1, –151/–1, –96/–1) of the mouse StAR gene were cloned into the pGL3 basic vector (Promega Corp., Madison, WI), upstream of a luciferase reporter gene utilizing the XhoI and HindIII sites as described previously (41, 43). The identities of the inserted fragments were verified by restriction endonuclease digestion and by automated sequencing with a PE Biosystem 310 Genetic Analyzer (ABI PRISM 310, PerkinElmer, Norwalk, CT). The wild-type CREB (WT-CREB) (41, 43), nonphosphorylatable CREB (CREB-M1, Ser¹³³Ala) (41, 43), dominant-negative CREB (A-CREB) (41), c-Jun, dominant-negative c-Jun (TAM-67) (44), SF-1 (69, 84), C/EBPß (69), GATA-4 (85), Sp1 (86), and SREBP-1 (87) expression plasmids have been previously described. An expression vector for the firefly luciferase reporter gene (pFR-Luc) was obtained from Stratagene (La Jolla, CA). The plasmid (pRL-SV40) containing the Renilla luciferase gene driven by simian virus 40 promoter (Promega) was used to normalize transfection efficiency.

Mouse Leydig tumor [mLTC-1; (88)] cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium (Invitrogen Corp., San Diego, CA), containing serum and antibiotics (34, 41). Transfection studies were carried out using FuGENE 6-transfection reagent (Roche Diagnostics, Indianapolis, IN) under optimized conditions (41, 43). In brief, cells were cultured in either 12 or six-well plates to 65–75% confluency and transfected using 1–2 µg of plasmid (or with 1:1 ratio where appropriate), in the presence of 12–20 ng of pRL-SV40. After 36 h of transfection, cells were treated as specified in different experiments.

Luciferase activity in the cell lysates was determined by the Dual-luciferase reporter assay (Promega). Briefly, after treatment cells were washed with 0.01 M PBS and 200–275 µl of the reporter lysis buffer was added to the plates. The cellular debris was pelleted by centrifugation at 14,000 x g at 4 C, and the supernatant was measured for relative light units (luciferase/Renilla) in a TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA), following the instructions of the manufacturer.

Isolation and Purification of Mouse Leydig Cells
Isolation and purification of testicular Leydig cells from adult mouse (C57BL/6 strain, 2–3 months of age) were carried out utilizing previously described procedures (89, 90). In brief, decapsulated interstitial cells were dispersed by collagenase (0.2%, 20 min, 34 C), filtered through sterile nylon gauze (0.5–0.8 mm mesh), and washed with DMEM/F12 medium to remove the collagenase. Cells were then purified using continuous Percoll density gradient (range 1.01–1.126 kg/liter) centrifugation. During centrifugation, cell types partitioning at approximately 1.07 kg/liter of Percoll were collected and washed, and 70–80% of the cells were Leydig cells as determined histochemically by 3ß-HSD staining. Cells were subcultured in DMEM/F12 medium containing antibiotics and used for experiments after 48 h.

RNA Extraction and Quantitative RT-PCR
Total RNA was extracted using Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA) from different treatment groups. Isolation and amplification of mouse StAR cDNA was carried out utilizing the following primer pairs: the sense, 5'-GACCTTGAAAGGCTCAGGAAGAAC-3', and the antisense, 5'-TAGCTGAAGATGGACAGACTTGC-3' (22), as described previously (41, 90). The primers used for DAX-1 (GenBank accession no. NM_007430) and SR-BI (GenBank accession no. U76205) were: DAX-1 sense, 5'-GCCGAGGGCCCCCTGGTGGGAC-3' and, DAX-1 antisense, 5'-TCCAGCATCATATCATCCATGCTGAC-3', and SR-BI sense, 5'-CTCATCCAAGCAGCAGGTGCTCAAGAA-3' and, SR-BI antisense, 5'-TGTTTGCCAACGGGTCCGTCTACCCACC-3', respectively. The variation in RT-PCR efficiency was assessed using the L19 ribosomal protein gene as an internal control, using the sense primer 5'-GAAATCGCCAATGCCAACTC-3' and the antisense primer 5'-TCTTAGACCTGCGAGCCTCA-3' (91).

