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Population Council (W.Y.L., C.Y.C.), Center for Biomedical Research, New York, New York 10021; and Department of Zoology (W.Y.L., W.M.L.), The University of Hong Kong, Hong Kong, China
Address all correspondence and requests for reprints to: C. Yan Cheng, Population Council, 1230 York Avenue, New York, New York 10021. E-mail: ycheng{at}popcbr.rockefeller.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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can induce disruption of
TJ-permeability barrier, possibly by reducing or altering the
distribution of ZO-1 or occludin at the sites of TJs in rat striatal
endothelium and mammary epithelium (for review, see 13), illustrating the crucial role of cytokines in TJ
regulation. Several studies have shown that cytokines such as TGF-ß, basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) are synthesized and expressed by Sertoli cells; they also regulate multiple testicular functions such as somatic and germ cell division (14, 15). TGF-ß has also been implicated in initiating spermatogenesis (16). The TGF-ß superfamily is composed of many multifunctional cytokines, which include TGF-ßs, activins, inhibins, bone morphogenetic proteins, and others, which exhibit wide ranges of effects on cell growth, differentiation, adhesion, tissue morphogenesis, and matrix production (for reviews, see 17, 18, 19, 20). TGF-ß1, TGF-ß2, and TGF-ß3 are highly conserved, but distinct, isoforms. TGF-ß isoforms display similar, although not identical, biological activity and differential tissue expression (16, 21). TGF-ß3 messenger RNA (mRNA) expression is the most abundant form of TGF-ß present in the testis; its expression peaked at an early pubertal stage, at 20 days of age, coinciding with the onset of spermatogenesis (16). In view of diversified effects of TGF-ß on testicular function, we sought to investigate if this cytokine participates in regulating the events of inter-Sertoli TJ assembly. In the present study, we assessed: 1) the expression of TGF-ß isoforms at the time inter-Sertoli cell junctions were assembled; 2) whether TGF-ß3 can perturb the assembly of inter-Sertoli TJ permeability barrier; and 3) its effects on the cellular expression of TJ-associated genes at the time of inter-Sertoli TJ assembly.
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Materials and Methods |
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Preparation of Sertoli cells for culture experiments
Sertoli cell cultures. Primary Sertoli cells were
isolated from 20-day-old Sprague Dawley rats as previously described
(5). Freshly isolated Sertoli cells were cultured at high
cell density (0.5 x 106
cells/cm2) on Matrigel (Collaborative Research, Inc., Bedford, MA)-coated 12-well dishes (effective
surface area, 3.83 cm2; containing 3 ml medium,
2 x 106 Sertoli cells in serum-free Ham’s F12
nutrition mixture and DMEM (F12/DMEM) (1:1, vol/vol) supplemented with
gentamicin (20 mg/liter), 15 mM HEPES, sodium bicarbonate
(1.2 g/liter), bovine insulin (10 µg/ml), human transferrin (5
µg/ml), epidermal growth factor (2.5 ng/ml), and bacitracin (5
µg/ml). Cells were then incubated at 35 C in a humidified atmosphere
of 95% air-5% CO2 (vol/vol) and treated as
cultures at time zero. To obtain Sertoli cell cultures with purity
greater than 98%, cells were hypotonically treated with 20
mM Tris (pH 7.4), for 2.5 min, to lyse contaminating germ
cells (22) 36 h after plating. The wells were washed
twice with F12/DMEM. Media were replaced every 24 h, and cells
were incubated for an additional 6–7 days. In those experiments where
we correlate cellular gene expression and the assembly of inter-Sertoli
TJs, cells were terminated at each time point within a set of
experiments for RNA extraction, and all samples within an experiment
were processed simultaneously for semiquantitative RT-PCR to eliminate
interexperimental variations. Total RNA was extracted from Sertoli
cells using RNA STAT-60 (Tel-Test "B" Inc.,
Friendswood, TX). To obtain Sertoli cell-enriched culture medium
(SCCM), Sertoli cells were cultured at 5 x
104 cells/cm2 in 100-mm
Petri dishes (4.5 x 106 cells/9 ml
F12/DMEM/100-mm dish) and incubated in a humidified atmosphere of 95%
air-5% CO2 (vol/vol) at 35 C for 4 days. Spent
media were collected, centrifuged at 800 x g for 30
min to remove residual Sertoli cells, followed by 3000 x g
for 45 min, to remove cellular debris, concentrated by a
Millipore Corp. (Bedford, MA) Minitan tangential
ultrafiltration unit equipped with eight Minitan plates
(Mr cut-off at 10K), and filtered through
0.2-µm filter units as described (28, 29). Protein
estimation was performed by Coomassie blue-dye binding assay using BSA
as a standard (23).
