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First published online 18 February 2009
doi: 10.1242/dev.029926
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1 Orthopaedic Hospital/UCLA Department of Orthopaedic Surgery, David Geffen
School of Medicine at UCLA, Los Angeles, CA 90095, USA.
2 Department of Biological Chemistry, David Geffen School of Medicine at UCLA,
Los Angeles, CA 90095, USA.
3 Department of Molecular, Cell and Developmental Biology, University of
California Los Angeles, Los Angeles, CA 90095, USA.
* Author for correspondence (e-mail: klyons{at}mednet.ucla.edu)
Accepted 2 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: BMP, Smad, Growth plate, Chondrogenesis, Mouse
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Yoon and Lyons, 2004;
Minina et al., 2005;
Minina et al., 2001;
Solloway et al., 1998;
Yoon et al., 2006). BMPs are
required at early stages for condensation, maintaining Sox9 expression and
matrix production (Haas and Tuan,
1999; Hatakeyama et al.,
2004). BMPs also promote proliferation and differentiation at
later stages, and are required for induction of type II and X collagen
(Fujii et al., 1999;
Shukunami et al., 2000;
Drissi et al., 2003;
Leboy et al., 2001;
Valcourt et al., 2002).
BMPs transduce signals by binding to complexes of type I and II
serine/threonine kinase receptors. Ligand binding induces phosphorylation of
the receptors, which then activate canonical signaling via receptor Smads
(R-Smads) 1, 5 and 8 (Smad8 is also known as Smad9 - Mouse Genome Informatics)
(Massague et al., 2005).
R-Smads contain two domains connected via a linker region. R-Smads are
phosphorylated at the C-terminus by the activated type I receptor. They then
complex with Smad4, triggering nuclear translocation.
Overexpression of BMPs leads to increased chondrocyte proliferation and
fused skeletal elements (Brunet et al.,
1998; Duprez et al.,
1996; Wijgerde et al.,
2005). Conversely, mice lacking BMP receptors exhibit almost
complete loss of cartilage (Yoon et al.,
2005). In contrast to these severe phenotypes, cartilage-specific
loss of Smad4 results in only mild defects
(Zhang et al., 2005). These
divergent phenotypes raise the possibility that canonical Smad signaling is
largely dispensable in the growth plate, or that R-Smads signal independently
of Smad4. BMPs trigger non-Smad (noncanonical) pathways in chondrocytes in
vitro by inducing Tgfβ-activated kinase (Tak1; Map3k7), activating p38
MAPK (Mapk1). The relative roles of canonical versus noncanonical pathways,
and whether they act independently, cooperate
(Qiao et al., 2005;
Reilly et al., 2005;
Stanton et al., 2004) or
antagonize (Hoffmann et al.,
2005) each other in chondrocytes, are unknown.
FGFs inhibit chondrocyte proliferation
(Ornitz, 2005;
Murakami et al., 2004;
Raucci et al., 2004;
Sahni et al., 2001), and the
BMP and FGF pathways antagonize each other in cartilage
(Minina et al., 2005;
Yoon et al., 2006). FGFs
reduce Bmp4 and Ihh expression through undefined pathways
(Chen et al., 2001;
Naski et al., 1998).
Phosphorylation of the Smad linker region represents one potential mechanism
of FGF-mediated antagonism. The linker region contains consensus sites for
phosphorylation by Erk1/2 (Mapk3/1), leading to inhibition of Smad activity
(Fuentealba et al., 2007;
Kretzschmar et al., 1997;
Pera et al., 2003;
Sapkota et al., 2007).
The secreted factor Indian hedgehog (Ihh) is expressed in the
prehypertrophic zone, and maintains chondrocyte proliferation by promoting
Pthrp (Pthlh) expression in distal cells of the cartilage
anlagen. PTHrP binds to the PTHrP receptor (PPR; Pth1r) and negatively
regulates Ihh expression in a feedback loop
(Kronenberg, 2003). The
Ihh/PTHrP pathway acts cooperatively with BMPs
(Kronenberg, 2003;
Minina et al., 2001;
Pathi et al., 1999;
Grimsrud et al., 2001;
Pateder et al., 2000), and BMP
receptor Smads can directly activate the Ihh promoter
(Seki and Hata, 2004).
