Hepatocyte growth factor activator HGFA: a serineprotease that links tissue injury to activation of hepatocyte growth factor Keiji Miyazawa Department of Biochemistry, Interdisciplinary
Trang 1Hepatocyte growth factor activator (HGFA): a serine
protease that links tissue injury to activation of
hepatocyte growth factor
Keiji Miyazawa
Department of Biochemistry, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Japan
Introduction
In tissues of multicellular organisms, different types of
cells are intricately and precisely arranged to perform
specific functions Once such tissue architecture is
destroyed, the regeneration system works to restore the
structure as well as the function of the damaged tissue
Liver regeneration has long been a subject of active
research, because it displays a dramatic form of organ
regeneration It also represents a good in vivo model
for understanding the regulation of cell growth:
hepatocytes are usually in a quiescent state, but most
of them enter the cell cycle during liver regeneration Humoral factors that trigger liver cell growth have been detected in the blood circulation of liver-injured animals, and many researchers have tried to isolate these factors
Hepatocyte growth factor (HGF) was originally identified as a potent mitogen for hepatocytes in pri-mary culture during studies of liver regeneration [1] Studies showed that HGF was induced in the blood plasma and liver in response to liver injury Therefore,
Keywords
hepatocyte growth factor; plasminogen;
proteolytic activation; tissue injury
Correspondence
K Miyazawa, Department of Biochemistry,
Interdisciplinary Graduate School of
Medicine and Engineering, University of
Yamanashi, 1110 Shimokato, Chuo,
Yamanashi 409-3898, Japan
Fax ⁄ Tel: +81 55 273 6784
E-mail: keiji-miyazawa@umin.ac.jp
(Received 26 November 2009, revised
3 February 2010, accepted 26 February
2010)
doi:10.1111/j.1742-4658.2010.07637.x
Growth factors are a group of proteins that regulate a wide variety of cel-lular processes, including proliferation, differentiation, motility, adhesion, and apoptosis of target cells They play crucial roles in the formation and maintenance of tissue architecture in embryonic development and adult tissue homeostasis Because aberrations in growth factor signaling often result in pathological conditions, the activities of growth factors are tightly controlled by extracellular and intracellular regulators Hepatocyte growth factor (HGF) is a mesenchymal cell-derived growth factor that affects various target cells, including epithelial and endothelial cells HGF is synthesized and secreted as a latent form, and is proteolytically activated in response to tissue injury, thus participating in tissue regeneration and repair Interestingly, HGF has a unique structural feature: it is homologous
to plasminogen, a key enzyme in the fibrinolytic system Elucidation of the regulatory mechanisms of HGF activity has revealed that a blood coagula-tion factor XII-like serine protease, hepatocyte growth factor activator, efficiently converts HGF from the latent form to the active form Hepato-cyte growth factor activator itself is activated downstream of the blood coagulation cascade, and links tissue injury to activation of HGF HGF thus has structural as well as functional relevance to the blood coagulation⁄ fibrinolytic system
Abbreviations
FXII, coagulation factor II; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; tPA, tissue-type plasminogen activator.
Trang 2HGF was regarded as the humoral factor that triggers
liver regeneration HGF was subsequently shown to
have mitogenic, motogenic and morphogenic activities
on various target cells, including epithelial and
endo-thelial cells HGF is now thought to play a major role
in the repair and regeneration of various tissues,
including the liver, kidney, lung, and stomach [1]
A new question then arose Why is the liver
specifi-cally targeted to grow after liver injury? HGF and
its receptor, MET tyrosine kinase, are widely expressed
and distributed among normal as well as injured
tis-sues After liver injury, HGF is induced not only in
the liver but also in blood plasma and other uninjured
tissues, such as spleen [2] Although HGF is not
exclu-sively induced in the liver, its action is limited to the
injured liver Apparently, the amount of HGF is not
correlated with the activity of HGF, suggesting that
HGF is latent in normal states, and is activated
specifi-cally at the site of tissue injury The mechanism of
localized activation of HGF, however, had remained
unclear In this minireview, I describe the discovery of
a novel serine protease, HGF activator (HGFA),
which has revealed the link between tissue injury and
localized activation of HGF
Active and inactive forms of HGF
The first step in solving this puzzle was to understand
the nature of latent HGF Several examples of latent
growth factors have already been reported Insulin-like
growth factors are inactive when bound to insulin-like
growth factor-binding proteins Transforming growth factor-b is produced and secreted as a latent precursor form composed of the N-terminal ‘latency-associated peptide’ and the C-terminal mature form It is activated after the N-terminal portion of the precursor