Sertoli cells cultured with recombinant human TGF-ß3 and bFGF protein. Sertoli cells, prepared as described above, were cultured on Matrigel-coated 12-well dishes at a density of 0.5 x 106 cells/cm2. To study the effects of cytokines on cellular gene expression at the time of inter-Sertoli TJ assembly, recombinant human TGF-ß3 protein (0.1 and 3 ng/ml; Calbiochem, La Jolla, CA) or recombinant human bFGF protein (0.01 and 3 ng/ml; PeproTech Inc., Rocky Hill, NJ) were added to Sertoli cell cultures immediately after their isolation. To prepare the stock solution, TGF-ß3 (2 µg/ml) and bFGF (100 µg/ml) were resuspended in 4 mM HCl/0.1% BSA and 5 mM Tris (pH 7.6), respectively, and they were aliquoted to F12/DMEM to desired concentrations. Cultures were hypotonically treated 36 h thereafter. Media containing TGF-ß3 or bFGF were replaced every 24 h, both in the culture dishes. Each dish had 3-ml F12/DMEM. Cultures were terminated at specific time points by RNA STAT-60. Control cultures were exposed to a similar amount of vehicle used to resuspend the recombinant protein.
Effects of human recombinant TGF-ß3 or bFGF protein on the
assembly of inter-Sertoli TJs. Sertoli cells, isolated as
described above, were cultured at high cell density to allow the TJ
assembly. Briefly, 1.2 x 106
cells/cm2 were plated on Matrigel-coated HA
filters in the apical compartment of a bicameral unit (Millipore Corp.) and treated as day zero (6, 24). To assess
the assembly of inter-Sertoli TJs, transepithelial electrical
resistance (TER) across the Sertoli cell epithelia was quantified using
a Millicell electrical resistance system (Millipore Corp.). Briefly, a pulse of current (20 µA) was applied across
the Sertoli cell epithelial between two sliver-sliver chloride
electrodes, and the resistance was determined. The resistance was
multiplied by the areal surface of the filter (0.6
cm2) to yield the areal resistance (in
ohm·cm2). The net value of electrical
resistance was calculated by subtracting the background, which was
measured by the Matrigel-coated cell-free bicameral units (6, 9). In most experiments, the first TER measurement was taken
24 h after cells were plated (i.e. day 1) so that
Sertoli cells were not disturbed at the time of their attachment to the
substratum (Matrigel) when forming adherens junctions. Preliminary
experiments have also shown that when TER was taken from bicameral
units at 3–6 h after cell plating, the subsequent TER readings to
monitor the inter-Sertoli TJ-permeability barrier were at least 10
ohm·cm2 lower than cultures without having such
disturbance, unless these TER readings were taken in extra bicameral
units such as those shown in Fig. 1C
. This practice, however increased the
cost of each experiment. To assess the effects of TGF-ß3 on the
assembly of inter-Sertoli TJ permeability barrier, Sertoli cells were
exposed to TGF-ß3 at concentrations ranging between 0.005 and 3 ng/ml
or bFGF (0.01–3 ng/ml) soon after their isolation. Media containing
TGF-ß3 were replenished, every 24 h, in both the apical (0.5 ml)
and basal (0.5 ml) compartments. Controls included Sertoli cells
cultured alone in F12/DMEM without any cytokines or with vehicle (4
mM HCl/0.1% BSA). TER across the Sertoli cell
epithelia was determined at specific time points. Each time point had
replicate cultures, and each experiment was repeated 2–3 times using
different batches of Sertoli cells. In selected experiments, the
specificity of TGF-ß3 treatment was assessed. TGF-ß3 was removed by
washing cells, 2.5 days after their incubation with TGF-ß3, to
investigate whether its removal could restore the disrupted
inter-Sertoli TJ permeability barrier.
