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Regular Articles |
From the Departments of Clinical Chemistry*
and
Pathology,
Helsinki University Central
Hospital, Helsinki, Finland; and the Departments of
Urology
and
Surgery,§
Malmö University Hospital,
Malmö, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human semen consists primarily of the secretions of the sex accessory tissues which include the prostate, seminal vesicles, epididymis, vas deferens, ampullae, Cowper’s gland, and glands of Littre. These organs produce several proteolytic enzymes such as human kallikrein-2 (hK2)11 and prostate-specific antigen (PSA).12 PSA is a chymotrypsin-like serine proteinase,13 whereas hK2 has trypsin-like enzymatic activity.14 Recent studies have shown that a recombinant proform of PSA is activated by bovine trypsin and recombinant hK2 in vitro,15,16 but the physiological activators of proPSA and prohK2 are not known.
In this study, we have purified and characterized two trypsinogen isoenzymes from human seminal fluid. Furthermore, we have examined their expression and localization in various tissues of the male genital tract.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fresh tissue specimens of human prostate, seminal vesicles, vas deferens, epididymis, and testis obtained by surgery were frozen by immersion in liquid nitrogen and stored at -80°C until analyzed. For immunohistochemistry and in situ hybridization studies, tissues were fixed within 30 minutes after removal in Bouin’s fixative (for 4 to 18 hours) or 4% buffered paraformaldehyde (overnight). The specimens were obtained from patients undergoing transurethral resection of the prostate or transvesical prostatectomy because of benign enlargement of the prostate, cystoprostatectomy because of invasive cancer of the urinary bladder, or radical prostatectomy or orchidectomy as treatment for prostate cancer. For control purposes, normal pancreatic tissue was obtained at surgery from two patients undergoing resection of small pancreatic tumors. All tissues were histopathologically normal according to hematoxylin and eosin staining. The Helsinki Declaration regarding the use of human tissues was followed.
Human semen was collected and allowed to liquefy at room temperature, after which the sperm was removed by low-speed centrifugation (600 x g, 10 minutes, room temperature). The resulting seminal fluid was further clarified by high-speed centrifugation (35,000 x g, 20 minutes, 4°C) and stored at -20°C until analyzed.
Antibodies and Immunoaffinity Media
Monoclonal antibodies (mAb) to PSA (6C11) (Leinonen J, Stenman UH,
unpublished data), trypsinogen-1 (3E8 and 6D11),17
and
trypsinogen-2 (14F10 and 14D4)17
were produced by standard
procedures. A polyclonal antibody to
1-antichymotrypsin (ACT) was from DAKO A/S
(Glostrup, Denmark). For immunoaffinity chromatography, mAbs 14F10,
3E8, and 6C11 were immobilized separately on CNBr-activated Sepharose
4B (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the
manufacturer’s instructions. For immunoassays, mAbs 14F10 and 6D11,
and the polyclonal antibody to ACT were labeled with an
Eu3+ chelate as described.18
A
polyclonal antiserum against trypsinogen was produced as
described3
and a peroxidase-conjugated swine anti-rabbit
IgG was from DAKO A/S. Characteristics of the primary antibodies used
in immunohistochemistry are given in Table 1
. Biotinylated secondary antibodies,
anti-rabbit and anti-mouse IgGs were included in the DAKO ChemMate kit
(code K5003; BioTek Solutions, Carpenteria, CA) and anti-sheep
IgG (code AB360) was from Binding-Site (Birmingham, UK).
