Constitutive α-secretase cleavage of the β-amyloid precursor protein in the furin-deficient LoVo...

Journal of Neurochemistry, 2001, 76, 1532±1539

Constitutive a-secretase cleavage of the b-amyloid precursor

protein in the furin-de®cient LoVo cell line: involvement of the

pro-hormone convertase 7 and the disintegrin metalloprotease

ADAM10

Elvira Lopez-Perez,* Yue Zhang,² Stuart J. Frank,² John Creemers,³ Nabil Seidah§ andFreÂdeÂric Checler*

*IPMC du CNRS, UMR6097, Valbonne, France

²University of Alabama, Birmingham, Alabama, USA

³Centre for Human Genetics, Leuven, Belgium

§Laboratory of Biochemical Neuroendocrinology, IRCM, Montreal, Canada

Abstract

The b-amyloid precursor protein (bAPP) undergoes a physio-

logical cleavage triggered by one or several proteolytic

activities referred to as a-secretases, leading to the secretion

of sAPPa. Several lines of evidence indicate that the

a-secretase cleavage is a highly regulated process. Thus,

besides constitutive production of sAPPa, several studies

have reported on protein kinase C-regulated sAPPa secretion.

Studies aimed at identifying a-secretase(s) candidates

suggest the involvement of enzymes belonging to the pro-

hormone convertases and disintegrin families. The delineation

of respective contributions of proteolytic activities in consti-

tutive and regulated sAPPa secretion is rendered dif®cult by

the fact that the overall regulated response always includes

the basal constitutive counterpart that cannot be selectively

abolished. Here we report on the fact that the furin-de®cient

LoVo cells are devoid of regulated PKC-dependent sAPPa

secretion and therefore represent an interesting model to

study exclusively the constitutive sAPPa secretion. We show

here, by a pharmacological approach using selective inhibi-

tors, that pro-hormone convertases and proteases of the

ADAM (disintegrin metalloproteases) family participate in the

production/secretion of sAPPas in LoVo cells. Transfection

analysis allowed us to further establish that the pro-hormone

convertase 7 and ADAM10 but not ADAM17 (TACE, tumour

necrosis factor a-converting enzyme) likely contribute to

constitutive sAPPa secretion by LoVo cells.

Keywords: Alzheimer's disease, disintegrins, a1-PDX,

prohormone-convertases, sAPPa, a-secretase.

J. Neurochem. (2001) 76, 1532±1539.

The b-amyloid precursor protein (bAPP) undergoes various

proteolytic attacks by activities gathered under the generic

term of secretases. In Alzheimer's disease, the amyloido-

genic hypothesis favours the central role of an excess

production of Ab, a highly aggregative 39±43 amino acid

long peptides derived from bAPP processing by b- and

g-secretases (for review see Haass and Selkoe 1993), as the

major effector of the neuropathology. This concept implies

that any mechanism able to interfere with the amyloidogenic

pathway, i.e. to decrease or abolish Ab production, could

prove useful to prevent, slow down or arrest Alzheimer's

disease progression.

bAPP also undergoes an alternative cleavage by another

proteolytic activity called a-secretase that could be seen as a

physiological bAPP-processing enzyme since its action

leads to the production of sAPPa, a secreted bAPP

1532 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1532±1539

Received October 12, 2000; revised manuscript received November 6,

2000; accepted November 8, 2000.

Address correspondence and reprint requests to FreÂdeÂric Checler,

IPMC du CNRS, UMR6097, 660 route des Lucioles, 06560 Valbonne,

France. E-mail: [email protected]

Abbreviations used: ADAM, a disintegrin and metalloprotease;

bAPP, b-amyloid precursor protein; PBS, phosphate-buffered saline;

PC7, pro-hormone convertase 7; PDBu, phorbol 12,13-dibutyrate;

4aPDD, 4a-phorbol 12,13-didecanoate; PMA, phorbol 12-myristate

13-acetate; SDS±PAGE, sodium dodecyl sulphate±polyacrylamide gel

electrophoresis; TACE, tumour necrosis factor a-converting enzyme.

metabolite that was shown to display among other functions,

cytoprotective and neurotrophic effects (for review see

Mattson 1997). Interestingly, a-secretase activity targets

bAPP at a peptide bond located inside the Ab domain,

thereby reducing its production. In this context, among

several theoretical possibilities, targeting an increase of the

a-secretase pathway of bAPP maturation could be envi-

sioned as a therapeutic strategy.

