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First published online 28 February 2007
doi: 10.1242/dev.02816
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Cancer and Developmental Biology Laboratory, National Cancer Institute, Frederick MD 21702, USA.
* Author for correspondence (e-mail: stewartc{at}ncifcrf.gov)
Accepted 9 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Nuclear envelope, Vasculogenesis, Man1 (Lemd3), TGFß
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A
growing body of evidence suggests that the lamins, as well as other proteins
associated with the lamina and nuclear envelope, are crucial for interphase
nuclear architecture, the structural integrity of the nucleus, gene
regulation, DNA replication and chromatin organization. These activities are
also highlighted by the fact that at least nine different diseases, ranging
from muscular dystrophy to premature aging (the laminopathies), are linked to
mutations within the lamin A gene (Burke
and Stewart, 2002). In addition to the laminopathies, other
diseases are caused by mutations in proteins associated with the NE
(Gruenbaum et al., 2005;
Worman, 2005).
Some eighty different NE-associated proteins have been identified
(Schirmer et al., 2003), and
among these are members of the LEM domain family
(Lee and Wilson, 2004;
Schirmer et al., 2003). LEM
domain proteins contain a 43 amino acid motif facing the nucleoplasm, shared
by the prototype members of the LEM family, LAP2, emerin and MAN1 (LEMD3)
(Cai et al., 2001;
Laguri et al., 2001;
Lin et al., 2000;
Wolff et al., 2001). Although
mutated forms of emerin and possibly LAP2 result in muscular dystrophy and
cardiomyopathy (Melcon et al.,
2006; Morris,
2004; Taylor et al.,
2005), the functions of LEM domain proteins are still ill-defined.
In Xenopus, XMAN1/SANE functions as an embryonic neuralizing factor,
by antagonizing bone morphogenetic protein (BMP) signaling
(Osada et al., 2003;
Raju et al., 2003), whereas in
humans, mutations in MAN1 (LEMD3) result in bone and
connective tissue disorders (Hellemans et
al., 2006; Hellemans et al.,
2004).
TGFß/BMP/activin signaling pathways activate SMAD transcription
factors and are important in regulating mouse embryogenesis
(Goumans and Mummery, 2000).
Previous studies revealed that MAN1 binds to and regulates SMAD
transcriptional activity, suggesting a potentially significant role for MAN1
in regulating TGFß/BMP signaling
(Hellemans et al., 2004;
Lin et al., 2005;
Pan et al., 2005). To
determine the role of Man1, we analyzed the expression of
Man1 and used a gene-trapped (GT) embryonic stem (ES) cell clone to
derive mice with a functional mutation in the Man1 gene. We show that
Man1 is essential for embryonic vasculogenesis and that the
Man1GT/GT mutation results in hyperactivation of some
components in TGFß signaling pathways.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) in the
Man1 locus (BayGenomics, San Francisco, CA), was microinjected into
C57Bl/6 blastocysts as described (Stewart,
1993). The resulting Man1GT line was
maintained as heterozygotes and intercrossed to obtain homozygotes
(Man1GT/GT). All mice were maintained in our facility in
accordance with the procedures outlined in the Guide for the Care and Use of
Laboratory Animals (NIH Publication No. 86-23, 1985). Genotypes were determined by Southern blotting or RT-PCR. For Southern analysis, a 300 bp genomic fragment, generated using primers 5'-GCGCTGGGTTACTTTGTGTGCTG-3' and 5'-GCTTCCCGTTCACCACACTTCTG-3', was used to probe DNA digested with EcoRV. Embryos were genotyped by RT-PCR using forward primer 1 located within exon 4 (5'-GACCATGAATGTGGCAGTTCTA-3') and reverse primer 2 located within exon 5 (5'-CGTACATGTGGAATAGGCATGTAAGG-3') to obtain a 424 bp wild-type PCR product and primer 1 and reverse primer 3 located within the ß-geo sequence (5'-TCGTCTGCTCATCCATGACC-3') to obtain a 1.6 kb mutant PCR product.
