REVIEWA Brief History of Tumor Necrosis Factor α– converting Enzyme: An Overview of Ectodomain Shedding
Keisuke Horiuchi

Department of Orthopedic Surgery and Department of Anti-aging Orthopedic Research,
School of Medicine, Keio University, Tokyo, Japan(Received for publication on March 21, 2012)(Revised for publication on April 4, 2012)(Accepted for publication on May 18, 2012)

Many membrane-bound molecules are cleaved at the cell surface, thereby releasing their extracellulardomains. This process, often referred to as ectodomain shedding, has emerged as a critical post-translational mechanism for various membrane-bound ligands, receptors, and adhesion molecules. Tumor
necrosis factor α (TNFα)-converting enzyme (TACE/ADAM17) was originally identified as an enzyme
responsible for releasing the membrane-bound TNFα precursor. However, subsequent studies found an
exceptionally large number of target molecules of TACE, including the ligands for epidermal growth
factor receptor, L-selectin, CD44, and vascular growth factor receptor 2. Furthermore, in vivo studies
using TACE-conditional knockout mice demonstrated the crucial roles of TACE and ectodomain shedding under both physiological and pathological conditions. However, the potential clinical applicationof the manipulation of TACE activity remains to be investigated. (doi: 10.2302/kjm.2012-0003-RE;
Keio J Med 62 (1) : 29–36, March 2013)

Keywords: TACE/ADAM17, ectodomain shedding, TNFα

active soluble TNFα.4,5 However, studies over the past
decade have identified a wide range of membrane-boundproteins in addition to TNFα as substrates for TACE,1–3
including the ligands for epidermal growth factor receptor (EGFR),6 which are also produced as membranebound precursors and are cleaved to become fully functional. TACE is, therefore, an essential modulator of the
TNFα–TNFα receptor signaling pathway and the EGFR
ligand–EGFR signaling pathway in vivo.7 Because TNFαand EGFR are involved in the pathogenesis of many disorders, such as rheumatoid arthritis and breast cancer,
respectively, TACE has now emerged as a potential molecular target for treating these conditions.8,9 In this review, I will outline the history of TACE, beginning with
its identification, and summarize some of the recent findings uncovered using TACE mutant mice.

Introduction
Various membrane-bound proteins are proteolyticallycleaved to release their extracellular domain. This proteolytic mechanism, also called ectodomain shedding, may
appear inconsiderable, with no significant physiologicalrole. It is therefore not surprising that this phenomenon
has been overlooked in the past and has been considereda mere degradation process of membrane-bound proteins.
However, as I discuss in this review, studies during thepast two decades have revealed that ectodomain shedding
is an essential post-translational mechanism with critical roles during development, in homeostasis, and underpathological conditions in vivo.1–3
As its name suggests, tumor necrosis factor α (TNFα)converting enzyme (TACE), also known as a disintegrin
and metalloprotease 17 (ADAM17), was originally identified as an enzyme responsible for releasing the membrane-bound TNFα precursor to produce biologicallyReprint requests to: Keisuke Horiuchi, MD, PhD, Department of Orthopedic Surgery and Department of Anti-aging Orthopedic Research,
School of Medicine, Keio University, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan, E-mail: horiuchi@z3.keio.jp
Copyright © 2013 by The Keio Journal of Medicine
29

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Horiuchi K: The Biology of TACE

Fig. 1 ADAM gene family and structure.
(A) ADAM proteins contain a metalloprotease domain, a disintegrin domain, a cysteine-rich domain, an EGF-like domain, a transmembrane domain, and a cytoplasmic tail. The prodomain maintains the proteolytic activity in an inactive state until it is cleaved
by a furin protease in the trans-Golgi network before delivery of the ADAM protein to the cell surface. The metalloprotease domain
contains a zinc (Zn 2+)-binding domain that is highly conserved among the metalloprotease family. (B) Dendrogram depicting relative
amino acid sequence homologies among catalytically active and widely expressed ADAM proteins.

