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from the Rat Testis1
Department of Woman and Child Health, Pediatric Endocrinology Unit, and Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institute and Hospital, S-17176 Stockholm, Sweden; and Endocrinological Genetics Unit, Institute of Cytology and Genetics, 630090 Novosibirsk, Russia
Address all correspondence and requests for reprints to: Olle Söder, M.D., Ph.D., Department of Woman and Child Health, Pediatric Endocrinology Unit, Astrid Lindgren Children’s Hospital, S-17176 Stockholm, Sweden. E-mail: olle.soder{at}kbh.ki.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(proIL-1), constitutively expressed by the
normal adult rat testis. In addition to the classical 32K proIL-1
(32proIL-1) messenger RNA, the testis produced a shorter variant
encoding a putative protein of 24K (24proIL-1). In
situ hybridization demonstrated constitutive expression of the
splice transcript in the seminiferous tubules. This alternative
complementary DNA lacked the fifth exon, harboring the calpain cleavage
site essential for generation of mature 17K IL-1. This was verified
by calpain treatment, producing the expected cleavage products of
recombinant 32proIL-1, but not of 24proIL-1. Similarly,
expression in COS-7 cells demonstrated processing of 32proIL-1 to
the mature 17K form and secretion, whereas 24proIL-1 remained
unprocessed. Both 32proIL-1 and 24proIL-1 showed a dose-dependent
stimulatory effect in a thymocyte proliferation assay, although at
lower potency than mature 17K IL-1. In contrast, when tested on
hCG-stimulated Leydig cells in vitro, a dose-dependent
inhibition of testosterone production was obtained with mature 17K
IL-1 and at a lower potency with 32proIL-1, whereas 24proIL-1
was inactive. In conclusion, the three IL-1 bioactive proteins
described here contribute to IL-1 protein heterogeneity and may serve
as constitutive paracrine mediators in the testis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IL-1), first
discovered as a potent proinflammatory cytokine (1), is a
multifunctional polypeptide acting as a systemic and paracrine
messenger in the endocrine and central nervous systems (2, 3) and is also active as growth factor for several cell types,
such as male germ cells (4). IL-1 may also be involved
in oncogenesis by serving as an autocrine growth regulator of several
malignant cell types (5, 6).
The proIL-1 gene contains seven exons and six introns, of which
exons 2–6 and part of exon 7 encode a 32K IL-1 precursor protein
(32proIL-1) (7, 8). In activated macrophages, the
32proIL-1 is cleaved by calpain into the C-terminal mature 17K
IL-1 protein, and an N-terminal 16K product (9, 10).
This 16K product has recently attracted attention, because it may act
as an oncogene via a nuclear targeting pathway (11). Both
the 32proIL-1 and the mature 17K form of IL-1 are biologically
active as exogenous cytokines (12).
In contrast to the activated production in macrophages, IL-1 is
constitutively produced under noninflammatory conditions in multiple
tissues such as liver, skin, esophagus, proventricular stomach, and
testis (13, 14). Testicular IL-1 expression is confined
to Sertoli cells and is developmentally and stage dependently regulated
(15, 16, 17). Bioactive IL-1 has been recently isolated and
characterized from adult rat testis (18, 19, 20), where it was
found to consist of several distinct protein species with a size range
of 17–40K and charge heterogeneity. The molecular background of this
IL-1 protein heterogeneity in the testis is not clear. In our RT-PCR
analysis of coding DNA sequence (cds) for testicular IL-1 messenger
RNA (mRNA), we repeatedly found an additional smaller transcript, apart
from the expected proIL-1 mRNA. We here report characterization of
the corresponding protein for this alternative IL-1 transcript.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]Deoxy-CTP,
[3H]thymidine, and complementary DNA (cDNA)
synthesis kits were purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). pthioHisB and
pcDNA3.1+ plasmids were obtained from
Invitrogen (Groningen, The Netherlands). pPCRscript was
purchased from Stratagene. The ligation kit was obtained
from Promega Corp. (Madison, WI), and size markers were
obtained from Life Technologies, Inc. (Gaithersburg, MD).
All other reagents used were of analytical grade.
Animals
Adult Sprague Dawley rats and a locally bred highly
IL-1-responsive mouse strain (NMRI/KI) (21) were purchased
from B&K Laboratories (Sollentuna, Sweden). Animal experiments were
approved by the Northern Stockholm animal ethics committee.
