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From the Population Council, New York, New York.
| Correspondence to: Dr C. Yan Cheng, Population Council, Center for Biomedical Research, 1230 York Ave, New York, NY 10021 (e-mail: ycheng{at}popcbr.rockefeller.edu). |
| Received for publication September 11, 2003; accepted for publication November 6, 2003. |
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-actinin/actin complex but not to the
nectin-3/afadin or the ß1-integrin–mediated protein complexes.
Interestingly, zyxin, axin, and WASP are also structurally linked to vimentin
(an intermediate filament protein) and -tubulin (the subunit of a
microtubule), which suggests that they have a role (or roles) in the
regulation of the dynamics of the desmosome-like junction and microtubule.
These results illustrate that zyxin, axin, and WASP are adaptors in both AJs
and intermediate filament-based desmosome-like junctions. This raises the
possibility that classic cadherins are also associated with vimentin-based
intermediate filaments via these adaptors in the testis. While virtually no
N-cadherin was found to associate with vimentin in the seminiferous tubules,
it did associate with vimentin when testis lysates were used. Interestingly,
about 5% of the E-cadherin associated with vimentin in isolated seminiferous
tubules, and about 50% of the E-cadherin in the testis used vimentin as its
attachment site. These data suggest that cadherins in the testis, unlike those
in other epithelia, use different attachment sites to anchor the
cadherin/catenin complex to the cytoskeleton. The levels of zyxin, axin, and
WASP were also assessed during AF-2364–mediated AJ disruption of the
testis, which illustrated a time-dependent protein reduction that was similar
to the trends observed in nectin-3 and afadin but was the opposite of those
observed for N-cadherin and ß-catenin, which were induced. Collectively,
these results illustrate that while these adaptors are structurally associated
with the cadherin/catenin complex in the testis, they are regulated
differently.
Key words: Sertoli cells, testis, adaptors
Axin is another adaptor and signaling protein that takes part in the Wnt
signaling pathway. It recruits ß-catenin to glycogen synthase
kinase-3ß (GSK-3ß), which in turn regulates the phosphorylation and
degradation of cytosolic ß-catenin (for a review, see
Seidensticker and Behrens,
2000). Interestingly, the phosphorylated form of GSK-3ß was
recently shown to be a crucial signaling molecule in the tumor necrosis factor
alpha (TNF)-mediated tight junction regulation in Sertoli cells
(Siu et al, 2003a). An
increasing number of proteins are shown to be putative axin-binding proteins,
such as plakoglobin (Kodama et al,
1999), mitogen-activated protein kinase kinase 1 (MEKK1), and
casein kinase I, all of which are implicated in regulating the JNK signaling
pathway (Zhang et al, 1999,
2002). Like zyxin, axin is a
putative substrate of protein kinases.
Wiskott-Aldrich syndrome protein (WASP) is another adaptor protein that regulates actin dynamics via activation and recruitment of the Arp (actin-related protein) 2/3 complex and profilin-bound monomeric actin (for a review, see Caron, 2002). In its inactive state, WASP is folded, which masks some of its protein-binding sites (for reviews, see Mullins, 2000; Caron, 2002). Upon its activation by cell division cycle 42 (Cdc42) guanosine triphosphatase (GTPase), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and WASP-interacting SH3 protein, WASP unfolds, exposing its binding sites to different interacting proteins (for a review, see Takenawa and Miki, 2001), which include c-Src, actin, WASP-interacting protein, and Arp2/3 (for reviews, see Mullins, 2000; Takenawa and Miki, 2001). As such, the principal role of WASP in AJ functions apparently lies in its ability to recruit different proteins to the multiprotein complexes at the site of AJs, which in turn affects the underlying cytoskeletal network.
