The disintegrin and metalloproteinase ADAM12 contributes to TGF-beta signaling through interaction with the type II receptor.

Abstract

Transforming growth factor-beta (TGF-beta) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-beta type I receptor and the TGF-beta type II receptor (TbetaRII). Upon ligand binding, TGF-beta type I receptor activated by TbetaRII propagates signals to Smad proteins, which mediate the activation of TGF-beta target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-beta signaling pathway that acts through association with TbetaRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-beta signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TbetaRII in early endosomal vesicles and stabilizes the TbetaRII protein presumably by suppressing the association of TbetaRII with Smad7. These results define ADAM12 as a new partner of TbetaRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.

Full text

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T H E J O U R N A L O F C E L L B I O L O G Y
JCB: REPORT
© The Rockefeller University Press $15.00
The Journal of Cell Biology, Vol. 178, No. 2, July 16, 2007 201–208
http://www.jcb.org/cgi/doi/10.1083/jcb.200612046
JCB
201
Introduction
TGF-β superfamily members are multifunctional cytokines
that regulate a broad range of cellular functions, including cell
proliferation, differentiation, and apoptosis (Massague et al.,
2000; Derynck et al., 2001). TGF-β signals through a hetero-
meric complex of two types of transmembrane Ser/Thr kinases:
TGF-β type I receptor and TGF-β type II receptor (TβRII).
TGF-β binding to TβRII induces the recruitment and phos-
phorylation of TGF-β type I receptor, which, in turn, phos-
phorylates the receptor-regulated Smads (R-Smads) Smad2
and Smad3. Once phosphorylated, Smad2 and Smad3 associ-
ate with the common partner Smad, Smad4, and translocate to
the nucleus, where they regulate the expression of TGF-β tar-
get genes. In contrast to R-Smads and Smad4, the inhibitory
Smad, Smad7, appears to block signal transduction by prevent-
ing access of R-Smads to the TGF-β receptor or by recruiting
distinct E3 ubiquitin ligases that target the receptor–Smad7
complex for degradation (Kavsak et al., 2000; Ebisawa et al.,
2001; Seo et al., 2004).
Upon TGF-β stimulation, Smad2 is recruited to the recep-
tor complex by an adaptor molecule called Smad anchor for re-
ceptor activation (SARA). At steady state, SARA-bound Smad2
is localized in early endosomes to which the receptor is internal-
ized via clathrin-coated pits (Hayes et al., 2002; Di Guglielmo
et al., 2003). The importance of the clathrin-mediated endocytic
pathway in TGF-β signaling is also manifested by the recent
fi nding that cPML (cytoplasmic form of the promyelocytic leu-
kemia protein) mediates TGF-β signaling by facilitating recruit-
ment of the SARA–Smad2 complex and TGF-β receptors to
early endosomes (Lin et al., 2004).
In addition to clathrin, TGF-β receptors can also associate
with caveolin (Razani et al., 2001), which leads to their inter-
nalization into caveolin1-positive vesicles with subsequent deg-
radation through the proteasome pathway. Consistent with this
notion, the caveolin1-positive vesicles were found to associate
with Smad7 (Ito et al., 2004), which is known to mediate the
association of the E3 ligases Smurf1 and Smurf2 to receptors,
leading to their degradation.
The disintegrin and metalloproteinase ADAM12
contributes to TGF-β signaling through interaction
with the type II receptor
Azeddine Atfi ,1 Emmanuelle Dumont,2 Frédéric Colland,3 Dominique Bonnier,2 Annie L’Helgoualc’h,2 Céline Prunier,1
Nathalie Ferrand,1 Bruno Clément,2 Ulla M. Wewer,4 and Nathalie Théret2
1Institut National de la Santé et de la Recherche Medicale, Unite 673, Hôpital St-Antoine, 75571 Paris, France
2Institut National de la Santé et de la Recherche Medicale, Unite 620, Institut Federatif de Recherche 140, Université de Rennes I, 35043 Rennes, France
3Hybrigenics, 75014 Paris, France
4Department of Biomedicine and Biotech Research and Innovation Centre, University of Copenhagen, DK-1017 Copenhagen, Denmark
T
type I receptor and the TGF-β type II receptor (TβRII). Upon
ligand binding, TGF-β type I receptor activated by TβRII
propagates signals to Smad proteins, which mediate the
activation of TGF-β target genes. In this study, we identify
ADAM12 (a disintegrin and metalloproteinase 12) as a
component of the TGF-β signaling pathway that acts
through association with TβRII. We found that ADAM12
ransforming growth factor-β (TGF-β) regulates a
wide variety of biological processes through two
types of Ser/Thr transmembrane receptors: the TGF-β
functions by a mechanism independent of its protease
activity to facilitate the activation of TGF-β signaling, includ-
ing the phosphorylation of Smad2, association of Smad2
with Smad4, and transcriptional activation. Furthermore,
ADAM12 induces the accumulation of TβRII in early endo-
somal vesicles and stabilizes the TβRII protein presumably
by suppressing the association of TβRII with Smad7. These
results defi ne ADAM12 as a new partner of TβRII that
facilitates its traffi cking to early endosomes in which activa-
tion of the Smad pathway is initiated.
