Expression of sterol carrier protein 2 (SCP2) in human adrenocortical tissue
Toshihiko Yanase, Takayuki Hara1, Yoshiyuki Sakai, Ryoichi Takayanagi and Hajime Nawata
Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan; Department of Food and Nutrition1. Nakamura Gakuen College, Jonan-ku, Fukuoka, Japan
Yanase T, Hara T, Sakai Y, Takayanagi R, Nawata H. Expression of sterol carrier protein 2 (SCP2) in human adrenocortical tissue. Eur J Endocrinol 1996;134:501-7. ISSN 0804-4643
Sterol carrier protein 2 (SCP2) has been implicated in adrenal steroidogenesis by in vitro studies. In order to clarify the clinical significance of SCP2 in human steroidogenesis, we investigated the expression of SCP2 mRNA in various types of adrenocortical tissue and one testis and examined the correlation between the amounts of SCP2 and other values such as the free cholesterol content and the cholesterol side-chain cleavage (SCC) activity in the tissue mitochondria. The types of adrenocortical tissue examined included adrenocortical carcinomas (N = 3), adrenocortical adenomas from patients with Conn’s syndrome (N = 3) and from patients with Cushing’s syndrome (N = 3), non-functioning adrenocortical adenomas (N = 2) and normal adrenal glands (N = 2). Northern blot hybridization predominantly revealed a 1.8-kb SCP2 mRNA in all tissue specimens examined. The mRNA concentra- tions of SCP2 in two out of three adrenocortical carcinomas were relatively lower than those in other types of tissue. No other special tendency was observed regarding the mRNA expression levels in various tissue specimens. The mRNA concentrations of SCP2 correlated significantly with mitochondrial contents of free cholesterol (r = 0.67, p < 0.01), but was not correlated with the SCC activities in mitochondria measured by an in vitro enzyme assay. The mitochondrial SCC activities, however, were correlated significantly with the protein levels of mitochondrial P-450 scc determined by a Western blot analysis (r = 0.79, p < 0.01). The significant positive correlation between mRNA concentrations of SCP2 and the mitochondrial content of free cholesterol suggests that the central role of SCP2 in human steroidogenic tissues may be in part a translocation of cytoplasmic free cholesterol to the mitochondria, as demonstrated previously by in vitro studies.
Toshihiko Yanase, Third Department of Internal Medicine, Faculty of Medicine, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan
The biosynthesis of steroid hormones begins with the transport of the hydrophobic substrate cholesterol from lipid droplets in the cytoplasm to the mitochon- dria in the adrenal cortex and gonads. This transport involves two steps: transport to the outer mitochon- drial membrane and transport from the outer to the inner membrane. In the inner membrane of the mitochondria, the initial and rate-limiting step in the steroidogenic pathway, side-chain cleavage (SCC) of cholesterol, takes place to form pregnenolone. This step is catalyzed by a complex mitochondrial mixed- function oxidase system that includes P-450 scc, adrenodoxin reductase and adrenodoxin (1, 2).
In vitro studies utilizing rat adrenal subcellular fractions have revealed that sterol carrier protein 2 (SCP2), also called non-specific lipid transfer protein, facilitates the intracellular transfer of cholesterol from the lipid droplets to the mitochondria (3, 4), as well as from the outer to the inner mitochondrial membrane (5), thereby stimulating pregnenolone formation (6).
However, other proteins also appear to be involved in
the intramitochondrial translocation of cholesterol from the outer to the inner membrane (7-12). This process is rapidly stimulated by ACTH, and is acutely and reversibly inhibited by a protein synthesis inhibitor, cyclohexamide (13-15), which thus strongly suggests that a labile protein mediates this translocation process. A variety of candidate proteins have been proposed to be this labile protein: steroidogenesis activator peptide (SAP) (7, 8); endozepine, which binds peripheral-type benzodiazepine receptor (9, 10); and phosphoproteins of about 30kD (also called steroidogenic acute regulatory protein, StAR) (11, 12, 16). Among these proteins, StAR appears to be most essential for intramitochondrial cholesterol transport, because it has most recently been proved that an inherited defect in the StAR gene results in congenital lipoid adrenal hyperplasia, an autosomal recessive disorder characterized by the impaired production of all adrenal and gonadal steroids (17). Although SCP2 does increase pregnenolone synthesis in the adrenal mitochondria (6) as well as in COS 7
cells engineered to express both P-450 scc and adrenodoxin (18), SCP2 is not likely to be the labile protein involved in acute steroidogenesis, because the levels of SCP2 are not acutely affected by cyclohex- imide (19). However, like steroidogenic P-450s (1, 2), SCP2 may be involved in the long-term regulation of steroidogenesis, because ACTH or dibutyryl cyclic AMP causes a three- to four- fold increase in SCP2 synthesis in rat adrenocortical cells treated for 12- 48 h (20).
