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First published online 4 April 2007
doi: 10.1242/dev.02843
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Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, T2N 4N1, Canada.
* Author for correspondence (e-mail: jcross{at}ucalgary.ca)
Accepted 26 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mrj, Keratin, Aggregation, Hsp40 co-chaperone, Proteasome, Chorioallantoic attachment, Tetraploid aggregation, Placenta, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In many cases,
the role of protein aggregates in disease pathogenesis is unclear. Efforts to
understand aggregation disorders have focused mainly on mutations within the
disease-associated protein. However, mounting evidence suggests that a major
cause of protein inclusion body formation is failure of the protein
quality-control system (McClellan et al.,
2005). This surveillance mechanism recruits both molecular
chaperones and proteases to safeguard against protein aggregation by
maintaining the equilibrium between protein folding and degradation
(Hohfeld et al., 2001).
A malfunction in the ubiquitin-proteasome degradation pathway is
hypothesized to be one cause of Mallory body formation in the hepatocytes of
patients with chronic liver disorders
(Bardag-Gorce et al., 2003;
Bardag-Gorce et al., 2004;
Denk et al., 2000). Mallory
bodies are inclusions composed of the keratin intermediate filaments keratin 8
(K8; also known as Krt8) and keratin 18 (K18; also known as Krt18), as well as
ubiquitin, the proteasome complex and molecular chaperones, including heat
shock protein 70 (HSP70; HSPA1B) and heat shock protein 90 (HSP90)
(Coulombe and Omary, 2002).
Cells that have been treated with a chemical inhibitor of proteasome function
(Bardag-Gorce et al., 2004) or
that contain the UBB+1 ubiquitin mutation
(Bardag-Gorce et al., 2003)
form Mallory bodies due to an accumulation of non-degraded keratin.
Misfolded proteins also tend to aggregate. Proper protein folding within a
cell often does not occur spontaneously and, thus, molecular chaperones are
required for some proteins to reach its native state efficiently
(Hartl and Hayer-Hartl, 2002).
Hsp70 is a ubiquitous chaperone that, together with diverse co-chaperone
binding partners, facilitates protein folding and the presentation of proteins
to the proteasome for degradation (Esser
et al., 2004). Co-chaperones of the DnaJ (Hsp40) protein family
are characterized by a conserved Jdomain that regulates substrate binding and
release by activating the ATPase activity of Hsp70
(Fan et al., 2003). These
co-chaperones differ in their tissue and subcellular patterns of expression,
and convey substrate specificity to Hsp70
(Fan et al., 2003). However,
little is known about the precise substrates that are regulated in this
manner.
The Mrj (Mammalian relative of DnaJ; also known as Dnajb6
- Mouse Genome Informatics) gene encodes a co-chaperone that is widely
expressed throughout the adult mouse and during development of the embryo and
placenta (Chuang et al., 2002;
Dai et al., 2005;
Hunter et al., 1999;
Izawa et al., 2000;
Seki et al., 1999).
Mrj-/- embryos die at mid-gestation due to a failure of
chorioallantoic attachment during placental development
(Hunter et al., 1999).
Recently, a few diverse Mrj-interacting proteins have been identified,
including Huntington disease (HD) protein
(Chuang et al., 2002) and K18
(Izawa et al., 2000). Here, we
show that Mrj deficiency prevents normal keratin turnover, resulting
in the formation of large keratin aggregates that are toxic to trophoblast
cells, inhibiting their normal function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Mrj-/- embryos were produced by mating
Mrj+/- mice. K18/K19 double-null embryos
were generated by mating K18-/- mice
(Magin et al., 1998) with
K19-/- mice (Hesse et
al., 2000) (provided by T. Magin, University of Bonn, Bonn,
Germany) and then mating the resulting double heterozygous progeny.
Mrj-/-;K18-/- and
Mrj-/-;K18+/- embryos were generated
by mating Mrj+/- mice with K18-/- mice
and then mating their resulting
Mrj+/-;K18+/- progeny together.
Enhanced green fluorescent protein (Egfp) transgenic mice
(Hadjantonakis et al., 1998)
were maintained at heterozygosity in a CD-1 background. Experiments were
performed in accordance with the Canadian Counsel on Animal Care and the
University of Calgary Committee on Animal Care (Protocol no. M01025).
