THE LOW-DENSITY LIPOPROTEIN PATHWAY I N HUMAN FIBROBLASTS: RELATION BETWEEN CELL SURFACE RECEPTOR BINDING A N D ENDOCYTOSIS OF LOW-DENSITY LIPOPROTEIN* Michael S. Brown,? Y. K. HoJ and Joseph L. Goldstein8 Division of Medical Genetics Department of Internal Medicine University of Texas Health Science Center at Dallas Dallas, Texas 7.523.5

Studies in our laboratory over the past 2 years have defined a specific metabolic pathway by which cultured human fibroblasts metabolize low-density lipoprotein (LDL), the major cholesterol-carrying protein in human plasma (reviewed in References 1 & 2). The delineation of this metabolic pathway grew out of the initial observation that cholesterol synthesis in human fibroblasts was specifically suppressed by plasma LDL, which acted by reducing the activity of the rate-controlling enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA r e d u c t a ~ e ) .Our ~ . ~ studies were facilitated by the finding that fibroblasts cultured from the skin of patients with the homozygous form of familial hypercholesterolemia (FH) were resistant to the LDL-mediated feedback sup~ . ~ availability of these mutant cells has made pression of HMG CoA r e d u ~ t a s e .The possible a combined biochemical and genetic analysis of the critical steps in cellular LDL action.

PATHWAY FOR LDL METABOLISM By proceeding through a coordinated sequence of experiments, we have been able to deduce that at least six distinct and sequential steps participate in the pathway by which LDL regulates cholesterol metabolism (FIGUREI). The initial event in cellular LDL metabolism involves the recognition of LDL by a molecular component of the cell surface. The binding of LDL to this cell surface molecule exhibits saturability, high affinity, and specificity.6-10We have designated this LDL recognition site as an LDL receptor, because its behavior is analogous to that of certain hormone receptors. Hormone receptors have been defined operationally as “those molecules that specifically recognize and bind the hormone and as a consequence of this recognition can lead to other changes (or a series of changes) which ultimately result in the biological response.”l’ Once LDL binds to the LDL receptor, the lipoprotein becomes internalized and incorporated into endocytotic vesicles (endosomes) that fuse with lysosomes. Within *Supported by Grants HL 16024 and GM 19258 from the National Institutes of Health and by a grant from the American Heart Association. t Established Investigator of the American Heart Association. $Recipient of National Institutes of Health Postdoctoral FellowshipT22-GM 0021. §Recipient of United States Public Health Service Research Career Development Award GM 70.211.

244

Brown et af.: LDL Pathway in Human Fibroblasts

245

the lysosome, the protein component of the LDL is rapidly degraded to free amino acids that are then released into the culture medium.8.12The cholesteryl ester component of LDL is hydrolyzed by a lysosomal acid l i p a ~ e , ' ~and * ' ~the resultant unesterified cholesterol is transferred to the cellular compartment, where it is found largely associated with cell rnembranes.l5 The accumulation of unesterified cholesterol within the cell regulates the activities of two microsomal enzymes: it suppresses HMG CoA reductase, causing a reduction of cholesterol ~ y n t h e s i s , ~and . ~ ~it activates an acyl-CoA:cholesteryl acyltransferase, facilitating its own reesterification.17*18 The endogenously reesterified cholesterol is preferentially attached to the monounsaturated fatty acids, oleate and palmitoleate, in contrast to the cholesteryl esters of plasma LDL, which are rich in the polyunsaturated fatty acid lin~leate.'~