Earlier studies have shown that the adhesion of spermatogonia and
spermatocytes onto Sertoli cells at or near the basal compartment of the
seminiferous epithelium is mediated largely by desmosome-like intermediate
filament-based anchoring junctions
(Russell, 1977) composed of
vimentin, plectin, and other adhesion proteins (for reviews, see
Russell and Peterson, 1985;
Cheng and Mruk, 2002), whereas
the actin-based AJs, such as ectoplasmic specializations (ESs), are restricted
to the apical compartment between Sertoli cells and round and
elongating/elongate spermatids and between Sertoli cells in the basal
compartment (Russell and Clermont,
1976; Russell,
1979). Recent studies have shown that the apical ES is constituted
by the 6ß1- or 4ß1-integrin/laminin 
3, the
nectin/afadin, and the cadherin/catenin, whereas the basal ES is constituted
by the 6ß4-integrin/laminin and the cadherin/catenin complexes
(Chapin et al, 2001;
Siu and Cheng, 2004; for
reviews, see Vogl et al, 2000;
Cheng and Mruk, 2002; Lui et al, 2003b). The precise
attachment site of the classic cadherin/catenin complex remains a
controversial issue. Notably, studies using immunofluorescent microscopy
(Johnson and Boekelheide,
2002a,b)
and electron microscopy (Mulholland et al,
2001) suggest that the classic cadherin/catenin complex, an
actin-based junctional protein complex in other epithelia (for a review, see
Gumbiner, 2000), uses the
intermediate filament as the attachment site in the testis. It is unknown
whether desmocollins and desmogleins (both are desmosome-integral membrane
proteins) and desmoplakins, plakophilins, and plakoglobins (the latter 3 are
desmosome-peripheral proteins), which are the putative structural proteins of
desmosomes in other epithelia (for a review, see
Ishii and Green, 2001), are
found in the testis (for a review, see
Cheng and Mruk, 2002). A
recent study using immunoprecipitation with and without a cross-linker has
clearly demonstrated that the N-cadherin/ß-catenin complex associates
exclusively with actin, but not with vimentin, using lysates of seminiferous
tubules isolated from rat testes (Lee et
al, 2003). This is consistent with an earlier report that
colocalized both N-cadherin and ß-catenin to the same site at the ES by
immunohistochemistry (Wine and Chapin,
1999). In this report, we have also investigated whether the 3
selected adaptor proteins, which are also known to associate with both the
actin and intermediate filament cytoskeletal network, can act as linkers
between the classic cadherin/catenin complex and the intermediate
filament-based and microtubule-based cytoskeletal networks.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-actinin (N-19, Cat: sc-7453, Lot: D292),
nectin 3 (C-19, Cat: sc-14806, Lot: K261), ß1-integrin (M-106, Cat:
sc-8978, Lot: E221), vasodilator-stimulated phosphoprotein (VASP) (C-17, Cat:
sc-1853, Lot: G262), c-Src (B-12, Cat: sc-8056, Lot: C051), actin (H-196, Cat:
sc-7210, Lot: C222), -tubulin (TU-02, Cat: sc-8035, Lot: G182), and
vimentin (V9, Cat: sc-6260, Lot: J2802). For N-cadherin, E-cadherin,
-catenin, ß-catenin, axin, ß1-integrin, and actin, the
antigen used for polyclonal antibody production in rabbits was derived from
the corresponding human recombinant protein. The rabbit p120ctn
polyclonal antibody and the mouse c-Src monoclonal antibody were raised
against peptide fragments derived from mouse p120ctn and human
c-Src proteins. Anti-VASP, nectin-3, and -actinin antibodies were
affinity-purified goat polyclonal antibodies raised against peptide fragments
derived from the corresponding human recombinant protein. Afadin and WASP were
mouse monoclonal antibodies raised against human recombinant proteins.
Vimentin and -tubulin were mouse monoclonal antibodies raised against
porcine recombinant proteins. All antibodies used in this study cross-reacted
with the corresponding rat proteins as indicated by the manufacturer. The
corresponding secondary antibodies were purchased from Santa Cruz
Biotechnology.
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Primary Testicular Cell Cultures
Sertoli cells were isolated from 20-day-old rat testes and cultured in
vitro in Ham F12 medium (F12)/Dulbecco modified Eagle medium (DMEM) as earlier
described (Lee et al, 2003).
Sertoli cells were plated at a high cell density (0.5 x 106
cells/cm2) on Matrigel (1:8 with DMEM, vol/vol) (Collaborative
Research Inc, Bedford, Mass)-coated 12-well dishes (effective area, 
3.8
cm2 per well) with 3 mL of F12/DMEM per well. Sertoli cells were
incubated at 35°C in a humidified atmosphere of 5% CO2/95% air
(vol/vol). Cultures were designated D0 (time 0) at the time of cell plating.
About 36 hours after plating, cells were hypotonically treated with 20 mM
Tris, pH 7.4, for 2.5 minutes to lyse residual germ cells
(Galdieri et al, 1981) and
washed twice to remove cellular debris. The resulting Sertoli cells had a
purity of approximately 95%. Sertoli cells were terminated 24 hours after
hypotonic treatment. Germ cells were isolated from 20-, 60-, and 90-day-old
rats and terminated within 3 hours (Lee et
al, 2003). Cell purity was monitored by microscopic examination
(Lee et al, 2003). Germ-,
myoid-, Leydig-, and Sertoli cell-specific marker genes, such as c-Kit
receptor, fibronectin, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and
testin, respectively, were used to examine cell contamination by reverse
transcriptase-polymerase chain reaction (RT-PCR)
(Lee et al, 2003).
Seminiferous tubules were isolated from adult rat testes (300 gm body
weight [bw]) as previously described (Lee
et al, 2003).