Most of the studies on SCP2 have been conducted using animal systems. Little is known about the mode of expression and the physiological relevance of this protein in human steroidogenic tissue. This is the first report demonstrating the constitutive expression of SCP2 in various types of human adrenocortical tissue and testis. The physiological significance of SCP2 in these tissues was estimated by examining the correla- tion between the levels of expression of SCP2 mRNA and the free cholesterol contents or SCC activities in the mitochondria.
Materials and methods
Subjects
Three adrenocortical carcinomas, three adrenocorti- cal adenomas from patients with Conn’s syndrome, three adrenocortical adenomas from patients with Cushing’s syndrome and two non-functioning adrenocortical adenomas were obtained at surgery. The diagnoses were histologically confirmed. The age, sex and relevant baseline laboratory data are summarized in Table 1. In the three children with
adrenocortical carcinomas, the 24-h urinary excretion of 17-ketosteroids was extremely high, indicating a high production of adrenal androgens. All three girls manifested slight virilization, including clitoral hypertrophy. The three cases with Conn’s syndrome showed hypertension and hypokalemia. Before operation, 10 mg/day of long-acting nifedipine (Adalat L) (Bayer Ltd., Osaka) was administered to treat hypertension in these three patients, although the treatment did not completely normalize their blood pressure. They all had a high plasma aldosterone and a low plasma renin activity (PRA), which showed little response to a bolus injection of 40 mg of furosemide. After operation, both the hypertension and hypokalemia of these patients disappeared, while plasma aldoster- one and PRA were normalized. All three cases with Cushing’s syndrome clinically showed a Cushingoid appearance and hypertension, and these three patients were administered 10mg/day of long- acting nifedipine and/or 0.5-1.0 mg/day of cilazapril (Inhibase) (Eisai Ltd., Tokyo) to treat hypertension, but the treatment did not completely normalize blood pressures. They all had high concentrations of 24-h urinary 17-hydroxy corticosteroids and a serum cortisol level that was not suppressed even by 1- 8 mg of dexamethasone. Basal plasma ACTH was undetectable in all three patients with Cushing’s syndrome (<1.1 pmol/l). After operation, both the hypertension and hypokalemia in these patients disappeared. Two cases with non-functioning adre- nocortical adenomas were clinically asymptomatic and had normal adrenal function. In addition, two normal adrenal glands were obtained from two patients (a 72-year-old male and a 72-year-old
| Patient | Age/sex | Urine | Plasma | |||
|---|---|---|---|---|---|---|
| 17-OHCS 17-KS (pmol/day) | Cortisol (nmol/day) | Aldosterone (pmol/l) | DHEA-S (umol/l) | |||
| Carcinoma | ||||||
| 1 | 1/F | 25.4 | 305.1 | NI | ND | ND |
| 2 | 6/F | 42.2 | 40.2 | 1021 | 471 | 24.3 |
| 3 | 3/F | 28.4 | 284.9 | 450 | 1081 | ND |
| Conn's adenoma | ||||||
| 1 | 39/F | ND | ND | 225 | 1331 | ND |
| 2 | 43/F | 16.8 | 15.4 | 328 | 720 | ND |
| 3 | 38/F | 13.4 | 21.0 | 248 | 1632 | 2.65 |
| Cushing's adenoma | ||||||
| 1 | 44/F | 45.5 | 14.6 | 692 | 100 | 0.71 |
| 2 | 44/F | 19.8 | 15.9 | 598 | ND | 0.14 |
| 3 | 50/F | 47.4 | 17.6 | 405 | ND | 0.54 |
| Non-functioning adenoma | ||||||
| 1 | 68/F | 12.4 | 5.8 | 146 | 225 | 0.54 |
| 2 | 62/F | ND | ND | 242 | 128 | ND |
| (Normal ranges) | (7.4-20.1) | (8.3-38.1) | (137-551) | (80-660) | (1.1-4.1) | |
ª17-OHCS = 17-hydroxycorticosteroids; 17-KS = 17-ketosteroids; ND = not determined.
female) with renal carcinoma who underwent a unilateral nephrectomy. One testis was obtained from a 60-year-old patient who underwent an orchidectomy for treatment of prostatic cancer. Immediately following surgery, the tissue specimens were stored at -80℃ for a protein and mRNA analysis.
