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Matriptase is a type II transmembrane serine protease expressed in most human epithelia,

where it is coexpressed with its cognate transmembrane inhibitor, hepatocyte growth factor
activator inhibitor (HAI)-1. Activation of the matriptase zymogen requires sequential N-
terminal cleavage, activation site autocleavage, and transient association with HAI-1.
Matriptase has an essential physiological role in profilaggrin processing, corneocyte
maturation, and lipid matrix formation associated with terminal differentiation of the oral
epithelium and the epidermis, and is also critical for hair follicle growth. Matriptase and HAI
expression are frequently dysregulated in human cancer, and matriptase expression that is
unopposed by HAI-1 potently promotes carcinogenesis and metastatic dissemination in
animal models. Matriptase is a 80- to 90-kDa cell surface glycoprotein with a complex
modular structure that is common to all matriptases

 

    

  


Scientists at the National Institute of Dental and Craniofacial Research (NIDCR) and
colleagues report in animal studies that a single, scissor-like enzyme called matriptase, when
left to its own devices, can cause cancer. This finding, published in the current issue of the
journal Genes and Development, marks the first report of a protein-cleaving enzyme, or
protease, on the cell surface that can efficiently trigger the formation of tumor cells. The
authors also note that matriptase is the first known cell-surface protease that can act as an
oncogene, an umbrella term for mutated genes and their proteins that prompt cells to divide
too rapidly, a hallmark of tumor cells.

"What makes matriptase potentially such a good molecular target to treat cancer is its
accessibility," said NIDCR scientist Dr. Thomas Bugge, the senior author on the paper. "We
don't have to trick the tumor cell to internalize a drug, then hope it reaches its destination in
an appropriate concentration and duration. In this case, the bull's eye is right on the cell
surface." Bugge said the exact function of matriptase in healthy human cells remains a bit of
a mystery. Previous studies show that cells comprising the outer lining, or epithelium, of
nearly all human organs express the protease. They also suggest that matriptase might play a
role in activating other membrane-bound proteins on the cell surface that are involved in
signaling basic cellular activities, such as growth and motility.

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Since its discovery nearly 13 years ago, scientists also have suspected that matriptase might
have a dark side. It is overly abundant in a variety of epithelial-derived tumors, including
breast, prostate, ovarian, colon, and oral carcinomas. Then, in 2002, scientists reported
women with breast and ovarian cancer have poor prognoses if their tumors contain high
levels of matriptase. In fact, just two months ago, researchers reported for the first time that
increased expression of matriptase is associated with more serious forms of cervical cancer.

Still unanswered, however, was the larger question of whether the protease, when deregulated
and overexpressed, might actually cause cancer. To find the answer, Bugge and colleagues
produced mice that expressed the human version of the matriptase gene in a stable, readily
measurable manner. "After our initial round of experiments, we found that the skin of the
mice was quite sensitive to fluctuations in the levels of matriptase," said Dr. Roman Szabo, a
co-lead author on the study and an NIDCR scientist. "So much so, that all 10 of the mice that
produced too much matriptase developed distinctive, splotchy skin lesions within a year."
According to Szabo, that's when things took an unexpected turn. He and his colleagues
biopsied the lesions and, to their surprise, found that they were tumors that had already
advanced in most cases to a type of cancer called squamous cell carcinoma, a strong
indication that the excess matriptase was driving the process.

The scientists next wondered whether excess matriptase and sustained exposure to a chemical
carcinogen might be a dangerous combination, a scenario with obvious real world
implications. They applied various doses of the chemical DMBA, a well-characterized
carcinogen present in tobacco products, to a small area of skin on each of 40 newborn
matriptase overproducers. Within seven weeks, 95 percent of these mice developed tumors
compared to roughly 2 percent of normal, healthy mice. The group also found that the higher
the exposure to DMBA in the matriptase overproducers, the greater the chances were that the
tumors would turn cancerous.

"What we found is deregulated matriptase sends a strong and versatile pro-growth signal that
can travel along more than one route to the cell nucleus," said Dr. Karin List, the other lead
author and an NIDCR scientist. "But the key point is, like a classic oncogene, matriptase
initiates the erroneous growth signal. As further confirmation of this, when we turned off
matriptase, not only the tumors but the precancerous lesions never appeared in the mice."

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"What this work really shows is the current list of about 100 known oncogenes remains very
much a work in progress," said Bugge. "It's also clear that matriptase and the approximately
200 other distinct cell-surface proteases will have a lot more to tell us about human health
and disease in the coming years."

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1.‘ Gloria Velasco,
2.‘ Santiago Cal,
3.‘ Victor Quesada,
4.‘ Luis M. Sánchez and
5.‘ Carlos López-Otıғn

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We have identified and cloned a fetal liver cDNA encoding a new serine proteinase that has
been called matriptase-2. This protein exhibits a domain organization similar to other
members of an emerging family of membrane-bound serine proteinases known as type II
transmembrane serine proteinases. Matriptase-2 contains a short cytoplasmic domain, a type
II transmembrane sequence, a central region with several modular structural domains
including two CUB (complement factor C1s/C1r, urchin embryonic growth factor,bone
morphogenetic protein) domains and three low density lipoprotein receptor tandem repeats,
and finally, a C-terminal catalytic domain with all typical features of serine proteinases. The
human matriptase-2 gene maps to 22q12-q13, a location that differs from all type II
transmembrane serine proteinase genes mapped to date. Immunofluorescence and Western
blot analysis of COS-7 cells transfected with the isolated cDNA confirmed that matriptase-2
is anchored to the cell surface. Matriptase-2 was expressed in r


 , and the
purified recombinant protein hydrolyzed synthetic substrates used for assaying serine
proteinases and endogenous proteins such as type I collagen, fibronectin, and fibrinogen.
Matriptase-2 could also activate single-chain urokinase plasminogen activator, albeit with
low efficiency. These activities were abolished by inhibitors of serine proteinases but not by
inhibitors of other classes of proteolytic enzymes. Northern blot analysis demonstrated that
matriptase-2 transcripts are only detected at significant levels in both fetal and adult liver,
suggesting that this novel serine proteinase may play a specialized role in matrix remodeling
processes taking place in this tissue during development or in adult tissues.



