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Division of Biological Sciences, Hokkaido University Graduate School of Science, Sapporo 060-0810, Japan
Address all correspondence and requests for reprints to: Dr. Takayuki Takahashi, Division of Biological Sciences, Hokkaido University School of Science, Sapporo 060-0810, Japan. E-mail: ttakaha{at}sci.hokudai.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tissue or glandular kallikreins (Klks) are a subfamily of many structurally related serine proteases (3, 4, 5). In humans, this gene subfamily comprises at least 15 genes (5, 6, 7, 8, 9, 10, 11), including three well known members, namely, tissue kallikrein, human glandular kallikrein, and prostate-specific antigen (PSA). On the other hand, in the mouse, this subfamily is reportedly comprised of at least 27 genes (3, 4, 12, 13). Existing evidence indicates that about half of the mouse genes are functional and expressed (4). Some functional Klks have been biochemically characterized, whereas other such proteins remain unidentified. Mouse Klk21 (mKlk21), which is among the unidentified Klks species, is of interest because expression of this gene in the mouse testis has already been demonstrated (14).
In our attempt to elucidate the functional roles of proteolytic enzymes in the mammalian reproductive organs, we isolated and identified cDNAs encoding a variety of proteases expressed in the adult mouse testis. These proteases included mKlk21, mKlk24, and mKlk27 (13); of these, mKlk21 was expressed most abundantly in this organ. In the present study we determined the complete nucleotide sequence of mKlk21 and examined its testicular expression as well as its enzymatic properties to establish a basis for future physiological studies. The present data show that mKlk21 is indeed a serine protease expressed specifically in the Leydig cells of the adult mouse testis. Furthermore, the enzyme is shown to be capable of hydrolyzing certain extracellular matrix proteins as well as IGF-binding protein-3 (IGFBP-3). The current study also demonstrates that IGFBP-3 mRNA is notably expressed in the Leydig cells expressing mKlk21. These results suggest the possible involvement of mKlk21 in the regulation of Leydig cell function in the mouse testis after sexual dimorphism of expression occurs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture
Mouse B16 melanoma cells (RCB 0557) were obtained from the RIKEN
Cell Bank (Tsukuba, Japan) and were cultured as described previously
(13). The mouse testicular cell line TM3 (Leydig cells)
was obtained from American Type Culture Collection
(Manassas, VA). Cells were grown according to the manufacturer’s
instructions.
RNA isolation
Total RNAs were prepared from frozen tissues or confluent
monolayers of melanoma cells using the guanidine isothiocyanate-cesium
chloride method (15). Polyadenylated
[poly(A)+] RNAs were selected by
oligo(deoxythymidine)-cellulose column chromatography.
cDNA cloning of mKlk21
The first strand of cDNA was synthesized from
poly(A)+ RNA of mouse B16 melanoma cells using a
SuperScript preamplification system (Life Technologies, Inc., Tokyo, Japan), according to the manufacturer’s protocol.
Two degenerated oligonucleotide PCR primers were synthesized
based on the cDNA sequence for conserved regions in mouse Klks
(sense primer S1, 5'-TGT GAGAAGAATTCCCAACCC-3', which corresponds to
the amino acid sequence
NH2-Cys-Glu-Lys-Asn-Ser-Gln-Pro-COOH; antisense
primer AS1, 5'-ACCATCACAGATCAGTGGGCC-3', which corresponds to the amino
acid sequence
NH2-Gly-Pro-Leu-Ile-Cys-Asp-Gly-COOH). cDNAs were
amplified under the following conditions: 3 min at 94 C for
denaturation; 40 cycles of 1 min at 94 C, 1 min at 58 C for annealing,
and 1 min at 72 C for extension; followed by 9-min final extension at
72 C. Fragments between 0.5–0.6 kb in size were recovered from the PCR
products by agarose gel electrophoresis. The fragments were subcloned
into pBluescript (II) KS+
(Stratagene, La Jolla, CA) cut with EcoRV.
Thirty-two clones were obtained from the PCR products after
transformation of the recombinant plasmids into Escherichia
coli JM109 cells. Among these, one clone (579 bp) was found to be
a fragment of mKlk24 cDNA. This cDNA fragment was used as a probe for
further experiments.