FIGURE1 . Pathway of LDL metabolism in cultured human fibroblasts. Each of the sequential steps in this pathway is discussed in the text. The binding, endocytosis, and lysosomal hydrolysis of LDL can be bypassed by incubation of cells with cholesterol itself or one of its oxygenated derivatives, such as 25-hydroxycholesterol. When added to the cells in a nonlipoprotein form, 25-hydroxycholesterol regulates the two microsomal enzymes, HMG CoA reductase and acyl CoA:cholesteryl acyltransferase (ACAT), without a requirement for the LDL receptor or lysosomal function. The overall effect of the LDL receptor-mediated process is to transfer free and esterified cholesterol from LDL into the cell15and to produce a switch in the fatty acid composition of cholesteryl esters from a polyunsaturated to a more saturated form.4 Mutant cells from patients with the receptor-negative form of homozygous FH have as their primary genetic defect a deficiency in functional LDL receptors.6-'o As a result of their failure to bind LDL at this specific surface receptor site, these FH homozygote cells fail to take up the lipoprotein with high affinity and therefore fail to . ~ a~ consequence, .~~ LDL hydrolyze its protein or cholesteryl ester ~ o m p o n e n t s . ~As neither suppresses HMG CoA reductase a ~ t i v i t ynor ~ . ~activates the acyl CoA:cholesteryl acyltransferase enzyme. l 7 . I 8

246

Annals New York Academy of Sciences BINDINGOFLDL TO ITS CELLSURFACE RECEPTOR

A major problem in the study of all cell surface receptor-mediated processes involves the development of conditions in which receptor binding can be studied at the same time that a physiologic effect is being achieved so as to allow a direct demonstration that the observed binding is a required event for the observed effect. For the LDL pathway, simultaneous study of cell surface binding and a physiologic effect (such as suppression of HMG CoA reductase) is difficult, because the latter requires the endocytosis of the receptor-bound lipoprotein.s Thus, when 1251-labeledLDL is added to monolayers of fibroblasts under conditions in which it suppresses HMG CoA reductase, the amount of cell-bound [ lz51]LDL reflects both lipoprotein bound at the cell surface receptor site and lipoprotein that has been internalized through receptor-mediated endocytosis.'.8.'n Furthermore, some of the [Iz5I]LDL that is bound to the surface under these physiologic conditions is bound to sites other than the physiologically important LDL receptor, and, moreover, some of the (1251]LDL within the cell has been internalized through bulk phase pinocytosis that does not require the LDL receptor.'.*.I" To overcome these problems, we have utilized four different approaches for measurement of the specific cell surface LDL receptor. Three of these methods use intact fibroblast monolayers, and the binding studies are conducted under conditions in which [L251]LDLbinding can be directly correlated with its physiologic effects. That each method measures binding to the specific receptor of physiologic im-

FIGURE2. Immunofluorescent staining of receptor-bound LDL on the surface of normal FH homozygote (right) fibroblasts. Cells were seeded on glass cover slips and grown in petri dishes as described in the legend to FIGURE 3. On Day 7, each dish received 2 ml of medium that contained 10 rg/ml of LDL. After incubation at 4°C for 3 hr, each monolayer was washed by a previously described technique,'O after which 1 ml of phosphate-buffered saline that contained 140 pg of a monospecific rabbit antibody to human LDL was added to each dish. After incubation at 4°C for I hr, the standard wash procedure was repeated, and the cover slips were removed and placed in a solution that contained an antibody prepared against rabbit yglobulin and conjugated with fluorescein isothiocyanate. After incubation for 1 hr at 4°C. the cover slips were washed thoroughly in phosphate-buffered saline and then observed with a fluorescent microscope. The photographs were taken by Dr. James Gilliam. x 700.

