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First published online September 5, 2008
doi: 10.1242/10.1242/dev.025627
1 Laboratory of Developmental Genetics and Imprinting, The Babraham Institute,
Babraham Research Campus, Cambridge CB22 3AT, UK.
2 Department of Comparative Biology and Experimental Medicine, Faculty of
Veterinary Medicine, University of Calgary, 3330 Hospital Drive N.W., Calgary,
Alberta T2N 4N1, Canada.
3 Centre for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG,
UK.
* Author for correspondence (e-mail: myriam.hemberger{at}bbsrc.ac.uk)
Accepted 11 August 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-actin
and to disintegrate blood vessels. Consequently, conditional ubiquitous
overexpression of Cts8 leads to midgestational embryonic lethality
caused by severe vascularization defects. In addition, both cathepsins
determine trophoblast cell fate by inhibiting the self-renewing capacity of
trophoblast stem cells when overexpressed in vitro. Similarly, transgenic
overexpression of Cts7 and Cts8 affects trophoblast
proliferation and differentiation by prolonging mitotic cell cycle progression
and promoting giant cell differentiation, respectively. We also show that the
cell cycle effect is directly caused by some proportion of CTS7 localizing to
the nucleus, highlighting the emerging functional diversity of these typically
lysosomal proteases in distinct intracellular compartments. Our findings
provide evidence for the highly specialized functions of closely related
cysteine cathepsin proteases in extra-embryonic development, and reinforce
their importance for a successful outcome of pregnancy.
Key words: Cysteine cathepsins, Placenta, Trophoblast differentiation, Vascular remodelling
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Here, after implantation of the blastocyst, a specialized
trophoblast population termed giant cells initiates an invasive process during
which they penetrate deeply into the surrounding decidualized uterine stroma.
Invasion of trophoblast giant cells is tightly regulated and occurs only in
the area surrounding the ectoplacental cone. It is specifically targeted
towards maternal spiral arteries and results in a remodelling of the uterine
vasculature that is characterized by a number of remarkable features: as far
as several 100 µm outside the main placental border, trophoblast giant
cells displace the endothelial cell lining of maternal spiral arteries and
adopt pseudo-endothelial cell function
(Adamson et al., 2002;
Hemberger et al., 2003). The
perivascular smooth muscle layer and the elastic lamina of spiral arteries are
lost, which enables an extensive dilation of these vessels
(Adamson et al., 2002;
Pijnenborg et al., 2006).
Spatially correlated with the site of trophoblast invasion (and possibly
induced by trophoblast-produced factors), spiral arteries converge to form a
few large canals that funnel maternal blood into the placenta. Thus, giant
cell invasion initiates a cascade of processes that are essential for
development of the foeto-maternal circulatory interface of the placenta.
Trophoblast giant cells differentiate by exiting the mitotic cell cycle and
undergoing repeated rounds of endoreduplication. This process results in
extremely large, highly polyploid cells with a DNA content of up to 1000 N
whose chromosome arrangement may even be, at least in part, polytene
(Goncalves et al., 2003;
Varmuza et al., 1988). Based
on the gestational stage of their differentiation and on gene expression
patterns, giant cells can be grouped into at least four distinct classes
represented by (1) parietal giant cells surrounding the implantation site, (2)
giant cells associated with maternal spiral arteries and (3) with maternal
blood canals, and (4) giant cells within sinusoidal spaces in the placental
labyrinth (Simmons et al.,
2007). Within the uterine bed, invasive characteristics are
displayed only by giant cells associated with maternal spiral arteries. We are
particularly interested in determining the molecular framework that regulates
their differentiation and invasive properties. For this purpose, a cDNA
subtraction library specific for invasive trophoblast was generated and
extensively analysed by array technology
(Hemberger et al., 2001;
Hemberger et al., 2000). Two
of the genes identified from this screen were the cathepsin proteases
Cts7 and Cts8. Intriguingly, expression of these two
proteases was confined to extra-embryonic tissues and exhibited a strict
temporal and spatial correlation with trophoblast giant cell invasion
(Hemberger et al., 2000).