A mouse testis cDNA library was constructed in 
gt10 with 5
µg poly(A)+ RNA and was packaged using Gigapack
III packaging extract (Stratagene). Approximately 4.5
x 105 plaques from the library were transferred
to nylon membranes (Schleicher & Schuell, Inc., Dassel,
Germany) and hybridized at 65 C in a buffer containing 5x 0.15
M NaCl/8.65 mM
NaH2PO4/1.25 mM EDTA
(SSPE), 0.5% SDS, 5x Denhardt’s solution (Wako, Osaka,
Japan), and 100 µg/ml denatured salmon sperm DNA with the
32P-labeled 579-bp PCR fragment described above.
Filters were washed with increasing stringency, with a final wash of
0.1x SSC/0.1% SDS at 50 C. Phage DNA was subcloned into pBluescript
(II) KS+ for sequencing. The nucleotide sequence
was determined using the ABI automatic sequencer model 377 (PE Applied Biosystems, Foster City, CA).
Northern blot analysis
For preparation of the blot loading total RNA from various mouse
tissues, 30 µg total RNA were electrophoresed on a formaldehyde/1.2%
agarose gel and transferred to a Nytran Plus membrane (Schleicher & Schuell, Inc.). For preparation of the blot loading total RNAs
from mouse testes of various postnatal developmental stages, 25 µg
total RNA were electrophoresed on a formaldehyde/1.2% agarose gel and
transferred to a Nytran Plus membrane. The blots were hybridized with a
32P-labeled mKlk21-specific oligonucleotide
(5'-CATTGCTGTAGTCATCCTCAGGATGTGGGG-3' complementary to nucleotides
404–433), mouse IGF-IA cDNA fragments (GenBank accession no. X04480,
nucleotides 81–547), and mouse IGFBP-3 cDNA fragments (GenBank
accession no. X81581, nucleotides 478–893). For hybridization with the
oligonucleotide probe, blots were hybridized for 18 h at 37 C in a
buffer containing 5x SSC, 50 mM sodium acetate (pH 8.0),
0.1% SDS, 10x Denhardt’s solution, 0.01% sodium pyrophosphate, and
100 µg/ml denatured salmon sperm DNA. For hybridization with the cDNA
probes, blots were hybridized for 18 h at 42 C in 50% formamide,
5x SSPE, 1% SDS, 5x Denhardt’s solution, and 100 µg/ml denatured
salmon sperm DNA. The membranes were washed with increasing stringency,
with a final wash of 0.1x SSC/0.1% SDS at 37 C (oligonucleotide
probe) or 42 C (cDNA probe).
RT-PCR/Southern blotting
Testis, submaxillary gland, and Leydig cell cDNAs that had been
reverse transcribed according to the manufacturer’s protocol from 5
µg of each of the total RNAs using the SuperScript preamplification
system (Life Technologies, Inc.) were subjected to PCR
using mKlk21-specific primers (mKlk21S, 5'-CGCTACAACAAATATATA-3',
corresponding to nucleotides 205–222; mKlk21AS,
5'-CAGCAGATAATGTGACAT-3', complimentary to nucleotides 862–879). The
conditions for PCR were 94 C for 3 min, followed by 34 cycles (for
comparable PCR) or 25 cycles (for Leydig cells treated with steroid
hormones) at 94 C for 1 min, 49 C for 1 min, and 72 C for 1 min. To
compare the respective extents of expression of mKlk21 mRNA 5-µl
aliquots were removed from the PCR mixtures after every 4 cycles
starting at cycle 18. Amplified products were electrophoresed on a
1.5% agarose gel and transferred to a Nytran Plus membrane
(Schleicher & Schuell, Inc.). The blots were hybridized
with the 32P-labeled mKlk21-specific
oligonucleotide probe and processed as described above. Samples were
also tested for the expression of ß-actin to confirm the integrity
and quantity of RNA.
Preparation of recombinant mKlk21 in E. coli cells
The expression vectors for mKlk21 were constructed by inserting
its cDNA (amino acids 18–261), including the complete pro-enzyme
region of the mKlk21 gene, into the SalI and XhoI
sites of pET30a (Novagen, Madison, WI). PCR using Pfu
polymerase (Novagen) was performed with oligonucleotide primers to
create SalI sites at the 5'-ends and XhoI sites
at the 3'-ends. The reaction product was sequentially digested with
SalI and XhoI, gel-purified, and ligated in-frame
between the SalI and XhoI sites of expression
vector pET30a. After the orientation and sequence of the cDNA in the
pET plasmid were confirmed by DNA sequencing, the ligated vector was
transformed to E. coli strain BL21 (DE3) pLysS (Novagen).
