E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

The shedding activity of ADAM17 is sequestered in lipid rafts

Edwige Tellier, Matthias Canault, Laure Rebsomen, Bernadette Bonardo,

Irène Juhan-Vague, Gilles Nalbone, Franck Peiretti⁎
Inserm, U626, Marseilles, France
Université de la Méditerranée, Faculté de Médecine, 27 Boulevard Jean Moulin, Marseilles 13385 Cedex 5, France

ARTICLE INFORMATION ABS T R AC T

Article Chronology: The tumor necrosis factor-alpha (TNF) converting enzyme (ADAM17) is a metalloprotease-
Received 27 June 2006 disintegrin responsible for the cleavage of several biologically active transmembrane
Revised version received proteins. However, the substrate specificity of ADAM17 and the regulation of its shedding
28 August 2006 activity are still poorly understood. Here, we report that during its transport through the Golgi
Accepted 31 August 2006 apparatus, ADAM17 is included in cholesterol-rich membrane microdomains (lipid rafts)
Available online 5 September 2006 where its prodomain is cleaved by furin. Consequently, ADAM17 shedding activity is
sequestered in lipid rafts, which is confirmed by the fact that metalloproteinase inhibition
Keywords: increases the proportion of ADAM17 substrates (TNF and its receptors TNFR1 and TNFR2) in
ADAM17 lipid rafts. Membrane cholesterol depletion increases the ADAM17-dependent shedding of
TACE these substrates demonstrating the importance of lipid rafts in the control of this process.
TNF-alpha Furthermore, ADAM17 substrates are present in different proportions in lipid rafts, suggesting
TNF receptors that the entry of each of these substrates in these particular membrane microdomains is
Cholesterol specifically regulated. Our data support the idea that one of the mechanisms regulating
Shedding ADAM17 substrate cleavage involves protein partitioning in lipid rafts.
© 2006 Elsevier Inc. All rights reserved.

Introduction [5]), imposing this enzyme as a centerpiece in mammalian cell

ADAMs (proteins containing A disintegrin and metallopro-
tease domain) are a large family of type I transmembrane
glycoproteins. ADAMs have been implicated in numerous
cellular processes [1,2], some of which involve shedding of
cell-surface molecules. The best-characterized ADAM protein-
ase is ADAM17 (also known as tumor necrosis factor-alpha
(TNF) converting enzyme (TACE)), originally discovered as the
processing protease of precursor TNF [3,4]. Subsequently,
ADAM17 was shown to be involved in the cleavage of a wide
diversity of ligands and receptors (reviewed by Smalley et al.
biology. The fact that the genetic inactivation of ADAM17 was
perinatally lethal for homozygous knockout mice [6] exempli-
fied its key role in cell biology and organism development.
No consensus sequence elements were found after align-
ment of the cleavage sites of ADAM17 substrates, which would
indicate that the sequence specificity is not primordial for the
cleavage of ADAM17 substrates. A model was proposed in
which ADAM17 would cleave an ectodomain as long as the
distance of the segment between the membrane plane and the
globular portion of the substrate ectodomain allows for
accessibility to the cleavage site [7]. Such a model could

⁎ Corresponding author. Inserm U626, Faculté de Médecine, 27 Boulevard Jean Moulin, Marseilles 13385 Cedex 5, France. Fax: +33 4 91 25 43
36.
E-mail address: Franck.Peiretti@medecine.univ-mrs.fr (F. Peiretti).

0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.yexcr.2006.08.027
3970 E XP E RI ME N TA L CE LL RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
explain why the transfer of the cleavage site from one protein
to another may either result in spontaneous or largely
diminished shedding [8,9]. In these conditions, one way to
consider a specific regulation of ADAM17 substrate release is to
envisage a precise regulation of the spatial and temporal
expression of ADAM17 and its substrates. A substrate will be
released only if, at a given time point, it is colocalized with
activated ADAM17. Thus, the sequestration of the activated
enzyme together with the substrate in particular cell compart-
ments could be considered as an efficient way to regulate the
shedding process. In favor of this notion, we previously
demonstrated that an increased transport of ADAM17 sub-
strates from their Golgi apparatus pool towards the cell surface
plays a major role in the stimulated shedding of ADAM17
substrates [10]. Recently, it was shown that the shedding of
some ADAM17 substrates like the amyloid precursor protein
[11], CD30 [12], interleukin-6 receptor [13], L-selectin [14] and
collagen XVII [15] could be increased by cholesterol-lowering
drugs, which suggests that membrane microdomains rich in
cholesterol may play a crucial regulatory role in the ectodo-
main shedding of ADAM17 substrates.
The cholesterol-rich membrane microdomains, also called
“lipid rafts”, are assemblies of sphingolipids and cholesterol
within the membrane. They are fluid but tightly packed and
well ordered, representing the liquid ordered phase that floats
around in the liquid disordered matrix of the entire membrane
[16]. Proteins exhibit different association capacities to these
cholesterol-rich microdomains, and their incorporation in
lipid rafts is a dynamic process [17]. The distribution of specific
membrane proteins between the lipid raft and non-raft phases
and their concentrations in lipid rafts regulate their interac-
tions and influence their functions [18].
In this work, we show that the furin-dependent maturation
of ADAM17 occurs in the cholesterol-rich membrane micro-
domains. As a consequence, the mature form of ADAM17 is
concentrated in lipid rafts, where its substrates (TNF and its
receptors TNFR1 and TNFR2) are cleaved. The importance of
lipid rafts in the regulation of ADAM17 substrate shedding is
revealed by the fact that lipid rafts disruption increases the
shedding of TNF, TNFR1 and TNFR2.