Cloning of IL-1 cDNA
Total RNA was extracted from testis and activated macrophages
(17) from adult Sprague Dawley rats by the
guanidinium/phenol method (22). Two micrograms of total
RNA were reverse transcribed to cDNA using the first strand cDNA
synthesis kit (Pharmacia Biotech), following the
manufacturer’s instructions. PCR of 100 µg cDNA was performed on a
Perkin-Elmer Corp./Cetus DNA Thermal Cycler 9600 (Palo
Alto, CA) in a reaction volume of 50 µl in the presence of 200
nM primers, 0.2 mM deoxy-NTP, 1.5
mM MgCl2, and 2.5 U Pfu
DNA polymerase (Stratagene). The upstream primer
(5'-CGCTTGAGTCGGCAAAGAAATC-3') corresponded to positions 37–59, and
the downstream primer (5'-CACATGCCATGCGAGTGATTAG-3') was
complementary to bases at position 955–978 in the published sequence
(GenBank accession no. D00403) (23). The cycling
conditions were 95 C for 4 min, and then 38 cycles of 95 C for 35 sec,
56 C for 35 sec, 72 C for 3 min, and a final extension at 72 C for 7
min. The PCR products were tailed with Taq polymerase and
cloned into pGEM-T Easy Vector according to the manufacturer’s
description (Promega Corp.). The clones were verified by
sequencing, using BigDye Terminator Ready Reaction Kit
(Perkin-Elmer Corp./Cetus).
Expression analysis by in situ hybridization
In situ hybridization was performed as previously
described (17). To detect the splice variant, a specific
44-mer oligonucleotide probe complementary to the splicing region was
synthesized, which consisted of sequences in genomic DNA corresponding
to part of exons 4 and 6, complementary to nucleotides 327–346 and
521–544 of the two exons, respectively. A probe that detects both
versions of IL-1 in testis (17) was also used for
comparison as well as a random probe with the same GC contents as
negative control. The hybridization and washing conditions were highly
stringent to avoid annealing of the probe with only one of either
exons. Briefly the probes were radiolabeled with
[35S]deoxyadenosine
5'-[-thio]-triphosphate (NEN Life Science Products)
at the 3'-end, as previously described. The 14-µm-thick tissue
sections were hybridized for 16–18 h at 45 C and rinsed five times for
15 min each time in 1 x SSC (standard saline citrate) at 63 C,
dehydrated in ethanol, and dipped in photographic emulsion (NTB-2,
Kodak, Stockholm, Sweden). The sections were developed and
examined under darkfield and phase contrast microscopy.
Expression and isolation of recombinant protein from Escherichia
coli
The coding DNA for both 32proIL-1 and the alternative splice
variant (24proIL-1) were amplified from their pGEM-T clones using an
internal set of primers. The upstream primer corresponds to position 62
of the published rat IL-1 cDNA sequence
(5'-ATGGCCAAAGTTCCTGACTTG-3'), and the downstream primer starts from
position 893 (5'-TGTCAGCACTTCTCAAGAAAGTAG-3'). The amplification was
performed using the reaction conditions outlined above, with 100 pg
plasmid.
The resulting PCR fragment of 32proIL-1 was inserted into the
NcoI site of the pthioHisB plasmid and filled up with Klenow
polymerase. The PCR-amplified fragment of 24proIL-1 was inserted
into the Asp718 site of the pthioHisB and
pretreated with mung bean nuclease1 following the manufacturer’s
instructions. To improve the affinity of the expressed fusion protein
to nickel affinity chromatography column, a histidine tag was inserted
into the thioredoxin part.
E. coli (strain BL21, Stratagene) were
transformed with the recombinant plasmid harboring 32proIL-1 or
24proIL-1. Bacteria transformed with intact pthioHisB plasmid served
as controls for the purification steps and the functional studies. Five
milliliters of overnight cultures were inoculated into fresh 100 ml
Luria Bertoni medium and grown until the cells reached an
OD600 of 0.6–1.0. Bacteria were induced with 1
mM
isopropyl-beta-D-thiogalaetopyranoside at 37 C, for
2 h, and then collected and lysed using 0.01
M Tris-HCl buffer, pH 8, containing 0.5
M NaCl, 8 M urea, and 0.1%
Triton X-100 at room temperature. Clear lysates were obtained after
centrifugation at 12,000 x g. The lysates were loaded
onto a Ni-NTA agarose column (QIAGEN, via KEBO, Stockholm,
Sweden). Proteins were eluted as a function of pH, following the
manufacturer’s instruction. The presence of expressed proteins was
then checked in cell lysates and purified fractions by PAGE/Western
analysis as outlined below.