Proteolytic enzymes play crucial roles in the development and maintenance of an organism as
well as in a number of pathological conditions including the progression of malignant tumors
(1). Most studies on cancer-associated proteinases have focused on matrix metalloproteinases
(MMPs), 1 a family of zinc-dependent endopeptidases that collectively degrade all major
protein components from extracellular matrix and basement membranes (2, 3). However,
enzymes from other catalytic classes such as cysteine, aspartyl, and serine proteinases have
been also implicated in different aspects of tumor progression. Among them, an emerging
group of membrane serine proteinases, called TTSPs and containing a complex organization
of domains, have raised recent interest because of their potential ability to participate in
matrix-degrading processes associated with cancer (reviewed in Ref. 4). To date, 11 distinct
human TTSPs have been described and characterized at the amino acid sequence level. They
include enteropeptidase, hepsin, human airway trypsin-like protease (HAT), corin,
matriptase/MT-SP1, epitheliasin/TMPRSS2, TADG-12/TMPRSS3, TMPRSS4, MSPL
(mosaic serineprotease large form), spinesin/TMPRSS5, and DESC1 protease
(differentially expressedsquamous cell carcinoma gene 1) (4-6). All of them share a number
of structural features: a short N-terminal cytoplasmic domain, a type II transmembrane
sequence, a central region of variable length containing modular structural domains, and a C-
terminal catalytic region with all of the characteristic features of serine proteinases. TTSPs
have been found in a wide variety of mammalian tissues as well as in other eukaryotic
organisms including ë 
     (7) and
   (8).
Although the physiological roles of most TTSPs are still unclear, there are some cases in
which their participation in specific functions has been suggested or demonstrated. This is the
case of enteropeptidase that is involved in the proteolytic activation of trypsinogen to trypsin,
which subsequently activates other digestive enzymes such as chymotrypsinogen or
procarboxypeptidases (9,10). Likewise, matriptase/MT-SP1 has been proposed to initiate
signaling and proteolytic cascades through their ability to activate cell surface-associated
proteins like pro-uPA and protease-activated receptor 2 (11). Matriptase has also been
suggested to participate in the control of intestinal epithelial turnover by regulating the cell-
substratum adhesion (12). Hepsin has been involved in mammalian cell growth,
developmental processes such as blastocyst hatching, and initiation of blood coagulation (13-
15). Corin, a TTSP family member isolated from human heart, has been found to act as an 
 activator of pro-atrial natriuretic peptide, a cardiac hormone essential for the regulation
of blood pressure (16, 17). HAT, originally isolated from the sputum of patients with chronic
airway diseases, may be involved in the host defense system on the mucous membrane (18).

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Recently, a HAT-related protease isolated from rat tissues has been found to cleave pro-Ȗ-
melanotropin at the adrenal cortex, stimulating the mitogenic actions of this peptide (19).
Spinesin, predominantly expressed at synapses, may play specific roles in neural functions
(20). Finally, insertion of ȕ-satellite repeats into the gene encoding TMPRSS3 causes a form
of autosomal recessive deafness, suggesting a role for this protease in the development or
maintenance of the inner ear or in the turnover of the protein contents of the perilymph and
endolymph (21).
The expression of virtually all TTSPs characterized to date is widely deregulated during the
development and progression of tumor processes. Thus, matriptase/MT-SP1 was originally
identified in breast cancer cells and is highly expressed in breast, prostate and colorectal
cancers (22-25). Inhibition of this protease abolishes both primary tumor growth and
metastasis in a murine model of prostate cancer (24,26), whereas stabilization of active
matriptase through glycosylation by x-acetylglucosaminyltransferase V is associated with the
prometastatic effects of this enzyme (27). Hepsin is overexpressed in ovarian and prostate
carcinomas (28-31), and its expression correlates inversely with measures of patient
prognosis (32). Epitheliasin is also overexpressed in prostate carcinomas, and a mutated form
of this protease has been found in a case of aggressive disease (33-35). TMPRSS3/TADG-12
is overexpressed in ovarian cancer (36), and TMPRSS4 is overexpressed in pancreatic cancer
(37). Finally, the recently described DESC1 was identified as a consequence of its differential
expression in squamous cell carcinoma of the head and neck (6).
These recent findings have stimulated the search for new TTSPs potentially associated with
some of the proteolytically mediated processes taking place during normal or pathological
conditions and especially during tumor progression. In this work, and as part of our studies
on tumor proteinases, we have examined the possibility that additional members of this
family of membrane proteinases could be produced by human tissues, with the discovery of a
novel family member named matriptase-2. We describe the molecular cloning and complete
nucleotide sequence of a cDNA coding for this protein and report an analysis of its
expression in human tissues. We also report the production of recombinant matriptase-2
in r


 and perform an analysis of its enzymatic activity against synthetic and
endogenous substrates. Finally, we demonstrate that matriptase-2 is bound to the cell
membrane.

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