(/eyf)and

Brown et al.: LDL Pathway in Human Fibroblasts

241

portance is indicated by the fact that the observed binding in each case is markedly reduced in FH homozygote cells that are resistant to the physiologic effects of LDL. FIGURE 2 shows the results obtained with one of these approaches, indirect immunofluorescence. Monolayers of normal and FH homozygote cells were incubated at 4°C with LDL, washed, exposed to an antibody to LDL, and finally to an antibody to y globulin that was conjugated with fluorescein. The normal cells show a specific pattern of surface fluorescence that is absent in the FH cells, which indicates an inability of LDL to bind to the surfaceof these mutant cells. A more quantitative approach for studying cell surface receptor binding involves incubation of fibroblast monolayers with [1251]LDL.A distinction between [lZ5I]LDL bound to the surface and lipoprotein that has been internalized was made possible through the use of heparin, a sulfated glycosaminoglycan that is known to form soluble ionic complexes with LDL.Ig Formation of such complexes allows heparin to release [lZ5I]LDLfrom its cell surface receptor.'O FIGURE 3 shows the results of an experiment in which [L251]LDL was incubated with cells either a t 4°C (where little to no endocytosis occurs) or at 37°C (where the receptor-bound LDL is internalized through endocytosis). In normal cells a t 4"C, the total cellular uptake of [lzs1]LDL reached completion at about 2 h r (FIGURE 3,A). At all time points, at least 75% of the cell-bound [1251]LDL could be released by subsequent exposure to heparin, which indicates that most of the bound material remained on the cell surface. At 37"C, the total cellular uptake of [1251]LDLwas much greater than at 4"C, but the fraction released by heparin was much smaller (FIGURE 3,C). Despite the difference in total uptake at 4" and 37"C, the absolute amount of heparin-releasable [Iz5I]LDLwas similar after incubation at both temperatures (FIGURE 3, inserf).These data suggest that the amount of cell surface binding of [lZ5II]LDLwas similar at 4" and 37°C but that at the higher temperature the lipoprotein was continually interiorized and replaced a t the cell surface receptor site with*fresh molecules of [1251]LDL.This temperature-dependent uptake rendered the [Iz5I]LDLthat had entered the cell no longer releasable by heparin. In the FH homozygote cells, the amount of heparinreleasable [Iz51]LDLwas markedly diminished a t both temperatures, and interiorization therefore did not occur (FIGURE 3,B & D). A comparison of the saturation kinetics for heparin-releasable [Iz5I]LDLbinding 4. In the normal cells, a t 4"C in normal and FH homozygote cells is shown in FIGURE saturation of the binding sites appeared to occur at an LDL concentration of about 5pg/ml. This high-affinity process was not detected in the FH cells. At LDL concentrations above 5 pg/ml, a slight progressive increase in heparin-releasable [lZ5I]LDLbinding occurred in both cell strains. The nature of this apparently nonsaturable process is not known. Of the 21 FH homozygote cell strains studied in our laboratory, 12 have demonstrated a lack of high-affinity cell surface [Iz5I]LDL binding that has correlated in each case with an absence of high-affinity LDL uptake, an absence of LDLmediated suppression of HMG CoA reductase activity, and an absence of LDLdependent cholesteryl ester formation. These cell strains have been designated receptor-negative FH homozygotes.20The remaining nine cell strains, designated receptor-defective FH homozygotes, show a slight degree of cell surface [1251]LDL binding at high concentrations of lipoprotein that correlates with kinetically abnormal biologic responses in regard to cellular uptake of [1251]LDL,suppression of HMG CoA reductase, and activation of cholesteryl ester formation. A similar finding of genetic heterogeneity among FH cell strains has been reported by Breslow e f al. By use of the heparin release assay as an index of cell surface [L251]LDL binding, it was possible to show that unlabeled LDL, but not high-density lipoprotein (HDL),

248

Annals New York Academy of Sciences NORMAL

I A . 4'

16. 4 '

loot

BINDING IN NORMAL CELLS '

750t

H O "'

'TOTAL

.-.-.

BINDING

0

2 4 HOURS

6

t

; I-

HEPARIN- RELEASABLE

2

5

OO

c

;