Northern analysis
Total RNA was extracted using the RNeasy Kit (Qiagen, Valencia, CA). Ten
micrograms of RNA were separated on 1% formaldehyde 1% agarose gels and
transferred to Hybond membranes (Amersham Biotech, Piscataway, NJ). The mouse
cDNA corresponding to nucleotides 1261-1898 bp of the Man1 transcript
(I.M.A.G.E. Consortium clone ID 455955) was used as a probe and a
Gapdh cDNA as a loading control. Tissue and embryonic blots were
obtained from Seegene (Rockville, MD).
Derivation of Man1GT/GT gene-trapped cells
Blastocysts from Man1 heterozygous intercrosses were used to
derive homozygous Man1GT/GT ES cells as described
(Abbondanzo et al., 1993;
Stewart et al., 1995). ES
clones were genotyped by northern analysis (see above). Embryoid bodies (EBs)
were prepared by growing ES cells in suspension. Two-week-old EBs were plated
onto 35 mm culture dishes to allow formation of explants.
Homozygous Man1GT/GT embryonic stem cells were injected
into blastocysts and Man1GT/GT fibroblasts were derived
from chimeric embryos at embryonic day 13 (E13) as described
(Escalante-Alcalde et al.,
2003). Man1GT/+ and wild-type fibroblasts were
derived from embryos of heterozygous crosses as described
(Escalante-Alcalde et al.,
2003). Primary fibroblasts were immortalized by retroviral
infection with SV40 large T antigen (Zhu
et al., 1991). For SMAD translocation experiments, PMEFs were
infected with retroviral vector (pBabe-Puro) containing a full-length MAN1
cDNA tagged with a Flag epitope (kind gift of Howard Worman, Columbia
University, New York, NY).
Tetraploid rescue
Wild-type tetraploid embryos were prepared by incubating fertilized
(C57Bl/6xC3H F1) oocytes overnight in KSOM (Specialty Media) with 0.25
µg/ml cytochalasin D (Sigma), which suppresses the first cleavage division,
making the zygotes tetraploid. The embryos were washed four times in KSOM to
remove the cytochalasin D and allowed to progress to the two-cell stage.
Two-cell-stage embryos were transferred to the oviducts of pseudopregnant
recipients for an additional 24-48 hours for further development. Tetraploid
blastocysts were recovered and injected with Man1GT/GT ES
cells. Embryos were transferred to pseudopregnant B6CBAF1 recipients with the
embryos being recovered on E9.5-12.
Histological staining
Embryos aged E8.0-10.0 were stained for lacZ expression and
photographed as previously described
(Escalante-Alcalde et al.,
2003). Benzidine staining to detect blood islands was performed as
described (Orkin et al.,
1975).
Embryos were whole-mount immunostained with antibodies to platelet
endothelial cell adhesion molecule (PECAM-1; also known as PECAM1) and FLK-1
(KDR - Mouse Genome Informatics) (BD Biosciences, San Diego, CA), as described
(Byrd et al., 2002;
Schlaeger et al., 1995). For
immunohistochemistry, embryos were fixed in 4% paraformaldehyde, embedded in
paraffin, sectioned and stained using indicated antibodies. Immunofluorescence
using an antibody to fibronectin was performed as described
(Zwijsen et al., 1999).
EB explants were immunostained with PECAM-1 as described
(Escalante-Alcalde et al.,
2003). For fluorescence activated cell sorting (FACS) analysis to
quantify expression of PECAM-1 in EB explants, 7-dayold explants were
trypsinized, immunostained with an antibody to PECAM-1 conjugated to
phycoerythrin (PE, BD Biosciences) and analyzed on an LSR1 flow cytometer
using CellQuest software (Becton Dickinson). Unstained cultures were used as
negative controls.
Immunofluorescence
Mouse embryonic fibroblasts (MEFs) were fixed with 4% paraformaldehyde,
stained with primary antibodies to SMAD1 (Santa Cruz, CA), SMAD2/3 (BD
Biosciences) and Flag (Affinity Bioreagents, Golden, CO) and then with
secondary antibodies conjugated to Alexa 488 or Alexa 568 (Molecular Probes,
Eugene, OR). After immunolabeling, coverslips were mounted in Vectashield
Mounting Medium (Vector Laboratories, Burlingame, CA) and visualized using a
Zeiss Axiophot inverted microscope.