The TNFα Precursor is a Membrane-bound Protein
and Must be Shed from the Cell Surface to Becomean Active Cytokine
Before discussing the major topic of this review, I will
introduce some of the early work on the proteolytic cleavage of pro-TNFα that ultimately led to the identificationof TACE. TNFα is a central component of the immune
system and also plays critical roles in the pathogenesisof inflammatory diseases such as rheumatoid arthritis,
Crohn’s disease, and asthma. Because TNFα was initially found in the serum as a soluble protein, TNFα wasoriginally thought to be produced as a soluble cytokine.10
However, the determination of its cDNA nucleotide sequence unexpectedly revealed that TNFα is initially produced as a type-2 membrane-bound protein that has tobe proteolytically cleaved to become soluble.11,12 This
discovery led to the idea that the activity of TNFα could
be suppressed by inhibiting this proteolytic activity, thus
opening up a potential approach to treating patients with
TNFα-related diseases.
Therefore, the mechanism underlying the processingof the TNFα precursor was intensively investigated bypharmaceutical companies in the 1990s. In 1994, it was
shown that the production of soluble TNFα can be greatly
suppressed by ethylenediaminetetraacetic acid (a chelator
used for depleting metal ions) and metalloprotease inhibitors, indicating that the cleavage of TNFα is metalloprotease dependent.13–15 In addition, Mohler et al.15 showedthat treatment with a metalloprotease inhibitor provides

strong protection against lipopolysaccharide (LPS)-induced endotoxin shock in mice. That study showed forthe first time that ectodomain shedding is involved in thefunctional modification of membrane-bound proteins invivo and that the bioactivity of TNFα can be manipulated
by inhibiting TNFα cleavage. The results also indicatedthat the pharmaceutical suppression of TNFα shedding
could be beneficial for treating TNFα-related disorders.
Later, the importance of ectodomain shedding in the regulation of TNFα function was also confirmed by a studyof a mutant mouse line in which pro-TNFα was genetically modified to be resistant to proteolytic cleavage.16 Thesecleavage-resistant TNFα mutant mice exhibited very
similar immune defects to those found in Tnfα-null mice,demonstrating that the soluble form, not the membranebound pro-form, is responsible for the major functions of
TNFα. Taken together, these observations confirmed that
ectodomain shedding is an indispensable mechanism forthe functional activation of TNFα in vivo.
Identification of TNFα-converting Enzyme, TACE
Three years after publication of the reports establishingthat the enzyme involved in TNFα cleavage was metalloprotease dependent,13–15 two independent groups identified an enzyme capable of cleaving pro-TNFα in vitro.4,5The enzyme, TACE, was a membrane-bound protein
and, as predicted by previous research, had a characteristic Zn2+-binding motif (HExxHxxGxxH), a highly
conserved motif among metalloproteases. On the basis

Keio J Med 2013; 62 (1): 29–36

of amino acid homology, TACE was found to belong to
the ADAM gene family (Fig. 1); therefore, TACE is also
called ADAM17.
The identification of TACE marked the first time thata potential link between a sheddase and its target molecule (in this case TACE and TNFα, respectively) had
been demonstrated and is undoubtedly one of the mostimportant discoveries in the field of ectodomain shedding. However, it remains elusive why TNFα is produced
as a membrane-bound protein and not as a soluble protein, as other cytokines are. It may be that this machinery
has evolved to release emergency signaling molecules
as rapidly as possible (the cleavage of membrane-boundproteins is generally more rapid than synthesizing them).
Nevertheless, because of two important studies publishedin 1997,4,5 the newly identified TACE gene emerged as
one of the most promising candidate genes responsible
for ectodomain shedding of the TNFα precursor. The next
step was to generate TACE-deficient mice and to confirm
whether TACE really is the relevant sheddase for TNFαin vivo. However, as described in the next section, the abrogation of the Tace gene in mice unexpectedly resulted
in early lethality, and the mice could not be used to analyze TNFα activity in vivo.
TACE Proved to Be an Enzyme with
Many Substrates
Although TACE had emerged as a candidate enzyme
responsible for the cleavage of pro-TNFα, other proteases, including matrix metalloprotease (MMP) 7,17
ADAM10,18 ADAM19,19 and protease 3,20 had also been
implicated in releasing TNFα, raising questions about theidentity of the relevant TNFα convertase in vivo. Because
both the MMP gene family and the ADAM gene family contain a large number of members, it seemed likelythat several different proteases could be involved in theprocessing of TNFα, and the generation and analysis of
TACE knockout mice was expected to clarify this issue.
Mice lacking TNFα or its receptors (TNF receptor 1 and
2) had been generated and were shown to be viable andfertile, even though they all showed similar immune system defects.21,22 Therefore, it was assumed that TACEnull mice would be viable without any overt defects. Contrary to this hypothesis, a study published in 1998 showedthat the disruption of TACE results in perinatal lethality
and a highly complex phenotype.23
Among the defects found in the TACE-null mice, themost striking and unexpected were open eyelids (miceare normally born with their eyelids closed) and abnormal skin and fur, which were nearly identical to the phenotypes of EGFR-null mice.24,25 Seven different ligands
had been identified for EGFR, and all of these ligands
were produced as membrane-bound precursors that must
be cleaved to become soluble growth factors, as is also thecase for TNFα.26 However, the enzyme responsible for31