RT-PCR
RT-PCR was performed as described (Lee
et al, 2003). Briefly, 2 µg of total RNA was reverse
transcribed into complementary DNAs using 1 µg oligo(dT)15 with
a Moloney murine leukemia virus RT kit (Promega Corp, Madison, Wis) in a final
reaction volume of 25 µL. Thereafter, PCR was performed by combining 2
µL of the RT product and 0.6 µg each of sense and antisense primers of a
target gene (coamplified with either rat ribosomal S-16 or rat ß-actin
using 0.01 µg each of the sense and antisense primer) (Table) and the
remaining reaction mixture as described
(Lee et al, 2003). The cycling
parameters for PCR were as follows: denaturation at 94°C for 1 minute,
annealing at 46°C–61°C for 2 minutes, and extension at 72°C
for 3 minutes, for a total of 20–29 cycles. The linearity of the PCR
products was assessed in preliminary experiments as detailed elsewhere
(Lee et al, 2003).
Treatment of Rats With AF-2364 to Induce Germ Cell Loss From the Seminiferous Epithelium
Rats weighing between 250 and 300 g were treated with 50 mg of AF-2364/kg
bw by gavage as described (Cheng et al,
2001; Grima et al,
2001) to perturb cell adhesion function in the seminiferous
epithelium (Chen et al, 2003;
Lau and Mruk, 2003;
Lee et al, 2003;
Lui et al, 2003a;
Siu et al, 2003b). Testes were
removed at specified time points for lysate preparation as described
(Lee et al, 2003).
Polyacrylamide Gel Electrophoresis and Immunoprecipitation
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under
reducing conditions was performed as described
(Lee et al, 2003). In brief,
cells and tubules were resuspended in immunoprecipitation buffer (10 mM Tris,
0.15 M NaCl, 2 mM PMSF, 2 mM EDTA, 2 mM N-ethylmaleimide, 1% NP-40 [vol/vol],
1 mM sodium orthovanadate [a protein tyrosine phosphatase inhibitor, PTPi],
0.1 µM sodium okadate [a protein Ser/Thr phosphatase inhibitor], and 10%
glycerol [vol/vol], pH 7.4, at 22°C) with a cell:buffer and a
tissue:buffer ratio of 2 x 106 cells:500 µL and 0.1 g
tissue:500 µL, respectively. Samples were sonicated twice for 10 seconds
with a 30-second interval in melting ice. Clear lysates were obtained by
centrifugation at 12 000 x g for 15 minutes at 4°C. The
total protein concentration was determined by the Coomassie blue dye binding
assay (Bradford, 1976) using
bovine serum albumin as a standard. For immunoprecipitation, 400 µg of
protein from the seminiferous tubule lysates was first pretreated with the
corresponding normal serum at 1:150 dilution for 4 hours at room temperature
in a rotator. Thereafter, 10 µL of Protein A/G-PLUS agarose (Santa Cruz
Biotechnology) was added to each sample and incubated at room temperature for
4 hours. Centrifugation was performed to separate the nonspecific complexes
that were bound to serum proteins and to the Protein A/G-PLUS agarose from
those proteins in the lysates. Then, the resulting lysates in supernatant were
incubated with anti-zyxin, axin, or WASP antibodies (1: 150) overnight in a
rotator to allow coimmunoprecipitation. Thereafter, 20 µL of Protein
A/G-PLUS agarose was added to each sample and incubated for 4 hours to
precipitate the immunocomplexes. After washing 4 times with
immunoprecipitation buffer (1000 x g, 5 minutes), the resulting
immunocomplexes were extracted from the agarose using a SDS-sample buffer
[0.125 M Tru, pH 6.8 at 22°C containing 1% SDS (w/v), 1–6%
-mercaptoethanol (v/v), 1 mM EDTA, and 10% glycerol (v/v)], denatured
at 100°C for 5–10 minutes, and resolved by SDS-PAGE as described
(Lee et al, 2003). A
chemiluminescent ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ) was used
to detect the immunoreactive target protein band with Kodak BioMax Light
Films. Blots were reprobed with a second antibody after the initial antibody
was removed by a stripping buffer (62.5 mM Tris-HCl, pH 6.7, at 22°C
containing 100 mM 2-mercaptoethanol and 2% SDS [wt/vol]) at 55°C for 30
minutes as described (Lee et al,
2003).
Immunohistochemistry
Testes were isolated from normal (control) and AF-2364–treated rats
and frozen in liquid nitrogen. Eight-micrometer sections were cut and mounted
on poly-L-lysine–coated slides in a cryostat. All sections
from one experiment were mounted onto a single slide to ensure that all
sections were subjected to the same incubation and color development times in
order to minimize interexperimental variations. Each experiment was repeated
at least twice using the testes from 2 sets of rats, and representative
results are shown. Sections were air dried and fixed in 4% paraformaldehyde
(wt/vol in phosphate-buffered saline [PBS], 10 mM sodium phosphate and 0.15 M
NaCl, pH 7.4, at 22°C) for 10 minutes at room temperature. Endogenous
peroxidase activity was blocked by incubating sections in 1% hydrogen peroxide
(in methanol [vol/vol]) for 20 minutes. Nonspecific antibody binding sites
were blocked by preincubating the sections with 10% normal goat serum (vol/vol
in PBS) (Zymed Laboratories Inc, San Francisco, Calif) for 1 hour. Thereafter,
sections were incubated with a goat anti-zyxin antibody (1:150) overnight in a
humidified chamber at 35°C. Sections were then incubated with biotinylated
rabbit anti-goat immunoglobulin G (IgG; Zymed) for 30 minutes and then with a
streptavidin-peroxidase complex (Zymed) for 15 minutes.
