Báo cáo khóa học: Soluble LDL-R are formed by cell surface cleavage in response to phorbol esters pdf

van der Westhuyzen2 1 Division of Medical Biochemistry, University of Cape Town, South Africa;2Department of Internal Medicine, University of Kentucky Medical Center, and Department of V

Soluble LDL-R are formed by cell surface cleavage in response

to phorbol esters

Michael J Begg1,*, Edward D Sturrock1and Deneys R van der Westhuyzen2

1

Division of Medical Biochemistry, University of Cape Town, South Africa;2Department of Internal Medicine, University of Kentucky Medical Center, and Department of Veteran Affairs Medical Center, Lexington, Kentucky, USA

A 140-kDa soluble form of the low density lipoprotein

(LDL) receptor has been isolated from the culture medium

of HepG2 cells and a number of other cell types It is

produced from the 160-kDa mature LDL receptor by a

proteolytic cleavage, which is stimulated in the presence of

4b-phorbol 12-myristate 13-acetate (PMA), leading to the

release of a soluble fragment that constitutes the bulk of the

extracellular domain of the LDL receptor By labeling

HepG2 cells with [35S]methionine and chasing in the

pres-ence of PMA, we demonstrated that up to 20% of

LDL-receptors were released into the medium in a 2-h period

Simultaneously, the level of labeled cellular receptors was

reduced by 30% in those cells treated with PMA compared

to untreated cells, as was the total number of cell surface

LDL-receptors assayed by the binding of125I-labeled

anti-body to whole cells To determine if endocytosis was

required for cleavage, internalization-defective LDL-recep-tors were created by mutagenesis or deletion of the NPXY internalization signal, transfected into Chinese hamster ovary cells, and assayed for cleavage in the presence and absence of PMA Cleavage was significantly greater in the case of the mutant receptors than for wild-type receptors, both in the absence and presence of PMA Similar results were seen in human skin fibroblasts homozygous for each of the internalization-defective LDL receptor phenotypes LDL receptor cleavage was inhibited by the hydoxamate-based inhibitor TAPI, indicating the resemblance of the LDL receptor cleavage mechanism to that of other surface released membrane proteins

Keywords: internalization signal; LDL-R; low density lipo-protein; ectodomain shedding

The low density lipoprotein (LDL) receptor (LDL-R) is a

cell surface protein that mediates the uptake and clearance

of the cholesterol-rich lipoprotein LDL from the plasma [1]

The LDL-R plays an important role in regulating plasma

LDL and cellular cholesterol levels and the activity of this

receptor has a direct bearing on plasma cholesterol levels

The LDL receptor is regulated at the transcriptional level

in response to intracellular sterol levels by means of sterol

sensitive transcription factors SREBP1 and SREBP2 [2,3],

however, it can also be upregulated by numerous cytokines

[tumor necrosis factor (TNF)-a, interleukin (IL)-1,

trans-forming growth factor (TGF)-b, oncostatin M,

platelet-derived growth factor and basic fibroblast growth factor

(bFGF) [4–8], hormones (insulin and estradiol) [9,10] and

second messenger systems [11,12] Although much is known about the transcriptional mechanisms that control LDL-R expression, little is known about the mechanism(s) that control the turnover and degradation of this important protein Earlier studies established that the LDL-R degra-dation mechanism(s) is a nonlysosomal process that is dependant on short lived mediator protein(s) and unaffected

by the presence of ligand and/or sterol [13,14] Some evidence suggests that degradation may also be able to modulate LDL-R number Ness et al [15] showed that degradation of LDL-R in hepatocytes is increased in the presence of cholesterol synthesis inhibitors Kraemer et al [16] reported that the half-life of LDL-R in rat adipocytes is decreased by 40% in the presence of insulin The proteolytic mechanism responsible for such regulated LDL-R degra-dation is not known

A regulatory mechanism common to many cell surface proteins is a proteolytic cleavage of the membrane anchor that releases a soluble form into the extracellular medium [17] Examples include TGF-a, TNF-a, TNF-receptor, angiotensin converting enzyme (ACE), amyloid precursor protein,L-selectin, IL-6 receptor and also members of the LDL receptor gene family, LRP and VLDL-R [17–22] In addition, this process has been observed to be highly regulated by second messenger systems [23] A common feature is the release of the extracellular domain via a single proteolytic cleavage at a site just extracellular to the membrane spanning domain In some cases further proteo-lytic processing of the extracellular domain occurs The function of these soluble forms varies In the cases of ACE, TGFa and other mitogens the soluble protein has the

Correspondence to E D Sturrock, Division of Medical Biochemistry,

University of Cape Town, Observatory 7925, South Africa.

