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Endocrinology, doi:10.1210/en.2003-1034
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Endocrinology Vol. 145, No. 4 1700-1707
Copyright © 2004 by The Endocrine Society

Molten Globule Structure and Steroidogenic Activity of N-218 MLN64 in Human Placental Mitochondria

Robert C. Tuckey, Himangshu S. Bose, Inez Czerwionka and Walter L. Miller

Department of Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences (R.C.T., I.C.), The University of Western Australia, Crawley, Western Australia 6009, Australia; and the Department of Pediatrics and the Metabolic Research Unit (H.S.B., W.L.M.), University of California San Francisco, San Francisco, California 94143

Address editorial correspondence to: Prof. Walter L. Miller, M.D., Department of Pediatrics and The Metabolic Research Unit, University of California San Francisco, 1466 4th Avenue, Building MR 4, San Francisco, California 94143-0978. E-mail: wlmlab{at}itsa.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Progesterone synthesis by the human placenta requires the conversion of mitochondrial cholesterol to pregnenolone by cytochrome P450scc. Most steroidogenic tissues use the steroidogenic acute regulatory protein (StAR) to deliver cholesterol to the inner mitochondrial membrane where P450scc is located, but StAR is not expressed in the human placenta. However, the human placenta does express MLN64, which has a C-terminal domain homologous to StAR that can also transport cholesterol. We investigated the ability of bacterially expressed N-218 MLN64 and N-62 StAR to transport cholesterol between artificial membranes and to its inner membrane site of use in placental mitochondria. Urea denaturation experiments show that N-218 MLN64 undergoes a pH-dependent and denaturant-dependent structural transition to a molten globule state, as reported previously for N-62 StAR. N-218 MLN64 stimulated cholesterol transfer between artificial phospholipid vesicles with an initial rate of 6.5 mol/min·mol N-218 MLN64. Both N-218 MLN64 and N-62 StAR stimulated cholesterol transfer to the inner mitochondrial membrane, as evidenced by a 6-fold stimulation of pregnenolone synthesis with saturating transporter. This stimulation was seen only after the endogenous cholesterol in the steroidogenic pool of the isolated mitochondria was first depleted. No stimulation was observed by N-218 MLN64 or N-62 StAR when 20{alpha}-hydroxycholesterol was added as substrate for P450scc, confirming that these proteins stimulate P450scc activity by enhancing cholesterol transport. MLN64 levels in placental JEG-3 cells were unresponsive to stimulation by 8-bromo-cAMP over 24 h. These data show that human N-218 MLN64 and N-62 StAR have similar biophysical and functional properties and are able to stimulate steroidogenesis in a human placental system, which normally lacks StAR. The results reveal that with saturating MLN64, steroidogenesis by placental mitochondria proceeds at near-maximal rate.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN THE SECOND and third trimesters of gestation the human placenta is the major site for synthesis of the progesterone required to maintain pregnancy (1). Cholesterol, the substrate for steroid hormone synthesis, must be transported to the inner mitochondrial membrane of the placenta where the cytochrome P450scc that catalyzes the cholesterol side chain cleavage reaction is located. The P450scc converts cholesterol from the inner mitochondrial membrane to pregnenolone, which is then rapidly converted to progesterone by type 1 3ß-hydroxysteroid dehydrogenase (2, 3, 4). In most steroidogenic tissues such as the corpus luteum, adrenal cortex, and testis, cholesterol translocation to the inner mitochondrial membrane is mediated by the steroidogenic acute regulatory protein (StAR), and this step is acutely regulated by tropic hormones (5, 6). Progesterone synthesis by the placenta is not acutely regulated (2, 7, 8, 9, 10), and the human placenta does not express StAR (11), nor do mutations in StAR affect placental steroidogenesis (12). However, the human placenta does express the StAR-like protein MLN64 (13, 14), which has a C-terminal domain (START domain) homologous to StAR and other lipid transfer proteins (15, 16, 17). MLN64 was first identified in certain breast cancers where it is overexpressed (18). The full-length form of the protein (445 residues) has four membrane-spanning domains at its N terminal that attach it to the late endosome (19) via tubules containing the NPC-1 protein (20). The N-terminal domain, which resembles the recently described MENTHO protein (21), induces cholesterol accumulation in lyosomes (20). The C-terminal START domain of MLN64 (residues 218–445) shares significant homology with StAR, can bind cholesterol, and also functions in cholesterol transport (13, 14, 15, 20). MLN64 with 218 amino acids that contain the membrane-binding domain deleted from the N terminal (N-218 MLN64), can stimulate pregnenolone synthesis in both transfected cell systems and in isolated MA-10 cell mitochondria with approximately 60% of the activity seen with N-62 StAR (14). The START domain of MLN64 has also been shown to stimulate progesterone synthesis by placental mitochondria (20). The steroidogenic activity of N-218 MLN64, which lacks mitochondrial targeting sequences, is consistent with StAR’s activity on the outer mitochondrial membrane (22, 23), an activity that does not require mitochondrial protein import (23). Western blotting of placental mitochondria shows that the 445-amino-acid MLN64 protein is cleaved to a fragment the same size as N-218 MLN64, suggesting the active form containing the START domain is present in the human placenta (14). There is also some evidence that the full-length form of MLN64 is associated with placental mitochondria (24). We now show that N-218 MLN64 undergoes a transition to a molten globule state that is similar to that of N-62 StAR (25) and compare the ability of bacterially expressed N-218 MLN64 and N-62 StAR to stimulate cholesterol transfer in artificial phospholipid membranes and placental mitochondria.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression and purification of N-62 StAR and N-218 MLN64
Human StAR lacking 62 amino acids at the amino terminus (N-62 StAR) and a corresponding inactive mutant of StAR where arginine 182 is replaced by leucine (R182L N-62 StAR) were expressed in Escherichia coli and purified as described (26). Human MLN64 lacking 218 amino acids at the amino terminus (N-218 MLN64) was also expressed in E. coli and purified as described (14). The purified protein was stored in 50% glycerol. The concentrations of N-218 MLN64 and N-62 StAR were determined spectroscopically using molar absorption coefficients of 30,620 (14) and 26,934 (26), respectively.