The cells were grown at 37 C, induced with
isopropyl-1-thio-ß-D-galactoside. Cells were
harvested by centrifuge and were treated in a freeze-thaw process. The
materials were washed twice with 0.5% Triton X-100 and solubilized by
being dissolved and then incubated in 25 ml 50 mM
Tris-HCl (pH 7.6) containing 6 M urea and 0.5
M NaCl for 12 h at room temperature. The
solubilized sample was fractionated on a
Ni2+-chelate column (5 ml; Novagen) under
previously described conditions (13). The eluted protein
was dialyzed twice against 2 liters 20 mM
Tris-HCl (pH 7.6). The recombinant protein was synthesized as a fusion
protein containing 51 extra amino acids, all of which originated from
the plasmid sequence, at the NH2-terminus. This
fusion protein, which was enzymatically inactive, was then treated at
37 C for 60 min with trypsin immobilized on Sepharose 4B in 20
mM Tris-HCl (pH 7.6) to produce an active mKlk21
by cleaving at the activation site
(Arg24-Ile25 in Fig. 1
). The immobilized protease was removed
by filtration, and the resulting filtrate was directly applied to a
column of soybean trypsin inhibitor (SBTI)-Sepharose 4B (1 ml bed
volume) previously equilibrated with 50 mM
Tris-HCl (pH 7.6) containing 0.2 M NaCl. The
retained materials were eluted with 0.1 M
glycine-HCl buffer (pH 3.0). Fractions of 1 ml were collected in tubes
to which 0.2 ml each of 0.1 M Tris-HCl buffer (pH
9) had been added. The enzyme activity of mKlk21 was assayed by a
4-methylcoumaryl-7-amide (MCA)-containing synthetic substrate,
Pro-Phe-Arg-MCA. The procedure described above worked satisfactorily
for the preparation of active recombinant mKlk21.
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Zymographic analysis using casein and gelatin was performed according to previously described methods (18).
The assay for the degradation of fibronectin, laminin, type I collagen, and type IV collagen by mKlk21 was performed as previously described (18).
The actions of mKlk21 and mKlk27 on IGFBP-3 were determined by Western blot analysis. A 25-ng sample of human IGFBP-3 (Genzyme, Cambridge, MA) was incubated with 10–100 ng of either recombinant mKlk21 or mKlk27 in 20 µl 100 mM Tris-HCl (pH 8.0) at 37 C. The reaction was stop by heating the sample in the presence of SDS sample buffer. The reaction mixtures were separated on 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). The blotted membrane was incubated with rabbit antihuman IGFBP-3 antibody (American Research Products, Inc., Belmont, MA) at a 1:1000 dilution and subsequently with goat antirabbit IgG antibody (Amersham Pharmacia Biotech, Tokyo, Japan). Immunoreactive signals were detected using an ECL Western blot detection kit (Amersham Pharmacia Biotech) according to the protocol provided by the manufacturer.
Kinin-releasing activity was determined by measurement of the amount of kinin liberated from human low mol wt kininogen (Athens Research and Technology, Inc., Athens, GA) using a Markit bradykinin detection kit (Dainippon Seiyaku, Osaka, Japan).
In situ hybridization analysis of mKlk21 and IGFBP-3 mRNA
Antisense and sense RNA probes of mKlk21 were prepared by
in vitro transcription of reverse transcriptase fragments of
mKlk21 cDNA (nucleotides 251–722) with T3 or T7 RNA polymerase using a
digoxigenin (DIG) RNA labeling kit (Roche, Mannheim,
Germany). Antisense and sense RNA probes of mouse IGFBP-3 were prepared
by in vitro transcription of reverse transcriptase fragments
of IGFBP-3 cDNA (nucleotides 478–893 of GenBank clone X81581) obtained
using RT-PCR from testis total RNA. Eight-week-old mouse testis
sections (10 µm) were cut on a cryostat, thaw-mounted onto slides
coated with silan, and subjected to in situ hybridization as
previously described (18) for mKlk21. For IGFBP-3, the
sections were treated with 100 ng/ml proteinase K (Roche),
postfixed, and acetylated. Prehybridization was performed with
hybridization buffer (50% formamide, 6x SSPE, 5x Denhardt’s, and
500 ng/ml tRNA) for 3 h at room temperature. Hybridization was
carried out in hybridization buffer containing 200 ng/ml cRNA probes at
60 C for 18 h. The sections were washed three times for 20 min
each time in 0.2x SSC at 60 C. The hybridization probes were detected
using a DIG nucleic acid detection kit (Roche).
