©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sterol Carrier Protein-2 Is Involved in Cholesterol Transfer from the Endoplasmic Reticulum to the Plasma Membrane in Human Fibroblasts (*)

(Received for publication, May 8, 1995; and in revised form, May 30, 1995)

Luigi Puglielli (1) Attilio Rigotti (1)(§),   Aldo V. Greco (3) Manuel J. Santos (2) Flavio Nervi (1)(¶)

From the (1)Departamento de Gastroenterolog&ıacute;a y Centro para la Prevención y Tratamiento del Cáncer Digestivo, Facultad de Medicina and (2)Departamento de Biolog&ıacute;a Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica, Casilla 114-D, Santiago, Chile and (3)Istituto di Medicina Interna, Facoltá di Medicina e Chirurgia, Universitá Cattolica del Sacro Cuore, 00198 Rome, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cellular mechanism of cholesterol transport from the endoplasmic reticulum to the plasma membrane is currently unknown. To assess the possibility that sterol carrier protein-2 (SCP-2) is involved in this transport, we studied the time course of newly synthesized cholesterol incorporation in the plasma membrane of normal and SCP-2-deficient (Zellweger syndrome) human fibroblasts.

Cholesterol transfer was rapid, cytoskeleton-independent, and Golgi-independent in normal cells, but it was slower, cytoskeleton-dependent, and Golgi-dependent in SCP-2-deficient cells. After SCP-2 antisense oligonucleotides treatment of normal fibroblasts, the rapid transport was reduced by 81% with a simultaneous increase of the slower one.

These results suggest that in normal fibroblasts the major fraction of newly synthesized cholesterol is transported to the plasma membrane by a SCP-2-dependent mechanism. In contrast, in SCP-2-deficient cells, newly synthesized cholesterol leaves the endoplasmic reticulum by a cytoskeleton/Golgi-dependent mechanism.


INTRODUCTION

Intracellular cholesterol trafficking may occur through several mechanisms, including aqueous diffusion, vesicle budding and fusion, and protein-mediated transport through the cytosol(1, 2, 3, 4) . These transport systems are ultimately responsible for the high compartmentalization of cholesterol between the intracellular organelles and the plasma membrane. In several cell types, over 80% of total unesterified cellular cholesterol is associated with the plasma membrane (2, 4) but the mechanism of this segregation is unknown. Immediately after its synthesis, cholesterol is transferred from the endoplasmic reticulum to the plasma membrane with a reported t of 10-20 min(5, 6, 7) . The exact mechanism of this transfer is presently unknown, although it has been shown to be not affected by several cytoskeleton- and Golgi-disrupting drugs(8, 9) , and to be both temperature-dependent (8, 9) and energy-dependent(5) .

The 13.2-kDa sterol carrier protein 2 (SCP-2) (^1)has been postulated to facilitate the movement of sterols within cells(10, 11, 12) . However, evidence that SCP-2 participate in cholesterol transfer to the plasma membrane in vivo is lacking. Antibodies against rat liver SCP-2 recognize the 13.2-kDa polypeptide, as well as 40- and 58-kDa polypeptides(11, 12) , which are thought to be SCP-2 precursors(12) . The processing of SCP-2 precursors depends on the presence of functional peroxisomes(12) . These precursors are rapidly degraded with a marked decrease of SCP-2 in cells that are deficient in peroxisomes, such as Zellweger syndrome fibroblasts(11, 12) .

In this study, we examined the transfer of newly synthesized cholesterol to the plasma membrane in normal and SCP-2-deficient fibroblasts. Our results provide evidence for the involvement of SCP-2 in a rapid and direct transfer of cholesterol from the endoplasmic reticulum to the plasma membrane.


EXPERIMENTAL PROCEDURES

Materials

[^14C]Acetic acid (sodium salt, 56 mCi/mmol) was purchased from Amersham Life Science (Buckinghamshire, United Kingdom). Culture media and fetal calf serum were obtained from Life Technologies, Inc. Thin layer chromatography plates were obtained from Merck (Darmstadt, Germany). All the other chemical agents were obtained from Sigma.

Cell Culture

Human normal (IMR 90) and Zellweger syndrome (GM 4340) fibroblasts, obtained from the Mutant Cell Repository (Camden, NJ), were cultured in 5% CO(2) at 37 °C, in Dulbecco's modified Eagle's medium (DMEM) plus 10% (v/v) fetal calf serum. On day 0, cells were plated in DMEM plus 10% (v/v) fetal calf lipoprotein-deficient serum (13) (medium A) to obtain 1.0-1.2 10^6 cells on the day of the experiment (day 3).