Urea denaturation of N-218 MLN64
For urea unfolding experiments at pH 7.0, N-218 MLN64 was equilibrated overnight with different concentrations of urea buffered with sodium phosphate, and the circular dichroism (CD) spectrum was recorded between 205 and 240 nm in a JASCO-720 spectropolarimeter. The urea concentration (C) was determined before and after recording the CD spectra according to the equation , where {Delta}N is the difference between the refractive index of the denaturant solution and water at the sodium D line (27). For urea unfolding experiments at pH 4.0, N-218 MLN64 was treated as above except that sodium acetate buffer was substituted for sodium phosphate buffer. The equilibrium constant (KD) and free energy ({Delta}G) were calculated according to the equation


where N and D represent native and denatured state, and values of y characteristic of the native state (yn) and of the denatured state (yd) were obtained in the transition region by extrapolation from the linear portions (27).

Measurement of cholesterol transfer between phospholipid vesicles
The transfer of cholesterol from synthetic cholesterol-rich phospholipid vesicles to cholesterol-free acceptor vesicles was measured by two procedures. The first used cytochrome P450scc in the acceptor vesicles as a reporter system (28). The activity of P450scc in acceptor vesicles reflects the pool of cholesterol in these vesicles rapidly transferred from donor vesicles in the presence of N-218 MLN64 or N-62 StAR. Donor vesicles comprised dioleoyl phosphatidylcholine (510 µM) and 0.2 mol cholesterol/mol phospholipid. Acceptor vesicles (510 µM phospholipid) comprised dioleoyl phosphatidylcholine containing 0.15 mol cardiolipin/mol total phospholipid and 0.05 µM P450scc. The second procedure measured [4-14C]cholesterol transfer from acidic donor vesicles comprising dioleoyl phosphatidylcholine, 15 mol % cardiolipin, and 10 nCi [4-14C]cholesterol to neutral dioleoyl phosphatidylcholine acceptor vesicles. N-218 MLN64 or N-62 StAR was preincubated with donor vesicles (1.02 mM) for 5 min at 35 C, and the transfer was started by the addition of acceptor vesicles (1.02 mM). After incubation at 35 C for 2–25 min, acidic donor vesicles were removed by chromatography on diethylaminoethyl (DEAE)-Sepharose as before (28), and the radioactivity associated with the neutral acceptor vesicles passing through the column was determined by scintillation counting.

