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Testis |
a Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430
b Geriatric Research, Education and Clinical Center, VA Palo Alto Health Care System, Palo Alto, California 94304
c Department of Pediatrics and the Metabolic Research Unit, University of California, San Francisco, San Francisco, California 94143-0978
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cyclic adenosine monophosphate, Leydig cells, progesterone, steroid hormones, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R2C cells are a rat Leydig tumor cell line [6], which, unlike MA-10 cells, secrete large amounts of progesterone in the absence of hormonal stimulation [7] and constitutively express high levels of StAR protein [8]. Although the rate-limiting step in steroid hormone biosynthesis is the transfer of cholesterol from the outer to the inner mitochondrial membrane, the constitutive expression of StAR in R2C cells alone does not appear to be sufficient to elicit the extremely high steroidogenic response observed. Indeed, transfection of both MA-10 and COS-1 cells (rendered steroidogenic by cotransfection with P450scc enzyme system) with StAR cDNA show only a modest increase in steroid production (3- to 10-fold, respectively) [4, 9], while trophic hormone (or cAMP analog) stimulation of MA-10 cells results in a robust (500 or greater fold) induction of steroid hormone synthesis. These observations provide further credence to the idea that additional factors may be required to maintain a constant supply of cholesterol precursor to support high rates of steroid synthesis.
In rodents, much of the precursor cholesterol for steroid hormone synthesis is derived from plasma lipoproteins in the form of cholesteryl esters (CE) [10–12]. Once CEs have entered steroidogenic cells, they are transported to intracellular storage sites (lipid droplets) for subsequent hydrolysis and mobilization by cholesteryl ester hydrolase (CEH or hormone sensitive lipase [HSL]) [11, 13]. The resulting free cholesterol is then transported into the mitochondria, where it undergoes side-chain cleavage as the first step in steroid hormone biosynthesis [5].
Rodent steroidogenic cells preferentially obtain most of their cholesterol for steroid production and cholesterol storage via the selective uptake pathway [14]. This pathway differs from the classic endocytic (B/E) receptor pathway [15] in that circulating lipoproteins (e.g., high-density lipoprotein [HDL]) bind to the cell surface and release cholesterol esters into the cells in the absence of internalization of the apolipoprotein particles [12, 16, 17]. The scavenger receptor class B, type 1 (SR-B1) is recognized as an authentic HDL receptor that binds various lipoprotein particles, facilitates selective transport of lipoprotein (notably HDL)-derived CE into the cell [18, 19], and provides substrate (cholesterol) for steroidogenesis [20].
The current studies were initiated to identify the cellular and molecular events responsible for conferring a constitutively active steroidogenic phenotype to the R2C cell line. Given the importance of lipoprotein-derived CE in supporting steroidogenesis and the established link between the selective pathway and SR-B1, we hypothesized that SR-B1 played a central role in maintaining the exceptionally high steroid production seen in R2C cells. To test this hypothesis, we studied the differences in capacity of MA-10 and R2C cell lines to take up and mobilize cholesterol for steroid hormone synthesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Na 125I (carrier free, 643.8 GBq/mg; 17.4 Ci/mg), was supplied by NEN (Boston, MA). [1
, 2(N)-3H]cholesteryl oleolyl ether (1.78 TBq/mmol; 48.0 Ci/mmol) and ECL Western blotting reagents were purchased from Amersham (Arlington Heights, IL). (Bu)2cAMP (dibutyryl cyclic AMP), cholesteryl oleate, egg phosphatidylcholine, cholesterol, and progesterone were obtained from Sigma (St. Louis, MO). Anti-rat HSL antibody was kindly supplied by Dr. Fredrick B. Kraemer (Stanford University School of Medicine, Stanford, CA). Anti-rat SR-B1 antiserum was prepared and characterized as described previously [21]. Mouse StAR cDNA was obtained as described previously [2, 22]. Rabbit anti-human antibody against StAR protein was obtained as described [23]. Rat SR-B1 was cloned as described previously [24] and the cDNA subcloned into the HindIII and XbaI endonuclease restriction sites of the pcDNA3.1 vector (Invitrogen, Carlsbad, CA).
Secretion of Steroids
MA-10 and R2C cells were cultured and maintained as described previously [8]. To assay steroidogenesis, triplicate culture dishes containing R2C and MA-10 cells were washed two times with PBS and incubated with or without (Bu)2cAMP (1 mM) at 37°C for an additional 4 h. Samples of the incubation medium were frozen and stored until analyzed for progesterone. Progesterone was quantified by RIA using specific antiserum as described previously and is expressed as nanograms per milliliter per microgram protein [8].