Calpain cleavage of proIL-1
Cell lysates of the bacteria expressing recombinant 32
proIL-1 and 24 proIL-1 proteins were prepared in a nondenaturing
buffer (50 mM Tris-HCl, pH 7.9, containing 0.1% Triton
X-100 and 600 mM NaCl). Clear supernatants were obtained
after centrifugation at 12,000 x g, and were used
directly for the cleavage reaction. Lysates were incubated in a
reaction mixture containing 10 mM
ß-mercaptoethanol, 5 mM
CaCl2, and 1.5 U calpain II enzyme for 0.5–1 h
at 37 C. An appropriate control was also included. The samples obtained
were analyzed by PAGE/Western analysis.
Expression of proIL-1 in monkey COS-7 cells
The coding sequences of both forms of proIL-1 were
reamplified from the pGEM-T clones with addition of a ribosome binding
site in the 5'-primer and subcloned into the pPCRscript, following the
manufacturer’s instructions. The inserts were excised from the
pPCRscript as a BamHI/NotI fragment and ligated
into pcDNA3.1, digested with BamHI/NotI.
Subconfluent monkey COS-7 cells (ECACC, European cell culture
collection) were transfected with pcDNA constructs of both forms of
proIL-1, using FuGENE 6 (Roche Molecular Biochemicals).
After 24 h of culture, the incubation medium was replaced with
serum-free medium and incubated for an additional 24 h. For
Western blot analysis, the cells were lysed in SDS loading buffer, and
secreted proteins were acetone precipitated from the culture medium and
resuspended in SDS loading buffer.
PAGE/Western analysis
Isolated protein and extracts from E. coli and COS-7
cells in SDS loading buffer were applied to a 12% (wt/vol)
polyacrylamide gel together with kaleidoscope size markers
(Bio-Rad Laboratories, Inc.). The gel was run at 100 V for
1.5 h in a Mini Protean II electrophoresis apparatus
(Bio-Rad Laboratories, Inc.). The proteins were then
transferred to a nitrocellulose membrane at 80 V for 45 min. After
blocking with 5% milk in Tris-buffered saline and 0.1% Tween-20 for
1 h, the membranes were incubated with goat antirat IL-1
C-terminal antibodies (Santa Cruz Biotechnology, Inc., via
Scandinavian Diagnostics, Stockholm, Sweden), diluted 1:5000 in
blocking buffer, for 16 h and finally with antigoat antibodies
conjugated with horseradish peroxidase (1:2000 in blocking buffer). The
ECL (Amersham Pharmacia Biotech) system was used for
detection, and chemiluminescence was monitored by a CCD camera (LAS
1000, Fuji Photo Film Co., Ltd.).
IL-1 bioassay
The IL-1 bioactivity of the purified recombinant 32proIL-1
and 24proIL-1 was determined by a murine thymocyte proliferation
assay (19), using a highly sensitive, IL-1-responsive,
locally bred mouse stain (NMRI/KI) (21). Rat recombinant
17K IL-1 (R&D Systems via Novakemi AB, Stockholm, Sweden) and a
crude rat testicular extract (13) were used as standards.
Thioredoxin fusion protein was used as a vector control.
Leydig cells steroidogenic assay
The assay was carried out following a previously reported method
(24). Forty-day-old Sprague Dawley rats were used as
testis donors, as the testis at this age contains a mixture of mature
and immature Leydig cells. When isolated from 80-day-old rats, we noted
a 10% higher response of the Leydig cells to hCG, but the effects of
the IL-1 isoforms were the same as those with 40-day-old Leydig cells.
Testes were decapsulated and incubated with collagenase (0.25 mg/ml)
for 20 min at 37 C to produce crude interstitial cells. Three
milliliters of the cell suspension were loaded on the top of a
discontinuous Percoll gradient, composed of cushions of 20%, 40%,
60%, and 90% Percoll, and centrifuged at 800 x g for
20 min. Fractions containing Leydig cells were collected and washed
with DMEM/Ham’s F-12 medium with 0.1% BSA. To further purify Leydig
cells, the recovered cells were centrifuged through a 60% Percoll
gradient at 20,000 x g for 30 min at 4 C. Fractions
with a density greater than 1.068 g/ml (purified Leydig cells) were
collected. 3ß-Hydroxysteroid dehydrogenase staining revealed
approximately 80% positively stained cells (25). The
purified Leydig cells (2 x 105 cells/ml)
were cultured in 96-well plates (Falcon) and incubated with or without
recombinant IL-1 isoforms (0.01–100 ng/ml) for 24 h. The culture
medium was replaced with fresh medium containing the IL-1 isoforms and
hCG (10 ng/ml) and incubated for another 24 h. Culture media were
collected and stored at -20 C until assayed for testosterone with a
Coat-a-Count RIA kit (Diagnostic Products, Los Angeles,
CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA from rat testis and
macrophages repeatedly gave two PCR products of similar intensity, one
of the expected size (941 bp) and a shorter product (767 bp; Fig. 1
). In situ hybridization with
a probe specific for the smaller transcript revealed a distinct
expression in the periphery of the seminiferous tubules (Fig. 2, a and b), with the same pattern
as previously reported for the full-length transcript. The negative
control with a random probe lacked specific signal (Fig. 2, c and
d).