Immunohistochemical detection of IGFBP-3 in a mouse testis
section
Frozen testis sections, 10 µm thick, were prepared by using a
cryostat, fixed with 100% methanol at -20 C for 10 min, and treated
with 3% H2O2 in PBS. After
being blocked with BlockAce (Dainippon Seiyaku) for 1 h at room
temperature, each section was incubated with antimouse IGFBP-3
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted
1:100 in PBS for 12 h at 4 C. Bound antibodies were detected using
an avidin-biotin-peroxidase kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s
instructions. Immunocomplexes were detected using diaminobenzidine.
Preparation of mouse Leydig cells
For the preparation of mature Leydig cells, the testes from
8-wk-old mice were decapsulated and digested by 0.25 mg/ml collagenase
(Life Technologies, Inc.) After sedimentation, Leydig
cells contained in the supernatant were purified on a Percoll gradient
(19), yielding a preparation containing more than 90%
Leydig cells, as judged by
5-3ß-hydroxysteroid dehydrogenase
staining.
In the experiments designed to investigate the hormonal regulation of mKlk21 expression in vitro, immature Leydig cells were isolated without Percoll gradient purification from 2-wk-old mice. Decapsulated testes were incubated for 15 min at 37 C in DMEM/Ham’s F-12 (Life Technologies, Inc.) containing 0.25 mg/ml collagenase. The cells were plated into six-well culture dishes. After 1 h, the Leydig cells had attached to the surface of the plate, whereas the contaminating cells (Sertoli and germ cells) remained unattached. At this time, the medium was discarded, and the wells were washed three times to remove contaminating cells. Fresh medium was added, and the cells were cultured at 37 C in humidified incubator in 95% O2 and 5% CO2.
Hormonal stimulation experiments
Immature Leydig cells from mouse testes were prepared as
described above. Twenty-four hours before the experiments, the primary
Leydig cell and TM3 cell cultures were placed in fresh medium. For the
stimulation experiments, various hormones dissolved in 100% ethanol
were added to the culture medium at a final concentration of
10-7 M. Cells stimulated with 100%
ethanol were included as controls. The cells were cultured for 48
h and then were harvested for RT-PCR/Southern blot analyses.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The nucleotide and the deduced amino acid sequence of mKlk21 cDNA are
shown in Fig. 1
. The nucleotide sequence of 921 bp contains a 5'-region
of 75 bp, a putative single open reading frame of 786 bp, and a
3'-untranslated region of 60 bp containing a polyadenylation signal
sequence and a poly(A) tail. The open reading frame encodes a protein
of 261 amino acids, which includes a hydrophobic signal peptide of 17
amino acids and an NH2-terminal activation
peptide of 7 amino acids. The calculated molecular masses of the
proprotein and mature protein are 26.7 and 26.0 kDa, respectively. The
amino acid residues comprising the active site catalytic triad
(His65, Asp120, and
Ser213) are all conserved in mKlk21. The protein
contains a potential N-glycosylation site
(Asn102).
The amino acid sequence of mKlk21 was homologous to those of known mouse Klks. The identity was over 74%. The greatest homology (89% identity) was observed for mKlk27 (13). It was also highly homologous to the amino acid sequences of mKlk24 (88% identity; AB039277), mKlk11 (83% identity) (20), and epidermal growth factor-binding protein A (80% identity) (21).
Expression of mKlk21 mRNA in various mouse tissues
To selectively detect the mKlk21 transcript, an mKlk21-specific
oligonucleotide probe was synthesized as described in Materials
and Methods. The specificity of this probe was tested by Southern
blot analysis using mKlk21, mKlk24, and mKlk27 cDNA. Although the
latter two nucleic acid sequences exhibit the greatest sequence
homology to mKlk21, the probe was confirmed to hybridize only with
mKlk21 cDNA (Fig. 2A). Using this
oligonucleotide probe, specific signals were detected with total RNAs
isolated from adult mouse kidney and testis (Fig. 2B).
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). Taking
the signal intensities of the amplified products up to 26 cycles of
PCR, mKlk21 mRNA level in mouse testis was shown to be less than 1/10th
that in the submaxillary glands.
Expression of mKlk21 mRNA in the testis during postnatal development
was also examined (Fig. 2D). The mKlk21 transcript was detectable at 4
wk after birth and thereafter became more prominent.