Western analysis
Whole cell lysates of MEFs were immunoblotted with a rabbit polyclonal
antiserum raised against a peptide (aa898-911) at the C-terminus of MAN1.
Antibodies used were for fibronectin (1:300, ICN), p21 (1:100, BD Pharmingen),
SMAD2 (1:250, Zymed, San Francisco, CA), pSMAD2 (1:250, Upstate,
Charlottesville, VA), pSMAD1 (Cell Signaling, St Louis, MO), and actin
(1:1000, Santa Cruz). Detection was performed using Chemiluminescence Reagent
Plus (PerkinElmer, Boston, MA). Cytoplasmic and nuclear fractions were
prepared using the NE-PER kit (Pierce, Rockford, IL) according to the
manufacturer's instructions.
Transient transfection and luciferase reporter assays
The pTP3-Lux construct containing a TGFß1-responsive Plasminogen
Activated Inhibitor I (PAI-1) promoter linked to a luciferase cDNA
(Wrana, 2000) (a kind gift of
Joan Massague, Memorial Sloan-Kettering Cancer Center, New York, NY) was
transfected into MEFs using Fugene 6 (Roche, Indianapolis, IN) according to
the manufacturer's instructions. To normalize for transfection efficiency,
cells were co-transfected with 0.5 µg pTK-RL (Promega, Madison, WI). After
42 hours following the transfection, cultures were incubated for 8 hours with
hrTGFß1 (R&D Systems) at indicated concentrations. Luciferase
activities were measured using the Dual-Luciferase Assay according to the
manufacturer's instructions (Promega).
Cell proliferation assay
MEFs were seeded onto 96-well culture dishes at a concentration of
5x103 cells per well and treated with indicated
concentrations of hTGFß1 (R&D Systems). The endothelial cell line
SVEC (ATCC #CRL-2181) was previously characterized
(O'Connell and Edidin, 1990).
After 48 hours, cells were incubated overnight with MTT using the Cell
Proliferation Kit I (Roche) according to the manufacturer's instructions.
Quantification of the formazan reaction product in active cells was performed
using the VersaMax Plate reader (Molecular Devices).
Real-time analysis
RNA from MEFs and EB explants was extracted using the RNeasy Kit and 1
µg was reverse-transcribed using the First-Strand cDNA Synthesis Kit
(Roche). Real-time PCR was performed on an ABI Prism 7000 using SYBR Green
Master Mix (AB, Warrington, UK).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Northern
analysis of RNA from heterozygous fibroblasts revealed a 7.7 kb
Man1-ß-geo hybrid transcript in addition to the
wild-type 4.7 kb transcript (Fig.
1C), indicating that the ß-geo sequence is in frame
with the Man1 transcript and the first 1702 bp of the endogenous
Man1 transcript is retained. No additional transcripts were observed,
indicating additional alternative splicing to exons downstream of the
gene-trap insertion into Man1 did not occur. This analysis predicts
that the gene-trapped Man1GT allele produces a fusion
protein consisting of 567 N-terminal MAN1 amino acids fused to ß-geo and
lacking the C-terminal sequence downstream of exon 4
(Fig. 1D). Western analysis
using an antibody derived from the C-terminus of MAN1 confirmed that the
C-terminus was no longer recognized (Fig.
1E).
Man1 expression during embryogenesis
The expression of Man1 was initially determined by northern
analysis (not shown). In embryonic tissues, the signal intensity of
Man1 was low at stages E4.5 through E8.5 and increased in intensity
in later developmental stages from days E9.5 through E18.5. Low levels of the
Man1 transcript were also observed in most adult tissues, whereas
robust expression was seen in the brain, testes and placenta. We used the
ß-galactosidase activity of the gene-trapped fusion protein product to
further determine the expression of Man1 during development.
Expression of Man1GT was first detectable in the
ectoplacental cone and yolk sac at E8.5
(Fig. 2A). At E9.5,
lacZ expression increased initially in the gut epithelium and the
floor plate (Fig. 2B) and by
E11 spread to the skin and throughout the embryo
(Fig. 2C), consistent with the
widespread expression of Man1 detected by northern analysis. At E13,
expression was widespread, with robust staining observed in the central
nervous system (Fig. 2E-G),
including the midbrain and hindbrain, neural tube, pons and cerebellum.
