Division of Biological Sciences (J.O., W.H., D.N., H.M., A.K.,
T.T.) Graduate School of Science Hokkaido University
Sapporo 060-0810, Japan
Laboratory of Molecular and Cellular
Pathology (E.O., M.J., H.S., K.N.) Hokkaido University School
of Medicine CREST, JST (Japan Science and Technology) Sapporo
060-8638, Japan
Institute of Biological Sciences and Gene
Experimental Center (K.N.) Tsukuba University Ibaraki 305-8572,
Japan
Division of Morphogenesis (H.S.) Department of
Developmental Biology National Institute for Basic Biology
Okazaki 444-8585, Japan
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ABSTRACT |
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INTRODUCTION |
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Currently, the number of members of the MMP gene family has been growing, and at present more than 20 genetically distinct human MMPs have already been cloned and characterized. They can be classified into two major subfamilies according to their primary structure, and into six subfamilies according to their substrate specificity (5, 6). Based on their primary structural features, one is the soluble type of MMP, and the other is the transmembrane-type MMP (MT-MMP). Subfamilies based on the substrate specificity, consist of: (I) collagenases: collagenase-1, -2, and -3 (MMP-1, -8, and -13, respectively); (II) gelatinases: gelatinase A and B (MMP-2 and -9, respectively); (III) stromelysins: stromelysin-1 and -2 (MMP-3 and -10, respectively); (IV) stromelysin-like: matrilysin (MMP-7), metalloelastase (MMP-12) and MMP-19, (V) MT-MMPs: MT1, 2, 3, 4-MMP (MMP-14, -15, -16, and -17, respectively), MT5, 6-MMP (MMP-24, -25, respectively); (VI) others: stromelysin-3 (MMP-11) and enamelysin (MMP-20). All of the reported MMP members contain two typical common domain structures: the cysteine switch region of the propeptide and the zinc-binding site of the catalytic domain. Furthermore, another superfamily has been designated as the metzincin family, which contains the MMP family, members of the ADAM (a disintegrin and metalloprotease) family, and snake venom metalloproteinases with the distinctive zinc-binding consensus motif HExxH (7).
The adult female reproductive tract in mammals is a uniquely dynamic organism in which rapid and extensive degree of tissue development and tissue remodeling occur normally during each estrous cycle. MMPs are believed to be primary contributors to this matrix remodeling and are expressed in a highly regulated manner in the process of reproductive events, including menstruation, ovulation, implantation, and postpartum uterine involution (8). To gain insight into the role of MMPs in the tissue remodeling processes of the female reproductive organs, we performed RT-PCR with a pair of degenerate primers designed for the two conserved regions of the MMP family using RNA isolated from gonadotropin-primed immature rat ovaries. Six different MMP cDNAs were cloned including a MMP-like clone not yet identified during the course of the project. Due to its predominant expression in both rat and human female reproductive tract, we tentatively named it MIFR (metalloproteinase in the female reproductive tract) with the GenBank accession numbers of AB010960 and AB010961 for rat and human clones, respectively (9, 10, 11, 26).1 Recently, the human homolog of this clone designated as MMP-21/22 (9) and MMP-23 (10) and the mouse counterpart termed CA-MMP (11) were also reported. At present these clones have been designated as MMP-23 (6). In this report, we describe the molecular cloning and expression of the membrane-anchored type of rat MMP-23, which shows several unique structural features and an expression pattern distinct from all other MMPs so far characterized. Interestingly, in situ hybridization analysis revealed a conditioned switching of MMP-23 gene expression from granulosa cells to theca externa/fibroblasts in rat ovarian follicles, which depends on the state of follicle maturation in response to gonadotropin action. Furthermore, using serum-free cultures of rat granulosa cells and theca-interstitial cells, we showed that the MMP-23 gene expression was regulated in a cell type-specific manner during gonadotropin-induced differentiation via the cAMP signaling pathway.
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RESULTS |
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Isolation of Rat and Human MMP-23 cDNAs
A preliminary Northern blot analysis of various rat tissues was
conducted using a 498-bp fragment of the novel MMP-like gene as a
probe. The result indicated that the corresponding mRNA transcript
migrated as a single 1.4-kb transcript and was found to be expressed at
the highest levels in ovary and uterus. We therefore decided to use the
rat ovary for further cDNA cloning experiments. The isolated clone of
1,444-bp length appeared to be full-length and to potentially encode a
391-amino acid protein (Fig. 1A). The
deduced amino acid sequence of the rat clone showed that it contained
the sequence from Gly53 to
Leu218 corresponding to the 498-bp RT-PCR
amplified MMP-like product. This sequence included the consensus
sequence HExGHxx involved in the zinc binding at the catalytic site of
MMPs. However, another conserved sequence, the so-called cysteine
switch, located in the profragment, was not found at positions 53 to 59
or at any other positions within the amino-terminal region of the
putative catalytic domain. Alignment of nucleotide sequences of the
1.4-kb cDNA and the 498-bp RT-PCR product revealed that the highly
homologous sequences for annealing the forward primer used for the
RT-PCR were located at nucleotides 331 to 351 within the 1.4-kb cDNA
(Fig. 1C
). It is postulated that a single thymine residue insertion at
nucleotide 340 would shift the reading frame to cause the lack of a
cysteine in this region. To further confirm this unique structural
feature, a 1,265-bp cDNA, highly homologous to the rat sequence, was
isolated from a human uterus 5'-STRETCH cDNA library (CLONTECH Laboratories, Inc., Palo Alto, CA). It contained an entire open
reading frame that encodes a protein of 390 amino acids.