We show here that ablation of Smad1 and Smad5 in mice
results in a nearly complete block in chondrocyte differentiation, and in
imbalances in signaling cross-talk between the BMP, FGF and Ihh/PTHrP
pathways. This is in marked contrast to the mild phenotype in mice lacking
Smad4 in cartilage (Zhang et al.,
2005). These results demonstrate that canonical Smad signaling is
the major mechanism of BMP signal transduction in endochondral bone, and that
Smad1 and Smad5 are key regulators of BMP canonical signaling in the growth
plate. The data also demonstrate that Smad4 is not required for the majority
of canonical BMP signaling. Finally, we provide evidence that linker
phosphorylation of Smads represents a physiologically significant mechanism
regulating BMP signaling in the growth plate, but that the inhibitory effects
of FGFs are likely to be mediated through different mechanisms.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and Smad5
(Umans et al., 2003) floxed
mice were crossed with Col2-Cre mice
(Ovchinnikov et al., 2000) to
generate Smad1fx/fx;Col2-Cre and
Smad5fx/fx;Col2-Cre mice (referred to as
Smad1CKO and Smad5CKO, respectively;
CKO, cartilage-specific knockout), and intercrossed with
Smad8-/- mice (a gift from Richard Behringer, M. D.
Anderson Cancer Center, Houston, TX, USA).
Smad1fx/+;Smad5fx/+;Smad8+/-;Col2-Cre
mice were intercrossed to generate
Smad1fx/fx;Smad5fx/fx;Smad8-/-;Col2-Cre
mice (Smad1/5CKO;Smad8-/-).
Histology
Skeletal preparations were generated as described
(Ivkovic et al., 2003;
Yoon et al., 2006). Alcian
Blue/nuclear Fast Red staining was performed as described
(Luna, 1992). Von Kossa
staining was performed by incubation in 1% silver nitrate under UV light for
20 minutes and counterstaining with nuclear Fast Red. Safranin O staining was
performed by staining in Weigert's iron hematoxylin solution for 10 minutes,
followed by Fast Green (0.001%) and Safranin O (0.1%) for 5 minutes each.
For immunofluorescence, sections were boiled for 15 minutes in citrate
buffer (Ivkovic et al., 2003).
Sections were blocked with 5% goat or donkey serum for 1 hour and incubated
with primary antibody overnight at 4°C, followed by incubation with
secondary antibody for 1 hour at room temperature, then with fluorophore for
30 minutes at room temperature. Primary antibodies were as follows:
phospho-Smad1/5/8 and phospho-Smad1/5 (Cell Signaling Technology); type II
collagen and Pth1r (Abcam); type I collagen (Southern Biotech); type X
collagen (a kind gift from Robin Poole, Shriners Hospitals for Children,
Montreal, Québec, Canada); aggrecan (Developmental Studies Hybridoma
Bank, Iowa City, USA); Pcna (Zymed); Fgfr1 and Stat1 (Sigma); phospho-Smad1L
(a kind gift from Eddy De Robertis, University of California, Los Angeles, CA,
USA). Secondary antibodies were conjugated with AlexaFluor-555 and
AlexaFluor-488. Sections were counterstained with DAPI (Vectashield). For
TUNEL staining, the fluorescein In Situ Cell Death Detection Kit (Roche) was
used according to the manufacturer's protocol. In situ hybridization was
performed as described (Song et al.,
2007).
Limb culture
Embryos were harvested at 16.5 days of gestation (E16.5). Forelimbs were
isolated and cultured as described (Minina
et al., 2001; Minina et al.,
2002). The contralateral limb was cultured in the presence of
recombinant human FGF18 (10 ng/ml; Invitrogen) or the FGFR inhibitor SU5402
(10 µM; Calbiochem). In all cases, the right forelimb served as the
untreated control. A total of six limbs were examined for each condition, in
two separate experiments.
RT-PCR and western analysis of growth plate cartilage
RNA was extracted from proximal humeri using the RNeasy Kit (Qiagen).
Synthesis of cDNA was performed with Superscript III (Invitrogen). Reverse
transcriptase (RT)-PCR reactions comprised 35-42 cycles of 95°C for 1
minute, 55°C for 1 minute, 72°C for 1 minute. For western blotting,
growth plate cartilage was isolated and homogenized in RIPA buffer.
Whole-tissue lysates were run on 10-15% SDS-polyacrylamide gels.
Cell culture
Rat chondrosarcoma (RCS) cells were cultured and transfected as described
(Yoon et al., 2006). The 1.8
kb fragment of the mouse Msx2 promoter has been described previously
(Brugger et al., 2004). The
mouse proximal 2HC8 and 994 bp Ihh promoters were gifts from Akiko
Hata (Seki and Hata, 2004) and
Toshihisa Komori (Yoshida et al.,
2004). The Smad1 expression constructs were gifts from Eddy
DeRobertis (Pera et al.,
2003). Cells were stimulated with recombinant human BMP2, FGF2
(R&D Systems), ERK inhibitor (PD98059, 10 µM; Calbiochem) or p38
inhibitor (SB202190, 10 µM; Calbiochem). BMP2 was used at 100 ng/ml. Unless
otherwise stated, FGF2 was used at 10 ng/ml. Induction was measured by the
dual luciferase assay. All experiments were performed in triplicate and
repeated three times; representative experiments are shown. Statistical
significance was assessed by Student's t-test;
*P<0.05.
