Molecular Cell
Article
Direct Activation of TACE-Mediated Ectodomain
Shedding by p38 MAP Kinase Regulates EGF
Receptor-Dependent Cell Proliferation
Pinglong Xu1 and Rik Derynck1,*
1Department of Cell and Tissue Biology, Programs in Cell Biology and Developmental Biology, University of California,
San Francisco, San Francisco, CA 94143, USA
*Correspondence: rik.derynck@ucsf.edu
DOI 10.1016/j.molcel.2010.01.034
SUMMARY
Inflammatory stimuli activate ectodomain shedding
of TNF-a, L-selectin, and other transmembrane
proteins. We show that p38 MAP kinase, which is activated in response to inflammatory or stress signals,
directly activates TACE, a membrane-associated
metalloprotease that is also known as ADAM17 and
effects shedding in response to growth factors and
Erk MAP kinase activation. p38a MAP kinase interacts
with the cytoplasmic domain of TACE and phosphorylates it on Thr735, which is required for TACE-mediated ectodomain shedding. Activation of TACE by
p38 MAP kinase results in the release of TGF-a family
ligands, which activate EGF receptor signaling,
leading to enhanced cell proliferation. Conversely,
depletion of p38a MAP kinase activity suppresses
EGF receptor signaling and downstream Erk MAP
kinase signaling, as well as autocrine EGF receptordependent proliferation. Autocrine EGF receptor
activation through TACE-mediated ectodomain
shedding intimately links inflammation and cancer
progression and may play a role in stress and conditions that relate to p38 MAP kinase activation.
INTRODUCTION
Ectodomain shedding results in the release of extracellular
domains of transmembrane proteins, including cytokines,
growth factors, and cell surface receptors. This process is mediated by membrane-anchored metalloproteases and often regulates the activities of the substrate proteins. Thus, shedding
results in the release of soluble cytokines and growth factors
that activate receptors on cells at a distance, regulating cell
proliferation and migration. (Blobel, 2005; Seals and Courtneidge, 2003). Ectodomain shedding plays roles in inflammation
by controlling the cleavage of inflammatory regulators, such as
TNF-a and the adhesion protein L-selectin (Seals and Courtneidge, 2003). Shedding of L-selectin disrupts leukocyte binding
to endothelial cells, preventing their recruitment at sites of
inflammation, and systemic release of TNF-a greatly contributes
to cachexia (Beutler et al., 1986).
Inflammation has also been implicated in cancer. Chronic
inflammation due to infection or irritation enhances initiation of
tumor development (Mantovani et al., 2008). Conversely, cellintrinsic changes that contribute to carcinogenesis are often
accompanied by inflammatory signaling or production of inflammatory mediators that contribute to tumor promotion and
progression (Coussens and Werb, 2002; Greten et al., 2004;
Mantovani et al., 2008; Pikarsky et al., 2004). Inflammatory cytokines, pathogenic stimuli, and cell stress activate signaling
leading to p38 MAP kinase (MAPK) through MKK3, MKK4, or
MKK6, which, in turn, are activated by MAPK kinase kinases
(Ono and Han, 2000). p38 MAPK contributes to inflammation,
e.g., by activating cytokine expression and modulating their
signaling (Dong et al., 2002). Among the four p38 MAPK isoforms, p38a is best characterized and crucial for inflammatory
cytokine production. The p38 MAPK pathway can contribute to
cancer progression through effects on cell proliferation (Dolado
et al., 2007; Ono and Han, 2000; Ventura et al., 2007) and its roles
in inflammation, e.g., in inducing VEGF and metalloprotease
expression (Coussens and Werb, 2002; Mantovani et al.,
2008). Further, inflammatory mediators that induce p38 MAPK
activation can stimulate cell proliferation and contribute to malignant progression (Moore et al., 1999; Vidal-Vanaclocha et al.,
2000). Yet, the p38 MAPK pathway can also exert a tumor
suppressor effect by modulating cell-cycle regulation, senescence, and DNA damage responses (Han and Sun, 2007) and
triggering apoptosis (Dolado et al., 2007).
In carcinomas, EGF receptor (EGFR) activation aids the
progression by promoting cell proliferation and cell survival
(Yarden and Sliwkowski, 2001), and interference with EGFR activation is the basis for therapies (Zhang et al., 2007). Autocrine
EGFR signaling is enhanced by increased receptor and/or
TGF-a family ligand expression, as observed in cancers of neuroectodermal origin (Hynes and Lane, 2005). Accordingly,
increased release of soluble ligands, such as TGF-a and amphiregulin, through ectodomain shedding (Blobel, 2005; Sahin et al.,
2004) enhances the EGFR activity and cancer progression (Borrell-Pagès et al., 2003; Kenny and Bissell, 2007; Zhou et al., 2006).
Among the metalloproteases, TACE, also known as ADAM17,
mediates ectodomain shedding of inflammatory cytokines and
TGF-a family ligands (Seals and Courtneidge, 2003). TACE
expression is often enhanced in inflammation (Patel et al.,
1998) and in carcinomas, especially in breast cancers (Mochizuki
and Okada, 2007). The release of TGF-a proteins by TACE stimulates cell proliferation and cancer progression (Borrell-Pagès
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TACE Mediates p38 MAPK-Induced Cell Proliferation
et al., 2003; Kenny and Bissell, 2007; Zhou et al., 2006). How
TACE is activated is of great interest. Erk MAPK signaling, often
observed in carcinomas, activates TACE and may activate
TACE-mediated ectodomain shedding in response to growth
factors (Dı́az-Rodrı́guez et al., 2002; Fan and Derynck, 1999;
Gechtman et al., 1999). Further, EGFR activation by G proteincoupled receptors involves Src-dependent phosphorylation of
TACE, resulting in release of amphiregulin (Zhang et al., 2006).
Whereas stimuli that activate p38 MAPK induce ectodomain
shedding, the responsible metalloproteases that are activated
by this pathway have remained unclear. In fact, it has been
proposed that stimuli that activate p38 MAPK do not act through
TACE (Dı́az-Rodrı́guez et al., 2002; Hinkle et al., 2003; Takenobu
et al., 2003). Using transmembrane TGF-a as model, we show
that p38 MAPK directly activates TACE through targeted phosphorylation of its cytoplasmic domain. Activation of TACE by
p38 MAPK in breast carcinoma cells leads to a release of
TGF-a ligands, resulting in increased EGF receptor activation
and proliferation. Our results establish a mechanism for the regulation of EGF receptor and Erk MAPK signaling, as well as cancer
cell proliferation, by the p38 MAPK pathway.
RESULTS
Transmembrane TGF-a as Reporter of Ectodomain
Shedding
We compared the ectodomain shedding responses to extracellular inducers of Erk or p38 MAPK activation using Ca cells,
a CHO cell derivative that expresses TGF-a. TGF-a undergoes
N-glycosylation and cleavage of the prodomain, giving rise to
three transmembrane forms: (1) an 22 kD form I that corresponds to the precursor with its N-terminal unglycosylated prodomain and is primarily located in the endoplasmic reticulum, (2)
an 28 kD form II with N-glycosylation of the prodomain, which
occurs primarily in the Golgi, and (3) an 16 kD form III with the
prodomain proteolytically removed (Bringman et al., 1987). Ectodomain shedding releases soluble TGF-a and thereby generates
a truncated transmembrane protein that is not recognized by
anti-TGF-a antibody. Cell surface protein biotinylation revealed
that transmembrane TGF-a forms II and III are at the cell surface
(Figure 1A). This scenario of TGF-a maturation allows quantification of ectodomain shedding based on the decrease of transmembrane TGF-a levels at the cell surface or release of soluble
TGF-a in the medium.
Extracellular Cues that Activate p38 MAP Kinase Induce
Ectodomain Shedding
Ca cells were treated with PMA, serum, anisomycin, UV light,
osmotic shock or ionomycin. Transmembrane TGF-a in cell
lysates was visualized by western blotting (Figure 1B), and
soluble TGF-a was measured in the media by ELISA (Figure 1C).
All treatments resulted in a decrease of the TGF-a forms II and III
at the cell surface and release of soluble TGF-a, indicating ectodomain shedding. We correlated these results with activation of
Erk or p38 MAPK signaling. PMA and serum activated Erk
MAPK, and anisomycin, UV light, and osmotic shock activated
p38 MAPK with only a minimal effect on Erk MAPK (Figure 1B).
These data suggested that p38 MAPK activation, like Erk
MAPK signaling, induces TGF-a shedding in Ca cells.