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The concentration of PSA was determined by the Delfia EQM PSA kit (Wallac, Turku, Finland) which recognizes free PSA and total PSA equally. The complex between PSA and ACT (PSA-ACT), was determined as described.19,20 Trypsinogen-1 and -2 were determined by specific time-resolved immunofluorometric assays.17 The detection limit was 0.01 µg/L for PSA, 0.2 µg/L for PSA-ACT,19,20 and 0.1 and 0.3 µg/L for trypsinogen-1 and -2, respectively.17 The inter- and intra-assay coefficients of variation were both 2 to 4% for the assay of PSA, 5 to 10% and 8 to 13% for PSA-ACT,19,20 10 to 15% for trypsinogen-1, and 10 to 12% for trypsinogen-2.17
Purification of Trypsinogen-1 and -2 from Human Seminal Fluid
Human seminal fluid (200 ml) was sequentially precipitated with ammonium sulfate at 30 and 70% saturation for 20 minutes at 4°C. The precipitate formed at 70% saturation was collected by centrifugation (5,000 x g, 20 minutes, 4°C), and dissolved in 50 ml of 50 mmol/L Tris-HCl, pH 7.4, containing 8 mmol/L NaN3, and 10 mmol/L benzamidine (buffer A). The resulting solution was clarified by centrifugation (35,000 x g, 20 minutes, 4°C), and applied to anti-trypsinogen-1 and -2 affinity columns (10 ml) connected in series. The columns were washed with buffer A containing 1 mmol/L NaCl and 0.1% (v/v) Triton X-100 until the absorbance at 280 nm in the flow-through fraction was <0.001. The two columns were separated and the bound proteins were eluted with 0.1% (v/v) trifluoroacetatic acid. Two-ml fractions were collected and immediately neutralized by adding 0.2 ml of 1 mol/L Tris-HCl, pH 8.0. Fractions containing trypsinogen-1 or -2 immunoreactivity were pooled and dialyzed against 10 mmol/L Tris-HCl, pH 8.0, containing 8 mmol/L NaN3 and 2 mmol/L benzamidine (buffer B), and applied to an anion exchange Resource Q column (6 ml) (Amersham Pharmacia Biotech) equilibrated with buffer B containing 0.5 mol/L NaCl (buffer C). The column was washed with five bed volumes of the same buffer. Bound proteins were eluted with a linear gradient consisting of 60 ml of buffer B and 60 ml of buffer C. Flow rate was 2 ml/min and fractions of 1 ml were collected. Trypsinogen in the fractions was detected by immunofluorometric assays. The fractions in each peak were pooled and dialyzed against 10 mmol/L Tris-HCl, pH 8.0, containing 8 mmol/L NaN3 before measuring the enzyme activity of trypsin.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Blotting
For Western blotting, 1 µg of trypsinogen-1 and -2 purified by immunoaffinity chromatography from seminal fluid were separated on a 2-mm thick 3 to 16% gradient sodium dodecyl sulfate-polyacrylamide gel under reducing conditions21 and electrophoretically transferred to an Immobilon-P membrane (Millipore, Bedford, MA).22 Nonspecific binding was blocked with 1% (w/v) bovine serum albumin (Sigma, St. Louis, MO) in phosphate-buffered saline (PBS) (50 mmol/L sodium phosphate, 150 mmol/L NaCl, pH 7.4) at 4°C (overnight). The membrane was incubated with a polyclonal antibody against trypsinogens3 (100 µg/ml) for 2 hours at 37°C followed by peroxidase-conjugated swine anti-rabbit IgG (DAKO A/S) diluted 1:1,000 for 4 hours at room temperature. After washing with PBS, the blot was developed with a solution of 0.3 mg/ml 3,3'-diaminobenzidine tetrahydrochloride (Sigma) and 0.08% H2O2 in PBS.
Measurement of the Enzymatic Activity of Trypsin
The enzymatic activity of trypsinogen fractions purified by immunoaffinity and anion exchange chromatography from seminal fluid were measured using a chromogenic peptide substrate N-benzoyl-L-isoleucyl-glutamyl-L-arginine-p-nitroanilide (S-2222) (Kabi, Stockholm, Sweden). Trypsinogen (0.9 nmol/L) and S-2222 (0.5 mmol/L) were incubated in the presence or absence of enteropeptidase (Sigma) (5 ng) in 1 ml of 50 mmol/L Tris-HCl buffer, pH 8.0, containing 10 mmol/L CaCl2 and 1% (v/v) Triton X-100 at room temperature. Substrate hydrolysis was followed for 110 minutes by reading the absorbance at 405 nm on a microplate reader (Labsystems Multiskan Bichromatic; Labsystems, Helsinki, Finland).
Inhibition of trypsin-2 purified from urine of pancreatitis patients3 (0.7 nmol/L) by zinc was studied using the substrate S-2222 (100 µmol/L) and ZnCl2 concentrations varying from 25 µmol/L to 1 mmol/L in 50 µl of 50 mmol/L Tris-HCl, pH 8.0, containing 20 mmol/L CaCl2 and 0.01% (v/v) Triton X-100. Tumor-associated trypsin inhibitor purified from urine of pancreatitis patients3 (1.5 nmol/L) was used as a control. Substrate hydrolysis was followed for 20 minutes at 37°C starting 10 minutes after mixing the enzyme with ZnCl2 by reading the absorbance at 405 nm on a microplate reader.
