Bioch#nica et Biophysica Acta, 1()84( 1991) 7-14
7
© 1991 ElsevierScience Publishers n.v. {Hx)5-276(}/91/$o3.5o ADONIS 000527609100220L
BBAL1P 53711
Effiux of phospholipid from fibroblasts with normal and elevated ! ....
in
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J o h n K. Bielicki, W i l l i a m J. J o h n s o n , J a n e M. Glick a n d G e o r g e H. R o t h b l a t Department of Physiology and Biochemist~'. The Medical College of Pcnnsyh'ania. Philadelphia. PA (U.S.A.)
(Received 17 April 1991)
Key words: Fibroblast:Cholesterol; Phospholipid;Efflux;HDL To addi-,~ss ;he h.~0othesis that phospholipid ettlux from cells contributes to lipoprotein structure, we have examined the elllux of biosynthetically labeled [~ZP]pbospbolipid from cells to lipoproteins. With normal human skin fihroblasts in monolayer culture, high density lipopratein (HDL s) promoted the effiux of phospholipid in a concentration-dependent manner. As analyzed by TLC, :he major phospholipids released from fibroblasts were phosphatidylcholine, sphingomyelin, lysophosphatldyleholine and phosphatidylethanolamine. At identical concentratlons, HDL a and dimethylsuberimidate treated-HDL 3 promoted similar etllux, suggesting that efflux did not depend on specific binding of HDL 3 to the cell surface. When the content of cholesterol in fibroblasts was doubled by pre-ineubation with LDL and cholesterol-rich liposomes, the fractional efflux of phospbolipid to HDL 3 and other aeceptors was stimulated about 2-fold. Most of this stimulation was due to enhanced release of phosphatidylcboline. Similar effects of enrichment were found with FuSAH rat hepatoma cells, but not with J774 mouse macruphages. The results support the hypothesis that the elnux of phospholipid from cells contributes to the phospholipid content of HDIL. This may enhance the ability of ItDL to remove cholesterol from cells, the initial step in reverse cholesterol transport.
Introduction
The movement of cholesterol from extrahepatic cells to high density lipoprotein (HE)L) is thought to be the first step in reverse cholesterol transport. The ability of HDL to promote cholesterol efflux from cells is related to the phospholipid content of the particle [!,2]. A few studies have documented that the exposffrc of cells in culture to s e r : m lipoproteins results in the efflux of cell phospholi)id. With macrophages, phospholipid efflux appears to be linked to the secretion of apolipoprotein E [3]. However, in other cell types such
Abbreviations: PL. phospholipld;PC, phosphatidylcholine;SM, sphingomyelin; lyso-PC, lysophosphatidylcholine; PE. phosphatidylethanolamine; FC, free cholesterol; DMS, dimethylsuherimidate; LCAT, lecithin:cholesterol acyltransferase; HDL, high density lipoprotein; LDL, low density lipoprotein; BSA, bovineserum albumin; PBS, phosphate-bufferedsaline; LSC. liquid scintillation counting, TLC. thin-layer chromatography; MEM. minimum essential medium; FBS, fetal bovineserum. Correspondence: J.K. Bielicki0 Department of Physiologyand Biochemistry, The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129. U.S.A.
as smooth muscle cells [4], epithelial cells [5] and fibroblasts [6], phospholipid efflux is stimulated by the presence of exogenous lipoproteins. In the case of smooth muscle cells, low density lipoprotein (LDL) and HDL have been shown to promote phospholipid release [4]. Yet, the contribution of phospholipid efflux to lipoprotein structure and to the process of cholesterol efflux from cells is unknown. Phospholipid-rich HDL has been isolated from plasma [7] and from the lymph of humans and dogs [8-10]. The pre-fl HDL-like particles described by Fielding [7,11,12] are thought to contribute to the reverse cholesterol transport process by serving as efficient acceptors of cellular cholesterol. The HDL particles isolated from lymph are larger and contain more unesterified cholesterol and phospholipid compared to HDL from the vascular compartment. The high phospholipid content of interstitial lipoproteins may be due to interactions of these particles with cells and transfer of phospholipid from cells to lipoproteins. In this study, we monitored the release of biosynthetically labeled cellular phosphoBpid to lipoproteins. The efflux of phospholipid to HDL 3 was examined using fibroblasts, rat hepatoma cells and macrophages.
Cells were enriched with unesterified cholesterol to determine whether cellular cholesterol content alters phospholipid efflux. We found that an elevation in cellular cholesterol content enhances the movement of phospholipid from cells to lipoproteins. This process may contribute to reverse cholesterol transport by enlarging interstitial lipoproteins and thereby enhancing cholesterol efflux from cells. Materials and Methods
Materials. Lysophosphatidylcholine (lyso-PC), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), unesterified cholesterol, heat inactivated fetal bovine serum (FBS) and bovine serum albumin (BSA, essentially fatty acid free) were purchased from Sigma (St. Louis, Me). Organic solvents were reagent grade and obtained from Fisher Scientific (Pittsburgh, PA). Anasil (3, 250 tzm TLC plates were from Ana!abs (North Haven, CT). Carrier free [32p]orthophosphoric acid was purchased from New England Nuclear (Boston, MA). Dimethylsuberimidate was obtained from Pierce Chemical (Rockford, IL). HeparinSepharose was from Pharmacia LKB Biotechnology (Piscataway, N J). Tissue culture flasks and plates were obtaincd from Falcon (Lincoln, N J) and Coming Glass Works (Corning, N'Y), respectively. Gentamicin was from Tri Bio Labs (State College, PA) and all other antibiotics, trypsin, culture media and calf serum were purchased from Gibco (Grand Island, NY). Sandoz compound 58035 was a gift from Dr. John Heider. Cell culture. GM3468A normal human skin fibreblasts were grown as monolayers in minimum essential medium (MEM) supplemented with 10% FBS (v/v). J774 mouse macrophage cells were maintained in RPMI containing 10% FBS (v/v) and Fu5AH rat heparoma cells were grown in MEM plus 5% calf serum (v/v). Stock cultures of cells were grown in T-75 culture flasks and all media contained 50 ~g of gentamicin/ml. Cells grown in bicarbonate-buffered media were maintained at 37 o C in a humidified 95% air/5% CO 2 atmosphere, whereas media buffered with Hepes were incubated in humidified air. Lipoproteins and lipid dispersions. Lipoproteins were obtained from fresh human plasma by sequential ultracentrifugation [13]. Lecithin:cholesterol acyltransferase (LCAT) activity was inhibited with N-ethylmaleimide [2]. The HDL 3 fraetien (d= 1.125-1.21) was chromatographed on heparin-Sepharose to remove particles containing apoprotein E [14]. Derivitization of HDL 3 with dimethylsuberimidate (DMS) was performed according to Chacko et al. [15]. Before use, HDL 3 and DMS-HDL 3 were dialyzed against 0.15 1,4 NaCI, 2 mM sodium phosphate (pH 7.4), supplemented with 50 units of penicillin G / m l and 50 g g of strepto-
mycin sulfate/ml. Final dialysis of this material was against 30-50 volumes of MEM buffered with 14 mM Hepes. Phospholipase activity of LCAT was not detected in HDL 3 preparations, as judged by the distribution of lyso-PC and PC in 24 h efflux medium that had been incubated overnight in the absence of cells (data not shown). LDL ( d = 1.019-1.063) was dialyzed exhaustively against 0.15 M NaCI with a final dialysis against MEM buffered with Hepes. All lipoproteins were sterilized by filtration (0.45 /zm) and stored at 4°C. Lipoprotein-deficient serum (LPDS) was obtained from density > 1.21 g/ml. Human serum was obtained with informed consent from healthy, malt donors, and stored at 4 ° C overnight prior to use. Dispersions containing free cholesterol and egg phosphatidylcholine (2-3:1, tool : tool) were prepared by sonication, as described by Arbogast et al. [16]. Phospholipid liposomes were prepared with egg phosphatidylcholine by the same procedure except th,-.t cholesterol was omitted. Experimental conditions. Ceils from stock cultures were plated on 22, 35 or 100 mm culture dishes and maintained in growth media until confluent. To label cellular phospholipid, monolayers were incubated with growth medium supplemented with 5 ~ C i / m i of [3ZP]phosphate for 3 days prior to the initiation of efflux studies. The 22, 35 and 100 mm culture dishes contained 0.5, 1.0 and 8.0 ml of labeling media, respectively. Preliminary experiments demonstrated that the distribution of ['~2p]phosphate among phospholipid subclasses did not change after 2 days of labeling. Lipoproteins were diluted to final concentrations with MEM, and BSA (2 mg/ml) was added as indicated in the text. Unless otherwise indicated, incubation time for measurement of phospholipid efflux was 24 h. After incubation with cells, efflux media were removed from the cells and spun at 3000 × g for 10 rain to pellet any cells which had become dislodged from the plates. The lipids in the supernatant were extracted by the method of Bligh and Dyer [17]. Lipids from media were redissolved in chloroform/methanol (2:1) and aliquots taken for liquid scintillation counting (LSC) and separation of phospholipid subclasses by thin-layer chromatography (TLC). The TLC plates were developed by a two-step, one-dimensional procedure as follows: the first solvent system consisted of chloroform/ methanol/H 20 (65 : 25 : 4, v/v), and, after the plate was fully developed, it was allowed to dry at room temperature for approx. 30 min before a second development in a system of c h l o r o f o r m / a c e t o n e / methanol/acetic a c i d / H 2 0 (50:20:10:10:5). Approximate R F values for various lipids were as follows: lyso-PC = 0.17, SM = 0.27, PC = 0.36 and PE = 0.59. The cell monolayers were rinsed with phosphatebuffered saline (PBS) and the lipids were extracted with isopropanol [18]. The isopropanol was transferred
9 from the culture dish to a separate tube, the monolayers were rinsed once with additional isopropanol, and the combined extracts were dried under nitrogen. The dried lipid residue was partitioned by the method of Bligh and Dyer [17]. The chloroform phase from this partition was dried, and then redisso!ved in chlorof o r m / methanol (2:1). Aliquots wece removed for LSC and TLC as described for the media samples. The data are expressed as either release of total lipid radioactivity (cpm) or as the percentage of [32p]phospholipid released from cells: % efflux = (cpm in medium after 24 h / c p m in cellular lipids at t = 0). 100. The distribution of radioactivity among the phospholipid subclasses, as separated by TLC, was measured using a Radiomatic Imaging TLC Scanner. This fractional distribution was multiplied by the total radioactivity obtained for cells and media to calculate the radioactivity associated with each phospholipid subclass and the subsequent release of this radioactivity from cells. Enrichment of cells with unesterified cholesterol. The experimental protocol was modified to enrich cells with free cholesterol (FC). The growth and plating of the cells were conducted as described above. When cells on 22 mm culture dishes reached confluency, the medium was replaced with MEM supplemented with 10 rag protein/ml of LPDS and 5 p,Ci/ml of [32p]phosphate. After a 48 h incubation, the cell monolayers were incubated an additional 24 h with one of the following media: (1) a FC-loading medium [19] containing 1 p.g/ml of Sandoz compound 58035 to inhibit cholesterol esterification, 100 p,g F C / m l of FC: PC dispersions ( > 2:1, tool:tool) and 50 p.g protein/ml of LDL, or (2) a control medium [19] containing 1 t~g/ml of 58035 and PC liposomes added at the same phospholipid concentration as in the FC enrichment medium. Both media preparations also contained 5 p,C i / m l [3ZP]phosphate and 2 m g / m l BSA. Sandoz compound 58035 was added to the cultures in DMSO, and the final c~ncee;ration of solvent was 0.5% (v/v). This procedure resulted in at least a 2-fold increase in cellular unesterified cholesterol content in each of the cells studied. Cholesteryl ester was not detected in cells. Efflux of labeled phospholipids was examined immediately after enrichment.
