©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Cellular Internalization and Degradation of Hepatic Lipase Is Mediated by Low Density Lipoprotein Receptor-related Protein and Requires Cell Surface Proteoglycans (*)

Maria Z. Kounnas (1)(§), David A. Chappell (2), Howard Wong (3), W. Scott Argraves (1), Dudley K. Strickland (1)(¶)

From the (1) Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, (2) Department of Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, and (3) VA Wadsworth Medical Center, Lipid Research Laboratory and the Department of Medicine, UCLA, Los Angeles, California 90073

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Hepatic lipase (HL) and lipoprotein lipase (LpL) are structurally related lipolytic enzymes that have distinct functions in lipoprotein catabolism. In addition to its lipolytic activity, LpL binds to very low density lipoproteins and promotes their interaction with the low density lipoprotein receptor-related protein (LRP) (Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). In vitro binding assays revealed that HL also binds to purified LRP with a Kof 52 n M. Its binding to LRP is inhibited by the 39-kDa receptor-associated protein (RAP), a known LRP antagonist, and by heparin. I-Labeled HL is rapidly internalized and degraded by HepG2 cell lines, and approximately 70% of the cellular internalization and degradation is blocked by either exogenously added RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly diminished capacity to internalize and degrade HL when compared to control fibroblasts. These data indicate that LRP-mediated cellular uptake of HL accounts for a substantial portion of the internalization of this molecule. Proteoglycans have been shown to participate in the clearance of LpL, and consequently a role for proteoglycans in HL clearance pathway was also investigated. Chinese hamster ovary cell lines that are deficient in proteoglycan biosynthesis were unable to internalize or degrade I-HL despite the fact that these cells express LRP. Thus, the initial binding of HL to cell surface proteoglycans is an obligatory step for the delivery of the enzyme to LRP for endocytosis. A small, but significant, amount of I-HL was internalized in LRP deficient cells indicating that an LRP-independent pathway for HL internalization does exist. This pathway could involve cell surface proteoglycans, the LDL receptor, or some other unidentified surface protein.


INTRODUCTION

Hepatic lipase (HL)() is a member of a family of structurally and functionally related enzymes that include pancreatic lipase and lipoprotein lipase (LpL) (Ben-Zeev et al., 1987) (for reviews, see Nilsson-Ehle et al. (1980) and Olivecrona and Bengtsson-Olivecrona (1990)). The three-dimensional crystallographic structure of human pancreatic lipase (Winkler et al., 1990) shows that this molecule consists of two domains; an N-terminal domain (amino acids 1-335), which contains the active site of the enzyme that is dominated by a central parallel -sheet structure, and a smaller C-terminal domain (amino acids 336-449) that forms a sandwich structure. The overall folding patterns of HL and LpL are thought to be similar to that of pancreatic lipase (Derewenda and Cambillau, 1991).

HL is secreted by hepatocytes and becomes bound to both hepatocytes (Marteau et al., 1988; Belcher et al., 1988; Hornick et al., 1992) and endothelial cells that line hepatic sinusoids (Kuusi et al., 1979a). Accumulated evidence has implicated HL in the metabolism of high density lipoproteins (HDL), intermediate density lipoproteins (IDL), and chylomicron remnants (Shirai et al., 1991; Laboda et al., 1986; Nilsson et al., 1987; Jensen et al., 1982; Shafi et al., 1994). Deficiencies of this enzyme in either HL-null transgenic mice (Homanics et al., 1995) or human patients (Hegele et al., 1993; Huff et al., 1993) result in increased plasma levels of HDL. Some patients lacking HL accumulate cholesterol-rich, very low density lipoproteins (-VLDL) which are abnormal lipoproteins that are enriched in apolipoprotein E (apoE). On the other hand, overexpression of HL in transgenic rabbits results in a lowering of plasma HDL and IDL levels (Fan et al., 1994). It has been speculated that HL may serve to promote hepatic clearance of apoE-containing lipoproteins by either hydrolyzing/remodeling them to a form that exposes an apoE receptor binding site or by binding to lipoproteins and serving as a recognition site for hepatic receptors (Ji et al., 1994b). This latter model is similar to a putative role played by LpL, which binds to the low density lipoprotein receptor-related protein (LRP) and promotes the interaction of lipoproteins with this receptor (Beisiegel et al., 1991; Chappell et al. 1992, 1993; Nykjaer et al., 1993). The ability of LpL to promote the LRP-mediated cellular internalization and degradation of lipoproteins may not require the catalytic site of LpL, since the carboxyl-terminal domain of LpL, which lacks this site, binds to LRP and promotes the LRP-mediated catabolism of lipoproteins (Williams et al., 1994).