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Preparation of SCCM and Sertoli cell lysates
Sertoli cells, prepared as described above, were cultured on
Matrigel-coated 12-well dishes, at a density of 1 x
106 cells/cm2, for a period
of up to 7 days, to allow the assembly of inter-Sertoli TJs. A set of
two dishes was terminated each day to collect SCCM and to prepare cell
lysate. To obtain cell lysates, cultures were briefly rinsed with 1 ml
lysis buffer (17 mM MOPS, pH 6.0, containing 250
mM sucrose, 25 mM EDTA, 1.0% Triton X-100, 0.2
µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 10
mM dithiothreitol, and 5 mM
MnCl2). Cells were then resuspended in 1 ml lysis
buffer and incubated at 4 C for 5 min to burse cells and solubilize
membrane proteins. Samples were centrifuged at 15,000 x
g for 5 min at 4 C. The clear supernatant was collected and
used as total cell lysates. Protein estimation was performed by
Coomassie blue-dye binding (23) using BSA as a
standard.
Immunoblot analysis
Protein (10–200 µg) derived from SCCM and Sertoli cell
lysates was resolved onto 15% T SDS-polyacrylamide gels
under reducing conditions, along with recombinant TGF-ß3, which is a
known product of Sertoli cells (16). After
electrophoresis, proteins were electroblotted onto nitrocellulose
paper. The presence of TGF-ß3 protein was detected by immmunoblots,
using a rabbit antihuman TGF-ß3 antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), essentially as previously
described (28, 29).
Detection of multiple-target gene mRNAs and their changes during
the assembly of inter-Sertoli TJs, with and without cytokines, by
sequential use of RT-PCR
Semiquantitative RT-PCR was performed, essentially as
previously described (4, 5, 6, 7, 8, 30), to detect changes in the
expression of selected target genes. Briefly, approximately 2 µg
total RNA was reverse-transcribed into complementary DNA (cDNA)s with 5
µg oligo(dT)15 using a Moloney murine leukemia
virus RT kit (Promega Corp., Madison, WI) in a final
reaction vol of 25 µl. PCR was routinely performed by combining 3
µl of the RT product with 0.4 µg each of the sense and antisense of
selected target gene primer pairs coamplified with the rat ribosomal
S16 primer pair (Table 1).
Coamplification with S16 was included to ensure that equal amounts of
RNA were reverse transcribed and amplified in each reaction tube. These
reagents were mixed with 5 µl 10x PCR buffer, 3 µl
MgCl2 (25 mM),
8 µl deoxynucleotide triphosphates (200 µM each of
deoxy-ATP, deoxy-GTP, deoxycytidine triphosphate, and thymidine
5'-triphosphate), 2.5 U Taq DNA polymerase (Promega Corp.), and sterile double-distilled water, to a final vol of 50
µl. The cycling parameters for PCR reaction were as follows:
denaturation at 94 C for 1 min, annealing at 56-63 C (depends on the
target genes) for 2 min, and extension at 72 C for 3 min, for a total
of 21–24 cycles, which were followed by an extension period at 72 C
for 15 min in a Perkin-Elmer Corp. (Norwalk, CT)
thermal cycler. To enhance the detection limit and to yield data for
semiquantitative analysis after densitometric scanning, PCR was
performed by the inclusion of trace amounts of
[
-32P]-labeled primers. Briefly, the sense
primer of target genes and S16, was labeled at the 5'-end with
[-32P]-deoxy-ATP (specific activity, 6000
Ci/mmol, Amersham Pharmacia Biotech, Arlington Heights,
IL) using T4 polynucleotide kinase
(Promega Corp.). Approximately, 1 x
106 cpm were used per PCR reaction. To ensure the
linearity in the synthesis of both target genes, such as TGF-ß and
S16 in the PCR, 10-µl-aliquots of PCR products at 18, 20, 22, 24, and
26 cycles were withdrawn and resolved onto 5% T
polyacrylamide gels using 0.5x TBE (44.5 mM
Tris-borate, 1 mM EDTA, pH 8.0) as a running
buffer in preliminary experiments. Also, different concentrations of RT
products and primer pairs were used in preliminary experiments to
assess the appropriate conditions to be used, so that the production of
both target genes and S-16 was in the linear phase. After gel
electrophoresis, PCR products were visualized by ethidium bromide
staining, and autoradiography was performed using X-OMAT AR x-ray film
(Eastman Kodak Co., Rochester, NY). The authenticity of
the PCR products, which include TGF-ß2, TGF-ß3, bFGF, ZO-1, and
occludin, claudin-11, was verified by direct nucleotide sequencing
after their subcloning in pGEM-T vector as previously described
(8, 10). Autoradiograms from 3–4 separate experiments
were densitometrically scanned at 600 nm using an Ultroscan XL Laser
Densitometer (Model LKB 2222–020; Amersham Pharmacia Biotech), normalized against S-16, and used for statistical
analysis.