To study the roles of the Erk and p38 MAPK pathways in the
induction of ectodomain shedding, we treated Ca cells with
the MEK inhibitor U0126, preventing activation of Erk MAPKs,
or with the p38 MAPK inhibitor SB203580. Because anisomycin,
UV light, and osmotic shock also activate JNK MAPK, we also
tested the effect of a JNK MAPK pathway inhibitor. Further,
because PMA activates protein kinase C, we examined the effect
of the protein kinase C inhibitor BIM. Figure 1D shows that TGF-a
shedding in response to anisomycin or UV light was unaffected
by inhibitors of the Erk or JNK MAPK pathways or protein kinase
C. In contrast, inhibition of p38 MAPK blocked shedding and
increased the cell surface levels of TGF-a forms III and II.
PMA-induced shedding was blocked by U0126, as reported
(Fan and Derynck, 1999; Gechtman et al., 1999), but not by the
p38 MAPK inhibitor. These results indicate that p38 MAPK activates ectodomain shedding induced by anisomycin or UV light
and that Erk MAPK or protein kinase C signaling is not involved.
We evaluated whether ectodomain shedding responded to the
same signals in HeLa cells ectopically expressing TGF-a (Figures
S1A and S1B available online) and T4-2 breast carcinoma cells
(Kenny and Bissell, 2007) (Figure 1E). PMA, anisomycin, and
UV light induced TGF-a shedding in HeLa cells. We could not
detect the low endogenous levels of transmembrane TGF-a in
T4-2 cell lysates, but ELISA revealed the release of TGF-a in
the medium in response to PMA or anisomycin. As in Ca cells,
PMA-induced shedding in HeLa or T4-2 cells was inhibited by
U0126, and anisomycin-induced shedding was inhibited by
SB203580 (Figures 1E, S1A, and S1B). The cytokine interleukin
(IL)-1b, which activates p38 MAPK signaling (Ono and Han,
2000) and is frequently expressed in human breast cancers (Nicolini et al., 2006), also induced TGF-a shedding in T4-2 cells,
albeit at a lower level than anisomycin, and this induction was
similarly inhibited by SB203580 (Figure 1E). These data using
Ca, HeLa, and T4-2 cells indicate that p38 MAPK activation
results in ectodomain shedding.
p38a MAP Kinase Activates Anisomycin- and UV
Light-Induced Ectodomain Shedding
All four p38 MAPKs—p38a, p38b, p38g, and p38d—are activated by MKK3 or MKK6 (Ono and Han, 2000). Two of these,
p38a and p38b, are inhibited by SB203580. Activation of p38
MAPK and TGF-a ectodomain shedding in response to anisomycin or UV light were inhibited by SB203580 with similar sensitivities (Figure 2A). The nearly complete inhibition at 625 nM
SB203580 was close to the reported IC50 of 600 nM SB203580
for p38 MAPK inhibition (Cuenda et al., 1995), suggesting that
p38a or p38b MAPK mediate anisomycin- and UV light-induced
ectodomain shedding.
We isolated p38a MAPK cDNAs from CHO cell mRNA and designed siRNA based on this sequence. Transfection of the siRNA
downregulated p38a MAPK expression in Ca cells and inhibited
p38 MAPK activation in response to anisomycin or UV light, as
apparent from the minimal p38 MAPK phosphorylation
(Figure 2B). Downregulation of p38a MAPK expression also prevented the induction of ectodomain shedding and release of
TGF-a into the medium in response to anisomycin or UV light,
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TACE Mediates p38 MAPK-Induced Cell Proliferation
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Figure 1. Activation of the p38 MAP Kinase Pathway Induces Ectodomain Shedding of TGF-a
(A) Ca cells express transmembrane TGF-a forms I, II, and III, as shown by immunoblotting of cell lysates. Forms II and III are visualized by cell surface protein
biotinylation.
(B and C) Ca cells were treated as indicated, and ectodomain shedding of TGF-a was examined by immunoblotting of cell lysates for transmembrane TGF-a
(B, top) or quantifying TGF-a released into the medium by ELISA (C). In (B), the middle and bottom panels show activation of Erk (anti-pErk) or p38 MAPK
(anti-pp38) versus total Erk or p38 MAPK. *p < 0.01, compared with control.
(D) Effects of pathway inhibitors on TGF-a shedding. Ca cells were treated with PMA, anisomycin, or UV light without or with the indicated inhibitor. Immunoblotting of cell lysates shows transmembrane TGF-a and activated Erk (anti-pErk) or p38 MAPK (anti-pp38) versus total Erk or p38 MAPK.
(E) Ectodomain shedding of TGF-a in T4-2 cells treated with PMA, anisomycin, or IL-1b without or with U0126 or SB203580. TGF-a released into the medium was
quantified by ELISA. *p < 0.001, compared with control. **p < 0.01, compared with corresponding PMA, anisomycin, or IL-1b stimulation.
Error bars show the standard deviations based on triplicate values of each data set.
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TACE Mediates p38 MAPK-Induced Cell Proliferation
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Figure 2. p38a MAP Kinase Mediates Anisomycin- and UV Light-Induced Ectodomain Shedding
(A) Ca cells were treated with anisomycin, UV light, or PMA and increasing doses of SB203580, and shedding was visualized by immunoblotting for transmembrane TGF-a. The bottom panels show p38 MAPK activation using antiphospho-p38 MAPK immunoblotting.
(B and C) Ca cells were transfected with p38a MAPK siRNA or control siRNA. Ectodomain shedding of TGF-a was induced by PMA, anisomycin, or UV light
and evaluated by immunoblotting of cell lysates for transmembrane TGF-a (B) or ELISA of TGF-a released in the medium (C). Immunoblotting for p38 MAPK
or phospho-p38 MAPK revealed the p38a MAPK activation. *p < 0.01, compared with control. **p < 0.01, compared with corresponding control siRNA
transfection.
(D) Ca cells were treated with PMA, anisomycin, or UV light without or with cycloheximide or brefeldin A. Shedding was assessed by immunoblotting of cell lysates
for transmembrane TGF-a.
554 Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc.
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TACE Mediates p38 MAPK-Induced Cell Proliferation
assessed in Ca (Figures 2B and 2C) and HeLa cells (Figure S2A).
Downregulation of p38a MAPK expression did not affect shedding in response to PMA, as apparent by western blotting of
transmembrane TGF-a (Figure 2B) and ELISA of TGF-a in the
medium (Figures 2C and S2A). These results indicate that p38a
MAPK activates anisomycin- or UV light-induced ectodomain
shedding.
p38 MAP Kinase-Activated Shedding Does Not Require
Protein Synthesis or Reactive Oxygen Species
Cycloheximide did not inhibit PMA-induced shedding but slightly
inhibited ectodomain shedding in response to anisomycin or UV
light (Figure 2D, lanes 5–8). Brefeldin A, which disrupts transport
through the Golgi apparatus, enhanced the level of immature
TGF-a form I, consistent with the disruption of protein transport
through the Golgi, which occurs prior to ectodomain shedding of
cell surface proteins. However, it did not affect shedding of the
cell surface TGF-a form III in response to PMA and slightly
affected shedding induced by anisomycin or UV light
(Figure 2D, lanes 8–12). These data indicate that new protein
synthesis is not required for activation of ectodomain shedding
by the Erk or p38 MAPK pathways.
Reactive oxygen species (ROS) and nitrogen oxide (NO) have
been proposed to play a role in activating TACE by inducing
dissociation of the inhibitory prodomain from active TACE
(Zhang et al., 2001) or activating p38 MAPK signaling (Dolado
et al., 2007; Fischer et al., 2004). However, the antioxidant
MNTmPyP did not inhibit anisomycin- or PMA-induced ectodomain shedding of TGF-a, nor did the NO scavenger PTIO and
iNOS inhibitor L-NIL (Figure 2E). Our data suggest that ROS
and NO do not mediate p38 MAPK- or Erk MAPK-activated
ectodomain shedding.
TACE Mediates TGF-a Release in Response
to Activation of p38 MAP Kinase
To address the role of TACE, we transfected siRNAs, based on
the Chinese hamster sequence of the catalytic domain of
TACE, in Ca cells. Silencing TACE expression blocked shedding
of transmembrane TGF-a (Figure 3A) and decreased the release
of soluble TGF-a (Figure 3B) in response to anisomycin or UV
light, similarly to its effect on PMA-induced shedding. A similar
inhibition of TGF-a release was seen in HeLa cells expressing
TGF-a (Figure S2B) or T4-2 cells expressing endogenous
TGF-a (Figure 3C), using siRNA targeting the human TACE
sequence.
CHO-derived M2 cells lack induction of ectodomain shedding
in response to PMA or serum due to an inactivating Cys600
mutation in TACE (Li and Fan, 2004). Anisomycin and UV light
were, similarly to PMA or serum, unable to induce shedding.
However, introduction of TACE conferred TGF-a shedding in
response to PMA, as well as anisomycin or UV light, and this
rescue was not seen with the catalytically inactive Glu406Ala
TACE (Figure 3D). These results indicate that TACE mediates
shedding in response to p38 MAPK activation.