Several studies have clearly documented that the

a-secretase pathway could be up-regulated by protein kinase

C agonists. Protein kinase C activation concomitantly leads

to decreased Ab production in various cell systems (for

review Checler 1995), as well as in transgenic mice (Savage

et al. 1998). In line with the above hypothesis, these

observations reinforce the interest in the identi®cation of the

a-secretase candidate(s).

There exists a network of biochemical and anatomical

clues suggesting that the term of a-secretase includes a set

of distinct activities. First, cell biology approaches have

clearly indicated that an a-secretase activity could be

detected at the cell surface (Sisodia 1992) but that an

intracellular pool of a-secretase occurs earlier in the late

compartments of the secretory pathway (Sambamurti et al.

1992; Kuentzel et al. 1993; De Strooper et al. 1993).

Attempts to identify the a-secretases activities have recently

led to the description of several proteolytic enzymes

belonging to distinct families. Thus, three studies indicated

that proteinases of the ADAM (a disintegrin and metallo-

protease) family, a group of enzymes involved in the

shedding of membrane-bound proteins could participate in

sAPPa production. Lammich et al. (1999) suggested that

ADAM10 was involved in the constitutive and regulated

a-secretase pathways in human kidney 293 (HEK293)

cells. By contrast, Buxbaum et al. (Buxbaum et al. 1998)

indicated that TACE (tumour necrosis a-converting

enzyme or ADAM17) selectively contributed to the

regulated a-secretase pathway in primary embryonic

®broblasts and CHO cells. Finally, Koike et al. (1999) also

demonstrated that MDC9 displays constitutive and regulated

a-secretase-like activity after coexpression with bAPP695

in CHO cells.

We established by a pharmacological approach

combined with transfection analysis that pro-hormone

convertase 7 (PC7), a novel enzyme of the subtilase

family (Seidah et al. 1996), contributed to the a-secretase

pathway in several cell systems (Lopez-Perez et al. 1999).

Thus, overexpressing the enzyme led to enhanced

secretion of sAPPa and concomitant reduction in the Ab

recovery, both effects being reversed by the convertase

inhibitor a1-PDX (Lopez-Perez et al. 1999). Finally,

Komano et al. reported on the involvement of a

glycosyl-phosphatidylinositol-anchored aspartyl protease

responsible for a-secretase-like activity in yeast (Komano

et al. 1998).

The above observations suggest a complex set of activities

contributing either alone, or in combination, to distinct

constitutive or regulated sAPPa secretion. The de®nite

delineation of the proteolytic enzymes involved in these

pathways is often dif®cult because regulated sAPPas

production always includes the basal constitutive counter-

part that can not be selectively abolished. Here we show

that LoVo cells de®cient in the furin activity (Takahashi

et al. 1993) represent a useful cell system with which to

study the constitutive sAPPa secretion processes. Thus, we

demonstrate that these cells are totally devoid of

regulated sAPPa secretion. By a pharmacological

approach using selective inhibitors, we demonstrate that

constitutive secretion of sAPPa can be blocked to various

extents by the pro-hormone convertase inhibitor a1-PDX

and by TAPI and BB3103, two distinct blockers of

ADAM-like activities. By transient transfection, we show

that overexpressing PC7 and ADAM10 triggers

increased recoveries of sAPPa while TACE remains

ineffective.

Materials and methods

Antibodies, inhibitors and pharmacological effectors

The anti a1-PDX antibody was from Sigma. MP1, a polyclonal

antibody recognizing mice and rat PC7 was obtained as described

(van de Loo et al. 1997). Antifurin (human monoclonal MON-152)

is from Alexis Biochemicals. The rabbit polyclonal AL45 labelling

TACE was previously described (Zhang et al. 2000). ADAM10

was detected with a polyclonal antibody from Euromedex. sAPPa

was immunoprecipitated with the 207 antibody (provided by

Dr M. Savage, Cephalon) and revealed by western blot with the

monoclonal 10D5 (provided by Dr D. Schenk) that recognizes the

Ab1±12 and Ab1±16 sequences that correspond to the C-terminus

of sAPPa (and that do not label the sAPPb), respectively. PMA

(phorbol 12-myristate 13-acetate), PDBu (phorbol 12,13-dibuty-

rate) and 4aPDD (4a-phorbol 12,13-didecanoate) were from

Sigma. BB3103 (hydroxamic acid-based zinc metalloprotease

inhibitor) was provided by British Biotech and TAPI (TNFa-

converting enzyme protease inhibitor was a gift from Immunex).