Fax: + 27 21 406 6470, Tel.: + 27 21 406 6312,

E-mail: sturrock@curie.uct.ac.za

Abbreviations: LDL, low density lipoprotein; LDL-R, low density

lipoprotein receptor; sLDL-R, soluble low density lipoprotein

receptor; PMA, 4b-phorbol 12-myristate 13-acetate; LPDS,

lipopro-tein deficient serum; CHO, Chinese hamster ovary; HSF, human skin

fibroblasts; TAPI, TNF-a protease inhibitor; TNF, tumor necrosis

factor; IL, interleukin; TGF, transforming growth factor; ACE,

angiotensin converting enzyme; PKC, protein kinase C.

*Present address: Ribotech Pty Ltd, Biopolymer Unit, 15 A Mail

Street, Western Province Park, Good wood 7460, South Africa.

(Received 3 September 2003, revised 27 November 2003,

accepted 2 December 2003)

potential of carrying out its function at sites remote from the

original cell surface [19] Another function may be the rapid

downregulation of the cell associated protein In other cases

the released soluble protein has been associated with

pathological conditions such as Alzheimer’s disease where

aberrant cleavage of the amyloid precursor protein forms

the soluble b-amyloid peptide which deposits in plaques

resulting in neuronal degeneration In almost all cases the

formation of these soluble proteins is greatly enhanced by

phorbol esters and inhibited by hydroxamate-based

inhi-bitors such as TAPI (TNF-a protease inhibitor) and

batimastat

In this report, we have investigated the degradation of

LDL-R in HepG2 cells and show that LDL-Rs are degraded

in part by a proteolytic mechanism which cleaves the receptor

close to the transmembrane domain, resulting in the release

of the extracellular domain as a soluble LDL-R (sLDL-R)

Like the release of many other transmembrane proteins, the

cleavage of LDL-R is markedly activated by phorbol esters

Investigations using mutant LDL-R and protease inhibitors

suggest that cleavage takes place at the cell surface and that

the mechanism is closely related to that which generates

soluble derivatives of other transmembrane proteins

Experimental procedures

Materials

All tissue culture media was from Highveld Biological,

Kelvin, South Africa Fetal bovine serum was from delta

bioproducts, Kempton Park, South Africa Human LDL

(density, 1.019–1.063 gÆmL)1) and lipoprotein deficient

serum (LPDS) (density > 1.25 gÆmL)1) were prepared

from whole, male blood, and iodinated by the iodine

monochloride method as described previously [24] IgG-C7

was prepared as described [24] from hybridoma cells

obtained from the American Type Culture Collection

(CRL 1691) Goat anti-mouse (IgG subfraction) was

from Cappel Research Products (Durham, NC, USA.) [35S]Methionine as Tran35S labelTM, and methionine/cys-teine free media were from ICN Radiochemicals (Irvine,

CA, USA) Protease inhibitors and 4b-phorbol 12-myristate 13-acetate (PMA) were from either Sigma Chemical Co or Boehringer Mannheim TAPI was from Immunex Mutant fibroblasts GM2408 (HSF-JD) and FH683 (HSF-792stop) and the monoclonal antibodies 4A4 (raised against LDL-R cytoplasmic domain) and HL-1 (raised against LDL-R ligand binding domain) were a kindly supplied by M S Brown and J L Goldstein (Dallas, TX, USA)

Generation of the cDNA constructs Construction and expression of LDL-R mutants To investigate whether endocytosis plays a role in the ecto-domain shedding of the LDL receptor, three cytoplasmic domain mutants were made (Fig 1) The mutant

792-LDL-R has a truncated cytoplasmic domain of only two amino acids due to a single base substitution (TGGfi TGA), which converts Trp792 to a stop codon The mutant receptor, JD-LDL-R (Tyr807Cys), has a single amino acid substitution that disrupts the NPVY internalization signal

To assess whether other signals in the LDL-R cytoplasmic tail influence cleavage, we constructed a third mutant (812-LDL-R), which contains a functional NPVY internalization sequence but lacks the last 27 amino acids of the cytoplasmic domain

Mutagenesis of the LDL-R cytoplasmic tail was carried out according to the method described [25] Briefly, a 2.1-kb EcoR1/Sma1 fragment was subcloned from the LDL-R expression plasmid pLDL-R2 into the bacteriophage vector, M13mp18 Mutagenic oligonucleotides and primer exten-sion were used to generate the mutated double-stranded vector according to the method of Kunkel et al [26] After sequencing, to confirm the mutations, a 1.1-kb BglII–SmaI fragment containing the cytoplasmic, transmembrane and O-linked sugar domains was subcloned into pLDL-R2

Fig 1 LDL-R constructs LDL-R mutations JD-LDL-R, 812-LDL-R and 792-LDL-R are depicted in relation to the wild-type (WT) receptor Each of the mutants is shown as an expanded view of the juxtamembrane, transmembrane and cytoplasmic domains in which all these mutations occur Above the linear bar diagram of the WT-LDL-R is the amino acid sequence with a cysteine which has been substituted for Tyr807 in JD-LDL-R indicated by an asterisk.