Activation of proPSA by Trypsin
Activation of proPSA by trypsin was studied by using proPSA
purified from LNCap cell medium and trypsin-1 and -2 purified from
seminal fluid, and further by using recombinant proPSA.16
ProPSA was purified from LNCap cell medium by immunoaffinity
chromatography as described earlier.23
Intact mature PSA
(B isoenzyme) was purified from seminal fluid as
described.24
Purified PSA (10 µg) was incubated with
0.05 µg or 10 µg of trypsinogens purified from seminal fluid (molar
ratio 
1:200 and 1:1, respectively) in the presence or absence of
enteropeptidase (5 ng; Sigma) in 100 µl of 50 mmol/L Tris-HCl
containing 150 mmol/L NaCl, 8 mmol/L NaN3, 1%
bovine serum albumin, pH 7.4, for 1 hour at 37°C. Then 80 µg of
1-antichymotrypsin (Athens Research Technology
Inc., Athens, Georgia) (molar ratio to PSA 4:1) in 1 ml of the
above-mentioned buffer was added and the incubation continued for 12
hours at room temperature. The concentration of PSA and PSA-ACT in
aliquots taken at 1, 4, and 12 hours was determined by specific
immunoassays. Formation of PSA-ACT complex was taken as a measure of
proPSA activation.
Activation of recombinant proPSA16 (kindly provided by Dr. Janita Lövgren, University of Turku, Finland) (0.7 µmol/L) by trypsin-2 purified from urine of a pancreatitis patient3 (40 nmol/L) was studied using a chromogenic peptide substrate 3-carbomethoxy-propionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanilide (S-2586) (Kabi) (0.5 mmol/L) in 50 µl of 50 mmol/L Tris-HCl, pH 7.5, containing 150 mmol/L NaCl and 1 mmol/L CaCl2. ProPSA and trypsin incubated alone with the substrate were used as negative controls. Substrate hydrolysis was followed for 4 hours at 37°C by monitoring the absorbance at 405 nm on a microplate reader.
Immunohistochemistry
Immunohistochemistry was performed using a detection kit (DAKO
ChemMate Detection Kit Peroxidase/Carbazole, Rabbit/Mouse) and a
staining machine (DAKO TechMate 500/1000 Instrument; BioTek
Solutions). Briefly, the sections were deparaffinized in xylene,
rehydrated, and treated with 0.3%
H2O2 in methanol for 30
minutes at room temperature to quench endogenous peroxidase activity.
For antigen retrieval, tissue sections were first incubated with sodium
citrate (10 mmol/L, pH 6.0) and boiled in a microwave oven at 750 W for
2 x 3 minutes, and then digested with proteinase K (20 µg/ml in
20 mmol/L Tris-HCl, 2 mmol/L CaCl2, pH 7.5) for
25 minutes at 37°C. The sections were incubated with primary
antibodies17,25,26
(Table 1)
for 60 minutes at room
temperature, after which they were incubated with either biotinylated
secondary antibodies against rabbit or mouse IgG, which were included
in the ChemMate kit (code K5003; BioTek), or with an anti-sheep IgG
(Binding-Site), diluted 1:200 for 60 minutes at room temperature. The
immunoreactivity was visualized using the manufacturer’s protocol for
the peroxidase/3-amino-9-ethyl-carbazole reagent in the ChemMate kit.
The sections were counterstained with Mayer’s hematoxylin solution. As
a negative control, adjacent tissue sections were processed by
replacing the primary antibody with nonimmune mouse IgG1 diluted 1:200
(code X0931; DAKO) or nonimmune rabbit IgG diluted 1:8,000 (code X0936;
DAKO). Pancreatic tissue was used as a positive control.