Genotyping
Mrj genotypes were determined by PCR using primers
5'-CCAATAGCAGCCAGTCCCTTCCC-3' and
5'-TTCGGCTATGACTGGGCACAACA-3' to detect the neomycin-resistance
gene of the ß-geo cassette (226 bp, mutant allele); and
5'-ACAGGGTCTTGCTACAAGTAGTGC-3' and
5'-TTTCTCTGTCCATGAAGGACTGGG-3' to detect the ß-geo
gene-trap insertion site in Mrj intron 2 (463bp, wild-type allele).
The PCR parameters were 30 seconds at 94°C, 30 seconds at 63°C and 30
seconds at 72°C for 30 cycles. Genotyping for K18 and
K19 (Krt19) mice was performed as previously described
(Hesse et al., 2000;
Magin et al., 1998).
Generation of tetraploid aggregation chimeras
Tetraploid aggregation chimeras were generated as described previously
(Nagy et al., 2003). Briefly,
tetraploid embryos were formed by electrofusing two-cell Egfp
embryos, which were then aggregated with eight-cell diploid embryos from
Mrj heterozygous matings. Aggregated chimeric embryos were allowed to
develop to the blastocyst stage and were then transferred into the uteri of
pseudopregnant CD-1 females [noon of the day that the vaginal plug is detected
was defined as embryonic day (E)0.5]. Embryos were dissected at E9.5. The
existence of wild-type tetraploid cells within the placenta was determined by
the presence or absence of enhanced GFP (EGFP) expression using a GFP filter
(41017 EN GFP, Leica). The yolk sac and embryo tissue was removed for
Mrj genotyping. Placentas were fixed in 4% paraformaldehyde (PFA;
VWR) in 1x phosphate buffered saline (PBS) and embedded in paraffin.
Tissue sections were stained with hematoxylin and Eosin (both Sigma).
Cell cultures
Wild-type (line 6-3) and Mrj homozygous mutant (lines 1-4 and 4-1)
trophoblast stem (TS) cell lines were derived from littermate blastocysts
generated from Mrj heterozygote matings. Blastocysts were allowed to
outgrow on embryonic feeder cells in TS cell medium
(Tanaka et al., 1998). These
TS cell lines and the Rs26 TS cell line (provided by J. Rossant,
Hospital for Sick Children, Toronto, Canada)
(Tanaka et al., 1998) were
maintained in 25 ng/ml basic fibroblast growth factor (bFGF), 1 µg/ml
heparin in TS cell medium, 70% of which was pre-conditioned by embryonic
fibroblasts, at 37°C under 95% humidity and 5% CO2. Cells were
differentiated by withdrawing bFGF, heparin and fibroblast-conditioned medium
(Tanaka et al., 1998).
Chorion-ectoplacental cones were dissected at E7.5 from Mrj heterozygous crosses in 1x PBS. Explants were placed in 0.125% trypsin/EDTA (Gibco) in PBS for 10 minutes at 37°C. Dispersed cells were then cultured on coverslips in fibroblast-conditioned TS cell medium with bFGF and heparin for 5 days at 37°C in 95% humidity, 5% CO2, with daily medium change. Yolk sacs were removed during dissection for genotyping.
X-gal and immunofluorescence antibody staining
Mouse conceptuses were dissected at E8.25 and E9.5. For X-gal staining,
tissue was fixed, stained as whole mount and was then paraffin embedded as
previously described (Hunter et al.,
1999). For antibody staining, tissue was fixed in 4% PFA and
embedded in paraffin. Paraffin sections (7 µm) were rehydrated in ethanol,
treated with trypsin tablets (Sigma) in PBS for 10 minutes and blocked in 1%
bovine albumin serum (BSA; Sigma), 5% host serum for 1 hour. Cell cultures
were fixed in 4% PFA for 10 minutes, treated with 0.1% Triton X-100 (Fisher
Biotech) for 10 minutes and blocked in 1% BSA for 30 minutes. Primary
antibodies and dilutions used include: anticytokeratin 18 (Ks18.04, 1:100;
RDI), anti-keratin 8 (Ks8.7, 1:100; Progen), anti-keratin 19 (Troma.3, 1:100;
a gift from T. Magin), anti-pan-cytokeratin (1:100; DAKO), anti-desmoplakin
(DP1&2, neat; RDI), anti-20S proteasome core subunits (1:100; Calbiochem)
and anti-Mrj (020417, 1:100; a gift from M. Inagaki, Aichi Cancer Centre
Research Institute, Aichi, Japan) (Izawa
et al., 2000). Secondary antibodies were used at a dilution of
1:300 and included: goat anti-rabbit Cy3, goat anti-rat Cy3, goat anti-mouse
Cy3 and goat anti-rabbit FITC (Jackson ImmunoResearch Laboratories), and goat
anti-mouse Alexa Fluor 488 (Molecular Probes). Antibody dilutions were made in
1% BSA, 5% host serum. DNA was counterstained with 1:10,000 bisbenzimide
(Sigma).