Testicular expression of mKlk21 relative to other Klks
We performed RT-PCR using primers corresponding to the conserved
regions of all mouse mKlks. RT-PCR amplified products with a size of
about 230 bp were gel-purified and subcloned into pBluescript (II)
KS+, and the recombinant plasmids were
transformed into E. coli, strain JM109. One hundred clones
were randomly picked for the nucleotide sequence analyses. Based on
these experiments, seven different mKlks were identified. The types and
numbers of identified Klks were as follows: mKlk21, 46; mKlk24, 33;
mKlk27, 11; mKlk8, 5; mKlk6, 2; mKlk22, 2; and mKlk5, 1. These results
indicate that mKlk21 is a dominant mKlk species in the mouse
testis.
Enzymatic properties of mKlk21
Under reducing and nonreducing conditions, SDS-PAGE analysis of
the active enzyme sample eluted from the SBTI-Sepharose 4B column gave
a single polypeptide band (Fig. 3).
Apparent molecular masses were estimated to be 30 kDa (reducing) and 24
kDa (nonreducing). In our recent study (13) a similar
molecular mass difference was observed by SDS-PAGE analysis of mKlk27
under these two conditions.
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). Pro-Phe-Arg-MCA was rapidly
hydrolyzed by the enzyme. The enzyme also significantly hydrolyzed
Z-Phe-Arg-MCA. Some synthetic tripeptide substrates containing arginine
at the P1 position were only slightly cleaved by mKlk21. The enzyme
showed no activity in the presence of Bz-Arg-MCA, Lys-containing
substrate (Boc-Val-Leu-Lys-MCA), or chymotrypsin substrates
(Suc-Ala-Ala-Pro-Phe-MCA and Suc-Leu-Leu-Val-Tyr-MCA). The same result
was obtained when a different batch of mKlk21 enzyme preparation was
used.
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-p-tosyl-L-lysine
chloromethyl ketone (0.1 mM),
N-p-tosyl-L-phenylalanine
chloromethyl ketone (0.1 mM), chymostatin (0.01
mM), or pepstatin (0.01
mM). Assays with two different batches of enzyme
preparation gave the same results. The results described above indicate
that mKlk21 is a serine peptidase with trypsin-like substrate
specificity.
Activity of mKlk21 in the presence of protein substrates
The hydrolysis of denatured casein and gelatin with mKlk21 was
demonstrated by zymographic analysis (Fig. 4A). Fibronectin was also degraded with
the enzyme, producing four detectable bands, corresponding to 210, 190,
170, and 75 kDa (Fig. 4B).
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). The human IGFBP-3 preparation used in
this experiment was a glycosylated form (95%). Therefore,
SDS-PAGE/Western blot analysis apparently gave a single band at a
position of 41 kDa for intact IGFBP-3. mKlk21 degraded the intact
protein in a time-dependent manner (Fig. 5A) as well as in a
dose-dependent manner (Fig. 5B), with concomitant production of the
31-kDa fragment. IGFBP-3 degradation by mKlk21 was examined as a
function of pH (Fig. 5C). The band intensities of the intact 41-kDa
polypeptide were apparently weak at pH 7–9. On the other hand, the
31-kDa degraded form of IGFBP-3 was visualized at pH values in the
range 4–10, with no detectable band at pH 8–9. These results indicate
that mKlk21 optimally degrades IGFBP-3 at pH 8–9. Disappearance of the
31-kDa polypeptide at these pHs is perhaps due to its further
degradation by the enzyme. IGFBP-3 hydrolysis with mKlk21
was inhibited by serine protease inhibitors, such as leupeptin, DFP,
PMSF, and SBTI, but not by chymostatin or aprotinin (Fig. 5D). We also
compared the action of mKlk21 on IGFBP-3 with that of mKlk27, which has
recently been demonstrated to hydrolyze the protein (13).
In a preliminary experiment, it was found that IGFBP-3 was degraded by
mKlk27 much faster than by mKlk21; specifically, when incubation was
conducted at the same substrate/enzyme ratio as in Fig. 5A, mKlk27
completely degraded the intact IGFBP-3 in 30 min. Figure 5E shows the
result of IGFBP-3 degradation by mKlk27 under reaction conditions in
which the enzyme was reduced to 1/10th. The size of the product was
estimated to be 29 kDa, which was slightly smaller than that produced
by mKlk21. The above results strongly suggest that mKlk21 and mKlk27
are both active against the naturally occurring glycosylated form of
IGFBP-3.