The pro-hormone convertase inhibitor decanoyl-RVKR-CMK was

from Bachem.

Cell culture and transfections

HEK293 and LoVo cells were cultured as previously described

(Ancolio et al. 1999), Logeat et al. 1998). Transient transfection

of LoVo cells with cDNA encoding a1-PDX, PC7, TACE or

ADAM10 (1 mg) were carried out with Lipofectamine (Lopez-

Perez et al. 1999). Three days after transfection, expression of the

proteins was assessed by western blot with the above described

antibodies. Immunological complexes were probed with protein A-

or adequate antimouse or rabbit antibodies-coupled to peroxidase

then revealed with ECL as described previously (Lopez-Perez et al.

1999).

a-Secretase cleavage of b-amyloid precursor protein 1533

q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 76, 1532±1539

sAPPa secretion and detection

HEK293 or LoVo cells were treated for 2 h in absence (control) or

in the presence of various pharmacological effectors then cells were

left for 5 h at 378C. Secretates were collected and sAPPa was

immunoprecipitated with the 207 antibody (1 mL/mL, overnight).

After addition of protein A-sepharose and centrifugation, pellets

were resuspended and analysed by 8% sodium dodecyl sulphate±

polyacrylamide gel electrophoresis (SDS±PAGE) then western

blotted by means of 10D5 as described below.

SDS±PAGE and western blot analysis

Dried samples and standards were resuspended in 30 mL of 50 mm

Tris, pH 6.8 containing 2% sodium dodecyl sulphate (SDS), 10%

glycerol and 5% of b-mercaptoethanol (Laemmli buffer). Samples

were then heated for 5 min at 958C, electrophoresed for 2 h (at 48C)

at 100 V then proteins were blotted onto nitrocellulose sheets

(Hybond-C super, Amersham). Membranes were then incubated for

1 h in PBS20.05% Tween containing 5% skimmed milk, then

exposed overnight with antibodies in PBS-0.05% Tween containing

5% skim milk. Nitrocellulose sheets were rinsed in phosphate-

buffered saline (PBS; 3 � 5 min) and immunological complexes

were revealed as previously described (Lopez-Perez et al. 1999).

Concentration of proteins

Concentration of proteins was carried out by the Bio-Rad method

(Bradford 1976).

Results

Lack of phorbol esters-stimulated sAPPa secretion in the

furin-de®cient LoVo cell line

As previously reported (Marambaud et al. 1997), HEK293

cells secrete a 110±120 kDa product (Fig. 1a), the immuno-

logical characterization of which clearly indicated that it

corresponded to sAPPa, the N-terminal bAPP metabolite

generated upon a-secretase attack of bAPP. As expected,

the secretion of sAPPa is highly enhanced (more than 250%

above control, Fig. 1b) by treatment of HEK293 cells by

two distinct phorbol esters, PMA and PDBu but not by the

inactive analogue a-PDD (Figs 1a and b). Interestingly, the

furin-de®cient LoVo cell line secreted an identical 110±

120 kDa bAPP-metabolite, the production of which is not

statistically signi®cantly affected by the two phorbol esters

(Figs 1c and d). Altogether, these data show that LoVo cells

are devoid of protein kinase C-regulated a-secretase path-

way and therefore, represents a suitable cellular model to

study in depth the proteolytic events responsible for basal

constitutive sAPPa secretion.