Specific activity determinations of labeled phospholipid subclasses. Normal skin fibroblasts plated on 100 mm culture dishes were grown to confluency, labeled with 5 p,Ci/ml of [3ZP]phosphate for 3 days, and enriched with cholesterol for 24 h as described above. The specific activity of the labeling medium was 13 250 cpm/nmol phosphate. Cell monolayers were rinsed with PBS, lipids extracted and then separated by TLC. The areas of the plate corresponding to the major phospholipid subclasses were scraped into test tubes, and the phospholipid was ext,acted from the silica gel
as described by Bamberger et al. [1]. Aliquots were taken for 32p quantitation and lipid phosphorus determination. Chemical analyses. Cholesterol was quantitated by gas liquid chromatography using cholesteryl methyl ether as an internal standard [20]. Phospholipid phosphorus was determined by the method of Sokoloff and Rothblat [21]. The protein content of lipid extracted monolayers was measured by the procedure of Markwell [22] using BSA as a standard. Radioactivity was measured with either a Beckman LS1801 or LSS000TD liquid scintillation counter using Scintiverse BD counting fluid. Statistical analysis. Triplicate measurements were taken for efflux and for cellular lipid and protein determinations. When percentages are calculated, the S.D. were derived from cellular and medium determinations of radioactivity. Student's t-test was used and P < 0.05 was the criterion for statistical significance. Results
E.fflwc of phospholipid from normal skin fibroblasts Initial studies were conducted to determine the dependence of phospholipid efflux on HDL 3 concentration. Maximal effiux of radiolabeled phospholipid was obtained at concentrations > I00 p.g HDL 3 protein/ml (Fig. 1). To address the specificity of the acceptor for promoting phospholipid release, a number of different acceptors were tested (Fig. 2). BSA alone in the medium stimulated phospholipid efflux to only a small extent (1.8 + 0.2%/24 h) compared to the basal release of phosphoiipid to protein-free medium (1.1 _+
co
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.... C o n ¢ ~ t r ( l t l o ¢ ~ 01 HDL 3 . ~
Droteln /ml
Fig. 1. Dependence of pho~pholipid release from fibroblasts on HDL~ concentration. Normal skin fibroblasts were grown to confluency on 35 mm dishes and then labeled for 3 days with 5 pCi/ml of [~-Plphosphate in media containing 10% FBS. Cells were incubated
an additional 24 h with MEM supplementedwith 2 mg of BSA/ml and increasing amountsof HDL3. The data are expressed as the percentage of the total [3ZP]phospholipid released from cells, normalizing to the total radioactivity initially present in cellular lipids.
The open circle on the vertical axis represents the basal release of phospholipid in the presenceof 2 mg BSA/mL Values are means± S.D., n = 3. Where not shown,the error bars are withinthe symbols.
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and the ACAT inhibitor Sandoz 58035, as described under Materials and Methods. This enrichment protocol has been used extensively in elucidating the mechanism of cholesterol movement between cells and HDL 3 [19]. Increasing the cellular cholesterol content 2- to 3-fold did not result in a significant increase in the phospholipid content; thus, the free cholesterol/ phospholipid (mol/mol) ratio more than doubled, from 0.23 + 0.08 to 0.49 + 0.12 (Table 1). Although the phospholipid content was not affected by the cholesterol treatment, the incorporation of [32p]phosphate iato
Fig, 2. Release of p h o s p h n l i p i d s from fibroblasts to various acccp-
tots. Normal skin fibroblasts were grown and labeled with [32p]phosphate as described in Fig. 1. The efflux medium consisted of M E M or ME M supplemented with either 2 mg B S A / m l , 500/~g p r o t e i n / m l of H D L 3, 500 p g p r o t e i n / m l of D M S - H D L 3, or 15% human serum (v/v). The data are expressed as the percentage of [ 32P]phospholipid released from cells in 24 h, normalizing to the total radioactivity initially present in cellular lipids. Values are means + S.D n = 3 .
0.1%/24 h). The role of specific HDL3 binding was tested by using HDL 3 cross-linked with dimetbylsuberimidate, a treatment which greatly reduces the ability of HDL 3 to associate with high affinity binding sites on cells [15]. This treatment had no effect on the ability of I-IDL 3 to promote phospholipid efflux (Fig. 2). indicating that specific binding to membranes is not required to obtain phospholipid release from fibroblasts. The results obtained with 15% human serum were similar to the results obtained with HDL3, added at a concentration of 500 p.g protein/ml. The kinetics of efflux of phospholipid from fibroblasts with normal levels of cellular cholesterol are presented in Fig. 3A. These data demonstrate that the total recovery of [32p]phospholipid from the system (cellular plus medium lipid radioactivity) decreases during the course of the experiment. Approx. 35% of the initial radiolabel is lost from the cellular phospholipid pool in 24 h. This decline in total recovery of [32p]phospholipid is probably due to degradation of phospholipid in cells. Approx. 6% of the cellular phospholipid is released from cells during a 24 h incubation with 500/.¢g protein/ml of HDL3. The 6% value was calculated by dividing the amount of radioactivity released from cells by the amount of radiolabeled phospholipid initially in cells. This value underestimates efflux because the specific activities of cellular phospholipid, and that of the phospholipid released from cells, are probably decreasing during the experiment.
Efflux of phospholipid from cholesterol-enriched fibroblasts To study the effect of cholesterol enrichment on phospholipid efflux, cells were treated with a combination of cholesterol-rich phospholipid liposomes, LDL
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Tir~ (h) Fig. 3. T i m e course of H D L y s t i m u l a t e d phospholipid release f r o m control and cholesterol-enriched fibroblasts. C e l l s w e r e p l a t e d o n 22 m m c u l t u r e dishes and grown to confluent monolayers. The l a b e l i n g
of cells with 32P and enrichment" of cells with unesterified cholesterol were performed as described in Materials and Methods. To initiate efflux, monolayers were rinsed extensively with MEM-Hepes and the cells were then incubated for various lengths of time with MEM containing 2 mg/ml of BSA plus 500/,tg protein/ml of HDL 3, Panel
A contains kinetic data from control cells (open symbols).Panel B contains kinetic data fromcholesterol-enrichedcells(closedsymbols). Symbol descriptions are as follows: 0 B, total recovery of [32p]phospholipid from cells and medium at each time point; ~, - ~., recoveryof 32p from cells; 0 o, recoveryof 32p from medium.Panel C showsthe resultse~pressedas the percentage of phospholipid released from cells to H D L 3, normalizing to the total radioactivity initially present in cellular lipids. Values are means+ S.D., n = 3.