Western Blot Analysis of StAR Protein, Cholesteryl Esterase/Hormone-Sensitive Lipase, and SR-BI Proteins
MA-10 and R2C cells were cultured in 100-mm culture dishes and incubated in the presence or absence of 1 mM (Bu)2cAMP for 6 h at 37°C in serum-free Waymouth medium. The cells were subsequently washed with PBS and then used to prepare mitochondria, membrane fractions, or HSL-enriched fractions for Western blot analysis. Mitochondria, isolated from cultured cells as described earlier [8], served as the starting material for the determination of StAR protein expression. To assess cholesteryl esterase/HSL expression, cells were homogenized in 0.5 ml of Tris-sucrose-EDTA homogenization buffer [25] and centrifuged at 5500 x g for 10 min. The resultant supernatant was enriched for HSL protein by extracting the HSL activity with diethyl ether [25]. Membrane preparations to measure SR-B1 levels were prepared as described previously [26]. The presence and amount of StAR, cholesteryl esterase/HSL, and SR-B1 were assessed by Western blot analysis using methods described previously [2, 21, 25].
Northern Blot Analysis
Total RNA was extracted from 5–7 x 106 control or 1 mM (Bu)2cAMP-treated MA-10 and R2C cells incubated at 37°C for 6 h using Trizol reagent (Gibco-Life Technologies, Carlsbad, CA). Northern blot analysis of StAR and SR-B1 mRNAs was performed as described previously using the nonradioactive, North2South Hybridization kit (Pierce, Rockford, IL) [27]. The blots were hybridized with biotin-labeled StAR and SR-BI cDNA probes and exposed to Hyperfilm TM MP (Amersham Biosciences, Piscataway, NJ) following washes for appropriate lengths of time.
Complementary AMP Enzyme-Linked Immunoassay (EIA)
About 105 MA-10 and R2C cells were plated in each well of 96-well plates at 37°C. After 16 h, the cells were washed with PBS and the culture medium was replaced with 200 µl of serum-free Waymouth medium. The cells were incubated for an additional hour at 37°C and solubilized directly in the wells using lysis buffer (2.5% dodecyltrimethyl ammonium bromide in 0.05 M acetate buffer, pH 5.8, containing 0.02% BSA) for 10 min. The quantity of cAMP in the lysed samples was determined according to the manufacturer's instructions (cAMP EIA system, Amersham Biosciences) using rabbit anti-cAMP antibody and expressed as femtomoles of cAMP per microgram of protein.
Assay of cAMP-Dependent Protein Kinase A Activity
PKA activity was measured using Signa TECT (Promega, Madison, WI) as described previously [28]. In brief, R2C and MA-10 cells were cultured in six-well plates and treated with or without 0.5 mM (Bu)2cAMP and 30 µM H-89 for 6 h, rinsed with PBS, harvested by scraping, and homogenized in 200 µl of Tris-EGTA-based extraction buffer. The cytosolic fractions of the lysates (5 µl of each sample) were incubated with 20 µl of reaction mixture containing 100 µM PKA substrate, biotinylated Kemptide (LRRASLG) at 30°C for 5 min in the Signa-TECT kit supplied buffer containing [
-32P]ATP. The reaction was terminated and 10 µl of the reaction mixture was applied to streptavidin-coated membrane supports and washed extensively to remove unincorporated [-32P]ATP. The washed membranes were counted for bound radioactivity using a liquid scintillation counter. The PKA activity was expressed as picomoles of 32P incorporated per minute per milligram of protein.
Reverse Transcription-PCR for StAR mRNA
Total RNA was prepared using Trizol reagent (Gibco-Life Technologies) and 4 µg of RNA from control, 0.5 mM (Bu)2cAMP-treated (4 h) or 0.5 mM (Bu)2cAMP + 30 µM of the PKA inhibitor. H-89-treated (4 h) MA-10 and R2C cells were used to prepare cDNA according to the manufacturer's instructions (First Strand cDNA Synthesis Kit, Amersham Biosciences) in a 15-µl reaction. An aliquot of the cDNA was then used for polymerase chain reaction (PCR) using the following cycling parameters: 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 25 cycles. The PCR products were resolved on 1% agarose gel and the DNA visualized with ethidium bromide staining. The primers used for amplification were, for StAR forward, 5' TTC AAG CTG TGT GCT GGG AGC TCC TA 3' and, for StAR reverse, 5' TTA ACA CTG GGC CTC AGA GGC AGG GCT GG 3'.