Riboprobe Synthesis for in Situ Hybridization
In vitro transcriptions of sense and antisense probes were made by fluorescein-UTP riboprobe synthesis using the RNA color kit (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. As a template, we used a 627-bp long trypsinogen-2 cDNA fragment (corresponding to nucleotides 42 to 688, accession number M2760227 ), cloned from COLO 205 cells by RT-PCR using the TA Cloning Kit (InVitrogen, San Diego, CA) and the following primers: 5'-TGC TGT TGC TGC CCC CTT TG-3' (sense) and 5'-GCA CAG CCA TAG CCC CAG GAG-3' (antisense). The integrity and length of the probes was determined by gel electrophoresis.
In Situ Hybridization
All reagents were purchased from Sigma and Amersham Pharmacia Biotech. Tissue specimens were fixed, paraffin-embedded, sectioned (4 µm), dried for 2 hours at 65°C and mounted on SuperFrost plus slides (Menzel-Gläser) under RNase-free conditions. The sections were deparaffinized in xylene and rehydrated, after which they were first treated with 0.2 mol/L HCl to abolish endogenous enzyme activity, and then digested with proteinase K (20 µg/ml in 20 mmol/L Tris-HCl, 2 mmol/L CaCl2, pH 7.5) for 25 minutes at 37°C. The slides were then incubated in 0.25% acetic anhydride containing 0.1 mol/L triethanolamine and 0.9% NaCl, and then equilibrated in 2x standard saline citrate (SSC, 1x contains 150 mmol/L NaCl and 15 mmol/L sodium citrate, pH 7.0). After prehybridization with 40 µl of hybridization buffer containing 50% (v/v) formamide, 10 mmol/L Tris-HCl, pH 7.6, 1x Denhardt’s solution (bovine serum albumin, polyvinylpyrrolidone and Ficoll, all at 0.2 mg/ml), 2x SSC, and 0.4 µg/ml salmon sperm DNA at 55°C for 1 hour, the slides were hybridized with 40 µl of 250 ng/ml antisense or sense probe in hybridization buffer first for 8 minutes at 85°C and then for 16 hours at 55°C.
After hybridization, the slides were washed in 1x SSC at room temperature (2 x 5 minutes), 0.1x SSC at 60°C (4 x 15 minutes), 1x SSC at room temperature (10 minutes), and then equilibrated in Tris-buffered saline (TBS) (100 mmol/L Tris-HCl, 0.4 mol/L NaCl, pH 7.5). For detection of hybridization signals, tissue sections were first incubated in blocking reagent, 0.5% (w/v) in TBS, for 1 hour at room temperature, rinsed in TBS, and subsequently incubated with anti-fluorescein alkaline phosphatase conjugate (Amersham Pharmacia Biotech) diluted 1:1,000 in TBS containing 0.5% (w/v) bovine serum albumin for 2 hours at room temperature. After washing in TBS, the sections were equilibrated in detection buffer (100 mmol/L Tris-HCl containing 100 mmol/L NaCl, 50 mmol/L MgCl2, pH 9.5) (5 minutes), and developed in detection buffer containing 1 mmol/L levamisol, 0.33 mg/ml nitroblue tetrazolium chloride, and 0.175 mg/ml 5-bromo-4-chloro-3-indolyl phosphate. The color reaction was stopped after 2 to 8 hours by incubating the sections in stop buffer (10 mmol/L Tris-HCl containing 10 mmol/L ethylenediaminetetraacetic acid, 0.9% NaCl, pH 7.5) for 10 minutes. The slides were coverslipped using Faramount mounting medium (DAKO).