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Since the mRNA is predominantly expressed in ovary and uterus (Fig. 2, BD), we originally termed both rat
and human cDNA sequences as MIFR and deposited these as such in the
GenBank data base [accession numbers AB010960 and AB010961,
respectively (9, 10, 11, 28)]. Although ovary and uterus are the two major
sites of expression of this protein, significant levels of these mRNAs
were also detected in the human male reproductive tract, including
testis and prostate (Fig. 2D
). During the course of this project, the
human homolog of this clone named MMP-21/22 (9) and MMP-23 (10) and the
mouse counterpart, designated CA-MMP (11), were reported. At present,
all of these reported clones including MIFR have been designated as
MMP-23 (6).
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Function of the Hydrophobic Stretch Present in the Amino Terminus
of MMP-23
Using the program of ExPASY via Internet at
http://www.expasy.ch/tools/#transmem, it is predicted that the
hydrophobic stretch of MMP-23 may serve as a type II signal anchor
(14, 15, 16). To analyze this possibility, we constructed cytomegalovirus
(CMV)-driven expression plasmids with the epitope-tagged sequences at
the carboxyl-terminal end of MMP-23 (Fig. 3A). As analyzed by Western blotting
experiments using the monoclonal antibodies 3F10 for hemagglutinin (HA)
epitope tag and M2 for FLAG tag, both human and rat MMP-23 were
detected in membrane fractions prepared from transiently transfected
COS-1 cells (Fig. 3B
, lanes 4 and 5 and 12 and 13).
Epitope-tagged proteins were synthesized as 51 kDa and 56 kDa forms in
rat and human, respectively, both being larger than those theoretically
calculated from the primary structure (46 kDa and 45 kDa for rat and
human, respectively). Reduction of the molecular size of both proteins
by endoglycosidase H treatment indicates that differences in molecular
size reflect the glycosylation of MMP-23 (Fig. 3B
, lanes 7
and 8 and 15 and 16). These data clearly showed, for the first time,
that rat and human MMP-23 were both synthesized as membrane-associated
glycoproteins with type II topology.
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Cleavage Analysis of MMP-23 by Furin
Both rat and human MMP-23 possess the unique dibasic motif (RRRR),
a putative recognition site for furin (17), between the type II signal
anchor and the catalytic domain. This sequence was previously
identified and proven to mediate the intracellular activation of
pro-MT-MMPs (18, 19), prostromelysin-3 (20, 21, 22), and ADAMs (23) in a
furin-dependent manner. Accordingly, it could be anticipated that
MMP-23 is converted intracellularly from a membrane-anchored form to
its soluble form by a proprotein convertase or by ectodomain shedding.
Indeed, a soluble form of the FLAG-tagged human MMP-23 (54 kDa) was
processed into the culture medium by COS-1 cells (Fig. 3B, lanes 9 and
10). In contrast, no significant soluble form of the HA-tagged rat
MMP-23 was detected (Fig. 3B
, lanes 1 and 2). Moreover, coexpression of
mouse furin with the epitope-tagged MMP-23 did not enhance the
formation of the soluble form, indicating that furin may not be
involved in this event (Fig. 3B
, lanes 2 and 10).
Nevertheless, no secreted form of human MMP-23 was detected in the
culture medium of the mutant in which the predicted furin cleavage site
was eliminated by changing the critical amino acids
RRRR78 to RRNG78 by
site-directed mutagenesis (data not shown). This result indicated that
the RRRR78 motif was required for human MMP-23
secretion.
Chromosomal Mapping of Human MMP-23
By using the Stanford G3 radiation hybrid panel, the human MMP-23
gene was determined to be located at D1S2565 (SHGC-4723) within the
terminal end of the short arm of chromosome 1, placing it between the
markers D1S243 and D1S253 within the band 1p36.3 (Fig. 2E; Ref. 24).
The obtained result is well in agreement with two recent reports by
Gururajan et al. (9) and Velasco et al. (10).