Materials and methods
Materials
Metalloproteinase inhibitor RU 36156 active on ADAM17 [19]
was donated by Dr. S. Roman-Roman (Aventis Pharma,
Romainville, France). Phosphatase inhibitor cocktail 2, α-
cyclodextrin, methyl-β-cyclodextrin (CD), cholesterol-loaded
cyclodextrin, filipine III, cholesterol oxidase, sphingomyeli-
nase, PMA, calcium ionophore A23187 and LPS from Escherichia
coli serotype 026:B6 (LPS) were from Sigma. Protease inhibitor
cocktail Complete EDTA-free was from Roche. The proprotein
convertase inhibitor decanoyl-RVKR-chloromethylketone was
from Bachem. ADAM17 polyclonal antibody, TIMP3, recombi-
nant human ADAM17 and the fluorogenic peptide III were from
R&D Systems. Caveolin-1 and Flotilin-1 polyclonal antibodies
were from Santa Cruz Biotechnology. FLAGm2 polyclonal
antibody was from AffinityBio Reagents.
Isolation of lipid rafts
Lipid rafts were isolated by sucrose density gradient centrifu-
gation of cells treated with non-ionic detergents. Approxi-
mately, 9 × 106 cells were washed with ice-cold PBS and then
harvested by scraping in PBS. The cells were collected by
centrifugation, suspended in 900 μl of “raft buffer” [20 mM
HEPES, pH 7.4, 150 mM NaCl, 1 mM EGTA, with protease,
phosphatase and metalloproteinase inhibitors], snapped fro-
zen in liquid nitrogen and thawed on ice. The suspension was
passed 10 times through a 25-gauge needle. Lysis with Triton
X-100 or Tween 20 was performed by adding these detergents
at a final concentration of 1% and 2% (v/v), respectively. The
cell suspension was then incubated on ice for 20 min under a
gentle agitation. Lysis with Brij 98 was performed by equili-
brating the cell suspension at 37°C, followed by addition of Brij
98 to a final concentration of 1% (w/v). The incubation was
carried out at 37°C for 7 min, then the sample was kept on ice.
One milliliter of the lysate was combined with an equal
volume of 90% (w/v) sucrose, transferred to 12 ml ultracentri-
fuge tubes and overlaid with a discontinuous sucrose gradient
comprised of 4 ml of 35% sucrose, followed by 2 ml of 5%
sucrose. Separation of the low-density lipid rafts was achieved
by centrifugation at 280,000×g in a swinging bucket rotor for
18–22 h at 4°C. Following centrifugation, 0.5 ml fractions were
harvested from the top of the gradient. Aliquots of selected
fractions were analyzed by immunoblot or by ELISA. Separa-
tion of lipid rafts was also performed from organs collected
from mice (C57/BL6j). Investigations were approved by the
local ethic committee of the Faculté de Médecine of Marseille.
Mice were killed by excessive anesthesia using isoflurane and
liver, heart and spleen were collected. They were frozen in
liquid nitrogen, pulverized using a steel beads shaker,
incubated 1 h at 4°C with the “raft buffer”. The homogenate
was treated as described above for lipid raft preparation using
Brij 98. In some cases, instead of performing lipid raft
separation on sucrose density gradient, the Brij 98 cell lysate
was centrifuged (1,000×g, 10 min, 4°C) to remove nuclei and
cellular debris. Then, the resulting supernatant was submitted
to high-speed centrifugation (100,000×g, 1 h, 4°C ) to pellet the
Brij 98 insoluble material corresponding to the total lipid raft
fraction. The pellet was washed with the “raft buffer”, then
solubilized in the SDS–PAGE sample buffer for immunoblot
analyses or in the “raft buffer” containing 60 mM of octyl beta-
D-glucopyranoside for ELISA analyses.
Isolation of the cytoskeleton fraction
Cytoskeleton-rich fraction was isolated as previously de-
scribed [20]. This fraction was solubilized in the SDS–PAGE
sample buffer for immunoblot analyses or incubated with 1 ml
of “raft buffer” containing 0.6 M KI (30 min, 4°C) in order to
depolymerize actin filaments [21], mixed to 1 ml of 90%
sucrose solution and submitted to lipid raft isolation.
Expression vectors
Expression vectors for human TNF, TNFR1 and TNFR2 were
described elsewhere [10,22]. Expression vector for human
furin-flag (pcDNA3) was given by Dr. G. Thomas (Portland, OR).
 E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
Cell culture, microvesicle preparation and transfection
The monkey kidney fibroblast cell line COS-7, the rat
cardiomyoblast cell line H9C2, the human colonic epithelial
cell lines HT29 and LoVo cells and the human monocytic cell
line THP-1 were cultured as suggested by ATCC (http://www.
lgcpromochem-atcc.com/). Microvesicles (MVs) were genera-
ted by stimulating THP-1 (5 × 106 cells/ml) with 5 μM calcium
ionophore A23187 for 15 min and isolated as previously
described [23]. Transient transfections were performed with
Polyfect reagent (Qiagen) essentially as described by the
manufacturer. The stable ectopic expression of TNF in the
human endothelial cell line ECV-304 (ECVTNF) and their culture
conditions were described previously [10].
Fluorogenic assays of metalloprotease activity
THP-1 MVs were added to the activity buffer [25 mM Tris/HCl pH
8.0, 2.5 μM ZnCl2] containing 10 μM of pro-TNF mimetic fluo-
rogenic peptide III that was designed as an ADAM17 substrate.