3,3'-Diaminobenzidine tetrahydrochloride was used as a substrate to
visualize zyxin in the seminiferous epithelium. After counterstaining with
hematoxylin and dehydration in 70% ethanol, 90% ethanol, 100% ethanol, and
xylene, sections were mounted and examined under a microscope.
Immunohistochemistry studies were limited to zyxin, because we could not
locate antibodies to WASP or to axin commercially that did not yield multiple
bands in addition to the antigen band. The anti-zyxin antibody yielded a
prominent immunoreactive band that corresponded to zyxin in immunoblot
analysis.
Immunofluorescent Microscopy
Immunofluorescent microscopy was performed as earlier described
(Lee et al, 2003;
Siu et al, 2003b). In brief,
testes removed from adult rats were fixed in Bouin fixative, embedded in
paraffin, and sectioned to 8-µm thickness. Following the removal of the
paraffin by the use of xylene, sections were incubated with a mouse
anti-N-cadherin antibody (Cat: 33-3900, Lot: 11268187, Zymed) (1:100 dilution)
and then with a goat-antimouse IgG-Cy3 (Cat: 81-6515, Lot: 11067429, Zymed).
For fluorescent immunocytochemistry, Sertoli cells cultured for approximately
2 days in F12/DMEM at 5 x 104 cells/cm2 were fixed
in Bouin fixative, permeabilized with Triton X-100, and stained with DAPI
(4',6-diamidino-2-phenylindole) (Molecular Probes, Eugene, Ore)
(specific staining for DNA in the nucleus) and a goat-anti-zyxin antibody, to
be followed by a rabbit anti-goat IgG-fluorescein isothiocyanate (FITC)
(Zymed) antibodyconjugate complex. Sections or cells were then mounted in
Vectashield (Vector Laboratories, Burlingame, Calif), and micrographs were
obtained using an Olympus BX40 microscope equipped with Olympus UPlanF1
fluorescent optics (Melville, NY). Controls included sections incubated with
either 1) normal mouse serum or 2) a mouse anti-human 1-antitrypsin
previously characterized in our laboratory
(Silvestrini et al, 1990) at
the same dilution as that of the primary antibody.
Electron Microscopy
Seminiferous tubules were removed from the testes, fixed in 2.5%
glutaraldehyde (vol/vol in 0.1 M sodium cacodylate buffer, pH 7.4) for
2–4 hours at room temperature, and processed for electron microscopy as
earlier described (Lee and Cheng,
2003).
Statistical Analysis
The Student's t test was performed using the GB-STAT Statistical
Analysis Software Package (Version 7.0; Dynamic Microsystems Inc, Silver
Spring, Md).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Relative Levels of Zyxin, Axin, and WASP in Sertoli and Germ Cells
To compare the relative levels of zyxin, axin, and WASP in Sertoli and germ
cells, these cells were isolated from 20-, 60-, and 90-day-old rat testes. All
3 adaptors, both mRNAs and proteins, namely zyxin
(Figure 2A and D), axin
(Figure 2B and D), and WASP
(Figure 2C and D), were found
in 20-day Sertoli cells. The levels of zyxin
(Figure 2A and D) and WASP
(Figure 2C and D) were higher
in 20-day Sertoli cells than in 20-day germ cells, whereas axin was more
predominant in germ cells (Figure 2B and
D). Zyxin was virtually undetectable in 20-day-old germ cells, yet
it was detected in germ cells from 60- and 90-day-old rats
(Figure 2A and D).
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Immunohistochemical Localization of Zyxin in the Rat Seminiferous Epithelium and in Sertoli Cells Cultured In Vitro
The localization of zyxin in the seminiferous epithelium of adult rat
testes was examined by immunohistochemistry. A negative control was performed
using normal goat serum as a substitute for the zyxin antibody. Negligible
staining was detected, as demonstrated by the stage V tubule shown in
Figure 3A. Yet when sections of
testes were incubated with an anti-zyxin antibody, zyxin was localized
predominantly in spermatocytes at stages V–VII
(Figure 3C and D vs
Figure 3B and E) with some
staining between elongating/elongate spermatids and Sertoli cells, consistent
with its localization at the site of apical and basal ES
(Figure 3B through E vs
Figure 3A).