Primary sternal chondrocytes were isolated as described
(Lefebvre et al., 1994). Cells
were seeded at 1x106 cells/well in 6-well plates, and
cultured in MEMalpha supplemented with 10% FBS and pen/strep. For western blot
and RT-PCR, cells were serum starved in MEMalpha containing 1% FBS overnight,
then stimulated the next day with 50-100 ng/ml BMP2, 10 ng/ml FGF2, or 10
ng/ml noggin (R&D Systems) for 60 minutes. For immunocytochemistry, cells
were trypsinized and reseeded at 4x105 cells/well into an
8-well chamber slide overnight.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Mice deleted for individual Smads, or harboring heterozygous allelic combinations, were recovered in Mendelian ratios and showed no abnormalities (Fig. 2). Mice harboring combined deletions of Smad1 and Smad8 (Smad1CKO;Smad8-/-), and mice with only one functional allele of Smad5 (Smad1CKO;Smad5fx/+;Smad8-/-) were also normal (Fig. 2, and data not shown), demonstrating that a single allele of Smad5 is sufficient to transduce BMP signals in cartilage.
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).
Skeletal preparations revealed the absence of an axial skeleton in double and
triple mutants (Fig. 2).
Vertebral bodies were replaced by fibroblasts and loose aggregates of
chondrocytes (Fig. 3A,B),
similar to the phenotype seen in mice lacking Bmpr1a and Bmpr1b
(Yoon et al., 2005). MicroCT
analyses of newborn mice demonstrated severely reduced mineralization.
Consistent with the later onset of Col2-Cre expression in
appendicular elements, chondrogenesis proceeded to a greater extent in long
bones than in axial elements in mutants. Shortened long bones consisting of a
thin cartilage rod surrounded by a thick bone collar were seen.
Intramembranous elements (skull and clavicles) ossified normally, confirming
that Smad1/5 deletion was restricted to the expression
domain of Col2-Cre (Fig.
3C,D). Skeletal preparations revealed no major differences between Smad1/5CKO double and Smad1/5CKO;Smad8-/- triple mutants (Fig. 2). Similarly, no additional defects were seen in Smad1CKO;Smad8-/- and Smad5CKO;Smad8-/- mice as compared with Smad1CKO and Smad5CKO mice (not shown). However, cartilage condensations were slightly smaller in Smad1/5CKO;Smad8-/- triple mutants as compared with Smad1/5CKO;Smad8+/- mice (Fig. 3E-G). Thus, although Smad8 may play a role in chondrogenesis, its contribution is minor.
Smad1 and Smad5 are required for limb development
Because Smad8 has a minor role in chondrogenesis, subsequent analysis
focused on Smad1/5CKO mice. In WT embryos, concentric
layers of elongated fibroblasts surround the cartilage
(Fig. 4A,B). There were fewer
layers around condensations in mutants, suggesting that the initial stages of
condensation require canonical Smad signaling for cell recruitment.
Long bones were shorter in E14.5 Smad1/5CKO mutants than in the WT. Cells in the center of the cartilage anlagen exhibited a hypertrophic morphology, but were smaller than in WT littermates (Fig. 4C,D), and DAPI staining (Fig. 4E,F) revealed that cells in mutant growth plates were more densely packed. This can be attributed to impaired type II collagen production. Moreover, a thicker type I collagen-producing perichondrium was seen in mutants (Fig. 4F).
Distinct zones of resting, columnar and hypertrophic chondrocytes can be seen in WT growth plates by E16.5 (Fig. 4G). Loss of Smad1/5 led to growth plate disorganization and loss of hypertrophic chondrocytes (Fig. 4H). It is likely that the hypertrophic chondrocytes seen at E14.5 in mutants are descendents of cells that were committed to differentiation prior to completion of Cre-mediated excision of all four alleles of Smad1 and Smad5, and by E16.5 these cells are cleared from the growth plate.
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There was no evidence of stratification of chondrocytes in Smad1/5CKO mutants up to P0 (Fig. 4M,N). The lack of trabecular bone and vascular invasion persisted in mutants. Cortical bone extended into the marrow cavity and surrounded the rudimentary cartilage template (Fig. 4M,N). Hence, loss of canonical Smads leads to a failure in osteoblast invasion into the cartilage. Consistent with the presence of chondrocytes embedded in the thickened periosteum (Fig. 4L), ectopic cartilage formed at the edge of the bone collar in mutants (Fig. 4M,N).