Involvement of a Dec-RVKR-CMK and a1-PDX-sensitive

convertase-like activity in the constitutive

sAPPa-secretion by LoVo cells

In order to examine the possible involvement of conver-

tase(s)-like activities in the sAPPa secretion in LoVo cells,

Fig. 1 LoVo cells are devoid of phorbol

esters-regulated sAPPa secretion. HEK293

cells (a and b) and LoVo cells (c and d)

were cultured as described in the methods

in the absence or in the presence of the

indicated effector. sAPPa secretion was

monitored by immunoprecipitation with 207

antibody, western blot with 10D5C and

revelation of the immunological complexes

as described in the Materials and methods

section. Densitometric analyses (b and d)

correspond to the means ^SEM of seven

experiments and values are expressed as

the percent of sAPPa recovered in control

conditions (taken as 100). PMA (phorbol

12-myristate 13-acetate, 1 mM), PDBu

(phorbol 12,13-dibutyrate, 1 mM), 4aPDD

(4a-phorbol 12,13-didecanoate, 1 mM).

Fig. 2 Effect of Dec-RVKR-CMK and a1-PDX expression on consti-

tutive sAPPa-secretion by LoVo cells. LoVo cells were cultured in

absence (c) or in the presence of the indicated concentrations of

Dec-RVKR-CMK (panel a) or transiently transfected with pcDNA3

empty vector (±) or containing (1) a1-PDX cDNA (panel b). sAPPa

secretion was monitored as in the Fig. 1. a1-PDX expression

(b, upper panel) was assessed by western blot as described in the

Methods section.

1534 E. Lopez-Perez et al.


we examined the effect of Dec-RVKR-CMK, a general

convertase inhibitor (Sato et al. 1999). Figure 2(a) shows

that Dec-RVKR-CMK strongly reduces the recovery of

sAPPa. We also overexpressed a1-PDX in LoVo cells

(Fig. 2b). This inhibitor of protein convertases of the

subtilisin/kexin family drastically diminished the recovery

of sAPPa (Fig. 2b). The two convertase-blocking agents did

not modify full length bAPP-like-immunoreactivity (not

shown). Altogether, these data ®rmly indicate that a pro-

hormone convertase, other than furin, likely contributes to

the a-secretase constitutive pathway in LoVo cells. It should

be noted that a high concentration of Dec-RVKR-CMK or

overexpression of a1-PDX did not fully abolish sAPPa-

secretion (Fig. 2).

Overexpression of PC7 leads to a drastic increase

of phorbol esters-insensitive sAPPa secretion by

LoVo cells

We have overexpressed PC7 in LoVo cells (Fig. 3a). In non

stimulated conditions, overexpression of PC7 increases by

more than two times, the recovery of sAPPa (Figs 3b and c).

Figure 3(b) further con®rms the lack of effect of PMA and

PDBu and indicates that overexpression of PC7 does not

restore the responsiveness of LoVo cells to phorbol esters

(Figs 3b and c). Altogether, these data indicate that PC7

could participate to constitutive sAPPa secretion in LoVo

cells.

Effect of TAPI and BB3103 on the constitutive sAPPa

secretion by LoVo cells

Several inhibitors of proteases belonging to the disintegrin

family have been examined as putative blockers of sAPPa

secretion by LoVo cells. TAPI (IC3) was documented as a

potent inhibitor of various proteases belonging to either

ADAM or matrixin families (Black et al. 1997) and

BB3103 is an hydroxamate-based inhibitor of zinc

metalloproteases. Both inhibitors dose-dependently inhibit

the secretion of sAPPa by LoVo cells (Fig. 4a) without

affecting full-length bAPP-like immunoreactivity (not

shown). BB3103 drastically reduced sAPPa recovery at a

100 mm concentration while TAPI appears less potent

(Fig. 4a). This is further supported by the comparison of

the effect of submaximally effective doses (10 mm) of the

two inhibitors (Figs 4b and c). Furthermore, our data

indicate that in the latter conditions, BB3103 and TAPI did

not trigger additive effects (Fig. 4b and c), indicating that

the two agents likely target an identical proteolytic activity.

Here again, it should be emphasized that even at saturating

concentrations, the inhibitors did not completely prevent

sAPPa secretion.

Fig. 3 Effect of PC7 overexpression on constitutive sAPPa secre-

tion by LoVo cells. LoVo cells were cultured and transiently trans-

fected with empty pcDNA3 vector (M) or PC7 cDNA (T) as

described in the Materials and methods section. Three days after

transfection, cells were incubated for 5 h in absence (basal) or in

the presence of the indicated effector then sAPPa was monitored as

in Fig. 1 (panel b). Cells were checked for their PC7 expression by

western blot as described in the Materials and methods section

(panel a). Bars in c represent the mean densitometric analyses of

sAPPa recoveries ^SEM of three experiments expressed as the

percent (taken as 100) of sAPPa obtained in basal conditions.