Differential Distribution of MMP-23 in the Rat Female Reproductive
Tract
In situ hybridization analysis was performed to
determine which cell types are responsible for the synthesis of MMP-23
in individual ovarian follicles, oviduct, and uterus during the
periovulatory period. In ovaries of untreated immature rats (Fig. 4, A and B), MMP-23 expression was found
to be strictly confined to granulosa cells (black arrow) of
preantral and small antral follicles. No significant signal was
observed in theca-interstitial cells during these stages. When immature
rats were administered eCG followed by hCG treatment, the follicles
increased in size due to the proliferation of granulosa and theca cells
and an enlargement of the antrum (Fig. 4
, CF). During the transition
from the small antral to the large antral follicles, and developing
corpus luteum, the intensity of the signal for MMP-23 mRNA in granulosa
cells greatly diminished to a baseline level. Instead, strong staining
for MMP-23 mRNA was observed in theca-externa/fibroblasts (black
arrowhead, Fig. 4, D and F) and in ovarian surface epithelium
(white arrowhead, Fig. 4F). Weak but clearly evident
signal was also detected in part of the stroma of the ovary from
hCG-treated rats.
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Hormonal Regulation of MMP-23 Gene Expression in Primary Cultures
of Rat Granulosa Cells and Theca-Interstitial Cells
Serum-free primary cell cultures of rat granulosa cells and
theca-interstitial cells were used to investigate the conditioned
switching mechanism of MMP-23 expression observed in vivo.
Both types of prepared cells showed a cell type-specific regulation in
response to gonadotropins accompanied by the accumulation of steroid
hormones and cAMP during differentiation. Treatment with FSH stimulated
cAMP and progesterone production in granulosa cells (Fig. 5, A and B), while LH-induced
differentiation of theca-interstitial cells caused a marked enhancement
of cAMP and androstenedione synthesis. An LH dose of 5 ng/ml produced a
response in thecal progesterone production. It should be mentioned that
LH-treated theca-interstitial cells produced progesterone at levels
lower than that observed for androstenedione at time points greater
than 24 h (Fig. 5
, E and F). Using these conditioned cells, we
performed detailed time course studies of MMP-23 gene expression by
semiquantitative multiplex RT-PCR. Figure 5
, C and D, shows an
accumulation of MMP-23 mRNA in untreated granulosa cells during 48
h of culture. FSH treatment of granulosa cells repressed MMP-23
expression after 24 h of culture and caused a drastic fall in mRNA
accumulation up to approximately 80% below the control levels at
48 h. In contrast, MMP-23 mRNA levels increased in
theca-interstitial cells regardless of the presence of LH during
culture. However, MMP-23 mRNA levels in LH-treated cells were somewhat
lower (
20%) than those of the unstimulated controls at any of the
investigated periods (Fig. 5
, G and H).
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DISCUSSION |
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Transient expression experiments of an epitope-tagged MMP-23 revealed that the unique dibasic motif might not function as a recognition site for furin. This is comparable to a previous example reported by Cao et al. (25) in which coexpression of the recombinant full-length membrane-bound MT1-MMP with furin in COS-1 cells had no effect on the processing of the membrane-associated proteinase. However, we found that the soluble form of FLAG-tagged human MMP-23 was processed endogenously in COS-1 cells and that processing was definitely abolished by introducing the mutation into the RRRR78 motif, suggesting this proprotein convertase recognition site is required for MMP-23 secretion. Recently, Pei et al. (26) provided direct evidence that the mouse counterpart CA-MMP/MMP-23 is processed at the RRRR79 motif before being secreted by MDCK cells stably expressing the FLAG-tagged recombinant protein. Alternatively, another proprotein convertase, e.g. PACE4, PC4, PC6A, PC6B, or a potential candidate protease, may be required for the efficient processing of MMP-23 (17).
Glycosylated forms of both rat and human MMP-23 were demonstrated to be
highly sensitive to endoglycosidase H and representative of the high
mannose-type glycoproteins. Consequently, overexpression of MMP-23 in
COS-1 cells may cause an impaired processing of the mannose-rich
N-linked glycan proteins in the Golgi, leading to the
inefficient conversion of the membrane-anchored forms into the soluble
species by proprotein convertases in the trans-Golgi
network, which usually accompanies the secretion of MMP-23. This may be
one possible explanation for the differences in the amount of the
recombinant MMP-23 secretion that was observed for the rat and human
proteins. Failure to detect the soluble form of rat recombinant protein
may have resulted from a large intracellular accumulation of immature
endoglycosidase H-sensitive protein, since much higher levels of the
rat protein expression were observed than that of the human in COS-1
cells (Fig. 8). Otherwise, there may exist some cell type-specific
factors or mechanisms that facilitate the intracellular targeting and
processing of MMP-23 (27). Our current efforts to clarify detailed
molecular events of processing and secretion of MMP-23 have been
performed using several ovarian cell lines endogenously expressing
MMP-23.
Immunohistochemical analysis of rat MMP-23 in the ovary demonstrated
the distinct localization patterns of MMP-23 protein and its mRNA
during the follicle maturation (Figs. 9 and 10
). Notably, significant
levels of immunopositive signals for MMP-23 were observed in
theca-interna, which did not correspond to regions of mRNA synthesis.