Figure 3F is a representative
fluorescent micrograph showing the immunocytochemical localization of zyxin in
Sertoli cells that were cultured in vitro from 2 different experiments. The
prominent nucleus of a Sertoli cell was stained with DAPI
(Figure 3F). Zyxin was
visualized as discrete patches of green fluorescence in the Sertoli cell
cytoplasm, which is consistent with its role as an adaptor that recruits other
proteins to the AJ site. Figure
3G shows the result of an immunoblotting experiment in which a
testis lysate was resolved by SDS-PAGE under reducing conditions; only a
prominent band corresponding to the electrophoretic mobility of zyxin,
approximately 82 kDa, was detected. Attempts were also made to localize axin
and WASP in the seminiferous epithelium; however, these 2 antibodies failed to
yield satisfactory results. Also, both of these antibodies were shown to
cross-react with other proteins by immunoblotting (data not shown).
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Zyxin, Axin, and WASP Structurally Interact With the Cadherin/Catenin/Actin Complex, Intermediate Filaments, and Microtubules in the Seminiferous Epithelium by a Coimmunoprecipitation Technique
Coimmunoprecipitation was performed using zyxin-, axin-, or WASP-specific
antibodies, with seminiferous tubule lysate as the starting material.
N-Cadherin, an AJ-integral membrane protein, and its binding partners, such as
ß-catenin, p120ctn, and -actinin, were shown to
associate with axin, zyxin, and WASP
(Figure 4). Yet nectin-3,
afadin, and ß1-integrin were not shown to structurally associate with
zyxin, axin, or WASP (Figure
4). Interestingly, c-Src, an AJ-associated signaling molecule, was
shown to associate with zyxin, axin, and WASP
(Figure 4). Furthermore, actin,
vimentin (an intermediate filament component), and -tubulin (a
component of microtubules) interacted structurally with axin, zyxin, and WASP
(Figure 4). Unexpectedly, VASP,
a zyxin adaptor protein found in other epithelia (for a review, see
Reinhard et al, 2001), such as
the liver, was not found in the testis
(Figure 4). Collectively, these
results suggest that zyxin, axin, and WASP structurally associate with the
cadherin/catenin/ actin complex but not the nectin/afadin and the
integrin/laminin complexes. They possibly recruit AJ-signaling molecules, such
as c-Src, to regulate the adhesion function of the cadherin/catenin
complex.
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Further Structural Analysis of the Association of Zyxin with N-Cadherin, E-Cadherin, Actin, and Vimentin in the Testis
Immunoprecipitation results reported in
Figure 4 have shown that the 3
AJ adaptors—namely, zyxin, axin, and WASP—associate with actin and
vimentin, which suggests that they serve as a linker between N-cadherin and
vimentin. To expand this observation, coimmunoprecipitation was performed
using antibodies specific to N-cadherin and E-cadherin, 2 classic cadherins
that are found in the testis. It is known that the classic cadherins that are
found in the brain are actin based (for a review, see
Gumbiner, 2000). Thus, brain
lysate served as a control as shown in
Figure 5. Virtually all
N-cadherin and E-cadherin (95%) in the seminiferous epithelium indeed
were shown to associate with actin filament
(Figure 5A and B), and 5% or
less of E-cadherin, but not N-cadherin, was found to associate with vimentin
(Figure 5B). However, a
substantial amount of both cadherins associated with vimentin in the testis,
suggesting that the cadherin/catenin complex outside the blood tumor barrier
uses both actin and an intermediate filament as attachment sites. On the
contrary, only a negligible amount of vimentin interacted with N-cadherin and
E-cadherin in the brain (Figure
5) (note: cadherins in the brain are actin based; see
Gumbiner, 2000). This also
confirms the results of our earlier immunoprecipitation analysis
(Lee et al, 2003). Interestingly, zyxin was also shown to link to N-cadherin and E-cadherin in
testes and seminiferous tubules (Figure
5).
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To further validate these data that N-cadherin indeed is restricted to the
ES in the seminiferous epithelium, which is an actin-based testis-specific AJ
type, we used immunofluorescent microscopy and paraffin sections to revisit
its localization in the testis. In this connection, it is notable that our
earlier studies using frozen sections for immunohistochemistry have
consistently localized N-cadherin to the basal ES only, consistent with an
earlier report by Wine and Chapin
(1999). However, Johnson and
Boekelheide
(2002a,b)
reported that they could also localize N-cadherin to the apical ES, suggesting
that N-cadherin in the seminiferous epithelium uses actin as its attachment
site, which is consistent with an earlier report
(Lee et al, 2003). Using
frozen sections, N-cadherin and E-cadherin indeed were restricted to the basal
ES. However, when paraffin sections were used, N-cadherin indeed was also
found to be localized to the site of apical ES between elongating spermatids
and Sertoli cells and was highest in stages V–VI
(Figure 6A and B), which is
consistent with earlier reported results (Johnson and Boekelheide,
2002a,b).
