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Department of Internal Medicine I, University of Ulm, 89081 Ulm, Germany [C. W., S. H., F. M-P., G. A., T. M. G.]; Hungarian Academy of Sciences, University of Budapest, 1450 Budapest, Hungary [B. B.]; Department of Medicine B, University of Münster, 48129 Münster, Germany [M. M. L., M. R.]; Miyazaki Medical College, Kiyotake, Miyazaki 889-1692, Japan [T. I.]
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Introduction |
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In the present study, we report the cloning and characterization of a novel serine protease identified in a recent cDNA-RDA4 approach (3) . This study was designed to isolate gene fragments highly overexpressed in pancreatic cancer compared with normal pancreas and chronic pancreatitis tissue. From the 16 gene fragments isolated in this study, we selected the 313-bp gene fragment RDA12 (GenBank accession no. U54603) for further characterization. Database comparison revealed a moderate homology to a number of serine proteases, indicating that RDA12 may be a fragment of a novel protease with cancer-specific expression.
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Materials and Methods |
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The human pancreatic cancer cell lines were obtained from the following suppliers: PATU-8988S and PATU-8988T (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany); PANC-1 and MIA-PaCa-2 (European Collection of Animal Cell Cultures, Salisbury, United Kingdom); HPAF (Metzgar, Durham, NC); Capan-1, Capan-2, and AsPC-1 (Cell Lines Service, Heidelberg, Germany); Patu II (Elsässer, Marburg, Germany); PC2 (Bülow, Mainz, Germany); SUIT-2 (S2-007, S2-013, S2-020, and S2-028; Iwamura, Miyazaki, Japan; Ref. 4 ); and SKPC2 and IMIM-PC2 (P. Real, IMIM, Barcelona, Spain).
Cloning of a New Serine Protease cDNA.
In a recent screen for differentially expressed genes in pancreatic
carcinoma, the 313-bp gene fragment RDA12 (accession no. U54603) was
isolated by cDNA-RDA (3)
; this fragment encodes the
putative motif of a new serine protease. The RDA12 fragment was
used to screen 
20,000 clones of an oligo(dT)-primed cDNA library
from a pancreatic cancer cell line by hybridization. Both strands of
the longest cDNA clone, RDA12/2, were sequenced by primer walking. For
stable transfection in mammalian cells, the cDNA clone RDA12/2 was
cloned in sense and antisense orientation into the BamHI
site of the mammalian expression vector pHß-Apr1-neo
(5)
. A COOH-terminal-tagged TMPRSS3 expression
vector was constructed by insertion of a 1427-bp fragment (nucleotides
96–1522) containing the open reading frame of TMPRSS3
into the BstXI site of the mammalian expression vector
pcDNA6/V5/His B (Invitrogen, San Diego, CA).
Northern Blot Analyses.
The expression of TMPRSS3 was studied by hybridizations
using Northern blots containing 30 µg each of total RNA from normal
pancreas tissue, chronic pancreatitis tissue, different carcinoma
tissues, and cell lines. The Northern blots containing RNA of different
human tissues were purchased from Clontech (Heidelberg, Germany).
Cell Culture and Transfection.
For functional analysis of TMPRSS3, the S2-020 pancreatic
cancer cell line, which expresses no endogenous TMPRSS3
mRNA, was transfected with the TMPRSS3-pHß-Apr1-neo
construct in sense and antisense orientation using DMRIE-C (Life
Technologies, Inc., Eggenstein, Germany). Several clones were picked
that showed various degrees of stable TMPRSS3
sense/antisense mRNA expression. Two of each sense and antisense clones
were used for functional assays.
HEK-293 cells were plated at 1.5 x 106 cells/10-cm dish and grown overnight in DMEM supplemented with 10% FCS. Cells were transiently transfected with the TMPRSS3-pcDNA6/V5/His plasmid DNA by use of the calcium phosphate protocol.
Preparation of Cell Extracts and Subcellular Fractionation.
Forty-eight h after transient transfection with V5-tagged
TMPRSS3 into HEK-293 cells, protein extracts were prepared
by resuspending pelleted cells in 1% Triton X-100, 1% sodium
deoxycholate, 0.1% SDS, 150 mM NaCl, 50
mM Tris-HCl (pH 7.2) supplemented with 5 µg/ml
Aprotinin, 5 mM Pefabloc, and 10 µg/ml
Pepstatin. For immunopurification of the epitope-tagged protein, cell
lysates were incubated with V5 antibody conjugated to protein G-agarose
beads at 4°C for 4 h on a shaker. The agarose beads were
pelleted by centrifugation and washed twice with 150
mM NaCl, 5 mM EDTA, 50
mM Tris + 0.1% NP40. The washed
pellets were resuspended in 150 mM NaCl, 5
mM EDTA, 50 mM Tris + 0.1% NP40 for PNGase F treatment.