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In situ detection of mKlk21 mRNA in the adult mouse testis
Tissue localization of mKlk21 mRNA in the mouse testis was
analyzed by in situ hybridization using a DIG-labeled mKlk21
antisense RNA probe. The expression of mKlk21 RNA was specifically
localized in the interstitial tissues and was not detected in the
seminiferous tubules (Fig. 6A). To
identify positive cells, further examinations were conducted by
staining the neighboring sections with hematoxylin-eosin. Based on the
morphological observation that positive cells had eosinophilic
cytoplasm (data not shown), we assumed that cells expressing mKlk21
mRNA were Leydig cells situated between the seminiferous tubules. Use
of the sense probe produced no significant signals (Fig. 6B).
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). During maturation, the abundance of
the 7-kb transcript drastically decreased, whereas the expression of
the 1.8- and 0.7-kb species apparently remained unchanged. The relative
levels of these IGF-I transcripts in the testis were similar to those
observed in the liver of the adult mouse (22). In
situ detection of IGF-I mRNA using sections of adult mouse testis
showed that positive signals were localized at the seminiferous
epithelium and Leydig cells in almost equal intensities (data not
shown). Expression of IGF-I mRNA in the seminiferous epithelium was
previously reported by Baker et al. (23).
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). However,
Leydig cells isolated from 8-wk-old mice were likely to express more
abundant IGFBP-3 transcripts than immature Leydig cells, as
demonstrated by RT-PCR analysis (Fig. 7C). To examine the
intratesticular localization of IGFBP-3 mRNA, an in situ
detection analysis was conducted using slices of adult mouse testis. As
shown in Fig. 8A, the IGFBP-3 transcript
was detected in limited areas of both interstitial tissues and
seminiferous tubules. Concerning IGFBP-3 expression associated with the
interstitial tissues, part of the Leydig cells appeared to express
IGFBP-3 mRNA at detectable levels. No significant signals were detected
when the sense probe was used. To determine where IGFBP-3 is localized
in the testis, the same IGFBP-3 antibody used in the Western blot
analysis was used to stain mouse testis sections collected from adult
male mice. Immunostaining for IGFBP-3 was observed in interstitial
tissues as well as in seminiferous tubules (Fig. 8B).
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Effects of various hormones on mKlk21 gene expression
To determine the factors controlling testicular mKlk21 expression,
the effects of various hormones were examined using immature Leydig
cells isolated from 2-wk-old mouse testes. As the expression level of
mKlk21 mRNA in the cells at this stage was very low, we investigated
its expression by the RT-PCR/Southern blot analysis method. When
immature Leydig cells were treated with T for 48 h, the intensity
of the mKlk21 PCR fragment was remarkably enhanced (Fig. 9, upper panel). Treatment of
the cells with E2 and progesterone caused no significant change.
Inclusion of T4 in the culture strongly reduced
the expression of the mKlk21 gene. In contrast to the primary immature
Leydig cells, the mouse testicular Leydig cell line TM3 prominently
responded to E2 treatment (Fig. 9, middle panel). Moreover,
T significantly increased the level of mKlk21 gene expression. Images
of amplified ß-actin confirmed the integrity and quantity of the RNA
(Fig. 9, lower panel). These results clearly indicate that
mKlk21 expression is under control of the male sex hormone T in Leydig
cells isolated from immature mouse testes.
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, incubation of IGFBP-3 with the
medium isolated from a 2-wk-old immature mouse testis produced no
degraded form of the binding protein. On the other hand, medium from
the Leydig cell culture of an 8-wk-old mouse testis produced the 31-kDa
IGFBP-3 fragment. The production of this fragment was completely
inhibited by the serine protease inhibitor PMSF. Leupeptin also
inhibited the IGFBP-3-degrading activity of the medium, but chymostatin
had no effect. These results indicate that mature mouse testis Leydig
cells secrete a leupeptin-sensitive, chymostatin-insensitive serine
protease capable of degrading a glycosylated form of IGFBP-3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mKlk21 was shown to be a serine protease with trypsin-like cleavage
specificity. Examination of the enzyme activity with a variety of
synthetic peptide substrates demonstrated that mKlk21 specifically
hydrolyzes internal peptide bonds at the carboxyl side of arginine, but
not lysine residues. Trypsin-like specificity of mKlk21 was expected
due to the fact that the amino acid residue at the position 6 residues
to the NH2-terminus from the active site serine
is aspartic acid (Asp207 in Fig. 1); Asp is
thought to be responsible for directing cleavage after basic amino
acids (24). However, the present data clearly indicate
that the active site of mKlk21 functions such that only arginine at the
P1 position of various substrates would be available for hydrolysis. In
support of this idea, the serine protease inhibitor
N-p-tosyl-L-lysine
chloromethyl ketone was not observed to affect mKlk21 activity, whereas
leupeptin and antipain were potent inhibitors of the enzyme.