Figure 6C and D are the
corresponding controls of the seminiferous epithelium at stages V and VI,
respectively, where the primary antibody was replaced with normal rabbit serum
and a monoclonal antibody (IgG1 subclass) against human 1-antitrypsin
(Silvestrini et al, 1990),
illustrating that the staining shown in
Figure 6A and B was specific to
N-cadherin. We had tried to perform similar experiments using 2 commercially
available E-cadherin antibodies without success, possibly because the titer of
these antibodies was low. Figure
6E is an immunoblot using testis lysate resolved by SDS-PAGE and
stained with the anti-N-cadherin antibody, which was used for the
immunofluorescent microscopy study shown in
Figure 6A and B. Only a single
immunoreactive band corresponding to the electrophoretic mobility of
N-cadherin (Mr 127 kDa) was detected, illustrating that the specificity of
this antibody and the staining shown in
Figure 6A and B indeed
represent N-cadherin staining.
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Changes in the Protein Levels of the 3 Adaptors and Their Associated Proteins at the AJ Site During AF-2364–Induced AJ Disruption in the Seminiferous Epithelium
Rats were fed AF-2364 (50 mg/kg bw) as described in "Materials and
Methods" to induce germ cell loss from the seminiferous epithelium by
perturbing the cell adhesion function
(Cheng et al, 2001; Grima et al, 2001). While
earlier biochemical and molecular studies using markers of the AJs, such as
testin, have shown that the anchoring junctions between Sertoli and developing
germ cells are the primary targets of AF-2364 (Grima et al,
1997,
2001;
Grima and Cheng, 2000;
Cheng et al, 2001), its
ultrastructural effects in the seminiferous epithelium at the electron
microscopy level remain obscure. Figure
7A shows the cross section of an intact seminiferous epithelium
from a control rat testis. Four hours after AF-2364 treatment, the
intercellular space between germ cells, particularly round spermatids, and
Sertoli cells became clearly visible, which was not found in the control rat
testis (Figure 7B vs A). By day
5, more intercellular spaces were detected between Sertoli cells and round
spermatids, and this pattern was typical throughout the entire seminiferous
epithelium in more than 95% of the tubules examined
(Figure 7C). This study clearly
illustrates that AF-2364 exerts its effects at the cell adhesion sites between
Sertoli and germ cells. Yet the basal lamina apparently was not affected by
this treatment (Figure 7B and
C).
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During AF-2364–induced AJ disruption in the testis, a time-dependent
reduction in the protein levels of zyxin, axin, and WASP was detected
(Figure 8A). This pattern of
reduction was similar to those observed for nectin-3 and afadin
(Figure 8A). An induction of
N-cadherin was detected at 16 hours posttreatment and persisted until day 7
(Figure 8A), which was
consistent with the results of a recent report
(Lee et al, 2003). The levels
of actin and -tubulin remained relatively stable during
AF-2364–induced germ cell loss from the seminiferous epithelium, but a
mild increase in vimentin was detected
(Figure 8A). Immunohistochemistry was performed using testes from control and
AF-2364–treated rats for localizing zyxin. These tests confirmed the
results from the immunoblotting analysis
(Figure 8B). Virtually no
immunoreactive zyxin was detected in the spermatocytes from the seminiferous
epithelium 7 days after the AF-2364 treatment. At this time, the
elongating/elongate spermatids, round spermatids, and spermatocytes had become
detached from the epithelium and were released to the seminiferous tubule
lumen.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-catenin,
c-Src, cortactin, integrin-linked kinase, and focal adhesion kinase (FAK) are
components of the multiprotein complexes at the apical ES site in the testis
(Wine and Chapin, 1999;
Chapin et al, 2001; Mulholland et al, 2001;
Lee et al, 2003;
Siu et al, 2003b). Subsequent
in vitro and in vivo studies have shown that AJ dynamics are regulated, at
least in part, via the integrin/cadherin/testin/pFAK/PI 3-kinase
(phosphatidylinositol 3-kinase)/p130cas (an adaptor protein of Mr
130 kd encoded by the Crkas gene, a Crk-associated protein, with an
SH3 domain) (Chen et al, 2003;
Lee et al, 2003; Siu et al, 2003b) and the
integrin/RhoB/ROCK/LIMK/cofilin (Lui et
al, 2003a) signaling pathways. As such, a thorough understanding
of the molecular architecture of the AJ complexes, particularly of the
adaptors that can recruit peripheral signaling molecules to the site of the
ES, will be crucial to the study of the regulation and biology of AJ dynamics.
Hereby, we have shown that zyxin, axin, and WASP indeed associate with the
cadherin/catenin complex but not with the nectin/afadin complex or the
ß1-integrin–mediated complex in the seminiferous epithelium. This
study used coimmunoprecipitation to show that zyxin structurally associates
with -actinin, which implies that it indirectly links to the
cadherin/catenin complex via its binding with -actinin, which in turn
binds to -catenin. Such linkage is likely because zyxin has been shown
to interact directly with -actinin in other epithelia
(Li and Trueb, 2001). Axin and
WASP may interact with components of the cadherin/catenin complex directly or
via other associated proteins.