Electron microscopy
E8.25 implantation sites from Mrj heterozygous matings were fixed
in 2% EM-grade glutaraldehyde (Sigma), 2% PFA in 0.2 M sodium cacodylate (pH
7.4; Sigma) overnight at 4°C, and were post-fixed in 1% osmium tetroxide
(EM Sciences) in 0.2 M sodium cacodylate (pH 7.4) for 2 hours at 4°C.
Tissue was dehydrated in increasing concentrations of ethanol up to 70%,
treated in 2% uranyl acetate (ProSciTech) in 70% ethanol, and further
dehydrated in increasing ethanol concentrations (70-100%). Tissue was treated
with propylene oxide (EM Sciences) and was resin embedded (EMBED 812 kit, EM
Sciences). Yolk sacs were removed for genotyping prior to fixation.
Proteasome inhibition assay
Rs26 TS cells were differentiated by withdrawal of bFGF, heparin and
fibroblast-conditioned media for 4 days on coverslips. Cells were exposed to
10 µM clasto-lactacystin ß-lactone (Invitrogen) in differentiating TS
medium overnight at 37°C under 95% humidity and 5% CO2. Cells
were washed with 1xPBS, fixed in 4% PFA and immunostained as described
above.
Equipment and software
A Leica DMR light microscope (for fluorescent and light images), Leica DMIL
inverted light microscope (for cell culture images) and Leica MZ95 dissection
microscope (for dissected embryo/placenta images) with Photometrics Coolsnap
cf camera and Open Lab 2.2.2 imaging software program were used to obtain
micrographs. Filters used were from Chroma: DAPI (31000), TRITC (31002) and
FITC (31001). De-convolved images were captured using an Axioplan 2 Imaging
(Zeiss) microscope and Axiovision 4.1 acquisition software. Electron
micrographs were taken on a Hitachi 7000 transmission electron microscope.
Minimal image processing was performed using Adobe Photoshop 7.0 and figures
were constructed using Canvas X.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Stable
attachment is required for allantoic spreading along the surface of the
chorion and for the formation of trophoblastic villi (reviewed by
Watson and Cross, 2005).
Mrj mutant conceptuses fail to undergo chorioallantoic attachment and
as a result do not form a mature placenta, dying by E10.5
(Hunter et al., 1999).
Mrj mutant allantoises appear to both expand and elongate normally
compared to wild type at E8.0 (Hunter et
al., 1999), and grow across the exocoelomic cavity to contact the
chorion. However, despite the close proximity of the allantois to the chorion,
subsequent firm attachment to the chorion is not observed. Our initial
observations have been confirmed in careful dissections of conceptuses after
E8.5, showing the inability of the allantois to stay attached to the chorion
in all mutants (n=81). Presumably as a consequence of failing to
attach the chorion, the allantois rapidly collapsed and shrunk back to form a
bud of tissue at the posterior end of the embryo by E9.5 (see Fig. S1 in the
supplementary material). This phenomenon has been observed in other mutants
with defects in chorioallantoic attachment
(Hildebrand and Soriano, 2002;
Tetzlaff et al., 2004;
Yang et al., 1995).
|
). Using
ß-galactosidase activity driven from the Mrj genetrap allele
(Fig. 1A) and Mrj antibody
staining (Fig. 1B) on E8.25
histological sections, we detected Mrj expression throughout the chorionic
trophoblast. However, expression within the chorionic mesothelium was
indeterminable because of the thinness of this cell layer.
To address better whether Mrj is required within the chorionic trophoblast
and/or the mesothelium for chorioallantoic attachment, we used tetraploid
aggregation, a technique that has previously been shown to produce chimeric
conceptuses with genetically distinct chorionic cell compartments
(Fig. 1C)
(Tarkowski et al., 1977).
Wild-type tetraploid embryos were aggregated with diploid embryos derived from
Mrj heterozygous matings. The resulting conceptuses developed so that
the chorionic mesothelium, embryo proper and allantois were almost exclusively
composed of diploid cells (Tarkowski et
al., 1977). By contrast, wild-type tetraploid cells contributed to
the trophoblast lineage, resulting in a chorionic trophoblast layer that
contains wild-type cells (Tarkowski et
al., 1977). Therefore, we expected that, if Mrj is required only
within chorionic trophoblast cells, and not in the mesothelium or allantois,
then chorioallantoic attachment would be rescued in
Egfp
Mrj-/- (tetraploiddiploid)
chimeric conceptuses.