~

,

6 HOURS

0

I

2

2-4

6

HOURS

FIGURE 3. Time course of total and heparin-releasable binding of [lzsl]LDLby normal ( 0 9 ) and FH homozygote (A+) fibroblast monolayers at 4' and 37"C. Cells were seeded (Day 0) at a concentration of l o 5 cells/dish into 60 x 15-mmdishes (Falcon) that contained 3 ml of growth medium with 10% fetal calf serum.Io On Day 3, the medium was replaced with 3 ml of fresh growth medium that contained 10% fetal calf serum. On Day 5, when the cells were not yet confluent, each monolayer was washed with 3 ml of phosphate-buffered saline, after which 2 ml of fresh medium that contained 10%(v/v) human lipoprotein-deficient serum' was added (final protein concentration, 5 mg/ml). On Day 7 of cell growth, after the cells had been incubated for 48 hr in the presence of growth medium that contained 10% lipoprotein-deficient serum, each dish received 2 ml of medium that contained 10 &ml of [Iz51]LDL(441 cpm/ng). After incubation for the indicated time at either 4 ° C (Expt. A & B) or 37°C (Expt. C & D), each monolayer was washed by a previously described technique,I0 after which 2 ml of buffer that contained 10 mg/ml of sodium heparin was added to each dish.I0All dishes were then incubated at 4'C for 60 min, after which the heparin-containing butTer was removed, and an aliquot was counted to determine the amount of [1251]LDLreleasable by heparin treatment and therefore bound at the cell surface (.,A). The cells were dissolved in I ml of 0. I N NaOH,I0 and an aliquot was counted to determine the amount of ['z51]LDLthat remained resistant to heparin treatment and therefore appeared to be within the cells. The total cellular binding of ['*'I]LDL ( 0 , ~ represents ) the sum of the heparin-releasable radioactivity and the heparin-resistant radioactivity. The inset compares the heparin-releasable radioactivity after binding at 4 ° C (0)and at 37°C (m) in the normal cells. Each value represents the average of duplicate incubations. Data from Reference 10.

Brown et al.: LDL Pathway in Human Fibroblasts FIGURE4. Saturation curves for heparin-releasable ['zSI]LDLbinding at 4'C in normal ( 0 ) and FH homozygote (A) fibroblasts. Cell monolayers were prepared as described in the legend to FIGURE3. On Day 7, each dish received 2 ml of medium that contained the indicated concentration of [Lz51]LDL (104 cpm/ng). After incubation at 4'C for 2 h r , each monolayer was washed by the standard technique and the amount of heparin-releasable [12s1]LDLwas determined. Each value represents the average of duplicate incubations. Data from Reference 10.

249

2

;7 5

9 50

L d

25

md

A~@A-

'0

10

,

20 30 40 LDL Ipq/rnl)

50

competed for receptor occupancy (FIGURE5). The specificity of binding to the heparin-releasable site a t 4 ° C was therefore similar to the specificity for intact cell binding and uptake measured a t 37°C.6*8 The ability of heparin to release [Iz5I]LDLfrom its receptor has provided a useful tool in distinguishing the [Iz5I]LDL bound to the receptor from [Iz5I]LDL that adheres nonspecifically to other components in the culture dish. A striking example of this effect is seen in the data shown in FIGURE 6. In this experiment, [IZ5I]LDLwas incubated in petri dishes under normal binding conditions a t 4"C, except that the dishes did not contain any cells. After 2 hr, the medium was removed and the dishes were treated by either of two methods that have previously been used to determine cell surface [i251]LDLbinding. In the first procedure, the dishes were washed six times in rapid succession with phosphate-buffered saline and were then incubated In the second procedure, with 0.5% trypsin to release surface-bound [1251]LDL.22.23 the dishes were washed six times with an albumin-containing buffer and were then incubated with heparin to release surface-bound [i2sI]LDL.10The results demonstrate that despite extensive washing with phosphate-buffered saline, a great deal of [IZ51]LDLremained bound to the empty dish, and this material was released by the trypsin treatment. In contrast, the albumin wash procedure removed much more of the nonspecifically bound [12sI]LDL,and subsequent treatment with heparin removed only small amounts of [IZ5I]LDLbound nonspecifically to the dish. This experiment illustrates that sticking of [1251]LDLto a variety of substances can occur and that misleading results can be generated if one does not directly correlate the observed binding with a physiologic function. For the LDL receptor described herein, a great deal of effort has been taken to show that this particular receptor molecule not only binds LDL but that its function is also critical to the observed LDL-mediated regulation of cellular cholesterol metabolism. I - 1 " . ' p - 1 7

0 0 % 0 ~ 0 % 7 f L )

UNLABELED LIPOPROTEIN ( p q l m l )

FIGURE5. Ability of native LDL and H D L to compete with [Iz5I]LDLfor cell surface binding in normal fibroblasts. Cell monolayers were prepared as described in the legend to FIGURE 3. On Day 7, each dish received 2 ml of medium that contained 13 pg/ml of [:2SI]LDL(404 cpm/ng) and the indicated amount of one of the following unlabeled human lipoprotein fractions: m, no unlabeled lipoprotein; 0 , human LDL (d = 1.019-1.063); and A, human HDL (d = 1.085-1.21). After incubation at 4°C for 2 hr, each monolayer was washed by the standard technique,Io and the amount of heparin-releasable ['Z51]LDLwas determined as described in the legend to FIGURE 3.