Cysteine cathepsins are a main component of the proteolytic breakdown
machinery in lysosomes. In addition to this general proteolytic function,
cathepsins are also implicated in a variety of specific cellular processes,
such as apoptosis, angiogenesis, cell proliferation and invasion
(Turk et al., 2001). They play
essential physiological roles in antigen presentation, bone remodelling and
epidermal homeostasis, and several family members have been associated with
tumour development and metastasis (Joyce
and Hanahan, 2004). Cts7 and Cts8 belong to a
placenta-specific group of papain-like cysteine cathepsins
(Deussing et al., 2002;
Sol-Church et al., 2002). This
group consists of eight closely related genes that are located in a dense
cluster on mouse chromosome 13, and that may have arisen by repeated gene
duplication of cathepsin L on the same chromosome. Evolution of a
placenta-specific cathepsin group and high expression levels of ubiquitously
expressed family members in trophoblast tissues
(Afonso et al., 1997;
Varanou et al., 2006) suggests
the importance of cysteine cathepsins for placental development. Direct
evidence for their requirement in extra-embryonic tissues has been provided by
the administration of cysteine protease inhibitors into pregnant mice and
rats. This treatment causes embryonic lethality associated with a failure of
extra-embryonic tissues to develop (Afonso
et al., 1997; Freeman and
Lloyd, 1983). Cathepsins have also come into the spotlight for
their clinical significance in human pregnancy-associated disorders. Recurrent
spontaneous miscarriage has been associated with increased decidual levels of
cathepsins B and H (Nakanishi et al.,
2005), and placentas from pre-eclamptic pregnancies exhibited
de-regulated expression levels of cathepsins B, L and L2
(Varanou et al., 2006).
Despite their abundance in trophoblast tissues, the precise function of
cysteine cathepsins in placental development remains elusive. Giant
cell-expressed cathepsins B and L have been knocked out, but are dispensable
during gestation even in compound mutants
(Felbor et al., 2002). Although
none of the eight placenta-specific cathepsins has been studied in knockout
experiments yet, close evolutionary and sequence similarity has suggested
overlapping functions. Such redundancy was further suggested by co-expression
of: Cts7 and Cts8 in invading giant cells
(Hemberger et al., 2000);
Ctsm, Ctsq, Ctsr and possibly Cts6 and Ctsj in
sinusoidal giant cells in the labyrinth layer
(Ishida et al., 2004;
Nakajima et al., 2000;
Simmons et al., 2007); and
Ctsm, Cts3 and Ctsr in the spongiotrophoblast of the mature
mouse placenta (Bode et al.,
2005; Ishida et al.,
2004). Because of this likely functional overlap, an
overexpression system was preferable to a knockout strategy for elucidating
the function of individual cathepsins. Hence, we chose to pursue a
gain-of-function analysis in trophoblast stem cells and conditional
overexpression in mice to gain insights into the roles of Cts7 and
Cts8 during development. Our data show that these two highly related
cathepsins play distinct roles during embryogenesis and contribute critically
to trophoblast proliferation, differentiation and remodelling of the uterine
vasculature.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). All constructs were sequence verified.
Cell culture
TS cell lines TS-GFP, TS-Rs26 and TS-B6 were grown under standard
conditions (Tanaka et al.,
1998). For transfections, Lipofectamine 2000 reagent (Invitrogen,
Paisley, UK) was used. Immunofluorescence staining was performed with an
anti-FLAG antibody (Sigma, Dorset, UK) diluted 1:250. Relative cell sizes were
measured using the `Cell' software (Olympus, Watford, UK).
Transgenic mouse production
TgLacZ/Cts8 mice were generated by pronuclear injection
of the linearized plasmid into C57BL/6xCBA F1 zygotes.
TgLacZ/Cts7 strains were obtained by electroporation of
E14 ES cells that were subsequently used for blastocyst injections.
Cre-expressing lines used were CMV-Cre and Sox2-Cre, which both confer
ubiquitous transgene induction upon maternal transmission
(Hayashi et al., 2003;
Schwenk et al., 1995), as well
as the spongiotrophoblast-specific Tpbp-Cre and pan-trophoblast Cyp19-Cre
lines (Wenzel et al.,
2007).