the cleavage of the membrane-bound precursors of EGFR
ligands had remained unidentified. On the basis of thesimilarity of the skin and fur defects among the TACE, EGFR-, and transforming growth factor α (TGFα; oneof the EGFR ligands)27-deficient mice, Peschon et al.23
examined whether proteolytic activity against pro-TGFα
was inhibited in fibroblasts lacking TACE and found that
this was indeed the case. Similarly, the shedding activity of another EGFR ligand, heparin-binding EGF-likegrowth factor (HB-EGF), was also found to be abrogatedin TACE-deficient fibroblasts; in addition, nearly identical defects in the heart valves were found in TACE-, HBEGF-, and EGFR-deficient mice.6,28 In short, the analysis of TACE-deficient mice had serendipitously revealed
TACE to be the enzyme responsible for processing EGFR
ligands. The importance of this study using TACE-deficient mice cannot be overstated: it proved ectodomain
shedding to be an essential post-translational mechanism
involved in the functional regulation of membrane-boundproteins in vivo. Of note, in addition to the TNFα and
EGFR ligands, numerous membrane-bound proteins havebeen identified as TACE substrates (for a list of selected
TACE substrates, please refer to Dreymueller et al.3 andMurphy1), indicating that TACE is a critical enzyme involved in ectodomain shedding in vivo. However, becauseof the early lethality of TACE-deficient mice, analysis of
the physiological functions of TACE in adult animals remained unaddressed.
TACE Is the Principal Enzyme for Releasing
Soluble TNFα in Vivo
As described above, the early lethality of TACE-deficient mice was an obstacle in understanding the rolesof TACE in vivo, and the relevance of TACE as a TNFα
sheddase in vivo remained to be clarified. To circumvent
this issue, I generated a conditional knockout mice using
the Cre-LoxP system (Taceflox/flox mice).29 Using Taceflox/
flox mice, I first generated conventional knockout mice
(Tace−/−) and found that they suffered early lethality, withmultiple defects in the skin, heart valves, and fur, as previously observed in TACE mutant mice.23 In a strict sense,
the TACE mutant mice reported by Peschon et al.23 were
not TACE null (Tace−/−). The mice were generated bytargeting the exon that encodes the Zn2+-binding motif,
and because the expression of a truncated non-functional
TACE protein was detected, the mice are often referred
to as TaceΔZn/ΔZn. Because monocyte/macrophage lineage
cells are the major source of TNFα in vivo,30 I crossed
Taceflox/flox mice with LysM-Cre transgenic mice,31 inwhich Cre is expressed under the control of the promoterof monocyte/macrophage-specific lysozyme, to specifically disrupt TACE in the monocyte/macrophage lineage.
The macrophages obtained from Taceflox/flox/ LysM-Cre
mice expressed low levels of TACE and released lower
levels of TNFα on stimulation with LPS in vitro com-