Annals New York Academy of Sciences

250

1

p -

C Proleolylic Degradation

10

N

PARAFORMALDEHYDE isb)

PARAFCRMALDEHYDE

(%I

PARAFORMALDEHYDE (%)

FIGURE 7. Effect of paraformaldehyde on cell surface binding, cellular uptake, and proteolytic degradation of [12"1]LDL in normal fibroblasts. Cell monolayers were prepared as described in the legend to F I G U R E 3. On Day 7, the medium from each monolayer was removed and replaced with 2 ml of phosphate-buffered saline that contained the indicated concentration of paraformaldehyde. After incubation at 4 ° C for 45 min. the paraformaldehyde was removed, and each monolayer was washed four times ( 3 ml/wash) with an albumin-NaCI-Tris buffer.x Each dish then received 2 ml of growth medium that contained 10 pg/ml of ['251]LDL (534 cpm/ng). After incubation at 37'C for 2 hr. the medium from each dish was removed, and its content of '*"I-labeled trichloroacetic-soluble protein material was measured (Expt. C ) as previously described." The cell monolayers were then washed by the standard technique,'" and the amounts of heparin-releasable [lZ'I]LDL (Expt. A ) and heparin-resistant ['z'I]LDL (Expt. B) were determined as described in the legend to F I G U R E 3.

Brown et al.: LDL P a t h w a y in Human Fibroblasts

25 1

A more direct approach to the study of cell surface binding was made possible by the observation that fixation of fibroblast monolayers with paraformaldehyde did not affect the binding of [12sI]LDLto its surface receptor in normal cells (FIGURE 7,A), even though it eliminated all subsequent endocytosis. Thus, cell surface binding could be demonstrated to occur in normal cells at 37°C under conditions in which no endocytosis and proteolytic degradation of the lipoprotein occurred (FIGURE 7,B & C). Again, as under all other conditions, cell surface binding was not observed in cells from FH homozygotes. FIGURE 8. Saturation curves for [1251]LDLbinding to membranes from normal ( 0 ) and FH homozygote (A) fibroblasts. Cells were seeded (Day 0) a t the concentration of 2 x lo5 cells/dish into 100 x 20-mm dishes that contained 7 ml of growth medium with 10%fetal calf serum. On Day 3, the medium was replaced with 7 ml of fresh growth medium that contained 10% fetal calf serum. On Day 5, each monolayer was washed with 5 ml of phosphate-buffered saline, and 7 ml of fresh medium that contained 5% (v/v) human lipoprotein-deficient serum was added. On Day 7, the cells from eight dishes were scraped with a rubber policeman into 10 ml of Eagle's minimum essential medium that contained 5% lipoprotein-deficient serum, and the pooled cells were collected into a pellet by centrifugation (ZOO0 rpm, 10 min, 24°C). Each cell pellet was resuspended in 0.8 ml of medium that contained 5% lipoproteindeficient serum, after which the cells were disrupted in a Dounce homogenizer (200 strokes with a tight-fitting pestle, 24"C). Each binding assay contained the following components in a final volume of 100 pl of growth medium: 250 pg of human lipoprotein-deficient serum, the indicated concentration of [1251]LDL(125 cpm/ng), and 95-145 pg of extract protein. After incubation a t 37°C for 30 min, membrane-bound [Iz51JLDL was isolated by centrifugation. Seventy-five microliters of each reaction mixture were first layered onto 300 PI of cold fetal calf serum contained within a Beckman microfuge tube, and each tube was then centrifuged (12,000 rpm, 2 min, 4°C). The supernatant was discarded, and the membrane pellet was washed twice with 300 pl of an albumin-NaC1-Tris buffera that contained 1 m M CaCI,. T h e membrane pellet was reisolated after each wash by centrifugation. After the final wash, the bottom of the tube that contained the membrane pellet was cut off, counted for radioactivity, and then used for determination of protein content. Each value represents the average of duplicate determinations.