RT and PCR were run sequentially in the same assay tube using 2–2.5 µg of total RNA as specified in the figure legends, and the parameters including the number of cycles used for StAR, DAX-1 and SR-BI were optimized to be in the exponential phase (41, 67, 90). The cDNAs generated were further amplified by PCR using the primer pairs listed above. The molecular sizes of StAR, DAX-1, SR-BI, and L19 were determined on 1.2% agarose gels, which were vacuum dried and exposed to x-ray film (Marsh Bio Products, Inc., Rochester, NY) for 1–3 h. The levels of StAR, DAX-1, SR-BI, and L19 signals were quantified using a computer-assisted image analyzer (Visage 2000, BioImage, Ann Arbor, MI).

Immunoblotting
Immunoblotting studies were carried out with either total cellular or mitochondrial proteins. Cells were sonicated in lysis buffer [50 mM Tris; 150 mM NaCl; 1% Nonidet P-40 (pH 7.4)], containing protease inhibitors (1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin) at 4 C. Mitochondria were isolated by differential centrifugation in TSE buffer (10 mM Tris; 250 mM sucrose; 100 mM EDTA, pH 7.4) containing protease inhibitors, as described previously (22, 41, 84).

Protein (20–30 µg) was solubilized in sodium dodecyl sulfate (SDS) sample buffer and loaded onto a 12% SDS-polyacrylamide gel (Mini Protean II System, Bio-Rad Laboratories, Inc., Hercules, CA), as described by Laemmli (92), with minor modifications (41, 90). Electrophoresis was performed at 200 V for 1 h, and the proteins were electrophoretically transferred onto Immuno-Blot PVDF Membrane (Bio-Rad). The membranes were incubated overnight at 4 C in blocking buffer (Tris-buffered saline containing 0.2% Tween 20 and 4% Carnation nonfat dry milk) and incubated for 2 h with primary antibodies to StAR protein (aa 88–98) (22) and phospho-StAR (aa 190–199) generated in rabbits. It should be noted that the former Ab cannot distinguish between nonphosphorylated and phosphorylated StAR and thus detects total StAR protein. The Ab detecting StAR phosphorylation was generated against a peptide conjugated to keyhole limpet hemocyanin corresponding to aa 190–199 of mouse StAR with Ser194 phosphorylated. This was produced on a fee-for-service basis (Bethyl Laboratories, Montgomery, TX), and serum samples were double affinity purified using nonphosphorylated and phosphorylated peptides. Other primary antibodies used in immunoblotting were obtained from the following sources: P450scc (Chemicon International, Inc., Temecula, CA); 3ß-HSD (a generous gift from Dr. C. R. Parker, University of Alabama at Birmingham, Birmingham, AL); phospho ERK1/2, phospho CREB, and CREB (Cell Signaling Technologies, Beverly, MA); ERK1/2 (BD Biosciences Pharmingen, San Diego, CA); phospho c-Jun (Upstate Biotechnology, Inc., Lake Placid, NY); and c-Jun (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After incubation with primary antibodies, the membranes were washed and incubated with appropriate secondary antibodies for 1.5 h and washed again, after which immunodetection of proteins was performed with the Chemiluminescence Imaging Western Lightning Kit (PerkinElmer, Boston, MA). The membranes were exposed to x-ray films (Marsh Bio Products), and the intensity of immunospecific bands was quantified by scanning (Visage 2000). Expression of phosphorylated and total proteins was assessed using identically processed membranes; however, where appropriate, the same membranes were also analyzed by stripping and reprobing.