On the other hand, theca-externa/fibroblasts expressing high levels of
MMP-23 mRNA in antral follicles showed very weak immunoreactive
signals. These results may provide indirect evidence that
membrane-anchored MMP-23 is processed in vivo to the soluble
form and is secreted and diffused through extracellular spaces in the
follicles.
In situ hybridization analysis revealed a conditioned
switching of MMP-23 gene expression in the ovary of gonadotropin-primed
immature rats (Fig. 4, AF), whereas the RNase protection assay
demonstrated that expression levels of MMP-23 transcripts were
apparently constant at various stages of follicular development (Fig. 2A
). These results clearly indicate that the expression of MMP-23 mRNA
is likely to be regulated spatially and temporally by gonadotropins in
the ovary and that this regulation is associated with the state of the
development of each follicle. This in vivo phenomenon was
confirmed by in vitro experiments using primary cultures of
granulosa cells and theca-interstitial cells prepared from immature
ovaries. The down-regulation of MMP-23 expression in granulosa cells
induced by FSH (Fig. 5
, C and D) and the accumulation of its mRNA in
LH-treated theca-interstitial cells in vitro (Fig. 5
, G and
H) was found to be in good agreement with the in vivo
results demonstrated by in situ hybridization (Fig. 4
, AF). These results indicate that the serum-free culture system of
ovarian cells used in this study is the preferable in vitro
tool to reflect and characterize in vivo profiles of MMP-23
expression.
A note of caution must be made concerning the accumulation of MMP-23
mRNA observed in theca-interstitial cells regardless of the presence of
LH during culture, despite the fact that LH treatment showed slightly
repressive effect on MMP-23 expression. The level of MMP-23 repression
by LH treatment was evidently much lower than that observed in
forskolin or 8-Br-cAMP treated cells (Fig. 5, G and H, and Fig. 6
, E
and F). Additionally, Fig. 6
, E and F, showed that 8-Br-cAMP repressed
MMP-23 expression in theca-interstitial cells, in a dose-dependent
manner. These results may have been caused by the quite low level of LH
receptor expressed in theca-externa/fibroblasts corresponding to the
site of MMP-23 synthesis (28, 29). Unfortunately this is
difficult to prove because of the technical difficulty in separating
theca-interna and theca-externa at present. Although, it can be
postulated that LH could not produce sufficient amounts of cAMP in
theca-externa/fibroblasts to repress MMP-23 expression to a level
similar to that observed in the forskolin or 8-Br-cAMP-treated cells.
In both types of the granulosa cells and theca-interstitial cells,
8-Br-cGMP essentially had no effect on MMP-23 gene expression (Fig. 6
, B, C, E, and F), indicating that at least the transcriptional
repression of MMP-23 is directly associated with the cAMP signaling
pathways.
Previous reports have demonstrated that progesterone represses MMP-1,
-3, -7, -9 and -11 mRNA in cultured endometrial tissue (8). As shown in
Fig. 5, BD, the time course of MMP-23 repression apparently coincided
with that of progesterone synthesis in FSH-treated granulosa cells. As
evidenced by the observation in theca-interstitial cells (Fig. 6
, DF), a degree to which MMP-23 expression was repressed seems to be
correlated with the increased extracellular progesterone levels.
However, in the present study no significant effects on MMP-23
expression were observed in either granulosa or theca-interstitial
cells cultured for 48 h in the presence of progesterone or the
progesterone antagonist mifepristone (RU486, data not shown).
Furthermore, it is well recognized that progesterone receptor
expression is selectively induced and localized in granulosa cells, not
theca-interstitial cells of preovulatory follicles within 57 h of the
LH surge in rats (30, 31). A more indirect role for progesterone may
not be excluded, although it is unlikely that the observed
transcriptional repression of MMP-23 in both granulosa cells and
theca-interstitial cells could be attributable to the direct action of
progesterone on the MMP-23 gene regulatory elements through the
progesterone nuclear receptor.
It is noteworthy that a marked elevation of MMP-23 transcripts was observed in untreated granulosa cells and theca-interstitial cells cultured for 48 h, indicating that an autonomous system to enhance MMP-23 gene expression is present in both types of cells. Interestingly, similar expression profiles have been reported as an example of angiotensin II type 2 receptor expression in the granulosa cells cultured in the absence of FSH (32, 33). Since culturing the granulosa cells in serum-free conditions without FSH has been shown to lead to the onset of spontaneous apoptosis, it may be proposed that MMP-23 plays some role in the formation and/or maintenance of atretic follicles (33).
The exact nature of the conditioned switching mechanism of MMP-23 gene expression during follicle maturation remains unclear, but this complex expression pattern could indicate that MMP-23 has different roles in granulosa cells and theca cells of the follicle during its development, ovulation, and corpus luteum formation. Additionally, the presence of high levels of MMP-23 transcripts in primary follicles as early as 7 days after birth indicates that this proteinase in granulosa cells may be involved in early follicular development (E. Ohnishi and J. Ohnishi, unpublished data). The molecular mechanisms underlying the ovarian cell type-specific profile of MMP-23 gene expression is currently under intense investigation.