Staining was also observed in the placenta
(Fig. 2D,E) and yolk sac
(Fig. 2E,H,I) in the visceral
endoderm, endothelial cells and mesoderm but excluded from blood cells
(Fig. 2H,I).
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The yolk-sac abnormality in Man1GT/GT mice is not due to defective placental function
Abnormal development and lethality of embryos around E8.5-9.5 is frequently
due to primary defects in placental development
(Rossant and Cross, 2001). To
establish whether the Man1GT/GT phenotype was due to
placental defects, we performed a tetraploid rescue experiment. Diploid
Man1GT/GT ES cells were microinjected into wild-type
tetraploid blastocysts and transferred to pseudopregnant recipients.
Postimplantation embryos were recovered on E9.5 or E12. Of the 36 blastocysts
transferred, three embryos were found to resemble the
Man1GT/GT phenotype
(Table 2), and their genotype
was confirmed by RT-PCR. Of these three, two were delayed approximately 36
hours and blood vessels had not formed from the primitive plexus. The third
embryo contained irregularly shaped blood vessels and had undergone embryonic
axial rotation but was edematous and small compared to E9.5 wild-type embryos.
No embryos were observed to develop beyond the Man1GT/GT
phenotype and none were recovered at E12. As the wild-type tetraploid cells
form the placenta and visceral endoderm layer of the yolk sac, our results
indicate that embryonic lethality was probably not due to a defect in
placental function.
|
). In wild-type yolk sacs, the endoderm and mesoderm are
tightly apposed and contain many developing blood vessels and discreet blood
islands (Fig. 5A). In
Man1GT/GT yolk sacs, the endoderm and mesoderm were poorly
attached to each other, resulting in large lacunae containing blood cells
(Fig. 5B). Immunostaining with
antibodies to PECAM-1 and FLK-1 revealed that although endothelial cells were
present in Man1GT/GT yolk sacs, many remained rounded,
were misaligned or had failed to incorporate into the vessel walls
(Fig. 5B,C).
Formation of branched blood vessels during vasculogenesis requires the
proliferation and recruitment of smooth muscle cells to line the vessel walls.
TGFß1 is a potent inhibitor of vascular smooth muscle cell (VSMC)
proliferation (Feinberg et al.,
2004). We analyzed sections immunostained with an antibody for the
VSMC marker 
-smooth muscle actin (ACTA2)
(Fig. 5D). Yolk sacs from
wild-type embryos contained a large number of VSMCs, whereas VSMCs appeared to
be less abundant in the Man1GT/GT embryos. Quantification
of Acta2 transcripts in E9.5 yolk sacs by RT-PCR revealed an
approximately twofold reduction in transcript levels in the
Man1GT/GT yolk sacs compared with wild type (data not
shown). This reduction in VSMCs is probably due to their decreased
proliferation and defective recruitment into developing blood vessels,
resulting in the subsequent failure of nascent capillaries to form functional
blood vessels.
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).
Thus, despite the formation of ECs in Man1GT/GT embryos
and EBs, further organization into branched structures is defective.
Quantitation of PECAM-1+ ECs in the EB explants was performed using
FACS analysis. During differentiation, ECs first express low levels of
PECAM-1, then expression of PECAM-1 increases as ECs differentiate from
precursors (Levenberg et al.,
2002). In accordance, two peaks of PECAM-1+ cells were
observed by FACS analysis. No difference was observed between wild-type and
Man1GT/GT cultures in the fraction of low-expressing
PECAM-1+ precursor cells, while the fraction of high
PECAM-1-expressing EC cells was reduced in Man1GT/GT
cultures compared with wild types (
30% of total cell population was high
PECAM-1+ in wild-type EB cultures compared with only 7% of cells in
the Man1GT/GT cultures)
(Fig. 6C).
Previous evidence suggested that Man1 regulates cell
proliferation. TGFß1 inhibits fibroblast proliferation, and
overexpression of Man1 decreases the anti-proliferative effect of
TGFß1 (Lin et al., 2005).