BMP canonical Smad signaling is required for chondrocyte proliferation, survival and differentiation
Mutant chondrocytes were rounder and more densely packed in mutants than in
WT littermates (Fig. 5A,B), and
the hypertrophic zone was absent (Fig.
5C,D). Pcna staining revealed little proliferation in mutants
(Fig. 5E,F), and was confined
to the perichondrial cells. Histomorphometric analysis revealed no differences
in the percentage of Pcna-positive cells in the perichondrium of WT and mutant
littermates (data not shown). Apoptosis is normally confined to the
hypertrophic zone (Fig. 5G).
However, apoptosis was expanded in mutant cartilage
(Fig. 5H). Thus, loss of BMP
canonical Smad signaling leads to reduced chondrocyte proliferation and
increased apoptosis.
Cartilage-specific extracellular matrix (ECM) proteins are required for
growth plate organization (Gustafsson et
al., 2003; Li and Schwartz,
1995; Watanabe and Yamada,
1999). Smad1/5CKO mutants exhibited no defects
in proteoglycan production as assessed by Alcian Blue or Safranin O staining
(Fig. 4;
Fig. 5;
Fig. 6A,B); however, whereas
aggrecan is present in the WT growth plate
(Fig. 6C), it was sporadic and
intracellular in mutants (Fig.
6D). Smad1/5CKO mice also exhibited a severe
reduction in type II collagen deposition, suggesting that mutant chondrocytes
are not fully differentiated (Fig.
6E,F). Type X collagen is produced in hypertrophic chondrocytes
(Fig. 6G), but little could be
seen in Smad1/5CKO mutants
(Fig. 6H), suggesting a defect
in terminal differentiation.
The transcription factor Sox9 is required for chondrocyte survival and
expression of ECM components (Bell et al.,
1997; Bi et al.,
1999; Lefebvre et al.,
1997). RT-PCR analysis revealed decreased levels of Sox9
in mutant cartilage (Fig. 6I),
and that the deficits in collagens II and X in mutants occur at the
transcriptional level. The detectable, albeit decreased, expression of
Col10a1 and Runx2 (Fig.
6I) suggests that at least a few cells with the characteristics of
hypertrophic chondrocytes were present in mutants. However, these cells were
not organized into a distinct layer (Fig.
6A,B). Expression of alkaline phosphatase was examined to test
whether the paucity of hypertrophic chondrocytes in mutants is due to
accelerated conversion of these cells to late hypertrophic chondrocytes. The
decreased level of alkaline phosphatase in mutants
(Fig. 6I) argues against this
possibility, and supports the alternative hypothesis that chondrocyte
maturation is blocked. We tested this by examining Ucma expression.
Ucma is a marker for upper (resting) chondrocytes
(Tagariello et al., 2008;
Surmann-Schmitt et al., 2008).
Ucma was expressed as robustly in mutant as in WT cartilage
(Fig. 6I). Hence,
Smad1/5CKO chondrocytes are impaired in their ability to
undergo terminal differentiation and retain characteristics of resting
chondrocytes.
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Loss of canonical Smad proteins disrupts expression of BMP signaling components
The Smad1/5CKO phenotype is similar to that of
Bmpr1aCKO;Bmpr1b-/- mice
(Yoon et al., 2005),
suggesting that the canonical pathway is the major transducer of BMP signals
in cartilage. Unexpectedly, the Smad1/5CKO phenotype is
markedly more severe than the Smad4CKO phenotype
(Zhang et al., 2005), even
though Col2-Cre was used to drive cartilage-specific excision in both
models. These divergent phenotypes challenge the dogma that Smad4 is required
to mediate canonical Smad signaling in chondrogenesis.
Smad4 and BMP receptor expression was examined in mutant cartilage to
investigate the basis for this divergence
(Fig. 6J). RT-PCR analysis
indicated that Smad4 is expressed in mutant cartilage. Therefore, the
severe chondrodysplasia in Smad1/5CKO mice does not
correlate with altered Smad4 expression. However, the expression of
type II (Bmpr2) and type I (Bmpr1a and Bmpr1b) BMP
receptors was reduced. Thus, the similarity in the receptor-deficient and
Smad-deficient cartilage phenotypes might be a consequence of reduced BMP
receptor expression in Smad1/5CKO mutants. The results
reveal the presence of a positive-feedback loop involving BMP receptors and
BMP receptor Smads. Acvr1, the gene encoding the type I BMP receptor
ActRI (Alk2), is expressed at apparently normal levels in mutants, but ActRI
alone is unable to support chondrogenesis in vivo
(Yoon et al., 2005).