Incorporation of Newly Synthesized Cholesterol in the Plasma Membrane

On day 3, cells were pulsed with 25 µCi of [^14C]acetate in ethanol (<1% final concentration) for 7 min at 37 °C. The medium was then removed and the label was chased by the addition of fresh medium A containing 10 mM sodium acetate for the indicated times. Cells were then washed twice in the assay buffer (310 mM sucrose, 1 mM MgSO(4), 0.5 mM sodium phosphate, pH 7.4) at 25 °C. Cholesterol oxidase (0.5 IU/ml) was added in the assay buffer, and the cells were incubated for 3 min at 25 °C(14) . The assay buffer was removed, and the cells were immediately extracted with chloroform/methanol (2:1). The chloroform phase was dried under nitrogen, resuspended with chloroform/methanol (1:1), and applied together with standards to a Silica Gel-G TLC plate. Plates were developed in hexane/ethyl ether/acetic acid (87:20:1, v/v) and visualized with I(2) vapor. Spots were scraped and counted in a liquid scintillation counter to reach 10,000 counts.

Treatment of Cells with Antisense/Sense Oligonucleotides

Phosphodiester oligonucleotides, including SCP-2 antisense (5`-AAAACCCATCTTGTA-3`), sense (5`-TACAAGATGGGTTTT-3`), and missense (5`-AATTCAACTCTGTTC-3`), were synthesized by Chiron Corporation (Emeryville, CA) and purified on reverse-phase high-performance liquid chromatography. Confluent normal fibroblasts were cultured in serum-free DMEM, and SCP-2 antisense, sense, or missense were added directly to the cultured medium at 10 µM final concentration. This treatment was repeated once daily for up to 5 days.

Immunoblotting

Cell monolayers were harvested by trypsinization, rinsed with phosphate-buffered saline, and then dissolved in SDS gel loading buffer. Aliquots containing 50 µg of proteins were analyzed by Western blot. Polyclonal anti-rat SCP-2 antisera (^2)binding was visualized using the enhanced chemiluminescence procedure (Amersham Life Science).

Other Methods

Protein was determined by the method of Lowry et al. (15) using bovine serum albumin as standard. Cholesterol was quantitated by enzymatic methods(16) .

Data Analysis

Data were analyzed using Student's t test.


RESULTS

The possibility that SCP-2 serves as a carrier protein for newly synthesized cholesterol to the plasma membrane was investigated in normal and SCP-2-deficient (Zellweger syndrome) fibroblasts. After 72 h of culture in medium A, normal and SCP-2-deficient cells showed similar rates of cholesterol synthesis. The maximal values of newly synthesized cholesterol in the plasma membrane of normal fibroblasts were observed 10 min after the beginning of the pulse, decreasing rapidly thereafter (Fig.1). By contrast, the maximal values of radiolabeled cholesterol were found only 20 min after the beginning of the pulse in the plasma membrane of SCP-2-deficient fibroblasts. It is noteworthy that the same amount of [^14C]cholesterol was found 20 min after the pulse in both normal and SCP-2-deficient fibroblasts (Fig.1, inset). Immunoblot analysis, using a specific antiserum against SCP-2, confirmed the absence of SCP-2 in these Zellweger syndrome fibroblasts (Fig.2).


Figure 1: Time course of newly synthesized cholesterol transport to the plasma membrane. Confluent fibroblasts were pulsed with [^14C]acetate for 7 min at 37 °C and chased for the indicated times. [^14C]Cholesterol incorporation in the plasma membrane was determined using the cholesterol oxidase assay. The maximal incorporation rates of [^14C]acetate into total cell cholesterol were similar in control and SCP-2-deficient fibroblasts (2564 ± 301 versus 2832 ± 409 counts min/10^6 cells, respectively). Results are the mean ± S.D. of four different experiments and are expressed as the relative and total (inset) percentage of cell [^14C]cholesterol/10^6 cells that was accessible to cholesterol oxidase. 1 10^6 cells corresponded approximately to 600 µg of protein. black square, normal fibroblasts; , SCP-2-deficient fibroblasts.