TABLE
I
Cholesterol and l~ho.vpholipid composition of control and cholesterol-enriched )"P lah,'h'd human skin fibroblasts
Cell type
Control FC-enriehed
Unesterified cholesterol (.ttg/mg cell protein) :' 19-+ 4 44:1:10 c
Phospholipid (#.g/mg cell protein)
FC: PL (rnol:mol) ~
168+_27 182_+18
0.23 +_0.08 1i.49_+0.12 c
[~-"P]PL specific activity (cpm/nmol PL) h 3127 +_356 3748_+382 ¢
a values are means-+S.D., n = 6. l, values are means±S.D., n = 9. P < 0.01. compared to control cells.
total cellular phospholipid increased by approx. 20% (Table I). W h e n the recovery of [aEp]phospholipid from cholesterol-enriched fibroblasts was analyzed, the degradation of [aZp]phospholipid was identical to that obtained with the control cells (Fig. 3A vs. B). The bottom p a n e l of Fig. 3 (Fig. 3C) c o m p a r e s the efflux of pho~pholipid from control and cholesterol-enriched fibroblasts. Increasing cell cholesterol content enhances phospholipid release from fibroblasts to H D L 3. The it/2 for this efflux of 32 p was estimated using a two-pool model for the exchange of lipid between cells and lipoproteins as described for cholesterol [l,19]. The i l l 2 for efflux from control cells r a n g e d from 165 to 270 h, whereas, with cholesterol-enriched cells, the t l / 2 was reduced to 65 and 83 h in two experiments (Table
Distribution o f phospholipid subclasses
Cellular phospholipids were separated by TLC to analyze the distribution of 32 p a m o n g individual phospholipid subclasses. Phosphatid~lcholine (PC) and phosphatidylethanolamine ( P E t accounted for approx. 80% of the cellular phospholipids. W h e n cells were enriched with cholesterol, there was a shift in the distribution of PC and PE. The content of cellular 3-'P in PC increased by about 20%, whereas, a 44% de-
IHo,,
Hi. The concentration d e p e n d e n c e for the efflux of phospholipid to H D L 3 and L D L using control and cholesterol-enriched fibroblasts is p r e s e n t e d in Fig. 4). W i t h control cells, phospholipid ¢fflux was maximal at concentrations of H D L a >_ 100 /Lg p r o t e i n / m l . This was similar to the results o b t a i n e d in Fig. 1, Cholesterol e n r i c h m e n t of fibroblasts e n h a n c e d the release of phospholipid at every concentration of H D L a. This release also reached its maximal value at 1 0 0 / z g prot e i n / m l of H D L a . Qualitatively similar results were o b t a i n e d with L D L (Fig. 4). Thus, the e n h a n c e d release of phospholipid observed with cholesterol enrichment does not require a specific lipoprotein in the medium.
4 =/I
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Concentration of hpoproteln, pg protein / ml
TABLE I! Estimated tt/z for efflux of [)2P/phospholipid cholesterol.enriched fibroblasts to HDL j Experiment
1 2
from control and
11/2 (h) a
control
FC-enriched
270±111 165± 43
t"~± 20 83-+ 17
values are mcans±S.D., n = 3; concentration of HDL = 500 /~g protein/ml.
Fig. 4. Concentration dependence of HDL~- and LDL-stimulated phospholipid release from control and cholesterol-enriched fibroblasts, Normal skin fibroblasts were plated on 22 mm culture dishes and grown to confluency in M E M supplemented with I0% FBS. Control cells and cholesterol-enriched cells w e r e prepared as described in Materials and Methods, Cells were incubated for 24 h with increasing amounts of H D L 3 ( o , e) or L D L (zx, & ) provided in
MEM containing 2 mg of BSA/ml. The data are expressed as the percentage of [~2P]phospholipidreleased from cells, as described in Fig. 3. Data from the control cells are the open symbols and from cholesterol-enriched cells are the closed symbols. Values are means + S.D., n = 3.
12 TABLE Ill Distribution of [t2Plphosphate among phospholipid subclasses in control and cholesterol.enriched fibroblasts
TABLE VI Efflw¢ of [ 32p]phospholipid from control and cholesterol-enriched cells Recovevy of 32p (%) ,,
Cell type Control FC-enriched
[32P]label (%) ~ lyso-PC SM 6.8+_2.2 6.7_+_1.3 8.5+_2.0 5.6-+0.9
PC 53 + 2.6 64+_3.9b
PE 27+_3.6 15+_1.1 b
" values are means+_S.D., n = 16. b p < 0.001 compared to control cells.
TABLE IV Specific acticities of phospholipid subclasses in control and cholesterolenriched fibroblasts Cell type
cpm/nmol phospholipid a lyso-PC
SM
PC
PE
Control 3236_+728 2082_+ 320 4153+131 2578:~409 FC-enriched 3880+871 2943±1232 5349_+298 h 2754+_77 " values are means_+S.D., n = 6. b p < 0.001 compared to control.
c r e a s e in the fraction o f P E w a s o b s e r v e d ( T a b l e !11). This c h a n g e in distribution c o r r e s p o n d s to a g r e a t e r specific activity o f P C in c h o l e s t e r o l - e n r i c h e d cells, c o m p a r e d to t h a t in c o n t r o l cells ( T a b l e IV). T h e specific activity o f P E w a s the s a m e in b o t h c o n t r o l a n d c h o l e s t e r o l - e n r i c h e d cells, T h e p r e d o m i n a n t r a d i o l a b e l e d p h o s p h o l i p i d detected in the m e d i u m w a s PC, a c c o u n t i n g for 5 2 % o f the total p h o s p h o l i p i d r e l e a s e d f r o m c o n t r o l cells (Table V). C h o l e s t e r o l e n r i c h m e n t o f cells h a d the largest effect on PC release to H D L 3. T h e efflux o f P C w a s s t i m u l a t e d 2.7-fold w h e n cells w e r e e n r i c h e d with cholesterol, c o m p a r e d to the a m o u n t of P C r e l e a s e d f r o m control cells. Efflux o f phospholipid f r o m different cell types P h o s p h o l i p i d effiux studies w e r e c o n d u c t e d u s i n g a n u m b e r o f different cell types ( T a b l e VII. In t h e s e experiments, we c o m p a r e d the ability o f H D L 3 to
Efflux of s.,p (%/24 h)
BSA ~
HDL3 d
BSA ~
Fibroblasts h control FC-enriched control FC-enriched
77.8+_ 7.8 72.0+- 6.5 74.9_+ 8.8 70.11_+5.3
67.5_+ 5.3 70.6_+ 9.8 67.3+ 7.1 65.6_+ 6.1
3.5+-0.4 5.6+-2.0 2.9_+0.6 9.5_+1.8 4.8:t:0.7 8.7_+2.0 5.5_+0.4 13.0+-2.0
HDL3 d
FuSAH cells b control FC-enriched control FC-enriched
56.8+_ 2.9 57.5_+ 4.9 61.8_+ 4.4 74.3::1:4.2
63.4:t: 2.0 64.0:t: 3.4 67.5_+ n.9 81.7+_ 5.7
2.2+0.1 4.75:0.3 3.3-+0.1 8.3:!:6.5 3.05:0.2 8.65:0.8 6.0_+0.3 15.0_+1.1
J774 cells h control FC-enriched control FC-enriched
64.7+_ 5.8 74.3_+!0.4 79.1:[: 9.0 83.5± 9.3
66.5+_ 4.1 72.05:6.7 79.1± 9.4 85.4:t:14.2
2.7+_0.3 4.0+_0.6 2.1 _+0.2 3.9_+0.3 2.3±0.2 3.9±0.3 1.9±0.1 4.5+0.7
a percentage of initial 32p recovered in cells and medium after 24 h. b tWO experiments were performed each comparing control and cholesterol-enriched cells, all values are means + S.D., n = 3. c concentration of BSA was 2 mg/ml. o medium contained 2 mg/ml BSA plus 500 /zg protein/ml of HDL 3. s t i m u l a t e p h o s p h o l i p i d efflux a b o v e t h a t o b t a i n e d with B S A , a n d we d e t e r m i n e d t h e e f f e c t o f c h o l e s t e r o l e n r i c h m e n t ' o n p h o s p h o l i p i d efflux to b o t h B S A a n d H D L 3. In all cells h a v i n g n o r m a l levels o f c h o l e s t e r o l , H D L 3 s t i m u l a t e d t h e efflux o f l a b e l e d p h o s p h o l i p i d a b o v e t h a t o b s e r v e d with BSA. T h e b a s a l release o f p h o s p h o l i p i d in the p r e s e n c e o f B S A a l o n e w a s u n a f f e c t e d w h e n t h e cells w e r e e n r i c h e d with c h o l e s t e r o l . W h e n H D L 3, at a c o n c e n t r a t i o n o f 5 0 0 / ~ g p r o t e i n / m l , w a s a d d e d to t h e m e d i u m , c h o l e s t e r o l e n r i c h m e n t s t i m u l a t e d p h o s p h o l i p i d efflux f r o m fibroblasts a n d F u 5 A H r a t h e p a t o m a cells, c o m p a r e d to t h e H D L 3s t i m u l a t e d r e l e a s e o f p h o s p h o l i p i d f r o m c o n t r o l cells. This, however, w a s not the ease with t h e J 7 7 4 m o u s e macrophages, wherein cholesterol enrichment had no effect o n the efflux o f p h o s p h o l i p i d . Discussion
TABLE V Efflvx of phospholipid subclassesfronl control and chok'sterol.enriched fibroblasts Cell type
Efflux of phospholipid (cpm dish- t
Control FC-enriched
lyso-PC SM PC 883 ± t~l 865:1:68 2729+332 1596+_36 1239_+168 7371_+ 96
2 4 h - t)
PE 742£ 72 582_+30
values are means±S.D., n = 3; cell protein was 88.6±5.2/.~g/dish in con'rol ceils, and 90.9+_0.2 ~g/dish in cholesterol-enriched cells.