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MATERIALS AND METHODS |
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Animals
Immature (21-day-old) female Sprague Dawley rats were
purchased from Charles River Breeding Laboratory (Yokohama,
Japan) and housed at a constant temperature on a 14-h light, 10-h dark
cycle and fed rat chow and tap water ad libitum. At 25 days
of age, the rats were injected sc with 10 IU of eCG
(Sigma) to initiate follicle development and 48 h
later with 10 IU of hCG (Sigma) to induce ovulation.
Ovulation took place 1016 h after hCG administration. While under
deep halothane/fluorothane (Takeda Chemical Industries, Ltd., Osaka,
Japan) anesthesia, animals were killed by decapitation at
different intervals after hormone treatment, and ovaries and uteri were
quickly removed for RNA preparation and in situ
hybridization. Animals were treated in accordance with the principles
and procedures outlined in the "Guidelines for Care and Use of
Experimental Animals," as approved by the Animal Care and Use
Committee at Hokkaido University School of Medicine.
RT-PCR
cDNAs were synthesized using 5 µg of total RNA, which was
prepared from ovaries removed at 0, 2, 4, 8, and 12 h after hCG
injection, by SuperScript Preamplification System (Life Technologies, Inc.) and used as templates for the PCR performed
with two degenerate oligonucleotides. The forward primer was
5'-(A/C)G(A/G/C)TGTGG(A/T)GT(C/G/T)CC(A/T/C)GATGT-3', and the reverse
primer was 5'-AGGG(A/C)(G/A)TGGCCAA(G/A)(C/T)TCATG-3'. Both were
designed for highly conserved sequences among the MMP family
corresponding to the cysteine switch domain PRCG(N/V)PD and the
zinc-binding region HExGHxx, respectively. The PCR was carried out with
40 cycles of denaturing (94 C, 1 min), annealing (55 C, 2 min), and
extension (72 C, 3 min).
Multiprobe RNase Protection Assay
Multiprobe RNase protection assay was performed using RNase
Protection Kit (Roche Molecular Biochemicals,
Indianapolis, IN). To prepare cRNA probes, cDNA fragments of rat
MMP-23 (nucleotides 5321,049) and ribosomal protein L32 (nucleotides
237380, X06483) were subcloned into appropriate sites of a
pBluescript II SK (+) vector (Stratagene, La Jolla, CA),
and transcribed in vitro in the antisense direction by T7 or
T3 RNA polymerase in the presence of
[-32P]UTP using RNA Transcription Kit
(Promega Corp., Madison, WI). Twenty micrograms of total
RNA were incubated with two labeled cRNA probes (20,000
50,000 cpm
per reaction for each probe) in a single tube at 60 C for 16
h.
cDNA Cloning and Sequence Analysis of MMP-23
Approximately 4.5 x 105 clones of
rat ovarian cDNA libraries constructed in gt10-EcoRI arms
were screened by hybridization with a 32P-labeled
498-bp RT-PCR fragment of the novel MMP-related clone mentioned above.
For isolating the human counterpart, approximately 7.0 x
105 plaques from the human uterus 5'-STRETCH cDNA
library (CLONTECH Laboratories, Inc.) were screened with a
32P-labeled rat MMP-23 full-length cDNA.
Transient Expression of the Epitope-Tagged Rat and Human
MMP-23
Carboxyl-terminally tagged expression plasmids of rat MMP-23
with an influenza virus hemagglutinin (HA) epitope sequence and human
MMP-23 with a FLAG tag were constructed by ligating each cDNA fragment
into pMH vector (Roche Molecular Biochemicals) and
pCMVTag4 vector (Stratagene), respectively. The HA-tagged
rat MMP-23 and FLAG-tagged human MMP-23 mutants at the putative
furin-cleavage site were made by using the QuikChange site-directed
mutagenesis kit (Stratagene). Constructed plasmids were
prepared for transfections into COS-1 cells (Riken Cell Bank, Tsukuba,
Japan) by using Endofree Plasmid Maxi Kit (QIAGEN,
Chatsworth, CA). Transient transfections of the expression plasmids
into COS-1 cells were performed using FuGENE6
(Roche Molecular Biochemicals) according to the
manufacturers instructions. pcDNA3 (Invitrogen, San
Diego, CA) was used to equalize the total amount of CMV-derived
plasmids per each dish.
Indirect Immunofluorescence Analysis
COS-1 cells grown on glass coverslips were transfected with
MMP-23 expression plasmids or pcDNA3 (MOCK) using
FuGENE6 and cultured for 24 h in DMEM with
10% FBS. Cells were fixed with 3% paraformaldehyde in PBS for 30 min
on ice, permeabilized with 0.1% Triton X-100 for 7 min on ice, and
treated with 1 µg/ml rat anti-HA high affinity antibody 3F10
(Roche Molecular Biochemicals) or 20 µg/ml of mouse
anti-FLAG M2 antibody (Stratagene) in HBSS containing 5%
crystallized BSA (Wako Pure Chemical Industries Ltd.) for
1 h at room temperature. After washing with PBS, cells were
incubated with Cy3-conjugated antirat or antimouse IgG (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 h at room
temperature in HBBS containing 5% crystallized BSA. The coverslips
were mounted with Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA) and observed on a
fluorescence microscope.