Figure 2: Western blot analysis of normal and Zellweger syndrome fibroblasts. 50 µg of proteins of cell extracts were analyzed by Western blot. Polyclonal anti-rat SCP-2 antisera binding was visualized using the enhanced chemiluminescence procedure. Lane1, normal human fibroblasts; lane2, Zellweger syndrome fibroblasts.



Next, we evaluated the effects of colchicine, cytocalasin B, monensin, and brefeldin A, which are well known cytoskeleton- and Golgi-disrupting drugs(18, 19, 20, 21) , on newly synthesized cholesterol incorporation in the plasma membrane of normal and SCP-2-deficient cells. None of these drugs altered cholesterol transfer in normal fibroblasts (Fig.3A). In contrast, the same drugs, used at equivalent concentrations, reduced significantly (37-66%) the transport of newly synthesized cholesterol to the plasma membrane in SCP-2-deficient cells (Fig.3B).


Figure 3: Effect of colchicine, cytocalasin B, monensin, and brefeldin A on newly synthesized cholesterol transport. Confluent normal (A, ) and SCP-2-deficient (B, ) fibroblasts were pulsed with [^14C]acetate and chased for the indicated times. 60 µM colchicine (black square), 50 µM cytocalasin B (), and 15 µM monensin (bullet) were added 90, 30, and 60 min before the pulse, respectively(8) . Brefeldin A () was added at a final concentration of 1 µg/ml 30 min before the pulse(9) . Cells preincubated with 0.1% ethanol or with 0.5% methanol, which were used to add the drugs(8, 9) , served as internal controls (). The maximal incorporation rates of [^14C]acetate into total cell cholesterol were similar in control and SCP-2-deficient fibroblasts and were not affected by pretreatment with these drugs. Results are the mean ± S.D. of four different experiments and are expressed as the relative and total (insets) percentage of cell [^14C]cholesterol/10^6 cells that was accessible to cholesterol oxidase. *, p < 0.001 (versus control).



In the last series of experiments, we treated normal fibroblasts with SCP-2 antisense oligonucleotides corresponding to the 5` initiation region of the SCP-2 coding sequence(17) . SCP-2 antisense treatment markedly reduced the total amount of both SCP-2 and its 58- and 40-kDa related proteins (Fig.4), whereas treatment with control oligonucleotides (sense and missense) did not affect SCP-2 expression. In agreement with these results, the biosynthesis of SCP-2 and its related proteins was significantly lowered by 95%, 80%, and 91%, respectively, at 5 days of antisense treatment (data not shown). After 5 days of oligonucleotides treatment, cells were pulsed with [^14C]acetate. As shown in Fig.5, the relative percentage of [^14C]cholesterol transported to the plasma membrane in the first 10 min was reduced by 81%, with a simultaneous increase of the radiolabeled cholesterol incorporation at 20 min after the beginning of the pulse. Such effect was not observed in sense- or missense-treated cells. When SCP-2 antisense-treated cells were incubated with colchicine, before the pulse with [^14C] acetate, the values of [^14C]cholesterol incorporated in the plasma membrane 20 min after the beginning of the pulse were reduced by 45% (Fig.5).


Figure 4: Effect of SCP-2 antisense oligonucleotides on SCP-2 expression in normal fibroblasts. Confluent normal fibroblasts were cultured in serum-free DMEM. SCP-2 antisense, sense, or missense were added directly to the cultured medium at 10 µM final concentration for 5 days, without a change of medium. Cells cultured without oligonucleotides served as internal controls. Immunoblot analysis of SCP-2 content of cell lysates was carried out as described in the legend of Fig.2.




Figure 5: Effect of SCP-2 antisense oligonucleotides on newly synthesized cholesterol transport to the plasma membrane. Confluent normal fibroblasts received SCP-2 antisense, sense, or missense, for 5 days as described in the legend of Fig.4. Cells cultured without oligonucleotides served as internal controls. The day of the experiment, cells were pulsed and chased for the indicated times. Experiments were conducted as described in Fig.1. The maximal incorporation rates of [^14C]acetate into total cell cholesterol of the different groups were (expressed as counts min/10^6 cells): control cells, 3682 ± 405; antisense-treated cells, 3562 ± 701; antisense + colchicine-treated cells, 3428 ± 388; sense-treated cells, 3875 ± 241. Results are the mean ± S.D. of three different experiments. *, p < 0.0005 (antisense versus control); &, p < 0.0005 (antisense + colchicine versus antisense).