We also incubated T4-2 cells with anisomycin, IL-1b, or lipopolysaccharide (LPS)—which is also known to activate p38
MAPK (Ono and Han, 2000)—immunoprecipitated TACE, and
assessed its activity in vitro by cleavage of a substrate peptide
spanning a known TACE cleavage site. As shown in Figure 3E,
anisomycin, LPS, and IL-1b increased the activity of TACE,
and those increases were abolished by incubating the cells
with p38 MAPK inhibitor SB203580 prior to isolation of TACE.
We conclude that p38 MAPK activation results in increased
enzymatic activity of TACE. In these assays, all TACE was immunoprecipitated, and not only the TACE at the cell surface that
accounts for only a small fraction of TACE in the cell lysate and
is likely the population that is activated by p38 MAP kinase.
TACE Is Directly Phosphorylated by p38a MAP Kinase
Because activation of p38a MAPK resulted in ectodomain shedding by TACE, we explored whether p38 MAPK interacts with
TACE. Using Ca cells expressing tagged TACE and p38a
MAPK, we showed that p38a MAPK associated with TACE. In
contrast, the kinase-inactive AF mutant of p38a MAPK did not
associate. Also, p38b MAP kinase, but not its kinase-inactive
mutant, associated with TACE (Figure 4A).
Using purified tagged proteins translated in vitro, we phosphorylated wild-type and kinase-inactive p38a MAPK with
MKK6. These were then immunopurified and incubated with
g32P-ATP and purified tagged TACE, which, due to lack of glycosylation, was smaller than TACE made in mammalian cells. TACE
was phosphorylated by activated wild-type p38a MAPK, but not
its inactive mutant (Figure 4B). Similar results were obtained
using proteins that were expressed in Ca cells. Thus, p38a
MAPK or its kinase-inactive mutant were coexpressed with activated MKK6, and immunopurified p38a MAPK was incubated
with tagged TACE that was also immunopurified from transfected cells. Figure 4C shows phosphorylation of TACE by
p38a MAPK, which was inhibited by SB203580. These data illustrate that TACE is a kinase substrate for p38a MAP kinase.
We evaluated whether p38 MAPK phosphorylates TACE in
response to anisomycin using in vitro kinase assays with tagged
p38a or p38b MAPK and TACE that were coexpressed in Ca
cells and then purified. Anisomycin induced TACE phosphorylation to a level similar to coexpression of activated MKK6
(Figure 4D). Kinase-inactive p38a MAPK did not phosphorylate
coexpressed TACE, and p38b MAPK only weakly phosphorylated TACE when compared with p38a MAP kinase, either in
response to anisomycin or when coexpressed with activated
MMK6 (Figure 4D). Thus, even though p38a and p38b MAPK
associated similarly with TACE in vitro (Figure 4A), TACE was
the preferred substrate of p38a MAP kinase.
In Ca cells, TACE was phosphorylated in vivo in response to
anisomycin by endogenous p38a MAPK, as apparent by the inhibition by SB203580 (Figure 4E). We also examined the phosphorylation of endogenous TACE in T4-2 cells. Because p38
MAPK is a proline-directed Ser/Thr kinase, we used anti-phosphoThr-Pro to detect phospho-TACE by immunoblotting
(E) Ectodomain shedding was induced with PMA or anisomycin without or with MNTmPyP, PTIO, or L-NIL and was evaluated by immunoblotting of cell lysates
for TGF-a.
Error bars show the standard deviations based on triplicate values of each data set.
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TACE Mediates p38 MAPK-Induced Cell Proliferation
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Figure 3. TACE Effects TGF-a Release in Response to p38 MAP Kinase Activation
(A and B) Ca cells were transfected with TACE siRNA or control siRNA and were stimulated with PMA, anisomycin, or UV light, and shedding of TGF-a was evaluated by immunoblotting cell lysates for transmembrane TGF-a (A) or by ELISA of TGF-a in the medium (B). Immunoblotting cell lysates with anti-TACE or antiphospho-p38 MAPK antibodies showed silencing of TACE expression or activation of p38 MAPK in response to anisomycin or UV light. *p < 0.05, compared with
corresponding transfection with control siRNA.
(C) T4-2 cells, transfected with TACE siRNA or control siRNA, were treated with PMA, anisomycin, or UV light, and TGF-a released in the medium was quantified
by ELISA. Silencing of TACE expression was apparent by immunoblotting of cell lysates for TACE (bottom). *p < 0.05, compared with corresponding transfection
with control siRNA.
(D) M2 cells lacking active TACE were transfected to express TGF-a and TACE or inactive E406A TACE mutant. Shedding of TGF-a was visualized by immunoblotting of cell lysates for transmembrane TGF-a. The bottom panel shows p38 MAPK activation, assessed by immunoblotting for phospho-p38 MAPK.
(E) In vitro proteolytic activity of TACE immunoprecipitated from T4-2 cells that were treated with anisomycin, LPS, or IL-1b in the absence or presence of
SB203580. *p < 0.05, compared with DMSO control.
Error bars show the standard deviations based on triplicate values of each data set.
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TACE Mediates p38 MAPK-Induced Cell Proliferation
(Figure 4F). SB203580 inhibited the low basal level of phosphorylation, and anisomycin and IL-1b enhanced the immunoreactivity of TACE. The lower level of immunoreactivity of TACE
upon IL-1b stimulation, when compared to anisomycin, is
consistent with the lower level of p38 MAPK activation
(Figure 4G) and shedding (Figure 1E). The enhanced phosphorylation of endogenous TACE upon stimulation was abolished by
SB203580 (Figure S3) or when cells were transfected with siRNA
to p38a MAPK (Figure 4F). Finally, the ability of p38a MAPK to
associate with TACE and increased phosphorylation of TACE
in response to anisomycin or IL-1b suggest that p38 MAPK interacts with TACE upon stimulation. Even though kinase-substrate
interactions are notoriously difficult to detect by coimmunoprecipitation, we detected a low-level interaction of endogenous p38
MAPK and TACE in T4-2 cells in response to anisomycin or IL1b; again, the effect of anisomycin was stronger than that of
IL-1b. These data show that TACE is a substrate for p38a MAP
kinase and that p38a MAPK phosphorylates TACE on Thr in
response to anisomycin and IL-1b.
TACE Phosphorylation at Thr735 Is Required for
Activation of Shedding by p38 MAP Kinase
A previous study reported that Erk MAPK phosphorylates Thr735
in the TACE cytoplasmic domain (Dı́az-Rodrı́guez et al., 2002). In
addition, Ser819 is phosphorylated and Ser791 is dephosphorylated in response to growth factor stimulation (Fan et al.,
2003). To assess whether these sites play roles in TACE phosphorylation by p38a MAPK, we expressed wild-type TACE,
TACE mutants with either site replaced by alanine, and two
double mutants in transfected cells. Immunopurified wild-type
and mutant TACE were subjected to in vitro phosphorylation
by p38a MAPK that was activated through coexpression with
activated MKK6 (Figures 5A and S4). The 32P labeling of TACE
correlated with phosphoThr-Pro immunoreactivity. Mutation of
Thr735 strongly decreased the TACE phosphorylation by p38a
MAPK and abolished the immunoreactivity, whereas mutation
of Ser819 mildly decreased its phosphorylation. In contrast, the
Ser791 mutation conferred increased phosphorylation. Because
only Pro-Gln-Thr735-Pro in the TACE cytoplasmic domain has
Thr in a Thr-Pro dimer and the Thr735Ala mutant is not phosphorylated by p38a MAP kinase, we conclude that the phosphoProThr antibody recognizes phosphoThr735 and that activation of
p38 MAPK results in TACE phosphorylation at Thr735. This is
consistent with Pro-Gln-Thr735-Pro as a predicted p38 MAPK
target
sequence
(http://scansite.mit.edu).
Accordingly,
Thr735Ala TACE had a much lower level of phosphorylation
than wild-type TACE, both in vitro using purified proteins
(Figure 4B) and in Ca cells in response to anisomycin (Figure 4E).
To examine the role of TACE phosphorylation in response to
p38 MAPK activation, we coexpressed each TACE mutant individually with TGF-a in M2 cells that lack active TACE expression
and measured ectodomain shedding by the amount of TGFa released into the medium (Figure 5B). Without TACE coexpression, very little TGF-a was detected in the medium, and no
increase was observed upon p38 MAPK activation. TACE
expression enhanced the level of TGF-a in the medium, consistent with a basal shedding that is apparent when TACE is
overexpressed. The TGF-a release increased in response to
anisomycin and decreased when the cells were treated with
SB203580 or expressed the dominant-negative AF mutant of
p38a MAPK, suggesting a role of p38a MAPK in the basal shedding activity. The Thr735Ala mutant of TACE had a much lower
level of basal shedding, similar to the level when p38 MAPK
was inhibited, and its activity was only marginally enhanced in
response to anisomycin. The Ser819Ala mutation conferred
a lower overall activity when compared to wild-type TACE,
whereas TACE Ser791Ala had an activity profile similar to that
of wild-type TACE.