Its fluorescence-related enzymatic cleavage was monitored at
320 nm excitation and 405 nm emission wavelength using a
microplate fluorescence reader (Chameleon™, Hidex). Recom-
binant human ADAM17 was used as the positive control. Blank
(buffer and MVs and substrate, separately) was subtracted from
sample measurements for calculations.
Protein assays
Total amounts (free and bound forms) of transmembrane and
soluble forms of human or mouse TNF, TNFR1 and TNFR2 and
transmembrane human ICAM-1 were assayed according to
the specifications of their respective enzyme-linked immu-
nosorbent assays (ELISA) kits (R&D Systems).
Statistical analyses
Isolations of lipid rafts by sucrose density gradient centrifu-
gation were repeated at least three times and a representative
experiment is presented. All other experiments were per-
formed in triplicate and repeated at least three times. All data
are expressed as the mean ± SD. Treatments were compared
with their respective controls and statistical calculation of the
mean differences was performed with the two-tailed t-test. A
value of p < 0.05 was considered statistically significant.
Results
The mature form of ADAM17 is contained in lipid rafts
On a physicochemical point of view, lipid rafts could be
defined as membrane domains that are resistant to extraction
in mild neutral detergents and that float in the upper half of a
5–30% sucrose density gradient. However, it is well admitted
that lipid rafts obtained with different detergents differ
considerably in their protein and lipid contents [24]. To avoid
misinterpretation linked to the ability of the detergent to
selectively solubilize membrane proteins, we separated lipid
rafts from ECVTNF cells (endothelial cells overexpressing TNF)
3971
using three different detergents. The very selective Triton X-
100, the less selective Tween 20 and the intermediate Brij 98
[24]. When Triton X-100 was used, the majority of the proform
and mature form of ADAM17 as well as the widely used lipid
raft marker caveolin-1 were detected in the gradient fractions
of the highest density (Fig. 1A). Minute amounts of the mature
form of ADAM17 and caveolin-1 were detected in the light
density gradient fractions corresponding to the region of lipid
raft flotation (Fig. 1A). The large amount of caveolin-1 in the
high-density gradient fractions suggests that Triton X-100
solubilized a main part of the proteins contained in lipid rafts.
Using Tween 20 or Brij 98, we found that most of the mature
form of ADAM17 and caveolin-1 were detected in the light
density gradient fractions and minor amounts of these
proteins were detected in the highest density gradient
fractions (Fig. 1A). The large amount of caveolin-1 in the
light density gradient fractions suggests that these two
detergents maintain the lipid raft cohesion and are thus
adapted for lipid raft separation. The proform of ADAM17 was
exclusively detected in gradient fractions of the highest
density. These results strongly suggest that a part of the
mature form of ADAM17 is contained in lipid rafts. In the
Fig. 1 – The mature form of ADAM17 is sequestered in lipid
rafts. (A) ECVTNF cell lysates were prepared as indicated in the
Materials and methods section using Triton X-100, Tween 20
or Brij 98. Where indicated, cells were treated for 30 min with
1% methyl-β-cyclodextrin (CD) prior the harvesting. After
fractionation by sucrose density gradient centrifugation,
ADAM17 and caveolin-1 (Cav1) were detected by immunoblot
in the indicated gradient fractions. (B) Cells were left
untreated (Cont) or treated for 3 h with 80 nM of PMA, 10 μM
of RU 36156 (RU) or both (PMA+RU), prior the harvesting.
Indicated fractions were pooled and ADAM17 and caveolin-1
(Cav1) were detected by immunoblot. Pro and mature forms
of ADAM17 are indicated on the right hand side of the blots by
p and m, respectively.
3972 E XP E RI ME N TA L CE LL RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
course of the work, separations of lipid rafts were made using
Brij 98 because it solubilizes disordered membrane at 37°C,
which suggests that isolated lipid rafts possess the biochem-
ical characteristics of those present in the cell membrane at
the physiological temperature [25].
To prove that the mature form of ADAM17 detected in the
light density gradient fractions was indeed integrated in
cholesterol-rich membrane microdomains, we disrupted lipid
rafts and analyzed the repartition of the mature form of
ADAM17 in a sucrose gradient. For this purpose, cells were
incubated for 30 min with the cholesterol lowering/raft
disrupter agent methyl-β-cyclodextrin (CD), which resulted
in the loss of about 60% of the cellular cholesterol (data not
shown) without altering the viability of the cells within this
time period. In this situation, the mature form of ADAM17 as
well as caveolin-1 were detected in gradient fractions of a
higher density than that originally containing lipid rafts (Fig.
1A), which supports the concept that these two proteins are
integrated in lipid rafts. To further identify the protein detected
by the anti-ADAM17 antibody, we treated cells with phorbol-
12,13-myristate acetate (PMA), which reduces the amount of
the mature form of ADAM17 [26], a phenomenon inhibited by
metalloproteinase inhibition. The signal detected with the
anti-ADAM17 antibody in lipid rafts prepared from PMA-
treated cells was reduced compared with that detected in
lipid rafts prepared from untreated cells and this effect was
reversed by the metalloproteinase inhibitor RU 36156 (Fig. 1B),
strongly suggesting that the protein detected in lipid rafts by
the anti-ADAM17 antibody is actually the mature form of
ADAM17. RU 36156 alone was without effect on the distribution
of ADAM17 and caveolin-1 (Fig. 1B).