is degraded or dissociated from the C-terminal portion
In the case of HGF, its unique primary structure pro-vided a valuable clue for solving the puzzle of HGF latency
Mature HGF consists of two polypeptide chains, a heavy a-chain (62 kDa) and a light b-chain (32–34 kDa), which are held together by a disulfide bond [3] In 1989, cDNA of human HGF was cloned, and the primary structure was elucidated [4,5] HGF is synthesized and secreted as a single-chain precursor [6], and is extracellularly processed to the two-chain form by proteolytic cleavage at a specific site The heavy chain consists of four tandem repeats of a krin-gle domain, and the light chain has a structure similar
to that of the catalytic domain of serine proteases (Fig 1A) [5] HGF, however, has no proteolytic activ-ity, because two of the conserved catalytic triad resi-dues of the serine protease domain are substituted The domains of HGF are very similar to those of pro-teases in the blood coagulation and fibrinolytic system HGF shows the highest similarity to plasminogen (about 40% amino acid similarity)
Plasminogen is synthesized as a single-chain form that consists of five tandemly repeated kringle domains and a serine protease domain [1] It is activated upon cleavage at a specific site between the fifth kringle
Heavy chain ( α-chain) Light chain ( β-chain)
Signal
1
(K) (N)
(N)
NK1
I
II
(K)
A
B
Hairpin region Kringle domain Serine
protease-like domain
Fig 1 Schematic structure of HGF (A) and
its variants (B) Circles denote amino acids,
and lines denote disulfide bonds
Arrow-head 1 denotes the cleavage site of the
signal sequence Arrowhead 2 denotes the
cleavage site for activation Roman numbers
denote kringle domain numbers.
Trang 3domain and the serine protease domain by the action
of either urokinase or tissue-type plasminogen
activa-tor (tPA) After this site-specific cleavage, the protease
domain changes conformation and becomes active
Plasmin then degrades fibrin clots and extracellular
matrices, and activates matrix metalloproteases, thus
contributing to the process of tissue remodeling
Similarly, the single-chain form of HGF was shown
to be a latent form We found that single-chain HGF
was inactive in the presence of serine protease
inhibi-tors in a hepatocyte proliferation assay, whereas the
two-chain form was active even in the presence of the
inhibitors [7] Furthermore, an HGF mutant, in which
Arg494 was replaced by glycine, and which was thus
resistant to proteolytic processing, was not active at all
[7,8] HGF needs be processed to the two-chain form
to exert its biological activity Recently, proteolytic
processing of HGF was shown to induce a
conforma-tional change in the serine protease-like domain, which
is required for functional interaction of HGF with its
receptor, MET, through a region corresponding to the
‘active site’ and ‘activation domain’ of serine proteases
[9,10] HGF and serine proteases thus share a similar
activation mechanism
Proteolytic activation of HGF in
response to tissue injury
In order to elucidate the in vivo roles of proteolytic
processing of HGF, we examined the molecular forms
of HGF by immunoblotting using a heavy
chain-specific monoclonal antibody [11] The antibody reacts
with both the single-chain and two-chain forms of
HGF, giving positive bands at 92 and 62 kDa,
respec-tively By scanning of the 92 and 62 kDa bands,
the ratio of the single-chain form (inactive) to the
two-chain form (active) can be quantified
We first found that HGF from various normal rat
tissues (liver, kidney, lung, and spleen) was present
exclusively in the inactive single-chain form We next
administered hepatotoxin or nephrotoxin to rats to
induce tissue injury HGF was extracted from injured
and uninjured tissues and then analyzed After
intraga-stric administration of carbon tetrachloride, liver tissue
was severely injured and the other tissues were
mini-mally affected The amount of HGF was dramatically
increased in the liver and spleen tissue, but not in the
kidney or lung tissue HGF was converted to the
active form only in the liver, which was injured in this
experimental model Similar results were obtained
when d-galactosamine was used to induce liver injury
through a different mechanism When we injected
mercuric chloride to induce renal injury in rats, HGF
in the kidney increased in quantity and was activated
In contrast, HGF in the liver and spleen increased in quantity but was not activated These findings indicated that HGF in uninjured tissue exists as the inactive form, even though it sometimes increases in amount HGF is therefore activated exclusively in injured tissues by proteolytic processing, and thus seems to contribute to the process of tissue regenera-tion and repair [11]
The fact that the proteolytic conversion of HGF is specific to injured tissues suggests that HGF-convert-ing enzyme(s) should work exclusively in the injured tissues We found that HGF-converting activity was induced in the injured liver but not in the normal liver tissue [11] This activity seemed most likely to repre-sent a key enzyme regulating the action of HGF in injured tissues We thought that identification of the enzyme would be crucial to understanding the control