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; Qiao et al.,
2005). RT-PCR analysis revealed reduced Tak1 and
Mekk3 expression in mutants (Fig.
6J). The basis for this decrease is unclear, but might be related
to reduced BMP receptor expression (Fig.
6J). Regardless of the mechanism, the results suggest that loss of
canonical signaling does not lead to compensatory upregulation of TAK-mediated
noncanonical pathways.
The BMP canonical Smad pathway is required for the Ihh/PTHrP signaling loop in vivo
Ihh and PTHrP form a signaling loop in the growth plate that regulates
chondrocyte proliferation and differentiation. This loop is modulated by BMP
and FGF pathways; transgenic mice overexpressing FGFs in cartilage display
decreased expression of Bmp4, Ihh and PTHrP receptor (PPR)
(Chen et al., 2001;
Naski et al., 1998). FGFs also
inhibit BMP- and Ihh-mediated proliferation in limb cultures
(Minina et al., 2002). We
showed previously that Ihh signaling is positively regulated by BMPs in vivo
(Yoon et al., 2006). We
extended this analysis by examining Ihh signaling in primary chondrocytes and
Smad1/5CKO mutants. RT-PCR analysis showed that BMPs
induce both Ihh and PPR expression. By contrast,
Pthrp expression was only moderately increased by BMPs
(Fig. 7A). We then tested
whether the Ihh signaling loop is impaired in mutant cartilage. Pthrp
mRNA levels were only slightly reduced in Smad1/5CKO
cartilage, but Ihh and PPR mRNAs were not detected
(Fig. 7B). These results
confirm that Ihh is a BMP target in the growth plate, and suggest
that PPR is as well.
Pthrp transcripts are present in chondrocytes underlying the
articular surface in WT elements at E165, as reported previously
(Chen et al., 2006)
(Fig. 7G,H). Pthrp
mRNA was also seen in Smad1/5CKO mutants
(Fig. 7I,J). Ihh is
expressed in the prehypertrophic zone of WT growth plates; however, no
Ihh mRNA was detectable in mutants
(Fig. 7C-F). Retention of
Pthrp expression in the apparent absence of Ihh in
Smad1/5CKO mutants was unexpected, as it has been shown
that Ihh is normally required to maintain Pthrp expression
(Vortkamp et al., 1996;
Lanske et al., 1996;
Chung et al., 1998;
St Jacques et al., 1999;
Chung et al., 2001). Hence, we
examined Ihh expression at E14.5 in mutants to test the possibility
that Ihh might be present at higher levels at earlier stages. Cells
resembling prehypertrophic and hypertrophic chondrocytes were present in
Smad1/5CKO mutants (see Fig. S2A-D in the supplementary
material). Low levels of Ihh expression were detectable in the E14.5
mutant tibia, and Pthrp was expressed in periarticular cells (see
Fig. S2C,D in the supplementary material). Ihh mRNA was also detected
in E14.5 WT and mutant digits (see Fig. S3 in the supplementary material). We
conclude that Ihh is expressed in mutants, but that the level of
expression declines during development, most likely owing to the gradual loss
of the prehypertrophic cells that had formed prior to complete Cre-mediated
recombination. Ihh expression might be too restricted and/or
disorganized to detect at E16.5, but still present at a sufficient level to
maintain Pthrp expression. To test this latter possibility, we
performed in situ hybridization for patched 1 (Ptch1), a sensitive
readout of Ihh signaling. In WT growth plates, Ptch1 is
expressed in proliferating chondrocytes, as well as in adjacent perichondrium
(Fig. 7M,N). Consistent with
decreased Ihh signaling, Ptch1 mRNA was detectable, albeit at much
lower levels, in the mutant growth plate
(Fig. 7O,P).
PPR was not expressed at detectable levels in
Smad1/5CKO growth plates
(Fig. 7K,L), but was readily
detected in mutant osteoblasts. Hence, Ihh and PPR, which are highly
induced by BMPs in vitro (Fig.
7A), require canonical Smads for expression in cartilage in vivo.
The greatly diminished expression of PPR and Ihh, which are expressed
in late columnar and prehypertrophic chondrocytes
(MacLean and Kronenberg,
2005), is consistent with a block in chondrocyte differentiation
beyond the resting phase in mutants.