The mature form of ADAM17 was detected in lipid rafts
prepared from several cell types (see throughout the text),
suggesting that its integration in lipid rafts is a general
phenomenon.
To further prove that the integration of the mature form of
ADAM17 in lipid rafts was not the consequence of our method
of preparation, we took advantage of the ability of THP-1 cells
to generate microvesicles (MVs) rich in lipid rafts [27] and we
analyzed the ADAM17 content of these MVs. As expected, after
the separation of lipid rafts from THP-1 cells, the mature form
of ADAM17 and the raft marker flotilin-1 were detected in the
light density gradient fractions whereas the proform of
ADAM17 was excluded from these fractions (Fig. 2A). MVs
originated from THP-1 cells were largely enriched in the
mature form of ADAM17 and flotilin-1 compared with the
original cells (Fig. 2B). The densitometric analysis of four
independent immunoblots showed that the ratio mature
form/proform of ADAM17 was significantly higher (p = 0.002)
in MVs (5.5 ± 09) than in THP-1 cells (0.67 ± 0.3). Furthermore, a
metalloproteinase activity that can be significantly inhibited
at 50% by the ADAM17 inhibitor TIMP3 was measured at the
surface of these MVs suggesting that the mature form of
ADAM17 is active (Fig. 2C).
A subset of lipid rafts containing the mature form of ADAM17
is associated with the cytoskeleton
We recently described that the mature form of ADAM17 was
more prone to bind the cytoskeleton than its proform [20].
Fig. 2 – THP-1 microvesicles are enriched in the active form of
ADAM17. (A) THP-1 cell lysate was prepared as described in
the Materials and methods section using Brij 98. After
fractionation by sucrose density gradient centrifugation,
ADAM17 and flotilin-1 (Flot1) were detected by immunoblot
in the indicated gradient fractions and (B) in lysates of THP-1
and THP-1 microvesicles (MVs) with similar protein content.
(C) Fluorogenic assay of the metalloprotease activity of
human recombinant ADAM17 (rADAM17) and of MVs was
performed as described in the Materials and methods section
in presence or absence of TIMP3 (100 nM). Pro and mature
forms of ADAM17 are indicated on the right hand side of the
blots by p and m, respectively. *Significant versus the
situation without TIMP3 (p < 0.001).
Furthermore, it was previously shown that the attachment of
lipid rafts to the cytoskeleton increased their density [28]. We
thus hypothesized the existence of very high-density lipid
rafts attached to the cytoskeleton and containing the mature
form of ADAM17. This hypothesis is sustained by the
following observations: (i) when more material was used for
the detection of caveolin-1, we noticed its presence in the
high density gradient fractions (Fig. 3A); and (ii) a small
amount of the mature form of ADAM17 was detected in the
gradient fraction of the highest density (Fig. 1A). After low-
speed centrifugation of the cell lysate, the mature form of
ADAM17 was mostly recovered in the fraction containing the
cytoskeleton which also contained most of the actin and
caveolin-1 (Fig. 3B). The proform of ADAM17 was detected in
the cytoskeleton-free fraction that contains cytosolic proteins
and membrane proteins solubilized by Brij 98. To verify that
the mature form of ADAM17 that cosedimented with the
cytoskeleton was indeed sequestered in lipid rafts, we
pelleted the cytoskeleton by low-speed centrifugation and
performed lipid raft separation on the resulting supernatant
(cytoskeleton-free) and pellet (cytoskeleton). In the cytoske-
leton-free preparation, the mature form of ADAM17 and
caveolin-1 was mostly detected in the lipid raft fractions (Fig.
3C). Actin was detected in the lipid raft region, but most of the
actin was found in the gradient fraction of the highest density
(Fig. 3C). This result shows that polymerized actin is still
present in the sample after cytoskeleton elimination. Before
 E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
Fig. 3 – Lipid rafts containing ADAM17 are linked to the
cytoskeleton. (A) Caveolin-1 (Cav1) was detected by
immunoblot as described in Fig. 1, except that 7-fold more
material was loaded onto the gel. (B) ADAM17, actin and
caveolin-1 (Cav1) were detected by immunoblot in the total
ECVTNF cell lysate (lane 1), the cytoskeleton fraction (lane 2)
and the cytoskeleton-free fraction (lane 3). (C) Rafts were
isolated from the cytoskeleton-free fraction and the
cytoskeleton fraction treated with KI (0.6 M) to depolymerize
actin filaments. ADAM17, actin and caveolin-1 (Cav1) were
detected by immunoblot. Pro and mature forms of ADAM17
are indicated by p and m, respectively.
performing lipid raft separation on the cytoskeleton prepara-
tion, the actin-based cytoskeleton was depolymerized by KI
treatment. Actin was only detected in the light fractions of the
gradient together with the mature form of ADAM17 and with
most of the caveolin-1 (Fig. 3C). Altogether, these results show
that the mature form of ADAM17 is contained in lipid rafts
and that a part of these lipid rafts is associated with the actin-
based cytoskeleton. The proform of ADAM17 is not integrated
in lipid rafts and is not associated with the cytoskeleton.
Furin cleaves the prodomain of ADAM17 in lipid rafts
Analysis of lipid rafts by sucrose density gradient centrifuga-
tion necessitates a large number of expressing cells. Due to the
low efficiency of transfection, experiments using transient
ectopic expression are not compatible with such a technique.