By contrast, decreased Man1 expression enhances the
anti-proliferative activity of TGFß1
(Pan et al., 2005). To
determine whether disruption of Man1 increased the sensitivity of
cells to the anti-proliferative effect of TGFß1, we compared cell
proliferation in response to TGFß1 treatment in
Man1GT/GT and wild-type MEFs
(Fig. 6D). Treatment of
wild-type MEFs with 1.6 ng/ml of TGFß1 resulted in a 10% suppression of
cell proliferation. By comparison, we also found that TGFß1 inhibited
proliferation of the immortalized mouse endothelial SVEC cell line to a
similar extent. By contrast, treatment of Man1GT/GT MEFs
with TGFß1 resulted in a significantly increased suppression of cell
proliferation by 25%. These results confirm that TGFß1-mediated
inhibition of proliferation is more effective in Man1GT/GT
cells.
To further characterize the reduction in the proliferation of endothelial
cells, the expression of endothelial cell markers and cell cycle mediators was
quantitated in the EB explants. Real-time PCR analysis showed that expression
of endothelial markers Flt-1 and Alk5 (Tgfbr1 -
Mouse Genome Informatics) were both reduced in Man1GT/GT
cultures (Fig. 6E). Consistent
with the observations in the yolk-sac sections
(Fig. 5D), the expression of
Acta2 was reduced by 60% relative to wild-type EB explants
(Fig. 6E). By contrast,
increased expression of the cell cycle inhibitors p15ink4b
(Cdkn2b - Mouse Genome Informatics) and p27kip1
(Cdkn1b - Mouse Genome Informatics) was observed, consistent with the
suppression of cell proliferation in the Man1GT/GT EB
explants. Both p15ink4b and Timp1 are direct transcriptional
targets of TGFß signaling (Edwards et
al., 1987; Hannon and Beach,
1994), and their increased expression suggests that TGFß
signaling is upregulated in the Man1GT/GT EB explants.
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). As MAN1 antagonizes BMP and TGFß1 signaling by
interacting with R-SMADs (Hellemans et
al., 2004; Lin et al.,
2005; Osada et al.,
2003; Pan et al.,
2005; Raju et al.,
2003), we analyzed whether SMAD activity was disrupted in
Man1GT/GT cells. We examined R-SMAD localization in
TGFß1-treated Man1GT/GT MEFs using antibodies to
R-SMADs (Fig. 7Aa-i). Untreated
wild-type MEFs express equivalent levels of SMAD2/3 in the nucleus and
cytoplasm (Fig. 7Aa). Treatment
of wild-type MEFs with TGFß1 resulted in an increase in SMAD2/3 signal in
their nuclei (Fig. 7Ab). In the
Man1GT/GT MEFs, high levels of nuclear SMAD2/3
immunofluorescence were observed in untreated cells
(Fig. 7Ac), and this nuclear
signal further increased following treatment with TGFß1 (not shown).
Expression of a full-length Flag-tagged MAN1 in wild-type MEFs prevented the
nuclear accumulation of SMAD2/3 (Fig.
7Ab,e insets). However, in Man1GT/GT MEFs
expressing Flag-MAN1, SMAD2/3 was equally distributed between the cytoplasm
and nucleus (Fig. 7Ac,f inset),
indicating that increased nuclear SMAD accumulation in
Man1GT/GT MEFs can be reduced by the reexpression of
wild-type MAN1. The increased accumulation of nuclear SMAD2 was quantified by immunoblotting cytoplasmic and nuclear fractions from Man1GT/GT and wild-type MEFs (Fig. 7B). Treatment with TGFß1 resulted in a more robust nuclear localization of SMAD2 in Man1GT/GT MEFs (upper panel). By contrast, in Man1GT/GT MEFs expressing a full-length MAN1, the nuclear localization of SMAD2 in Man1GT/GT MEFs was significantly reduced (Fig. 7B lower panel).
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Transcriptional targets of TGFß1 signaling are misregulated in Man1GT/GT cells
To examine whether the transcription of TGFß1-regulated genes is
affected in Man1GT/GT cells, we performed luciferase
reporter assays in Man1GT/GT and wild-type MEFs
(Fig. 7D) transfected with
p3TP-Lux (Wrana, 2000).