In ~his s t u d y we h a v e o b s e r v e d t h a t c h o l e s t e r o l e n r i c h m e n t o f n o r m a l h u m a n skin fibroblasts a n d F u 5 A H r a t h e p a t o m a cells e n h a n c e s the efflux o f r a d i o l a b e l e d p h o s p h o l i p i d to e x t r a c e l l u l a r l i p o p r o t e i n s . O n e e x p l a n a t i o n for this o b s e r v a t i o n is t h a t excess cell c h o l e s t e r o l m a y c h a n g e the s t r u c t u r e of the p l a s m a m e m b r a n e , t h e r e b y allowing p h o s p h o l i p i d s to m o r e easily d e s o r b f r o m the m e m b r a n e into the a q u e o u s p h a s e . F u l l i n g t o n et al. [23] have s h o w n t h a t the i n c o r p o r a t i o n o f c h o l e s t e r o l into mixed bile acid : p h o s p h o lipid vesicles i n c r e a s e d the s p o n t a n e o u s t r a n s f e r o f
phospholipid between donor and acceptor vesicles. This transfer is thought to occur by an 'aqueous diffusion' mechanism which is rate-limited by the energy barrier for the desorption of phospholipid from the vesicle surface. Bittman et al. [24] have shown that lipid phase transitions of plasma membranes isolated from Mycoplasma alter the transfer of phospholipid to acceptor vesicles. Whether excess cholesterol reorganizes the membrane enabling increased phospholipid efflux by such mechanisms has yet to be determined. Phospholipid efflux experiments performed with isolated plasma membrane vesicles obtained from control and cholesterol-enriched cells will be valuable in assessing this possibility. The phospholipid subclasses released from cells are those phospholipids which are thought to be present on the outer leaflet of the plasma membrane [25,26], Choline-containing phospholipids are also the most abundant lipids found on circulating HDL 3 [27], and these lipids have been shown to exchange between cells and iipoproteins [4-6]. These observations are consistent with the hypothesis that cholesterol enrichment of membranes enhances the transfer of plasma membrane phospholipid. The lack of efflux of acidic phospholipids (data not shown), which are thought to be localized on the inner leaflet of the plasma membrane [25,26], and the lack of effect of cholesterol enrichment on the efflux of PE indicates that the efflux observed is probably not the result of membrane vesiculation or fragmentation. Cross-linking the apolipoproteins of HDL 3 with dimethylsuberimidate to inhibit the specific binding of H D L 3 to cells did not block the efflux of phospholipid from cells. Therefore, it seems unlikely that interaction of H D L 3 with high affinity binding sites is required for phospholipid efflux. Moreover, LDL and human serum also promoted the efflux of phospholipid from normal fibroblasts. LDL was also able to support the enhanced release of phospholipids observed when ceils were enriched with cholesterol. Thus, a specific lipopfotein does not seem to be required for promoting the enhanced efflux of phospholipid from cholesterol-enriched cells. However, these results do not rule out the possibility that lipoproteins interact with cells nonspecifically in mediating phospholipid release from cells. Such a mechanism might involve the formation of a transient 'collision complex' between cells and lipoproteins. This type of mechanism would also result in the exchange of the phospholipid subclasses which were detected in the medium. Phospholipid compartmentalization may also contribute to the pattern of phospholipid efflux observed. Such compartmentalization has been described by Vance et al. [28,29] who showed that phospholipid used for VLDL synthesis in hepatocytes is derived from a synthetic pathway that is different from that of
bulk phospholipid in these cells. Additionally, high levels of cellular cholesterol have been associated with increased incorporation of biosynthetic precursors into phospholipid [30,31] and increased activity of CTP:phosphocholine cytidyltransferase, the enzyme catalyzing the rate limiting step in PC biosynthesis [32]. Our observation that the specific activity of phosphatidylcholine is higher in cholesterol-enriched cells is consistent with these observations. If newly synthesized phosphatidylcholine is compartmentalized in the plasma membrane, the difference in phospholipid efflux between control and cholesterol-enriched cells may be the result of the selective efflux of this pool whose specific activity may be influenced by cholesterol. The fact that BSA does not support the enhanced efflux of phospholipid from cholesterol-enriched cells indicates that lipoproteins are required for the deso~tion of phospholipid from this specific pool. To address the possibility that lipoproteins stimulate the efflux of phospholipid from a specific phospholipid pool, it will be necessary to measure the specific activity of whole cell and plasma membrane phospholipid, as well as the specific activity of phospholipid released from cells. The latter measurement will require the presence of a lipid-free acceptor able to promote the efflux of phospholipid from cells. Such experiments will also be important in determining whether cholesterol-enrichment enhances the net mass efflux of phospholipid. The reason for the lack of effect of cholesterol enrichment on phospholipid efflux in the .1774 mouse macrophages is unclear. These cells may be able to sequester relatively more cholesterol into intracellular membranes compared to fibroblasts, which are thought to have most of their unesterified cholesterol in the plasma membrane [33]. It may be that a certain critical amount of excess cholesterol must accumulate in the plasma membrane before membrane structure is altered to the point where phospholipid efflux is increased. The 2- to 3-fold enrichment of cholesterol in J774 macrophages may not be enough to reorganize the plasma membrane. The efflux of phospholipid from cells may contribute to reverse cholesterol transport. Phospholipid released from cells could enlarge interstitial lipoproteins making them better cholesterol acceptors. Consistent with this idea are the observations that lymph lipoproteins tend to be rich in unesterified cholesterol and phospholipid [8-10]. Moreover, small amounts of cholesterol and phospholipid are released from cells to lipid-free apolipoproteins [34]. Fielding et al. [7,11,12] have characterized a pre-/3 migrating H D L particle that appears to be the initial acceptor of cellular cholesterol in a transport chain leading to the equilibration of cholesterol among other lipoproteins. Although the origin of this phospholipid-rich particle is also unknown, it is reasonable to speculate that phospholipid efflux from
14 cells m a y c o n t r i b u t e to t h e b i o g e n e s i s o f l i p o p r o t e i n particles. Acknowledgments W e t h a n k D a v i d W. S t e p p a n d D r . F l o r e n c e H. M a h l b e r g for t h e i r e n c o u r a g e m e n t a n d v e r y h e l p f u l discussions. T h i s r e s e a r c h w a s s u p p o r t e d by P r o g r a m Project Grant HL-22633 and Training Grant HL-07443 from the National Heart, Lung and Blood Institute of the National Institutes of Health. References l Bamberger. M., Lund-Katz, S.. Phillips. M.C. and Rothblat. G.H. (1985) Biochemistry 24, 3693-3701. 2 Johnson, W.J., Bamberger. M.J., Lana, R.A., Rapp, P.E., Phillips, M.C. and Rothblat, G.H. (1986) J. Biol. Chem. 261, 5766-5776. 3 Basu. S.K., Goldslein, J.L. and Brown, M,S. (1983) Science 219, 871-873. 4 Blumenfeld, O.O., Schwartz, E. and Adamany, A.M. (1979) J. Biol. Chem. 254, 7183-7190. 5 lllingworth, D.R., Portman, O.W., Robertson, Jr., A.L and Magyar, W.A. (1973) Biochim. Biophys. Acta 306, 422-436. 6 Peterson, J.A. and Rubin, H. (1969) Exp. Cell. Res. 58, 365-378. 7 Castro, G.R. and Fielding, C.J. (1988) Biochemistry 27, 25-29. 8 Reichl, D. (1990) Eur. Heart J. 11,230-236, 9 Roheim. P,8., Dory, L., Lefevre, M. and Sloop, C.H. (1990) Eur. Heart J. 11,225-229. 10 Sloop, C.H., Dory, L. and Roheim. P.S. (1987) J. Lipid Res. 28, 225-237. ll Fielding, C,J. and Fielding, P.E. (1981) Proc. Natl. Acad. Sei. USA 78. 3911-3914. 12 Francone, O.L. and Fielding, C.J. (1990) Eur. Heart J. 11,218224. 13 Hatch, F.T. and Lees, R.S. (1968) Adv. Lipid Res. 6. 1-68.