3973
Thus, to study the membrane localization of furin, one of the
proprotein convertases that catalyzes the cleavage of ADAM17
prodomain [22], we ectopically expressed a furin-flag in COS-7
cells, separated the total lipid raft fraction from the lipid raft-
free fraction by direct high-speed centrifugation (100,000×g,
1 h) of the 1% Brij 98 cell lysates and performed immunoblots
on each fraction. Furin was enriched in the total lipid raft
fraction (Fig. 4A). This fraction was also enriched in the mature
form of ADAM17 compared with its proform and caveolin-1
(Fig. 4A). This result suggests that furin, like the mature form of
ADAM17, is contained in lipid rafts.
Incubation of ECVTNF cells with the proprotein convertase
inhibitor decanoyl-RVKR-chloromethylketone resulted in less
TNF released from the cells (data not shown) and in the
appearance of the proform of ADAM17 in lipid rafts (Fig. 4B).
Furthermore, the proform of ADAM17 was detected in lipid
rafts isolated from the furin-deficient human colon epithelial
cell line LoVo whereas only the mature form of ADAM17 was
detected in lipid rafts from another human colon epithelial
cell line HT29 (Fig. 4C). Altogether, these results indicate that
furin cleaves the prodomain of ADAM17 in lipid rafts, although
this maturation process is not necessary for its integration in
lipid rafts.
Fig. 4 – Furin activates ADAM17 in lipid rafts. (A) Furin-flag
was ectopically expressed in COS-7 cells. 48 h
post-transfection, cells were harvested and lysed. ADAM17,
furin-flag (Furin) and caveolin-1 (Cav1) were detected by
immunoblot in the total cell lysate (lane 1), the lipid raft-free
fraction (lane 2) and the total lipid raft fraction (lane 3). (B)
ECVTNF cells were left untreated (Cont) or incubated for 24 h
with 50 μM of decanoyl-RVKR-chloromethylketone (CMK),
then treated as indicated in Fig. 1 and ADAM17 was detected
by immunoblot in indicated gradient fractions. (C) HT-29 and
LoVo cells were treated as described in Fig. 1, indicated
fractions were pooled and ADAM17 was detected by
immunoblot. Pro and mature forms of ADAM17 are indicated
on the right hand side of the blots by p and m, respectively.
3974 E XP E RI ME N TA L CE LL RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0

TNF, TNFR1 and TNFR2 are normally processed by ADAM17
in lipid rafts
Because the mature form of ADAM17 is sequestered in lipid
rafts, we hypothesized that the cleavage of ADAM17 sub-
strates occurs in this place. TNF, TNFR1 and TNFR2 were
previously described as being ADAM17 substrates [3,4,6,29].
We first analyzed the repartition of these substrates between
the lipid raft and non-raft regions of the membrane and
defined the localization of their cleavage under basal condi-
tions. A small proportion of TNF from ECVTNF cells was
detected in the light density fractions of the gradient (11% of
the total TNF was contained in fractions 3 to 7). Cholesterol
depletion by CD treatment reduced this proportion (4% of the
total TNF was contained in fractions 3 to 7) and increased that
in the fractions of the gradient of higher density (Fig. 5A). This
result suggests that a small proportion of TNF is contained in
lipid rafts. Inhibition of ADAM17 by RU 36156 increased the
proportion of TNF detected in lipid rafts and conversely
reduced that in non-raft fractions (Fig. 5B) without affecting
the sedimentation profile of ADAM17 and caveolin-1 (data not
shown). Metalloproteinase inhibition also significantly in-
creased the percentage of TNF associated with the total lipid
raft fraction isolated by direct high-speed centrifugation of the
1% Brij 98 cell lysates (Fig. 5B, inset). This result corroborates
that obtained by separation on sucrose gradient and validates
the use of this faster although less discriminative method to
study the membrane repartition and cleavage of ectopically
expressed TNFR1 and TNFR2. We showed in two different cell
lines (COS-7 and H9C2) that metalloproteinase inhibition
significantly increased the proportion of ectopically expressed
TNFR1 and TNFR2 in the total lipid raft fraction (Fig. 5C),
suggesting that TNFR1 and TNFR2 are cleaved by ADAM17 in
lipid rafts. By comparison, ICAM-1, that does not behave like
an ADAM17 substrate [10,30], was detected in lipid rafts, but its
amount was not modified by the treatment with the metallo-
proteinase inhibitor (Fig. 5D). We previously described that
PMA stimulation of ECVTNF cells increased the synthesis and
the ADAM17-dependent shedding of ectopically expressed
TNF [10]. However, TNF distributed similarly in the sucrose
gradient whether these cells were treated or not with PMA (Fig.
6A). Inhibition of metalloproteinases increased the proportion

Fig. 5 – The cleavage of TNF, TNFR1 and TNFR2 occurs in lipid rafts. (A) ECVTNF cell were left untreated or treated for 30 min with
1% CD, then lysates were prepared as described in Fig. 1. After fractionation by sucrose density gradient centrifugation,
TNF was measured by ELISA in each fraction. (B) Same as panel A except that ECVTNF cells were treated or not for 3 h with 10 μM
of RU 36156 (RU). (B, inset) Total lipid raft fractions from ECVTNF cell lysates were prepared by high-speed centrifugation.
(C) COS-7 and H9C2 cells transiently expressing ectopic TNFR1 and TNFR2 were treated as in (B, inset). TNF, TNFR1 and TNFR2
were measured by ELISA in the raft and non-raft fractions. The % of ADAM17 substrate present in the total lipid raft fraction is
given by the formula: [substrate]raft × 100 / ([substrate]raft + [substrate]non-raft). (D) Same as panel B except that ICAM-1 was
measured (AU: arbitrary unit).