Subcellular fractions were prepared from transiently transfected HEK-293 cells as reported previously (6) . The plasma membrane-enriched fraction, which was prepared using sucrose density gradient centrifugation, the cytosolic fraction, and concentrated culture medium were studied by Western blot analysis.
Glycosylation.
For PNGase F treatment, immunopurified protein was incubated overnight
with 2 units of PNGase F supplemented with 10 mM EDTA at
37°C. Inhibition of N- and mucin-like
O-glycosylation was performed by cultivating
TMPRSS3-expressing HEK-293 cells for 24 h in DMEM, 10% FCS
containing either 2.5 µg/ml tunicamycin (7)
or 2
mM
phenyl-N-Acetyl-
-D-galactosaminide
(8)
. Thereafter, cells were harvested for protein
extraction.
Functional Assays.
Nude mouse experiments were done by injecting 2 x 106 S2-020 cells stably transfected with
TMPRSS3 sense/antisense constructs, both s.c. and in the
tail vein of female nu/nu mice. Five weeks after the tail
vein injections, the lung, spleen, and liver were used for standard
histological analysis to identify the presence or absence of metastatic
lesions. Subcutaneous tumors were measured and used for histological
analysis.
In vitro matrigel invasion assays were done by seeding 105 transfected cells in medium + 1% FCS in the upper chamber of Matrigel-coated 8-µm transwell plates. The lower chamber was filled with medium + 10% FCS. The number of invading cells adhering to the lower side of the porous membrane was counted after fixation with 4% paraformaldehyde and staining with methylene blue.
The proteolytic activity in TMPRSS3 sense/antisense-transfected S2-020 cells and transiently transfected HEK-293 cells was determined fluorometrically in native lysates and lysates treated with enterokinase for activation, using oligopeptide substrates for elastase-like (Ala-Ala-Ala-Ala) and trypsin-like (Ile-Pro-Arg) serine proteases as described previously (9) .
Chromosomal Mapping of the TMPRSS3 Gene Locus.
The chromosomal localization of TMPRSS3 was determined by
screening the GeneBridge4 radiation hybrid panel (Research Genetics,
Huntsville, AL), using the TMPRSS3-specific primers
5'-CATGTGGTGGGCATCGTTA-3' and 5'-CCAGTTGAGATAGGCTGAG-3'.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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). Translation of the open reading frame suggests that the cDNA codes
for a putative polypeptide of 437 amino acids with an estimated
molecular mass of 48.202 kDa. The NH2-terminal
region of the hypothetical protein contains a putative signal-anchor
sequence characteristic for group II integral membrane proteins. The
highly hydrophobic region of 22 amino acids may serve as a
transmembrane domain that is involved in anchoring the protease to the
cell membrane. According to the charge difference rule
(10)
, it can be assumed that the COOH terminus of the
protein with its protease module is located on the extracellular
surface.
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50% similarity was
found to members of the serine protease family known as the human
transmembrane proteases, TMPRSS1/hepsin (11)
or
TMPRSS2 (12)
. Thus, our new protease is the
third member of a family of transmembrane-bound serine proteases.
Consequently, this new gene was named TMPRSS3 for
transmembrane protease, serine 3. Sequence homology was high in the
domains containing the three principal active-site amino acids
H245, D290, and
S387, required for peptide bond hydrolysis. The
arrangement of the catalytic residues in the linear sequence defines
the membership of TMPRSS3 to the S1 family of the chymotrypsin clan SA
of serine-type peptidases (2)
. The prototype of this
family is chymotrypsin, and the three-dimensional structures of some of
its members have already been resolved (12)
.
TMPRSS3 is predicted to cleave in a trypsin-like manner after lysine or
arginine residues because it contains D381 at the
base of the specificity pocket that binds the substrate
(13)
. In addition, the novel protein shares considerable
structural similarities of the TMPRSS family, including the putative
NH2-terminal membrane anchor and the conserved
cysteine residues, which by homology most likely form the disulfide
bonds C196–C310,
C230–C246,
C356–C372, and
C383–C410. Serine
proteases are most commonly synthesized as inactive proenzymes, which
are activated by extracellular, proteolytic removal of a
propeptide. At the NH2-terminal part of the
protease domain, TMPRSS3 contains the peptide sequence RVVGG, which is
typical for the proteolytic activator site of many protease zymogens.