Histology and in situ hybridization
Pregnant females were dissected at the gestational age indicated, counting
the morning of the vaginal plug as E0.5. For lacZ staining, embryos
were fixed in 0.25% glutaraldehyde and stained for β-galactosidase
according to standard protocols. For embedding, tissues were fixed overnight
in 4% paraformaldehyde and processed for routine paraffin histology. In situ
hybridization was performed with digoxigenin-labelled riboprobes according to
a standard protocol. Signals were detected with an anti-DIG alkaline
phosphatase-conjugated antibody (Roche, Basel, Switzerland), followed by
colour reaction using NBT and BCIP (Promega, Madison, WI, USA) and
counterstaining with nuclear Fast Red (Sigma, Dorset, UK).
Immunohistochemistry and immunofluorescence
Immunostaining was carried out on paraffin sections treated with 100
µg/ml Proteinase K or boiling in 0.01 M sodium citrate (pH 6.5). Antibodies
and dilutions were: anti-CTS7 (MAB1499; R&D Systems, Minneapolis, MN, USA)
1:200; anti-Laminin (Sigma, Dorset, UK) 1:200; anti-phosphohistone H3-S10
(#06-570; Upstate, Charlottesville, VI, USA) 1:200; anti-Ki-67 (ab15580;
Abcam, Cambridge, UK) 1:100; anti-FLAG M2 (F3165; Sigma, Dorset, UK) 1:300;
and peroxidase-conjugated isolectin BSI-B4 (L5391; Sigma, Dorset,
UK) 1:60. Smooth muscle -actin was detected with the IMMH-2 kit (Sigma,
Dorset, UK). Sections were counterstained with DAPI or Hematoxylin.
Quantitative expression analyses
Total RNA was prepared using Trizol Reagent (Invitrogen, Paisley, UK).
Total RNA (20-40 µg) was electrophoresed in 1% formaldehyde gels and
processed for northern blotting according to standard procedures
(Sambrook et al., 2001).
Signals were quantified against 18S ribosomal RNA (rRNA). For qRT-PCRs,
random-primed reverse-transcribed cDNA was used and PCRs were carried out on
an ABI 7700 cycler. mRNA levels were normalized against three housekeeping
genes (Gapdh, Dync11:2, Sdha).
Western blotting
Cells were lysed in PBS/1% Triton X-100 or in 1x RIPA buffer in the
presence of 20 mM dithiothreitol and protease inhibitors (Sigma, Dorset, UK).
Routine SDS-PAGE electrophoresis and western blotting were carried out using
10% Bis-Tris polyacrylamide gels (Invitrogen, Paisley, UK)
(Sambrook et al., 2001).
Anti-FLAG antibody (Sigma, Dorset, UK) was used at 1:5000. The processing
pattern of endogenous and transfected CTS7 was confirmed with the anti-CTS7
antibody used at 1:1000. Signals were detected using the ECL Plus Detection
system (GE Healthcare, Chalfont, UK).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To
determine the onset and sites of expression more precisely, serial gestational
stages were analysed from early post-implantation development until the second
half of pregnancy (E5.5-E14.5). Both cathepsins were found as early as E5.5 in
parietal trophoblast giant cells surrounding the implantation site, where they
demarcated a distinct subset when compared with the giant cell marker
placental lactogen-I (Prl3d1)
(Fig. 1A-C). Cts7- and
Cts8-expressing giant cell populations largely overlapped, although
Cts8 expression was somewhat weaker and confined to fewer giant
cells. Coinciding with the onset of trophoblast invasion, a striking shift in
expression was observed towards invasive giant cells around the ectoplacental
cone (Fig. 1D-L). This effect
was particularly pronounced for Cts8, whereas Cts7
expression extended slightly further distally to parietal giant cells
positioned around the midline of the implantation site
(Fig. 1G,H). Again, only a
subpopulation of all giant cells in the ectoplacental cone region was labelled
when compared with the marker genes Prl3d1 and proliferin
(Prl2c2) (Fig. 1F,L
inset). Remarkably, this population of Cts7- and
Cts8-expressing invasive trophoblast giant cells was always
associated with maternal blood vessels
(Fig. 1I). It is also important
to note that this expression pattern seemed to be unique to Cts7 and
Cts8, and was not shared by other placental cathepsins
(Deussing et al., 2002),
including the presumptive ancestral Ctsl
(Fig. 1L). Expression levels
declined sharply after E9.5 with only very few giant cells at the lateral
edges of the placenta remaining positive for Cts7 and Cts8
(not shown). A second site of expression was observed in late-stage placentas
from 
E14.5 onwards in labyrinth trophoblast, albeit at much reduced
levels [not shown (Hemberger et al.,
2000)]. Thus, expression of Cts7 and Cts8 was
most abundant in giant cells associated with maternal arteries during the
phase of trophoblast invasion.