In situ hybridization analysis using a 472-bp antisense probe complementary to nucleotides 251–722 of mKlk21 in the testis detected strong signals in association with the Leydig cells. The results were consistent with an observation by Penschow et al. (14). It should be noted that the mKlk21 472-bp nucleotide probe is highly homologous to the corresponding parts of two other mKlks, namely, mKlk24 and mKlk27. These latter two Klks are also expressed simultaneously in the same cells (13). Therefore, one may suspect that Leydig cell-associated signals detected in the current in situ hybridization analysis exhibit a combined expression of mKlk mRNAs, including the mKlk21 transcript itself. We tentatively think that our in situ hybridization signals probably represent the mRNA localization of mKlk21.
In a systematic study of the expression of 12 mouse Klk genes using various tissues from 22-d-old mice, only mKlk21 mRNA was detected in the testis (14). On the other hand, RT-PCR analysis identified 7 different mKlk transcripts in the adult mouse testis. These data together with the present finding that mKlk21 mRNA is expressed most abundantly in the adult testis indicate that mKlk21 remains after puberty in the testis as a dominant type of mKlk.
Our present observation strongly suggests that mKlk21 is synthesized in and secreted from these cells into the extracellular environment, and that the physiological substrate(s) of mKlk21 might be present in the testicular interstitial tissue surrounding Leydig cells. Fibronectin, the extracellular adhesion protein that contributes to organizing the extracellular matrix and helps cells attach to it, is degraded by mKlk21 in vitro. Thus, mKlk21 may be involved in the in vivo turnover of fibronectin. A recent immunohistochemical study by Loveland et al. (25) demonstrated the presence of fibronectin in the seminiferous tubule basement membrane and interstitial matrix of the rat testis throughout development. In the adult testis this adhesion protein was found abundantly and uniformly in the interstitial spaces where Leydig cells are localized. These observations suggest that mKlk21 secreted from Leydig cells readily comes into contact with fibronectin. We tentatively assume that by controlling fibronectin turnover, mKlk21 may play a role in the extracellular matrix remodeling of testicular interstitial tissues.
IGF-I is a polypeptide mitogen that mediates the effects of GH. There is increasing evidence that IGF-I is involved in the autocrine and paracrine regulation of testicular function; IGF-I enhances the differentiated functions of Leydig cells (26). More recently, Baker et al. (23) reported that male mice lacking the IGF-I gene show drastically reduced levels of serum T and infertility. The authors of that study concluded that this hormonal deficiency is due to a significant delay in the differentiation of mutant mouse testis Leydig cells. The activity of IGF-I is known to be modified by binding proteins (27). It has also been reported that both IGF-I and its binding protein (IGFBP-2, -3, and -4) mRNAs are expressed in the Leydig cells of the rat testis and furthermore that IGFBP-3 is a potent inhibitor of IGF-I-induced T formation (28). The present study demonstrates that the mouse testis, like the rat testis, expresses IGF-I and IGFBP-3 genes at detectable levels throughout postnatal development. With respect to the latter gene, the protein was immunohistochemically localized at the interstitial tissues of the testis. These findings indicate that mKlk21, the gene expression of which starts a few weeks after birth, could interact with IGFBP-3 or IGFBP-3 complexed with IGF-I in the vicinity of Leydig cells of the adult mouse testis. Our observation that mKlk21 degrades IGFBP-3 tempts us to speculate that mKlk21 is involved in the maintenance of adult mouse testis Leydig cell function; that is, the mechanism of its actions would include prevention of IGF-I/IGFBP-3 complex formation. Consistent with this idea is the present finding that the culture medium of Leydig cells isolated from adult mouse testes degraded IGFBP-3. The nature of proteases responsible for the binding protein degradation remains to be determined, but it could be mKlk21 for the following two reasons. 1) The relative molecular mass of the major product generated by the putative enzyme was 31 kDa, exactly the same size observed with the recombinant mKlk21. 2) Inhibitor profiles of IGFBP-3 degradation were essentially the same between recombinant mKlk21 and the culture medium. It will be of future interest to determine whether mKlk21 is able to degrade IGFBP-3 bound to IGF-I. In the present study we failed to detect IGFBP-3 in the culture medium of adult mouse testicular Leydig cells by means of Western blot analysis. We presume that the current method was not sensitive enough to detect the binding protein.