Correspondence to Nathalie Théret: nathalie.theret@univ-rennes1.fr
Abbreviations used in this paper: ADAM, a disintegrin and metalloproteinase;
EEA1, early endosomal antigen 1; HSC, hepatic stellate cell; PAI-1, plasmino-
gen activator inhibitor-1; RD, Rhabdomyosarcoma; SARA, Smad anchor for
receptor activation; shRNA, short hairpin RNA; TβRII, TGF-β type II receptor.
The online version of this article contains supplemental material.

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JCB • VOLUME 178 • NUMBER 2 • 2007 202
To gain more insight into the regulation of TGF-β signal-
ing, we have performed yeast two-hybrid screens using TβRII
as bait. ADAM12 (a disintegrin and metalloproteinase 12) was
one of the TβRII interactors that exhibited specifi c and strong
binding to TβRII. ADAM12 belongs to the ADAMs family,
which are glycoproteins characterized by a multidomain struc-
ture comprised of pro-, metalloproteinase, disintegrin, cysteine-
rich, transmembrane, and cytoplasmic domains (Primakoff and
Myles, 2000; Seals and Courtneidge, 2003). ADAMs exhibit
proteolytic, cell adhesion, and signaling properties, and pertur-
bations of ADAM expression are associated with several human
diseases, including cancers (Duffy et al., 2003). In the present
study, we provide the fi rst evidence that ADAM12 interacts
with TβRII and enhances TGF-β signaling by controlling the
localization of TGF-β receptors to early endosomes. These re-
sults reveal a new role for ADAM12 in the regulation of TGF-β
receptor traffi cking.
Results and discussion
Using the extracellular domain of human TβRII as bait, we
performed a yeast two-hybrid screen of a human placental cDNA
library. Eight different fragments of ADAM12 were found to inter-
act with TβRII (Fig. 1 A). Two variants were previously described
for ADAM12: a transmembrane glycoprotein (Yagami-Hiromasa
et al., 1995) and a shorter secreted form (Gilpin et al., 1998).
The common sequences shared by the overlapping fragments of
the prey span the metalloproteinase and disintegrin domains
common to the two variants (Fig. 1 A).
To confi rm the association of ADAM12 with TβRII, a frag-
ment of ADAM12 isolated in the yeast two-hybrid screen (amino
acids 142–739 that include the metalloproteinase and cysteine-
rich domains; Fig. 1 A) was tagged with Flag and cotransfected
into 293 cells alone or in combination with HA-TβRII. Immuno-
precipitation with anti-Flag followed by immunoblotting with
anti-HA revealed that TβRII can interact with ADAM12, and this
interaction was not affected by TGF-β (Fig. 1 B). To provide fur-
ther evidence that ADAM12 interacts with TβRII, we examined
their colocalization by immunofl uorescence. As expected, TβRII
is localized predominantly in patched areas near the cell surface.
Interestingly, we found that ADAM12 extensively colocalized
with TβRII, confi rming their interaction (Fig. 1 C).
To examine whether the association of ADAM12 with
TβRII can occur under physiological conditions, we used
he patic stellate cells (HSCs), Rhabdomyosarcoma (RD), and
C2C12 cells, three cell lines that were previously described to
express detectable ADAM12 (Gilpin et al., 1998; Galliano et al.,
2000; Le Pabic et al., 2003). In immunoprecipitates prepared
with preimmune antisera, no TβRII was coprecipitated. How-
ever, in the anti-ADAM12 immunoprecipitates, we could clearly
detect TβRII coprecipitating with ADAM12 (Fig. 1 D). Forma-
tion of the endogenous ADAM12–TβRII complex was also
Figure 1. T훃RII interacts with ADAM12. (A) Schematic diagram of ADAM12 fragments that interact with TβRII in two-hybrid assays. (B) 293T cells were
transfected with HA-TβRII in the presence or absence of Flag-ADAM12. Cell lysates were subjected to anti-Flag immunoprecipitation (IP) followed by immuno -
blotting (IB) with anti-HA. In this and all of the following experiments, the expression of proteins was determined by direct immunoblotting. (C) C2C12 cells
transfected with GFP-ADAM12 and HA-TβRII were immunostained with anti-HA followed by TRITC-conjugated secondary IgG. The panels represent
three representative fi elds. (D–F) Cell extracts from HSC, RD, and C2C12 cells (D) or C2C12 cells (E and F) were immunoprecipitated with anti-ADAM12
(D and F) or anti-TβRII (E) and immunoblotted with the indicated antibodies.