The potential cleavage between R204 and
V205 would result in a new terminal -amino
group, which forms a salt bridge with D386 and
thereby leads to the assembly of the functional catalytic sites.
Therefore, the activated form would consist of a non-protease and a
protease subunit linked by a disulfide bond that most likely involves
C196–C310. Whether this
activation is mediated under physiological conditions by autocatalytic
cleavage or other proteases is not known. The TMPRSS3 gene
locus was localized to chromosome 11 at q23.3 between the markers
D11S4362 and D11S4387 by use of a radiation hybrid panel.
As anticipated, an overexpression of the 2.3-kb transcript was found in
9 of 13 primary pancreatic carcinoma tissues (Fig. 2
) and in 10 of 16 pancreatic carcinoma cell lines (not shown) by
Northern blot analysis. Because TMPRSS3 was not expressed in
normal pancreas (n = 6) and in chronic
pancreatitis (n = 6) tissue samples,
overexpression appears to be cancer-specific and not due to
inflammatory alterations in the stroma. No clear correlation was found
between the stage of pancreatic tumors and the expression of the
protease (Table 1)
. Northern blot analyses with RNA from a small number of other tumor
tissues revealed that TMPRSS3 overexpression is not
restricted to pancreatic cancer, but can also be found in gastric
(n = 4), colorectal (n = 7), and ampullary (n = 1) cancer. No
expression was found in one tissue sample each of soft tissue sarcoma
and breast cancer (Fig. 2
). TMPRSS3 transcripts were not
detectable in normal heart, brain, placenta, lung, liver, skeletal
muscle, uterus, and adipose tissue. A weak signal was found in tissues
of the normal gastrointestinal tract (esophagus, stomach, small
intestine, colon) and in some tissues of the urogenital tract (kidney
and bladder). Nevertheless, expression was much weaker than in the
corresponding tumors (data not shown). Furthermore, we analyzed the
expression of TMPRSS3 in the SUIT-2 clonal cell lines
S2-007, S2-013, and S2-028 (4)
. These subclones of the
human pancreatic cancer cell line SUIT-2 differ in their spontaneous
metastatic potential after s.c. injection in nude mice. In this setting
S2-007 regularly shows a high rate of metastases, whereas the other two
cell lines show a lower rate (S2-013) or no metastases at all (S2-028).
As shown in Fig. 2
, the strength of TMPRSS3 expression
correlated well to the metastatic potential of the SUIT-2 subclones,
which may serve as an indication that this serine protease is
associated with the promotion of metastasis.
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). According to the charge difference rule
(10)
, the transmembrane domain (amino acids 32–53)
anchors the protease to the cell membrane. Because of this anchorage,
the NH2-terminal domain (amino acids 1–31) would
appear to be located intracellularly, and the COOH-terminal region
(amino acids 54–437), which contains the catalytic domain, would be
located extracellularly (Fig. 3b
). The alleged subcellular
localization of the protease was confirmed using a V5-tagged
TMPRSS3 construct, which was transiently transfected into
HEK-293 cells. Membrane fractionation and Western blotting with the
corresponding anti-V5 antibody revealed a signal only in the plasma
membrane-enriched fraction, whereas no tagged TMPRSS3 protein was
detectable in the cytosol and in the culture medium (Fig. 4
).
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68 kDa, suggesting the presence of
potential carbohydrate moieties. The primary sequence of TMPRSS3
displays two consensus motifs for N-linked glycosylation
(N-X-T/S) at N130 and N178.
To confirm this N-glycosylation, epitope-tagged TMPRSS3 was
expressed in HEK-293 cells, immunoprecipitated, and treated with PNGase
F. This resulted in an increase in mobility on denaturing SDS-PAGE,
demonstrating N-glycosylation of TMPRSS3 (Fig. 4
).
Cultivation of transfected HEK-293 cells in the presence of
tunicamycin, an inhibitor of N-glycosylation, revealed the
same mobility shift of TMPRSS3 to a molecular mass of 60 kDa.
Phenyl-N-acetyl--D-galactosaminide,
which inhibits mucin-like O-glycosylation, had no effect on
the molecular mass (data not shown). The generation of recombinant
proteases frequently has been shown to be difficult or impossible
(14)
. Despite extensive and repeated efforts, we were
unable to successfully generate recombinant protein in
Escherichia coli and insect cells, possibly because TMPRSS3,
as many other proteases, had a cytotoxic effect on transfected cells.