Transfection of the mutant plasmids into LDL-R-negative

CHO cells (CHO-A7) and selection of positive clones

were carried out according to the procedures described by

Davis et al [27]

Metabolic labeling with [35S]methionine

Semi-confluent HepG2 cells were seeded into 35-mm dishes

at a split ratio of 1 : 4 and cultured at 37C in MEM

containing 10% (v/v) fetal bovine serum After 36 h,

LDL-R activity was upregulated by replacing medium with

MEM containing LPDS (2.5 mgÆmL)1) and culturing for a

further 12 h at 37C [24] Cells were then metabolically

labeled with [35S]methionine by incubating in methionine/

cysteine-free EMEM/LPDS for 30 min and then for

2 h in methionine/cysteine-free EMEM/LPDS containing

50 lCiÆmL)1 [35S]methionine The medium was changed

to complete MEM/LPDS containing 200 lM unlabeled

methionine and incubated at 37C for various chase times

in the presence or absence of PMA After the indicated

chase periods, the medium was removed from the cells

and spun at 15 000 g for 10 min before adding one-tenth

volume of 100 mMHepes pH 7.4, 500 mMNaCl, 20 mM

MgCl2, 0.5 mM leupeptin, 10 mM phenylmethanesulfonyl

fluoride and 10% (v/v) Triton X-100 The cells were then

washed in buffer A (10 mMHepes pH 7.4, 150 mMNaCl,

2 mM CaCl2), and cell associated LDL-R solubilized in

buffer B (10 mM Hepes pH 7.4, 200 mM NaCl, 2 mM

CaCl2, 2.5 mMMgCl2, 1 mMphenylmethylsulphonyl

flou-ride, 0.02 mM Leupeptin, 1% (v/v) Triton X-100) Both

medium and cells were immunoprecipitated using

pre-formed immune complexes of the monoclonal antibody

IgG-C7 as described [28] The immunoprecipitates were

separated by SDS/PAGE, enhanced with salicylate and

visualized by fluorography, or direct detection on a Packard

Instant Imager 2024 (Packard Instrument Company)

Surface LDL-R binding of125I-labeled LDL or

125

I-labeled IgG-C7

Human LDL (density, 1.019–1.063 gÆmL)1) and the

monoclonal antibody IgG-C7 were prepared and labeled

with 125I as described [24] HepG2 cells were seeded into

35-mm dishes at a split ratio of 1 : 4 and cultured in

MEM containing 10% (v/v) fetal bovine serum After

36 h, LDL-R activity was upregulated by incubating cells

in MEM/LPDS (2.5 mgÆmL)1) for a further 12 h Surface

LDL-R activity was measured by incubating cells with

1 mL ice cold MEM/LPDS buffered with 20 mM Hepes

pH 7.4, containing either 125I-labeled LDL (10 lgÆmL)1)

or125I-labeled IgG-IgG-C7 (1 lgÆmL)1) for 2 h at 4C as

described [24] This medium was removed and cells were

washed four times with 2 mL NaCl/Pi containing 0.2%

BSA, followed by three times with 2 mL NaCl/Pi The

specifically bound fraction of 125I-labeled LDL that

remained cell associated after washing was removed by

incubating cells with 0.4% heparin for 1 h at 4C and

counted Bound 125I-labeled IgGC7 was determined by

solubilizing cells in 1M NaOH and measuring the

associated counts Nonspecific counts, determined by

adding excess unlabeled ligand, was subtracted from total

counts to give specific binding

Results

Phorbol ester enhances the release of sLDL-R The LDL receptor typically has a half-life of
10–12 h in fibroblasts and Chinese hamster ovary (CHO) cells [13,27]

To determine if LDL-R is cleaved in a similar fashion to certain other cell surface proteins, HepG2 cells were pulse-labeled with [35S]methionine and chased in the absence or presence of PMA The cells (C) and medium (M) were then immunopreciptated with LDL-R-specific antibody IgGC7 and subjected to SDS/PAGE and autoradiography (Fig 2) Immunoprecipitates of labeled HepG2 cells with the IgG-C7 antibody showed the characteristic 120-kDa precursor LDL receptor present at the start of the chase period but not

at later time points when only the 160-kDa mature receptor

is seen The addition of PMA to labeled cells resulted in a marked increase in the levels of a 140-kDa immunopreci-pitable protein in the extracellular medium The presence of this protein in the medium of untreated cells was hardly noticable (Fig 2) Recognition of this protein by immune-complexes of the IgG-C7 antibody suggest that it is a soluble form of LDL-R Immunecomplexes of a second monoclonal antibody HL-1 demonstrated equivalent results

to the IgG-C7 antibody (data not shown)