Preparations of Membrane Fractions and Conditioned Medium, and
Western Blotting
COS-1 cells were transiently transfected with either human
or rat MMP-23 expression plasmid with or without mouse furin expression
vector pCMVmFur (36) using FuGENE6. After 24
h culture with 10% FBS, cells were washed twice with serum-free
DMEM/F12 and further cultured for 48 h with serum-free DMEM/F12
containing 15 mM HEPES, 1 x IST-X (10 mg/liter of
insulin, 5.5 mg/liter of transferrin, 6.7 µg/liter of sodium
selenite, and 2 mg/liter of ethanolamine, Life Technologies, Inc.) and 1 x lactoalbumin hydrolysate (Life Technologies, Inc.). The conditioned media were concentrated
approximately 100-fold using Centricon-10 (Millipore Corp., Bedford, MA). To isolate membrane fractions, cells were
lysed with 20 mM HEPES, pH 7.4, 0.25 M sucrose,
10 mM EDTA, 1 x Complete (Roche Molecular Biochemicals) for 30 min on ice, and then homogenized
by a Dounce homogenizer. After centrifugation at 700 x
g for 15 min at 4 C, the supernatant was centrifuged at
100,000 x g for 30 min at 4 C. The pellet was
solubilized for 1 h on ice in RIPA buffer (Roche Molecular Biochemicals) and used for Western blotting analysis as the
membrane fractions. Corresponding amounts of concentrated media and
membrane fractions were electrophoresed in 10% SDS-polyacrylamide gels
under reducing conditions and transferred to polyvinylidene
difluoride membranes (Immobilon P, Millipore Corp.). Blotted proteins were probed with 3F10 antibody at 0.1
µg/ml or M2 antibody at 1 µg/ml, and specific bands were visualized
using peroxidase-conjugated antirat or mouse IgG (Amersham Pharmacia Biotech) at 1:10,000. The ECL or ECL Plus Western
Blotting Kit (Amersham Pharmacia Biotech) was used
according to manufacturers directions.
For deglycosylation of epitope-tagged MMP-23, solubilized membrane fractions were treated with or without endoglycosidase H (Endo H, Roche Molecular Biochemicals) for 18 h at 37 C in 50 mM sodium acetate buffer (pH 5.5) containing 1% SDS and 0.1 M dithiothreitol according to the manufacturers protocol.
Cloning of the Human MMP-23 Gene and Genetic Mapping
Human genomic MMP-23 clones (GenBank accession number,
AB031068) were obtained by screening the human cosmid library
(CLONTECH Laboratories, Inc.) with the full length of
human MMP-23 cDNA as a probe. To determine the MMP-23 chromosomal
locus, the Stanford G3 RH panel with 83 radiation hybrid clones
(Research Genetics, Inc., Huntsville, AL) was analyzed by
PCR with the following pair of primers from human MMP-23 cDNA
sequences: 5'-CTTCAGCTTCCGCGAGGTGG-3' (forward primer) and
5'-CTGTCGTCGAAGTGGATGCCG-3' (reverse primer), which correspond to
nucleotides 430449 and 583663, respectively. The PCR conditions
were 30 cycles at 98 C for 10 sec, 68 C for 5 sec, and 72 C for 1 min
using Pfu DNA polymerase. The PCR product specifically
amplified under these conditions was 241 bp. The result was submitted
to the Radiation Hybrid Server of Stanford Human Genome Center
(http://shgc-www.stanford.edu) to calculate linkage of the MMP-23
gene to reference markers.
Primary Cell Cultures
Granulosa cells were isolated from immature female Sprague
Dawley rats primed with 1 mg diethylstilbestrol in 0.1 ml sesame oil
once daily for 3 days by a modification of previously published
techniques (38, 39). Briefly, the excised ovaries were suspended in
McCoys 5A medium (modified) supplemented with 25 mM
HEPES, 0.22% NaHCO3, 2 mM
L-glutamine, 100 µg/ml streptomycin, and 100 IU/ml
penicillin (M5A-H media) containing 6.8 mM EGTA. Ovaries
were punctured with a 27-gauge needle, and then incubated for 8 min at
37 C to disrupt the intracellular gap junctions. The released granulosa
cells and ovaries were centrifuged and incubated for an additional 4
min with 0.5 M sucrose and 1.8 mM EGTA in
M5A-H. Ovarian tissues were gently pressed through 42-mesh stainless
steel grid, washed three times, and resuspended in M5A-H. Cell
viability was determined by trypan blue exclusion and was normally
7080%. Viable cells (3x105) were pipetted
onto collagen-coated 24-well culture dishes (IWAKI, Tokyo, Japan) in a
total volume of 0.5 ml of M5A-H supplemented with 1xITS-x
(10 mg/liter of insulin, 5.5 mg/liter of transferrin, 6.7 µg/liter of
sodium selenite, and 2 mg/liter of ethanolamine).