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).
Both cathepsins were upregulated with the onset of giant cell differentiation
(-FGF/CM; Fig. 2A), indicating
that the normal regulation of expression is recapitulated in cultured TS
cells. In situ hybridization on TS cells confirmed giant cell-specific
expression of Cts7 and Cts8
(Fig. 2B); however, similar to
the situation in implantation sites, only a subset of giant cells was
labelled. Feasibly, it is this cathepsin-positive subset that endows TS cells
with the invasive capacity that is characteristic of trophoblast in vivo and
that is recapitulated by TS cells in vitro
(Hemberger et al., 2004).
CTS7 and CTS8 are mainly localized to endo-/lysosomes and can be secreted
Taking advantage of tagged expression constructs, the subcellular
localization of CTS7 and CTS8 was investigated in TS cells and in heterologous
COS-7 cells. Following the typical distribution that has been described for
other cathepsins (Wang et al.,
1998), the majority of CTS7 and CTS8 was localized to the
cytoplasm with a punctate staining pattern indicative of a predominant lyso-
and endosomal localization (Fig.
2C). As other cathepsins can be secreted
(Gottesman, 1978), we assessed
whether CTS7 and CTS8 also shared this characteristic. Indeed, both cathepsins
were readily detected in the supernatant of transfected TS and COS-7 cells,
whereas non-secreted control proteins were not found in the medium
(Fig. 2D). No difference in the
post-translational processing pattern was observed between the tagged
expression constructs and the endogenously expressed proteases, indicating
that the constructs were normally processed into active cathepsin enzymes.
These data suggested a potential dual role of CTS7 and CTS8 in the intra- and
extracellular environments.
Cts7 and Cts8 overexpression interferes with TS cell maintenance
To gain first insights into the roles of Cts7 and Cts8,
we pursued a gain-of-function strategy by transient transfections of TS cells.
As the transfection efficiency of TS cells is notoriously low, we used
bicistronic expression constructs that conferred concomitant cathepsin and GFP
expression to identify transfected cells. Assessing only GFP-positive and,
hence, transfected cells, we found that both cathepsins caused an increase in
cell and nuclear sizes as early as 24 hours post transfection, indicative of
the initiation of giant cell differentiation
(Fig. 2E,F). Co-cultured cells
and cathepsin-conditioned media showed no effect (not shown), demonstrating
that cell size enlargement was a cell-autonomous, intracellular function of
CTS7 and CTS8. Consistent with these findings, the clonal expansion of
Cts7/Cts8 transfected TS cells was limited and mitotic
indices were reduced by 1.5-fold to twofold (P<0.025). The reduced
proliferative capacity interfered with the generation of TS cell lines that
stably overexpressed these proteases. Thus, Cts7 and Cts8
reduced the proliferation rates of TS cells and primed them towards
differentiation.
|
) (Fig. 3A,B).
This system allowed us to investigate the roles of Cts7/Cts8
by gain-of-function analysis in trophoblast cells in their physiological
environment, including potential paracrine effects on neighbouring decidual or
extra-embryonic mesodermal cells. Importantly, it also enabled the examination
of potential effects at ectopic sites where functions of these proteases may
be revealed that are masked in trophoblast cells by the pre-existing
endogenous expression of both cathepsins.