Chimeric blastocysts (n=24) were transferred into the uteri of
pseudopregnant female mice. At E9.5, 1 day after chorioallantoic attachment is
normally complete, all 15 surviving chimeric embryos had undergone
chorioallantoic attachment. Genotyping revealed that two of these embryos were
Mrj mutants (Fig.
1D,E), indicating that Mrj is not required within the mesothelium
or allantois for chorioallantoic attachment to occur. Furthermore, development
proceeded normally up to E9.5 in EgfpMrj-/-
chimeric placentas, as indicated by the initiation of branching morphogenesis
(Fig. 1H), similar to
EgfpMrj+/+ and
EgfpMrj+/- littermates
(Fig. 1F and data not shown).
This is in contrast to Mrj-deficient chorions, which remained flat
(Fig. 1G). These data imply
that Mrj function was required exclusively within the trophoblast compartment
at the chorioallantoic interface.
Mrj is required for a normal keratin cytoskeleton in trophoblast giant cells
Previous studies have demonstrated that Mrj binds to both soluble and
filamentous K18 (Izawa et al.,
2000). Because, in our study, Mrj function in the placenta was
restricted to the trophoblast lineage, where keratin is also expressed, we
explored whether Mrj mutant cells had a defective keratin
cytoskeleton. Using immunofluorescence, we examined K18-containing filament
organization within Mrj-/- trophoblast cells derived from
primary cultures of explanted chorion-ectoplacental cone tissues. The
chorionic trophoblast and ectoplacental cones are thought to be composed of
multiple progenitor populations (Simmons
and Cross, 2005), yet in culture they spontaneously differentiate
primarily into trophoblast giant cells. Compared with 89.7% of wild-type
trophoblast giant cells (n=659), only 36.9% of Mrjdeficient
cells (n=634) had a normal, dense keratin filamentous network
(Fig. 2A,B). Strikingly, the
majority of Mrj-/- cells exhibited a collapsed keratin
network, in which large perinuclear inclusions of keratin were present (53.9%
compared with only 5.0% of wild-type cells). The remaining
Mrj-/- cells contained either sparse keratin filaments
(5.8%), or K18 levels were undetectable (3.4%).
All trophectoderm-derived cells express simple epithelial type I (K18 and
K19) and type II (K7 and K8) keratins (Lu
et al., 2005). Each keratin filament is composed of type I and
type II heterodimers. K8 can dimerize with both K18 and K19
(Owens and Lane, 2003).
Therefore, we also immunostained cells using K8, K19 and pan-cytokeratin
antibodies. Interestingly, the results were similar to the collapsed K18
cytoskeleton or sparse K18 filaments observed
(Fig. 2A,B). Ultrastructural
analysis of keratin aggregates using electron microscopy in vivo revealed that
they consisted of closely packed, parallel filaments
(Fig. 2C). From this, we
concluded that Mrj was required for the maintenance and/or turnover of the
keratin cytoskeleton, but not for the assembly of filaments per se.
The keratin cytoskeleton appeared normal in approximately a third of
Mrj-deficient trophoblast giant cells derived from explants
(Fig. 2B). However, we noted
that 37.4% of wild-type trophoblast giant cells (n=1478) had
undetectable levels of Mrj at E8.25 (Fig.
2D), indicating that not all trophoblast giant cells would be
expected to show a phenotype. This suggests an alternative method of keratin
regulation in Mrj-negative cells. Mrj was heterogeneously expressed
in cultured trophoblast giant cells, either within the nucleus, the cytoplasm
or both (Fig. 2E). This may be
attributed to the presence of several trophoblast giant cell subtypes that
appear both in vitro and in vivo (Simmons
et al., 2007).