of HGF action in vivo The activity of this enzyme, however, was not high enough to allow purification of the protein for identification Fortunately, we detected strong HGF-converting activity in human serum
HGFA – one of the key enzymes of HGF activation
We thus purified, from human serum, a novel serine protease of 34 kDa that activates HGF very efficiently
in vitro, and designated it as HGFA [12] The nucleo-tide sequence of HGFA cDNA showed that HGFA is derived from the C-terminal region of a precursor of
655 amino acids by proteolytic processing Interest-ingly, the precursor consists of multiple domains, a type II fibronectin homology region, two epidermal growth factor domains, a type I fibronectin homology region, a kringle domain, and a catalytic domain (Fig 2) These domains are homologous to those observed in blood coagulation factor XII (FXII), with
an overall amino acid similarity of 39% Analysis of genomic DNA coding for HGFA indicated a relation-ship between HGFA and FXII as well as urokinase and tPA, activators of plasminogen These four pro-teins therefore constitute a family (the PA–FXII– HGFA family) [13]
Other proteases, such as urokinase, tPA, FXII, factor XI, plasma kallikrein, matriptase, and hepsin, were subsequently reported to activate HGF in vitro [14–18] The first five of these are of blood plasma ori-gin, whereas the latter two are transmembrane serine proteases and may be involved in pericellular activa-tion of HGF Among these proteases, matriptase and hepsin activate HGF with comparable efficiency to that of HGFA in vitro [17,18] Although the activities
Trang 4of the other proteases on HGF are very weak in vitro,
they may be stimulated by a cofactor(s) or by a certain
microenvironment in vivo These proteases, as well as
HGFA, are thus candidates for the HGF-converting
enzyme in injured tissues
Recently, Itoh and Kataoka generated mice
defi-cient in hgfa, and directly demonstrated that HGFA
is the serum-derived protease responsible for
activa-tion of HGF [19,20] HGFA was also shown to be
required for tissue repair in experimental colitis
mod-els Both HGF activation and tissue regeneration
were markedly impaired in injured intestinal tissue of
HGFA-deficient mice The important function of
HGFA during tissue repair in vivo was thus
demon-strated
How is HGFA activity localized to the
site of tissue injury?
HGFA is produced in liver parenchymal cells, and
behaves as an acute-phase protein [21] HGFA is
found in the active form in serum, but in the inactive
form in plasma This precursor form was purified and
characterized [22] The inactive form of HGFA
(pro-HGFA) has a molecular mass of 96 kDa, and is
converted to its mature form by thrombin and
plasma kallikrein (KLKB1) [22] (Fig 2) The
process-ing by thrombin is required for activatprocess-ing HGFA,
whereas that by plasma kallikrein is not The role of
the processing by plasma kallikrein remains to be
elucidated
HGFA circulates in the bloodstream as an inactive
precursor, and is activated in response to tissue injury,
probably coupled with activation of the blood
coagula-tion system Immunoblotting analysis of HGFA from
normal and injured tissues indicated that HGFA is
activated exclusively in injured tissues [23] Thus,
injured tissue-specific activation of HGFA appears to
be involved in the localized activation of HGF HGF was originally identified as a potent mitogen for hepatocytes, but it was subsequently shown to induce angiogenesis in vivo, and to stimulate prolifera-tion⁄ migration of vascular endothelial cells in vitro [24,25] HGF thus appears to be linked to the blood coagulation and fibrinolytic system, not only structur-ally but also functionstructur-ally (Fig 3) The blood coagula-tion system is activated upon injury of blood vessels, leading to conversion of prothrombin to thrombin Thrombin processes fibrinogen and coagulation fac-tor XIII (plasma transglutaminase) to form stable blood clots and prevent further hemorrhage from the injured sites Concomitantly, thrombin induces activa-tion of HGF via HGFA HGF then stimulates prolif-eration and migration of endothelial cells to repair blood vessels It appears rational that the blood coagu-lation system triggers activation of a growth factor that promotes angiogenesis Therefore, the prototypic function of HGF may be to maintain the integrity of blood vessels Thrombin appears to be a bifurcation point for clotting and endothelial cell migration⁄ prolif-eration, and HGFA represents the link between tissue injury and activation of HGF
Perspectives
In 1995, Uehara et al and Bladt and coworkers [26,27] reported that the embryonic lethality of
HGF-knock-Kringle
Type I Type II
EGF EGF
1
(2) Serine protease
Fig 2 Schematic structure of HGFA Circles denote amino acids,
and lines denote disulfide bonds The names of the domains are
shown Type I and Type II denote the type I fibronectin homology
region and the type II fibronectin homology region, respectively.