Isolation of RNA, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), and Sequencing of the PCR Product
Total RNA was extracted from prostate tissue and seminal vesicle according to the method of Chomczynski and Sacchi.28 The oligonucleotide primers used for trypsinogen were constructed on the basis of the published sequence for trypsinogen-227 so that they also recognized trypsinogen-1 transcripts: 5'-CAT GAA TCT ACT CCT GAT CC-3' (outer sense), 5'-TGT CAT TGT CCA GAG TCC-3' (outer antisense), and 5'-CCC CTT TGA TGA TGA TGA C-3' (inner sense) 5'-AAC TGT TCA TTC CCC TCC-3' (inner antisense). The outer and inner primer pairs produced fragments of 323 and 213 bp, respectively. ß-actin primers were prepared on the basis of the published sequence:29 5'-CCC AGG CAC CAG GGC GTG AT-3' (sense) and 5'-TCA AAC ATG ATC TGG GTC AT-3' (antisense). They produced a fragment of 260 bp. Total RNA (1 µg) was transcribed into cDNA using SuperScript II-RT (GibcoBRL, Paisley, UK) according to the manufacturer’s instructions. Contamination of RNA samples with cDNA was excluded by using control reactions without reverse transcriptase. The reverse transcription product (1 µl) was amplified in a 40-µl reaction volume in 1x PCR buffer (10 mmol/L Tris-HCl, 1.5 mmol/L MgCl2, 50 mmol/L KCl, 0.1% Triton X-100, pH 8.8; Finnzymes, Espoo, Finland) containing 0.25 mmol/L of each dNTP, 20 pmol of both outer antisense and sense primers, and 1.6 U of Dynazyme DNA polymerase (Finnzymes) for 30 cycles at 94°C for 1 minute, 60°C for 1 minute, and 72°C for 30 seconds. After the first PCR round, 1 µl of the PCR product was further amplified using the inner primer pair and the same PCR conditions except the annealing temperature which was 53°C. RNA isolated from COLO 205 cells (ATCC, Rockville, MD) was used as a positive control and water as a negative control in all experiments. The PCR products were separated in a 1.5% agarose gel and stained with ethidium bromide. The PCR products were then purified using a kit for DNA Extraction (Amicon, Inc., Beverly, MA) and sequenced using the ABI Prism Dye Terminator Cycle Sequencing Core Kit with AmpliTaq DNA Polymerase and the ABI Prism 310 Genetic Analyser (PE Biosystems, Foster City, CA). The identity of the mRNA was determined by sequencing the PCR products and comparing the resulting sequences with the ones in the EMBL Nucleotide Sequence Database.
Northern Blotting
Nylon membrane blotted with 2 µg of poly A RNA (Clontech Laboratories, Inc., Palo Alto, CA) was used to study the expression of trypsinogen in the prostate and testis. The membrane was hybridized with a 627-bp long 32P-labeled cDNA probe (labeled with the Rediprime DNA-labeling system, Amersham Pharmacia Biotech) corresponding to nucleotides 42 to 688 of the human trypsinogen-2 cDNA sequence27 (see riboprobe synthesis for in situ hybridization) in ExpressHyb hybridization buffer (Clontech Laboratories) at 68°C for 16 hours. The membrane was washed in several changes of 2x SSC, 0.05% sodium dodecyl sulfate for 15 minutes at room temperature. For autoradiography, the membrane was exposed to Hyperfilm-MP film (Amersham Pharmacia Biotech) for 1 to 2 days. A glycaraldehyde-3-posphate dehydrogenase cDNA probe was used to quantify the amount and quality of loaded polyA RNA.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concentration of trypsinogen-1 in 24 samples studied ranged
from 0.12 to 1.6 mg/L (median, 0.4 mg/L), and that of trypsinogen-2
from 0.02 to 1.4 mg/ml (median, 0.5 mg/L). A pool of 200 ml of seminal
fluid containing 70 µg of trypsinogen-1 and 100 µg of
trypsinogen-2 was used as starting material for purification of
trypsinogens. Precipitation with ammonium sulfate resulted in a
2.6-fold purification (Table 2)
. When the
precipitate was reconstituted and applied to the immunoaffinity
columns, 90% of trypsinogen immunoreactivity was retained. After
disconnection of the columns, bound trypsinogen-1 and -2 were
separately eluted with 0.1% trichloroacetatic acid, neutralized, and
further fractionated by anion exchange chromatography on a Resource Q
column. Three peaks with trypsinogen-1 immunoreactivity, designated 1A
to 1C and four peaks with trypsinogen-2 immunoreactivity, designated 2A
to 2D, were obtained (Figure 1, A and B)
.
The major peak with trypsinogen-1 immunoreactivity (1C) represented
80% of the trypsinogen-1 immunoreactivity recovered and 26% of
that in the starting material. The major trypsinogen-2 peak (2C)
represented 50% of the trypsinogen-2 recovered and 30% of that in the
starting material (Table 2)
.
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Trypsinogen-1 and -2 purified by immunoaffinity chromatography
were subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and Western blotting with a polyclonal trypsin
antibody. Trypsinogens appeared as two major bands of 25 to 28 kd and
several minor fragments of smaller size (Figure 2)
.