|
; Uy et al.,
2002). Therefore, we derived both wild-type and
Mrj-deficient TS cell lines from littermate blastocysts generated
from Mrj heterozygote crosses. Approximately 80% of wild-type TS
cells (n=494) had normal K18 expression
(Fig. 3B), whereas the
remaining 20% had undetectable levels. By contrast, 78% of
Mrj-/- TS cells (n=469) lacked a normal keratin
network and contained keratin inclusions similar to, albeit smaller than,
those seen within primary cultures of Mrj-/- trophoblast
giant cells (Fig. 3C). K18
expression was less-apparent in histological sections of
Mrj-/- chorionic trophoblast cells in vivo compared with
wild type (Fig. 3E-H). However,
electron microscopy confirmed the presence of filamentous aggregates
(Fig. 3D). These data indicate
that Mrj mutant chorionic trophoblast cells show the same type of
keratin defects as trophoblast giant cells - both the lack of normal keratin
cytoskeleton and the presence of large keratin aggregates - albeit at a higher
frequency owing to the fact that a subset of giant cells do not express Mrj
protein. Next, we examined the ultrastructure of Mrj-deficient chorions to determine the effects of keratin aggregate formation on tissue integrity. In comparison to wild-type chorionic trophoblast cells (Fig. 3I,K), Mrj-/- cells were disorganized and rounded (Fig. 3J,L). This suggested that the adhesive properties of Mrj-null cells may have been altered. Therefore, we analyzed the expression of desmoplakin, a protein that links keratin filaments to desmosomes at the cell membrane. Remarkably, both Mrj-/- chorionic trophoblast cells and TS cells showed a significant reduction in desmoplakin immunofluorescence (Fig. 3O,P,R) compared with wild type (Fig. 3M,N,Q). Ultrastructurally, however, desmosomes appeared to be normal in Mrj-/- chorionic trophoblast cells (Fig. 3S,T). Together, these data imply that keratin inclusion bodies disrupt the organization of chorionic trophoblast cells, which in turn may affect the ability of the allantois to attach to the chorion. Notably, although they were disorganized, the Mrj-/- chorionic trophoblast cells showed no signs of cell death.
Proteasome degradation of keratin filaments requires Mrj
Keratin filaments can oligomerize without catalysts or co-factors
(Owens and Lane, 2003;
Quinlan et al., 1986).
Accordingly, keratin inclusions both in Mrj-deficient chorionic
trophoblast and in trophoblast giant cells contained filaments
(Fig. 2C,
Fig. 3D), indicating that Mrj
was not necessary for filament assembly. Therefore, we reasoned that Mrj may
have played a structural role or have been required for keratin turnover. We
found that Mrj protein did not co-localize with K18-containing filaments in
trophoblast giant cells (Fig.
4), implying that Mrj was not playing a direct role in the
organization of the keratin filamentous network.
It was previously shown that Mrj can interact with the soluble forms of
K8/K18 (Izawa et al., 2000).
Therefore, we next questioned whether Mrj regulated keratin by modulating the
activity of the proteasome. Immunofluorescence of Mrj and 20S proteasome core
subunits revealed that these two proteins co-localized
(Fig. 5A). We then inhibited
the proteasome complex in wild-type trophoblast giant cells by adding
lactacystin (10 µM) and found that proteasome-inhibited cells contained
large, perinuclear keratin inclusions (Fig.
5B) similar to Mrj-/- cells
(Fig. 2A,
Fig. 3C). Interestingly, Mrj
co-localized with keratin inclusions within proteasome-inhibited cells
(Fig. 5C). These data suggest
that, without Mrj, keratin filaments are not properly degraded by the
proteasome, leading to filament accumulation.
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;
Jaquemar et al., 2003;
Tamai et al., 2000).
Interestingly, only 6.8% of Mrj-deficient embryos (4/59) developed
placental site hemorrhages (see Fig. S2 in the supplementary material). No
hemorrhages were observed in wild-type or heterozygous conceptuses (0/147).
The rarity of placental hemorrhage in Mrj mutants indicated that the
consequences of Mrj deficiency differ from those of keratin
deficiency.
Keratin inclusion bodies are toxic to chorionic trophoblast cells and disrupt normal function
To investigate further whether keratin deficiency was responsible for the
placental phenotype in Mrj-/- embryos, we assessed the
occurrence of chorioallantoic attachment in keratin mutant mice.
Chorioallantoic attachment was normal in both K18-/-- and
K19-/--knockout embryos
(Fig. 6B,C and data not shown),
resulting in mice that are viable and fertile
(Hesse et al., 2000;
Magin et al., 1998;
Tamai et al., 2000). Although
K18-/-;K19-/- embryos have placental
hemorrhages (Hesse et al.,
2000), they all exhibited chorioallantoic attachment (n=8
double mutants from five litters) (Fig.