Treatment of Man1GT/GT and wild-type MEFs with TGFß1
for 8 hours resulted in approximately threefold higher levels of normalized
luciferase reporter activity than in untreated cultures
(Fig. 7D). However, basal
luciferase activity was approximately tenfold higher in
Man1GT/GT MEFs compared with wild types, indicating
increased basal levels of R-SMAD-dependent transcription in
Man1GT/GT MEFs. Together, these data reveal that R-SMAD
levels in Man1GT/GT cells are not only increased in the
nucleus, but also have increased functional activity.
We extended these findings to determine what effect the
Man1GT/GT mutation has on specific gene expression levels
in MEFs. Several known targets of TGFß1 signaling were analyzed using
quantitative real-time PCR in Man1GT/GT and wild-type MEFs
treated with TGFß1 for 48 hours (Fig.
7E). Transcript levels of Pai-1 (Serpine1 -
Mouse Genome Informatics) were upregulated in untreated
Man1GT/GT MEFs compared with wild-type MEFs and treatment
with TGFß1 resulted in a further fivefold increase. Similarly
transcript levels of Timp1 were upregulated in untreated
Man1GT/GT MEFs compared with wild-type MEFs and were
further increased by TGFß1 treatment. Previous reports using gene
profiling of MEFs demonstrated that inhibitor of DNA binding-3 (Id3),
a target of the BMP signaling pathway, is negatively regulated by TGFß1
stimulation (Karlsson et al.,
2005). Id3 levels were consistently decreased by the
TGFß1 treatment and showed decreased levels in
Man1GT/GT MEFs.
Proper yolk-sac formation also requires a balanced deposition in
extracellular matrix molecules (Baldwin,
1996). Analysis of fibronectin (Fn; also known as Fn1) expression
in yolk-sac sections by immunofluorescence showed that fibronectin levels are
increased in the mesoderm but reduced in the endoderm in the
Man1GT/GT yolk sacs, indicating abnormal extracellular
matrix deposition in the Man1GT/GT yolk sacs
(Fig. 5E). Consistently, we
found an upregulation of Fn mRNA levels in
Man1GT/GT EBs (Fig.
6E) and Man1GT/GT MEFs
(Fig. 7F) and an increase in Fn
protein levels in Man1GT/GT MEFs
(Fig. 7F upper panel). Because
the extracellular matrix plays an important role in regulating the
proliferation of endothelial cells during lumen formation and blood vessel
branching (Baldwin, 1996),
increased levels of fibronectin, as well as other extracellular matrix
proteins including PAI-1 and TIMP1 in Man1GT/GT embryos,
may have a direct impact on EC proliferation and migration, resulting in
defective vasculogenesis. In addition, expression of cell cycle regulator
p21waf1 (Cdkn1a - Mouse Genome Informatics) was increased in
Man1GT/GT MEFs (Fig.
7F, lower panel), indicating that increased TGFß1 signaling
and activation of cell cycle regulators may contribute to the decreased
proliferation of ECs in Man1GT/GT embryos.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Rossant and Howard, 2002).
|
;
Massague et al., 2005). One of
the more recently identified factors implicated in regulating TGFß
signaling is the nuclear-envelope-associated protein MAN1. Recent studies
showed that the nucleoplasmic RRM domain at the C-terminus of MAN1 binds, and
seemingly sequesters, SMADs1-3 to the nuclear envelope. Such sequestration
regulates SMAD nuclear translocation and transcriptional activity, by an as
yet undefined mechanism (Lin et al.,
2005; Pan et al.,
2005). Here we show that disruption of the Man1 gene
leads to the death of homozygous mouse embryos around the time vascularization
commences, probably by perturbing TGFß signaling in the yolk-sac
mesoderm.