Quantitation of SCP2 mRNA
Total RNA was isolated from tissue by a single-step extraction using the acid guanidium-thiocyanate- phenol-chloroform method (21). For the Northern blot analysis, samples containing 10 µg of total RNA were fractionated on a formaldehyde-1.5% agarose gel and transferred to nitrocellulose filters by capillary blotting (22). Probes for hybridization included the Eco RI-Eco RI insert of human SCP2 cDNA clone, pCMV5SCP2 (18), kindly donated by Dr JF Strauss III, and human 3-actin cDNA (Nippon Gene Co., Toyama, Japan). These probes were radiolabeled with [Q- 32p]dCTP using a nick translation kit (Amersham International plc, Buckinghamshire, UK). The prehy- bridization, hybridization and washing conditions have been described previously (22). The signal intensities of mRNA were quantified by densitometric scanning (FUSIX BAS 2000) and SCP2 expression was normal- ized to that of 3-actin.
Preparation of mitochondria
Mitochondrial fractions of the tissues were prepared as described previously (23).
Immunoblots of mitochondrial proteins
Immunoblots of mitochondrial proteins using anti- body against bovine cytochrome P-450 scc was carried out as described (24). The signal detection was conducted using an ECL kit (Amersham International plc, Buckinghamshire, UK). The signal intensities of immunodetectable P-450 scc protein were quantified by a densitometer system (Jookoo Co., Tokyo, Japan).
Side-chain cleavage activity and free cholesterol (FC) content in mitochondria
Cholesterol SCC activity was measured as described by Takeshima and Hara (25). The standard reaction mixture contained 100 mmol/l sucrose, 93 mmol/l KCI, 5 mmol/l L-malic acid, 0.1 mmol/l Trilostane, 2 mmol/l purified bovine adrenodoxin, 0.2 mmol/l adrenodoxin reductase (26), 1 mmol/l NADPH and 1 mg/ml mitochondria in a total volume of 0.2 ml. After the addition of NADPH, the reaction tubes were incubated at 37ºC for 60min. The reaction was terminated by adding 0.08 ml of methanol, and then
the tubes were kept in an ice-bath for 5 min. Then 1.4 ml of 0.1 mol/l phosphate buffer (pH 7.0) contain- ing 0.05% Tween 20 was added and the tubes were incubated at 37℃ for 10 min after the addition of 0.2 U of cholesterol oxidase kindly donated by Toyo Jozo (Ohita, Japan). This reaction was terminated by adding 3.5 ml of methanol.
Progesterone converted by cholesterol oxidase from pregnenolone was extracted by 5 ml of petroleum ether, and the extract was evaporated to dryness under a nitrogen stream. The dried extract was resolubilized with n-hexane and an aliquot was analyzed using a normal-phase HPLC column (Zorbax- Sil, 4.6 mm × 6 × 250 mm) equilibrated with a mixture of n-hexane and isopropanol (82 : 18) and eluted with the same solvent at a flow rate of 1 ml/min. The peak was monitored at 240 nm. The free mitochondrial cholesterol content was measured by HPLC profile of 4-cholestane-3- one, a metabolized product of cholesterol by cholesterol oxidase without incubation at 37°C for SCC reaction.
Statistics
Pearson’s correlation coefficient was calculated to analyze the correlations among several parameters; P < 0.05 was accepted as being statistically significant.
Results
Northern blot hybridization revealed SCP2 transcripts corresponding to 1.8 kb in size in all the examined tissue specimens (Fig. 1). The amount of SCP2 mRNA, when normalized to 3-actin mRNA, was relatively lower in the adrenocortical carcinomas (N=3) and in the testis (N=1) than in the other tissue specimens. In fact, the amounts of SCP2 mRNA in two carcinomas (cases 1 and 2 in Fig. 1) were substantially lower than those of the other tissue specimens. The protein levels of P-450 scc in mitochondrial fractions determined by Western blot analysis varied in different tissue specimens (Fig. 2). The relative expressions of SCP2 mRNA and P-450 scc protein in all types of tissue are summarized in Table 2. The mitochondrial free cholesterol content estimated by HPLC profile of 4-cholestane-3-one, a metabolized product of cholesterol by cholesterol oxidase, and SCC activity in mitochondria are also shown in Table 2. Because the mitochondrial free cholesterol contents differed substantially among the various types of tissue, the mitochondrial SCC activity was also measured in the presence of exogenous 25-hydroxy-cholesterol in order to ensure the cholesterol supply. However, the mitochon- drial SCC activities in the presence of 25-hydroxy- cholesterol were not essentially different from those in the absence of 25-hydroxy-cholesterol (data not shown), which thus indicated that the endogenous cholesterol content in the inner mitochondrial mem- brane is sufficient for the SCC reaction. The correla- tions observed between the levels of SCP2 mRNA and
Carcinoma
Conn’s Syn.