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2 x
106 cells/3 ml F12/DMEM, prepared as described
above, were harvested at specific time points by resuspending cells in
1 ml PBS (10 mM sodium phosphate, 0.15 M NaCl,
pH 7.4, at 22 C). Cells were then sonicated using an ultrasonic
homogenizer (Model CP50; Cole Parmer, Chicago, IL) at 40–60
amplitudes, 6 strokes, and 15 sec, on ice, in 1.5 ml
Eppendorf microcentrifuge tubes (VWR, Bridgeport,
NJ). A sample (1 ml) was obtained from each culture well. To 50 µl
of the sonicated sample (0.1 x 106
cells), 50 µl of 0.05-M sulfuric acid was added, and
hydrolysis was performed at 100 C for 1 h. After cooling for 5
min, 15 µl periodate reagent (0.1 M sodium metaperiodate
in 9 M phosphoric acid) was added to the above sample and
incubated at room temperature for 20 min. Thereafter, 100 µl arsenite
solution (0.5 M sodium sulfate, containing 10% sodium
arsenite and 0.05 M sulfuric acid) was added, vortexed, and
incubated at room temperature for 10 min. The samples were then mixed
with 300 µl thiobarbituric acid (0.6% thiobarbituric acid in 0.5
M sodium sulfate) and incubated at 100 C for 15 min. After
cooling the samples to room temperature, 500 µl isoamyl alcohol was
added, and they were mixed and centrifuged to facilitate separation of
the immiscible solvent. The pinkish top isoamyl alcohol layer
was then read at 490 nm.
Cloning and sequencing of rat claudin-11
While the mouse claudin-11 sequence is known, the rat
claudin-11 nucleotide sequence was lacking, we sought to clone it by
PCR. The cDNA cloning and sequencing strategy of the full-length rat
claudin-11 was essentially as previously described (7, 8).
A single fragment (corresponding to nucleotide 1–624) was synthesized
by PCR from a rat Sertoli cell cDNA expression library. The claudin-11
specific primers: 5'-ATGGTAGCCACTTGCCTTC-3' (sense primer corresponding
to nucleotides 1–19) and 5'-TTAGACATGGGCACTCTTGG-3' (antisense primer
corresponding to nucleotides 605–624), were designed according to the
known mouse claudin-11 full-length cDNA (32), which
yielded a PCR product of 624 bp. The cDNAs with the expected size were
electroeluted from the gel and subcloned into pGEM-T vector
(Promega Corp.) and sequenced by the dideoxynucleotide
chain termination method using Sequenase (Amersham Pharmacia Biotech) as described previously (7, 8). The entire
rat claudin-11 sequence was verified by using three different cDNAs
isolated in different PCR experimental sessions, and the differences
between the rat and mouse cDNA sequence shown in Fig. 2 did not seem to be the result of PCR
artifacts.