Determining the Accessibility of Mitochondrial Free Cholesterol for Steroidogenesis Using Recombinant StAR
Mitochondria were isolated from MA-10 and R2C cells as described previously [29]. Bacterially expressed wild-type N-62 StAR and the R182L mutant of N-62 StAR were prepared as described [30]. Ten micrograms of mitochondria were preincubated at 37°C for 15 min in a buffer containing 125 mM sucrose, 1 mM ATP, 1 mM NADH, 50 mM ADP, 2 mM DTT, 5 mM sodium succinate, 2 mM magnesium acetate, and 2 mM potassium dihydrogen phosphate, pH 7.4, along with 2 µg/ml of cyanoketone (inhibitor of 3ß-hydroxysteroid dehydrogenase) and 5 µg/ml of SU10603 (inhibitor of C17-hydroxylase) to prevent conversion of pregnenolone to its downstream metabolites. The incubation was continued for 2 h following the addition of wild-type 0.25 µM N-62 StAR or 0.25 µM R182L StAR in 100 µl reaction volume [30, 31]. The reaction mixture was assayed for pregnenolone using a specific RIA [29]. Buffer controls containing no exogenous recombinant protein were included in the assay.
Lipoprotein Preparation
High-density lipoprotein hHDL3 devoid of ApoE was isolated as described [32]. These human-derived lipoproteins were used exclusively because they are not recognized by the low-density lipoprotein (LDL) (B/E) receptor-mediated endocytic pathway. For uptake and internalization studies, hHDL3 preparations were conjugated with 125I-labeled dilactitol tyramine ([125I]DLT) and [3H]cholesteryl oleolyl ether ([3H]COE) [33].
Selective Uptake of hHDL3-Derived Cholesteryl Esters
For these experiments, cultured R2C and MA-10 cells were first exposed to lipoprotein-deficient sera (LPDS) at 24 h. Subsequently, they were replaced with fresh LPDS medium with or without (Bu)2cAMP and hHDL3 containing radiolabeled, nonreleasable apolipoprotein (ApoA-1) and cholesteryl ester tags that would accumulate within the cells even when degraded [21, 33]. Incubations were carried out with [125I]DLT-[3H]COE-hHDL3 (100 µg protein/ml) ± (Bu)2cAMP (2.5 mM) for 24 h at 37°C. At the end of incubation, the cells were washed and then solubilized in 2 ml of 0.1 N NaOH. One-milliliter aliquots were precipitated with an equal volume of 20% trichloroacetic acid to determine insoluble and soluble 125I radioactivity or extracted with heptane-isopropanol to determine 3H radioactivity [21, 33].
Under the conditions used, trichloroacetic acid-insoluble 125I radioactivity was assumed to represent 125I-labeled protein remaining bound to the cell surface as part of intact lipoprotein [33, 34], and trichloroacetic acid-soluble 125I radioactivity was taken to be internalized, degraded, and accumulated residualizing protein. The uptake of CE was calculated as described earlier [33, 34] and expressed as micrograms per milligram protein of 125I-labeled (a measure of endocytic uptake) or 3H-labeled (a measure of selective uptake) protein internalized, which is in turn determined from the specific activity of the labeled hHDL3 particle [33, 34]. To determine the net mass of CE internalized, 125I- and 3H-protein values were divided by the protein:cholesterol ratio of hHDL3 (i.e., 2.65).
Miscellaneous Techniques
Protein content of hHDL3 and double-labeled lipoprotein preparation were quantitated as described [35]. Cellular and mitochondrial CE, free cholesterol (FC), and total cholesterol (TC) content were determined as described [36]. Cellular lysates and mitochondria for estimation of CE and FC were prepared as described [8, 37]. Cholesterol in HDL preparations was measured as described [38]. Western blots and Northern blots were quantitated by measuring the integrated optical density of each band using the BioImage Visage 2000. Statistical analysis of the data was performed with ANOVA using the Statview SE system (Abacus Concepts, Berkeley, CA).