Cushing’s Non
Normal Normal -func. Ad.gl. Testis
Syn.
1 2 3 1 2 3 1 2 3 1 2 1 2 1
SCP2
28 S
18 S
B-actin
8
Ratio (SCP2/B-actin mRNA)
6
4
2
0
Carcinoma
Conn’s Cushing’s Nonfunc. Normal N
Syn.
Normal Testis
Syn.
Tumor
Adrenal gl.
other values, such as mitochondrial free cholesterol content and SCC activity, and between the levels of P- 450 scc protein and mitochondrial SCC activity are summarized in Table 3. The mRNA concentrations of SCP2 did correlate significantly with the mitochon- drial free cholesterol content, but did not correlate with the SCC activities in the mitochondria. The mitochondrial SCC activities did not correlate with the free cholesterol content in the mitochondria, but correlated significantly with the levels of mitochondrial P-450 scc.
Discussion
Sterol carrier protein 2 is thought to be one of the leading candidates for intracellular cholesterol transport (27, 28), and exhibits a tissue-specific distribution and expression that is closely correlated with the rate of cholesterol metabolism in each tissue (19).
A cDNA cloning of rat liver SCP2 has revealed that SCP2 is synthesized as a 15.3-kD protein with a 20- amino-acid N-terminal sequence that is subsequently cleaved to produce a 13.2-kD mature protein. In
| Conn's | Cushing's | Non | Normal Testis |
|---|---|---|---|
| Syn. | Syn. | -Func | Ad.gl. |
Carcinoma
1 2 3 1 2 3 1 2 3 1 2 1 2 1
addition, transcripts that encode proteins larger than pro-SCP2 (15.3 kD), including 30- and 58-kD proteins, have been disclosed (29, 30). Because these proteins have identical C-terminal sequences, it is presumed that the different SCP2 transcripts are produced either by transcription that originates at an alternative start site or by alternative splicing (31). In humans, two mRNA species, one of 3.2 kb, which may encode the 58-kD protein, and one of 1.8 kb, which may encode pro- SCP2. have been shown to be present in liver, while the predominance of 1.8 kb SCP2 mRNA in human tissue or cells has been reported in fibroblasts, placenta and JEG-3 choriocarcinoma cells (18), thus suggesting that the transcriptional regulation of the SCP2 gene differs from tissue to tissue.
The present study clearly demonstrated the expression of a predominant 1.8 kb SCP2 mRNA in various types of human adrenocortical tissue and a normal testis. These results thus suggest that SCP2 plays a specific role in the steroidogenesis of various tissues, as suggested by the findings of in vitro experiments (3-6, 18-20). Interestingly, the mRNA concentration of SCP2 in two adrenocortical carcinomas was found to be much lower than in other adrenal tumors. Because all three patients with carcinomas were infants, aged 1-6 years, one possibi- lity is that SCP2 mRNA expression may be influenced by age. While admittedly the sample size was very small in the present study, the more likely possibility is that SCP2 expression may also be regulated abnormally in some carcinomas as a result of tumorigenesis.
Although the subcellular localization of SCP2 is controversial, it has been shown in the rat liver and adrenal glands (32) that SCP2 is present mainly in peroxisomes rather than in the mitochondria or in cytosol. The significance of the relation between peroxisomal SCP2 and steroidogenesis, however, remains unclear. The present study was undertaken to determine the function of SCP2 in steroidogenesis by examining the correlation between the tissue amounts
of SCP2 mRNA and several other parameters. As a result, the amount of SCP2 mRNA correlated significantly with the free cholesterol content in mitochondria, and thus supports the hypothesis that SCP2 facilitates the intracellular transport of free cholesterol from the lipid droplets to the outer mitochondrial membrane (3, 4).