Theca-interstitial cells were prepared from immature intact female
Sprague Dawley rats (day 24 of age) by the modified procedure
originally described by Magoffin (34). Each of the ovaries were cut
into four to six pieces, washed twice with 10 ml of Hanks balanced
MEM supplemented with 25 mM HEPES, 0.035%
NaHCO3, 2 mM L-glutamine,
100 µg/ml streptomycin, and 100 IU/ml penicillin (H-MEM). Pieces of
ovaries were incubated for 60 min at 37 C in 0.25 ml per ovary of
collagenase A-DNase I solution including 2.5 mg/ml collagenase A
(Roche Molecular Biochemicals) and 100 µg/ml DNase I
(Roche Molecular Biochemicals) in H-MEM. The incubated
ovaries were flushed every 30 min by gently pipetting through a Pasteur
pipette. The dispersed cells were washed three times with H-MEM and
passed through 100-µm pore sizes of cell strainers (Becton Dickinson
Labware, Lincoln Park, NJ). Theca-interstitial cells were then
purified by a discontinuous density centrifugation procedure. Six
milliliters of 44% Percoll in H-MEM were carefully layered on top of
56% Percoll cushion in 17 x 100-mm sterile polystyrene Falcon
tubes. Dispersed cells (1025 x 106 cells
in 1.5 ml) were layered on top of a 42% Percoll solution and
centrifuged at 400 x g for 20 min at 4 C. After
centrifugation, the theca-interstitial cells was settled down into 44%
Percoll layer above the interface with 56% Percoll cushion. The
purified theca-interstitial cells phase was aspirated and then washed
three times with M5A-H media. The cells were 90% viable, as determined
by trypan blue exclusion. Viable cells (2x105)
were placed onto collagen-coated 24-well culture dishes in a volume of
0.5 ml of M5A-H supplemented with 1 x ITS-x. At selected times
during culture at 37 C in a humidified 95% air, 5%
CO2 environment, medium was removed, centrifuged,
and boiled for 10 min. After centrifugation, supernatant was frozen at
-80 C until assayed for steroid hormones and cAMP by ELISA.
Progesterone, androstenedione, and cAMP were measured with Progesterone EIA Kit (Cayman Chemical Company, Ann Arbor, MI), Androstenedione ELISA Kit (Oxford Biomedical Research, Inc., Oxford, MI) and cAMP EIA System (Amersham Pharmacia Biotech), respectively, according to manufacturers instructions.
In Situ Hybridization
Immature female rats were decapitated, and ovaries and uteri
were quickly removed by dissection at the indicated times. Tissues were
embedded in Tissue-Tec OCT compound (Miles Laboratories, Kankakee,
IL) and frozen in an isopentane-dry ice bath. Sections of 10
µm (ovaries) and 12 µm (uteri) in thickness were cut on a cryostat
and mounted on silane-coated microscope slides. Hybridization was
performed with digoxigenin-labeled riboprobes synthesized using a DIG
RNA Labeling Kit (Roche Molecular Biochemicals) under the
slightly modified conditions as described by Schaeren-Wiemers and
Gerfin-Moser (35). Briefly, sections were fixed in 4% paraformaldehyde
in PBS for 10 min at room temperature and acetylated for 10 min with
buffer containing 0.1 M triethanolamine and 0.25% acetate
anhydride. Prehybridization was performed at room temperature with 200
µl of hybridization buffer: 50% formamide, 5 x SSC, 5 x
Denhardts (Wako Pure Chemical Industries Ltd, Osaka, Japan), 500 µg/ml tRNA (Roche Molecular Biochemicals) per slide for overnight. Hybridization buffer
containing heat-denatured DIG-cRNA probe was spread over the sections
and covered with siliconized coverslips and sealed with DPX mounting
reagent (Fluka Chemical Co., Ronkonkoma, NY). The
hybridization was performed overnight at 72 C. Slides were washed in
0.2 x SSC at 72 C for 60 min and incubated for 1 h with an
anti-DIG antibody (Roche Molecular Biochemicals).
Visualization of the signal was performed with nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl-phosphate solution
containing 0.24 mg/ml levamisole for 3 days.
To examine the expression and localization of MMP-23 mRNA in cultured granulosa cells and theca-interstitial cells, 12 x 105 cells were each cultured for 48 h on the collagen-coated glass eight-well chamber slides (Becton Dickinson Labware) in 0.5 ml of McCoys 5a medium containing 1 x ITS-x with or without stimuli. The cultured cells were fixed in 4% paraformaldehyde in PBS. After fixation and acetylation, the slides were incubated in 1 x SSC for 5 min and permeabilized with 0.2 N hydroxyl chloride for 10 min. In situ hybridization was performed as described above.