Repeated efforts to generate peptide antisera failed as well (data not
shown), and a TMPRSS3 antibody was therefore not available for further
studies. Whereas the established physiological role of the chymotrypsin family of secreted serine proteases is primarily in protein catabolism, the function of serine proteases of the TMPRSS family is of special interest. Although the function of TMPRSS2 remains unknown (12 , 15) , TMPRSS1, also known as hepsin, frequently is overexpressed in ovarian tumors and may therefore contribute to the invasive nature or growth capacity of ovarian tumor cells (16) . Treatment of hepatoma cells with antihepsin antibodies or specific antisense oligonucleotides confirmed that hepsin plays an essential role in cell growth and maintenance of cell morphology (17) . It has also been shown that hepsin can proteolytically activate human coagulation factor VII and thereby contribute to the activation of the coagulation cascade (18) .
The correlation of TMPRSS3 expression with the metastatic potential of the SUIT-2 cell lines is a first indication that this new protease, in the same way as hepsin, may be involved in promoting metastasis formation and tumor invasion. To confirm this hypothesis in functional assays, stably transfected S2-020 cell lines were generated using the TMPRSS3 cDNA cloned in sense and antisense orientation into the pHß-Apr1-neo vector. Several clones were generated showing variable degrees of TMPRSS3 sense/antisense mRNA transcription. Two sense and two antisense clones were further characterized by s.c. injections in nude mice, in vitro Matrigel invasion assays, and biochemically for their capacity to hydrolyze substrates for trypsin and elastase. No significant differences could be observed between sense and antisense clones in any of the functional assays. There was no difference in tumor size and local invasiveness after s.c. injections, and there was no evidence of metastasis formation after tail vein injection with both sense and antisense cells. Similarly, we failed to show an effect on in vitro invasiveness and on proteolytic activity of native and enterokinase-treated lysates for a selection of serine protease substrates. Many factors may be responsible for the failure of TMPRSS3-transfected tumor cells to behave differently in these assay, including the necessity for a complex activation mechanism, processes that affect protein folding, or the absence of essential cofactors. Furthermore, although transiently transfected HEK-293 cells showed expression of the V5-tagged recombinant TMPRSS3 protein, we could not directly demonstrate expression of the protein in the transfected cells because we lacked a specific antibody. In the absence of final experimental proof, we can therefore only hypothesize, based on the structural characteristics and the expression pattern in cancer tissues and in the SUIT-2 subclones, that this new protease has a potential role for tumor progression, metastasis formation, and tumor invasion.
Proteases have an important function in the context of tumor growth, because they can break down the surrounding extracellular matrix components, they can pave the way for spreading tumor cells, and they can release and activate growth and angiogenic factors. Protease activity on the surface of tumor cells is required to allow malignant invasion through surrounding connective tissue, which is an important event in the multistep process of metastasis formation (19) . Thus, it is conceivable that TMPRSS3 may contribute to the invasive and metastatic potential of tumor cells. In this context, cell surface proteases such as TMPRSS3 may function as an activator of other extracellular proteases or act directly by degrading the extracellular matrix surrounding the tumor cells. Furthermore, TMPRSS3, as shown for many other proteases, may participate in the activation of hormones or growth factors by proteolytic cleavage of inactive proforms. Because the biochemical events required for the activation of this novel serine protease are unknown and the specific substrates have not yet been identified, the precise role of TMPRSS3 in carcinogenesis remains to be elucidated.
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Acknowledgments |
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FOOTNOTES |
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1 This work was supported by grants from the
Bundesministerium für Bildung und Forschung (01 GB9401), the
European Community (BMH4-CT98-3085), and the Deutsche
Forschungsgemeinschaft (SFB518, project B1; to T. M. G.). 
2 The nucleotide sequence in this report has been
submitted to the GenBank Data Library with accession no. AF179224.
3 To whom requests for reprints should be
addressed, at Department of Internal Medicine I, University of Ulm,
89081 Ulm, Germany. Phone: 49-731-5024385; Fax: 49-731-5024302; E-mail: thomas.gress{at}medizin.uni-ulm.de
4 The abbreviations used are: RDA,
representational difference analysis; PNGase F,
peptide-N-glycosidase F.
Received 12/17/99. Accepted 3/30/00.
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-chymotrypsin. 3. Crystallographic studies of substrates and inhibitors bound to the active site of -chymotrypsin. J. Mol. Biol., 46: 337-348, 1969.[Medline]
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indicates the active-site
residues histidine (H), aspartate (D),
and serine (S). Double underlines
indicate potential N-linked glycosylation sites.