LDL Ipp/ml

The fourth approach that we have used to distinguish receptor binding from cellular uptake has involved the use of membranes obtained from cells disrupted by 8 shows that, as with intact cells, high-affinity and Dounce homogenization. FIGURE saturable binding of [*251]LDLis markedly deficient in the membranes of cell-free extracts prepared from FH homozygote cells. A nonsaturable component of [Iz5I]LDL binding of similar magnitude is present in the extracts of both cell strains. SPECIFICITY OF THE

LDL RECEPTORDEFECTIN FH HOMOZYGOTE CELLS

An extensive series of studies in our laborabory has so fur failed to reveal any membrane abnormality in FH homozygote cells other than the inability to bind

25 2

Annals New York Academy of Sciences FIGURE 9. Total and heparin-releasable binding of [3H]poly-~-lysineby normal (0,o)and FH homozygote (A+) fibroblast monolayers at 4°C. Cell monolayers were prepared as described in the legend to 3. On Day 7, each dish received 2 FIGURE ml of medium that contained 10%human lipoprotein-deficient serum and the indicated concentration of [3H]poly-~-lysine (36,000 cpm/pg). After incubation at 4°C for 2 hr, each monolayer was washed by the standard technique, and the amount of heparin-releasable [3H]poly-~-lysinewas determined as described in the legend to FIGURE 3. The cells were dissolved in 1 ml of 10%Triton@X-100,and an aliquot was counted to determine the amount of [3H]poly-~-lysinewithin the cells. The total cellular binding of [3H]poly-~-lysine ( 0 , ~represents ) the serum of the heparin.) and the hepreleasable radioactivity (+ arin-resistant radioactivity. Each value represents the average of duplicate incubations.

LDL.24 Because negatively charged glycosaminoglycans, such as heparin, release LDL from its cell surface receptor in normal cells, the possibility was raised that the membrane defect in the FH cells involves the absence of negatively charged regions on the cell surface that normally bind to positive sites on the LDL particle. Consistent with this hypothesis is the observation that positively charged macromolecules, such as poly-D-lysine, poly-L-lysine, and poly-L-arginine, but not the single amino acids, bind to the normal cell in such a manner a s to block (1251]LDLbinding to the Despite their lack of an LDL receptor, however, the FH homozygote 9 cells are able t o bind just as much [3H]poly-D-lysine as do normal cells. FIGURE shows that under conditions in which poly-D-lysine is known to bind to cells and undergo absorptive endocytosis,26both the total uptake and the cell surface binding (as indicated by heparin-release) of [3H]poly-~-lysinewas the same in the normal and FH homozygote cells. Thus, if the LDL receptor does represent a negatively charged

BINDING OF [3H]CONCANAVALINA

IN

TABLE 1 NORMALA N D FH HOMOZYGOTE FIBROBLASTS* I3HlConcanavalinA Bound -

+ a-MMP

a-MMPt

Cell Strain

(ue/me of moteinl ~

Normal FH homozygote

2.64 2.03

~

0.45 0.35

*Cell monolayers were prepared as described in the legend to FIGURE 3. On Day 7, each dish received 2 ml of medium that contained 20 pg/rnl of [~ceryl-~H]concanavalin A (4000 cpm/pg) with or without 0.1 M a-methyl mannopyranoside as indicated. After incubation at 37°Cfor 30 min, each monolayer was washed repeatedly, and the amount of [3H]concanavalinA bound to the cells was determined as previously described.g fa-Methyl mannopyranoside.