We also generated derivatives of Ca cells expressing wildtype or mutant TACE at levels comparable to endogenous
TACE (Figure 5C). Because of the endogenous TACE activity,
anisomycin and PMA stimulated shedding in cells expressing
wild-type or Thr735Ala TACE, as apparent by the decrease in
the TGF-a forms II and III (Figure 5C). Silencing endogenous
TACE expression with siRNA that did not affect the transfected
human TACE expression blocked TGF-a shedding (Figure 5C).
In CaTACE cells that express wild-type human TACE, the endogenous TACE silencing did not affect the TGF-a shedding by
human TACE in response to Erk or p38 MAPK activation. In
contrast, the shedding in response to anisomycin was severely
impaired in Ca cells expressing Thr735Ala TACE. This was most
apparent from the minimal decrease in TGF-a form III, which
was comparable to the level in Ca cells that had endogenous
TACE expression silenced and did not express human TACE.
The shedding in response to PMA was much less affected by
the Thr735Ala mutation.
The induction of TGF-a shedding in these cells was confirmed
by ELISA for TGF-a in the medium (Figure 5D). When silencing
endogenous TACE expression, the strong increase of soluble
TGF-a in response to anisomycin was only observed when
wild-type human TACE was expressed, whereas its Thr735Ala
mutant conferred a limited response to p38 MAPK activation.
TGF-a shedding in response to Erk MAPK signaling in these cells
was largely unaffected by the Thr735Ala mutation (Figure 5D).
Together, these data illustrate that Thr735 phosphorylation is
required for TACE activation by p38 MAP kinase, but not by
the Erk MAPK pathway.
p38 MAP Kinase Signaling Increases the Presentation
of TACE at the Cell Surface
The molecular mechanism of TACE activation has largely remained elusive but been suggested to affect the secretory
transport (Soond et al., 2005). We therefore examined the
effect of p38 MAPK activation on the cell surface levels of
TACE in Ca cells (Figure 6A). As reported (Schlöndorff et al.,
2000), TACE appeared as two forms at the cell surface: proTACE that has an N-terminal prodomain and corresponds to
the bulk of TACE that resides inside cells and active TACE
(mTACE) that has this prodomain removed (Figure 6A).
Because cells in culture have basal p38 MAPK signaling, we
compared cells in which p38 MAPK signaling was inhibited
by SB203580 with cells treated with anisomycin to activate
p38 MAPK. p38 MAPK activation resulted in an increased
cell surface level of mTACE that was also seen in the presence
of the TACE inhibitor TAPI-1 and therefore does not depend
on its catalytic activity. The increase in cell surface
Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc. 557
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
A
B
p38a
p38a-AF
p38b-AF
p38b
TACE-Myc
–
–
–
–
+
+
–
–
–
+
–
+
–
–
+
–
–
+
–
+
–
–
–
+
+
IP: anti-Myc
IB: anti-Flag
p38b-Flag
p38a-Flag
IB: anti-Flag
p38b-Flag
p38a-Flag
TACE-Flag
TACE-Flag (T735A)
p38a
p38a-AF
MKK6-E
_
Autoradiography 98 _
62
IB: anti-Flag
kDa
+
–
–
–
+
+
–
+
–
+
+
–
–
+
+
–
+
+
–
+
pTACE
TACE-Flag
p38a-Flag
IB: anti-Flag
IB: anti-Myc
TACE-Myc
D
C
TACE-Flag
– +
+ +
p38a
– –
p38a-AF
MKK6-E
+ +
_
150
Autoradiography
_
100
IB: anti-Flag
kDa
DMSO
SB203580
+
+
–
+
+ + –
– – +
+
–
+
+
pTACE
TACE-Flag
+
–
–
+
–
–
–
–
TACE-Flag
p38a
p38a-AF
p38b
p38b-AF
MKK6-E
150 _
Autoradiography100 _
IB: anti-Flag
kDa
IB: anti-pp38
–
–
+
pTACE
TACE-Flag
a-tubulin
+
–
+
–
–
–
+
–
–
+
–
–
+
–
–
+
–
–
+
–
–
+
–
–
–
+
–
+
–
–
+
–
+
TACE-Flag
pp38
+ + – + + + + – + +
– – + – – – – + – –
M
–
–
+
+
+
–
–
–
+
–
+
+
IP: anti-TACE
IB: anti-pTP
pTACE
IP: anti-TACE
IB: anti-TACE
TACE
IB: anti-p38
p38
oc
C k
on
IL trol
-1
An b
is
om
yc
in
+ – –
Ca
Ca-TACE
– + +
Ca-TACE (T735A) – – –
IP: anti-Flag
150 _
Autoradiography 100 _
IB: anti-Flag
kDa
IB: anti-a-tubulin
+ + –
DMSO
– – +
SB203580
Anisomycin
+ + +
+
–
+
–
–
–
oc
C k
on
SB trol
2
IL 035
-1 8
An b 0
is
o
C my
on c
in
t
IL rol
-1 si
R
IL b + NA
-1 C
b on
+ tr
p3 ol
8a si
R
si N
R A
N
A
F
+
+
–
–
–
–
pTACE
DMSO
Anisomycin
E
+
+
–
–
–
–
M
G
IP: anti-TACE
IB: anti-p38
p38
IB: anti-p38
p38
IB: anti-TACE
TACE
IP: anti-TACE
IB: anti-TACE
IP supernatants
IB: anti-TACE
TACE
IB: anti-pp38
pp38
TACE
Figure 4. p38a MAP Kinase Interacts with and Phosphorylates TACE
(A) Ca cells were transfected to express Myc-tagged TACE with Flag-tagged p38a or p38b MAPK or inactive p38a-AF or p38b-AF. Cell lysates were
subjected to anti-Myc immunoprecipitation and anti-Flag immunoblotting. TACE and p38 MAPK expression were shown by immunoblotting of cell lysates.
(B) TACE, TACE (T735A) mutant, p38a MAPK, p38a-AF, and activated MKK6 (MKK6-E) were generated by in vitro transcription and translation. p38a MAPK was
then activated through phosphorylation by MKK6-E. Phosphorylated p38a MAPK and TACE were purified using anti-Flag beads and were incubated with
32
P-gATP in a kinase assay. The bottom panels show immunoblotting of the proteins in the kinase assays. TACE is smaller than in vivo-expressed TACE due
to lack of glycosylation.
(C) In vitro kinase assays as in (B) were performed with purified proteins from transfected Ca cells with SB203580 added to inhibit p38a MAPK.
558 Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc.
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
presentation of mTACE in response to anisomycin correlated
with ectodomain shedding of TGF-a, as apparent from the
strong decrease in cell surface levels of TGF-a forms II and
III (Figure 6A).
To examine whether TACE phosphorylation at Thr735 by p38
MAPK affects the cell surface presentation of TACE, we stably
transfected Ca cells to express a C-terminally Flag-tagged
form of human wild-type or T735A mutant TACE (Figure 6B).
Anisomycin induced increased cell surface levels of wild-type
mTACE when compared with cells in the presence of
SB203580 (Figure 6B), consistent with what we observed for
endogenous TACE (Figure 6A). However, anisomycin did not
significantly affect the cell surface levels of T735A mTACE. Cells
expressing T735A TACE also expressed two smaller Flag-tagged
proteins at the cell surface (Figure 6B). Their size and reactivity
with antibody to the C-terminal Flag epitope suggest that they
derive through proteolytic removal of the catalytic and disintegrin
domains. This process is likely to occur primarily at the cell
surface because total TACE in the cell lysate showed only low
levels of these fragments. We conclude that phosphorylation of
Thr735 by p38 MAPK regulates the cell surface levels and processing of TACE.
p38 MAP Kinase Regulates Cell Proliferation by
Shedding of EGF Receptor Ligands by TACE
TACE expression is often increased in carcinomas, especially
breast carcinomas, and has been implicated in EGFR-mediated
cancer progression by TGF-a family growth factors (BorrellPagès et al., 2003; Kenny and Bissell, 2007; Zhou et al., 2006).
Because p38 MAPK activates TACE and induces the release
of TGF-a, we studied the role of p38 MAPK in the proliferation
of T4-2 breast carcinoma cells. Like many carcinomas, the
proliferation and tumor formation of T4-2 cells depend, in part,
on autocrine EGFR-dependent stimulation, resulting from
ectodomain shedding of TGF-a and amphiregulin (Kenny and
Bissell, 2007).