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, B, D, and F; Fig. 4B; Fig. 5B; and Fig. 6B) and the effects of recombinant
TGF-ß3 or bFGF on the cellular expression of occludin, ZO-1, and
claudin-11 at the time of inter-Sertoli TJ assembly (Fig. 4, D and F;
Fig. 5, D and F; and Fig. 6D), statistical analyses were performed by
ANOVA with Tukey’s honestly-significant-difference test using the
GB-STAT statistical analysis software package (version 7.0) from
Dynamic Microsystems, Inc. (Silver Spring, MD). Using Tukey’s test for
ANOVA, results of individual samples were compared with the endogenous
mRNA level of the corresponding target gene at time zero, when cells
were plated, as well as samples within the same group with
(e.g. Fig. 4, D and F; Fig. 5, D and F; Fig. 6D) or without
(e.g. Fig. 3, B, D, and F; Fig. 4B; Fig. 5B; Fig. 6B)
cytokine treatment, over the course of the experiment. Each experiment
was repeated 2–3 times, and each time point had replicate cultures.
Data were expressed as mean ± SD (n =
4 or 6). For results shown in Fig. 1
and Fig. 4G, each data point in
treatment groups (or vehicle control group) was compared with the
corresponding control, by Student’s t test, using the
GB-STAT software package.
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Results |
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, A and B) and TGF-ß3
(Fig. 3, C and D), but not bFGF (Fig. 3, E and F), coinciding with the
assembly of inter-Sertoli TJs (Fig. 3 vs. Fig. 1A). When the
TER across the Sertoli cell epithelia was monitored, there was a steady
rise in TER between approximately 3 h post plating and days 3–4
(Fig. 1 A–C), illustrating the assembly of the inter-Sertoli
TJ-permeability barrier. On days 5–8, after the inter-Sertoli TJs had
been assembled, as manifested by a stable TER across the Sertoli cell
epithelia, the expression of TGF-ß2 and TGF-ß3 were shown to be
induced by as much as 2- to 3-fold (Fig. 3). However, there were no
significant changes in the expression of bFGF throughout the entire
culture period (Fig. 3, E and F), seemingly suggesting that TGF-ß2
and -ß3, but not bFGF, may be involved in regulation of the assembly
and maintenance of inter-Sertoli TJs.
Detection of TGF-ß3 protein in Sertoli cell-conditioned media
(SCCM) and Sertoli cell total cell lysates at the time of inter-Sertoli
TJ assembly
Because there was a change in TGF-ß3 expression at the
time of inter-Sertoli assembly, and that TGF-ß3 is a known Sertoli
cell product (16), it is worthwhile to investigate the
relative amounts of TGF-ß3 gene product at the time when TJs are
assembled. The presence of TGF-ß3 was assessed by immunoblots using
10–200 µg protein from SCCM and Sertoli cell lysates. However, no
immunoreactive TGF-ß3 could be detected by using as much as 200 µg
total proteins from SCCM, because of the inherent low detection limit
of immunoblots vs. RT-PCR (Fig. 3G vs. Fig. 3, A–F). As shown in Fig. 3G, it took as much as 50 ng recombinant
TGF-ß3 to be visualized by immunoblots (Fig. 3G, lanes 6–8) when
numerous proteins in SCCM or Sertoli lysates were detected by silver
staining (Fig. 3G, lanes 16 and 17), whereas PCR can amplify a single
copy rat sequence to as much as 0.1–1 µg DNA in 25–35 cycles
(33). In the next section, it showed that as little as 3
ng recombinant TGF-ß3 could perturb the inter-Sertoli TJ permeability
barrier by as much as 70%. As such, these protein data could not
negate results shown in Fig. 3, A–F, because of the limited
sensitivity of the immunoblot technique.
Effects of recombinant TGF-ß3 on the assembly of inter-Sertoli TJ
permeability barrier in vitro
Because there was a significant decrease in TGF-ß2 and -ß3
expression when inter-Sertoli TJs were assembled in vitro,
we sought to examine whether the presence of TGF-ß3 could perturb the
inter-Sertoli TJ assembly by quantifying TER across the Sertoli cell
epithelia at specified time points. It is noted that inter-Sertoli TJs
began to assemble soon after cells were plated in vitro, as
manifested by a rapid increase in TER across the epithelia (Fig. 1A).