6D), indicating that this process can occur in the absence of a
keratin cytoskeleton. This raised the possibility that keratin aggregation
causes the Mrj phenotype. To address this,
Mrj+/-;K18+/- mice were mated and the
resulting progeny (n=9 litters) were assessed for the presence of
placental hemorrhages and chorioallantoic attachment. The incidence of
placental hemorrhage in Mrj mutants was unaltered in
Mrj-/-;K18+/- (1/13) or
Mrj-/-;K18-/- (1/9) conceptuses. By
contrast, 77% of Mrj-/-;K18+/- embryos
(10/13) and 100% of Mrj-/-;K18-/-
embryos (9/9) exhibited rescue of chorioallantoic attachment
(Fig. 6E,F;
Table 1). Histological analysis
of rescued Mrj-/-;K18+/- and
Mrj-/-;K18-/- placentas at E9.5
confirmed that chorioallantoic attachment had occurred and that subsequent
villous formation was initiated. However, the chorionic plates remained
compact with little branching or fetal blood vessel growth
(Fig. 6H,I). Furthermore, fewer
branchpoints were seen in
Mrj-/-;K18+/- chorionic plates
(Fig. 6H) compared with those
of Mrj-/-;K18-/-
(Fig. 6I). This contrasted with
wild-type and Mrj+/-;K18+/+ littermate
placentas (data not shown and Fig.
6G, respectively), which had extensive branching and
vascularization within the labyrinth layers of their placentas.
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),
indicating that the 129Sv genetic background, itself, is not sufficient to
rescue chorioallantoic attachment in
Mrj-/-;K18+/- or
Mrj-/-;K18-/- embryos. Together, these
results suggest that, by reducing the amount of keratin expressed, and thus
aggregated, chorionic trophoblast cell function was restored and
chorioallantoic attachment was rescued. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells containing keratin inclusions, such as Mallory bodies, also have a
diminished keratin cytoskeleton (Denk et
al., 2000). To date, this has complicated our understanding of the
consequences of protein aggregation. In this study, we have shown directly
that keratin deficiency and keratin inclusion bodies have different effects.
In the developing placenta, complete deficiency of the keratin cytoskeleton
(e.g. K8 mutants or K18/19 double mutants) is associated
with normal chorioallantoic attachment, whereas keratin inclusions (e.g.
Mrj mutants) are associated with a failure in chorioallantoic
attachment.
An essential function of keratin is to protect the cell against mechanical
stresses (Owens and Lane,
2003). Mice that lack K8
(Jaquemar et al., 2003), both
K8 and K19 (Tamai et
al., 2000) or both K18 and K19
(Hesse et al., 2000) fail to
form keratin filaments in simple epithelial cells, such as trophoblast cells.
As a result, mutant embryos die at approximately E10.5 because of compromised
trophoblast cell integrity, which leads to placental site hemorrhages that are
particularly associated with trophoblast giant cells. Despite a compromised
keratin cytoskeleton in Mrj mutants, placental hemorrhage was rare,
indicating that Mrj deficiency is distinct from keratin deficiency.
One explanation for this is that Mrj is not expressed in all trophoblast giant
cells and, therefore, not all Mrjdeficient trophoblast giant cells
lack a normal cytoskeleton or contain aggregates. This implies the involvement
of another cochaperone in the regulation of keratin degradation within these
cells. As a result, trophoblast giant cells with a normal keratin cytoskeleton
are probably interspersed with affected cells. This arrangement may be
sufficient to maintain the integrity of the trophoblast giant cell layer
surrounding the implantation site, preventing hemorrhage.
|
).
Desmoplakin mutants die at E6.5 because unstable desmosomes lead to a loss of
integrity within extra-embryonic tissues
(Gallicano et al., 2001;
Gallicano et al., 1998).
Normal localization of desmoplakin at the desmosome within
K18-/-;K19-/- trophoblast cells
(Hesse et al., 2000) suggests
that, in the absence of keratin filaments, desmosomes remain relatively
stable. By contrast, Mrj-deficient chorionic trophoblast cells showed
a significant reduction of desmoplakin expression. As a result, desmosomes in
Mrj-/- trophoblast cells may be unstable, resulting in
disrupted chorionic organization. This implies further that keratin inclusion
bodies disrupt cell function independently of keratin deficiency. Presumably,
the reduction of K18 expression in
Mrj-/-;K18+/- and
Mrj-/-;K18-/- conceptuses decreased
the amount of keratin aggregated, which in turn salvaged chorionic
organization and rescued chorioallantoic attachment.