|
Interestingly, zyxin, axin, and WASP also have direct structural association with the underlying actin cytoskeleton, which implies that they have roles in the regulation of actin dynamics. Zyxin is known to bind VASP, which in turn docks profilin and monomeric actin to the junction site in other epithelia (for reviews, see Beckerle, 1998; Reinhard et al, 2001). We have examined whether VASP is an adaptor that tethers actin to the cadherin/catenin complex via zyxin. Surprisingly, no VASP was detected in the testis in a series of coimmunoprecipitation experiments. This result suggests that the testis is using another protein (or proteins) to substitute for the functional role of VASP, as found in other epithelia. It is likely that WASP is being used to replace the tethering function of VASP in the testis because of its structural association with the cadherin/catenin complex and its ability to bind profilin (for a review, see Mullins, 2000). Indeed, zyxin was shown to associate with WASP and vice versa, as reported herein. Furthermore, WASP can regulate actin dynamics via its interaction with and activation of the Arp2/3 complex. For instance, WASP in its native form is unfolded upon its activation by PI(4,5)P2 and Cdc42 GTPase. This exposes the binding sites for Arp2/3, which facilitates the assembly and branching of the actin filament (for reviews, see Mullins, 2000; Caron, 2002). Less is known about the functional role of axin in the actin network; it may act either as a scaffold to bring other peripheral proteins to the site of the actin filament or as a regulator of ß-catenin, controlling the stability of the cadherin/catenin/actin complex (for a review, see Seidensticker and Behrens, 2000). Collectively, these data clearly imply that zyxin, axin, and WASP regulate actin cytoskeleton dynamics at the AJ site in the testis.
Other studies have shown that AJ dynamics are regulated by kinases,
phosphatases, cytokines, proteases, and protease inhibitors, resulting in
either the assembly or the disassembly of the AJ complexes (for reviews, see
Cheng and Mruk, 2002;
Lilien et al, 2002). In
general, the dissolution of the AJ structural protein complexes, such as the
cadherin/catenin and integrin/laminin complexes, and the eventual uncoupling
from the actin cytoskeleton depend largely on their phosphorylation status
(for reviews, see Gumbiner,
2000; Cheng and Mruk,
2002). For instance, when pervanadate-treated leukemia cells with
enhanced tyrosine phosphorylation of cadherins and catenins (note: sodium
vanadate is a PTPi) were examined, a significant reduction in -catenin
binding to E-cadherin was detected (Ozawa
and Kemler, 1998). Furthermore, increased phosphorylation of
ß-catenin by recombinant c-Src can lead to cell-cell dissociation
(Roura et al, 1999).
Collectively, the results illustrate that the integrity of the cell adhesion
function of the AJ functional units (eg, the cadherin/catenin, the
nectin/afadin, and the integrin/laminin complexes) depends at least in part on
the phosphorylation status of the constituent proteins. Since zyxin
(Crawford and Beckerle, 1991), axin (Ikeda et al, 1998), and
WASP (for a review, see Oda and Ochs,
2000) are phosphoproteins per se and are putative substrates of
kinases and phosphatases, they are likely subjected to a similar regulatory
mechanism. The fact that these 3 adaptors are structurally associated with the
cadherin/catenin complex seemingly suggests that their role in regulating the
cell adhesion function occurs through changes in their phosphorylation status.
In turn, the binding of the adaptors with cadherin and its associating
proteins is affected, causing a disruption or an enhancement of the adhesion
function of the cadherin/catenin complex. Equally important, we have shown
that c-Src, an AJ nonreceptor protein tyrosine kinase, structurally associates
with zyxin, axin, and WASP, which further suggests that they are substrates of
this protein kinase at the site of AJs.
Zyxin, Axin, and WASP Are Structural Adaptors and Functional Linkers Between the Cadherin/Catenin Complex and the Actin Filaments, Intermediate Filaments, and Microtubules in the Seminiferous Epithelium
Actin microfilaments, intermediate filaments, and microtubules are the 3
cellular cytoskeletons in the testis. Unlike the actin network, less is known
about the regulatory functions of zyxin, axin, and WASP in the intermediate
filament-based and microtubule-based cytoskeletons. We have shown that zyxin,
axin, and WASP associate with vimentin (a structural component of the
intermediate filament) and -tubulin (a subunit of the microtubule).
Several recent studies have suggested that axin and WASP are crucial to the
regulation of microtubule dynamics. First, axin sequesters GSK-3ß away
from tau, a microtubule-associated GSK-3ß substrate, altering its
phosphorylation and microtubule dynamics
(Stoothoff et al, 2002).
Second, WASP is involved in the regulation of microtubules during podosome
formation in primary human macrophages
(Linder et al, 2000). On the
other hand, these adaptors also regulate the function of different
cytoskeletal networks simply by providing a direct linkage of these networks.
For instance, the direct interaction between fimbrin, an actin
filament-binding protein, and vimentin has been confirmed in macrophages by
studies of coimmunoprecipitation and colocalization
(Correia et al, 1999).