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RESULTS |
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R2C and MA-10 cells were incubated with or without (Bu)2cAMP in serum-free medium, and progesterone production was measured by RIA. As shown in Figure 1, MA-10 cells secreted only negligible amounts of progesterone whereas R2C cells synthesized large quantities of progesterone under basal incubation conditions. Addition of (Bu)2cAMP substantially stimulated progesterone production in MA-10 cells but had no effect on steroid production in R2C cells. These results confirm the constitutive nature of steroid production in the R2C cells.
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(Bu)2cAMP-Mediated Regulation of StAR Expression
Previous studies demonstrated that, in most steroidogenic systems, the cAMP-dependent PKA signaling cascade rapidly induces steroidogenesis and StAR protein expression through both transcriptional and posttranslational mechanisms [39, 40]. Thus, in an effort to study the mechanisms involved in the constitutive secretion of steroids in R2C cells, we first investigated the effects of (Bu)2cAMP stimulation on StAR expression in R2C and MA-10 cells. Both cell types were incubated with or without (Bu)2cAMP and analyzed for StAR protein and mRNA expression. Unstimulated MA-10 cells have essentially undetectable levels of StAR mRNA and protein, whereas R2C cells express high levels of both StAR mRNA and immunoreactive StAR protein (30-kDa band) (Fig. 2, A and B). Stimulation of MA-10 cells resulted in the anticipated increase in both StAR mRNA and StAR protein. Northern blot analysis performed on total RNA revealed, as expected, three (1.6, 2.7, and 3.4 kilobases [kb]) different mRNA transcripts in MA-10 cells but only two (1.6 and 3.4 kb) in R2C cells (Fig. 2B). Stimulation of MA-10 cells with (Bu)2cAMP greatly increased StAR protein and mRNA levels while treatment of R2C cells with (Bu)2cAMP induced only a slight increase in StAR protein and mRNA levels (Fig. 2, A and B).
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Intracellular Levels of cAMP and PKA Activity in R2C and MA-10 Cells
Because R2C cells constitutively express high levels of StAR, we considered the possibility that constitutive activation of the cAMP-PKA signaling cascade may be responsible for the observed StAR expression. To examine this possibility, we compared the intracellular cAMP levels and PKA activity in R2C and MA-10 cell lines. Basal levels of cAMP were comparable between the two cell types (1.28 ± 0.61 fmol/µg protein in R2C vs. 2.4 ± 0.54 fmol/µg protein in MA-10), but PKA activity, measured in the absence of exogenous cAMP, was approximately twofold greater in cell lysates from R2C cells (Table 1). In contrast, maximal cAMP-stimulated PKA activity was comparable in the two cell types (Table 1). Moreover, addition of the PKA inhibitor H-89 reduced the PKA activity in R2C cell extracts to a level that was comparable with the activity seen in MA-10 cells.
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Next, we determined whether PKA had a differential regulatory effect on StAR expression in the two cell types. As shown previously, (Bu)2cAMP treatment resulted in a robust induction of StAR protein in MA-10 cells, which is completely inhibited following H-89 treatment (Fig. 3A). In contrast, StAR protein expression in R2C cells did not change appreciably in response to (Bu)2cAMP treatment or treatment with (Bu)2cAMP + H-89. Interestingly, H-89 treatment decreased StAR mRNA levels in both MA-10 and R2C cells (Fig. 3B).
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Cellular and Mitochondrial Sterol Levels in R2C and MA-10 Cells
Regardless of the type of steroidogenic cells, the first step in steroid hormone synthesis is the cellular mobilization and transport of cholesterol to the inner mitochondrial membrane and its conversion to pregnenolone by the P450scc enzyme. To investigate whether high mitochondrial cholesterol levels are responsible for constitutive steroid secretion in R2C cells, we quantitated the sterol levels in R2C and MA-10 cells both under basal conditions and in response to cAMP. MA-10 cells contained four- to fivefold more cellular (stored) CE relative to R2C cells (Table 2). Similarly, MA-10 cells exhibited an approximately twofold greater total cellular cholesterol content than R2C cells.
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Quantitation of sterol levels in the mitochondrial fractions indicated that the levels of free cholesterol were fourfold higher in R2C than MA-10 mitochondria (Table 3). Also, R2C cell mitochondria contained significantly more CE than MA-10 cells. However, in both cell types, CE represented only a small fraction (
20%) of the total cholesterol values. (Bu)2cAMP treatment induced a significant increase (250% relative to control cells) in the levels of mitochondrial free cholesterol in MA-10 cells (Table 3). In contrast, mitochondrial free cholesterol levels in R2C cells did not increase following (Bu)2cAMP treatment (Table 3).