Fig. 4 Effect of BB-3103 and TAPI compounds on constitutive

sAPPa secretion by LoVo cells. LoVo cells were cultured in absence

(c) or in the presence of the indicated concentrations of BB-3103 or

TAPI (panel a). In b, cells were incubated without (±) or with BB-

3103 and TAPI that were used at a submaximal concentration

(10 mM), alone or in combination. sAPPa secretion was monitored as

in the Fig. 1. Densitometric analysis (c) of experiments described in

b corresponds to the means ^SEM of four experiments and values

are expressed as the percent of sAPPa recovered without inhibitors

(taken as 100).



The overexpression of ADAM10 but not TACE increases

BB3103-sensitive sAPPa secretion by LoVo cells

Transient transfections of ADAM10 and TACE cDNAs in

LoVo cells led to the overexpression of both proteins, the

immunological characterization and molecular weights of

which corresponded to ADAM10 (Fig. 5a) and TACE

(Fig. 5e), respectively. LoVo cells expressing ADAM10

secreted a higher amount (185% over control value) of

sAPPa (Figs 5b±d) while overexpressing TACE did not

signi®cantly affect sAPPa recovery (Figs 5f±h). Interest-

ingly, the ADAM10-induced increase in sAPPa secretion

was drastically lowered but not fully abolished by cell

treatment with both BB3301 (Fig. 5b) and TAPI

(Fig. 5c).

Discussion

The processing of the bAPP is a highly regulated process.

Several studies have clearly documented the fact that,

besides other regulatory mechanisms, phosphorylation

events contributed to the modulation of the a-secretase

cleavage of bAPP giving rise to sAPPa. Thus, numerous

independent works demonstrated that protein kinase C

agonists consistently increased the recovery of secreted

sAPPa in various cell lines (Caporaso et al. 1992; Gillespie

et al. 1992; Fukushima et al. 1993; LoÈf¯er and Huber 1993;

Slack et al. 1993; Dyrks et al. 1994; Efthimiopoulos et al.

1994). This a-secretase pathway appears of most impor-

tance because in most of the cases, the sAPPa increase is

accompanied by a concomitant decrease of Ab recovery

(Buxbaum et al. 1993, Gabuzda et al. 1993, Hung et al.

1993, Jacobsen et al. 1994). Thus, a strategy aimed at

amplifying the a-secretase cleavage by pharmacological

probes targeting putative upregulators of such pathway or

directly activating the a-secretase(s) themselves could be

envisioned. The identi®cation of the a-secretase(s) candi-

date(s) appears obviously a key milestone of such a

pharmacological approach.

Several papers suggest that there exist several a-secretase

activities that can be biochemically and anatomically

distinguished. Subcellular localization studies coupled to

cell biology approaches indicate that there are at least two

distinct pools of a-secretase, one located at the cell surface

while another counterpart occurs in late compartments of

the Golgi network (Checler 1995). Studies on the PKC-

regulated a-secretase pathway also revealed that proteolytic

activities involved in such a response can be distinguished

from the a-secretase responsible for constitutive sAPPa

secretion.

Recent papers have described protease candidates ful®lling

the properties of a-secretases. ADAM17 (TACE, tumour

necrosis factor a-converting enzyme) was shown to contri-

bute to the PKC-regulated a-secretase pathway (Buxbaum

et al. 1998). This was later contradicted by a study showing

that ADAM10 not only participates in the constitutive

a-secretase pathway but also contributes to the regulated

sAPPa secretion (Lammich et al. 1999). These two pro-

teolytic activities could account for the membrane bound

a-secretase activity as disintegrins are typical transmem-

brane proteases involved in proteins shedding (Turner and

Hooper 1999). Concerning the intracellular a-secretase

activity, we recently documented the fact that a novel pro-

hormone convertase, namely PC7 displays all the expected

Fig. 5 Effect of ADAM10 and TACE over-

expressions on constitutive sAPPa secre-

tion by LoVo cells. LoVo cells were cultured

and transiently transfected with empty

pcDNA3 vector (M), ADAM10 cDNA (T,

panels a±d) or TACE cDNA (T, panels

e±h) as described in the Methods section.