LRP binds diverse ligands and belongs to a family of endocytic receptors structurally related to the low density lipoprotein (LDL) receptor (for reviews, see Krieger and Herz (1994), Moestrup (1994), and Strickland et al. (1994)). Other mammalian members of the LDL receptor family include the very low density lipoprotein (VLDL) receptor, and glycoprotein 330 (gp330). LRP mediates the cellular internalization of proteinases, such as plasminogen activators, proteinase-inhibitor complexes, and apoE- and lipoprotein lipase-enriched lipoproteins. Ligand binding by LRP is blocked by a 39-kDa protein, termed the receptor-associated protein (RAP) (Herz et al., 1991; Williams et al., 1992). While the complete physiological role of LRP is not fully appreciated at this time, it is clear that one important function of LRP is to mediate the hepatic removal of ligands from the circulation, and LRP is thought to play an important role, along with the LDL receptor, in the hepatic removal of chylomicron remnants from the circulation (Willnow et al., 1994).

Recently, Ji et al. (1994b) transfected a rat hepatoma cell line with the HL cDNA and demonstrated that the transfected cells had an increased capacity to internalize chylomicron remnants. Since the receptor responsible for this process has not yet been identified, we initiated studies to determine if LRP will bind and mediate the cellular internalization of HL and to examine the role of proteoglycans in this process. The results of our studies indicate LRP binds to HL in vitro and mediates the cellular internalization of HL leading to its degradation in lysosomes. We also demonstrate that proteoglycans appear essential for the LRP-mediated catabolism of HL.


EXPERIMENTAL PROCEDURES

Proteins

LRP was isolated from human placenta as described by Ashcom et al. (1990). Human RAP, expressed in bacteria as a fusion protein with glutathione S-transferase, was prepared and purified as described previously (Williams et al., 1992). HL was labeled with [I]iodine to a specific activity ranging from 2 to 10 µCi/µg of protein using Iodogen (Pierce Chemical Co., Rockford, IL). Bovine serum albumin, fraction V, and ovalbumin were purchased from Sigma.

Production and Purification of Recombinant Rat HL

Roller cultures of Chinese hamster ovary (CHO)-K1 cells stably transformed with HL (Komaromy and Reed, 1991) were grown in Dulbecco's modified Eagle's medium supplemented with 1% Nutridoma (Boehringer Manneheim) until subconfluent. The medium was replaced with 30 ml of induction medium (Dulbecco's modified Eagle's medium containing Nutridoma, 30 µ M ZnSO, and 10 units/ml heparin) for 14 days, harvesting, and replacing the medium every 24 h. Harvested medium was spun at 1200 g for 20 min and stored at -80 °C until utilized. Rat HL was purified by a four-step procedure utilizing octyl-Sepharose, heparin-Sepharose, hydroxylapatite, and dextran sulfate-Sepharose columns. Elution and loading steps were linked to maximize enzyme recovery and prevent loss of activity. Briefly, rapidly thawed CHO medium (1 liter) was loaded onto an octyl-Sepharose column (80-ml bed volume) which had been previously equilibrated with 5 m M barbital, 0.35 M NaCl, 20% glycerol, pH 7.4 (Buffer A). Following a wash step to remove unbound proteins, the column was eluted with 200 ml of 1.2% Triton N-101 in Buffer A. This material was applied immediately to a column of heparin-Sepharose (50-ml bed volume), washed, and eluted with a 0-1 M NaCl step gradient. Fractions containing HL were then applied to a 2-ml hydroxylapatite column, washed with 0.1 M NaPO, pH 7.4, and eluted with 0.3 M NaPO. The eluate was applied directly to a dextran sulfate-Sepharose column (6 ml), washed with Buffer A, and eluted with a linear NaCl gradient (0.35-1.5 M). This procedure resulted in isolation of a homogeneous band as determined by silver staining of SDS-polyacrylamide gel electrophoresis gels (data not shown). The specific activity was indistinguishable from HL purified from rat liver perfusates (Ben-Zeev et al., 1987). Typically, 1 mg of recombinant HL was obtained from 2 liters of CHO cell medium.