The TER reached plateau within 4 days, with a reading of 55–65
ohm/cm2, which is consistent with previously
published results (6, 9). However, inclusion of TGF-ß3
perturbed the assembly of inter-Sertoli TJ permeability barrier
in vitro. The presence of TGF-ß3 yielded a dose-dependent
inhibitory effect on the assembly of inter-Sertoli TJs (Fig. 1A). The
effects of TGF-ß3 on the assembly of TJ permeability barriers
apparently were not the result of cell cytotoxicity, because Sertoli
cells were capable of reassembling the TJ permeability barrier on
removal of TGF-ß3, at day 2.5, by two successive washings (Fig. 1C).
Also, the effects of TGF-ß3 seem to be specific, because bFGF had no
effect on the assembly of inter-Sertoli TJs (Fig. 1B).
Effects of human recombinant TGF-ß3 on the expression of occludin
in cultured Sertoli cells when inter-Sertoli TJs were assembled in
vitro
We next sought to examine the mechanism by which TGF-ß3
mediates its effects on the inter-Sertoli TJ permeability barrier, as
shown in Fig. 1. We hypothesize that TGF-ß3 may induce disruption of
TJ-associated gene expression. Recent studies from this laboratory have
shown that there was a significant, but transient, increase in the
expression of occludin (Chung and Cheng, unpublished observations) and
ZO-1 (9, 10) at the time of inter-Sertoli TJ assembly
in vitro. If such timely expression can be halted by
TGF-ß3, it may be the cause of TJ-disruption, because these molecules
are required to maintain the dynamic nature of inter-Sertoli TJs. We
thus examined the effects of TGF-ß3 on the expression of several TJ
proteins, such as occludin, ZO-1, and claudin-11. As with ZO-1, there
was a 2-fold increase in occludin steady-state mRNA levels in rat
Sertoli cells cultured in vitro (Fig. 4, A and B) when
inter-Sertoli TJs were assembled (Fig. 1A). However, both 0.1
(data not shown) and 3 ng/ml TGF-ß3 were capable of suppressing the
transient increase in occludin expression when inter-Sertoli TJs were
assembled (Fig. 4, C and D vs. Fig. 4, A and B), but the
endogenous occludin steady-state mRNA level (i.e. the level
of occludin expression on day zero, at the time Sertoli cells were
plated) throughout the entire culture period was found not to be
affected by the presence of TGF-ß3 (Fig. 4, C and D vs.
Fig. 4, A and B). This TGF-ß3-induced suppression in occludin
expression probably was not the result of cell death induced by
TGF-ß, because the total DNA content in TGF-ß3-treated cells at all
time points was not significantly different from control cultures
without TGF-ß3 (Fig. 4G). And Sertoli cells cultured under the same
conditions, with the presence of bFGF (3 ng/ml) rather than TGF-ß3,
exhibited no inhibitory effect on the expression of occludin
steady-state mRNA levels (Fig. 4, E and F).
Effects of TGF-ß3 on the expression of ZO-1 in cultured Sertoli
cells when inter-Sertoli TJs were assembled in vitro
Recent occludin gene knockout studies illustrated that the
occludin-deficient epithelial cells are still capable of forming a
well-developed network of TJ strands (32), suggesting that
other TJ-associated proteins (such as claudin-11, ZO-1, and other
yet-to-be identified TJ proteins) can supersede its role in
constructing and maintaining TJs. Recent studies from this laboratory
have also illustrated a timely expression of ZO-1 coinciding with the
assembly of inter-Sertoli TJs (10). These results thus
suggest that ZO-1 also participates in TJ assembly. It is therefore
worthwhile to study the effects of TGF-ß3 in regulating the
expression of TJ-associated peripheral proteins such as ZO-1. The
expression of ZO-1 increased by at least 5-fold on days 1–2 (Fig. 5, A
and B) in control cultures consistent with an earlier paper
(10) reporting an induction of ZO-1 at the time TJs were
assembled, possibly by furnishing the building blocks needed to
assemble TJs. The presence of TGF-ß3 at 3 ng/ml, however, eliminated
the transient ZO-1 induction on days 1–2 (Fig. 5, C and D
vs. Fig. 5, A and B). Its presence also reduced the basal
ZO-1 steady-state mRNA level by as much as 5-fold (Fig. 5, C and D).