Canonical Smad signaling is required for BMP and FGF antagonism in chondrocytes
FGF signaling is elevated when BMP signaling is blocked through ablation of
Bmpr1a and Bmpr1b (Yoon
et al., 2006). The domain of Fgfr1 expression was expanded in
Smad1/5CKO cartilage
(Fig. 8A,B), as is the case in
Bmpr1a/bCKO mice (Yoon
et al., 2006). FGF receptor expression was also analyzed by RT-PCR
(Fig. 6J). Fgfr1
levels were increased in Smad1/5CKO cartilage, but
Fgfr2 and Fgfr3 levels were decreased. Fgfr1 mRNA
is expressed in resting/periarticular and hypertrophic chondrocytes,
Fgfr2 is expressed in proliferating chondrocytes, and Fgfr3
is expressed in proliferating and prehypertrophic chondrocytes
(Minina et al., 2005). Hence,
the absence of Fgfr2 and Fgfr3 expression in
Smad1/5CKO mutants is consistent with defective formation
of proliferating, prehypertrophic and hypertrophic chondrocytes. Elevated
expression of Fgfr1 mRNA and protein
(Fig. 6J;
Fig. 8B) and of Ucma
mRNA (Fig. 6I) in mutant
chondrocytes indicates that they retain characteristics of resting cells.
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;
Sahni et al., 1999). Stat1 was
observed in the hypertrophic zone and in the articular and lateral edges of
the growth plate in WT mice (Fig.
8C). An expanded domain of Stat1 expression was seen in mutants
(Fig. 8D). In its inactive
state, Stat1 is excluded from the nucleus; activation by FGFs and other
pathways leads to Stat1 phosphorylation and nuclear entry. In WT growth
plates, Stat1 staining is localized primarily in the cytoplasm and at the cell
membrane (Fig. 8E). By
contrast, nuclear localization of Stat1 was apparent in mutants, consistent
with activation of Stat1 signaling (Fig.
8F). In summary, FGF signaling is enhanced by loss of canonical
Smad signaling in Smad1/5CKO mutants.
FGF signaling exhibits functional antagonism with BMP signaling through ERK-mediated pathways
BMPs and FGFs are antagonistic in the growth plate. To investigate
potential mechanisms, we used a 1.8 kb Msx2 promoter fragment
(Brugger et al., 2004) linked
to luciferase (1.8 kb Msx2-luc) as reporter in chondrocytic RCS cells
(Fig. 9A). Levels of
BMP-mediated induction varied from 5-fold to 28-fold in different experiments.
Various factors may contribute to this variability, including the passage
number of the RCS cells and the batch of BMP used. BMP-mediated induction of
the 1.8 kb Msx2 promoter was also antagonized by FGF2 in a
dose-dependent manner (Fig.
9A). An ERK inhibitor (PD98059) had no effect on basal (not shown)
or BMP-mediated expression of 1.8 kb Msx2-luc, but was able to block
the ability of FGF2 to antagonize BMP2 induction of the promoter
(Fig. 9B). In fact, treatment
with PD98059 led to a synergistic increase in promoter activity in the
presence of BMP2 plus FGF2 (55-fold) as compared with BMP2 alone (6.5-fold).
The basis for this synergy is unknown, but might reflect an effect of PD98059
on other signaling pathways, and/or the ability of non-ERK/MAPK pathways
activated by FGF2 to synergize with BMP pathways to induce the 1.8 kb
Msx2 promoter. A p38 MAPK inhibitor (SB202190) had no effect on
BMP2-mediated induction. These experiments indicate that FGFs can antagonize
canonical BMP signals via ERK/MAPK pathways.
ERK/MAPK can phosphorylate the linker regions of Smads, leading to Smad
degradation (Fuentealba et al.,
2007; Pera et al.,
2003; Sapkota et al.,
2007). We overexpressed constructs encoding a WT Smad1 (Smad1WT),
or a version in which the Erk1/2 phosphorylation sites in the linker region
have been mutated so that they cannot be phosphorylated (Smad1LM)
(Fig. 9C), to test whether this
mechanism might account for ERK/MAPK-mediated inhibition of Msx2
promoter activity (Fig. 9D).
Smad1WT and Smad1LM enhanced BMP-mediated Msx2 promoter activity
2-fold (Fig. 9D). FGF2
treatment led to a slight induction in the experiment shown, but in other
cases no induction was seen (Fig.
9A,B, and data not shown). The presence of Smad1WT and Smad1LM did
not alter the effects of FGF (Fig.
9D). As expected, both Smad1WT and Smad1LM led to enhanced
responsiveness to BMP2, and no significant difference was noted in the ability
of either Smad construct to enhance this responsiveness. Moreover, both Smad
constructs antagonized the effects of FGF2 on BMP2-mediated induction of the
Msx2 promoter. In fact, the presence of Smad1LM led to a synergistic
activation of Msx2 promoter activity by FGF2 and BMP2 as compared
with BMP2 alone (Fig. 9D). This
result provides additional evidence that FGF-mediated pathways may have
positive effects on Msx2 promoter activity when ERK-mediated effects
on linker phosphorylation are prevented.