Antibodies

Rabbit polyclonal antibodies to the 515-kDa heavy chain of LRP (R777) and to a synthetic peptide from the cytoplasmic domain of the 85-kDa light chain of LRP (R704) have been described elsewhere (Kounnas et al., 1992b). R777 antibodies were affinity selected on a column of LRP-Sepharose. R704 antibodies were purified on protein G-Sepharose (Pharmacia Biotech Inc.). Both of the rabbit antibody preparations were dialyzed against isotonic PBS prior to use in cell assays.

Solid-phase Binding Assays

Homologous and heterologous ligand displacement assays were performed as described by Williams et al. (1992). Microtiter wells were coated with LRP or ovalbumin (5 µg/ml) in Tris-buffered saline, pH 8.0, 5 m M CaCl, and nonspecific sites were blocked with 0.5% ovalbumin, Tris-buffered saline, pH 8.0, 5 m M CaCl. Coated wells were incubated with radiolabeled ligand (2 n M) in 0.5% ovalbumin, Tris-buffered saline, 5 m M CaCl, 0.05% Tween-20 in the presence of varying concentrations of unlabeled competitor for 18 h at 4 °C. Binding data were analyzed by using the computer program LIGAND (Munson and Rodbard, 1980).

Cells

Human hepatocellular carcinoma (HepG2, ATCC HB 8065) cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and were grown in Eagle's minimal essential medium containing 10% bovine calf serum (Hyclone Labs, Logan, UT), 100 units/ml penicillin (Life Technologies, Inc.), 100 µg/ml streptomycin (Life Technologies, Inc.), and 1% L-glutamine (Life Technologies, Inc.). Three types of CHO cells were used in these studies. CHO 745 provided by Dr. J. Esko (University of Alabama, Birmingham, AL) are deficient in all types of proteoglycans (Esko et al., 1986), CHO C16 (ATCC CCL 61) are a wild type cell line, and CHO 13-5-1 are LRP-deficient cells prepared by toxin-mediated selection of mutagenized CHO C16 cells.() CHO cells were grown to approximately 80% confluence in Ham's F-12 medium supplemented with 5% bovine calf serum-optimized for CHO cells (Hyclone), 100 units/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. A normal mouse embryo fibroblast (MEF) line and a derivative of this cell line (Willnow and Herz, 1994) that is deficient in LRP biosynthesis (PEA13) were obtained from Dr. J. Herz (University of Texas Southwestern Medical Center, Dallas, TX) and maintained as described by Willnow and Herz (1994).

Cell-mediated Ligand Internalization and Degradation Assays

Cells were transferred to 12-well dishes (Corning, Corning, NY) at 2-3 10cells/well and allowed to grow for 24 h at 37 °C, 5% CO. Prior to performing assays, cultured cells were washed with medium and incubated in medium containing 1% Nutridoma (Boehringer Manneheim), 20 m M Hepes, penicillin/streptomycin, and 1.5% bovine serum albumin (assay medium). For assays with HepG2 cells, radiolabeled HL (4 n M) in assay medium was incubated with cells for 6 h at 37 °C, 5% COin the presence of increasing concentrations of RAP (0.2-1350 n M) or heparin (Sigma, 0.1-300 mg/ml). For assays using antibodies, cultured cells were preincubated for 1 h at 37 °C, 5% COwith assay medium containing various concentrations of antibodies. Fresh medium containing the I-labeled ligand and antibodies was then added and incubated with the cells for 6 h at 37 °C, 5% CO. For CHO cell assays, I-HL (4 n M) was incubated with cells in the presence of unlabeled heparin (100 µg/ml) or LRP antibodies (100 µg/ml) for the indicated time intervals. In assays utilizing cultured fibroblasts, I-HL (4 n M) was added in assay medium either alone or in the presence of RAP (450 n M), heparin (100 µg/ml), or chloroquine (0.1 m M, Sigma). Radioactivity appearing in the cell culture medium that was soluble in 10% trichloroacetic acid was taken to represent degraded ligand. Total ligand degradation was corrected for the amount of degradation that occurred in control wells lacking cells. The amount of radioligand that was bound to the cell surface or that was internalized by cells was determined as described previously (Chappell et al., 1992). Briefly, following incubation with the radioligand, cells were washed with phosphate-buffered saline and treated with a trypsin-EDTA, proteinase K solution. Surface-bound material was defined as the amount of radioactive ligand released by this treatment, and the amount of internalized ligand was defined as the amount of radioactivity which remained associated with the cell pellet following the treatment.