During chorioallantoic attachment, the allantois attaches directly to the
chorionic mesothelium (Downs,
2002). However, there are a handful of genes, including
Mrj, that are expressed only in the chorionic trophoblast and which,
when knocked-out, result in chorioallantoic attachment defects
(Hildebrand and Soriano, 2002;
Parr et al., 2001;
Saxton et al., 2001;
Tetzlaff et al., 2004). This
reveals that the mesothelium must interact with the trophoblast compartment
for it to become receptive to the allantois. Conceptuses deficient for
Wnt7b, a gene encoding a signaling protein secreted by chorionic
trophoblast cells, fail to express 
4-integrin (encoded for by
Itga4), a mesothelially expressed adhesion molecule required for
allantoic attachment (Parr et al.,
2001; Yang et al.,
1995). Interestingly, Wnt7b-/- chorions are
disorganized (Parr et al.,
2001), a phenotype reminiscent of Mrj deficiency.
Although it was previously shown that 4-integrin is expressed in
Mrj-/- chorionic mesothelium
(Hunter et al., 1999), it is
unknown whether it is properly presented and active at the cell surface.
Regardless, there are probably other unidentified adhesion mechanisms that are
required because the chorioallantoic attachment phenotype in
4-integrin-deficient embryos is incompletely penetrant
(Yang et al., 1995). This
suggests that the disorganization of the Mrj-/- chorionic
trophoblast layer may disrupt signaling between the trophoblast and
mesothelium. Without this interaction, the mesothelium may not be prepared to
receive the allantois, resulting in a failure of chorioallantoic
attachment.
Molecular chaperones are components of the protein-folding machinery and
can also be transformed into protein-degradation factors when bound to the
correct regulatory proteins (Esser et al.,
2004). The DnaJ co-chaperones not only regulate the ATPase
activity and substrate-binding specificity of Hsp70, but also play a
significant role in determining which chaperone pathway Hsp70 should pursue
(Esser et al., 2004). For
example, binding of Ydj1, a yeast homolog of DnaJ, to Hsp70 promotes the
proper folding of its substrate (Meacham
et al., 1999). By contrast, a human DnaJ homolog, HSJ1 (also known
as DNAJB2 - Human Gene Nomenclature Database), and HSP70 stimulate
substrate-ubiquitination and -sorting to the proteasome for degradation
(Westhoff et al., 2005). MRJ
can bind and activate HSP70 (Chuang et al.,
2002; Izawa et al.,
2000) yet, until now, determining whether this complex is involved
in protein-folding or -degradation had never been pursued.
A limited number of chaperones interact with intermediate filaments, giving
us few clues about how chaperone complexes regulate the keratin cytoskeleton
(Izawa et al., 2000;
Liao et al., 1995;
Liao et al., 1997;
Perng et al., 1999). The
keratin network is highly dynamic, but surprisingly little is known about its
regulation (Coulombe and Omary,
2002). A simple keratin filament, composed of heterodimers of type
I (K18 or K19) and type II (K7 or K8) keratins, can form spontaneously without
catalysts (Owens and Lane,
2003; Quinlan et al.,
1986). Hsp70 (Izawa et al.,
2000; Liao et al.,
1995) and Mrj (Izawa et al.,
2000) can associate with both soluble and filamentous forms of
K8/K18. However, our finding that keratin filaments do form, albeit ending up
in aggregates, within Mrj-/- trophoblast cells indicates
that Mrj is not required for either keratin folding or keratin-filament
assembly.
With the help of various proteins, keratin filaments bundle together and
attach to desmosomes at the cell membrane to become organized into a dense
network (Coulombe and Omary,
2002; Owens and Lane,
2003). We have shown here that Mrj is diffusely expressed
throughout the cytoplasm, but does not localize onto K18-containing filaments
in trophoblast cells. This suggests that Mrj is not involved in playing a
structural role, which contradicts previous findings that described Mrj
expression as filamentous (Izawa et al.,
2000). This difference may be explained by the use of different
cell types or by the resolution of imaging.