Preparation of placental mitochondria
Term placentas (37–40 wk) were obtained after spontaneous delivery from King Edward Memorial Hospital for Women, according to protocols approved by the hospital’s human ethics committee. Major blood vessels were removed and tissue washed three times in ice-cold 0.25 M sucrose. The tissue was homogenized in 7 vol of 0.25 M sucrose in a Potter-Elvehjem homogenizer with two low-speed passes of the Teflon pestle. A mitochondrial fraction was then isolated as described before (29).

Measurement of pregnenolone synthesis by mitochondria
Mitochondria (0.25–0.75 mg/ml) were incubated in buffer comprising 50 mM HEPES (pH 7.4), 0.25 M sucrose, 20 mM KCl, 5 mM MgSO4, 0.2 mM EDTA, 1 mg/ml BSA (fatty acid free), 5 mM isocitrate, and 50 µM reduced nicotinamide adenine dinucleotide phosphate (NADPH). Cyanoketone (8 µM) (Sterling-Winthrop Research Institute, Rensselaer, NY), a 3ß-hydroxysteroid dehydrogenase inhibitor (30), was added to prevent metabolism of the pregnenolone product. The substrate for pregnenolone synthesis was the endogenous cholesterol present in the mitochondrial membranes, exogenous cholesterol (200 µM), or 20{alpha}-hydroxycholesterol (25 µM). N-218 MLN64 was added from a glycerol stock (25–50%), resulting in 5% glycerol being present in the incubation. In incubations with N-218 MLN64, 5% glycerol was included in controls and at this concentration did not influence the rate of pregnenolone synthesis. Glycerol concentrations above 5% do inhibit pregnenolone synthesis by P450scc (14, 31). Tubes were preincubated at 37 C for 10 min with all components except NADPH and isocitrate, which were used to start the side-chain cleavage reaction. Aliquots (25 µl) were removed at various times (0–60 min) after starting the reaction and were added to 1 ml ice-cold ethanol. The pregnenolone content of samples was determined by RIA (32).

JEG-3 cell culture and blotting
JEG-3 cells were cultured alone or in the presence of 1 mM 8-bromo-cAMP as described before (33). A mitochondrial fraction was prepared as before except the washing steps were omitted (23). Western blotting was carried out using anti-StAR antiserum (25), which cross-reacts fully with N-218 MLN64 (14).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Urea denaturation of N-218 MLN64
Using CD and mass spectrometric analysis of proteolyzed samples, we previously reported that N-218 MLN64 is folded and cleaved similarly to N-62 StAR (14). Although we also detected pH-dependent changes in the far-UV CD spectra suggestive of a pH-dependent transition to a molten globule state, the results were not nearly so dramatic as those for N-62 StAR (25). Therefore, to examine the molten globule nature of N-218 MLN64 in more detail, we examined its denaturation in urea (Fig. 1Go). The use of urea rather than guanidine hydrochloride avoids potential complications due to the high ionic strength of guanidine hydrochloride and the potential complications from Cl- binding to positively charged residues (34). The unfolding of N-218 MLN64 at pH 7.0 follows a two-state transition (Fig. 1AGo): it begins to unfold at 2.5 M urea, reaches an intermediate state (midpoint) at 3.75 M urea, and becomes completely unfolded at 5.25 M urea. The calculated {Delta}G is -3.2 kCal/mol, suggesting that the protein remains in a stable condition at pH 7.0. Interestingly, at pH 4.0 the unfolding of N-218 MLN64 undergoes a three-stage transition (Fig. 1BGo). Unfolding begins at 2.0 M urea, similarly to its behavior at pH 7.0, suggesting that N-218 MLN64 retains a stable conformation at pH 4.0. With increasing concentrations of urea, N-218 MLN64 reaches a partially unfolded conformation at 3.25 M urea, remains at equilibrium up to 4.12 M urea, and undergoes a second transition to complete unfolding at 5.25 M urea. The calculated free energy at the first stage is -1.7 kCal/mol and at the second state is -0.9 kCal/mol. The plateau from 3.25 to 4.12 M urea may represent an intermediate stable conformation at pH 4.0. We consider this stage of equilibrium to represent a molten globule conformation of N-218 MLN64. Thus at pH 4.0 N-218 MLN64 exhibited unfolding behavior that was virtually indistinguishable from its native behavior at pH 7.0, with this first stage showing that the secondary structures were preserved with a partial loss of the weakened tertiary structure, and at higher urea concentrations achieved an intermediate state to that seen for the similar unfolding of StAR (25).