RESULTS

Hepatic Lipase Binds to LRP

To determine if HL can bind to LRP in vitro, homologous ligand displacement binding assays were conducted. The results of these experiments, shown in Fig. 1 ( Panel A), demonstrate that I-HL bound to purified LRP immobilized on microtiter plastic wells, but not to ovalbumin-coated microtiter wells. Its binding was prevented by excess cold HL, which is consistent with specific binding. Quantitative information from these data was derived using the program LIGAND, and the binding was adequately described by a model containing a single class of sites with a Kof 52 n M. This value is slightly weaker than the Kof 20 n M measured for the interaction of LpL with LRP (Chappell et al., 1993) using a similar assay. RAP, a known antagonist of ligand binding to LRP, inhibited the binding of HL to LRP (Fig. 1, Panel B). HL is known to bind heparin, and consequently the effect of heparin on its interaction with LRP was also measured. The results (Fig. 1, Panel B) revealed that heparin almost completely blocked the binding of I-HL to LRP.


Figure 1: Binding of I-HL to LRP. In Panel A, I-HL (2 n M) was incubated for 18 h at 4 °C with LRP-coated wells ( closed circles) or ovalbumin-coated wells ( open circles) in the presence of increasing concentrations of unlabeled HL. Following incubation, wells were washed and counted. The data were analyzed using the program LIGAND and the curve represents the best-fit data to a single class of sites with a K of 52 n M. In Panel B, I-HL (2 n M) was incubated as in Panel A in the absence of competitor or in the presence of either 450 n M RAP, or 100 µg/ml heparin. Plotted values represent means of duplicate experiments.



I-HL Degradation Is Greatly Diminished in LRP-deficient Fibroblasts

To investigate whether or not LRP is able to mediate the cellular internalization of HL, we utilized a MEF cell line, and a derivative of this cell line that is LRP-deficient (PEA13) (Willnow and Herz, 1994). As shown in Fig. 2, LRP-expressing MEF cells efficiently internalized and degraded I-HL. Approximately 80% of the degradation of I-HL was inhibited by RAP, while heparin completely blocked the degradation of this ligand. Chloroquine, a drug which effectively prevents lysosomal degradation by altering the pH of lysosomes, also blocked the degradation of I-HL in these cells, confirming that the degradation of this ligand requires lysosomal enzymes. In contrast, I-HL was degraded relatively poorly in LRP-deficient mouse fibroblasts (PEA13 cells) to a level of 20% of that found in MEF cells (Fig. 2, right four bars). I-HL degradation in PEA13 cells was largely insensitive to RAP, but was inhibited by heparin and chloroquine. The data indicate that LRP is the predominant mediator of HL degradation in fibroblasts. Since a small amount of degradation was observed in PEA13 cells, it does appear that an LRP-independent pathway for HL clearance exists in these cells.


Figure 2:I-HL degradation by LRP-deficient mouse fibroblasts. Control LRP-expressing fibroblasts (MEF cells, closed bars) and LRP-deficient fibroblasts (PEA13 cells, open bars) were incubated with I-HL (4 n M) for 8 h in the absence of competitor or in the presence of either RAP (450 n M), heparin (100 µg/ml), or chloroquine (0.1 m M). The levels of radioligand degradation were determined as described under ``Experimental Procedures.'' Plotted values represent means of duplicate experiments ± S.E.



I-HL Degradation in HepG2 Cells Is Partially Mediated by an LRP-independent Pathway

To characterize the cellular catabolism of HL further, we next investigated this process in HepG2 cells. These cells were observed to rapidly internalize and degrade I-HL. To identify the receptor(s) that are responsible for this process in these cells, the effect of RAP and heparin on I-HL catabolism were investigated. As shown in Fig. 3 A, heparin inhibited the surface binding of I-HL to HepG2 cells, while RAP slightly increased HL binding to HepG2 cell surfaces. However, concentrations of RAP sufficient to prevent binding of I-HL to LRP (Fig. 1, Panel B) blocked approximately 55% of the internalization and 60% of the degradation of I-HL in these cells (Fig. 3, Panels B and C). Exogenously added heparin completely blocked both processes. Experiments using both RAP and heparin together as competitors of HL binding, uptake, and degradation showed results similar to those obtained with heparin alone ( data not shown). Because RAP prevented HL binding to cells without preventing its surface binding, while heparin completely blocked both processes, the data suggest that HL binding to cells is largely independent of LRP and is perhaps mediated by cell surface proteoglycans.