The Man1GT/GT phenotype is mediated by defective TGFß signaling
In the Man1GT/GT embryos, abnormal yolk-sac
vasculogenesis is phenotypically similar to previously reported defects
arising from mutations in receptors, ligands and other components of the
TGFß signaling pathway (Agah et al.,
2000; Dickson et al.,
1995; Larsson et al.,
2001; Oshima et al.,
1996). Yolk-sac vasculogenesis consists of a proliferative
activation state and a final resolution state, which are mediated by the
coordinate activation by TGFß of two parallel receptor-mediated cellular
pathways (Oh et al., 2000).
TGFß1 acting through the ALK1 receptor and SMADs1/5/8, mediates the
activation state leading to proliferation of endothelial cells as well as
recruitment of smooth muscle cells; whereas TGFß1 acting through the ALK5
receptor and SMADs2/3, inhibits the proliferation of endothelial cells
(Bertolino et al., 2005;
Goumans et al., 2002;
Pepper, 1997). Supportive of
this model, functional deletion of Alk5 in mice leads to defects both
in hematopoiesis and vasculogenesis and death around day E10.5. Endothelial
cells derived from the deficient animals are over-proliferative and show
reduced levels of Fn in response to TGFß1
(Larsson et al., 2001).
However, mice deficient in Alk1 (Acvrl1 - Mouse Genome
Informatics) also develop defects in angiogenesis and show upregulation of the
ALK5-regulated Pai-1 (Oh et al.,
2000). Comparably, cells overexpressing Alk5 show
decreased proliferation, downregulation of inhibitory factor Id1 and
upregulation of Pai-1 (Goumans et
al., 2002). Finally, disruption of Endoglin, a component
of the TGFß1 receptor complex that antagonizes TGFß signaling
through the ALK5 receptor, also results in defective angiogenesis
(Arthur et al., 2000;
Li et al., 1999;
van den Driesche et al.,
2003).
Several lines of evidence from our studies suggest that the defect in
vascularization in Man1GT/GT yolk sacs is preferentially
mediated through an overactivated ALK5 pathway. Results from the western
analysis showed that ALK5-responsive SMAD2 and not ALK1-responsive SMAD1 is
hyperphosphorylated and more concentrated in the nucleus. In
Man1GT/GT MEFs and EBs, proliferation of ECs and VSMCs is
reduced, suggesting that vascularization in Man1GT/GT yolk
sacs is compromised due to insufficient numbers of cells being available to
remodel the existing capillary plexus. This reduction in cell number
correlated with upregulation in the expression of the cell cycle inhibitors
p15ink4b, p21waf1 and p27kip1. In addition, increased ECM
deposition (Goumans et al.,
1999), together with increased PAI-1 inhibit EC migration and
angiogenic branching (Ignotz and Massague,
1986; McIlroy et al.,
2006). Thus, increased Fn and Pai-1 levels in
Man1GT/GT embryos may have also exacerbated the effects on
EC proliferation and contributed to defective vasculogenesis in
Man1GT/GT yolk sacs.
During the review process of this paper, a complementary study of the
Man1GT/GT phenotype was published
(Ishimura et al., 2006).
Consistent with our findings, the authors demonstrated elevated nuclear levels
of SMADs2/3, as well as increased ECM deposition in the
Man1GT/GT mice. Interestingly, the authors attribute the
increase in ECM due to apoptosis, not cell proliferation, as suggested by an
apparent lack of increased phosphohistone 3 signal in the embryo sections.
Although not inconsistent with this observation, our conclusion from the
cellular proliferation analysis and upregulation of cell cycle inhibitor
proteins, suggests that decreased proliferation does play a role in the
vascular defect.
MAN1 regulates nuclear localization and phosphorylation of SMADs
R-SMADs are normally in equilibrium between the cytoplasm and the nucleus
(Risau and Flamme, 1995;
Xiao et al., 2001;
Xu et al., 2002). Stimulation
by TGFß1 increases the levels of phosphorylated R-SMADs in the nucleus.
Activated R-SMADs are subject to negative regulation by nuclear factors such
as c-Ski, SARA and SMURFs (Massague and
Chen, 2000). Gating the entry and exit of R-SMADs into the nucleus
is another means of regulating a balance of cellular response to TGFß
stimulation (Luo, 2004).