Ihh is a target of the BMP and FGF pathways in chondrocytes
(Naski et al., 1998;
Minina et al., 2002), and the
Ihh promoter contains Smad binding sites
(Seki and Hata, 2004;
Yoon et al., 2006). We
therefore examined whether Ihh induction might be antagonized by FGFs
via Smad linker phosphorylation. A 430 bp Ihh proximal promoter
fragment (2HC8-luc) that is Smad1/5-responsive in some cell types
(Seki and Hata, 2004) was
poorly responsive in primary and RCS chondrocytes (data not shown). However, a
longer, 994 bp Ihh promoter was responsive to BMP2, and was
antagonized by FGF (Fig. 9E).
Cells expressing Smad1WT or Smad1LM showed enhanced BMP2-mediated induction.
Higher Ihh promoter activity was seen in response to BMP in the
presence of Smad1LM than Smad1WT, and, in the case of Smad1LM, this induction
was resistant to FGF antagonism (Fig.
9E). Thus, BMP-mediated induction of Ihh might be
negatively regulated by ERK/MAPK through Smad linker phosphorylation.
Smad proteins are phosphorylated at both the C-terminus and the linker region in chondrocytes
The above results suggest that Smad1/5 linker phosphorylation accounts for
the antagonistic effects of FGFs. However, whether this occurs in the growth
plate is unclear. Therefore, we analyzed whether FGFs antagonize BMP signaling
at the level of Smad activity in cultured limbs. Levels of activated
(C-terminal-phosphorylated) nuclear Smads were highest in proliferating
chondrocytes and at the edges of the growth plate in unstimulated limbs
(Fig. 10A,C). FGF18 treatment
reduced C-terminal phosphorylation throughout the growth plate
(Fig. 10B), whereas inhibition
of endogenous FGF signaling using the FGF receptor antagonist SU5402 led to
activation (C-terminal phosphorylation) of Smad1/5
(Fig. 10D). Thus, FGFs inhibit
C-terminal phosphorylation of Smads.
|
).
pSmad1L was detected in the cytoplasm and nuclei of proliferating chondrocytes
and perichondrium (see Fig. S4A in the supplementary material). In contrast to
pSmad1/5, pSmad1L was not detected in epiphyseal and hypertrophic
chondrocytes. Thus, there are differences in the relative levels of pSmad1/5
and pSmad1L in different regions of the growth plate. No change in linker
phosphorylation was detected upon FGF18 stimulation
(Fig. 10E,F; see Fig. S4 in
the supplementary material). Furthermore, treatment with the FGFR inhibitor
SU5402 did not reduce pSmad1L levels (Fig.
10G,H). These results indicate that the activation of Smads by
C-terminal phosphorylation is negatively regulated in the growth plate by FGF
pathways. However, levels of Smad linker phosphorylation in the growth plate
are regulated independently of FGFs, or, alternatively, FGF-mediated
phosphorylation requires the activity of other factors.
Western blot analysis of primary chondrocytes confirmed that FGF
stimulation does not induce linker phosphorylation, even though Erk1/2
activity is elevated (Fig.
10I). Linker and C-terminal Smad1 phosphorylation was observed
only after BMP stimulation (Fig.
10I). These data are consistent with recent demonstrations that
BMP signaling directly induces Smad linker phosphorylation following
C-terminal phosphorylation and nuclear translocation
(Fuentealba et al., 2007;
Sapkota et al., 2007).
If Smad linker phosphorylation is catalyzed primarily by BMP pathways in chondrocytes, then nuclear colocalization of C-terminal- and linker-phosphorylated Smads is expected. Consistent with this prediction, Smad1 linker phosphorylation occurred primarily in the nucleus (Fig. 10J). Collectively, these data indicate that Smad linker phosphorylation is primarily a consequence of BMP-mediated canonical pathway activation in cartilage, and most likely represents a feedback mechanism to control the duration of BMP signaling. Moreover, despite widespread pSmad1/5 localization, modulation of pathway activity by linker phosphorylation appears to be restricted to the proliferative zone.
|
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Thus, although the conservation of Smad8 in
vertebrates indicates a function, mutational analyses have not yet revealed
this role. Developing cartilage is one of the tissues in which Smad8
is most highly expressed (Arnold et al.,
2006); the Smad1/5/8 triple knockout exhibits slightly
smaller cartilage condensations than those in Smad1/5CKO
mutants. It is conceivable that Smad8 plays a more prominent role in cartilage
in adults.