We recently reported that mKlk27 also degrades IGFBP-3 (13). Under the hydrolysis conditions of a fixed substrate/enzyme ratio (0.25), the degradation of IGFBP-3 by mKlk27 took place faster than that catalyzed by mKlk21. Based on the disappearance rates of intact 41-kDa IGFBP-3 polypeptide bands as a function of incubation time, the binding protein was determined to be degraded by mKlk27 approximately 10 times more efficiently than by mKlk21. However, this does not indicate that mKlk21 is less important in the process of IGFBP-3 degradation than mKlk27. It should be stressed that mKlk21 expression is apparently much greater than mKlk27 expression in the mouse testis.
A number of studies have previously documented that conditioned media
of several cell types, including human fibroblasts
(29, 30, 31), bone cells (32), breast cancer
cells (33), and rat neuroblastoma cells (34),
and biological fluids such as human seminal plasma (35)
and ovine ovarian follicular fluids (36) contain proteases
capable of degrading IGFBPs. With respect to IGFBP-3 proteolysis,
cathepsin D was involved in the case of human fibroblasts, osteoblastic
cells, and breast cancer cells (31); PSA in human seminal
plasma (35, 37); and mKlk27 in mouse testis Leydig cells
(13). Interestingly, PSA and mKlk27 are both
chymotrypsin-like serine proteases belonging to a Klk subfamily. In
contrast, mKlk21 is a trypsin-like serine protease. As nerve growth
factor-
, the mouse Klk member (mKlk3) characterized as a
trypsin-like serine protease, has recently been shown to cleave IGFBP-3
(38), mKlk21 is a second example of IGFBP-3 proteases
having trypsin-like specificity. Considering that no significant
expression of mKlk3 in the mouse testis has been detected, the
contribution of mKlk21 may be solely important in this organ. However,
as revealed by testicular gene expression analysis during postnatal
development, IGFBP-3 expression is greatest when expression of mKlk21
is very low, and expression of mKlk21 is greatest when that of
IGFBP-3 is at its lowest. This fact could argue against the
physiological significance of interactions between mKlk21 and IGFBP-3.
Further investigations are definitely required to establish the
relevance of interactions between these two molecules to the
physiological function of the testis.
Previous studies of hormonal regulation of Klk gene expression and/or enzyme activity in salivary glands point to the importance of androgen and thyroid hormones (4, 5, 14). In this study we also observed that mKlk21 mRNA levels in Leydig cells isolated from immature mouse testes were significantly enhanced by T. However, T4 treatment of the cells drastically reduced the expression of this gene, suggesting that this thyroid hormone exerts different effects on gene expression in mouse salivary glands and testicular Leydig cells. It is interesting to note that the commercially available Leydig cell line TM3 exhibited an mKlk21 expression pattern apparently different from that of the primary cultured Leydig cells. Unlike the isolated Leydig cells, ß-E2, but not T, was demonstrated to be the most effective inducer of mKlk21 expression, whereas T4 was without effect in TM3 cells. We observed that mKlk21 mRNA expression patterns in response to hormones vary with the cells used. Such findings indicate that a discussion of gene expression in vivo must proceed with caution. At the present moment, we presume that the mKlk21 expression pattern exhibited by Leydig cells isolated from immature mouse testes in response to various hormones may represent a pattern similar to that of in vivo expression.
In summary, we have characterized the ability of recombinant mKlk21 to degrade fibronectin and IGFBP-3 in vitro. The temporal and spatial expression studies of mKlk21 using mouse testes indicate that mKlk21 may play a role in the testicular Leydig cell function of adult mice. We tentatively suggest that mKlk21 is involved in the degradation of fibronectin and/or IGFBP-3 in the interstitial tissue surrounding the Leydig cells in the mouse testis. Future work directed at elucidating the biological role of this functional mKlk in the testis depends upon current findings with regard to its biochemical and enzymatic properties.
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Acknowledgments |
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Footnotes |
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Abbreviations: DFP, Diisopropylfluorophosphate; DIG, digoxigenin; IGFBP-3, IGF-binding protein-3; Klks, kallikreins; MCA, 4-methylcoumaryl-7-amide; mKlk, mouse Klk; PMSF, phenylmethylsulfonylfluoride; poly(A)+, polyadenylated; PSA, prostate-specific antigen; SBTI, soybean trypsin inhibitor; SSPE, 0.15 M NaCl/8.65 mM NaH2PO4/1.25 mM EDTA.
Received January 29, 2001.
Accepted for publication July 25, 2001.
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