Cts7 overexpression is compatible with embryonic development to term
Conditional overexpression of Cts7 was evaluated in three
independent transgenic lines after ubiquitous and trophoblast-specific
transgene activation using the CMV-Cre, maternally transmitted Sox2-Cre,
Tpbp-Cre and Cyp19-Cre lines (Hayashi et
al., 2003; Schwenk et al.,
1995; Simmons et al.,
2007; Wenzel et al.,
2007). Matings to CMV-Cre and Sox2-Cre females conferred ectopic,
ubiquitous tgCts7 expression to the embryos proper
(Fig. 3C) and strong (up to
eightfold) Cts7 overexpression to placentas where particularly high
Cts7 levels were observed in spongiotrophoblast, the chorionic plate
and in sinusoidal giant cells (Fig.
3D,E). By contrast, the efficiency of transgene activation was
incomplete when the trophoblast-specific Tpbp-Cre and Cyp19-Cre lines were
used, resulting in weak tgCts7 expression in target cells
(not shown). Importantly, live, healthy and fertile
Cts7+/Cre+ offspring were obtained at
Mendelian ratios, even from matings that yielded highest ubiquitous
tgCts7 expression, indicating that Cts7
overexpression did not interfere with embryonic development to term.
CTS7 affects trophoblast proliferation and differentiation
As there was no difference in survival between the various transgenic
lines, we analyzed the strongest tgCts7-overexpressing
line for subtle defects in trophobast differentiation. Consistently, these
placentas were characterized by a thinner spongiotrophoblast layer
(Fig. 3D). This phenotype
correlated with the spongiotrophoblast exhibiting strongest transgene
expression, and resulted in a downregulation of spongiotrophoblast-expressed
genes, such as Tpbpa, Prl2c2, proliferin-related protein
(Prl7d1) and placental lactogen-II (Prl3b1) relative to
controls at E10.5 and E12.5 (Fig.
3D,E). By E14.5, however, the proportional size of placental
layers was similar to that of controls and, accordingly, relative expression
of these genes reached comparable levels in wild-type and
tgCts7 placentas (Fig.
3E).
Because of the timing of this placental growth defect, we investigated the
differentiation of cell types that form after E10.5 in more detail. Thus, the
appearance of Prl3b1-positive sinusoidal giant cells as well as
glycogen cells was analyzed (Coan et al.,
2006), both representing cell types in which the transgene was
highly expressed. Consistent with reduced Prl3b1 expression levels,
fewer sinusoidal giant cells had formed by E12.5
(Fig. 4A). Similarly, initial
differentiation of glycogen cells was sparse in E12.5
tgCts7 placentas. Compared with wild-type controls and
placentas carrying the inactive
(tgLacZ/Cts7+/Cre-) transgene,
glycogen cells contributed only 32.82±2.32% to the spongiotrophoblast
layer compared with 47.22±2.43% in controls (P<0.00015;
Fig. 4B). Thus, although all
trophoblast cell types could be formed in principle, Cts7
overexpression caused a delay in their differentiation.