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Horiuchi K: The Biology of TACE

Fig. 2 TACE is essential for releasing TNFα from monocytes/macrophages.
(A) On stimulation with LPS, monocytes/macrophages produce membrane-bound pro-TNFα. Pro-TNFα is then delivered to the cellsurface and cleaved by TACE to become soluble and active. (B) In the absence of TACE, pro-TNFα remains on the cell surface and is
not fully functional.

pared with wild-type cells. Otherwise, the mutant mice
were indistinguishable from their wild-type littermates.
To determine whether TACE was responsible for the processing of pro-TNFα in vivo, I subjected the mutant mice
to a mouse model of LPS-induced endotoxin shock that
depends on soluble TNFα. As expected, the Taceflox/flox/
LysM-Cre mice were highly resistant to endotoxin shock,whereas most of the control littermates died within thefirst 16 h after treatment.29 The serum TNFα levels after
LPS injection were also significantly lower in the Taceflox/
flox/ LysM-Cre mice compared with the wild-type controls(Fig. 2).
Taken together, these observations showed that (1)TACE is the principal sheddase for TNFα in vivo, (2)TACE expressed in monocyte/macrophage lineage cells
plays a critical role in the pathogenesis of endotoxin
shock, and (3) the disruption of TACE in these cells confers strong protection against LPS-induced endotoxin
shock. The results of this study, I believe, finally provided
a conclusive answer regarding which enzyme is primarily
responsible for the activation of TNFα in vivo.
TACE Is Indispensable for Normal Growth andAdult Homeostasis
The first study using Taceflox/flox mice focused on thefunction of TACE expressed in monocyte/macrophagelineage cells, especially with regard to its relevance as
the sheddase for TNFα in vivo.29 However, the roles ofTACE in normal growth and adult homeostasis remained
to be elucidated. To address this question, I next crossed
the Taceflox/flox mice with Sox9-Cre transgenic mice, inwhich Cre is expressed under the control of the Sox9
promoter.32 SOX9 is an essential transcription factor forskeletal development; however, it is also expressed in awide range of tissues, including the skin, brain, and digestive tract,33,34 whereas there is little expression inhematopoietic cells and their progenitors. The Taceflox/
flox /Sox9-Cre mice (referred to as Tace/Sox9 mice) wereborn with open eyes, as observed in the Tace−/− mice, butthey did not show any defects in their heart valves, andmost survived to adulthood. Except for the eyelid defect,
Tace/Sox9 mice appear almost indistinguishable fromtheir wild-type littermates up to approximately 1 week
old. However, as they grow older, defects in the skin andhair and growth retardation become conspicuous. The
Tace/Sox9 mice exhibit a highly complex phenotype that
includes defects in the skeletal system (bone mass loss
and shorter long bones), hematopoietic system (splenomegaly, extramedullary hematopoiesis, and an increasein the number of hematopoietic stem cells), skin (atopic
dermatitis-like inflammation), and hair (short, wavy fur).
Presumably because of these defects, few mice survive
longer than 8 months. Interestingly, I found that the serum levels of granulocyte colony-stimulating factor (GCSF) were significantly increased in the Tace/Sox9 mice.
Because G-CSF is a potent stimulator of both granulopoiesis and bone resorption, it was assumed that the lackof TACE activity in non-hematopoietic cells (via poorly
understood mechanisms) led to the aberrant productionof G-CSF and, ultimately, resulted in the hematopoietic
defects and bone mass loss observed in Tace/Sox9 mice.
Because of the severity and complexity of the phenotype of Tace/Sox9 mice, the existence of a TACE loss-offunction genetic disorder in humans appeared unlikely,
and no such case has been reported in the literature. However, surprisingly, a recent study using high-throughput
sequencing technology revealed a 4-bp deletion muta-

Keio J Med 2013; 62 (1): 29–36

33

Table 1 Summary of the phenotypes of TACE mutant mice
Mouse

Phenotype

References

Tace-/-

Perinatal/embryonic lethality; open eyelids; misshaped heart valves23, 28, 29

TaceΔZn/ΔZn
Taceflox/flox/Mx1-Cre
Taceflox/flox/LysM-Cre
Taceflox/flox/Sox9-Cre
Taceflox/flox/Tie2-Cre
Taceflox/flox/HB9-Cre
Taceflox/flox/Vav-Cre