Brown et al.: LDL Pathway in Human Fibroblasts

253

region of the plasma membrane, its absence does not reduce the overall negative charge of the cell surface sufficiently to influence the total cellular binding of [3H]poly-~-lysine. A molecule that binds to specific carbohydrates on the membrane surface of fibroblasts, [3H]concanavalin A, also showed identical binding in normal and FH homozygote cells (TABLEI). UPTAKEOF LDL

A N D OTHERMACROMOLECULES I N NORMAL A N D FH HOMOZYGOTE CELLS

Previous studies have shown that LDL bound to its receptor is internalized by endocytosis and that the lipoprotein becomes degraded within I y s o ~ o m e s . ~Be~~~-~~ cause FH homozygote cells do not bind LDL at the surface receptor site, they cannot internalize it with high affinity.8.10However, the FH homozygote cells, like the normal cells, engage in a continuous process of bulk phase pinocytosis by which soluble macromolecules, including LDL, are ingested in proportion to their concentration in the fluid medium.8 LDL taken up by the nonspecific pinocytotic process is degraded in lysosomes just like the LDL taken up by the high-affinity receptor mechanism.8 However, under the conditions of our experiments, the LDL that is degraded by the bulk phase pinocytotic process, either in the normal cells or in the FH homozygote cells, does not contribute its cholesterol in a net sense to the cells and therefore does not suppress HMG CoA reductase a ~ t i v i t y . ~ ~ ' ~ That nonspecific bulk phase pinocytosis of LDL is normal in FH homozygote cells is indicated by the data in FIGURES10 and 11. When incubated with 100 Fg/ml of either [1251]LDLor [1Z51]y-globulin,the normal fibroblasts took up much more 10). The rapid uptake of (1251]LDLwas a [Iz5I]LDL than [1251]y-globulin(FIGURE result of its binding to the cell surface receptor, as indicated by the fact that this uptake was markedly inhibited by the inclusion of heparin in the incubation medium (FIGURE 10,A). Heparin did not affect the uptake of [1251]y-globulin (FIGURE 10,C). When the receptor-dependent uptake of [Iz5I]LDLwas inhibited by the presence of heparin, the uptakes of [1Z51]LDL and [1251]y-globulinwere equal in the normal cells (FIGURE10,A & C). In the FH homozygote cells, the uptake of [Iz5I]LDLwas the same as that of [1251]y-globulin.and the addition of heparin had only a slight inhibitory effect on the uptakeof [1251]LDL(FIGURE 10,B). When the uptakes of [lZ5I]LDLand [12sI]y-globulinwere compared in more detail at two concentrations in the FH homozygote cells, it was observed that the initial rate of uptake of each protein was linearly related to its concentrations in the medium and that both proteins were taken up at similar rates (FIGURE 11). The conclusion from these uptake studies is that both normal and FH homozygote cells take up [1*51]LDLby a nonspecific pinocytatic process just as they take up [1251]y-globulin.In addition, the normal cells, but not the FH homozygote cells, possess a high-affinity uptake mechanism, that is, the LDL receptor, that markedly and selectively enhances their ability to take up LDL. Our previous studies have shown that the LDL receptor defect leads to an impaired ability of the FH homozygote cells to take up and hydrolyze [3H]cholesteryl linoleate when it is contained within the LDL p a r t i ~ l e . 'As ~ a further test for the specificity of the LDL receptor defect, [3H]cholesteryl linoleate was incorporated into positively and negatively charged multilamellat liposomes that were then in12 shows that in both the normal and cubated with monolayers of fibroblasts. FIGURE FH homozygote cells, there was virtually no uptake of the [3H]cholesteryl linoleate from the negatively charged liposomes. On the other hand, there was extensive up-

Annals New York Academy of Sciences

254 7

FH HOMOZYGOTE CELLS

NORMAL CELLS

B

A w

1000 -

Y

750-

H

n

'

250

+ Heparin A H A -

A-A

A /~A

w Y

MINUTES

MINUTES

FIGURE10. Uptake of [Iz5I]LDL(A,B) and ["'l]y-globulin (C,D) by normal ( q o ) and FH homozygote (i ,A) fibroblast monolayers in the absence ( 0 , ~and ~ ) presence (*,A) of heparin. Cell monolayers were prepared as described in the legend to FIGURE 3. On Day 7, each dish received 2 ml of medium that contained one of the following: 100 fig/ml of [1251]LDL(101 cpm/ng) in the absence o r presence of 10 mg/ml heparin or 100 pg/ml of [1251]y-globulin(298 cpm/ng) in the absence or presence of 10 mg/ml heparin. After incubation for the indicated time a t 37"C, each monolayer was washed by the standard technique,'O and the total cellular content of lz51radioactivity that remained associated with the cells was determined as previously described.8 Each value represents the average of duplicate incubations.

take of [3H]cholesteryl linoleate from the positively charged liposomes. This uptake was nearly identical in bath cell strains.