 E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
Fig. 6 – The PMA-stimulated cleavage of TNF occurs in lipid
rafts. (A) ECVTNF cells were left untreated (Control) or
treated for 3 h with 80 nM of PMA in absence or presence of
10 μM of RU 36156 (PMA + RU). After fractionation by sucrose
density gradient centrifugation, TNF was measured by
ELISA in each fraction. (B) Same as panel A to simplify the
reading only the PMA and PMA + RU situations are shown.
TNF in each fraction is expressed as the percentage of the
total amount of TNF present in the gradient.
of TNF in the lipid rafts, reflecting ADAM17 inhibition, and also
in the non-raft regions (Fig. 6A), which reveals the PMA-
stimulated TNF synthesis. However, when the amount of TNF
present in each gradient fraction was expressed as the
percentage of the total amount of TNF contained in the entire
gradient (Fig. 6B), it is clear that under PMA stimulation,
metalloproteinase inhibition increased the proportion of TNF
only in the lipid raft region. This result demonstrates that the
PMA-stimulated TNF shedding by ADAM17 occurs in lipid
rafts.
In mouse organs, the mature form of ADAM17 and a fraction
of TNF and TNFRs are sequestered in lipid rafts
Freshly collected mouse organs were used to verify that the
above described distribution of ADAM17, TNF, TNFR1 and
TNFR2 in the lipid raft and non-raft regions of the membrane
was not restricted to cultured cells or due to the ectopic
expression. Mouse livers were collected and lipid rafts were
isolated by sucrose density gradient centrifugation. As
expected, the proform of ADAM17 was excluded from lipid
3975
rafts whereas a portion of the mature form of ADAM17 and
caveolin-1 were detected in lipid rafts (Fig. 7A). Comparable
results were obtained on the heart and the spleen (data not
shown). The relative high proportion of the mature form of
ADAM17 detected in the heavy fractions of the gradient could
result from the association of lipid rafts with the cytoskeleton
as shown above. To make TNF levels confidently measurable
in liver, mice were injected with LPS 5 h before organ
collection. This treatment did not alter the presence of the
mature form of ADAM17 in lipid rafts (data not shown). A large
proportion of TNF (40% of the total) was detected in lipid rafts
(Fig. 7B). TNFR1 and TNFR2 were measurable upon unstimu-
lated (data not shown) and LPS-stimulated conditions and
were present in lipid rafts (18% and 40% of the total,
respectively) (Fig. 7C). This result demonstrates that the
localization of the mature form of ADAM17, TNF, TNFR1 and
TNFR2 in cholesterol-rich membrane microdomains is a
physiological phenomenon that does not result from the cell
culture conditions or the ectopic expression.
Alteration of lipid raft integrity increases ADAM17 substrate
release
Because cholesterol depletion reduced the amount of the
mature form of ADAM17 present in lipid rafts (Fig. 1A), we
Fig. 7 – TNF, TNFR1 and TNFR2 are contained in lipid rafts
isolated from mouse organs. Mouse livers were prepared as
described under in the Materials and methods section and
lipid rafts were isolated by sucrose density gradient
centrifugation. (A) ADAM17 and caveolin-1 (Cav1) were
detected by immunoblot in the indicated fractions. (B) TNF
and (C) TNFR1 and TNFR2 were measured by ELISA in each
fraction.
3976 E XP E RI ME N TA L CE LL RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
Table 1 – Lipid raft disruption increases the release of TNF, TNFR1 and TNFR2
ECVTNF COS-7
TNF TNF TNFR1
Control 193.9 ± 96 4320.5 ± 888 98.9 ± 8.5
CD 385.4 ± 25 6171.1 ± 421 154.6 ± 14
t-test, p 0.0001 0.0005 0.0014
ECVTNF cells, COS-7 and H9C2 cells ectopically expressing TNF, TNFR1 and TNFR2 were left untreated (Control) or treated for 30 min with 1% CD.
TNF, TNFR1 and TNFR2 accumulated in the media over this period of time were measured by ELISA and expressed as pg/ml. The p values
(Control vs. CD) are indicated in the table.
suspected that it should also alter the release of its substrates.
Our results showed that an incubation time of 30 min with CD
significantly increased the release of TNF, TNFR1 and TNFR2
ectopically expressed in several cell lines (Table 1). It is unlikely
that CD increase the CMV-driven synthesis of ectopically
expressed TNF, TNFR1 and TNFR2 over such a short period of
time. Nevertheless, to rule out the possibility of an increased
TNF synthesis, ECVTNF cells were preincubated for 30 min with
cycloheximide before the treatment with CD. In this condition,
the CD-stimulated TNF release was not modified (data not
shown) indicating that it only involves post-translational
events. The post-translational origin of this regulation was
further proven by showing that the spontaneous release of TNF
from membranes isolated from ECVTNF was significantly
increased by a treatment with CD (control = 1002 ± 31 pg/ml;
CD = 1850 ± 200 pg/ml; p = 0.0019).
Fig. 8 – Modulation of cholesterol homeostasis alters the
release of ADAM17 substrates. (A) ECVTNF cells were left
untreated or treated for 30 min with 1% CD, α-cyclodextrin
(αCD), cyclodextrin loaded with cholesterol (Chol) or for 1 h
with 500 ng/ml of filipin III (Fil), 0.5 U/ml of sphingomyelinase
(SM) or with 5 U/ml of cholesterol oxidase (CO). (B) ECVTNF
cells were treated for 30 min with 1% CD, 400 nM of TIMP3 or
TIMP3 + CD. TNF released in the cell culture media was
measured by ELISA. *Significant versus control p < 0.0002.