14 BambergcL M.J., Glick. J.M. and Rothhlat, G.H. (1983)J. Lipid Res. 24, 869-876. 15 Chacko. G.K., Mahlberg. F.H. and Johnson, W.J. (1988) J. Lipid Res. 29, 319-324. 16 Arbogast, L.Y., Rolhblal. G.H., Leslie, M.H. and Cooper. R.A. (1976) Proc. Natl. Acad. 8ci. USA 73, 3680-3684. 17 Bligh, E.G. and Dyer, W.J. (1959) Can. J. Biochem. 37, 911-917. 18 Bernard. D.W., Rodriggez. A., Rothblat. G.H. and Glick. J.M. (1990) Arteriosclerosis 10, 135-144. 19 Johnson, W.J., Mahlberg, F.H.. Chacko, G.K., Phillips. M.C. and Rothblat, G.H. (1988) J. Biol. Chem. 263, 14099-14106. 20 Johnson, W,J., Chacko, G.K, Phillips, M.C. and Rothblat, G.H. (1990) J. Biol. Chem. 265, 5546-5553. 21 Sokoloff. L. and Rothblat, G.H. (1974) Proc. Soe. Exp. Biol. Med. 146. 1166-1172. 22 Markwell, M.A.K., Haas, S.M., Bicber, L.L. and Tolbert, N.E. (1978) Anal. Biochem. 87, 206-210. 23 Fullington, D.A., Shoemaker, D.G. and Nichols. J.W. (19901 Biochemistry 29, 879-886. 24 Bittman, R., Clejan, S. and Hui, S.W. (1990) J. Biol. Chem. 265, lSll0-15117. 25 Op den Kamp. J.A.F. (1979) Annu. Rev. Biochem. 48, 47-71. 26 Van Meer, G. (1989) Annu. Rev. Cell Biol. 5, 247-275. 27 5kipski, V.P. (1972) Blood lipids and lipoproteins: quantitation, composition and metabolism, pp. 471-583. Wiley-lnterscience, New York. 28 Vance, J.E. and Vance, D.E. (1986) J. Biol. Chem. 261, 44864491. 29 Vance, J.E. and Vance. D.E. (1986) FEBS Lett. 204, 243-246. 30 Slotte, J.P. and Lundberg, B. (1983) Med. Biol. 61,223-227. 31 Day, A.J., Fidge, N.H. and Wilkinson, G.N. (1966) J. Lipid Res. 7,132-140. 32 Lira, P.H.. Pritchard, P.H.. Paddon, H.B. and Vance, D.E. (1983) Biochim. Biophys. Acla 753, 74-82. 33 Lange, N., Swaisgood, M.H., Ramps, B.V. and Steck. T.L. (1989) J. Biol. Chem. 264. 3786-3793. 34 Stein, O. and Stein, Y. (1973) ['.ochim. Biophys. Acta 326, 232-244.
7
© 1991 ElsevierScience Publishers n.v. {Hx)5-276(}/91/$o3.5o ADONIS 000527609100220L
BBAL1P 53711
Effiux of phospholipid from fibroblasts with normal and elevated ! ....
in
,,~v,.,~ o f c h o l e s t e r o l
J o h n K. Bielicki, W i l l i a m J. J o h n s o n , J a n e M. Glick a n d G e o r g e H. R o t h b l a t Department of Physiology and Biochemist~'. The Medical College of Pcnnsyh'ania. Philadelphia. PA (U.S.A.)
(Received 17 April 1991)
Key words: Fibroblast:Cholesterol; Phospholipid;Efflux;HDL To addi-,~ss ;he h.~0othesis that phospholipid ettlux from cells contributes to lipoprotein structure, we have examined the elllux of biosynthetically labeled [~ZP]pbospbolipid from cells to lipoproteins. With normal human skin fihroblasts in monolayer culture, high density lipopratein (HDL s) promoted the effiux of phospholipid in a concentration-dependent manner. As analyzed by TLC, :he major phospholipids released from fibroblasts were phosphatidylcholine, sphingomyelin, lysophosphatldyleholine and phosphatidylethanolamine. At identical concentratlons, HDL a and dimethylsuberimidate treated-HDL 3 promoted similar etllux, suggesting that efflux did not depend on specific binding of HDL 3 to the cell surface. When the content of cholesterol in fibroblasts was doubled by pre-ineubation with LDL and cholesterol-rich liposomes, the fractional efflux of phospbolipid to HDL 3 and other aeceptors was stimulated about 2-fold. Most of this stimulation was due to enhanced release of phosphatidylcboline. Similar effects of enrichment were found with FuSAH rat hepatoma cells, but not with J774 mouse macruphages. The results support the hypothesis that the elnux of phospholipid from cells contributes to the phospholipid content of HDIL. This may enhance the ability of ItDL to remove cholesterol from cells, the initial step in reverse cholesterol transport.
Introduction
The movement of cholesterol from extrahepatic cells to high density lipoprotein (HE)L) is thought to be the first step in reverse cholesterol transport. The ability of HDL to promote cholesterol efflux from cells is related to the phospholipid content of the particle [!,2]. A few studies have documented that the exposffrc of cells in culture to s e r : m lipoproteins results in the efflux of cell phospholi)id. With macrophages, phospholipid efflux appears to be linked to the secretion of apolipoprotein E [3]. However, in other cell types such
Abbreviations: PL. phospholipld;PC, phosphatidylcholine;SM, sphingomyelin; lyso-PC, lysophosphatidylcholine; PE. phosphatidylethanolamine; FC, free cholesterol; DMS, dimethylsuherimidate; LCAT, lecithin:cholesterol acyltransferase; HDL, high density lipoprotein; LDL, low density lipoprotein; BSA, bovineserum albumin; PBS, phosphate-bufferedsaline; LSC. liquid scintillation counting, TLC. thin-layer chromatography; MEM. minimum essential medium; FBS, fetal bovineserum. Correspondence: J.K. Bielicki0 Department of Physiologyand Biochemistry, The Medical College of Pennsylvania, 3300 Henry Avenue, Philadelphia, PA 19129. U.S.A.
as smooth muscle cells [4], epithelial cells [5] and fibroblasts [6], phospholipid efflux is stimulated by the presence of exogenous lipoproteins. In the case of smooth muscle cells, low density lipoprotein (LDL) and HDL have been shown to promote phospholipid release [4]. Yet, the contribution of phospholipid efflux to lipoprotein structure and to the process of cholesterol efflux from cells is unknown. Phospholipid-rich HDL has been isolated from plasma [7] and from the lymph of humans and dogs [8-10]. The pre-fl HDL-like particles described by Fielding [7,11,12] are thought to contribute to the reverse cholesterol transport process by serving as efficient acceptors of cellular cholesterol. The HDL particles isolated from lymph are larger and contain more unesterified cholesterol and phospholipid compared to HDL from the vascular compartment. The high phospholipid content of interstitial lipoproteins may be due to interactions of these particles with cells and transfer of phospholipid from cells to lipoproteins. In this study, we monitored the release of biosynthetically labeled cellular phosphoBpid to lipoproteins. The efflux of phospholipid to HDL 3 was examined using fibroblasts, rat hepatoma cells and macrophages.