Plectin, an intermediate filament structural protein, was also shown to act as
a link that connects the intermediate filament to microtubules and actin
cytoskeletons (Svitkina et al,
1996). These observations clearly illustrate that one cytoskeletal
network can directly affect the other networks via these interacting linkage
proteins, such as zyxin, axin, and WASP. Collectively, these proteins may
provide the crucial linkages for the cross-talking that must occur in the
seminiferous epithelium among the 3 cytoskeletons—namely, the actin, the
intermediate filament, and the microtubule networks.
Does the Classic Cadherin/Catenin Complex in the Testis Use the Actin Filament and/or Intermediate Filament as an Attachment Site?
The actin filament is the underlying cytoskeleton and attachment site for
classic cadherins, such as N-cadherin and E-cadherin, in many organs and
tissues, including the brain (for a review, see
Gumbiner, 2000), as
illustrated in this report. In light of the complexity and uniqueness of the
junction structures and arrangements in the testis, whether classic cadherins
are actin based or vimentin based remains a subject of dispute. To date,
several transmembrane proteins have been identified at the ES site, which is a
testis-specific actin-based type of AJ. These proteins include
6-integrin, ß1-integrin, testin, cadherins, and nectins (for
reviews, see Cheng and Mruk,
2002; Takai and Nakanishi,
2003). However, classic cadherins have been shown to associate
with the intermediate filament-based desmosome-like junctions in the testis
(Mulholland et al, 2001;
Johnson and Boekelheide,
2002b). In contrast, 2 other reports have demonstrated that the
cadherin/catenin complex is actin based
(Wine and Chapin, 1999;
Lee et al, 2003). For
instance, studies using coimmunoprecipitation have shown that N-cadherin
structurally associates with ß-catenin, forming a functional
cadherin/catenin complex that uses actin filament as the attachment site
(Lee et al, 2003). In light of
these intriguing findings, we hypothesized that classic cadherin might indeed
structurally link to vimentin-based intermediate filament via the
vimentin-associating adaptors, such as zyxin, axin, and WASP. Indeed, we have
shown that a small amount of cadherin (eg, E-cadherin) is associated with
vimentin in the seminiferous epithelium using a Leydig cell-free seminiferous
tubule for lysate preparation. Surprisingly, a much higher amount of vimentin
can be retrieved with an anti-N-cadherin or E-cadherin antibody using testis
lysates. This suggests that cells (probably Leydig cells and others, such as
myoid cells, residing outside the seminiferous epithelium) are using vimentin
as the attachment sites for the classic cadherin/catenin protein complexes. In
conclusion, the data reported support our earlier finding that classic
cadherins within the seminiferous tubules are indeed largely actin based, with
only 5% or less associated with the vimentin-based intermediate filament,
whereas a larger portion (50%) of classic cadherins associate with the
intermediate filament component in the testis but outside the blood-testis
barrier.
Is the Loss of Zyxin, Axin, and WASP From the Seminiferous Epithelium Following AF-2364 a Result of Cytotoxicity?
While the physiological significance of the AF-2364–induced loss of
zyxin, axin, and WASP from the seminiferous epithelium at the time germ cells
are also depleted from the epithelium is not entirely understood, it does not
appear to be a result of cytotoxicity. First, an induction of N-cadherin, in
contrast to these adaptors, was detected in the seminiferous epithelium
following AF-2364 treatment. This is consistent with several earlier reports,
which showed that the loss of cell adhesion function between Sertoli and germ
cells that was induced by AF-2364 is also accompanied by an induction of
cadherins and catenins (Chen et al,
2003; Lee et al,
2003). Second, the levels of actin and tubulin remained relatively
stable throughout the treatment period. Third, and perhaps most significantly,
the use of AF-2364 at the dosing reported (50 mg/kg bw) or at dosings up to 20
times higher (ie, Irwin dose range: 100–1000 mg/kg bw, single dose) has
safely passed the acute toxicity test in mice and rats conducted by licensed
toxicologists (Cheng, unpublished data). Additionally, genotoxicity tests
performed by licensed toxicologists according to Food and Drug Administration
guidelines have shown that a single dose of AF-2364 (1, 10, 1000, or 2000
mg/kg b.w. by gavage) failed to induce a significant increase in
micronucleated polychromatic erythrocytes in either male or female mice. Thus,
AF-2364 is negative in the mouse micronucleus assay in vivo. Furthermore,
AF-2364 (at 12.5–75 µg/mL) is negative for the induction of numerical
chromosome aberrations in CHO cells in vitro in both the nonactivated and
activated test systems (Cheng, unpublished data). These results are also
consistent with recent serum microchemistry findings that AF-2364 is neither
nephrotoxic nor hepatotoxic to rats at doses (up to 50 mg/kg bw) effective
enough to induce reversible infertility
(Cheng et al, 2001;
Grima et al, 2001). It is
likely that AF-2364 suppresses these adaptors at the site of
Sertoli–germ cell AJs, disrupting the recruitment of AJ-associated
proteins to these sites and thereby perturbing the cell adhesion function in
the epithelium. However, this possibility must be vigorously investigated in
future studies.
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Acknowledgments |
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Footnotes |
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References |
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