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  		FIG. 1. Urea denaturation. N-218 MLN64 was equilibrated overnight with different concentrations of urea buffered either with Na2HPO4 (for pH 7.0) or NaOAc buffer (for pH 4.0). The ellipticity at 222 nm was recorded in a 1.0-mm path-length cuvette at 20 C in a spectropolarimeter and plotted against the urea concentration, as determined in a refractometer.

 
Cholesterol transfer in synthetic vesicles
To examine the ability of N-218 MLN64 to facilitate cholesterol transport we tested its activity in a defined system comprising donor vesicles containing cholesterol and acceptor vesicles containing P450scc but no cholesterol. We have previously used this system to characterize cholesterol transfer between membranes by N-62 StAR (28). N-218 MLN64 was as effective as N-62 StAR in transferring cholesterol from donor vesicles to acceptor vesicles containing P450scc, stimulating the basal rate of cholesterol transfer 7-fold (Fig. 2Go). Because the transfer of cholesterol measured using P450scc as a reporter is dependent on the P450scc concentration and this procedure measures the amount of cholesterol transferred (28), not the rate, we devised a new method for measuring the transfer using radiolabeled cholesterol in which P450scc was not necessary. The procedure makes use of DEAE-Sepharose to bind acidic vesicles containing cardiolipin enabling them to be quantitatively removed from neutral acceptor vesicles. N-218 MLN64 and N-62 StAR stimulated the amount of [4-14C]cholesterol transferred from acidic donor vesicles to neutral acceptor vesicles 9-fold and 8-fold, respectively, over the background (control) in a 5-min incubation (Fig. 3Go). The rate of transfer slowed with time as the cholesterol in the donor vesicles became depleted (Fig. 4Go). The initial rate of transfer was 6.5 mol cholesterol/min·mol N-218 MLN64.



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  		FIG. 2. Stimulation of cholesterol transfer from donor vesicles to acceptor vesicles by N-218-MLN64. Acceptor vesicles containing P450scc were incubated with N-218 MLN64 (1.7 µM) or N-62 StAR (5 µM) for 10 min at 37 C in the presence of adrenodoxin reductase (0.3 µM), adrenodoxin (15 µM), and NADPH (50 µM), and the cholesterol side-chain cleavage reaction was started by the addition of donor vesicles containing cholesterol. The pregnenolone produced at the indicated times was measured by RIA.

 


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  		FIG. 3. DEAE-Sepharose elution profile of [4-14C]cholesterol in acceptor vesicles after transfer from acidic donor vesicles. Acidic donor vesicles (1.02 mM phospholipid) with a molar ratio of radiolabeled cholesterol to phospholipid of 0.1 were incubated with neutral acceptor vesicles (1.02 mM phospholipid) in the absence or presence of N-218 MLN64 or N-62 StAR for 5 min at 35 C. The incubation mixture was applied to a 0.75 x 2.8-cm DEAE-Sepharose column, and neutral vesicles were washed through the column with 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol, with fractions being collected for scintillation counting.

 


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  		FIG. 4. Time course for transfer of [4-14C]cholesterol from acidic donor vesicles to neutral acceptor vesicles by N-218 MLN64. Conditions were as described for Fig. 3Go except that the molar ratio of cholesterol to phospholipid in donor vesicles was 0.2 and the time of incubation was varied.

 
Cholesterol transfer in placental mitochondria
To test the ability of N-218 MLN64 and N-62 StAR to transfer cholesterol to the inner mitochondrial membrane, human placental mitochondria were incubated with these proteins under conditions where P450scc was active in converting cholesterol to pregnenolone. Initially the cholesterol concentration in the steroidogenic pool in placental mitochondria is saturating for pregnenolone synthesis (2, 35). It was only after approximately 5 min, when this pool was depleted, that N-62 StAR (Fig. 5AGo) and N-218 MLN64 (Fig. 5BGo) began to stimulate steroidogenesis above the basal, control level. The presence of 200 µM exogenous cholesterol in the incubation had little effect on the rate of pregnenolone synthesis with 1.1 µM N-218 MLN64 but caused a small stimulation with 5 µM N-62 StAR. The addition of the R182L mutant of N-62 StAR to placental mitochondria did not stimulate pregnenolone synthesis (Fig. 6Go). This R182L mutation is one of several that cause congenital lipoid adrenal hyperplasia (12); this protein is incorrectly folded (26) and is inactive in transfected cell systems (12), and the mutant protein is inactive in isolated mitochondria (14, 23). Its lack of activity with placental mitochondria confirms that only the properly folded, biologically active protein is functional in cholesterol transport.