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Accessibility of Mitochondrial Free Cholesterol for Steroidogenesis in MA-10 and R2C Cells
As noted in Table 3, R2C cells contain significantly higher amounts of free cholesterol in their mitochondria even under basal conditions when compared with MA-10 cells. In order to determine whether this free cholesterol was readily available for steroidogenesis or whether it constituted a structural component of R2C mitochondrial membranes, the ability of MA-10 and R2C mitochondria to convert free cholesterol into pregnenolone was assayed in the presence of a constant amount of exogenously added recombinant StAR protein (N-62 StAR). Under the same experimental conditions, R2C mitochondria converted twice as much cholesterol to pregnenolone as MA-10 mitochondria (Fig. 4), suggesting that the higher free cholesterol content in R2C mitochondria is indeed available for steroidogenesis. In contrast, MA-10 and R2C mitochondria incubated with equal quantities of the mutant StAR protein (R182L N-62 StAR) produce negligible amounts of pregnenolone.
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Expression of HSL in R2C and MA-10 Cells
To address the mechanism by which R2C cells maintain high levels of mitochondrial cholesterol and constitutive steroid secretion, we examined the expression of HSL by Western blotting. HSL is rapidly phosphorylated in response to trophic hormone stimulation and catalyzes the hydrolysis of stored CE to free cholesterol. The newly released cholesterol is mobilized and transported into the mitochondria and converted to pregnenolone. As shown in Figure 5, R2C cell lysates contained a very strong anti-HSL immunoreactive band with a molecular mass of 84 kDa. However, there was no difference in the levels of HSL between control and cAMP-treated cells. In contrast, only weak immunoreactivity was detected in cell lysates from control or cAMP-treated MA-10 cells.
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Comparison of Lipoprotein-Derived Cholesteryl Esters in R2C and MA-10 Cells
The data in Tables 2 and 3 show that R2C cell mitochondria contain several-fold more free cholesterol than do MA-10 mitochondria. To test whether this high level of R2C mitochondrial cholesterol was due to increased uptake of lipoprotein-derived selective cholesteryl ester uptake, R2C and MA-10 cells were incubated with 125I/3H-labeled hHDL3 and assayed for cholesteryl ester uptake via the selective and endocytic uptake pathways. Under basal conditions, R2C cells selectively internalized 10-fold more HDL-CE as compared with MA-10 cells (Fig. 6). (Bu)2cAMP treatment caused a fourfold increase in the rate of HDL-CE selective uptake in MA-10 cells, although the maximal amount of (Bu)2cAMP-stimulated HDL-CE selectively taken up by the MA-10 cells was still less than 30% of that observed for R2C cells. In contrast, stimulation with (Bu)2cAMP had no discernible effect on HDL-CE selective uptake in R2C cells. Note that the CE uptake via the endocytic pathway was negligible when compared with selective uptake (Fig. 6).
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SR-B1 Expression in R2C and MA-10 Cells
The results presented in Figure 6 demonstrate that R2C cells have a far greater capacity to internalize HDL-CE by the selective uptake pathway compared with basal or (Bu)2cAMP-stimulated MA-10 cells. To examine the possibility that this robust HDL-CE-selective uptake was due to high levels of expression of SR-B1, R2C and MA-10 cells were stimulated with (Bu)2cAMP and subsequently analyzed for SR-BI protein and mRNA. Western blot analysis of the basal R2C and MA-10 cell membrane preparations indicated that R2C cells express 20-fold greater levels of SR-B1 protein than that seen in MA-10 cells (Fig. 7A). (Bu)2cAMP treatment enhanced the SR-B1 levels about twofold in MA-10 cells, while R2C cells were unresponsive to cAMP analog stimulation (Fig. 7A).