Figure 3: RAP and heparin inhibit I-HL uptake and degradation by HepG2 cells. HepG2 cells were incubated for 6 h at 37 °C with I-HL (4 n M) in the presence of increasing concentrations of unlabeled RAP (0.6-1350 n M) or heparin (0.11-300 µg/ml). Panels show binding ( A), internalization ( B), and degradation ( C) data, respectively. Plotted values represent means of duplicate experiments.



Since RAP can also block LDL receptor-mediated catabolism (Medh et al., 1995), additional experiments were performed using antibodies that specifically inhibit LRP function. Surface binding of I-HL to HepG2 cells was not inhibited by LRP antibodies or control antibodies (Fig. 4, Panel A). LRP-antibodies did, however, effectively block 80% of I-HL internalization (Fig. 4, Panel B) and 60% of its degradation (Fig. 4, Panel C). Control antibodies had little or no effect on I-HL binding, internalization, or degradation by HepG2 cells (Fig. 4). When assays were performed which included both heparin and LRP antibodies as competitors of I-HL binding, uptake, and degradation, the results paralleled those observed using heparin alone. These data are consistent with the observation that heparin competes for HL binding to LRP in vitro (Fig. 1, Panel B) as well as for HL binding to cell surface proteoglycans, a likely primary binding site for HL on the cell surface, and support the hypothesis that HL binds initially to cell surface proteoglycans and that its subsequent internalization and degradation are substantially mediated by LRP.


Figure 4: LRP-antibodies inhibit I-HL uptake and degradation by HepG2 cells. Wells containing 3 10HepG2 cells were incubated for 6 h at 37 °C with I-HL (4 n M) in the presence of increasing concentrations of affinity purified anti-LRP IgG (0.11-300 µg/ml), control IgG against the cytoplasmic domain of LRP (0.11-300 µg/ml), or heparin (0.11-300 µg/ml). Panels show binding ( A), internalization ( B), and degradation ( C) data, respectively. Plotted values represent means of duplicate experiments.



Proteoglycans Are Required for Cell-mediated Catabolism of HL

To further study the individual roles of LRP and proteoglycans in the catabolism of HL, cellular assays with I-HL were performed with normal CHO cells (C16), LRP-deficient CHO cells (13-5-1), or proteoglycan-deficient CHO cells (745) . The degradation of I-HL by normal CHO cells (Fig. 5, Panel A), was inhibited by LRP antibodies (65%) and heparin (100%), while control antibodies had no effect. In the LRP-deficient cells (Fig. 5, Panel B), I-HL degradation is not inhibited by LRP antibodies but was blocked by heparin. In the proteoglycan-deficient cells (Fig. 5, Panel C) only a small amount of degradation of I-HL was observed. These data indicate that proteoglycans are required for HL catabolism. In addition, it is apparent that, like LRP-deficient mouse fibroblasts, the LRP-deficient CHO cells possess an alternative pathway for HL clearance, albeit somewhat less than that mediated by LRP. It is interesting to note that the alternative pathway seems to play a greater role in the CHO cells when compared to either HepG2 or fibroblasts. This is evidenced by comparing the percent of total HL internalized and degraded in the presence and absence of LRP antagonists in the individual cell lines. Likely this reflects that fact that CHO cells contain far fewer LRP molecules than HepG2 cells or fibroblasts.


Figure 5: Proteoglycans are essential for I-HL catabolism. Normal CHO cells ( Panel A); LRP-deficient CHO cells ( Panel B); proteoglycan-deficient CHO cells ( Panel C) were incubated with I-HL (4 n M) for selected time intervals in the presence of control IgG (100 µg/ml), affinity-purified LRP antibodies (100 µg/ml), or heparin (100 µg/ml). At selected times, the medium was removed and the amount of radioligand degradation determined as described under ``Experimental Procedures.'' Plotted values represent means of duplicate experiments.