H9C2
TNFR2 TNF TNFR1 T NFR2
592.5 ± 42.6 579.2 ± 96 17.3 ± 0.4 83 ± 6.2
1243 ± 110 1144.2 ± 237 21.9 ± 1.7 152.4 ± 37.4
0.0001 0.0011 0.0056 0.0057
The responsibility of cholesterol depletion in the CD-
stimulated TNF release was confirmed by showing that α-
cyclodextrin and cholesterol-loaded cyclodextrin, that are
less efficient than CD to remove cholesterol, were unable to
significantly increase TNF release (Fig. 8A). Other molecules
susceptible of disrupting lipid rafts also affected TNF
release. Treatment of ECVTNF cells with the sterol binding
polyene antibiotic filipin III, with sphingomyelinase that
degrades sphingomyelin and liberates cholesterol from lipid
rafts or with cholesterol oxidase that oxidizes cellular
cholesterol leading to lipid raft degradation, all significantly
increased the release of TNF in the cell culture media (Fig.
8A) without significantly altering its cell-associated amount
(data not shown) ruling out an effect on its production. The
effect of cholesterol withdrawal on the release of endoge-
nous ADAM17 substrates was tested using human umbilical
vein endothelial cells. The release of endothelial TNFR1 and
TNFR2 was increased by a factor 1.4 (control = 74.97 ±
10.86 pg/ml; CD = 108.1 ± 6.1 pg/ml; p < 0.0001) and 1.7 (con-
trol = 23.81 ± 8.5 pg/ml; CD = 41.28 ± 7.8 pg/ml; p = 0.0007),
respectively.
Finally, the role of ADAM17 in the CD-stimulated TNF
release was confirmed by the inhibitory effect of TIMP3
(Fig. 8B).
Discussion
In this work we show conclusively that the shedding activity
of ADAM17 is sequestered in the cholesterol-rich membrane
microdomains called lipid rafts.
The mature form of ADAM17 was detected in lipid rafts
prepared from cell lines of different origins and from freshly
collected mouse organs. In our study, the detergent Brij 98 was
chosen for the preparation of lipid rafts because it respects
their integrity that exists at physiological temperature [25].
However, with the more selective detergent Triton X-100, the
mature form of ADAM17 was also detected in the detergent
resistant membrane fraction. The fact that the proform of
ADAM17 did not follow the same profile of flotation as the
mature form of ADAM17 is an argument in favor of the
discriminative capacity of our lipid raft separation method.
However, there is no standard method to prepare and to study
lipid rafts and it could be objected that the observed
localization of the mature form of ADAM17 in the lipid rafts
results from the selected method of separation. Thus, instead
of preparing lipid rafts by a conventional biochemical
approach, we took advantage of the ability of THP-1 cells to
 E XP E RI ME N TA L CE L L RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0 3977

generate MVs that are rich in lipid raft regions of the The lower proportion of ADAM17 substrates in lipid rafts
membrane [27]. Expectedly, we show that these MVs are compared with that in non-raft region should not preclude its
enriched in the mature and active form of ADAM17. role because this pool should be considered in a dynamic way:
The maturation of ADAM17 is not a prerequisite for its at every moment, molecules are cleaved in lipid rafts and
entry in lipid rafts, as demonstrated by experiments showing replaced by molecules coming from the non-raft region. The
that either the inhibition or the absence of furin, led to the effect of PMA argues in favor of this interpretation: it increases
appearance of the proform of ADAM17 in lipid rafts. Further- the synthesis and the shedding of TNF without altering its
more, furin was localized in lipid rafts, which is in accordance membrane repartition, suggesting a faster transit of TNF from
with data showing its interaction with caveolin-1 [31] and non-raft to the raft regions. A survey of the literature shows
suggests that the maturation of ADAM17 takes place in lipid that more than half of the identified ADAM17 substrates are
rafts. Because it was previously demonstrated that the localized in lipid rafts (Table 2). However, to our knowledge,
cleavage of ADAM17 prodomain by furin occurs in the medial our work originally describes the processing of ADAM17
Golgi [22,32], our data suggest that the proform of ADAM17 is substrates in lipid rafts.
integrated in lipid rafts present in the medial Golgi and is then The sequestration of the mature form of ADAM17 in
processed by furin. cholesterol-rich membrane microdomains suggests a poten-
Other secretases are integrated in lipid rafts, as it is the tial regulation of ADAM17 substrate shedding through the
case for the mature form of the beta secretase BACE [33] and modulation of cholesterol homeostasis. It was already
for all four components of the gamma secretase complex,
namely presenilin 1-derived fragments, mature nicastrin,
APH-1 and PEN-2 [34]. However, to our knowledge, this is the
Table 2 – ADAM17 substrates present in lipid rafts
first demonstration of the maturation of a secretase, herein
ADAM17, in lipid rafts. ADAM17 substrate Presence in
We previously demonstrated that the mature form of lipid rafts
ADAM17 is associated with the cytoskeleton [20]. Herein, we Amphiregulin
confirm this result because we find that a fraction of lipid rafts Amyloid-beta precursor protein [50]
containing the mature form of ADAM17 is associated with the AXL Receptor Tyrosine Kinase
cytoskeleton. The association of lipid rafts with the cytoske- CD30 [12]
CD40 [51]
leton increases their density [28], which could explain why a
CD117 (cKIT) [52]
previous report localized the mature form of ADAM17 in the Cellular prion protein PrP [53]
heaviest fractions of the sucrose gradient corresponding to the Collagen XVII [15]
supposed non-raft region of the cell membrane [12]. Two other Epiregulin
reports suggest that ADAM17 is not integrated in lipid rafts Fractalkine (CX3CL1) [54]
[15,35]. However, these works did not distinguish the mature Glycoprotein GP of Ebola virus [55]
Glycoprotein IB α [56]
form of ADAM17 from its proform.