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  		FIG. 5. Stimulation of mitochondrial pregnenolone synthesis by N-218 MLN64 and N-62 StAR in the presence and absence of exogenous cholesterol. Mitochondria (0.6 mg/ml) were incubated alone (control) or with 200 µM cholesterol, 5 µM N-62 StAR (A) or 1.1 µM N-218 MLN64 (B), and the pregnenolone produced at the indicated times was measured by RIA. The mitochondria used for A and B were from different placentas.

 


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  		FIG. 6. Mutant N-62 StAR does not stimulate pregnenolone synthesis by placental mitochondria. Mitochondria (0.75 mg/ml) were incubated with 2.7 µM R182L N-62 StAR or 1.5 µM N-62 StAR or alone (control), and pregnenolone synthesis was measured.

 
The effect of adding N-62 StAR at 35 min of incubation is shown in Fig. 7Go. At this time of incubation the endogenous cholesterol in the steroidogenic pool in the inner mitochondrial membrane has been depleted to a steady-state level, as evidenced by the linear rate of pregnenolone synthesis. Here the rate of pregnenolone synthesis equals (and is dependent on) the rate of cholesterol delivery to the mitochondria. After a lag of approximately 2 min, N-62 StAR increased the rate of pregnenolone synthesis to that seen for 20–60 min of incubation where the N-62 StAR was preincubated with the mitochondria for 10 min. Thus, preincubation of N-62 StAR with mitochondria causes an increase in the concentration of cholesterol in the steroidogenic pool causing the initial rate to be maintained for longer, before the rate slows to a linear phase that reflects the rate of cholesterol transport to the steroidogenic pool.



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  		FIG. 7. The effect of time of the addition of N-62 StAR on stimulation of pregnenolone synthesis. N-62 StAR (5 µM) was either preincubated with mitochondria (0.75 mg/ml) for 10 min before starting the cholesterol side-chain cleavage reaction with NADPH and isocitrate (zero time) or added 35 min after initiation of the reaction.

 
Stimulation of pregnenolone synthesis by N-62 StAR and N-218 MLN64, measured during the steady-state phase, was saturable with each data set giving a good fit to a hyperbolic curve (Fig. 8Go). The concentration of N-218 MLN64 causing half-maximal stimulation (1.16 ± 0.07 µM ± SE from curve fit) was the same as that for N-62 StAR (1.19 ± 0.12 µM). The extent of the stimulation above the basal rate with saturating transporter, determined from the fitted curves, was also similar for N-62 MLN64 (4.9-fold) and N-62 StAR (5.3-fold). We did not test concentrations of N-218 MLN64 above 3 µM because of its aggregation at higher concentrations (14).



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  		FIG. 8. The effects of N-218 MLN64 and N-62 StAR concentrations on pregnenolone synthesis by placental mitochondria. Rates of pregnenolone production by placental mitochondria were measured in the steady-state phase (30–60 min) where the rate of pregnenolone synthesis is dependent on the rate of cholesterol delivery to the P450scc. Control rates were subtracted from the rates measured with N-218 MLN64 or N-62 StAR, and data are expressed as a ratio to the control rate (fold stimulation over control).

 
Effect of hydroxysterol substrate
To demonstrate further that N-62 StAR and N-218 MLN64 increase pregnenolone synthesis by stimulating cholesterol movement from the outer mitochondrial membrane to the steroidogenic pool in the inner mitochondrial membrane, we tested the effect of these proteins on pregnenolone synthesis from 20{alpha}-hydroxycholesterol (Fig. 9Go). Being more water soluble than cholesterol, this sterol can rapidly move to the inner mitochondrial membrane without a transporter (36, 37). Cytochrome P450scc has a lower Michaelis-Menten constant (Km) for 20{alpha}-hydroxycholesterol than for cholesterol (38, 39), and hence it effectively saturates the P450scc and outcompetes the endogenous cholesterol. It causes a small lag in the time course, more noticeable in Fig. 9BGo, which was done at lower mitochondrial protein concentration, due to its competing with 20{alpha},22R-dihydroxycholesterol for the active site of P450scc (35, 40). No stimulation of pregnenolone synthesis was seen with either N62-StAR or N-218 MLN64 when 20{alpha}-hydroxycholesterol was present.