|
), whereas
H3S10-P is associated with chromosome condensation and accumulates in late G2
and M phase of the cell cycle (Hendzel et
al., 1997). Unexpectedly, a subtle increase was observed for both
markers in tgCts7 placentas (9% for Ki-67; 8%
for H3S10-P). However, closer examination revealed that this increase was
chiefly due to cells exhibiting a punctate H3S10-P staining pattern, whereas
fewer cells with a meta- or telophase arrangement of chromosomes were seen
(Fig. 4C,D). This phenotype was
particularly obvious at E9.5 but was still present at E12.5. Accordingly,
these placentas exhibited larger patches of Ki-67-positive cells in the
spongiotrophoblast and labyrinth. The Ki-67 staining pattern also highlighted
the absence of differentiated trophoblast cells in these areas
(Fig. 4E,F). These findings
indicated that Cts7 overexpression was associated with a slower
progression through the cell cycle, in particular through mitosis, and
provided a possible explanation for the observed delay in trophoblast
differentiation. Next, we aimed to determine whether the proliferation defect depended on the proteolytic activity and involved a direct nuclear function of CTS7. Such a nuclear role was feasible because the CTS7 propeptide sequence contains a predicted bipartite nuclear localization signal (defined by a motif consisting of two consecutive basic residues, 10 intervening amino acids and a minimum of three out of five additional basic residues; Prosite motif PDOC00015). Interestingly, we found that the increase in H3S10-P staining caused by Cts7 was abolished when point mutations were introduced into the catalytic site or the nuclear localization signal (Fig. 4G), thus indicating that proteolytic activity and nuclear localization were essential for the role of CTS7 in affecting cell proliferation. S-phase progression as assessed by BrdU incorporation rates was comparable for all transfected protein variants (not shown), reinforcing that the main effect of CTS7 was restricted to late G2 and/or M phase of the cell cycle.
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Cts8 overexpression enhances giant cell differentiation
For Cts8, one transgenic line was obtained that exhibited
ubiquitous lacZ expression and in which the floxed lacZ/Neo
reporter cassette could be efficiently removed by CMV-Cre and Sox2-Cre, while
transgene activation was again incomplete when the trophoblast-specific
Tpbp-Cre and Cyp19-Cre expressors were used. Analysis of placentas showed
approximately equal amounts of endogenous Cts8 and transgenic
Cts8-IRES-GFP mRNA upon strongest tgCts8
activation (Fig. 5A). When
hemizygous tgCts8 mice were mated to ubiquitous or inner
cell mass-specific (paternal transmission of Sox2-Cre) Cre expressors, no live
transgenic offspring were obtained. By contrast, foetuses survived to term
when Cts8 expression was induced trophoblast specifically. Hence,
lethality was due to Cts8 expression in the embryo and/or
extra-embryonic endo- and mesoderm. Consistent with this observation,
trophoblast differentiation occurred largely normally in conceptuses derived
from matings to all four Cre lines at mid-gestation, as determined by
expression and distribution of markers of giant cell differentiation
(Prl3d1, Prl3b1, Prl2c2, Cts7), ectoplacental cone/spongiotrophoblast
(Tpbpa) and extra-embryonic mesoderm (Peg1, Cdh5, F8c)
(Fig. 5B). However, late-stage
placentas of trophoblast-activated tgCts8 exhibited an
enlarged giant cell/spongiotrophoblast layer compared with control placentas
(Fig. 5C). The effect of
Cts8 on giant cell differentiation was particularly obvious when the
trophoblast layer of E9.5 CMV-CrextgCts8 placentas
was dissected and grown in culture for 2 days, a system where transgene
activation could be easily detected by the concomitantly induced GFP
expression. In this combination with in vitro culture, strikingly more and
larger giant cells (P<6x10-15) were observed in
the transgenic samples (Fig.
5D,E). These findings showed that, similar to our previous results
in overexpressing TS cells, Cts8 primed diploid trophoblast cells
towards differentiation into giant cells, but that (mild) overexpression in
trophoblast is not detrimental for normal development to term.
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CTS8 causes vascular breakdown by smooth muscle degradation
To explain the vascularization defect in Cts8 transgenic
conceptuses, the perivascular support lining was examined by staining for
smooth muscle -actin (SMA=Acta2). Whereas the vitelline
vessels of wild-type yolk sacs were surrounded by a thin, continuous layer of
smooth muscle cells, the enlarged blood spaces of tgCts8
yolk sacs were devoid of SMA staining (Fig.
6C). The same finding was observed in the chorioallantoic
vasculature of the placenta. Vessel outlines were present in transgenic
placentas, as detected by laminin staining; however, they appeared
disorganized. Ruptured blood vessels and free foetal blood cells were
frequently observed. Importantly, these areas were characterized by a striking
reduction of SMA staining (Fig.
6D,E). To determine whether the decrease in SMA amounts was
regulated on the transcriptional or protein level, quantitative RT-PCRs were
performed. These experiments demonstrated that mRNA levels remained unchanged
in transgenic embryos and placentas (Fig.