Cells were enriched with unesterified cholesterol to determine whether cellular cholesterol content alters phospholipid efflux. We found that an elevation in cellular cholesterol content enhances the movement of phospholipid from cells to lipoproteins. This process may contribute to reverse cholesterol transport by enlarging interstitial lipoproteins and thereby enhancing cholesterol efflux from cells. Materials and Methods
Materials. Lysophosphatidylcholine (lyso-PC), sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylethanolamine (PE), unesterified cholesterol, heat inactivated fetal bovine serum (FBS) and bovine serum albumin (BSA, essentially fatty acid free) were purchased from Sigma (St. Louis, Me). Organic solvents were reagent grade and obtained from Fisher Scientific (Pittsburgh, PA). Anasil (3, 250 tzm TLC plates were from Ana!abs (North Haven, CT). Carrier free [32p]orthophosphoric acid was purchased from New England Nuclear (Boston, MA). Dimethylsuberimidate was obtained from Pierce Chemical (Rockford, IL). HeparinSepharose was from Pharmacia LKB Biotechnology (Piscataway, N J). Tissue culture flasks and plates were obtaincd from Falcon (Lincoln, N J) and Coming Glass Works (Corning, N'Y), respectively. Gentamicin was from Tri Bio Labs (State College, PA) and all other antibiotics, trypsin, culture media and calf serum were purchased from Gibco (Grand Island, NY). Sandoz compound 58035 was a gift from Dr. John Heider. Cell culture. GM3468A normal human skin fibreblasts were grown as monolayers in minimum essential medium (MEM) supplemented with 10% FBS (v/v). J774 mouse macrophage cells were maintained in RPMI containing 10% FBS (v/v) and Fu5AH rat heparoma cells were grown in MEM plus 5% calf serum (v/v). Stock cultures of cells were grown in T-75 culture flasks and all media contained 50 ~g of gentamicin/ml. Cells grown in bicarbonate-buffered media were maintained at 37 o C in a humidified 95% air/5% CO 2 atmosphere, whereas media buffered with Hepes were incubated in humidified air. Lipoproteins and lipid dispersions. Lipoproteins were obtained from fresh human plasma by sequential ultracentrifugation [13]. Lecithin:cholesterol acyltransferase (LCAT) activity was inhibited with N-ethylmaleimide [2]. The HDL 3 fraetien (d= 1.125-1.21) was chromatographed on heparin-Sepharose to remove particles containing apoprotein E [14]. Derivitization of HDL 3 with dimethylsuberimidate (DMS) was performed according to Chacko et al. [15]. Before use, HDL 3 and DMS-HDL 3 were dialyzed against 0.15 1,4 NaCI, 2 mM sodium phosphate (pH 7.4), supplemented with 50 units of penicillin G / m l and 50 g g of strepto-
mycin sulfate/ml. Final dialysis of this material was against 30-50 volumes of MEM buffered with 14 mM Hepes. Phospholipase activity of LCAT was not detected in HDL 3 preparations, as judged by the distribution of lyso-PC and PC in 24 h efflux medium that had been incubated overnight in the absence of cells (data not shown). LDL ( d = 1.019-1.063) was dialyzed exhaustively against 0.15 M NaCI with a final dialysis against MEM buffered with Hepes. All lipoproteins were sterilized by filtration (0.45 /zm) and stored at 4°C. Lipoprotein-deficient serum (LPDS) was obtained from density > 1.21 g/ml. Human serum was obtained with informed consent from healthy, malt donors, and stored at 4 ° C overnight prior to use. Dispersions containing free cholesterol and egg phosphatidylcholine (2-3:1, tool : tool) were prepared by sonication, as described by Arbogast et al. [16]. Phospholipid liposomes were prepared with egg phosphatidylcholine by the same procedure except th,-.t cholesterol was omitted. Experimental conditions. Ceils from stock cultures were plated on 22, 35 or 100 mm culture dishes and maintained in growth media until confluent. To label cellular phospholipid, monolayers were incubated with growth medium supplemented with 5 ~ C i / m i of [3ZP]phosphate for 3 days prior to the initiation of efflux studies. The 22, 35 and 100 mm culture dishes contained 0.5, 1.0 and 8.0 ml of labeling media, respectively. Preliminary experiments demonstrated that the distribution of ['~2p]phosphate among phospholipid subclasses did not change after 2 days of labeling. Lipoproteins were diluted to final concentrations with MEM, and BSA (2 mg/ml) was added as indicated in the text. Unless otherwise indicated, incubation time for measurement of phospholipid efflux was 24 h. After incubation with cells, efflux media were removed from the cells and spun at 3000 × g for 10 rain to pellet any cells which had become dislodged from the plates. The lipids in the supernatant were extracted by the method of Bligh and Dyer [17]. Lipids from media were redissolved in chloroform/methanol (2:1) and aliquots taken for liquid scintillation counting (LSC) and separation of phospholipid subclasses by thin-layer chromatography (TLC). The TLC plates were developed by a two-step, one-dimensional procedure as follows: the first solvent system consisted of chloroform/ methanol/H 20 (65 : 25 : 4, v/v), and, after the plate was fully developed, it was allowed to dry at room temperature for approx. 30 min before a second development in a system of c h l o r o f o r m / a c e t o n e / methanol/acetic a c i d / H 2 0 (50:20:10:10:5). Approximate R F values for various lipids were as follows: lyso-PC = 0.17, SM = 0.27, PC = 0.36 and PE = 0.59. The cell monolayers were rinsed with phosphatebuffered saline (PBS) and the lipids were extracted with isopropanol [18]. The isopropanol was transferred
9 from the culture dish to a separate tube, the monolayers were rinsed once with additional isopropanol, and the combined extracts were dried under nitrogen. The dried lipid residue was partitioned by the method of Bligh and Dyer [17]. The chloroform phase from this partition was dried, and then redisso!ved in chlorof o r m / methanol (2:1). Aliquots wece removed for LSC and TLC as described for the media samples. The data are expressed as either release of total lipid radioactivity (cpm) or as the percentage of [32p]phospholipid released from cells: % efflux = (cpm in medium after 24 h / c p m in cellular lipids at t = 0). 100. The distribution of radioactivity among the phospholipid subclasses, as separated by TLC, was measured using a Radiomatic Imaging TLC Scanner. This fractional distribution was multiplied by the total radioactivity obtained for cells and media to calculate the radioactivity associated with each phospholipid subclass and the subsequent release of this radioactivity from cells. Enrichment of cells with unesterified cholesterol. The experimental protocol was modified to enrich cells with free cholesterol (FC). The growth and plating of the cells were conducted as described above. When cells on 22 mm culture dishes reached confluency, the medium was replaced with MEM supplemented with 10 rag protein/ml of LPDS and 5 p,Ci/ml of [32p]phosphate. After a 48 h incubation, the cell monolayers were incubated an additional 24 h with one of the following media: (1) a FC-loading medium [19] containing 1 p.g/ml of Sandoz compound 58035 to inhibit cholesterol esterification, 100 p,g F C / m l of FC: PC dispersions ( > 2:1, tool:tool) and 50 p.g protein/ml of LDL, or (2) a control medium [19] containing 1 t~g/ml of 58035 and PC liposomes added at the same phospholipid concentration as in the FC enrichment medium. Both media preparations also contained 5 p,C i / m l [3ZP]phosphate and 2 m g / m l BSA. Sandoz compound 58035 was added to the cultures in DMSO, and the final c~ncee;ration of solvent was 0.5% (v/v). This procedure resulted in at least a 2-fold increase in cellular unesterified cholesterol content in each of the cells studied. Cholesteryl ester was not detected in cells. Efflux of labeled phospholipids was examined immediately after enrichment.
Specific activity determinations of labeled phospholipid subclasses. Normal skin fibroblasts plated on 100 mm culture dishes were grown to confluency, labeled with 5 p,Ci/ml of [3ZP]phosphate for 3 days, and enriched with cholesterol for 24 h as described above. The specific activity of the labeling medium was 13 250 cpm/nmol phosphate. Cell monolayers were rinsed with PBS, lipids extracted and then separated by TLC. The areas of the plate corresponding to the major phospholipid subclasses were scraped into test tubes, and the phospholipid was ext,acted from the silica gel
as described by Bamberger et al. [1]. Aliquots were taken for 32p quantitation and lipid phosphorus determination. Chemical analyses. Cholesterol was quantitated by gas liquid chromatography using cholesteryl methyl ether as an internal standard [20]. Phospholipid phosphorus was determined by the method of Sokoloff and Rothblat [21]. The protein content of lipid extracted monolayers was measured by the procedure of Markwell [22] using BSA as a standard. Radioactivity was measured with either a Beckman LS1801 or LSS000TD liquid scintillation counter using Scintiverse BD counting fluid. Statistical analysis. Triplicate measurements were taken for efflux and for cellular lipid and protein determinations. When percentages are calculated, the S.D. were derived from cellular and medium determinations of radioactivity. Student's t-test was used and P < 0.05 was the criterion for statistical significance. Results
E.fflwc of phospholipid from normal skin fibroblasts Initial studies were conducted to determine the dependence of phospholipid efflux on HDL 3 concentration. Maximal effiux of radiolabeled phospholipid was obtained at concentrations > I00 p.g HDL 3 protein/ml (Fig. 1). To address the specificity of the acceptor for promoting phospholipid release, a number of different acceptors were tested (Fig. 2). BSA alone in the medium stimulated phospholipid efflux to only a small extent (1.8 + 0.2%/24 h) compared to the basal release of phosphoiipid to protein-free medium (1.1 _+
co
°!
.... C o n ¢ ~ t r ( l t l o ¢ ~ 01 HDL 3 . ~
Droteln /ml
Fig. 1. Dependence of pho~pholipid release from fibroblasts on HDL~ concentration. Normal skin fibroblasts were grown to confluency on 35 mm dishes and then labeled for 3 days with 5 pCi/ml of [~-Plphosphate in media containing 10% FBS. Cells were incubated
an additional 24 h with MEM supplementedwith 2 mg of BSA/ml and increasing amountsof HDL3. The data are expressed as the percentage of the total [3ZP]phospholipid released from cells, normalizing to the total radioactivity initially present in cellular lipids.
The open circle on the vertical axis represents the basal release of phospholipid in the presenceof 2 mg BSA/mL Values are means± S.D., n = 3. Where not shown,the error bars are withinthe symbols.
10
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and the ACAT inhibitor Sandoz 58035, as described under Materials and Methods. This enrichment protocol has been used extensively in elucidating the mechanism of cholesterol movement between cells and HDL 3 [19]. Increasing the cellular cholesterol content 2- to 3-fold did not result in a significant increase in the phospholipid content; thus, the free cholesterol/ phospholipid (mol/mol) ratio more than doubled, from 0.23 + 0.08 to 0.49 + 0.12 (Table 1). Although the phospholipid content was not affected by the cholesterol treatment, the incorporation of [32p]phosphate iato
Fig, 2. Release of p h o s p h n l i p i d s from fibroblasts to various acccp-
tots. Normal skin fibroblasts were grown and labeled with [32p]phosphate as described in Fig. 1. The efflux medium consisted of M E M or ME M supplemented with either 2 mg B S A / m l , 500/~g p r o t e i n / m l of H D L 3, 500 p g p r o t e i n / m l of D M S - H D L 3, or 15% human serum (v/v). The data are expressed as the percentage of [ 32P]phospholipid released from cells in 24 h, normalizing to the total radioactivity initially present in cellular lipids. Values are means + S.D n = 3 .