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  		FIG. 9. The effects of N-218 MLN64 and N-62 StAR on placental mitochondria in the presence of 20{alpha}-hydroxycholesterol. Mitochondria were incubated with 25 µM 20{alpha}-hydroxycholesterol in the presence of 5 µM N-62 StAR (A) or 0.61 µM N-218 MLN64 (B), and pregnenolone synthesis was measured. The mitochondrial protein concentration was 0.6 mg/ml (A) and 0.3 mg/ml (B).

 
Effect of cAMP
cAMP is an important regulator of StAR in tissues that display acute tropic stimulation of steroid synthesis such as the adrenal cortex and gonads (5, 6). Because the placenta appears to lack acute regulation of steroid synthesis and expresses MLN64 rather than StAR, we tested the effect of 8-bromo-cAMP on MLN64 levels in the JEG-3 choriocarcinoma cell line. No change in MLN64 levels were seen in either whole-cell extracts or a mitochondrial fraction after treatment of the cells with 1 µM 8-bromo-cAMP for 12 or 24 h.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study establishes that N-218 MLN64, like N-62 StAR, undergoes a pH-dependent transition to the molten globule state that is associated with StAR’s activity (25, 41). We confirm previous reports that N-terminally deleted MLN64 can effectively transfer cholesterol from the outer to the inner mitochondrial membrane of steroidogenic tissues (13, 14, 20) and extend those reports by showing that with saturating N-218 MLN64, P450scc becomes essentially saturated with substrate, the condition under which placental steroidogenesis appears to work in placental cells (2), and distinct from adrenal steroidogenesis. We have developed a new assay for measuring the rate of cholesterol transport between synthetic phospholipid membranes that does not require the presence of other proteins. Using this assay, we found that N-218 MLN64 can transfer cholesterol between synthetic phospholipid vesicles with an initial rate (turnover number) of transfer (6.5 mol/min·mol N-218 MLN64) that is higher than the turnover number of P450scc in placental mitochondria (3–4 mol/min·mol P450scc) (2). The ability of N-218 MLN64 to transport cholesterol between the membranes of artificial phospholipid vesicles indicates that no other proteins are necessary for the transport activity of N-218 MLN64.

We have previously shown that the 445-amino-acid MLN64 protein is cleaved in placental cells to a fragment the same size as N-218 MLN64, suggesting the active form containing the START domain is present in the human placenta (14). There is also some evidence that the full-length form of MLN64 is localized to placental mitochondria (24). Together these studies provide strong evidence for a key role of MLN64 in the provision of cholesterol for progesterone synthesis in the human placenta. The ability of N-62 StAR to enhance cholesterol delivery to P450scc in placental mitochondria, despite its lack of expression in this tissue (11), is not surprising. It has previously been shown that StAR constructs function in nonsteroidogenic COS-1 cells rendered steroidogenic by either transient (42) or stable (43) transfection with a vector encoding a monomolecular fusion protein comprising the three components of the cholesterol side-chain cleavage system (44). This study shows that N-62 StAR and N-218 MLN64 have comparable efficacy in stimulating cholesterol transport in placental mitochondria. Although StAR is not expressed in the placenta, there appears to be coexpression of StAR and MLN64 in human granulosa and thecal cells (13). MLN64 might provide a basal unregulated rate of cholesterol transport to the inner mitochondrial membrane in these cells with the potential for rapid stimulation of the transfer provided by StAR.

Mitochondria from the human placenta are an excellent system for studying cholesterol delivery to the inner mitochondrial membrane by both MLN64 and StAR. We have previously shown that the concentration of adrenodoxin reductase in placental mitochondria, and not cholesterol delivery, primarily limits the rate of placental progesterone synthesis (9, 10, 29). Under conditions of limiting adrenodoxin reductase where adrenodoxin and P450scc cannot be kept fully reduced, P450scc displays a lower Km for cholesterol than with saturating adrenodoxin reductase (10). This low Km for cholesterol makes it easier to achieve saturating cholesterol in the steroidogenic pool compared with other steroidogenic tissues and thereby facilitates the action of the cholesterol transporter in providing near-saturating cholesterol to the P450scc.