Semiquantitative Multiplex RT-PCR
Total RNA was prepared from cultured rat granulosa cells and
theca-interstitial cells by ISOGEN (Nippon Gene, Tokyo, Japan).
One microgram of total RNA was reverse transcribed with random hexamer
by using ThermoScript RT-PCR System (Life Technologies, Inc., Gaithersburg, MD). Specific primers were used to amplify
rat MMP-23 cDNA: sense, 5'-CAGGATTCTCTCCTTTCCCC-3'; antisense,
5'-GCCTCTTCATGAGCCTCTGG-3'. Each reaction contained the template DNA
corresponding to cDNA synthesized from 50 ng of total RNA, specific
primer sets for rat MMP-23, and Quantum RNA alternate 18S internal
standards (Ambion, Inc., Austin, TX), which was used as an
internal control at the 1:9 ratio of 18S primers to competimers.
Multiplex PCR reaction was carried out in 50 µl reaction volumes
using Pfu Taq polymerase with 30 cycles of
denaturing (97 C, 30 sec), annealing (60 C, 30 sec), and extension (72
C, 2 min). Aliquots of PCR product (5 µl) were electrophoresed
through 2% agarose 21 gels (Nippon Gene) in 0.5xTBE. The separated
DNA bands were stained with SYBR Green I (Molecular Probes, Inc., Eugene, OR), visualized, and analyzed by computerized
densitometric scanning of the images using a lumino image analyzer
LAS-1000 (Fuji Photo Film Co., Ltd., Tokyo, Japan).
MMP-23 expression was normalized to expression of 18S ribosomal RNA
presented as the ratio of fluorescence intensity in the MMP-23 and 18S
bands.
Immunological Characterization of MMP-23
Antihuman MMP-23 polyclonal antibodies were raised against a
synthetic peptide
(329KGKVYWYKDQEPLE342)
corresponding to amino acids 329342 in the carboxyl-terminal domain
of human MMP-23, whose sequence is encoded at amino acids 331344 in
rat ortholog. BLAST searching at the National Center for Biotechnology
Information web site provided no peptide with greater than 45% level
of identity except human, rat, and mouse MMP-23. Two rabbits were
immunized with the peptides coupled to keyhole limpet hemocyanin
(Sigma Genosys Japan, Hokkaido, Japan). Specificity
of the obtained antiserum GN2062 was determined by Western blotting
analysis of the membrane fractions prepared from COS-1 cells expressing
epitope-tagged human or rat MMP-23. A 1:1,000 dilution of GN2062
specifically recognized both human and rat MMP-23 (see Fig. 8). The
freshly obtained ovaries were embedded in Tissue-Tec OCT compound and
frozen in an isopentane-dry ice bath. Tissue sections of 10 µm
thickness were cut on a cryostat and mounted on silane-coated
microscope slides. The sections were fixed in 4%
periodate-lysine-paraformaldehyde for 10 min at 4 C, washed in PBS, and
incubated for 10 min at 90 C with 0.01 M citrate buffer (pH
6.0). After cooling to room temperature, the sections were quenched
twice with 50 mM ammonium chloride-PBS for 10 min. Blocking
was performed with 10% BSA in PBS containing 0.05% Tween 20
(Roche Molecular Biochemicals) for 30 min at room
temperature. Each of the sections was incubated overnight at 4 C with
the primary antibody GN2062 (1:200) in PBS containing 0.05% Tween 20
and 1% BSA. Subsequently, sections were washed with PBS containing
additional 0.5 M NaCl and 0.1% Tween 20, and endogenous
peroxidase activity was blocked by the treatment with 0.3% hydrogen
peroxide and 0.3% NaN3 in PBS for 10 min at room
temperature. After reacting with peroxidase polymer-conjugated
secondary antibody Envision (DAKO Corp., Carpinteria, CA)
for 30 min at room temperature, immunoreactive signals were visualized
with a mixture of 1 mg/ml diaminobenzidine, 0.65 mg/ml
NaN3, and 0.016% hydrogen peroxide. Sections
were counterstained with hematoxylin. Specificity of staining was
determined by comparing the serial sections incubated with an equal
protein amount of nonimmunized rabbit antiserum or with a specific
antiserum neutralized by preabsorbing an antiserum overnight at 4 C
with a 20-fold (by weight) excess of the synthesized peptide, which was
used as the immunogen.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education and Culture, Japan, and the Japan Society for the Promotion of Science, and by Research grants from the Nissan Science Foundation and the Kurata Foundation. E. Ohnishi, H.M. and A.K. are supported by Research Fellowships of the Japan Society for the Promotion of Science.
1 Data deposition: The sequences of MIFR
reported in this paper have been deposited in the GenBank database
(accession numbers, AB010960, AB010961, and AB031068).
Received for publication March 3, 2000. Revision received February 14, 2001. Accepted for publication February 20, 2001.
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REFERENCES |
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