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To test whether this increased expression of SR-B1 corresponds to changes at the mRNA level, the SR-B1 mRNA content of R2C and MA-10 cells was measured by Northern blotting (Fig. 7). R2C cells expressed a significant level of SR-B1 mRNA (2.4 kb size) under basal conditions, and interestingly, (Bu)2cAMP stimulation further up-regulated the SR-B1 mRNA (Fig. 7B). In contrast, SR-B1 mRNA was undetectable in unstimulated MA-10 cells and increased only slightly following hormone stimulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The predominant source of cholesterol for steroid hormone synthesis in the liver and nonplacental steroidogenic cells [41] is the selective uptake of cholesterol esters from circulating lipoproteins. Several lines of evidence have established a causal relationship between the expression of SR-B1 and the selective uptake of cholesterol esters at the microvillar membrane channels of steroidogenic cells [32, 42, 43]. Our study further highlights the importance of SR-BI expression and the efficient uptake of CE by the selective pathway in rendering the R2C cells constitutively steroidogenic. In addition, the R2C cells also express higher amounts of HSL and StAR proteins, which makes the R2C cellular background well equipped for sustained steroidogenesis.
The role of signaling pathways in determining physiological responses is indisputable. The cellular steroidogenic response has long been known to be dependent on the PKA signaling pathway, which is activated in response to trophic hormone stimulation via the production of cAMP. Activation of PKA leads to the up-regulation of the StAR gene both at the transcriptional and translational levels [44]. Considering the indispensable nature of the PKA pathway in determining the steroidogenic response, we then asked the question whether the constitutive nature of steroidogenesis could be attributed to the marginally elevated PKA activity in R2C cells. Our data using the PKA inhibitor H-89 (which functions by competing with the ATP-binding function of the kinase [45]) shows that StAR mRNA expression is under the direct regulatory control of PKA activity in both MA-10 and R2C cells. Treatment with H-89 also substantially reduces progesterone production in both the cell types (data not shown).
Furthermore, Y-1 cells (mouse adrenocortical cells) respond to 8-bromo-cAMP treatment with a 15-fold induction of SR-B1, while Kin 8 cells (adrenocortical cells with inactive PKA) show only a twofold induction [46]. This suggests that a functional PKA signaling pathway is essential for SR-B1 expression and its response to trophic hormones. This is further supported by our observation that SR-B1 mRNA is down-regulated following H-89 treatment in both MA-10 and R2C cells (data not shown). In addition, the tonic activation of PKA could also be responsible for the phosphorylation and mobilization of HSL to the lipid droplets in R2C cells, which in turn is a PKA-dependent function [25]. All these observations suggest that the cAMP pathway may indeed be responsible for up-regulating the steroidogenic response of R2C cells. It is tempting to speculate that several other genes involved in cholesterol homeostasis might also be coordinately up-regulated in R2C cells, a hypothesis that needs further experimentation.
Surprisingly, in contrast with the rapid down-regulation of StAR mRNA following H-89 treatment, we did not observe an appreciable decrease in the level of StAR protein in R2C cells over the course of several hours. While the half-life of the 30-kDa StAR is about 5–6 h, the constitutive nature of StAR expression appears to be able to maintain fairly high levels of this protein in R2C mitochondria (Fig. 3A). It should be pointed out that the mitochondrial matrix-bound 30-kDa StAR is biologically inactive but nevertheless serves as a reliable indicator of the levels of StAR protein synthesis. Also, one cannot rule out the possibility that the accumulation of the 30-kDa protein in the mitochondria of R2C cells is a consequence of a decreased degradation rate of 30-kDa StAR in R2C cells.
The PKA-dependent steroidogenic response is primarily mediated by the phosphorylation of cAMP-dependent transcriptional activators (e.g., SF-1, CREB, etc.) in various cell types, resulting in the transcriptional up-regulation of StAR and other steroidogenic genes [47, 48]. However, it remains to be seen whether multiple PKA-dependent pathways like the arachidonic acid pathway, the PI3-kinase, the PKB, or the MAPK pathways also contribute to the constitutive nature of R2C cell steroidogenesis [49, 50]. Considering the complexity of these individual pathways and their interdependence, it is highly likely that more than one signaling pathway determines the steroidogenic potential of R2C cells.
In summary, we propose that, in R2C cells, SR-B1 and the selective uptake pathway function in concert with increased levels of hormone-sensitive lipase and StAR protein to maintain high levels of free cholesterol in the mitochondria, which serves as a mechanism to support constitutive steroid hormone production. The PKA pathway appears to play an important role in conferring this phenotype to the R2C cells.
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FOOTNOTES |
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2 Correspondence. FAX: 806 743 2990; doug.stocco{at}ttmc.ttuhsc.edu
Received: 21 May 2002.
First decision: 17 June 2002.
Accepted: 17 July 2002.
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