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ADAM12 REGULATES TGF-β RECEPTOR TRAFFICKING • ATFI ET AL.203
demonstrated by anti-ADAM12 immunoblotting of anti-TβRII
immunoprecipitates (Fig. 1 E). The interaction of ADAM12
with TβRII is specifi c because we were unable to detect an in-
teraction between TβRII and ADAM10 or ADAM17 (Fig. 1 E),
which share the structure organization with ADAM12. Simi-
larly, we were unable to see an interaction between ADAM12
and the bone morphogenetic protein type II receptor (Fig. 1 F).
To explore the functional signifi cance of the interac-
tion between ADAM12 and TβRII, we investigated whether
the expression of ADAM12 may infl uence TGF-β–mediated
transcriptional esponses. For this, we made use of the TGF-β/
Smad2-responsive reporter ARE3-Lux (Labbe et al., 1998) and
found that the expression of ADAM12 resulted in an approxi-
mately fi vefold increase in TGF-β–induced transcription (Fig.
2 A). A similar effect of ADAM12 was observed with the TGF-β/
Smad3-responsive reporter CAGA9-Lux (approximately three-
fold in Fig. 2 B and sixfold in Fig. 2 C; Zawel et al., 1998).
Next, we attempted to confi rm the role of ADAM12 in
enhancing TGF-β signaling by investigating its effect on the ex-
pression of endogenous plasminogen activator inhibitor-1 (PAI-1),
which contains CAGA boxes in the promoter. The results showed
that the TGF-β–dependent expression of PAI-1 was increased
by the expression of ADAM12 (Fig. S1 A, avail able at http://
www.jcb.org/cgi/content/full/jcb.200612046/DC1). During the
course of these analyses, we also investigated the role of endoge-
nous ADAM12 in enhancing the transcriptional activation of
collagen I (COL1A2) by TGF-β. For this, HSC cells were
treated by ADAM12 antisense oligonucleotides before TGF-β
stimulation, and the expression of ADAM12 or COL1A2 was
analyzed. As we recently reported (Le Pabic et al., 2003), TGF-β
treatment induces an accumulation of ADAM12 mRNA and
protein, and this increase was reduced to the background level
by ADAM12 antisense. Similarly, treatment of cells with anti-
sense to ADAM12 attenuated the TGF-β–dependent induction
of COL1A2 mRNA (Fig. S1 B). To confi rm these results, we
depleted HSC, RD, and C2C12 cells from ADAM12 by RNAi.
When ADAM12 was targeted in these cells using a specifi c
short hairpin RNA (shRNA), both the steady-state levels and
the TGF-β–dependent accumulation of ADAM12 were reduced.
Interestingly, the knockdown of ADAM12 resulted in a decrease
in the TGF-β–induced expression of PAI-1 (Fig. 2 D). A similar
result was obtained with JunB (Fig. 2 D), the expression of which
Figure 2. ADAM12 increases TGF-훃 signaling. (A–C) HepG2 (A and B) and C2C12 (C) cells transfected with ARE3-Lux together with FAST1 (A) or CAGA9-
Lux (B and C) in the presence or absence of ADAM12 were treated with or without TGF-β. In these and all of the following reporter assays, luciferase activity
was determined and normalized, and results are expressed as means ± SD (error bars) of triplicates from fi ve independent experiments. (D) HSC, RD, or
C2C12 cells were transfected with the indicated combinations of pEGFP, ADAM12 shRNA, or scrambled shRNA. 36 h later, GFP-transfected cells were
sorted by FACS and exposed to TGF-β for 16 h. The expression of endogenous PAI-1, JunB, and ADAM12 was assessed by direct immunoblotting (IB).