Human skin fibroblasts from a receptor negative familial hypercholesterolemia (FH) patient (NS) were analyzed in the same manner for LDL-R cleavage (Fig 3) The 140-kDa protein was absent from the culture medium of PMA-treated FH fibroblasts but present in the medium

of HepG2 cells and normal fibroblasts Downregulation

of LDL-R in the normal cells by pretreatment with 25-hydroxycholesterol resulted in the absence of any detectable 140-kDa soluble protein (Fig 3) The normal LDL-R expressed in transfected CHO cells (CHO-715) displayed significant 140-kDa protein in the medium while the parent LDL-R-negative cells (CHO-A7) showed no immunoprecipitable protein in the chase medium Taken together, these data clearly suggest the identity of the 140-kDa protein as a soluble form of LDL-R

Fig 2 Characterization of the soluble form of LDL-R induced by PMA HepG2 cells were metabolically labeled for 2 h with [35S]methionine (lane 1) followed by a 4-h chase period (lanes 2–4) in the presence or absence of PMA (100 ngÆmL)1) Both cells (C) and medium (M) were collected and immunoprecipitated with IgG-C7, followed by SDS/PAGE and autoradiography.

Based on the mobility of the soluble protein on SDS/

PAGE (140 kDa for sLDL-R vs 160 kDa for the mature

LDL-R), and the fact that a monoclonal antibody (4A4)

directed against the cytoplasmic domain of LDL-R does not

recognize the 140-kDa protein (data not shown), it is likely

that the sLDL-R consists of the bulk of the extracellular

domain of the LDL receptor This protein could be

generated either by alternative splicing of LDL-R mRNA,

eliminating sequences responsible for anchorage in the

membrane, or by proteolytic cleavage of the transmembrane

LDL-R mRNA splicing is less likely as sLDL-R was

detected only in the medium and not in the associated

cells either during or after the pulse Furthermore, soluble

receptors were only readily detected once PMA was added

to the medium and this occurred even when labeling of

newly synthesized receptors had ceased, i.e., during the

chase period Incorporation of [35S]methionine into newly

synthesized receptors ceased within 15 min of commencing

the chase as no more 120-kDa precursor LDL receptor

protein could be detected after this time period Even after

a 4-h chase period the addition of PMA stimulated the

generation of labeled soluble receptors (data not shown)

This would not be the case if mRNA splicing was the source

of the truncated sLDL-R

The rate at which sLDL-R was released from

PMA-treated and unPMA-treated HepG2 cells was assessed as shown in

Fig 4 Cells treated with PMA (closed symbols) released

sLDL-R at a rate significantly faster than untreated cells

(open symbols) The accelerated release induced by PMA

lasted for about 2 h after which the rate of release tended to

slow down The slowdown in LDL-R cleavage was not due

to sLDL-R degradation in the medium as no significant

loss of sLDL-R was detected during a 20-h incubation of

sLDL-R-containing medium at 37C (data not shown) By

2 h, 18.7% (± 3.5; n¼ 5) of the total labeled LDL-R was

detected as soluble receptor in PMA-treated cells, compared

to 4.8% (± 2.1; n¼ 5) released from untreated cells in the

same time period

The regulation of cleavage was further characterized by

determining the effect of PMA on sLDL-R release (Fig 5)

No effect was detected at PMA concentrations

< 1 ngÆmL)1and sLDL-R release was increased between

1 ngÆmL)1and 10 ngÆmL)1PMA No significant increase in

sLDL-R is seen > 10 ngÆmL)1, although in one experiment maximum release was achieved only at 30 ngÆmL)1 The response of cells to the different PMA concentrations varied between experiments as indicated by the relatively large error bars This variation is thought to be systematic as all values were either high or low depending on the experiment The requirement for an active protein kinase C (PKC) in the PMA response was established by using the PKC inhibitor staurosporine (10 lM) which almost completely abolished the enhanced release at 100 ngÆmL)1 PMA (closed dia-mond, Fig 5)

The number of surface LDL-R is affected by PMA

To assess whether PMA-stimulated release of sLDL-R alters the number of LDL-Rs on the cell surface, HepG2 cells were incubated for various times in the presence of PMA after which the number of cell surface receptors was assessed by binding of an anti-LDL-R monoclonal Ig (IgG-C7) at 4C A 1-h treatment of HepG2 cells with PMA resulted in a 30% decrease in the number of cell surface LDL-R as measured by labeled antibody (Fig 6) Incuba-ting the cells with PMA for longer periods at 37C resulted

in a reversal of the decreased receptor number seen at 1 h, such that by 4 h the number of cell surface LDL-Rs had doubled This increase is in all likelihood due to PMA stimulation of the PKC dependent, p42/44MAPKinduction

of LDL-R transcription in HepG2 cells [29,30] The initial

Fig 4 Kinetics of sLDL-R release induced by PMA HepG2 cells, metabolically labeled for 2 h with [ 35 S]methionine, were chased in unlabeled MEM/LPDS for the indicated periods in the presence (d) or absence (s) of PMA (100 ngÆmL)1) After the indicated chase periods, the medium was removed, immunoprecipitated with IgG-C7, subjec-ted to SDS/PAGE and followed by autoradiography and quantifica-tion These data represent the mean (± SEM) of duplicates from three experiments.