6F). Therefore, SMA reduction occurred on the protein level
presumably by direct proteolytic degradation by CTS8. An important expectation
arising from these results was that, if perivascular smooth muscle degradation
was a general function of CTS8 in placentation, spiral arteries should be
devoid of SMA where they are in contact with trophoblast giant cells that
express Cts8 endogenously. Consistent with this expectation, the area
surrounding Cts8-positive giant cells of the ectoplacental cone was
characterized by a lack of SMA (Fig.
6G). Taken together, our approach of ectopic Cts8
expression proved to be a powerful tool to reveal additional activities of
this protease. Our findings strongly indicate that perivascular smooth muscle
degradation is a physiological function of CTS8 that contributes to the
remarkable artery remodelling capacity characteristic of invasive trophoblast
giant cells.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Freeman and Lloyd, 1983), but
despite their abundance in trophoblast tissues their molecular function during
placentation remains elusive. Here, we have characterized two
placenta-specific cathepsins, Cts7 and Cts8, and provide
evidence for their role in trophoblast proliferation, differentiation and
vascular remodelling at the site of trophoblast invasion.
Cts7 and Cts8 define a unique subset of trophoblast giant
cells that is generally characterized by an invasive behaviour and by
interaction with maternal spiral arteries. The previously underappreciated
diversity of trophoblast giant cells has recently been highlighted by the
description of four subtypes that can be defined by a combination of their
localization and gene expression (Simmons
et al., 2007). According to this classification, Cts7 and
Cts8 mark an exclusive set of parietal and spiral artery-associated
giant cells. This pattern suggests a common function between these cells and
establishes another giant cell subgroup, emphasizing the functional diversity
of this morphologically similar cell type.
Our data indicate that overexpression of CTS7 causes a prolongation of the
cell cycle in G2 and/or M phase, which may be due to a subtle defect in
chromosome condensation. The observed delay in differentiation of trophoblast
cell types, such as glycogen and sinusoidal giant cells, may be a direct
consequence of this mitotic slowdown. This effect may also account for the
inability to maintain the self-renewing, proliferative capacity of
Cts7-overexpressing TS cells. As Cts7 is expressed in newly
emerging giant cells during gestation, it is feasible that the delay in
chromosome condensation and cell cycle progression contributes to the switch
towards endoreduplication. In this context, it is interesting to note that
MENT (myeloid and erythroid nuclear termination stage-specific protein), a
nuclear protein that can act as an inhibitor of cathepsins L and L2, was
identified as a heterochromatin-associated protein mediating chromatin
condensation (Grigoryev et al.,
1999; Irving et al.,
2002). Hence, it is possible that a similar protease-inhibitor
relationship is disturbed in trophoblast cells upon CTS7 (over)expression and
interferes with the progression of mitotic chromatin condensation.
|
).
However, in contrast to CTSL, where alternate translation start sites give
rise to isoforms that lack a signal peptide, nuclear CTS7 localization is
achieved mainly by an internal nuclear localization signal that, when
abrogated, rescues the chromosome condensation defect and leads to
significantly weaker nuclear immunostaining signals.
As to CTS8, our data indicate that this cathepsin may have complementary
functions to CTS7 in promoting giant cell differentiation
(Fig. 7), an effect that was
particularly obvious when signals from surrounding cells such as decidual
stroma, maternal blood and/or endothelial cells were removed in an in vitro
context. Hence, within the uterine bed, the differentiation-promoting effect
of Cts8 is rather mild and an increase in fully differentiated giant
cells becomes only obvious in late-stage placentas upon prolonged
overexpression. This is consistent with the biological function of
Cts8-expressing giant cells that need to remain comparatively small
to exhibit their highly invasive properties
(Hemberger, 2008).
Most notably, however, our transgenic approach revealed a dramatic
functional difference between the closely related cathepsins CTS7 and CTS8, in
that (ubiquitous) Cts8 overexpression causes midgestational embryonic
lethality whereas Cts7 is tolerated even at high levels. The most
significant effect of CTS8 is that it interferes with normal embryonic and
vitelline angiogenesis, and leads to a reduction of the perivascular support
structure, most probably by direct degradation of smooth muscle -actin.