(E and F) 293T cells were transfected with myc-Smad2, Flag-ADAM12, and HA-Smad4 as indicated. For Smad2 phosphorylation (E), cell lysates were
immunoblotted with antiphospho-Smad2. For the association of Smad2 with Smad4 (F), cell lysates were immunoprecipitated (IP) with an anti-myc before
immunoblotting with anti-HA. (G) C2C12 cells were transfected with the indicated combinations of pEGFP, ADAM12 shRNA, or scrambled shRNA. 36 h
later, GFP-transfected cells were sorted by FACS and exposed to TGF-β for 30 min. The phosphorylation of endogenous Smad2 was assessed by immuno-
blotting with antiphospho-Smad2. White lines indicate that intervening lanes have been spliced out.

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JCB • VOLUME 178 • NUMBER 2 • 2007 204
is up-regulated by TGF-β through a mechanism similar to that
of PAI-1.
To investigate the mechanism underlying the effects of
ADAM12 on TGF-β signaling, we investigated whether the ex-
pression of ADAM12 may regulate the TGF-β–dependent phos-
phorylation of Smad2. We observed that exposure of cells to
TGF-β resulted in increased Smad2 phosphorylation, and this
effect was further enhanced by the expression of ADAM12 (Fig.
2 E). Consistent with this, the expression of ADAM12 enhanced
the ability of TGF-β to induce assembly of the Smad2–Smad4
complex (Fig. 2 F). In addition, the depletion of endogenous
ADAM12 by RNAi suppressed Smad2 phosphorylation (Fig.
2 G). Collectively, these data suggest that ADAM12 may func-
tion to enhance TGF-β signaling by facilitating Smad2 phos-
phorylation and its subsequent heterodimerization with Smad4.
At least six members of the ADAM family have been
demonstrated to have proteolytic activity, including ADAM12
(Loechel et al., 2000; Shi et al., 2000). In initial experiments,
we found that a truncated form of ADAM12 (ADAM12-tail),
which lacks the cytoplasmic domain, retains its ability to en-
hance TGF-β signaling (Fig. 3 A). Therefore, we sought to in-
vestigate whether the increase in TGF-β transcriptional activity
mediated by ADAM12 may involve its catalytic activity. To ap-
proach this question, we investigated the effect of phenanthro-
line, a specifi c metalloproteinase inhibitor, on the ability of
ADAM12 to enhance TGF-β transcriptional responses. Surpris-
ingly, exposure of cells to phenanthroline failed to suppress the
effect of ADAM12 on TGF-β–induced CAGA9-Lux (Fig. 3 B).
In another approach, we used ADAM12-E351Q, a protease in-
active mutant. As shown in Fig. 3 C, the expression of ADAM12-
E351Q enhanced TGF-β–induced transcription with an activity
similar to that of wild-type ADAM12. Together, these results
indicate that ADAM12 enhances TGF-β signaling through a
protease-independent mechanism.
During our immunofl uorescence analyses, we observed
that ADAM12 and TβRII are colocalized predominantly in
patched areas near the cell surface in C2C12 cells, but a substan-
tial fraction of both proteins can also colocalize in endosome
vesicle-like structures (Fig. 1 C). This pattern of colocalization
of ADAM12 and TβRII in the two compartments was also evi-
dent in Mv1Lu cells (Fig. 4 A), but their distribution is more
pronounced in endosomal vesicles when compared with C2C12
cells (Fig. 1 C). Based on the fi ndings that TβRII colocalizes
with early endosomal antigen 1 (EEA1), a marker of early endo-
somes (Di Guglielmo et al., 2003), we sought to investigate
whether ADAM12 colocalizes with TβRII in the EEA1-enriched
compartment using Mv1Lu cells that exhibit extensive staining of
these proteins in early endosomes (Fig. 4 A; Di Guglielmo et al.,
2003). As for TβRII, there is some colocalization of ADAM12
with EEA1 in Mv1Lu cells (Fig. 4 A and Fig. S2, available
at http://www.jcb.org/cgi/content/full/jcb.200612046/DC1), sug -
gesting that ADAM12 may accumulate in early endosomes to
which TβRII is internalized via clathrin-coated pits.
To examine whether the localization of ADAM12 in early
endosomes plays a role in TGF-β signaling, we examined the ef-
fect of inhibition of clathrin-mediated endocytosis by potassium
depletion, which was reported to prevent endosome- dependent
TGF-β signaling (Di Guglielmo et al., 2003). As shown in Fig.