After incubating placental mitochondria with an electron source for 20 min, the rate of pregnenolone synthesis from endogenous cholesterol falls to a basal rate of approximately 10% of the initial rate. Saturating amounts of N-218 MLN64 and N-62 StAR stimulated this rate 6-fold, maintaining it above 50% of the initial rate. The rate is even closer to the initial rate when exogenous cholesterol is present. The initial rate reflects a saturating concentration of cholesterol in the steroidogenic pool (2, 35). Thus, if there is a saturating concentration of MLN64 in placental mitochondria and ample cholesterol in the outer mitochondrial membrane, P450scc would be nearly saturated with its substrate, the condition observed in freshly isolated trophoblast cells (2). The degree of stimulation by MLN64 in this study exceeds that reported for N-218 or N-234 MLN64 previously in transiently transfected COS-1 cells or mitochondria from MA-10 cells (13, 14). The addition of exogenous cholesterol to placental mitochondria had little effect on the rate of pregnenolone synthesis with subsaturating amounts of the transporter and caused a small stimulation with near-saturating amounts of transporter. The concentration of cholesterol in the placental outer mitochondrial membrane is therefore sufficient to support nearly maximal rates of pregnenolone synthesis by isolated mitochondria for at least an hour, provided a transporter is present. When membrane-permeable 20{alpha}-hydroxycholesterol was used as substrate, N-218 MLN64 did not stimulate pregnenolone synthesis, illustrating that its mechanism of action is to stimulate cholesterol delivery to P450scc in the inner mitochondrial membrane. The requirement for a correctly folded transporter for stimulation of pregnenolone synthesis is shown by the lack of stimulation by R182L N-62 StAR. This protein contains one of the mutations causing congenital lipoid adrenal hyperplasia (12) and is nonfunctional in transfected cells and mitochondria.

A role for MLN64 in placental steroidogenesis is consistent with the lack of mitochondrial targeting sequences in MLN64, as the action of StAR and N-62 StAR is confined to the outer mitochondrial membrane, and protein import inactivates StAR (23). It is also consistent with studies of trypsin-treated placental mitochondria that suggested that cholesterol uptake by placental mitochondria is mediated by a protein (45) and with the lack of acute regulation of placental progesterone synthesis (2, 8, 9). In all tissues where acute regulation is observed, it is mediated by StAR, which is regulated by tropic hormones via cAMP. This acute regulation is associated with substantial changes in plasma steroid concentrations within minutes. The present study shows that MLN64 levels in placental JEG-3 cells are not influenced by 8-bromo-cAMP over a 24-h period, consistent with constitutive expression and with the finding that progesterone derived from the placenta shows little short-term fluctuation throughout the day during gestation (7). Little is known about the regulation of the MLN64 gene or the processing of the expressed protein. Long-term regulation of MLN64 expression and processing in the human placenta may play a role in maintaining a near-saturating concentration of cholesterol in the mitochondria as their steroidogenic output increases during gestation (46).


   Footnotes
 
Address all reprint requests to: Dr. Robert C. Tuckey, Biochemistry and Molecular Biology, University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia. E-mail: rtuckey{at}cyllene.uwa.edu.au.

This work was supported by a University of Western Australia grant (to R.C.T.), and by NIH Grants DK02762 (to H.S.B.) and DK37922 (to W.L.M.).

Abbreviations: CD, Circular dichroism; DEAE, diethylaminoethyl; StAR, steroidogenic acute regulatory protein.

Received August 12, 2003.

Accepted for publication January 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Miller WL 1998 Steroid hormone biosynthesis and actions in the materno-feto-placental unit. Clin Perinatol 25:799–817[Medline]
  2. Tuckey RC, Kostadinovic Z, Cameron KJ 1994 Cytochrome P-450scc activity and substrate supply in human placental trophoblasts. Mol Cell Endocrinol 105:103–109[CrossRef][Medline]
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  4. Beaudoin C, Bonenfant M, Tremblay Y 1997 Regulation of cytochrome P450 cholesterol side-chain cleavage, 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase type 1 and estradiol-17 ß-hydroxysteroid dehydrogenase mRNA levels by calcium in human choriocarcinoma JEG-3 cells. Mol Cell Endocrinol 133:63–71[CrossRef][Medline]
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