SUMMARY The studies reported here, coupled with our previous studies, indicate that LDL is ingested by cultured human fibroblasts in a process that resembles adsorptive endocytosis. The critical step is the binding of the lipoprotein to a high-affinity cell surface receptor. Uptake of LDL by this receptor-mediated process permits the cell

Brown et al.: LDL Pathway in Human Fibroblasts

layers. Cell monolayers were prepared as

255

-

F

A Normal Cells

B -

300 -

FH Homozygole Cells

200 Liposomes I00 Negatively-chorged

0

0

25

5 0 0

Negatively-charged Liposomes

25

50

CONCENTRATION OF LIPOSOMES IN MEDIUM (pg choleslerol/ml)

FIGURE12. Uptake of [3H]cholesteryl linoleate contained in multilamellar liposomes by normal (0,o) and F H homozygote (A,.) fibroblast monolayers. Cell monolayers were prepared as described in the legend to FIGURE3. On Day 7, each dish received 2 ml of medium that contained 10% human lipoprotein-deficient serum and the indicated concentration of either positively charged (.,A) or negatively charged ( 0 , ~liposomes. ) After incubation a t 37°C for 7 hr, each monolayer was washed by the standard technique,1° and the cellular content of [3H]cholesteryl linoleate was determined by thin-layer chromatography of ch1oroforrn:rnethanol extracts of the ce1ls.I" Each value represents the average of duplicate incubations. The multilamellar (unsonicated) liposomes were prepared by the method of Weissmann et al. by use of a swelling solution that contained 0.29 M glucose and 50 mM potassium phosphate (pH 7.4).27.28Each 1 ml of solution of positively charged liposomes contained 1.5 pmol (579 p g ) of cholesterol, 10 pmol of egg lecithin, 3 pmol of stearylamine, and 1.3 x lo6 cpm of [3H]cholesteryl linoleate (sp act 56 Ci/mmol). Each 1 ml of solution of negatively charged liposomes contained 1.5 pmol (579 pg) of cholesterol, 10 pmol of egg lecithin, 3 pmol of dicetyl phosphate, and 1.3 x lo6cpm of [3H]cholesteryl linoleate (sp act 56 Ci/mmol).

256

Annals New York Academy of Sciences

t o acquire cholesterol from the lipoprotein, and this acquisition, in turn, suppresses t he cell’s own cholesterol synthesis and activates t h e cell’s system for reesterification and storage of t h e incoming cholesterol. In cells from patients with t h e receptor-negative form of homozygous FH, t h e cell surface receptor is functionally absent. T h e absence of high-affinity binding t o this receptor produces a defective uptake of LDL and prevents the normal process of feedback regulation of cholesterol synthesis by t h e lipoprotein. T h e physiologic importance of the LDL pathway is indicated by t h e fact that patients who lack the LDL receptor (FH homozygotes) develop both profound hypercholesterolemia and fulminant atherosclerosis. It is likely that other defects in the LDL pathway account for o th e r forms of hypercholesterolemia and atherosclerosis in man.

REFERENCES

4. 5.

6.

7. 8. 9. 10. 1I.

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The low-density lipoprotein pathway in human fibroblasts: relation between cell surface receptor binding and endocytosis of low-density lipoprotein.

THE LOW-DENSITY LIPOPROTEIN PATHWAY I N HUMAN FIBROBLASTS: RELATION BETWEEN CELL SURFACE RECEPTOR BINDING A N D ENDOCYTOSIS OF LOW-DENSITY LIPOPROTEIN...
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