In cell culture, the proliferation of T4-2 cells was inhibited by
TAPI-1, an inhibitor of metalloproteases including TACE, the
EGFR inhibitor AG1478, or the MEK inhibitor PD98059, which
blocks activation of the Erk MAPK pathway downstream from
the EGFR (Figures 7A and 7B). These results indicate that T4-2
cell proliferation depends on TACE activity and EGFR signaling,
as reported (Kenny and Bissell, 2007). In addition, the proliferation was inhibited by SB203580, but not its negative control
SB202474, indicating an important role of p38 MAPK.
IL-1b enhanced the cell proliferation (Figures 7C and 7D),
which was inhibited by silencing p38a MAPK or TACE expression. Further, neutralizing antibodies to TGF-a and amphiregulin
or antibodies that prevent ligand binding to the EGFR inhibited
the basal and IL-1-induced proliferation (Figures 7C and 7D).
Like IL-1b, LPS also enhanced cell proliferation, which was inhibited by SB203580, as well as by TAPI-1 or the EGFR inhibitor
(Figure S5). Long-term treatment of cultured cells with anisomycin was toxic, and its effect was therefore not examined in these
assays. Together, these results indicate that the activation level
of p38 MAPK, which is enhanced in response to inflammatory
mediators, is a key determinant of the proliferation of T4-2 cells,
mediated at least in part by its effect on TACE activity and acting
through the EGFR.
We next studied whether p38 MAPK controls EGFR activation
as a consequence of the release of soluble EGFR ligand by
TACE. Although T4-2 cells express more amphiregulin than
TGF-a (Kenny and Bissell, 2007), we elected to score the
TGF-a release using an ELISA, considering that both ligands
are released through cleavage by TACE (Sahin et al., 2004).
Consistent with previous results (Figure 1E), IL-1b induced the
release of TGF-a, albeit to a lower extent than anisomycin (Figure 7E). Silencing the expression of p38a MAPK or TACE
repressed its release. LPS similarly induced TGF-a release,
and the basal and LPS-induced TGF-a release were inhibited
by SB203580 or TAPI-1 (Figure S5C).
We examined the autocrine EGFR activation under the same
conditions of IL-1 treatment as in Figure 7E. The EGFR activation, assessed by its level of Tyr phosphorylation (Figure 7F),
correlated with the secreted TGF-a levels (Figure 7E). IL-1b or
anisomycin induced EGFR activation, which was prevented by
silencing the p38a MAPK or TACE expression. As expected,
neutralizing TGF-a and amphiregulin antibodies or EGFR antibodies prevented the autocrine EGFR activation (Figure 7F).
Consistent with these results, LPS induced EGFR activation,
and the p38 MAPK inhibitor SB203580 and TACE inhibitor
TAPI-1 inhibited the basal and LPS-induced EGFR activation
(Figures S5C and S5D). Whereas the EGFR kinase inhibitor
AG1478 inhibited the autocrine EGFR activation rapidly,
SB203580 and TAPI-1 required a longer time to do so (Figures
S5C and S5D), as they act on the release of ligands that activate
the EGF receptor rather than on the EGFR itself. These data
suggest that the regulation of the ectodomain shedding by
TACE through p38 MAPK signaling defines the EGFR activity.
To further define the role of p38a MAPK in EGFR activation, we
silenced the TACE or p38a MAPK expression (Figure 7G, left). As
(D) Ca cells were transfected to express TACE with p38a or p38b MAPK, or p38a-AF or p38b-AF, with or without MKK6-E. Cells were treated for 30 min or not with
anisomycin and were lysed, and TACE and p38 MAPK proteins were purified using anti-Flag beads and incubated with 32P-gATP in a kinase assay. The proteins
were separated by SDS-PAGE, and autoradiography detected 32P-labeled TACE.
(E) Ca cells and Ca cells expressing TACE or T735A TACE were cultured in 32P-orthophosphate, treated with anisomycin, with or without SB203580, and lysed.
Immunoprecipitated, 32P-labeled TACE was visualized by SDS-PAGE and autoradiography. Anti-Flag immunoblotting showed the immunoprecipitated
TACE-Flag.
(F) T4-2 cells, transfected or not with control siRNA or p38a MAPK siRNA, were treated or not with SB203580, anisomycin, or IL-1b for 30 min, lysed, and subjected to anti-TACE immunoprecipitation and immunoblotting with anti-phosphoThr-Pro (anti-pTP) antibody to reveal phosphorylated TACE or with anti-TACE
antibody to reveal endogenous TACE. The bottom panel shows p38 MAPK levels, assessed by immunoblotting.
(G) Untransfected T4-2 cells were treated or not with IL-1b or anisomycin for 15 min, lysed, and subjected to anti-TACE or IgG (Mock) immunoprecipitation,
followed by anti-p38 MAPK immunoblotting. Immunoblotting shows endogenous TACE and p38 MAPK in cell lysates. Immunoprecipitated TACE was visualized
by immunoblotting, and immunoprecipitate supernatants were adsorbed to Con A-Sepharose followed by immunoblotting using anti-TACE antibody.
Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc. 559
Molecular Cell
A
B
M2 cells
IP: anti-Flag
IB: anti-Flag
p38a
MKK6-E
–
+
+
+
+
+
+
+
+
+
+
+
Ve
ct
TA or
C
E
TA w
C t
E
TA (T
C 73
E
5
TA (S A)
C 79
E
1
(S A)
81
9A
)
TACE Mediates p38 MAPK-Induced Cell Proliferation
TACE-Flag
+ +
+ +
IP: anti-Flag
150 _
Autoradiography 100 _
kDa
IP: anti-Flag
IB: anti-pTP
IB: anti-Flag
pTACE
pTACE
TACE-Flag
p38a-Flag
IB: anti-Flag
TA Ve wt S8 S7 T7 T7 T7
CE cto
19 91 35 35 35
A A A A, A,
r
S8 S7
19 91
A A
on
PM trol
A
An
is
C om
on y
c
PM trol in
A
An
is
om
yc
in
D
C
C
IB: anti-TACE
II
I Ca
III
TACE
IB: anti-a-tubulin
a-tubulin
IB: anti-TGF-a
IB: anti-TACE
II
I CaTACE
III
TACE
IB: anti-a-tubulin
a-tubulin
IB: anti-TGF-a
IB: anti-TACE
II
I CaTACE (T735A)
III
TACE
IB: anti-a-tubulin
a-tubulin
IB: anti-TGF-a
Control siRNA
TACE siRNA
+
–
+
–
+
–
–
+
–
+
–
+
Figure 5. Phosphorylation of TACE at Thr735 Is Required for Activation of Shedding by p38 MAP Kinase
(A) Wild-type or mutant TACE was expressed in Ca cells, purified using anti-Flag beads, and incubated with 32P-gATP and p38a MAPK, which was separately
coexpressed with MKK6-E and purified using anti-Flag beads. Phosphorylated TACE was visualized by autoradiography (top) or immunoblotting with anti-pTP
antibody (middle). Anti-Flag immunoblotting revealed the levels of TACE and p38a MAPK.
(B) M2 cells, transfected to express TGF-a and low-level wild-type or mutant TACE, were treated with anisomycin or SB203580 or were additionally transfected to
express p38a-AF. Ectodomain shedding was evaluated by measuring TGF-a in the medium. TACE was visualized by anti-Flag immunoblotting of proteins precipitated with anti-Flag M2 beads (top). *p < 0.01, compared with wild-type TACE control. **p < 0.01, compared with wild-type TACE or wild-type TACE with
anisomycin.
(C and D) Ca cells and Ca cells expressing human wild-type or T735A TACE were transfected with control or TACE siRNA to silence endogenous TACE expression. Ectodomain shedding was induced with PMA or anisomycin and evaluated by immunoblotting for transmembrane TGF-a (C) or measuring TGF-a in the
medium (D). TACE was visualized by immunoblotting; siRNA-mediated silencing of endogenous TACE could only be revealed in Ca cells that do not express
human TACE. *p < 0.01, compared with cells expressing wild-type TACE.
Error bars show the standard deviations based on triplicate values of each data set.
in Figure 7F, silencing TACE or p38a MAPK expression decreased the basal EGFR activation, resulting in decreased Erk
MAPK activation (Figure 7G, middle), a downstream effect of
EGFR signaling that regulates proliferation. These results illustrate that TACE and p38a MAPK regulate the activation of Erk
MAPK by EGFR signaling.
560 Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc.
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
activation of TACE and the release through ectodomain shedding of EGFR ligands and increased EGFR signaling.