Fig 3 Production of sLDL-R by different cell types HepG2 cells,

normal HSF and FH HSF, were either upregulated in DMEM/LPDS

(up) or downregulated in DMEM/fetal bovine serum + 1 lgÆmL)1

25OH-cholesterol (down) for 24 h prior to metabolic labeling with

[ 35 S] methionine CHO cells transfected with human LDL-R (715) and

its LDL-R negative parent cell line (A7) were maintained in full

medium All cells were pulse-labeled with [35S]methionine for 2 h and

chased in DMEM/LPDS in the presence of PMA (100 ngÆmL)1) for

4 h The medium was removed from the dishes and subjected to

immunoprecipitation and autoradiography described.

decrease in cell surface receptor number following PMA

treatment was also seen in CHO cells transfected with

human LDL-R (CHO-715); however, in these cells the

number of surface receptors remained below the control

for an extended period and no reversal of this effect was

detected (data not shown) This is probably because the

transfected gene is not under the control of its native

promoter The loss in LDL receptor surface binding was

supported by degradation studies, which demonstrate that

PMA enhances the loss of [35S]methionine-labeled receptors

significantly (Fig 7), such that by 2 h, PMA-treated cells

have 30% less labeled receptors than untreated cells Given

the lack of steady state conditions in this experiment, it was

not possible to determine accurately the half-life of LDL-R

following PMA treatment As an estimate, the apparent

half-life of LDL-R was
2 h in PMA-treated cells

compared to 5–6 h in untreated cells The discrepancy

between soluble receptor detected in the medium (18.7%) in

2 h vs the increased loss of total receptor following PMA

treatment (40% less labeled receptors in treated cells vs

untreated cells after 2 h) is an indication that other

proteolytic pathways are also stimulated by PMA Other

degradative pathways may include the generation of the

125-kDa Band X as reported by Lehrman et al [31]

Internalization deficient LDL-R undergo increased

cleavage

While sLDL-R of 140 kDa was readily detected in the

medium of PMA-treated cells, no protein of this size was

detected in cell lysates, suggesting that receptor cleavage takes place at or near the cell surface, possibly in the endosomal compartment In order to ascertain if endo-cytosis plays a role in cleavage, two mutant LDL-Rs (792-LDL-R and JD-LDL-R) were constructed which are unable to undergo endocytosis via coated pits The muta-tions were confirmed to be internalization defective as assayed by 125I-labeled LDL uptake, with the rates of internalization being 10% and 25% of normal for 792-LDL-R and JD-LDL-R, respectively The transfected CHO cell lines, CHO-792 and CHO-JD, were pulse-labeled with [35S]methionine and chased in the presence or absence

of PMA for 2 h (Fig 8A) Both 792-LDL-R and JD-LDL-R were cleaved to a significantly greater extent than the wild-type LDL-R After the 2-h chase period in the presence of PMA,
70% of the total population of labeled 792-LDL-R was present as sLDL-R in the medium, compared to 42% of JD-LDL-R and
20% of wild-type receptors (Fig 8B) In the absence of PMA, the degree of cleavage for 792-LDL-R, JD-LDL-R and wild-type LDL-R was 40%, 11% and 6%, respectively These results indicated that endocytosis via coated pits is not required for cleavage and in fact it may inhibit cleavage of LDL-R In addition it suggested that cleavage takes place on the cell surface

The LDL-R mutant 812-LDL-R that is truncated after Thr811 (Fig 1) was used to investigate whether other

Fig 6 Effect of PMAon surface LDL-R number HepG2 cells upregulated for 12 h in MEM/LPDS were incubated at 37 C in the presence (d) or absence (s) of PMA (100 ngÆmL)1) for the indicated time period They were then cooled to 4 C and incubated for 2 h in the presence of125I-labeled monoclonal antibody IgGC7 After sub-stantial washing the remaining cell associated label was determined and normalized to cell protein in each dish The graph represents the level of IgG-C7 binding to the cell surface as a percentage of the zero hour value Error bars represent the SEM of four values.

Fig 5 Effect of PMAdose on sLDL-R production HepG2 cells were

pulse-labeled with [ 35 S]methionine and chased for 4 h in the presence

(d) or absence (s) of indicated doses of PMA, or in the presence of

100 ngÆmL)1PMA and 10 l M staurosporine (r) The medium was

immunoprecipitated and subjected to SDS/PAGE as described The

dried gels were then exposed to electronic autoradiography and

quantitation The error bars represent the range of four data points

from two experiments, with the symbol representing the mean.

signals in the LDL-R cytoplasmic tail influence cleavage.