Resistance to LPS-induced shock; less soluble TNFα production on LPS stimulation; improved
29, 55, 56
insulin resistance and energy homeostasis on high-fat diet; resistance to abdominal aortic aneurysm
development
Resistance to LPS-induced shock lethality; less soluble TNFα production on LPS stimulation
29
Open eyelids; growth retardation; elongated growth plates; skin inflammation; reduced bone mass; 32
short, wavy hair; splenomegaly; increased myelopoiesis; early death
Reduced pathological neoangiogenesis; reduced tissue damage in acute lung inflammation
36, 57
Increased Schwann cell myelination
58
Decreased inflammation and alveolar neutrophil recruitment following LPS inhalation; greater59, 60resistance to E. coli infection

tion in the TACE gene in siblings (a girl and a boy) bornto consanguineous parents.35 The deletion introduces aframe shift and a premature stop codon, consequently resulting in knockout of the TACE gene. The surviving boy(the sister died at 12 years old) exhibited inflammatory lesions in the skin and intestine, left ventricular dilatation,
and wiry and disorganized hair. The apparent similarities
in the phenotype between the affected boy and Tace/Sox9
mice indicate that the roles of TACE are highly similar inhumans and mice and may further validate TACE mutant
mice as a relevant model for studying the functions ofTACE in humans. Nevertheless, the striking and complex
phenotype observed in the Tace/Sox9 mice unequivocallyshows that ectodomain shedding catalyzed by TACE is
indispensable for normal growth and adult homeostasis
and that a lack of its activity would lead to a disastrous
condition, as illustrated by Tace/Sox9 mice and the boy
with the TACE mutation.
Unresolved Issues and the Future Direction ofStudies on TACE
Studies in the past decade have established the importance of TACE under physiological conditions, during
development, and under many pathological conditions,
including cancer,9 endotoxin shock,29 and pathologicalneoangiogenesis36 (see Table 1 for a summary of the phenotypes of different TACE mutant mice). These studies
suggest that TACE may potentially be a target molecule
for the treatment of these disorders; however, considering its numerous substrates and diverse functions in vivo,
further studies are required to define the role of ectodomain shedding in vivo and to understand the biochemical
properties of TACE. Some of the fundamental but poorly
understood issues regarding the biology of TACE are described below.

How does TACE recognize its substrates?
The specificity of TACE for its substrates appears to
be partly dependent on the structure of the extracellulardomain and the amino acid sequence of the juxta-membrane domain of the target molecule.37 TACE cleaves
its target molecules approximately 12–16 amino acids
from the transmembrane domain; however, no consensus
amino acid sequences have been identified for the cleavage site in TACE substrates. Therefore, the substrates for
TACE cannot be determined by the amino acid sequenceof potential candidate genes, and the identification of thesubstrates primarily relies on biochemical analyses andcell-based assays (see Overall and Blobel38 for further
discussion). If inhibition of the ectodomain shedding ofa given molecule is to be used clinically, it will be necessary to devise a method to selectively inhibit the sheddingof the target molecule to avoid potential side effects. An
understanding of the mechanisms of substrate recognition for TACE will facilitate the pharmaceutical designof such an agent.
How is the activity of TACE regulated in vivo?
Before delivery to the cell surface, the prodomain of
TACE is cleaved by furin protease to allow TACE to
become proteolytically active (Fig. 1A). Interestingly,
recent studies have revealed that iRhom2, a proteolytically inactive member of the rhomboid protease family, isrequired to promote the trafficking of TACE in immune
cells,39,40 suggesting that iRhom2 is a critical regulator
involved in the maturation of TACE, at least in immune
cells. In addition, it is widely accepted that the shedding
activity of TACE can be rapidly up-regulated by various
stimuli and intracellular signaling, including a protein
kinase C stimulator (phorbol ester), ultraviolet light, osmotic pressure, and MAP kinases, without increasing theamount of mature TACE protein on the cell surface.37,41,42