As SMA ablation allows development into adulthood
(Schildmeyer et al., 2000),
CTS8 may well have additional targets whose proteolysis leads to the observed
embryonic lethality. The Cts8 gain-of-function phenotype resembles
that of knockout models of many factors required for normal vascular
development, haematopoiesis, cell adhesion and communication, and of the
TGFβ signalling network. Thus, these factors represent good candidates
for potential substrates of CTS8. Particularly noteworthy is the TGFβ
receptor Alk1 (activin receptor-like kinase 1) as
Alk1-deficient and tgCts8 conceptuses share
phenotypic similarities not only in the embryo and yolk sac, but also in the
placenta. As in Cts8-overexpressing placentas, allantoic mesoderm
invasion into the chorionic trophoblast is largely undisturbed, but
chorioallantoic vessels are severely dilated and fused
(Hong et al., 2007;
Oh et al., 2000). Moreover,
Alk1 deficiency leads to enhanced expression of a number of proteases
(Oh et al., 2000), and
TGFβ signalling is known to regulate cathepsins negatively
(Gerber et al., 2000). Hence,
Cts8 activation in TGFβ/Alk1 mutants could provide an
explanation for the almost identical phenotypes. As mutations of the TGFβ
signalling cascade also lead to disruptions in vascular smooth muscle support,
this defect may be a direct consequence of aberrant Cts8
expression.
Importantly, our approach of activating Cts8 expression at ectopic
sites revealed a major effect of this protease on the integrity of blood
vessels. As it is only Cts8-positive trophoblast giant cells that are
in contact with mature vessels in the early conceptus, this activity of CTS8
would not have been recognized by trophoblast-restricted overexpression only.
In the later placenta where foetal blood vessels are present in the labyrinth,
poor transgene inducibility in the lining syncytiotrophoblast layer III and
differences in the ultrastructure of labyrinthine vessels compared with spiral
arteries may explain the lack of an overt placental phenotype. Our findings
suggest that CTS8 produced by giant cells in direct contact with maternal
spiral arteries mediates their localized remodelling by degradation of
perivascular smooth muscle cells. Such a paracrine effect is feasible because
secreted cathepsins can function in the extracellular milieu as well as inside
exposed cells (Nielsen et al.,
2007). Weakening of the vessels could then facilitate endovascular
giant cell invasion and the formation of trophoblast-lined blood sinuses;
these are essential processes for the normal progression of pregnancy.
Partial loss of arterial smooth muscle cells starts in the metrial triangle
of the decidua, some distance away from trophoblast giant cells, and is
mediated by uterine natural killer (uNK) cells
(Adamson et al., 2002). In
addition, remodelling of the smooth muscle layer is incomplete in uNK
cell-deficient females (Croy et al.,
2000). Thus, a model emerges where in the environment of the
uterine bed, a concerted action of uNK cells and trophoblast giant cells is
necessary to mediate normal spiral artery remodelling
(Fig. 7). This dual regulation
from both the foetal and maternal side represents an intriguing control
mechanism to prevent excessive arterial wall degradation and uterine bleeding.
Having established this blood vessel-disintegrating function of CTS8 that is
not shared by the co-expressed CTS7, it will now be interesting to investigate
whether CTS8 is essential for trophoblast-mediated vascular remodelling in
knockout approaches.
Cysteine cathepsins are implicated in a variety of cellular processes and are involved in numerous pathological conditions that include extracellular matrix remodelling and invasion. Here, we provide molecular insights into the role of the two placenta-specific cathepsins, Cts7 and Cts8, in trophoblast differentiation and function. This knowledge will further our understanding of the physiological and pathological significance of cathepsins. Our data demonstrate that, despite their close ontological and genetic relationship, highly related placental cathepsin proteases have distinct non-redundant functions during development and contribute crucially to a successful outcome of pregnancy.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/19/3311/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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NLS) and active
site-mutant Cts7 (