The last 27 amino acids of the cytoplasmic domain are

required for receptor dimerization [32] and also contain a

phosphorylation site at Ser833 [33] These receptors were

shown to be internalization competent (data not shown)

Deletion of this domain had no significant effect on cleavage

(Fig 8B) showing that neither the phosphorylation site,

nor receptor dimerization appear to play any role in the

formation of the 140-kDa sLDL-R

Two of the three cytoplasmic-mutants, JD-LDL-R and

792-LDL-R occur in patients with FH To confirm the

cleavage process observed in the transfected cells, assays

were carried out using human skin fibroblasts (HSF) from

patients homozygous for these mutations Both HSF-JD

(GM2408) and HSF-792 (FH683) behaved in a very

similar fashion to their transfected CHO cell counterparts

when stimulated with PMA The percentage of labeled

receptors released as sLDL-R in a 2-h chase period

following PMA treatment was 85.9% (± 6.0; n¼ 4) for

HSF-792 compared to 70.1% (± 10.9; n¼ 6) for

CHO-792 Likewise the percentage release of the JD mutant

receptors was 42.2% (± 1.2; n¼ 4) in human skin

fibroblasts and 41.6% (± 8.9; n¼ 8) in CHO cells

Wild-type LDL receptor release was also very similar – 18.7%

for HSF and 20.7% for CHO cells The most notable

difference between CHO cells and HSF cells was that the

unstimulated release was much lower in HSF cells than in

their CHO counterparts For example, unstimulated

wild-type CHO cells released 6.6% of their LDL-R in 2 h

compared to 0.9% for wild-type HSF cells Similarly,

CHO-792 released 40.4% compared to 18.5% HSF The

phenotype of the cytoplasmic domain mutations is thus a

direct result of the mutation itself and cannot be ascribed

as an artifact of transfection and receptor overexpres-sion Furthermore, the cell surface cleavage mechanism appeared more sensitive to PMA regulation in HSF than

in CHO cells

Enzymes responsible for ectodomain shedding of cell surface proteins have remained largely unidentified except for a few proteases such as ADAM 10 and ADAM 17 (TNF-a converting enzyme or TACE) [34] From protease inhibitor studies these proteinases fall into two main classes: (a) elastase-like serine proteinases as for c-kit receptor ligands KL-1 and KL-2 [23] and (b) metalloproteinases as for ACE and TNF-a [35,36] To characterize the protease responsible for the generation of sLDL-R, HepG2 cells were pulse-labeled with35S-methionine and chased in the pres-ence of various protease inhibitors Table 1 shows the effects of multiple inhibitors on sLDL-R release Only the metalloproteinase inhibitors showed any significant inhibi-tion of release TAPI, a hydroxamate-based metallopro-teinase inhibitor, shown to inhibit the cleavage of TNF-a, IL-6R, ACE and others [37], inhibited sLDL-R production

by as much as 90% (Table 1) The other metalloproteinase inhibitors, EDTA and EGTA inhibited the release by 50–70%

Fig 8 Effect of cytoplasmic mutations on LDL-R cleavage LDL-R negative CHO cells (CHO-A7) were transfected with pLDL-R2 con-taining the requisite mutations, and stable clones were selected and seeded into 35-mm dishes After 24 h, cells were labeled with [35S]methionine for 2 h and chased in unlabeled medium in the pres-ence or abspres-ence of PMA for a further 2 h Immunoprecipitates of cells and medium were subject to SDS/PAGE and fluorography (A) Autoradiographs of respective mutants labeled as above; for each cell type: (lane 1) cells after 2-h pulse; (lanes 2 and 3) medium after 2-h pulse and 2-h chase in the absence (lane 2) or presence (lane 3) of PMA (B) The medium bands from B were quantified and expressed as a percent of total receptor label at time zero The data represents the mean (± SEM) of duplicates from four experiments.

Fig 7 Effect of PMAon cellular LDL-R turnover HepG2 cells labeled

for 2 h with [ 35 S]methionine, were first chased in unlabeled MEM/

LPDS for 1 h followed by a further chase in the presence (d) or

absence (s) of PMA (100 ngÆmL)1) for the indicated times, after which

the cells were solubilized, immunoprecipitated and subject to electronic

autoradiography and quantification The points reflect the quantity of

labeled LDL-R remaining in the cell as a percentage of the 0 time point

at the start of the chase period These data represent the mean

(± SEM) of duplicates from four experiments.