A
DMSO
SB203580
Anisomycin
TAPI-1
Biotin-Neutravidin
IB: anti-TACE
AP: Con A
IB: anti-TACE
+
–
–
–
–
+
–
–
–
–
+
–
–
+
+
–
–
–
+
+
DISCUSSION
proTACE (Surface)
mTACE (Surface)
TACE (Lysate)
II
Biotin-Neutravidin
IB: anti-TGF-a
III
IB: anti-a-tubulin
a-tubulin
B
DMSO
SB203580
Anisomycin
+ – –
– + –
– – +
+
–
–
– –
+ –
– +
kDa 150 –
100 –
Biotin-Neutravidin 75 –
IB: anti-Flag
50 –
proTACE-Flag
mTACE-Flag
TACE-Flag (Truncated)
37 –
TACE-Flag (Truncated)
25 –
TACE-Flag (Lysate)
IB: anti-Flag
TACE
wt
T735A
Figure 6. Activation of p38 MAP Kinase Enhances TACE Levels at
the Cell Surface
(A) Ca cells were treated with anisomycin, without or with SB203580, or
TAPI-1. ProTACE and active TACE (mTACE) were detected at the cell surface
by immunoblotting cell surface biotinylated proteins with anti-TACE antibody,
and total glycosylated TACE in the cell lysate was shown by immunoblotting
concanavalin A-adsorbed proteins. Cell surface biotinylation detected TGFa forms II and III at the cell surface and correlated the increased cell surface
mTACE level with increased shedding of TGF-a.
(B) Cell surface TACE, detected as in (A), in Ca cells stably expressing wildtype or T735A TACE and treated with SB203580 or anisomycin. ProTACE
and mTACE were detected, and truncated TACE forms were at the surface
of cells expressing T735A, but not wild-type TACE. The bottom panel shows
the total TACE in cell lysates.
We next compared the cell proliferation using BrdU uptake
(Figure 7G, right). The decrease in TACE or p38a MAPK expression inhibited cell proliferation by 50% and 36%, respectively,
consistent with the effects of TAPI-1 or SB203580 on cell
proliferation (Figures 7B and S5E). The stronger inhibition by
interfering with p38a MAPK activity, compared with TACE inactivation, may suggest that p38 MAPK signaling involves other
aspects of cell proliferation besides the activation of the EGFR
through TACE-mediated EGFR ligand shedding. The inhibition
of cell proliferation by interfering with p38a MAPK or TACE
activity was largely rescued by adding EGFR ligands (Figures
7G, right, and S5E).
Together, these results indicate that p38 MAPK activation,
e.g., in response to inflammatory mediators or stress, critically
regulates the proliferation of these cells. The induction of proliferation by p38 MAPK signaling results, at least in part, from the
Activation of Shedding in Response to p38 MAP Kinase
Signaling Is Mediated by TACE
We have shown that inducers of p38 MAPK signaling induce ectodomain shedding and that inhibition of p38 MAPK blocks
shedding in response to these inducers. Our results complement
reports that p38 MAPK inhibitors prevent shedding in different
contexts (Fan and Derynck, 1999; Fischer et al., 2004; Takenobu
et al., 2003). Thus, in addition to the Erk MAPK pathway, p38
MAPK activates ectodomain shedding. Among the p38 MAPKs,
shedding was specifically activated by p38a MAPK, which has
been linked to the activities of inflammatory cytokines (Dong
et al., 2002; Ono and Han, 2000). Thus, siRNA specific for
p38a MAPK prevented shedding in response to p38MAPK
inducers, and no response by other p38 MAPKs was apparent.
TACE was shown to mediate shedding in response to Erk
MAPK pathway activation. Which metalloprotease effects shedding in response to p38 MAPK activators has remained unclear,
and it has been proposed that shedding in response to such
inducers is mediated by other metalloprotease(s) (Dı́az-Rodrı́guez et al., 2002; Hinkle et al., 2003; Takenobu et al., 2003).
Our use of siRNA to silence TACE expression, as well as rescue
experiments using wild-type or mutant TACE, show that TACE
effects shedding in response to p38 MAPK activation. Further,
introduction of wild-type TACE in M2 cells that do not express
active TACE (Li and Fan, 2004) rescued ectodomain shedding
in response to Erk or p38 MAPK pathway activation. We
conclude that TACE is activated by either of these pathways
that are, in turn, activated by diverse stimuli.
Mechanism of TACE Activation by the p38 MAP
Kinase Pathway
The activation of TACE-mediated ectodomain shedding by p38
MAPK was rapid and did not require new protein synthesis, suggesting that TACE activation results from a posttranslational
event. Although ROS was proposed to play roles in activation
of p38 MAPK or TACE, the antioxidant MNTmPyP did not affect
shedding in response to anisomycin.
Wild-type, but not kinase-defective, p38a MAPK associated
with and phosphorylated TACE in response to stimuli that activate p38 MAPK, such as anisomycin and IL-1b. The phosphorylation site, Thr735, is located in a predicted p38 MAPK target
sequence, i.e., Pro-Gln-Thr735-Pro, and its mutation prevented
TACE phosphorylation by p38 MAPK and activation of shedding
in response to anisomycin or IL-1b. This finding directly links
extracellular cues that activate the p38 MAPK pathway with ectodomain shedding. The targeting of a metalloprotease by p38
MAPK adds to our understanding of the role of p38 MAPK, which
hitherto is known to primarily regulate transcription and translation (Ono and Han, 2000), and raises the possibility that p38
MAPK targets other ADAM metalloproteases.
The mechanism by which cell signaling activates TACE has
remained elusive. It has been proposed that the TACE prodomain contains a cysteine switch involved in TACE activation,
Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc. 561
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
B
A
DMSO
AG1478
SB203580
TAPI-1
PD98059
SB202474
D
C
Control + ctrl siRNA
IL-1b + ctrl siRNA
IL-1b + p38a siRNA
IL-1b + TACE siRNA
Co
IL-1b + EGFR Ab
IL-1b + IgG
E
F
C
I
I
I
I
I
I
ntr L-1b L-1b L-1b L-1b L-1b L-1b
ol
+ c + ctr + p3 + TA + TG + EG + IgG
8
l
C
F
F
trl
siR siRN a siR E si -a/A R A
NA A
NA RNA R A b
b
on
t
IL rol
-1 +
b ct
IL + rl
-1 ct siR
b r
IL + l siR NA
-1 p3 N
b
IL + 8a A
-1 TA siR
b
IL + CE NA
-1 TG s
iR
b
IL + F-a NA
-1 EG /A
b
An + FR R A
is IgG Ab b
om
yc
in
IL-1b + TGF-a/AR Ab
Co
A
I
I
I
I
I
ntr L-1b L-1b L-1b L-1b L-1b niso
ol
+ s + ctr + p3 + TA + TG + IgG myci
n
CE
FiRN l siR 8a
NA siRN siR a/AR
A
NA
A
Ab
IP: anti-EGFR
IB: anti-pY
pEGFR
IP: anti-EGFR
IB: anti-EGFR
EGFR
IB: p38
p38
IP supernatants
IB: anti-TACE
TACE
IB: anti-a-tubulin
a-tubulin
TACE
p38
pp38
a-tubulin
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EGFR
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Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
that TIMP3 acts as natural inhibitor of TACE, and that TACE
maturation and localization are involved in TACE activation.
Intriguingly, a phosphomimetic mutation of Thr735 in TACE
affects the subcellular localization of TACE toward the secretory pathway (Soond et al., 2005). We now show that p38
MAPK activation enhances the cell surface presentation of
active TACE and that this increase correlates with activation
of shedding. Replacing Thr735 with alanine that cannot be
phosphorylated resulted in high levels of two truncated TACE
forms at the cell surface, which are predicted to be nonfunctional. Possibly, Thr735 phosphorylation may regulate proteolysis of TACE at the cell surface, a possibility that needs further
exploration.
Erk MAPK has been reported to phosphorylate TACE on Thr735
(Dı́az-Rodrı́guez et al., 2002). However, mutation of Thr735 had
little effect on the PMA-induced activation of TACE, which
occurs through the Erk MAPK pathway. We have shown that
growth factors induce Ser819 phosphorylation and Ser791
dephosphorylation. Ser819 phosphorylation depends on Erk
MAPK pathway, whereas Ser791 dephosphorylation does not
(Fan et al., 2003). We now show that Ser791 mutation, mimicking
dephosphorylation, enhances TACE phosphorylation at Thr735
by p38 MAPK and that mutation of Ser819 decreased Thr735
phosphorylation by p38 MAPK, suggesting a crosstalk between
growth factor signaling and p38 MAPK activation of TACE.