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. The
two minor peaks, 1A and 1B, had no enzymatic activity and activity was
not induced by enteropeptidase. The four peaks of trypsinogen-2 showed
no enzymatic activity, but after incubation with enteropeptidase, the
major trypsinogen-2 fraction (2C) was activated (Figure 3B)
.
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The activation of LNCap-proPSA by seminal fluid trypsin was
monitored by analyzing the complex formation between PSA and ACT by
PSA-ACT immunoassay and recovery of PSA immunoreactivity. When
LNCap-proPSA was incubated with ACT for 12 hours at 37°C,
25% of the PSA formed a complex with ACT. After incubation of
LNCap-PSA with active trypsin-1C at a molar ratio of 1:200 for 1
hour at 37°C, 40% of the PSA formed a complex with ACT (Figure 4A)
. The recovery of total PSA
immunoreactivity was 90 to 100%. The proenzyme trypsinogen-2C had no
effect on the complexation of LNCap-PSA with ACT, but after addition of
enteropeptidase, the trypsin-2C formed increased the complexation of
LNCap-PSA with ACT from 25 to 37% (Figure 4B)
. When intact mature PSA
isolated from seminal fluid was incubated with active trypsin-1C or
-2C, there was no further increase in complex formation between PSA and
ACT. If PSA was incubated with trypsin-1C at a high molar ratio (1:1),
a significant loss (>50%) of PSA immunoreactivity was observed,
regardless of whether PSA purified from seminal fluid or from LNCap
cell medium was used, indicating degradation of PSA by trypsin.
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Inhibition of Trypsin-2 Activity by Zinc
Zinc inhibited trypsin-2 activity in a dose-dependent manner. At
0.025, 0.1, and 1 mmol/L Zn2+ concentrations,
trypsin-2 activity was inhibited by 7.6%, 18%, and 50%,
respectively. In a control experiment 1.5 nmol/L tumor-associated
trypsin inhibitor inhibited trypsin-2 activity by 90% (Figure 5)
.
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Trypsinogen immunoreactivity was detected in most tissue sections
examined, but the number of positive cells and staining intensity
varied (Figure 6)
. However, a similar
staining pattern was obtained with all antibodies used. In testis, a
small number of immunoreactive spermatocytes were found in a few
tubules. An intense immunostaining was detected in most epithelial
cells of cauda epididymis, but also in a subset of the basal and
luminal epithelial cells of the corpus and caput region. A vast
majority of the epithelial cells of the vas deferens (close to the
epididymis), ampulla vas deferens, seminal vesicles, utriculus
prostaticus, and ejaculatory ducts also stained positively for
trypsinogen. In the prostatic epithelium, luminal but not basal cells
were immunoreactive for trypsinogen. A majority of the luminal cells in
prostatic excretory ducts stained positively but in the acini and areas
of benign hyperplasia, only few immunoreactive cells were detected. The
epithelial cells of the prostatic part of urethra as well as the
luminal cells of periurethral glands were immunoreactive.
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For in situ hybridization, the fluorescein-labeled antisense
and sense probes were hybridized to adjacent tissue sections. In
pancreas and in all tissues from the male genital tract that were
studied, hybridization signals were generated by the antisense probe,
whereas the sense probe did not give rise to any positive signals
(Figure 7)
. The intensity of the
hybridization signals varied, but the results were in accordance with
the immunohistochemical findings (Figure 7)
.