In this study we have investigated LDL-R degradation in

HepG2 cells, and report that a 140-kDa sLDL-R is released

into the medium by a proteolytic mechanism sensitive to

phorbol-ester induction and inhibited by TAPI, a

metallo-protease inhibitor Such solubilizing proteolysis occurs for

a number of transmembrane proteins, including TGF-a,

TNF-a, TNF-R, ACE, amyloid precursor protein,

L-selectin and IL-6 receptor [17–20] The release of

sLDL-R into the medium after phorbol ester induction is

accompanied by a decrease in both surface LDL-R number

(Fig 6) and total cellular LDL-R (Fig 7) A similar loss of

LDL-R binding after PMA treatment has been reported in

U937 cells but the mechanism responsible for this is

unknown [38]

The formation of sLDL-R was found in various cell types

when stimulated with phorbol esters (Fig 3) Soluble

LDL-Rs have previously been reported to be in the medium of

CHO cells that are defective in O-linked glycosylation [39],

and to be produced by cells in response to interferon [40] In

the latter case, the 28-kDa soluble receptor, which consists

of the N terminus of the receptor, has marked antiviral

activity by interfering with vesicular stomatitis virus

assem-bly and budding This 28-kDa N-terminal domain is

contained within the 140-kDa soluble receptor reported in

this study Other members of the LDL receptor family, LRP

and VLDL-R also undergo surface proteolysis to generate

soluble ectodomains In the case of VLDL-R, the soluble

fragment, as well as the corresponding region of the

LDL-R, binds minor group rhinoviruses and inhibits viral

infection in HeLa cells [21,22] The important question is

whether the cleavage mechanism responsible for the

140 kDa sLDL-R is operative in vivo, and if so, what is

the function of such soluble receptors? Apart from potential

antiviral activity, a possible function of these sLDL-R

would be to bind ligand (LDL) and thus interfere with its

uptake and clearance from the plasma by cell bound LDL-R

This occurs in the case of soluble growth factor receptors and soluble cytokine receptors, where the soluble receptors have been shown to act as antagonists by binding to their respective ligands and thereby reducing their effects [19] Such soluble complexes of receptor and ligand are reported

to stabilize the cytokine or growth factor in the extracellular fluid [41] On the other hand, some cytokines and their receptors, such as IL-6 and its receptor, can act as potent agonists on cells [42] Cleaved membrane proteins are also involved in various diseases, such as Alzheimer disease and Heymann nephritis [43] In both of these the pathology is a result of deposition of solublized membrane proteins in plaques It is tempting to speculate that sLDL-R could become deposited in atherosclerotic plaques and act as a trap for LDL at these sites Further work is needed to establish to what extent this pathway is operative in vivo and what the potential in vivo activators of this pathway might be

Transferrin receptors and asialoglycoprotein receptors also display a decrease in cell surface number in response to PMA treatment of HepG2 cells [44] In the case of the latter two receptors, reduced binding is due to redistribution of receptors to intracellular compartments [44] This redistri-bution scenario does not hold true for the LDL-R as the loss

of surface binding is equivalent to the loss of total receptors; also, the mutant LDL-R that is unable to undergo endocytosis and is thus restricted to the cell surface is more susceptible to surface cleavage than the wild-type LDL-R These mutant receptors also demonstrate an equivalent loss

of surface binding (data not shown), indicating that redistribution from the cell surface is not required for the loss in surface receptor number Furthermore, the kinetics

of LDL-R binding loss do not match the much faster kinetics of transferrin receptor and asialoglycoprotein receptor redistribution [44]

The cytoplasmic domain of LDL-R appears to contain elements that are able to modulate cleavage Receptors with

a deletion of the entire cytoplasmic domain (792-LDL-R) or

Table 1 The effect of protease inhibitors on the release of sLDL-R into the medium HepG2 cells were labeled for 2 h with [35S]methionine and then chased in DMEM/LPDS plus 100 ng mL)1PMA for a further 2 h in the presence of various protease inhibitors The medium was immuno-precipitated and subjected to SDS/PAGE and quantitative autoradiography as indicated in the methods section The degree of release was calculated as a percentage of the zero inhibitor control NA, not applicable; n, Number of experiments performed; for each experiment duplicate dishes were used for each inhibitor; SEM, Standard error of the mean.

Protease inhibitors

Concentration

of inhibitor

sLDL-R release (% of control) SEM n

3,4-dichloroisocoumarin Serine 100 l M 128 NA 2

an amino acid subsitution in the NPVY internalization

signal (JD-LDL-R) become hypersensitive to solubilizing

cleavage (Fig 8), presumably due to prolonged residence

times on the cell surface and thus more frequent exposure to

a cell surface cleaving protease This hypothesis is supported

by an inverse correlation between the ability to internalize

via coated pits (internalization index) and the sensitivity of

LDL-R to cleavage 792-LDL-R which has the lowest

internalization index (10% of normal) is cleaved at the

highest rate, whereas JD-LDL-R is internalized more

efficiently than 792-LDL-R (25% of normal) and is less

sensitive to ectodomain cleavage than 792-LDL-R, but still

cleaved to a greater extent than wild-type receptors We

cannot, however, rule out that changes in the tertiary

structure of the cytoplasmic domain are responsible for the

enhanced release In any case, the modulatory element(s)

must reside in the N-terminal 22 amino acids of the

cytoplasmic domain (Lys790–Glu812) containing the

NPVY internalization signal, as a deletion of the last 27

amino acids of the cytoplasmic tail (812-LDL-R) has no

effect on cleavage The modulatory element may in fact

be the NPXY motif itself, as cytosolic adaptor proteins

containing phospho-tyrosine binding domains are known to

interact with the NPXY motif ARH and Disabled 1 are

two cytosolic proteins that have been shown to bind to the

LDL-R cytoplasmic domain [45,46] Recent studies have

demonstrated the interaction of numerous signal

transduc-tion proteins with the cytoplasmic domains of members of

the LDL-R gene family [47]