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cDNA. The cDNA sequence of the shorter
version lacked an internal DNA fragment with a size of 174 bp,
corresponding to nucleotides 322–469 of the 32proIL-1 cds and
comprising exon 5 of the human and mouse gene (Fig. 3a). Analysis of reading frames predicted
a corresponding putative 24K protein lacking 58 amino acids compared
with 32proIL-1 (Fig. 3b).
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. The
expressed 32proIL-1 and 24proIL-1 obtained after purification
through a nickel nitrilotriacetic acid column showed the
apparent molecular mass of fusion proteins as 48 kDa (16K thioredoxin
and 32K proIL-1) for 32proIL-1 and 40 kDa (16K thioredoxin and
24K proIL-1) for 24proIL-1 when assessed by PAGE/Western analysis
(Fig. 4a). In vitro calpain
cleavage of 2K proIL-1 released a 17K C-terminal protein detected by
PAGE/Western analysis using a C- terminal antiserum, whereas the
24K proIL-1 was resistant to calpain digestion (Fig. 4b).
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, we
compared the expression patterns of 32proIL-1 and 24proIL-1 in
transfected monkey COS-7 cells. PAGE/Western analysis confirmed the
production of the expected 24K and 32K proteins (Fig. 4c
) and showed
that 32proIL-1 was processed and secreted into the medium as 17K
IL-1 from the transfected cells, whereas 24proIL-1 was
unprocessed and was retained intracellularly (Fig. 4c). Further
evidence of this fact was provided by the observation that 24proIL-1
was exclusively localized to the nucleus in transfected COS-7 cells, as
revealed by PAGE/Western analysis of nuclear and cytoplasmic fractions
(not shown).
To investigate whether the expressed proteins were biologically active,
a highly IL-1-sensitive murine thymocyte proliferation assay was
employed. 32K proIL-1 showed a dose-dependent stimulatory effect in
the assay, although the potency was 30 times lower than that of mature
17K IL-1. 24proIL-1 also showed dose-dependent stimulatory
activity, but the potency was 800-fold less than that of mature 17K
IL-1, and thus more than 25-fold lower than that of 32proIL-1
(Fig. 5). Interestingly, mature rat 17K
IL-1ß also showed lower potency (
20 fold) in the presently used
bioassay compared with rat 17K IL-1. To determine whether the
recombinant rat IL-1 proforms have testis-specific function(s), their
effects on rat Leydig cell steroidogenesis in vitro were
examined. 32proIL-1 showed an inhibitory effect on testosterone
production by cultured Leydig cells, although with 200-fold lower
potency than mature 17K IL-1. In contrast, 24proIL-1 had no
effect (Fig. 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, is the result of
differential splicing of the primary transcript of 32proIL-1. The
transcript was expressed and localized in the seminiferous tubules with
a pattern indistinguishable from that of the full-length transcript
(17). Our previous results demonstrated a distinct
localization of 32proIL-1 mRNA in Sertoli cells in all stages except
stage VII (17). We have not performed a precise cellular
localization of 24proIL-1 transcript, which requires in
situ hybridization and cytological identification of squash
preparation from staged segments of seminiferous tubules
(17). However, the expression pattern shown here strongly
favors a Sertoli cell origin. Furthermore, as discussed below, this
conclusion is also obvious from the fact that the presence of the
24proIL-1 transcript is a consequence of processing of the classical
32proIL-1 mRNA.
From the sequence data the molecular charge of 24proIL-1 was
calculated to pI 5.2. We also confirmed the sequence identity of
32proIL-1 from the rat testis to the classical proIL-1 from
activated rat macrophages. The calculated molecular size and charge of
the precursor protein were 31K and pI 5.5, respectively. The calculated
pI of the rat mature 17K IL-1 was 5.6, corresponding well to our
previous finding with purified testicular 17K IL-1 protein
(18). Taken together, the results give an explanation for
the observed size and charge heterogeneity of bioactive IL-1 proteins
isolated from the rat testis.