Role of Regulation of TACE by p38 MAP Kinase in EGF
Receptor Function and Cancer
Increased autocrine EGFR activation by TGF-a ligands drives
carcinoma progression (Hanahan and Weinberg, 2000). Consistent with the notion that soluble TGF-a is more effective than
transmembrane TGF-a in carcinoma development (Blobel,
2005; Borrell-Pagès et al., 2003), human cancers show
increased TACE expression (Mochizuki and Okada, 2007). In
addition, increased TACE-mediated release of TGF-a or amphiregulin enhances EGFR-mediated cell proliferation and transformation, as observed in breast carcinoma cells (Kenny and
Bissell, 2007; Zhou et al., 2006). We therefore assume that
the increased Erk MAPK activity, observed in many carcinomas
(Sebolt-Leopold and Herrera, 2004), increases the TACEmediated release of TGF-a ligands and, consequently, the
EGFR activation.
Various studies suggest that the p38 MAPK pathway exerts
tumor suppression by modulating aspects of cell behavior.
However, inflammatory cytokines that activate p38 MAPK,
e.g., IL-1 or TNF-a, have cancer-promoting roles (Moore et al.,
1999; Vidal-Vanaclocha et al., 2000), and p38 MAPK promotes
the proliferation of cancer and immune cells (Dolado et al.,
2007; Ono and Han, 2000). Thus, the overall role of p38 MAPK
signaling in cancer is likely to depend on the tumor type and
cancer stage. Our observations that TACE is activated by p38
MAPK now link inflammatory signals to increased EGFR
signaling. Indeed, p38 MAPK activation, due to the inflammatory
cytokine release, activates TACE, resulting in the release of
TGF-a family ligands. These, in turn, activate the EGFR, leading
to increased Erk MAPK signaling and cell proliferation, possibly
contributing to carcinoma progression. Further, p38a MAPK
activity in autocrine EGFR-dependent cancer cells is required
for maintaining self-sufficient growth, a property acquired in
cancer progression (Hanahan and Weinberg, 2000). This
scenario allows for crosstalk between Erk MAPK and p38
MAPK signaling that may not only contribute to carcinoma
progression, but also be of relevance in inflammation that is
not associated with cancer.
EXPERIMENTAL PROCEDURES
Expression Plasmids, Reagents, and Antibodies
Plasmids encoding C-terminally Flag-tagged p38a or p38b MAPK, or their
kinase-inactive forms p38a-AF and p38b-AF (Huang et al., 1997), or constitutively active MKK6-E (Jiang et al., 1996) were described. Plasmids encoding
Flag- or Myc-tagged TACE were made by subcloning the TACE sequence
into pRK5 (Graycar et al., 1989). Plasmids encoding the TACE mutants
T735A, S791A, S819A, T735A-S791A, and T735A-S819A were made by QuikChange Site-Directed Mutagenesis (Stratagene) using pRK5-TACE-Flag. The
expression plasmids for TGF-a or TACE E406A mutant have been described
(Fan et al., 2003). Coding DNA sequences were verified by DNA sequencing.
PMA, anisomycin, IL-1b, TAPI-1, SB203580, SB202474, U0126, PD98059,
JNK inhibitor II, AG1478, Bisindolylmaleimide I (BIM), brefeldin A, MnTMPyP,
PTIO, L-NIL, EGF, and TGF-a were from Calbiochem. Cycloheximide, LPS,
and anti-Flag (M2) agarose were from Sigma. Protein A Sepharose was from
Amersham, ConA-Sepharose was from GE Healthcare, and EZ-link
Sulfo-NHS-LC-Biotin and Neutravidin beads were from Pierce. g-32P ATP
and 32P-orthophosphate were from Perkin Elmer.
Goat anti-TGF-a polyclonal antibody (R&D) was used for immunoprecipitation and neutralization, and the monoclonal anti-TGF-a ab-1 antibody (Calbiochem) was used for immunoblotting. The EGFR was immunoprecipitated or
Figure 7. p38 MAP Kinase Signaling Stimulates T4-2 Cell Proliferation by TACE-Mediated Shedding of EGF Receptor Ligands
(A and B) Effects of TAPI-1, the EGFR inhibitor AG1478, MEK inhibitor PD98059, SB203580, or negative control SB202474 on the proliferation of T4-2 cells. BrdU
incorporation was measured by ELISA and expressed relative to untreated control. *p < 0.01, compared with DMSO control.
(C and D) Effect of IL-1b on proliferation of cells transfected with p38a MAPK, TACE, or control siRNA or treated with neutralizing antibodies to TGF-a and
amphiregulin (AR), or the EGFR, or rabbit IgG. BrdU incorporation was measured by ELISA and expressed relative to untreated control. *p < 0.01, compared
with control. **p < 0.001, compared with IL-1b-treated cells transfected with control siRNA or treated with IgG.
(E and F) T4-2 cells transfected or not with p38a MAPK, TACE, or control siRNA were treated for 60 (E) or 30 min (F) with IL-1b or anisomycin without or with
neutralizing antibodies to TGF-a and amphiregulin (AR), or the EGFR, or rabbit IgG. TGF-a released in the medium was quantified by ELISA (E). In (F), antiEGFR immunoprecipitates were blotted with anti-phosphoTyr (anti-pY) to show EGFR activation or with anti-EGFR for EGFR expression. Endogenous p38
MAPK and TACE were visualized by immunoblotting of cell lysates or Con A-adsorbed proteins. *p < 0.001, compared with control siRNA. **p < 0.001, compared
with IL-1b stimulation of cells transfected with control siRNA or treated with IgG.
(G) T4-2 cells were transfected with TACE, p38a MAPK, or control siRNA. Immunoblotting of cell lysates showed downregulation of endogenous TACE or p38a
MAPK expression in siRNA-transfected T4-2 cells. EGF receptor activation and expression are shown as in (F). Erk MAPK activation was shown by immunoblotting for phospho-Erk MAPK (pErk) and total Erk MAPK. The right panel shows the proliferation with or without 5 ng/ml TGF-a or EGF. *p < 0.01, compared with
untreated cells transfected with control siRNA. **p < 0.01, compared with untreated cells transfected with TACE or p38a MAPK siRNA.
Error bars show the standard deviations based on triplicate values of each data set.
Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc. 563
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
neutralized using EGFR antibody clone 528 (Calbiochem), its Tyr phosphorylation was detected by immunoblotting using anti-pY antibody (Zymed Laboratories), and EGFR levels were detected by immunoblotting with anti-EGFR
antibody (Santa Cruz Biotechnology). Anti-amphiregulin goat polyclonal antibody (R&D) was used for neutralization. Anti-Erk, anti-pErk, anti-pp38
MAPK, and anti-pTP antibodies were from Cell Signaling; rabbit anti-p38
MAPK polyclonal antibody and anti-p38a/b MAPK monoclonal antibody
were from Santa Cruz Biotechnology. Rabbit anti-TACE polyclonal antibody
was from QED Bioscience. Anti-a-tubulin, streptavidin-HRP, anti-Flag (M2),
anti-FLAG (rabbit), and anti-Myc (9E10) antibodies were from Sigma.
Cell Culture and Transfections
CHO cells and derivative Ca (Fan and Derynck, 1999) and M2 cells (Li and Fan,
2004) were cultured in F-12 medium with 10% FBS. Ca cells were also supplemented with 200 mg/ml Geneticin (Roche) to maintain TGF-a expression. T4-2
cells were provided by Dr. M. Bissell (Lawrence Berkeley National Laboratory)
and cultured as described (Kenny and Bissell, 2007). HeLa cells expressing
TGF-a (Imhof et al., 2008) were maintained in DMEM, supplemented with
10% FBS and 2 mg/ml puromycin. Transfections of CHO cells were done using
Fugene 6 (Roche). Ca cells were stably transfected with plasmids for wild-type
TACE or T735A TACE in the presence of pRK7-Hyg, allowing selection with
600 mg/ml hygromycin B (Roche). Colonies were obtained by serial dilution,
and immunoblot analysis for TACE expression was performed using antiFlag M2 antibody.
Ectodomain Shedding Assay
Cells at 80% confluence were starved overnight, washed, and treated for
60 min with anisomycin (1 mM), LPS (2.5 mg/ml), IL-1b (60 ng/ml), UV light
(60 mJ), PMA (25 nM), and 20% FBS in serum-free medium or medium containing 200 mM NaCl (osmotic shock). Effects of an inhibitor or antibody
were evaluated by incubating the cells for 30 min prior to and during treatment.