DISCUSSION

In the present study we demonstrate that HL binds to purified LRP with a Kof 52 n M. Like all known LRP ligands, the interaction of HL with LRP is inhibited by RAP. Heparin, which is known to bind to HL, also prevents its association with LRP. An interaction of LRP with HL immobilized on nitrocellulose has been recently reported (Nykjaer et al., 1994) and is supported by findings in the present investigation. Cellular uptake experiments show that I-HL is rapidly internalized and degraded in mouse fibroblasts, HepG2 cells, and Chinese hamster ovary cells. Our studies identified LRP as a major receptor responsible for mediating the cellular internalization and degradation of I-HL. The role of LRP in HL catabolism is supported by the fact that, at concentrations known to completely block binding of HL and other ligands to LRP, RAP reduced the cellular catabolism of I-HL by 80 and 60%, in mouse fibroblasts and HepG2 cells, respectively. Further, anti-LRP IgG reduced the extent of cellular mediated degradation of I-HL by 60 and 65%, in HepG2 cells and Chinese hamster ovary cell lines, respectively. Finally, two mutant cell lines that do not express LRP show a greatly diminished capacity to internalize and degrade I-HL when compared to the control cell lines from which they were derived that do express LRP.

Since HL catabolism was completely blocked by heparin, we also investigated the role of cell surface proteoglycans in this process. For this purpose, cell lines deficient in proteoglycan biosynthesis were utilized (Esko et al., 1986). The results from these experiments reveal that cell surface proteoglycans appear essential for the LRP-mediated uptake of HL and that glycosaminoglycans are important for serving as the primary cell surface binding site for HL. One possible explanation of these data is that after binding to cell surface proteoglycans, HL is then transferred to LRP for subsequent internalization and degradation. Cell surface proteoglycans are also known to facilitate the LRP-mediated catabolism of LpL (Chappell et al., 1993). It seems likely that proteoglycans serve to concentrate HL or LpL on the cell surface, thereby enhancing their interaction with LRP. Whether or not the proteoglycans are internalized along with LRP remains to be determined. The LRP-mediated catabolism of several other molecules, such as tissue-factor pathway inhibitor (Warshawsky et al. 1994), and thrombospondin (Mikhailenko et al. 1995) have recently been demonstrated to also be facilitated by cell surface proteoglycans.

Experiments in the present study also confirm that HL is catabolized by a second pathway that does not involve LRP. This conclusion is supported by the fact that RAP or LRP antibodies are unable to completely block I-HL uptake and degradation in all cell lines examined and by the observation that I-HL is degraded in a heparin-dependent but RAP-independent manner in cell lines which lack LRP. While the receptor responsible for this second pathway has not been identified, it is not likely to be gp330 or the VLDL receptor, since RAP is an effective antagonist of ligand binding by these two receptors (Kounnas et al., 1992a; Battey et al., 1994). Further, gp330 is not expressed in any of the cell lines used in the current investigation. On the other hand, the LDL receptor has weak affinity for RAP ( K= 300 n M) and thus could be responsible for the low levels of uptake seen in these cells. The LDL receptor is present on HepG2 cells, fibroblasts, and CHO cells and is known to participate in the uptake of lipoprotein lipase.()Alternately, another unidentified receptor may be responsible for the catabolism of HL in these cell types. This receptor, like LRP, is likely to be dependent on proteoglycans for serving as the initial binding site for HL, since no specific uptake and degradation was observed in cells deficient in proteoglycan biosynthesis.