On the other hand, it has also been demonstrated that SCP2 increases pregnenolone formation by enhancing the transfer of cholesterol from the outer to the inner mitochondrial membrane (5). Although SCP2 may not be the putative high-turnover “labile protein” involved in acute steroidogenesis, it may well be involved in the chronic regulation of steroidogenesis by ACTH (20). In this respect, the correlation between the amounts of SCP2 mRNA and mitochondrial SCC activities were examined based on the assumption that steady-state mitochondrial SCC activity may be determined partly by the SCP2 amount if SCP2 truly enhances cholesterol transfer to the inner mitochon- drial membrane. Although our findings are not conclusive, this seems unlikely because mitochon- drial SCC activity did not correlate with the amount of SCP2 mRNA, while it did correlate significantly with the amount of mitochondrial P-450 scc. These results suggest that in the long-term regulation of steroidogenesis, mitochondrial SCC activity is chiefly dependent on the amount of mitochondrial P-450 scc.
Even if SCP2 facilitates the cholesterol transport to the mitochondria, the different levels of SCP2 expres- sion do not seem to have an essential effect on steroidogenesis, because the urinary excretion of 17- hydroxycorticosteroids and 17-ketosteroids did not decrease even in the two cases of adrenocortical carcinoma that had only a slight expression of SCP2 mRNA (nos. 1 and 2 in Fig. 1). A minimal expression of SCP2 may thus be sufficient to maintain the cholesterol supply to mitochondria for steroidogen- esis. Another possibility is that some other unknown factor(s) may also participate in cytosolic cholesterol
| SCP2/3-actin mRNA | FC (pmol/mg) | SCC activity (pmol mg~1 min-1) | P-450 scc protein | |
|---|---|---|---|---|
| Adrenal carcinoma | ||||
| 1 | 0.065 | 0.039 | 277.8 | 2.48 |
| 2 | 0.48 | 0.028 | 137.4 | 2.18 |
| 3 | 4.63 | 0.042 | 187.8 | 1.29 |
| Conn's adenoma | ||||
| 1 | 6.31 | 0.064 | 480.0 | 4.56 |
| 2 | 4.40 | 0.060 | 99.0 | 2.54 |
| 3 | 3.76 | 0.024 | 100.8 | 1.66 |
| Cushing's adenoma | ||||
| 1 | 6.30 | 0.042 | 319.6 | 2.73 |
| 2 | 2.69 | 0.014 | 330.1 | 2.75 |
| 3 | 7.18 | 0.082 | 360.2 | 1.76 |
| Non-functioning adenoma | ||||
| 1 | 6.74 | 0.078 | 115.2 | 2.18 |
| 2 | 3.14 | 0.039 | 531.6 | 4.70 |
| Normal adrenal gland | ||||
| 1 | 5.80 | 0.051 | 245.4 | 2.60 |
| 2 | 4.99 | 0.032 | 180.6 | 1.81 |
| Normal testis | ||||
| 1 | 1.00 | 0.036 | 128.4 | 1.00 |
a The ratio SCP2/3-actin mRNA indicates the relative expression of SCP2 mRNA to that of 3-actin based on the data in Fig. 1. The relative comparison of the amount of P-450 scc protein is based on the quantification of the signal intensities of immunodetectable table P-450 scc protein (Fig. 2) by densitometric scanning. When each value of the normal testis is given as 1.00, the relative values are indicated.
| SCP2/3-actin mRNA vs FC contents in Mt | r = 0.67 (p< 0.01) |
| SCP2/3-actin mRNA vs SCC activities in Mt | r = 0.19 (NS) |
| FC contents in Mt vs SCC activities in Mt | r = 0.12 (NS) |
| SCC activities in Mt vs amounts of P-450 scc | r = 0.79 (p< 0.01) |
a Each correlation was calculated based on the values shown in Table 2. SCP2 = sterol carrier protein 2; SCC = side-chain cleavage; Mt = mitochondrial fractions; FC = free cholesterol: NS = not significant.
transport. However, a complete lack of SCP2 due to peroxisome-deficient disorders, including Zellweger syndrome and adrenoleukodystrophy, may cause adrenal insufficiency (33, 34). Further studies are needed to establish the functional significance of SCP2 in steroidogenic tissue. A disruption of the SCP2 gene may be crucial in attempting to answer this question.
Acknowledgment. We are grateful to Dr JF Strauss II, University of Pennsylvania, for providing the human SCP2 cDNA clone PCMV5SCP2.
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Received September 19th. 1995
Accepted January 23rd, 1996