Path-detect Trans-reporting Assay
mLTC-1 cells were transfected with the transactivator plasmid (pFA2-c-Jun) in the presence of a reporter plasmid (pFR-Luc) using the FuGENE 6 transfection reagent (Roche Diagnostics), and the effect of IGF-I on c-Jun signaling was determined (Stratagene). The transactivator protein consists of the activation domain of c-Jun (aa 1–223) fused with the Gal4 DNA binding domain (aa 1–147), which were driven by the cytomegalovirus promoter. The pFR-Luc plasmid contains a synthetic promoter of the yeast Gal4 binding sites that control expression of the firefly luciferase gene. Upon cotransfection, the DNA binding domain of the fusion transactivator protein binds to the reporter plasmid at the Gal4 binding sites, and phosphorylation of the transcription activation domain of the fusion transactivator protein by IGF-I will activate transcription of the luciferase gene and indicate the involvement of this specific pathway. Cells were also cotransfected with a dominant-negative form of c-Jun, TAM-67, to determine the functional specificity of c-Jun in IGF-I signaling. After 36 h of transfection, cells were treated with 0–100 ng/ml of IGF-I for 6 h, and luciferase activity in the cell lysates was determined using a TD 20/20 Luminometer (Turner Designs).

Determination of DNA Synthesis
To determine the mitogenic effect of IGF-I, mLTC-1 cells were plated at a density of 1 x 10⁵ cells per well in six-well plates (89). Cells were cultured in the absence or presence of 10 and 100 mg/ml IGF-I for 6 h. [³H]thymidine (2 µCi/well; Amersham, Aylesbury, UK) was added to the cells, and incubation was continued for an additional 18 h. Thereafter, cells were washed with 0.01 M PBS, treated with 10% trichloroacetic acid at 4 C, lysed using 0.3 N NaOH containing 1% SDS, and counted in a ß-spectrometer (LKB Wallac OY, Turku, Finland).

Statistical Analysis
All experiments were carried out in triplicate or quadruplicate and were repeated three to five times. The results are expressed as the mean ± SE. Data were analyzed by ANOVA followed by Fisher’s least-significant difference tests using Statview 5.1 program (Abacus Concepts, Inc., Berkeley, CA). Differences were regarded as significant at P < 0.05.

	ACKNOWLEDGMENTS

 
We thank Dr. C. R. Parker for the gift of 3ß-HSD Ab. We also thank Dr. T. F. Osborne (University of California, Irvine, CA) for providing us the SREBP-1, Dr. J. Jiang (Baylor College of Medicine, Houston, TX) for the Sp1, and Dr. R. J. Schawrtz (Baylor College of Medicine) for the GATA-4 expression constructs, respectively.

	FOOTNOTES

 
This work was supported by funds from National Institutes of Health (NIH) Grant HD-17481 and the Robert A. Welch Foundation Grant B1-0028 (to D.M.S.); the Academy of Finland and the Sigrid Jusélius Foundation (to I.T.H.); and NIH Grant DK61548 and the Lalor Foundation (to S.R.K.).

First Published Online September 15, 2005

Abbreviations: aa, Amino acid; Ab, antibody; AP-1, activator protein 1; (Bu)₂cAMP, N,O'-dibutyrl-cAMP; C/EBPß, CCAAT/enhancer-binding protein ß; CRE, cAMP response element; CREB, cAMP response-element binding protein; DAX-1, dosage-sensistive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1; GFX, GF-109203X; hCG, human chorionic gonadotropin; HSD, hydroxysteroid dehydrogenase; LTC-1, Leydig tumor cells type 1; P-ATF-1, phosphorylated activating transcription factor 1; PKA, protein kinase A; MEK, MAPK/ERK; PKC, protein kinase C; PD, PD98059; PMA, phorbol 12-myristate 13-acetate; P450scc, cytochrome P450 side-chain cleavage enzyme; P-StAR, StAR phosphorylation; SDS, sodium dodecyl sulfate; SF-1, steroidogenic factor 1; Sp1, specific protein 1; SR-BI, scavenger receptor class B type I; SREBP, sterol regulatory element-binding protein; StAR, steroidogenic acute regulatory protein; U0, U0126.

Received for publication December 20, 2004. Accepted for publication September 6, 2005.

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