4 B, potassium depletion decreased the ability of ADAM12 to
enhance TGF-β–induced transcription. Potassium depletion
also decreased TGF-β signaling in the absence of transfected
ADAM12, but this effect seems to depend on ADAM12 be-
cause it was lost in cells depleted from endogenous ADAM12
by RNAi. In a control experiment, we found that potassium
depletion can further decrease TGF-β–induced transcription in
cells depleted from Smad3 (Fig. 4 C), supporting the hypothesis
that potassium depletion may inhibit TGF-β signaling by spe-
cifi cally interfering with ADAM12 function. To provide further
Figure 3. Up-regulation of TGF-훃 signaling by ADAM12 does not involve its cytoplasmic domain or its protease activity. (A–C) HepG2 cells were trans-
fected with CAGA9-Lux, ADAM12, ADAM12-tail, and ADAM12.E351Q as indicated. For A and C, cells were treated with or without TGF-β for 16 h
before lysis. Dose effects are shown in insets. For B, cells were treated with 1.10 phenanthroline in the presence or absence of TGF-β. In all cases, luciferase
activity was determined. Error bars represent SD. WT, wild type.

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ADAM12 REGULATES TGF-β RECEPTOR TRAFFICKING • ATFI ET AL. 205
evidence that ADAM12 functions in TGF-β signaling by facili-
tating the traffi cking of TβRII to early endosomes, we examined
the localization of SARA, which has been shown to interact
with TβRII at the plasma membrane and in EEA1-positive early
endosomes (Hayes et al., 2002; Itoh et al., 2002). We observed
that the expression of ADAM12 caused the redistribution of the
TβRII–SARA complexes from the plasma membrane into early
endosomes (Fig. 4 D). This effect is likely to be direct because
the expression of ADAM12 had no effect on the association of
TβRII with several transmembrane proteins that could poten-
tially prevent or enhance its traffi cking (Fig. S3, available at
http://www.jcb.org/cgi/content/full/jcb.200612046/DC1).
To provide further evidence that ADAM12 facilitates the
localization of TβRII in early endosomes, we tested the effect of
nystatin, a sterol-binding antibiotic that is known to induce the
redistribution of TGF-β receptors into EEA1-positive endosomes
by affecting the raft structures (Di Guglielmo et al., 2003). We
reasoned that if we induce the majority of TβRII to accumulate
in early endosomes by an alternative approach, such as the treat-
ment of cells with nystatin, ADAM12 should have no further
effect on TGF-β–mediated transcription. As shown in Fig. 4 E,
exposure of cells to nystatin caused a considerable increase in
the TGF-β–mediated activation of CAGA9-Lux, and this in-
crease was not affected by the expression of ADAM12. Under
these experimental conditions, the expression of Smad3 can syn-
ergize with nystatin to enhance TGF-β–induced transcription,
arguing against the possibility that the lack of ADAM12 effect is
caused by the ability of nystatin to elicit the maximum threshold
level of TGF-β signaling in this cell system. Collectively, these
results suggest that ADAM12 may function as an important
component in TGF-β signaling by modulating the traffi cking of
the TGF-β receptor.
Figure 4. ADAM12 facilitates clathrin-
dependent TGF-훃 signaling. (A) MvLu1 cells
transfected with GFP-ADAM12 and HA-TβRII
were immunostained with rabbit anti-HA and
mouse anti-EEA1 followed by secondary stain-
ing with TRITC-conjugated anti–rabbit and Cy5-
conjugated anti–mouse IgG. The subcellular
localization of GFP-ADAM12 (green), TβRII (red),
and EEA1 (blue) was analyzed by a confo-
cal microscope. Colocalization of EEA1 with
ADAM12, ADAM12 with TβRII, and EEA1 with
TβRII appears as turquoise, yellow, and purple,
respectively. The panels represent three inde-
pendent representative fi elds. Representative ar-
eas from cells that display TβRII and ADAM12
in the EEA1 compartments are enlarged in the
insets. (B and C) HepG2 cells were transfected
with the indicated combinations of CAGA9-Lux,
ADAM12 shRNA, ADAM12, Smad3 shRNA, or
scrambled shRNA. Cells were potassium de-
pleted before stimulation with TGF-β, and luci-
ferase activity was examined. (D) COS7 cells
were transfected with HA-TβRII and Flag-SARA
with or without ADAM12. Cells were immuno-
stained with rabbit anti-HA and mouse anti-EEA1
followed by secondary staining with TRITC-
conjugated anti–rabbit and Cy5-conjugated
anti–mouse IgG. For the detection of Flag-SARA,
cells were incubated with FITC-conjugated anti-
Flag. Flag-SARA, TβRII, and EEA1 appear as
green, red, and blue, respectively. (E) HepG2
cells transfected with CAGA9-Lux and the indi-
cated expression vectors were treated with nys-
tatin at 10 μM for 1 h before stimulation with
TGF-β, and luciferase activity was examined.
Error bars represent SD.