0.1%/24 h). The role of specific HDL3 binding was tested by using HDL 3 cross-linked with dimetbylsuberimidate, a treatment which greatly reduces the ability of HDL 3 to associate with high affinity binding sites on cells [15]. This treatment had no effect on the ability of I-IDL 3 to promote phospholipid efflux (Fig. 2). indicating that specific binding to membranes is not required to obtain phospholipid release from fibroblasts. The results obtained with 15% human serum were similar to the results obtained with HDL3, added at a concentration of 500 p.g protein/ml. The kinetics of efflux of phospholipid from fibroblasts with normal levels of cellular cholesterol are presented in Fig. 3A. These data demonstrate that the total recovery of [32p]phospholipid from the system (cellular plus medium lipid radioactivity) decreases during the course of the experiment. Approx. 35% of the initial radiolabel is lost from the cellular phospholipid pool in 24 h. This decline in total recovery of [32p]phospholipid is probably due to degradation of phospholipid in cells. Approx. 6% of the cellular phospholipid is released from cells during a 24 h incubation with 500/.¢g protein/ml of HDL3. The 6% value was calculated by dividing the amount of radioactivity released from cells by the amount of radiolabeled phospholipid initially in cells. This value underestimates efflux because the specific activities of cellular phospholipid, and that of the phospholipid released from cells, are probably decreasing during the experiment.
Efflux of phospholipid from cholesterol-enriched fibroblasts To study the effect of cholesterol enrichment on phospholipid efflux, cells were treated with a combination of cholesterol-rich phospholipid liposomes, LDL
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Tir~ (h) Fig. 3. T i m e course of H D L y s t i m u l a t e d phospholipid release f r o m control and cholesterol-enriched fibroblasts. C e l l s w e r e p l a t e d o n 22 m m c u l t u r e dishes and grown to confluent monolayers. The l a b e l i n g
of cells with 32P and enrichment" of cells with unesterified cholesterol were performed as described in Materials and Methods. To initiate efflux, monolayers were rinsed extensively with MEM-Hepes and the cells were then incubated for various lengths of time with MEM containing 2 mg/ml of BSA plus 500/,tg protein/ml of HDL 3, Panel
A contains kinetic data from control cells (open symbols).Panel B contains kinetic data fromcholesterol-enrichedcells(closedsymbols). Symbol descriptions are as follows: 0 B, total recovery of [32p]phospholipid from cells and medium at each time point; ~, - ~., recoveryof 32p from cells; 0 o, recoveryof 32p from medium.Panel C showsthe resultse~pressedas the percentage of phospholipid released from cells to H D L 3, normalizing to the total radioactivity initially present in cellular lipids. Values are means+ S.D., n = 3.
TABLE
I
Cholesterol and l~ho.vpholipid composition of control and cholesterol-enriched )"P lah,'h'd human skin fibroblasts
Cell type
Control FC-enriehed
Unesterified cholesterol (.ttg/mg cell protein) :' 19-+ 4 44:1:10 c
Phospholipid (#.g/mg cell protein)
FC: PL (rnol:mol) ~
168+_27 182_+18
0.23 +_0.08 1i.49_+0.12 c
[~-"P]PL specific activity (cpm/nmol PL) h 3127 +_356 3748_+382 ¢
a values are means-+S.D., n = 6. l, values are means±S.D., n = 9. P < 0.01. compared to control cells.
total cellular phospholipid increased by approx. 20% (Table I). W h e n the recovery of [aEp]phospholipid from cholesterol-enriched fibroblasts was analyzed, the degradation of [aZp]phospholipid was identical to that obtained with the control cells (Fig. 3A vs. B). The bottom p a n e l of Fig. 3 (Fig. 3C) c o m p a r e s the efflux of pho~pholipid from control and cholesterol-enriched fibroblasts. Increasing cell cholesterol content enhances phospholipid release from fibroblasts to H D L 3. The it/2 for this efflux of 32 p was estimated using a two-pool model for the exchange of lipid between cells and lipoproteins as described for cholesterol [l,19]. The i l l 2 for efflux from control cells r a n g e d from 165 to 270 h, whereas, with cholesterol-enriched cells, the t l / 2 was reduced to 65 and 83 h in two experiments (Table
Distribution o f phospholipid subclasses
Cellular phospholipids were separated by TLC to analyze the distribution of 32 p a m o n g individual phospholipid subclasses. Phosphatid~lcholine (PC) and phosphatidylethanolamine ( P E t accounted for approx. 80% of the cellular phospholipids. W h e n cells were enriched with cholesterol, there was a shift in the distribution of PC and PE. The content of cellular 3-'P in PC increased by about 20%, whereas, a 44% de-
IHo,,
Hi. The concentration d e p e n d e n c e for the efflux of phospholipid to H D L 3 and L D L using control and cholesterol-enriched fibroblasts is p r e s e n t e d in Fig. 4). W i t h control cells, phospholipid ¢fflux was maximal at concentrations of H D L a >_ 100 /Lg p r o t e i n / m l . This was similar to the results o b t a i n e d in Fig. 1, Cholesterol e n r i c h m e n t of fibroblasts e n h a n c e d the release of phospholipid at every concentration of H D L a. This release also reached its maximal value at 1 0 0 / z g prot e i n / m l of H D L a . Qualitatively similar results were o b t a i n e d with L D L (Fig. 4). Thus, the e n h a n c e d release of phospholipid observed with cholesterol enrichment does not require a specific lipoprotein in the medium.
4 =/I
~o o
'iI"°" ~. 12
Concentration of hpoproteln, pg protein / ml
TABLE I! Estimated tt/z for efflux of [)2P/phospholipid cholesterol.enriched fibroblasts to HDL j Experiment
1 2
from control and
11/2 (h) a
control
FC-enriched
270±111 165± 43
t"~± 20 83-+ 17
values are mcans±S.D., n = 3; concentration of HDL = 500 /~g protein/ml.
Fig. 4. Concentration dependence of HDL~- and LDL-stimulated phospholipid release from control and cholesterol-enriched fibroblasts, Normal skin fibroblasts were plated on 22 mm culture dishes and grown to confluency in M E M supplemented with I0% FBS. Control cells and cholesterol-enriched cells w e r e prepared as described in Materials and Methods, Cells were incubated for 24 h with increasing amounts of H D L 3 ( o , e) or L D L (zx, & ) provided in
MEM containing 2 mg of BSA/ml. The data are expressed as the percentage of [~2P]phospholipidreleased from cells, as described in Fig. 3. Data from the control cells are the open symbols and from cholesterol-enriched cells are the closed symbols. Values are means + S.D., n = 3.
12 TABLE Ill Distribution of [t2Plphosphate among phospholipid subclasses in control and cholesterol.enriched fibroblasts
TABLE VI Efflw¢ of [ 32p]phospholipid from control and cholesterol-enriched cells Recovevy of 32p (%) ,,
Cell type Control FC-enriched
[32P]label (%) ~ lyso-PC SM 6.8+_2.2 6.7_+_1.3 8.5+_2.0 5.6-+0.9
PC 53 + 2.6 64+_3.9b
PE 27+_3.6 15+_1.1 b
" values are means+_S.D., n = 16. b p < 0.001 compared to control cells.
TABLE IV Specific acticities of phospholipid subclasses in control and cholesterolenriched fibroblasts Cell type
cpm/nmol phospholipid a lyso-PC
SM
PC
PE
Control 3236_+728 2082_+ 320 4153+131 2578:~409 FC-enriched 3880+871 2943±1232 5349_+298 h 2754+_77 " values are means_+S.D., n = 6. b p < 0.001 compared to control.