Three days after transfection, cells were

incubated for 5 h in absence (control) or in

the presence of BB-3103 (b and f) or TAPI

(c and g) then sAPPa was monitored as in

Fig. 1 (panels b, c, f and g). Cells were

checked for their ADAM10 (a) or TACE (e)

expression by western blot as described in

the Methods section. Bars in d and h repre-

sent the mean densitometric analyses of

sAPPa recoveries ^SEM of three experi-

ments expressed as the percent (taken as

100) of sAPPa obtained in basal conditions

for ADAM10 (d) and TACE (h) transfected

cells.



properties of the intracellular a-secretase (Lopez-Perez et al.

1999). However, the identity and relative contribution of the

above putative a-secretases candidates remain to be ®rmly

established.

We established that LoVo cells that are de®cient of furin

activity display the remarkable property of being devoid of

phorbol ester-stimulated sAPPa secretion (Fig. 1). This

unique feature allowed us to examine deeply and exclu-

sively the constitutive a-secretase pathway with no kinase-

mediated contribution. We showed clearly that enzyme(s) of

the convertase family, other than furin, are involved in the

constitutive secretion of sAPPa. First, a general inhibitor of

convertases, Dec-RVKR-CMK, diminishes sAPPa secretion

by LoVo cells. Second, overexpressing the mammalian

dibasic subtilase inhibitor a1-PDX also led to drastic reduc-

tion of sAPPa. These data agree well with our previous

work, demonstrating that a1-PDX prevented constitutive

sAPPa-secretion in HEK293 cells. Furthermore, the fact

that a1-PDX inhibitor increases Ab secretion concomitantly

with decreased sAPPa in HEK293 cells (Lopez-Perez et al.

1999), indicates that a1-PDX effect on the latter product

was not simply due to a general perturbation of a secretory

process that would lead to a general decrease in secreted

proteins.

The non exclusive pharmacological spectrum of this

serpin did not allow per se the formal identi®cation of the

convertase(s) involved, since HEK293 display a panel of

various a1-PDX-sensitive convertases. Among them, furin

appeared as a possible candidate but the fact that a1-PDX

was also effective in the furin-de®cient LoVo cell line

suggests that another convertase could be involved. The

characterization of the set of convertases present in LoVo

cells has revealed that only PACE4 and PC7 were the known

identi®ed convertases (Seidah et al. 1994; Seidah and

ChreÂtien 1999). It is striking that PC7 was shown to increase

sAPPa secretion in HEK293 cells, in an a1-PDX-sensitive

manner (Lopez-Perez et al. 1999). This prompted us to

overexpress PC7 in LoVo cells leading to a drastic

potentiation of the constitutive sAPPa secretion. Therefore,

our data clearly document that an a1-PDX-sensitive

convertase, distinct from furin, contributes to the constitu-

tive sAPPa secretion in LoVo cells and that PC7, that is one

of the few convertases present in the LoVo cells, displays

a-secretase-like activity. It is important to emphasize the

fact that we demonstrated that PC7 overexpression does not

restore the sensitivity of sAPPa secretion to phorbol esters,

further supporting the fact that endogenous PC7 was not

involved in the PKC-regulated a-secretase response.

Preliminary data from our group indicated that complemen-

tation of the furin de®ciency by overexpression of the

enzyme to some extent restores the phorbol ester-sensitive

response in LoVo cells (data not shown).

BB-3103 and TAPI are two inhibitors, originally

described as blockers of metalloproteases, in particular the

two zinc metalloenzymes belonging to the disintegrin family

ADAM10 and TACE (Turner and Hooper 1999). In LoVo

cells, both agents trigger a dose-dependent reduction of con-

stitutive sAPPa secretion. The effect of the two inhibitors on

sAPPa secretion was not additive, indicating that in LoVo

cells, an identical activity similarly sensitive to BB3103 and

TAPI (although the latter agent was slightly less effective)