The significance of the interaction of HL with LRP likely centers around the important role HL plays in lipoprotein catabolism. HL is a lipolytic enzyme that has both triglyceride lipase and phospholipase activities and participates in the metabolism of remnants, IDL, and HDL particles. Remnant lipoprotein particles are generated from chylomicrons and VLDL during lipolysis of their triglycerides by LpL. The smaller, cholesterol-rich remnant particles are then removed from the circulation by liver parenchymal cells. This process is thought to occur by an initial interaction of the lipoproteins with cell surface proteoglycans, followed by internalization via a receptor-mediated process (Ji et al. 1994a). Efficient catabolism of lipoproteins requires apoE to be present on the particle surface and can be mediated by the LDL receptor. However, since these lipoproteins are efficiently catabolized in patients with homozygous familial hypercholesterolemia who have severe deficiencies of LDL receptor activity (Goldstein and Brown, 1989), another receptor must assume this function in these patients. Recent studies in mice in which the LDL receptor expression was prevented by gene targeting suggest that LRP is a candidate for this receptor (Willnow et al., 1994). It is important to point out that LRP may not directly bind these remnant particles; in vitro experiments document that both remnant-like particles, and native VLDL require ``activation'' in order to bind to LRP. This can occur either by the addition of exogenous apoE (Kowal et al. 1989; Beisiegel et al., 1989) or LpL (Chappell et al., 1993). A secretion-recapture model for remnant uptake in the liver has been proposed (Brown et al. 1991; Ji et al. 1994a) in which remnants entering the sinusoidal space of the liver are exposed to high concentrations of proteoglycan-bound apoE that has been secreted by hepatocytes. After enrichment of the remnants with apoE, they are active ligands for LRP, which can then mediate their internalization and subsequent degradation. HL and to a lesser extent LpL are also present in the hepatic sinusoids, and thus these molecules may also play an important role in remnant catabolism.

The potential role of LpL in lipoprotein catabolism was first suggested by Felts et al. (1975) who proposed that LpL promotes lipoprotein catabolism by providing a binding site for an endocytic receptor to internalize LpL-lipoprotein complexes. Using cross-linking experiments, Beisiegel et al. (1991) demonstrated that LpL could be cross-linked to a large cell surface receptor thought to be LRP. LpL was subsequently shown to bind to LRP and promote the LRP-mediated internalization and degradation of I-labeled VLDL (Chappell et al. 1992, 1993; Nykjaer et al., 1993). Thus, in addition to its capacity to hydrolyze triglycerides on the surface of lipoproteins, LpL serves as a proteoglycan-anchored ligand for remnant binding and promotes the interaction of the lipoprotein with the endocytic receptor, LRP.

It is possible that HL also promotes the LRP-mediated cellular internalization of lipoproteins by a similar mechanism. HL is abundant on the luminal surface of endothelial cells and hepatocytes within hepatic sinusoids and can be released from proteoglycan attachment sites by intravenous heparin. Injection of HL antibodies into animals resulted in an increase in remnant particles (Kuusi et al., 1979b; Goldberg et al., 1982; Daggy and Bensadoun, 1986; Shafi et al., 1994), suggesting a role for HL in remnant clearance. Infusion of HL into rabbits fed a high cholesterol diet reduced the levels of -VLDL which had accumulated in their plasma (Chang and Borensztajn, 1993). In addition, studies have shown that cells transfected with HL cDNA bind 3-fold more -VLDL than nontransfected cells (Ji et al. 1994b). Further, Ji et al. (1994b) demonstrated that transfected cells displayed an increased capacity to internalize -VLDL, presumably via a receptor-mediated process. While the receptor responsible for this process has not been identified, LRP is a likely candidate since our studies have confirmed that HL interacts with LRP.

In summary, we have identified LRP as a primary endocytic receptor for HL in HepG2 cells, fibroblasts, and CHO cells. Our studies indicate that proteoglycans are critical for initial binding of HL to the cell surface. Studies are underway to determine if HL bound chylomicron remnants are also endocytosed in association with HL via LRP.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL50787 (to D. K. S.), GM42581 (to D. K. S.), HL49264 (to D. A. C.), DK45598 (to W. S. A.), and VA Merit Review (to H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of Individual National Research Service Award HL08744 from the National Heart, Lung, and Blood Institute.

To whom correspondence should be addressed: American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. Tel.: 301-738-0726; Fax: 301-738-0794; E-mail: strickland@hlsun.red-cross.org.

The abbreviations used are: HL, hepatic lipase; LpL, lipoprotein lipase; HDL, high density lipoprotein; LDL, low density lipoprotein; IDL, intermediate density lipoprotein; VLDL, very low density lipoprotein; apoE, apolipoprotein E; LRP, low density lipoprotein receptor-related protein; CHO, Chinese hamster ovary; MEF, mouse embryo fibroblast.

Fitzgerald, D. J., Fryling, C., Zdanovsky, A., Saelinger, C., Kounnas, M., Winkles, J., Strickland, D., and Leppla, S. (1995) J. Cell Biol., in press.

J. Medh, S. Bower, G. Fry, M. Pladet, M. Andracki, I. Inoue, J-M. Lalouel, D. Strickland, and D. Chappell, manuscript in preparation.


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