Keratin filaments can be remodeled or degraded by varying the levels of
phosphorylation and ubiquitination
(Coulombe and Omary, 2002;
Ku and Omary, 2000). Our study
demonstrates the involvement of Mrj in keratin degradation. As indicated
above, Mrj does not colocalize to K18-containing filaments. However, it was
previously demonstrated that Mrj can interact with the soluble fraction of
K8/K18 proteins (Izawa et al.,
2000). Furthermore, keratin inclusions in
Mrj-/- cells are ultrastructurally similar to Mallory
bodies that appear in the hepatocytes of patients with chronic liver disorders
(Denk et al., 2000). A defect
in the degradation mechanism of the proteasome is one cause of Mallory body
formation because cells treated with inhibitors of proteasome function
(Bardag-Gorce et al., 2004) or
cells that contain the UBB+1 ubiquitin mutation
(Bardag-Gorce et al., 2003)
accumulate K18 and K8. We have shown here that Mrj protein co-localizes to the
proteasome in trophoblast giant cells. When we chemically inhibited proteasome
function in these cells, keratin aggregates formed that were similar to those
in Mrj-deficient cells, suggesting that, in the absence of Mrj, K18
is not targeted to and/or degraded by the proteasome.
Mrj deficiency was associated with effects on the entire keratin
cytoskeleton, such that K8-, K18- and K19-containing filaments aggregated.
Previously, Mrj was shown to specifically interact with K18 but not with K8
(Izawa et al., 2000); however,
whether Mrj associates with K19 is unknown. Because K18 and K19 are both type
I keratins, it is plausible that Mrj also directly regulates K19-filament
degradation. Alternatively, what may start as a primary effect on K18 protein
aggregation may indirectly impair the activity of the ubiquitin-proteasome
system, causing a vicious circle of protein aggregation
(Bence et al., 2001;
Esser et al., 2004). Another
hypothesis suggests that there is interdependence between K8/K18 and K8/K19
filaments such that, if one type of filament aggregates, the other collapses
into the inclusion body as well.
Our study clearly supports the hypothesis that the formation of keratin inclusion bodies in Mrj-deficient trophoblast cells is detrimental to cellular function rather than protective. If non-degraded keratin is sequestered to protect the cell, we would expect that the chorioallantoic attachment defect observed in Mrj-/- conceptuses is due to a non-functioning keratin cytoskeleton. However, K18-/-;K19-/- embryos underwent chorioallantoic attachment. Instead, by genetically reducing or eliminating K18 expression in Mrj-deficient embryos (Mrj-/-;K18+/- or Mrj-/-;K18-/-), and thus reducing the amount of keratin aggregated, we observed that chorionic trophoblast cell function was restored and chorioallantoic attachment was rescued. This suggests that the keratin inclusions themselves are toxic to cells. Although we have not documented when keratin aggregates first appear in the trophoblast giant cells of Mrj mutants, it is remarkable that implantation occurs and that post-implantation development progresses as far as it does.
There are many mutants within the literature with defects in
chorioallantoic attachment, yet several of these display incomplete penetrance
(reviewed by Watson and Cross,
2005). Interestingly, mutant conceptuses that achieve
chorioallantoic attachment will often exhibit later defects in morphogenesis
of the labyrinth layer of the placenta (reviewed by
Watson and Cross, 2005).
Although chorioallantoic attachment was rescued in the majority of
Mrj-/-;K18+/- and
Mrj-/-;K18-/- placentas, the labyrinth
layer remained under-developed such that little villous branching occurred
within the chorionic plate and only a small amount of fetal vascularization
was apparent. Furthermore,
Mrj-/-;K18+/- labyrinths were
more-severely affected compared to those of
Mrj-/-;K18-/-, suggesting that the
continued presence of keratin aggregates, even at a reduced level, affects
branchpoint morphogenesis and the subsequent development of the labyrinth.
Reduced villous formation in
Mrj-/-;K18-/- placentas compared to
wild type suggests that Mrj function may be required for labyrinth development
independent of its role in keratin turnover. This implies that Mrj probably
interacts with another substrate during this stage in placental
development.
Mrj deficiency may affect a variety of tissues that express
keratins, apart from the placenta, because Mrj is widely expressed
both during later embryonic development and in the adult
(Chuang et al., 2002;
Hunter et al., 1999;
Seki et al., 1999). In
addition, Mrj has been shown to interact with other substrates
(Chuang et al., 2002;
Dai et al., 2005), notably the
HD protein (Chuang et al.,
2002). In Huntington's disease, mutant HD proteins with expanded
N-terminal repeats aggregate within neurons
(DiFiglia et al., 1997). In
vitro, the Mrj-Hsp70 complex can reduce the accumulation of mutant HD
aggregates (Chuang et al.,
2002). These data are consistent with a general role for Mrj in
preventing intracellular inclusion bodies, probably by promoting protein
degradation through the proteasome.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1809/DC1
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ACKNOWLEDGMENTS |
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