The presence of bFGF in Sertoli cell cultures was shown to have no
inhibitory effect on the expression of ZO-1 steady-state mRNA levels
(Fig. 5, E and F vs. Fig. 5, A and B).
Effects of TGF-ß3 on the expression of claudin-11 in cultured
Sertoli cells when inter-Sertoli TJs were assembled in vitro
Claudins (Mr, 22K) are TJ-integral
proteins, and at least 20 claudins have been identified in various TJs
in different epithelia (for reviews, see 34, 35).
Previous studies by Northern analyses have shown that testes express at
least 7 claudins, including claudin-1, -3, -4, -5, -7, -8, and -11
(36, 37, 38, 39). Claudin-11 was selected in this study because it
is found abundantly in the testis and was localized to the TJ strands
of Sertoli cells when examined by immunogold electron microscopy
(32). Also, claudin-11 was detected exclusively in the
brain (32, 38) and the choroids plexus and the collecting
ducts in the kidney (38), besides in testis. Unlike other
TJ-associated proteins, such as occludin and ZO-1, the claudin-11
steady-state mRNA level remained relatively steady throughout the
entire culture period, even when TJs were being assembled (Fig. 6, A
and B). Also, there was a consistent decline in claudin-11 expression
on day 7–9, when inter-Sertoli TJs were already assembled (Fig. 6, A
and B vs. Fig. 1A). Addition of TGF-ß3 at 3 ng/ml,
however, suppressed the expression of claudin-11 by as much as 4-fold
in all time points throughout the experiment (Fig. 6, C and D) in a
pattern similar to that of occludin and ZO-1 (Fig. 6 vs.
Figs. 4 and 5), whereas TGF-ß3 (at a lower dose of 0.1 ng/ml) had no
apparent effects on claudin-11 expression (data not shown). The
presence of bFGF in Sertoli cell culture was shown to have no
inhibitory effect on the expression of claudin-11 steady-state mRNA
levels (Fig. 6E).
Nucleotide sequence and primary structure analysis of rat
claudin-11
Because the claudin-11 sequence in the rat is not known, we have
isolated the full-length rat claudin-11 cDNA by PCR. Its nucleotide
sequence and the deduced amino acid sequence are shown in Fig. 2. This
claudin-11 cDNA has an open reading frame of 621 bp coding for a
207-amino acid polypeptide. At the amino acid sequence level, rat
claudin-11 displayed a 98.6% homology with the mouse homolog with
differences only in 3 amino acid residues (His63,
Ala78, and Gly95) in the
rat vs. Tyr63,
Val78, and Ala95 in the
mouse; whereas, at the nucleotide level, the rat protein displayed
96.1% identity with the mouse protein (Fig. 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The primary amino acid sequence of rat claudin-11 shows a high homology with the mouse homolog. Out of the 207 amino acids, rat claudin-11 differed by 3 residues, when compared with the mouse counterpart. Such striking conservation reflects the unique function of claudin-11. Previous studies revealed that claudin-11 null mice exhibited both neurological and reproductive deficits (38). Slow CNS nerve conduction and weak hind limb are found in claudin-11 null mice, and male null mice are sterile (38). Claudin-11 is the mediator of parallel-array TJ strands, which distinguishes this protein from other intrinsic TJ-membrane proteins (38). The fact that claudin-11 is required to express at a constant level vs. occludin and ZO-1 suggests its pivotal role in the junction restructuring events. These results, coupled with the effects of TGF-ß in eliminating the timely expression of occludin and ZO-1, at the time when inter-Sertoli TJs were being assembled suggest that the intricate interaction between these molecules contributes significantly to the event of TJ assembly.