contributed to sAPPa secretion. This suggests that these

inhibitors do not display an exclusive selectivity towards a

given proteolytic activity and therefore do not per se identify

the secretase candidate. In this context, we overexpressed

ADAM10 and TACE in LoVo cells to examine a potential

in¯uence on sAPPa secretion. Our data clearly show that

ADAM10 expression enhanced the recovery of sAPPa

while TACE was totally ineffective. It is interesting to note

that the ADAM10-induced increase in sAPPa secretion was

prevented by both BB3103 and TAPI, indicating that

besides its expected pharmacological spectrum, TAPI also

behaves as an inhibitor of ADAM10, thus reinforcing the

hypothesis of a contribution of ADAM10 in the endogenous

constitutive sAPPa secretion by LoVo cells. These data

agree well with previous studies indicating that ADAM10

contributes to constitutive sAPPa secretion in HEK293 cells

(Lammich et al. 1999). Furthermore, Buxbaum et al. (1998)

showed that constitutive sAPPa secretion was not affected

by TACE gene disruption in primary embryonic ®broblasts,

in agreement with a study by Parvathy et al. (1998) demon-

strating that ADAM17 did not contribute to constitutive

a-secretase-mediated breakdown of bAPP.

Altogether, our data suggest that PC7 and ADAM10

contribute to constitutive secretion of sAPPa in LoVo cells.

It is interesting to note that ADAM10 occurs mainly at the

plasma membrane level (Lammich et al. 1999) while PC7 is

located intracellularly in the trans-Golgi network compart-

ment, but could cycle to the cell surface and back (Seidah

et al. 1996). Therefore, ADAM10 and PC7 are present in

cell compartments reminiscent of those suspected to display

membrane-bound and intracellular a-secretase, respectively.

Are PC7 and ADAM10 cleaving bAPP itself? This

question is in fact virtually impossible to solve conclusively.

Thus, whatever the type of approach, i.e transgenesis,

knock-in or knock-out, both direct or indirect PC7- or

ADAM10-mediated proteolytic event ultimately would

lead to an identical phenotypic increased APPa secretion.

Because PC7 is a proprotein converting enzyme, it could be

hypothesized that this enzyme targets an intracellular

intermediate located upstream to a-secretase such as a

pro-a-secretase. In this context, it is noteworthy that

ADAM10 and TACE exist as pro-proteases that can be

activated by convertase-like activities (Seidah et al. 1998).

The fact that LoVo cells did not display the phorbol esters'

sensitive sAPPa secretion could be due to the lack of

pro-TACE maturation in absence of furin. By contrast,

the increase of sAPPa recovery upon overexpression of



pro-ADAM10 indicates that the proteolytic machinery

necessary for its maturation is present in LoVo cells and

that the convertase involved in this process is distinct from

furin. This maturation occurs likely in the Golgi apparatus, a

cell compartment enriched in PC7 (Seidah et al. 1996).

Therefore, we cannot rule out the possibility that PC7 acts

upstream to a-secretase as a pro-ADAM10 converting

enzyme. In this case, as a1-PDX does not fully prevent

sAPPa secretion, this would imply either that this serpin

cannot completely block this activity or the occurrence of

another proteolytic activity independent of the convertase

activation. This hypothesis agrees well with the fact that

BB3103 and TAPI do not trigger complete inhibition of

sAPPa secretion. Alternatively, it remains possible that PC7

processes an unknown proteolytic activity distinct from

ADAM10 and insensitive to BB3103 and TAPI. Work is

currently in progress in our laboratory to address these

issues.

Acknowledgements

TAPI and TACE cDNA were generously provided by Dr

R. Black (Immunex) and BB3103 was a kind gift from British

Biotech. ADAM10 cDNA was a kind gift from Dr C. Lunn

(Sherring Plough). We are indebted to Dr M. Savage

(Cephalon) and Dr D. Schenk (Elan Pharmaceutical) form

providing us with the 207 and 10D5 antibodies, respectively.

aPDX was kindly provided by Dr G. Thomas (Volum Institute,

Portland) and LoVo cells were kindly given by Dr A. Israel

(Institut Pasteur, Paris). ELP is a recipient of a training grant

from the European Community (N8 BMH4±98±5082). NGS

was supported by a grant from the Medical Research Council of

Canada (MT-14466) which is part of an MRC grant (MGC

11474). SJF is supported in part by a Merit Review grant from

the US Department of Veterans Affairs. This work was

supported by the Institut National de la SanteÂ et de la

Recherche MeÂdicale and the Centre National de la Recherche

Scienti®que.

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Constitutive α-secretase cleavage of the β-amyloid precursor protein in the furin-deficient LoVo...

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