DISCUSSION

The present results provide the first evidence in vivo for the involvement of SCP-2 in a rapid transport of newly synthesized cholesterol to the plasma membrane in normal fibroblasts. When the expression of SCP-2 is abolished, such as occurs in peroxisomes-deficient cells, or after SCP-2 antisense treatment, newly synthesized cholesterol reaches the plasma membrane through a slower, cytoskeleton- and Golgi-dependent route.

Previous reports have shown that transfer of newly synthesized cholesterol to the plasma membrane of animal cells is not affected by cytoskeleton- and Golgi-disrupting agents(8, 9) . This is consistent with present results in normal human fibroblasts. However, we found that both the cytoskeleton and the Golgi apparatus are partially required for cholesterol transport in SCP-2-deficient cells. These results support the possibility that two different mechanisms are involved in the transport of newly synthesized cholesterol to the plasma membrane of normal and SCP-2-deficient cells. This possibility is supported by the evidence that, in normal cells, SCP-2 antisense treatment induced a shift of the maximal values of [^14C]cholesterol transport from 10 to 20 min, and that this shift was reduced by colchicine. These observations suggest that the endoplasmic reticulum may modulate the two routes and, when SCP-2 is absent, cholesterol leaves this organelle by the cytoskeleton/Golgi-dependent route. The molecular mechanisms by which the endoplasmic reticulum may direct cholesterol to one or the other route are unknown. It is noteworthy that the same amount of radiolabeled cholesterol was found 20 min after the pulse in both normal and SCP-2-deficient cells. In agreement with this result, normal and SCP-2-deficient fibroblasts showed comparable amounts of free cholesterol in the whole cell and in the plasma membrane (results not shown). We do not know if SCP-2-deficient cells remove cholesterol by increasing the number of vesicles leaving the endoplasmic reticulum, the mass of cholesterol per vesicle, or both.

SCP-2 antisense treatment reduced the total amount of both SCP-2 and its 58- and 40-kDa related proteins. Both the 58- and 40-kDa proteins represent N-terminal extensions of SCP-2(11) , but only the 13.2-kDa moiety has the functional capacity to transfer cholesterol(2, 4) . We cannot rule out the possibility that SCP-2 is involved in cholesterol transport only for its affinity for sterols while other factors (i.e. docking proteins or cytoskeleton-independent vesicles) are carrying the cholesterol-SCP-2 complex to the final destination.

Pre-Golgi cholesterol-enriched vesicles have been isolated previously (8, 9) . Moreover, the Golgi apparatus contains substantial amounts of cholesterol, which is particularly enriched in the trans Golgi(22) . Our evidence that cholesterol may be transported by a Golgi-dependent route is consistent with these previous studies (8, 9, 22) and supports the possibility that this route may also serve as a mechanism for the sorting of proteins, as proposed recently(23) .

Plasma membrane cholesterol is not evenly distributed throughout the bilayer but is instead localized into cholesterol-rich and cholesterol-poor domains(24) , which show different exchange rates in the presence of lipoprotein acceptors(25) . We do not know if the SCP-2-dependent and the Golgi/cytoskeleton-dependent routes are carrying cholesterol to any specific plasma membrane domains, and if they are playing a specific role in reverse cholesterol transport. These possibilities would imply the presence of docking proteins, or receptors in the plasma membrane, and would give a functional role to the two different routes of intracellular cholesterol transport.


FOOTNOTES

*
This work was supported by Ministero degli Affari Esteri d'Italia and Istituto per la Cooperazione Universitaria, Italy, and Fondo Nacional de Ciencia y Tecnologia 1940620 and 1940686, Chile. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Biology Dept., Rm. 68-483, Massachusetts Institute of Technology, Cambridge, MA 02139.

To whom correspondence should be addressed: Departamento de Gastroenterolog&ıacute;a, Facultad de Medicina, Pontificia Universidad Católica, Casilla 114-D, Santiago, Chile. Fax: 56-2-6331457.

^1
The abbreviations used are: SCP-2, sterol carrier protein-2; DMEM, Dulbecco's modified Eagle's medium.

^2
Polyclonal antibody against SCP-2 (26) was a generous gift of Dr. Charles L. Baum (Department of Medicine, Gastroenterology Section, University of Chicago, Chicago, IL).


ACKNOWLEDGEMENTS

We thank L. Nuñez, L. Amigo, M. E. Kawada, V. Volrath, and Dr. J. F. Miquel for technical support, and E. Brandan and P. P. Rosso for editorial reading of the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.