Although TGF-ß3 is capable of suppressing the cellular expression of ZO-1, occludin, and claudin-11 when inter-Sertoli TJs are being assembled in vitro, its inclusion in the bicameral unit can only perturb the assembly of TJ permeability barrier but not completely abolish its formation. These observations suggest that, whereas TGF-ß3 is crucial to the assembly of inter-Sertoli TJs, it is not the only paracrine factor that participates in the regulation of TJ assembly. Also it is increasingly clear that the TJs are composed of multiple gene products, such as junction-associated molecules, occludin, and claudins (for reviews, see 34, 35, 42). As such, a TGF-ß3-induced disruption of de novo synthesis of ZO-1 and occludin cannot completely dissociate the inter-Sertoli TJs; rather, it can only perturb the TJ-barrier.
Both Sertoli and germ cells are capable of synthesizing and secreting
multiple growth factors such as FGF, EGF, interferon- and -, and
insulin-like growth factor, which all play a role in regulating
testicular function (for reviews, see 15, 43, 44, 45). For instance, bFGF and EGF have been shown to affect
Sertoli cell plasminogen activator inhibitor-1 expression
(46). EGF can also change cell shape and reorganize the
cytoskeleton, leading to cell adhesion (12). Thus, it is
possible that other yet-to-be-identified cytokines, together with
TGF-ß3, contribute to the assembly of inter-Sertoli TJs via different
regulatory pathways.
The assembly of TJs in other epithelial cells is known to be modulated by a number of signaling molecules, which include cAMP, Ca2+, protein kinase C, G proteins, phospholipase C, and diacylglycerol (47, 48, 49, 50). In addition, the family of Ras-related small GTPase, such as RhoA and Rac1, are involved in modulating the gate and fence functions of the TJ, and they also participate in the spatial organization of TJ proteins (51). Although the signaling molecules involved in the TJ assembly have been partially identified, how these signaling molecules are being triggered, leading to TJ assembly (in particular, their role in regulating junction assembly in the testis) is entirely unknown.
Previous studies have shown that TGF-ß3 can exert its effects via the mitogen-activated protein kinase pathway. The mitogen-activated protein kinase pathway consists of the extracellular signal-regulated kinase pathway and two stress-activated pathways: those of p38 and the c-Jun N-terminal kinase (52, 53). Also, small G proteins, such as Ras and Rho-like proteins (e.g. Rac and Cdc42), provide the link between growth factor signaling and reorganization of the actin cytoskeleton (for reviews, see 54, 55), which is essential for cell adhesion and cell movement. Moreover, it has been reported that EGF and TGF-ß are possible regulators of the Rho GTPase-mediated pathway (12, 56). These small GTPases are also implicated in the signaling pathways that regulate the initiation and turnover of cell-cell adhesion, cell-substratum contact, and junction assembly via their effects on the cellular cytoskeleton network (for reviews, see 57, 58). Current investigation in our laboratory has demonstrated that both Sertoli and germ cells express RhoB, Rac2, and Cdc42 GTPases (Lui and Cheng, unpublished observations), indicating that Sertoli and germ cells are equipped with the needed machinery to link TGF-ß surface receptors to the organization of the actin cytoskeleton network, which, in turn, triggers junction assembly (56).
Apart from the MAP kinase signaling pathway, TGF-ß3 is also known to initiate its signaling process by binding to a serine/threonine kinase receptor II, a constitutively protein kinase. It then recruits, phosphorylates, and signals through type I receptor. The type I receptor kinase then activates intracellular substrates known as SMAD proteins. The receptor-regulated SMADs become phosphorylated by type I receptor. The phosphorylated receptor-regulated SMADs then form complexes with Co-SMAD, translocate into the nucleus, and induce gene transcription via specific transcription factors (18, 19, 20). Work is now in progress to use specific kinase inhibitors to identify the possible signaling pathway of TGF-ß3 in the regulation of TJ-associated protein expression during inter-Sertoli TJ assembly.
Data presented herein demonstrated that TGF-ß3 is an important cytokine in the initiation of the downstream signal transduction pathway in regulating TJ assembly. The evaluation of the function of this cytokine in the testis (in particular, its role in TJ assembly) will yield new insights into understanding testicular physiology and spermatogenesis.
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Received August 22, 2000.
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