In contrast to the large release measured for 792-LDL-R

in both CHO and HSF cells, Lehrman et al [31] did not

detect any soluble receptor in the medium of HSF-792 This

may be due to the immunoprecipitation protocol used,

as we have found that immunoprecipitation with IgG-C7

and Protein A sepharose does not precipitate any product

from the extracellular medium whereas precipitation with

immunecomplexes of IgG-C7 and goat antimouse IgG, or

IgG-C7 linked to sepharose beads precipitates significant

amounts of sLDL-R

Of the proteases responsible for the solubilizing of

surface proteins, the best characterized is TACE [48–50]

In the main, the solubilizing enzymes responsible for the

large array of ectodomain cleavages have not been

identified Two potential classes of solubilizing proteases

appear to exist, a serine class with elastase-like sequence

specificity [23], and a metalloproteinase class, of which

TACE is an example In our study, only

metalloprotein-ase inhibitors inhibited cleavage, with TAPI inhibiting

cleavage by up to 90% TAPI and other closely related

hydroxamate-based inhibitors have proved useful in

inhibiting the cleavage of proteins such as TNF-a,

L-selectin, p60 TNF receptor and IL-6 receptor as well

as a number of other cell surface proteins [36,51–54] The

specificity with which TAPI inhibits the cleavage of LDL-R

suggests a close relationship between the LDL-R cleaving

protease and the family of protease’s of which TACE is a

part This family belongs to a subgroup of adamalysin-like

metalloproteinases known as ADAMs, which contain both

a disintegrin and a metalloproteinase domain [55,56]

Our data are supported by Guo et al [57] who demonstrate

the accumulation of amongst others, sLDL-R in the

medium of TACE–/– DRM cells transfected with TACE

No sLDL-R is detected in the medium of the untransfected TACE–/– cells [57]

Recent evidence also suggests that reactive oxygen species, including nitric oxide (NO) are responsible for mediating the PMA induced activation of enzymes such as TACE, thus providing a potential natural mechanism for inducing cleavage of LDL-R in vivo [58,59]

Turnover of LDL-R in HepG2 cells is rapid with a half-life in the order of 4 h (Fig 7) compared to turnover in CHO cells and fibroblasts (t½, 10–12 h) [13] This difference may be significant in the overall regulation of cholesterol homeostasis within the liver, as the faster the rate of turnover of a protein, the more rapid is the response to transcriptional downregulation In contrast to our findings, Tam et al [60] measured the half-life of LDL-R in HepG2 cells to be 9–10 h We are unable to explain the differences

in half-life measured in the same cell type; however, evidence from circadian rhythms in rat liver LDL-R expression suggests a LDL-R half-life of
6 h in the liver [61] The enhanced LDL-R degradation measured in Fig 7 in the presence of phorbol ester, could not be completely accoun-ted for by the increased release of receptors into the medium, as the increase in [35S]methionine labeled sLDL-R

in the medium was approximately 2.5-fold less than the decrease of [35S]methionine labeled cellular LDL-R over the same time period In addition there was no significant degradation of sLDL-R in the medium We therefore conclude that PMA induces more than one degradation mechanism for LDL-R, of which the formation of sLDL-R contributes about 50% Another degradation pathway may involve the production of a 125 kDa LDL-R degradation intermediate referred to a band X, reported by Lehrman

et al [31], which is not recognized by the anti-N-terminal Ig (IgG-C7) used in this study, and is presum-ably due to cleavage of the N terminus from the remainder

of the receptor This potential pathway requires further investigation

In conclusion, LDL receptor number, at least in HepG2 cells, can be regulated not only by transcriptional control but also by enhanced turnover in response to unknown stimuli that signal via PKC This enhanced turnover involves at least two proteolytic systems, one of which involves cell surface cleavage and the release of soluble receptors into the extracellular medium Cell surface clea-vage of the LDL-R represents the first described mechanism that can regulate LDL-R expression post-translationally

Acknowledgements

We thank S Jones and L Gatterdam for excellent technical assistance This work was supported by American Heart Association grant

9750284 N (D R v.d.W.), and by the South African Medical Research Council (E D S and D R v.d.W).

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