The RRM domain of MAN1 physically interacts with R-SMADs, so regulating
SMAD activity (Lin et al.,
2005; Osada et al.,
2003; Pan et al.,
2005). We demonstrated that disruption of MAN1 results in enhanced
SMAD2/3 nuclear localization and transcriptional activity, leading to the
altered expression of genes regulated by TGFß1. Although the
nucleoplasmic localization of pSMAD2 was increased in
Man1GT/GT MEFs in the absence of TGFß1 addition, this
was probably due to stimulation by endogenous TGFß/BMPs in the serum in
which the cells were cultured (Ying et
al., 2003). However, treatment of Man1GT/GT
MEFs with TGFß1 further increased the nuclear concentration and activity
of R-SMADs, suggesting that MAN1 regulates the entry/exit of SMADs in the
nucleus. Our observations also suggest that MAN1 regulates the nuclear
localization and transcriptional activity of SMADs by affecting SMAD
phosphorylation. Although MAN1 has not been shown to possess any intrinsic
phosphatase activity, the C-terminal domain may act as a scaffold or binding
site that brings phosphorylated SMADs into an association with SMAD
phosphatases (Chen et al.,
2006; Knockaert et al.,
2006; Lin et al.,
2006). A similar role for the nuclear lamina affecting the
phosphorylation state of SMADs was recently proposed, as loss of the A-type
lamins may affect TGFß1-mediated SMAD phosphorylation by regulating
protein phosphatase 2A (Van Berlo et al.,
2005). Whether the lamin A-mediated pathway is independent of MAN1
in regulating SMAD phosphorylation and activity remains to be
demonstrated.
Role of MAN1 may differ between species
Functional analysis of MAN1 in different species has revealed differing
roles, possibly reflecting tissue specificity
(Hellemans et al., 2004;
Osada et al., 2003). In
Xenopus embryos, a reduction in XMAN1 disrupted the formation of
anterior neural structures, suggesting that in XMAN1 antagonizes BMP signaling
(Osada et al., 2003).
Formation of the neural crest and anterior neural structures appeared to be
overtly normal in Man1GT/GT embryos before their death.
However, as Man1 transcripts are abundantly expressed in the adult
brain, we cannot rule out that Man1 may still play an undefined role
in later development of the nervous system. In humans, loss-of-function
mutations in MAN1 result in osteopoikilosis, melorheostosis and
Buschke-Ollendorf syndrome (Hellemans et
al., 2004). Although we performed X-ray scans on the bones from
adult Man1GT/+ mice, we did not detect any evidence of
hyperostotic lesions, and the bone density in Man1GT/+
mice was similar to that of wild-type mice (data not shown). It is unclear
whether failure to detect bone defects in the heterozygous mice is due to
possible differences in the age of onset of lesions between humans and mice,
or due to differences between the types of mutation between the human gene and
the gene-trap. As the N-terminal sequence of MAN1 is still present in
Man1GT mice, and targeting to the NE is undisturbed, it is
possible that the remaining sequence, including the LEM domain, may prevent
Man1GT/GT embryos from developing additional
phenotypes.
The nuclear envelope/lamina in development
Our findings and other recent evidence strengthens the notion that the
NE/lamina is an important nuclear compartment regulating chromatin
organization and gene expression. Studies in yeast revealed the importance of
the NE in gene silencing and telomere function
(Taddei et al., 2004). Recent
studies have shown that the lamina may be important for the sequestration, and
to some extent function, of crucial regulatory factors such as the c-Fos and
Retinoblastoma (Rb) proto-oncogenes
(Ivorra et al., 2006;
Markiewicz et al., 2002;
Melcon et al., 2006). Also,
silencing in the expression of tissue-specific transcription factors such as
MyoD, and the activity of the chromatin insulator gypsy, is
associated with their localization to the nuclear periphery and lamina,
respectively (Capelson and Corces,
2005; Lee et al.,
2006). In this capacity, MAN1, a transmembrane protein of the INM
that is differentially expressed during development and in adult tissues, may
act as a scaffold protein regulating SMAD phosphorylation, localization and
activity. The NE and lamina may therefore have many additional, as yet
undiscovered, roles in regulating cell differentiation and function during
development.
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ACKNOWLEDGMENTS |
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REFERENCES |
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