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RT-PCR and sequencing of the PCR products showed that
trypsinogen-1 and -2 transcripts are expressed in normal prostatic
tissue and trypsinogen-1 in seminal vesicles (Figure 8)
. Sequencing of the PCR products
revealed the reported cDNA sequences of trypsinogen-1 and -2,
respectively.27
By Northern blotting, trypsinogen
transcripts were detected in the prostate, but not in the testis (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Trypsin hydrolyzes peptide bonds at the carboxyl side of arginine and
lysine residues, and it efficiently activates various serine
proteinases and metalloproteinases (MMPs) involved in digestion,
fibrinolysis, and tumor invasion. Trypsin is more potent than plasmin
and other serine proteinases in activating the latent forms of many
MMPs, including MMP-2, MMP-9, and MMP-7 (matrilysin).30,31
Trypsin is also able to activate membrane receptors such as the
proteinase-activated receptor-2.32,33
These findings
suggest that extra-pancreatic trypsin may be involved in tissue
remodeling and tumor invasion. The widespread distribution of
trypsinogen in the male genital tract suggests that this proteinase
plays a physiological role also in reproduction, and it is tempting to
speculate that this function is related to the activation of other
proteinases such as PSA and hK2 which are present in seminal plasma at
high concentrations. ProPSA has been shown to be activated by bovine
trypsin15
and we could show that trypsin purified from
seminal plasma activated proPSA produced by LNCap cells. PSA is one of
the most abundant serine proteinases in seminal fluid with an average
concentration of 1.0 mg/ml34
The main function of PSA
is thought to be the dissolution of the sperm-entrapping gel formed
immediately after ejaculation by cleavage of the gel forming proteins
semenogelin I and II.13
Multiple cleavages result in the
release of progressively motile sperm. In seminal fluid, no proforms of
hK2 and PSA are detected. Most PSA is free and enzymatically
active34
whereas hK2 occurs in complex with activated
protein C inhibitor.11
It is most likely that proPSA and
prohK2 are activated by an enzyme with trypsin-like specificity,
because the cleavage of the activation peptide occurs at the carboxyl
terminal side of an arginine.16
On the other hand, 30
to 35% of the PSA in seminal fluid is partially cleaved. The cleavage
sites occur after the basic amino acids Arg-85, Lys-145, and
Lys-182.24,35,36
Isoforms of hK2 with multiple internal
cleavages at Arg-101, Arg-145, and Arg-226 have also been
observed.11,16,37
All these are typical cleavage sites for
trypsin. They are also potential cleavage sites for hK2, but PSA is not
degraded by hK2.16
Thus trypsin may contribute not only to
the activation of prohK2 and proPSA, but also to their degradation in
seminal plasma.
Another potential physiological activator of proPSA is hK2.16 The average concentration of hK2 in seminal fluid is 6 µg/ml,38 which is approximately six times higher than that of the trypsins. However, zinc, that is present in the prostate at very high concentrations (9 mmol/L),39 inhibits hK2 more efficiently than trypsin: 100 µmol/L zinc causes a 90% reduction in hK2 activity,40 but only an 18% reduction in trypsin activity. This difference in inhibition of proteinase activity suggests that trypsin may function as an activator of proPSA under circumstances in which hK2 is inhibited. It is also possible that trypsin is an initiator of a proteinase cascade that leads to the activation of both hK2 and PSA.
Of the male sex glands, only the prostate expresses PSA and hK2. In the prostate the distribution of PSA and hK2 is different from that of trypsinogen. PSA and hK2 are highly expressed in secretory cells of the acini, whereas trypsinogen is predominantly found in the luminal cells of the prostatic excretory ducts. Ampulla vas deferens and seminal vesicles, that also store trypsinogen, empty into ejaculatory ducts passing through the prostate. These findings suggest that trypsin is admixed to the prostatic fluid during ejaculation and that it exerts its activity at this step. The fact that PSA is active also in patients with aplasia of the seminal vesicles and the deferent duct13 suggests that trypsin produced in the prostate may be sufficient for initiation of a proteinase cascade leading to activation of PSA.
The results of the present study show that trypsinogen is widely produced in the male genital tract. Being a highly potent proteinase, it may play a physiological role in reproduction. This function may comprise both activation and degradation of other proteinases. It remains to be studied whether trypsinogen is also expressed in prostate cancer.
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Acknowledgements |
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Footnotes |
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Supported by the Finnish Cancer Foundation; Sigrid Juselius Foundation; Finnish Academy of Sciences; Helsinki University Central Hospital; University of Helsinki; Swedish Cancer Society Research Fund (Project no. 4292-B99-01XAB); Cancer Research Fund at Malmö University Hospital; Foundation for Urology Research in Malmö; and the Thulefjord Foundation.
Accepted for publication August 20, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5ß1-dependent adhesion to fibronectin and proliferation of human gastric carcinoma cells through activation of proteinase-activated receptor-2. J Biol Chem 2000, 275:4592-4598This article has been cited by other articles:
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x 174/HaeIII. The
integrity of RNA was checked by amplifying all samples with ß-actin
primers producing a fragment of 260 bp.