Sequence analysis of 24proIL-1 transcript revealed that it lacked an
internal sequence corresponding to exon 5 in the human gene,
constituting the N-terminal part of secreted mature 17K IL-1 and
harboring the calpain cleavage site of 32proIL-1. Failure of calpain
to cleave 24proIL-1 in vitro and lack of processing in
COS-7 cells verified these observations. Our findings are further
supported by recent studies showing the presence of a proIL-1
differential splice transcript in other animal species such as dogs,
cats, and pigs. These studies showed up-regulation of both long and
short proIL-1 transcripts in response to infection
(26). However, the present study is the first to show that
the alternative splice product can be translated into a functional
protein.
Recently, the N-terminal 16K fragment of 32proIL-1 has attracted
attention as a potential oncogenic transforming factor. It is
interesting to note that the 24proIL-1 retains this N-terminal
propiece, which has a nuclear localizing sequence and potential
cell-transforming activity (11), which is normally absent
in mature 17K IL-1. It is nonfunctional when expressed in the
context of 32proIL-1, but can act as potential oncogene once cleaved
to the N-terminal 16K form (11). The cleavage, in turn,
depends on the level of the processing enzyme, calpain, the activity of
which is regulated by phosphorylation, and a specific inhibitor,
calpstatin. The splice variant lacks the cleavage site for calpain,
which liberates it from regulatory control of the cleavage enzyme. It
remains to be determined, however, whether 24proIL-1, in which this
16K protein part cannot be released by calpain, retains any of these
oncogenic functions. Its predominant localization to the nucleus
suggests involvement in the regulation of such cellular functions.
Previous data have shown inhibitory effects of 17K IL-1 on Leydig
cell steroidogenesis (27). The present study confirms
these results and further shows that rat 32proIL-1, but not
24proIL-1, has a similar function. The functional difference between
the two forms of proIL-1 in the Leydig cell assay indicates
alternative functions of the splice variant that need further
exploration.
24proIL-1 displayed bioactivity in the thymocyte proliferation
assay, indicating a receptor-mediated action on thymocytes. This
finding is not surprising, as 24proIL-1 shares C-terminal
receptor-binding sites with 32proIL-1 and 17K IL-1.
Interestingly, the potency of rat 17K IL-1 in the thymocyte
proliferation assay was 1 order of magnitude greater than that of rat
IL-1ß. This is in contrast to most other studies with human and mouse
IL-1, where IL-1ß has shown to be more potent. Further, our results
confirmed previous findings with human 32proIL-1 (12),
demonstrating that the corresponding rat 32K IL-1 precursor protein
is also bioactive, although with much lower potency than mature 17K
IL-1.
Of the two types of IL-1 receptors described, only type I has been
reported to be signaling (28, 29). Whether 24proIL-1
binds to both the type I and type II receptors is presently unknown,
but would be interesting to explore. Little is known about the presence
of IL-1 receptors in the testis, although some reports have indicated
expression in testicular somatic cells (30). Therefore,
more detailed analysis of IL-1 receptor expression and action of the
different IL-1 proteins are needed to more precisely define the exact
role of IL-1 in testicular physiology. From the present data,
however, it is possible to conclude that all three IL-1-related
proteins described here may have paracrine functions in the testis.
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Acknowledgments |
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Footnotes |
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and
24proIL-1 have been deposited in the EMBL database with accession
numbers AJ245642 and AJ245643, respectively. This work was supported by
the Swedish Medical Research Council (Projects 8282 and 11412), the
Children Cancer Fund, the Swedish Cancer Foundation, Frimurare
Barnhuset in Stockholm, H.R.H. Crown Princess Lovisa Society of
Pediatric Health Care, the Society for Child Care, and the Karolinska
Institute. 
Received June 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation of spermatogonial proliferation
in vivo. Reprod Fertil Dev 1:85–87[CrossRef][Medline]
. Nucleic Acids Res 14:3167–3179. Proc
Natl Acad Sci USA 87:5548–5552 is a transforming nuclear
oncoprotein. Proc Natl Acad Sci USA 94:508–513 precursor but not the interleukin-1ß
precursor. J Biol Chem 262:2941–2944. Endocrinology 129:1613–1623
messenger ribonucleic acid in rat Sertoli cells is dependent upon
interaction with germ cells. Endocrinology 140:3755–3761 cDNA. J Biochem 105:351–357. Gene 236:273–280[CrossRef][Medline]
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