Glycoprotein V [57]
The cleavage of ADAM17 prodomain by furin is a prere-
Growth hormone receptor [58]
quisite to its activation, although it is not the only event Heparin binding epidermal growth factor
involved because dissociation of the prodomain [36], phos- Human epidermal growth factor receptor 4 [59]
phorylation events [37,38] and association with other proteins Human Meprin
[20,39,40] were described to be likely important. However, the IL-6 receptor α [13]
presence of the mature form of ADAM17 in lipid rafts suggests Interleukin-1 receptor 2
Interleukin-15 receptor α
that its cleavage activity occurs in these membrane micro-
Low-density lipoprotein receptor [60]
domains. We studied this aspect by analyzing the lipid raft
L-selectin [61]
repartition and cleavage of three substrates of ADAM17: TNF, Macrophage colony stimulating factor receptor
TNFR1 and TNFR2. The presence of these proteins was Mucin 1 [62]
established in lipid rafts from different cell lines and mouse Nectin 4
organs. Moreover, the proportion of these proteins in lipid Neuregulins [63]
rafts was increased upon metalloproteinase inhibition, sug- Neurogenic locus notch homolog protein
P75 neurotrophin receptor [64]
gesting that the cleavage of TNF, TNFR1 and TNFR2 occurs in
Sortilin-related receptor
lipid rafts. For some substrates, ADAM10 was found to be Transforming growth factor-α
involved in the constitutive shedding whereas ADAM17 was TNF-related activation-induced cytokine
involved in the stimulated shedding [41,42]. However, this Transforming tyrosine kinase protein [65]
situation cannot be generalized to all ADAM17 substrates. Tumor necrosis factor-α
ADAM17 was implicated in both the constitutive and stimu- Tumor necrosis factor receptor I [66]
Tumor necrosis factor receptor II [67]
lated shedding of several substrates [43] and ADAM10 was not
Vascular cell adhesion molecule 1
shown to be responsible for the constitutive or stimulated
shedding of several others [44,45], in particular that of TNF The list of ADAM17 substrates is from [5] and actualized for
[46]. Our result showing that the PMA-stimulated shedding of collagen XVII [47] and nectin-4 [48]. References indicate the
TNF also occurs in lipid rafts further arguments in favor of the presence of the ADAM17 substrate in lipid rafts. The absence of
reference indicates that no data is available except for interleukin-
sequestration of ADAM17 activity in these membrane
15 receptor α that was described to be a non-raft protein [49].
microdomains.
3978 E XP E RI ME N TA L CE LL RE S E A RCH 3 1 2 ( 2 00 6 ) 3 9 6 9 –39 8 0
described that lipid raft disruption by different means
increased the release of ADAM17 substrates like CD30
interleukin-6 receptor and collagen XVII [12,13,15]. Herein,
we show that lipid raft disruption also increases the release of
TNF, TNFR1 and TNFR2. The precise mechanism at the basis of
this effect was not studied. However, we showed that
disruption of lipid rafts displaced the mature form of
ADAM17 in the non-raft region of the membrane that contains
the major part of ADAM17 substrates. We propose that the
cleavage of ADAM17 substrates outside the lipid rafts likely
accounts for a dysregulated and increased release of these
substrates induced by cholesterol depletion and it could be
predicted that a drastic cholesterol depletion would increase
the release of an ADAM17 substrate as long as it is mostly
present in the non-raft region of the membrane. Therefore, in
normal conditions, the sequestration of the mature form of
ADAM17 in lipid rafts can be considered as the rate-limiting
process of its shedding activity. Indeed, outside of lipid rafts,
ADAM17 substrates are not accessible to the enzyme and the
fine tuning of their processing could imply a regulation of their
entry in lipid rafts. In favor of a substrate-specific regulation of
the entry in lipid rafts we showed that, in presence of a
metalloproteinase inhibitor that prevents their release, differ-
ent proportions of ectopically expressed TNFR1 and TNFR2
were present in lipid rafts (Fig. 5C). Lipid rafts are thus an
obligatory membrane platform that participates to the control
of the proteolytic cleavage of ADAM17 substrates.
In conclusion, we found that the maturation of ADAM17
occurs in lipid rafts and that its shedding activity is seques-
tered in these cholesterol-rich membrane microdomains.
Furthermore, TNF, TNFR1 and TNFR2 are present in different
proportions in lipid rafts, which suggests a specific regulation
of their entry into these structures. However, the identification
of the posttranslational modifications that could modulate the
entry of ADAM17 substrates in lipid rafts was out of the scope
of this present work. These findings point out a key role of lipid
rafts in the regulation of ADAM17-dependent shedding events.
They also offer new perspectives about the possibility of
modulating ADAM17 substrate shedding by altering the fatty
acid and/or cholesterol content of the membrane.
Acknowledgments
E.T. is a recipient of the Ministère de l'Enseignement Supér-
ieur, de la Recherche et de la Technologie. M.C. is a recipient of
Nouvelle Société Française d'Athérosclérose (Paris). This work
was supported by funds of Inserm (Paris) and Université de la
Méditerranée (Marseilles).
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