T4-2 cells were also incubated with 0.5 mg/ml anti-EGFR neutralizing antibody
for 15 min before stimulation to prevent ligand binding to the EGFR. Inhibitors
and neutralizing antibodies include the p38 MAPK inhibitor SB203580 (10 mM),
its negative control SB202474 (10 mM), MEK1/2 inhibitor U0126 (10 mM), JNK
inhibitor II (50 mM), protein kinase C inhibitor Bisindolylmaleimide I (BIM, 1 mM),
metalloprotease inhibitor TAPI-1 (10 mM), EGFR kinase inhibitor AG1478
(1 mM), NO scavenger PTIO (100 mM), iNOS inhibitor L-NIL (50 mM),
anti-oxidant MnTMPyP (50 mM), brefeldin A (10 mM), cycloheximide (10 mM),
anti-TGF-a (1 mg/ml), anti-amphiregulin (5 mg/ml) neutralizing antibodies,
anti-EGFR neutralizing antibody (1 mg/ml), and rabbit IgG (6 mg/ml). After treatment, cells were lysed in MLB (20 mM Tris-Cl, 200 mM NaCl, 10 mM NaF,
1 mM NaV2O4, 1% NP-40, 20 mM b-glycerophosphate, 2 mM Lefabloc,
0.5 mM leupeptin, and 1 ug/ml aprotinin [pH 7.5]), and lysates were analyzed
by 12.5% SDS-PAGE and immunoblotting with anti-TGF-a antibody to visualize transmembrane TGF-a. Released TGF-a was quantified by ELISA (Quantikine Immuno Assay kit; R&D Systems) of medium that was cooled on ice and
then cleared at 5000 rpm for 10 min to remove debris.
Coimmunoprecipitations and Western Blotting
Ca cells were transfected with 0.2 mg plasmids for Flag-tagged p38a or p38b
MAPK, or their mutants p38a-AF or p38b-AF, or Myc-tagged TACE. At 48 hr
after transfection, cells were lysed using MLB, and lysates were subjected
to immunoprecipitation using anti-Myc monoclonal antibody 9E10 and protein
A Sepharose beads for 2 hr. The beads were washed with MLB, and immunoprecipitated proteins were analyzed by 4%–20% SDS-PAGE and immunoblotting using anti-Flag antibody M2.
In Figures 4F and 4G, endogenous TACE and p38 MAPK were coimmunoprecipitated from T4-2 cells that were incubated for 30 min with SB203580
or were transfected with p38a MAPK siRNA or control siRNA for 48 hr and
treated with IL-1b or anisomycin for 15 min. Cells were then lysed using
MLB with 150 mM NaCl, and lysates were preincubated with rabbit IgG and
protein A Sepharose and subjected to immunoprecipitation using anti-TACE
polyclonal antibody (QED) and protein A Sepharose for 2 hr. Immunoprecipitated proteins were analyzed by 4%–20% SDS-PAGE and immunoblotting
using anti-p38a/b MAPK mIgG1 antibody. Immunoprecipitation supernatants
were subjected to Con A Sepharose, and the remaining TACE was visualized
by immunoblotting using anti-TACE antibody to ensure effective immunoprecipitation. Fractions of the cell lysates were subjected to SDS-PAGE and
immunoblotting to control for protein expression.
Cell Surface Protein Biotinylation
Cells were washed with ice-cold PBS and incubated with EZ-link Sulfo-NHSLC-Biotin (0.5 mg/ml in PBS) at 4 C for 60 min. Biotinylation reactions were
stopped with glycine (100 mM in PBS), and cells were lysed in MLB. Cell
lysates were incubated 2 hr with Neutravidin beads (Pierce). The beads were
washed with MLB, and the adsorbed proteins were analyzed by SDS-PAGE
and blotting with Streptavidin (Sigma) or anti-TGF-a (ab-1), anti-TACE (QED),
or anti-Flag antibody (Sigma).
RNA Interference
siRNAs (QIAGEN) to silence TACE expression targeted the Chinese hamster
TACE mRNA sequence 50 -GAGGAUUUAAAGGUUAUGGAA-30 (nucleotides
900-920; ref: AY313173Z) or the human TACE sequence 50 -AAGAAACAGAGU
GCUAAUUUA-3 (Hs_ADAM17_8) (nucleotides 2642-2662; ref: NM_003183.4)
in CHO or T4-2 cells, respectively. siRNA to silence p38a MAPK expression in
CHO cells targeted the sequence 50 -UCGUGAAGUGUCAGAAGCUUA-30
(Rn_Mapk14_4), derived from the CHO cell p38a cDNA (Ref: GU371300)
that we generated by RT-PCR using primers for nested PCR. siRNA for human
p38a MAPK targeted the sequence 50 -CUCAGUGAUACGUACAGCCAA-30
(Hs_MAPK14_7) (nucleotides 1933-1953; ref: NM_001315). Control siRNA
was used to control for possible nonspecific effects of RNA interference.
Transfections of siRNA were done using Lipofectamine RNAiMAX (Invitrogen).
Reverse transfection was used for HeLa and T4-2 cells to reach optimal efficiency. Cells were used at 48 hr after transfection.
TACE Proteolytic Activity Assay
TACE activity was measured using the InnoZyme TACE activity kit (CalBiochem). T4-2 cells were seeded in a 12-well plate and treated for 1 hr with anisomycin (1 mM), LPS (2.5 mg/ml), or IL-1b (60 ng/ml) without or with SB203580
(10 mM). Cells were lysed with MLB, and a portion of cell lysates was subjected
to the TACE activity assay. In brief, TACE in cell lysates was captured by antiTACE monoclonal antibody, and its activity was measured using an internally
quenched fluorescent substrate. Cleavage of amide bond of substrate
released the fluorophore, resulting in increased fluorescence, which is directly
related to the activity of TACE.
In Vitro Kinase Assay
In vitro kinase assays used proteins generated by in vitro translation or in transfected Ca cells. Plasmids encoding MKK6-E, Flag-tagged p38a MAPK, or
Flag-tagged TACE were subjected to in vitro transcription from the T7 or
SP6 promoter and translation using the TNT Quick-Coupled Transcription/
Translation kit (Promega). p38a MAPK, generated in vitro, was activated by
incubation with in vitro-translated MKK6-E in the presence of 20 mM ATP at
30 C for 30 min. To obtain proteins expressed in transfected cells, Ca cells
were transfected with 0.2 mg of plasmid expressing p38a or p38b MAPK, or
their mutants, MKK6-E, or wild-type or mutant TACE. TACE and p38a MAPK
from in vitro translation or cell lysates were immunoprecipitated using antiFlag M2 agarose beads and washed with MLB before in vitro kinase assay.
In vitro kinase assays were done with g-32P ATP (Perkin Elmer) in 20 mM
HEPES, 10 mM MgCl2, 1 mM DTT, 50 mM NaCl for 20 min at 30 C and
analyzed by 4%–20% SDS-PAGE, transferred to PVDF membrane, and subjected to autoradiography or western blotting.
In Vivo 32P-Orthophosphate Labeling
Ca, CaTACE, and CaTACE(T735A) cells at confluence were washed with
phosphate-free medium and incubated in phosphate-free medium for 3 hr
and then in phosphate-free medium with 0.5 mCi/ml 32P-orthophosphate (Perkin Elmer) for 2 hr. Anisomycin (1 mM) was added for the last 30 min before cell
lysis in MLB. Proteins, immunoprecipitated with anti-Flag agarose, were
analyzed by 4%–20% SDS-PAGE, transferred to PVDF membrane, and
analyzed by autoradiography or immunoblotting with anti-Flag antibody.
Parallel cell lysates without isotope were analyzed by SDS-PAGE and immunoblotting with anti-a-tubulin antibody.
564 Molecular Cell 37, 551–566, February 26, 2010 ª2010 Elsevier Inc.
Molecular Cell
TACE Mediates p38 MAPK-Induced Cell Proliferation
Cell Proliferation Assay
Cell proliferation was quantified by BrdU incorporation. T4-2 cells, in some
cases 24 hr after transfection with siRNA to TACE or p38a MAPK, or control
siRNA were seeded in a 48-well plate and treated with SB203580 (10 mM) or
its negative control SB202474 (10 mM), TAPI-1 (10 mM), EGFR inhibitor
AG1478 (1 mM), MEK inhibitor PD98059 (10 mM), LPS (2.5 mg/ml), or IL-1b
(60 ng/ml), anti-TGF-a (1 mg/ml) and anti-amphiregulin (5 mg/ml), or antiEGFR neutralizing antibody (1 mg/ml), or rabbit IgG (6 mg/ml) for 72 hr. Cells
were imaged using a LEICA DMI4000B microscope, and cells were incubated
with BrdU for 3 hr before ELISA. In the rescue analyses, EGF or TGF-a (5 ng/ml)
was added. BrdU incorporation assays were done using the BrdU Cell Proliferation Assay kit (Calbiochem).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures
and five figures and can be found with this article online at doi:10.1016/
j.molcel.2010.01.034.
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ACKNOWLEDGMENTS
This research was sponsored by grants RO1-CA63101 and PO1-HL60231
(Project III) to R.D. We are grateful to J. Han and R.J. Davis for plasmids encoding p38 MAPK, MKK3, or MKK6 and to M.J. Bissell for T4-2 cells.
Received: June 5, 2009
Revised: November 12, 2009
Accepted: January 23, 2010
Published: February 25, 2010
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