The arrowhead denotes the cleavage site of the signal sequence.
Arrow 1 denotes the site of cleavage by thrombin, kallikrein
1-related peptidase 4, and kallikrein 1-1-related peptidase 5, which is
required for activation of HGFA Arrow 2 denotes the site of
cleav-age by plasma kallikrein EGF, epidermal growth factor.
Cell growth, migration
Pro-HGF HGF
Cell
FXa
Pro-HGFA HGFA Thrombin
FVa Prothrombin
FXIII Fibrinogen fibrin
FXIIIa
Cross-linked fibrin clot
Fig 3 Activation of HGF in response to tissue injury Activation of the blood coagulation cascade, through either an intrinsic or an extrinsic pathway, results in conversion of prothrombin to thrombin Thrombin processes fibrinogen and factor XIII (FXIII; plasma trans-glutaminase) to form stable blood clots and prevent further hemor-rhage from the injured sites Concomitantly, thrombin induces activation of HGF via HGFA, which leads to endothelial cell growth ⁄ migration and contributes to repair of the blood vessel structure HGF also stimulates the proliferation and migration of epithelial cells to repair the tissue architecture FVa, factor Va; FXa, factor Xa; FXIIIa, activated FXIII.
Trang 5out mice was caused by dysfunction of the placenta
and liver, indicating that HGF plays important roles
during embryonic development We can explain the
activation of HGF in injured tissues in adults by the
scheme illustrated in Fig 3, but it remains unclear
how HGF is activated during embryonic development
in which there is no apparent tissue injury In the
Drosophila embryo, the signal transduction pathway
that establishes the dorsal–ventral pattern is temporally
and spatially regulated through the proteolytic cascade
to activate Spa¨tzle, the Toll receptor ligand [28] It is
likely that a proteolytic cascade also plays an
impor-tant role in the regulation of HGF activity during
mammalian development Known proteases that
acti-vate HGF in vitro do not appear to be the key
prote-ases activating HGF during embryonic development,
because mice lacking these proteases are not
embry-onic lethal Alternatively, truncated variant forms of
HGF, NK1, and NK2 (Fig 1B), which are abundantly
expressed in embryo-derived cells, might contribute to
HGF activity during embryonic development These
variants exhibit weak agonistic activity, although they
lack the serine protease-like domain [29,30], and do
not appear to require proteolytic activation Further
progress in this field would answer this question
In chronic liver diseases, liver dysfunction often
leads to disorders in blood coagulation, because the
plasma levels of blood clotting enzymes, which are
produced in hepatocytes, are decreased Similarly,
HGFA is produced in hepatocytes [21], and its
produc-tion is impaired in a rat model of liver cirrhosis,
caus-ing decreased efficiency of HGF activation [31]
Kaibori et al [32] demonstrated that local
administra-tion of HGFA promotes conversion of HGF from the
inactive form to the active form, leading to
accelera-tion of liver regeneraaccelera-tion In other cases, aberrant
activation of HGF may result in overactivity of HGF,
which is implicated in malignancy as well as some
types of kidney disease (including polycystic disease,
glomerulosclerosis, and renal tubular hyperplasia) [33]
It is important to identify HGF-activating enzymes in
each pathogenic situation that is associated with
aber-rant HGF function Selective inhibitors against those
enzymes, probably including HGFA, could be
candi-dates for clinical drugs for treatment of such diseases
with HGF overactivity
We identified HGFA as a protease that links tissue
injury to activation of HGF However, HGFA may
activate HGF in other contexts in vivo Notably, the
kallikrein 1-related peptidases KLK-4 and KLK-5
were recently shown to activate HGFA [34] These
proteases cleave HGFA at the same site as thrombin
KLK-4 and KLK-5 are implicated in the activation of
HGFA in tumor tissues Further investigation of HGFA activation in vivo, as well as regulation of HGFA activity by endogenous protease inhibitors, including hepatocyte growth factor activator inhibitor-1 (HAI-1) and protein C inhibitor [35–37], will be important for understanding the pathophysiological processes regulated by the HGF–HGFA system
Acknowledgements
I apologize to colleagues in the field for not citing many important papers, because of the limitation of the length of this review article I would like to thank
T Shimomura, D Naka and T Kawaguchi of Mitsu-bishi Chemical Corp., A Okajima and A Kitamura of Kansai Medical University and N Kitamura of Tokyo Institute of Technology for their contributions to the study of HGFA
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