34

Horiuchi K: The Biology of TACE

These observations indicate that TACE can be rapidly
activated by various stimuli from the surrounding milieu, presumably by changing its protein conformation.
Of note, a recent study showed that the transmembrane
domain, but not the cytoplasmic domain, of TACE is required for this activation, suggesting that the activation of
TACE is not dependent on intracellular signaling via thephosphorylation of its cytoplasmic domain.43 Regardless,the mechanism by which TACE recognizes stimuli andbecomes catalytically active remains poorly understood.
Because suppression of the stimulated shedding of TACE
and inhibition of the maturation of TACE are potential
therapeutic targets, it will be necessary to understandhow and under what circumstances TACE is activatedand processed under both physiological and pathologicalconditions.
What is the function of ectodomain shedding in general?
For TNFα and EGFR ligands (such as TGFα and HBEGF), the consequence of ectodomain shedding by TACE
is relatively clear. These proteins are, in principle, inactive in their membrane-bound pro-from and are activatedupon shedding. However, there are several cytokines (suchas the cell surface isoform of colony stimulating factor-1)
that are active in both their membrane-bound form andtheir soluble form.44,45 Some receptors, including vascular growth factor receptor 2 and c-Kit, are also subjected
to ectodomain shedding,46,47 and in theory, the cleavageof these receptors results in the down-regulation of theavailability of these receptors and, consequently, signaling through these receptors. In contrast, the interleukin
6 receptor, which has also been shown to be released by
TACE, acts as a functional soluble receptor via binding
to interleukin 6 and gp130.48 Moreover, there are several
cases [e.g., TNFα and its receptors (TNFR1 and TNFR2)] in which both the membrane-bound ligand and its receptor are cleaved by TACE. Thus, the consequences of
ectodomain shedding are highly context dependent andcan be different or even opposite when different targets
are involved. Therefore, the outcome of TACE inhibitionin disorders suspected to be causally related to aberrant
TACE activity may not always be as predicted theoretically. For these reasons, it will be essential to understandthe roles of TACE in such disorders in as much detail as
possible before attempting any clinical trial.
Concluding Remarks
TACE, an enzyme once thought to be solely involved in
the processing of pro-TNF, is now considered to be one of
the central components of ectodomain shedding in vivo,
with many identified target molecules, and most likely
even more unidentified target molecules. In addition,

ADAM10, one of the ADAM gene family members mostclosely related to TACE (Fig. 1B), has also emerged as amajor player in the field of ectodomain shedding.3,49–51
Although these two ADAM proteins appear to have common target molecules, it is becoming clear that each has
specific substrates and functions. Emerging data suggest that ADAM10 plays central roles in the activation of
Notch signaling and the processing of amyloid precursor
protein and cadherins.50–54
As described in this review, studies during the past two
decades have launched the field of ectodomain shedding;however, we are left with more questions than answers.
Further studies are required to address the unresolved
issues in this rapidly evolving field. Considering the diverse and fundamental functions of TACE and ADAM10
and their target molecules (e.g., EGFR ligand, TNFα,
Notch, and amyloid precursor protein), it is certain that
ectodomain shedding is involved in development and homeostasis and also in the pathology of many disorders.
Therefore, further investigation and a deeper understating of the roles of ectodomain shedding will be of great
importance and relevance in both basic and biomedical
research in the coming years.
Acknowledgments
The author would like to thank Dr. Carl Blobel, Dr. Yoshiaki Toyama, and Dr. Yasunori Okada for their mentorship and invaluable advice and is grateful to colleagues atthe Hospital for Special Surgery (NY, USA) and School of
Medicine, Keio University (Tokyo, Japan), for their help
and encouragement. The author’s studies on ectodomain
shedding were supported in part by the Uehara Memorial Foundation, the Mochida Memorial Foundation, theTakeda Science Foundation, the Nakatomi Foundation,
Keio University Kanrinmaru Project, and MEXT Kakenhi (21390424 and 19591765).
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