c r e a s e in the fraction o f P E w a s o b s e r v e d ( T a b l e !11). This c h a n g e in distribution c o r r e s p o n d s to a g r e a t e r specific activity o f P C in c h o l e s t e r o l - e n r i c h e d cells, c o m p a r e d to t h a t in c o n t r o l cells ( T a b l e IV). T h e specific activity o f P E w a s the s a m e in b o t h c o n t r o l a n d c h o l e s t e r o l - e n r i c h e d cells, T h e p r e d o m i n a n t r a d i o l a b e l e d p h o s p h o l i p i d detected in the m e d i u m w a s PC, a c c o u n t i n g for 5 2 % o f the total p h o s p h o l i p i d r e l e a s e d f r o m c o n t r o l cells (Table V). C h o l e s t e r o l e n r i c h m e n t o f cells h a d the largest effect on PC release to H D L 3. T h e efflux o f P C w a s s t i m u l a t e d 2.7-fold w h e n cells w e r e e n r i c h e d with cholesterol, c o m p a r e d to the a m o u n t of P C r e l e a s e d f r o m control cells. Efflux o f phospholipid f r o m different cell types P h o s p h o l i p i d effiux studies w e r e c o n d u c t e d u s i n g a n u m b e r o f different cell types ( T a b l e VII. In t h e s e experiments, we c o m p a r e d the ability o f H D L 3 to
Efflux of s.,p (%/24 h)
BSA ~
HDL3 d
BSA ~
Fibroblasts h control FC-enriched control FC-enriched
77.8+_ 7.8 72.0+- 6.5 74.9_+ 8.8 70.11_+5.3
67.5_+ 5.3 70.6_+ 9.8 67.3+ 7.1 65.6_+ 6.1
3.5+-0.4 5.6+-2.0 2.9_+0.6 9.5_+1.8 4.8:t:0.7 8.7_+2.0 5.5_+0.4 13.0+-2.0
HDL3 d
FuSAH cells b control FC-enriched control FC-enriched
56.8+_ 2.9 57.5_+ 4.9 61.8_+ 4.4 74.3::1:4.2
63.4:t: 2.0 64.0:t: 3.4 67.5_+ n.9 81.7+_ 5.7
2.2+0.1 4.75:0.3 3.3-+0.1 8.3:!:6.5 3.05:0.2 8.65:0.8 6.0_+0.3 15.0_+1.1
J774 cells h control FC-enriched control FC-enriched
64.7+_ 5.8 74.3_+!0.4 79.1:[: 9.0 83.5± 9.3
66.5+_ 4.1 72.05:6.7 79.1± 9.4 85.4:t:14.2
2.7+_0.3 4.0+_0.6 2.1 _+0.2 3.9_+0.3 2.3±0.2 3.9±0.3 1.9±0.1 4.5+0.7
a percentage of initial 32p recovered in cells and medium after 24 h. b tWO experiments were performed each comparing control and cholesterol-enriched cells, all values are means + S.D., n = 3. c concentration of BSA was 2 mg/ml. o medium contained 2 mg/ml BSA plus 500 /zg protein/ml of HDL 3. s t i m u l a t e p h o s p h o l i p i d efflux a b o v e t h a t o b t a i n e d with B S A , a n d we d e t e r m i n e d t h e e f f e c t o f c h o l e s t e r o l e n r i c h m e n t ' o n p h o s p h o l i p i d efflux to b o t h B S A a n d H D L 3. In all cells h a v i n g n o r m a l levels o f c h o l e s t e r o l , H D L 3 s t i m u l a t e d t h e efflux o f l a b e l e d p h o s p h o l i p i d a b o v e t h a t o b s e r v e d with BSA. T h e b a s a l release o f p h o s p h o l i p i d in the p r e s e n c e o f B S A a l o n e w a s u n a f f e c t e d w h e n t h e cells w e r e e n r i c h e d with c h o l e s t e r o l . W h e n H D L 3, at a c o n c e n t r a t i o n o f 5 0 0 / ~ g p r o t e i n / m l , w a s a d d e d to t h e m e d i u m , c h o l e s t e r o l e n r i c h m e n t s t i m u l a t e d p h o s p h o l i p i d efflux f r o m fibroblasts a n d F u 5 A H r a t h e p a t o m a cells, c o m p a r e d to t h e H D L 3s t i m u l a t e d r e l e a s e o f p h o s p h o l i p i d f r o m c o n t r o l cells. This, however, w a s not the ease with t h e J 7 7 4 m o u s e macrophages, wherein cholesterol enrichment had no effect o n the efflux o f p h o s p h o l i p i d . Discussion
TABLE V Efflvx of phospholipid subclassesfronl control and chok'sterol.enriched fibroblasts Cell type
Efflux of phospholipid (cpm dish- t
Control FC-enriched
lyso-PC SM PC 883 ± t~l 865:1:68 2729+332 1596+_36 1239_+168 7371_+ 96
2 4 h - t)
PE 742£ 72 582_+30
values are means±S.D., n = 3; cell protein was 88.6±5.2/.~g/dish in con'rol ceils, and 90.9+_0.2 ~g/dish in cholesterol-enriched cells.
In ~his s t u d y we h a v e o b s e r v e d t h a t c h o l e s t e r o l e n r i c h m e n t o f n o r m a l h u m a n skin fibroblasts a n d F u 5 A H r a t h e p a t o m a cells e n h a n c e s the efflux o f r a d i o l a b e l e d p h o s p h o l i p i d to e x t r a c e l l u l a r l i p o p r o t e i n s . O n e e x p l a n a t i o n for this o b s e r v a t i o n is t h a t excess cell c h o l e s t e r o l m a y c h a n g e the s t r u c t u r e of the p l a s m a m e m b r a n e , t h e r e b y allowing p h o s p h o l i p i d s to m o r e easily d e s o r b f r o m the m e m b r a n e into the a q u e o u s p h a s e . F u l l i n g t o n et al. [23] have s h o w n t h a t the i n c o r p o r a t i o n o f c h o l e s t e r o l into mixed bile acid : p h o s p h o lipid vesicles i n c r e a s e d the s p o n t a n e o u s t r a n s f e r o f
phospholipid between donor and acceptor vesicles. This transfer is thought to occur by an 'aqueous diffusion' mechanism which is rate-limited by the energy barrier for the desorption of phospholipid from the vesicle surface. Bittman et al. [24] have shown that lipid phase transitions of plasma membranes isolated from Mycoplasma alter the transfer of phospholipid to acceptor vesicles. Whether excess cholesterol reorganizes the membrane enabling increased phospholipid efflux by such mechanisms has yet to be determined. Phospholipid efflux experiments performed with isolated plasma membrane vesicles obtained from control and cholesterol-enriched cells will be valuable in assessing this possibility. The phospholipid subclasses released from cells are those phospholipids which are thought to be present on the outer leaflet of the plasma membrane [25,26], Choline-containing phospholipids are also the most abundant lipids found on circulating HDL 3 [27], and these lipids have been shown to exchange between cells and iipoproteins [4-6]. These observations are consistent with the hypothesis that cholesterol enrichment of membranes enhances the transfer of plasma membrane phospholipid. The lack of efflux of acidic phospholipids (data not shown), which are thought to be localized on the inner leaflet of the plasma membrane [25,26], and the lack of effect of cholesterol enrichment on the efflux of PE indicates that the efflux observed is probably not the result of membrane vesiculation or fragmentation. Cross-linking the apolipoproteins of HDL 3 with dimethylsuberimidate to inhibit the specific binding of H D L 3 to cells did not block the efflux of phospholipid from cells. Therefore, it seems unlikely that interaction of H D L 3 with high affinity binding sites is required for phospholipid efflux. Moreover, LDL and human serum also promoted the efflux of phospholipid from normal fibroblasts. LDL was also able to support the enhanced release of phospholipids observed when ceils were enriched with cholesterol. Thus, a specific lipopfotein does not seem to be required for promoting the enhanced efflux of phospholipid from cholesterol-enriched cells. However, these results do not rule out the possibility that lipoproteins interact with cells nonspecifically in mediating phospholipid release from cells. Such a mechanism might involve the formation of a transient 'collision complex' between cells and lipoproteins. This type of mechanism would also result in the exchange of the phospholipid subclasses which were detected in the medium. Phospholipid compartmentalization may also contribute to the pattern of phospholipid efflux observed. Such compartmentalization has been described by Vance et al. [28,29] who showed that phospholipid used for VLDL synthesis in hepatocytes is derived from a synthetic pathway that is different from that of
bulk phospholipid in these cells. Additionally, high levels of cellular cholesterol have been associated with increased incorporation of biosynthetic precursors into phospholipid [30,31] and increased activity of CTP:phosphocholine cytidyltransferase, the enzyme catalyzing the rate limiting step in PC biosynthesis [32]. Our observation that the specific activity of phosphatidylcholine is higher in cholesterol-enriched cells is consistent with these observations. If newly synthesized phosphatidylcholine is compartmentalized in the plasma membrane, the difference in phospholipid efflux between control and cholesterol-enriched cells may be the result of the selective efflux of this pool whose specific activity may be influenced by cholesterol. The fact that BSA does not support the enhanced efflux of phospholipid from cholesterol-enriched cells indicates that lipoproteins are required for the deso~tion of phospholipid from this specific pool. To address the possibility that lipoproteins stimulate the efflux of phospholipid from a specific phospholipid pool, it will be necessary to measure the specific activity of whole cell and plasma membrane phospholipid, as well as the specific activity of phospholipid released from cells. The latter measurement will require the presence of a lipid-free acceptor able to promote the efflux of phospholipid from cells. Such experiments will also be important in determining whether cholesterol-enrichment enhances the net mass efflux of phospholipid. The reason for the lack of effect of cholesterol enrichment on phospholipid efflux in the .1774 mouse macrophages is unclear. These cells may be able to sequester relatively more cholesterol into intracellular membranes compared to fibroblasts, which are thought to have most of their unesterified cholesterol in the plasma membrane [33]. It may be that a certain critical amount of excess cholesterol must accumulate in the plasma membrane before membrane structure is altered to the point where phospholipid efflux is increased. The 2- to 3-fold enrichment of cholesterol in J774 macrophages may not be enough to reorganize the plasma membrane. The efflux of phospholipid from cells may contribute to reverse cholesterol transport. Phospholipid released from cells could enlarge interstitial lipoproteins making them better cholesterol acceptors. Consistent with this idea are the observations that lymph lipoproteins tend to be rich in unesterified cholesterol and phospholipid [8-10]. Moreover, small amounts of cholesterol and phospholipid are released from cells to lipid-free apolipoproteins [34]. Fielding et al. [7,11,12] have characterized a pre-/3 migrating H D L particle that appears to be the initial acceptor of cellular cholesterol in a transport chain leading to the equilibration of cholesterol among other lipoproteins. Although the origin of this phospholipid-rich particle is also unknown, it is reasonable to speculate that phospholipid efflux from
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