|
; Ross and Hill,
2008). The severe and lethal growth plate phenotype of
Smad1/5CKO mice compared with the mild and viable
phenotype of Smad4CKO mice
(Zhang et al., 2005) was thus
unexpected. In vitro (Sirard et al.,
2000; Subramaniam et al., 2004) and in vivo
(Wisotzkey et al., 1998;
Chu et al., 2004) studies have
shown that some Tgfβ/Smad-dependent processes occur in the absence of
Smad4. R-Smads can form homotrimers that do not contain Smad4
(Wu et al., 1997;
Chacko et al., 2001), but the
signaling capacity of these complexes is unknown. A recent report described a
Smad2-Smad3-Tif1
transcriptional complex that has a function distinct
from Smad2-Smad3-Smad4 heterotrimers (He
et al., 2006), but another report characterizes Tif1
(Trim33) as a Smad4 E3 ubiquitin ligase
(Dupont et al., 2005). Thus,
mechanisms underlying Smad4-independent canonical signaling in Tgfβ
pathways remain unknown.
Even less is known about the requirement for Smad4 in canonical BMP
pathways. Genetic studies reveal that some BMP receptor Smad-dependent
processes occur in the absence of Smad4
(Chu et al., 2004;
Wisotzkey et al., 1998). BMPs
induce R-Smad nuclear translocation in Smad4-null colon cancer cells
(Liu et al., 1997). A recent
report demonstrated that BMPs transduce signals in a Smad4-independent manner
through interaction of Smad1 with the Drosha complex, which promotes microRNA
(miRNA) processing (Davis et al.,
2008). The roles of specific miRNAs in chondrogenesis are unknown,
however, and loss of the major miRNA processor, dicer 1, in cartilage leads to
a less severe chondrodysplasia than is observed in
Smad1/5CKO mice
(Kobayashi et al., 2008),
indicating that other mechanisms underlie the Smad4-independent effects in
Smad1/5CKO mice.
C-terminal-phosphorylated Smad1/5/8 staining is most prominent at the
lateral edges of the growth plate (e.g.
Fig. 10A,C,D). This is
consistent with studies demonstrating lateral expression of Bmp2, 4 and 7, and
medial expression of the BMP inhibitor noggin
(Minina et al., 2005). Taken
together, these findings suggest that BMP ligands produced in the
perichondrium signal through canonical pathways in a lateral-to-medial
gradient.
Antagonism between BMP and FGF signaling pathways
This study demonstrates that canonical Smad signaling is required for
growth plate formation and cross-talk with FGF and Ihh/PTHrP pathways, and
that FGFs target canonical pathways by affecting the phosphorylation status of
Smads. C-terminal Smad1/5 phosphorylation is reduced by FGF stimulation and
elevated by FGF inhibition in limb cultures
(Fig. 10). We demonstrate that
FGF signaling can activate ERK/MAPK pathways, which antagonize canonical Smad
signaling in chondrocytes. On the other hand, we found that Msx2
promoter activity can be activated by non-ERK/MAPK pathways downstream of FGF.
Hence, the interaction between BMP and FGF pathways can be both positive and
negative for some target genes. The Ihh promoter is also a target of
canonical Smads, and mutation of Smad linker phosphorylation sites attenuated
FGF-mediated antagonism of Ihh in vitro. However, we saw no
difference in the levels or localization of linker-phosphorylated Smad1 upon
FGF stimulation (Fig. 10G-I),
suggesting that although ERK/MAPK-mediated linker phosphorylation limits BMP
signaling in vitro, FGFs might antagonize canonical pathways through a
different mechanism in the growth plate. For example, FGFs might inhibit
expression of BMP ligands and/or receptors, or activate a phosphatase that
inactivates C-terminal-phosphorylated Smads in the growth plate.
Recent findings have shown that BMPs induce sequential Smad
phosphorylations, first at the C-terminus and then in the linker region
(Fuentealba et al., 2007;
Sapkota et al., 2007).
BMP-induced linker phosphorylation occurs independently of MAPK, and localizes
to the nucleus, in contrast to MAPK-induced linker phosphorylation, which is
seen mainly in the cytosol (Sapkota et
al., 2007). The predominantly nuclear localization of
linker-phosphorylated Smads in the growth plate, and the lack of a clear
effect on this localization in response to FGF stimulation or inhibition, thus
suggest that the majority of linker phosphorylation in the growth plate is
mediated directly by BMPs. The high levels of nuclear linker-phosphorylated
Smad1 in the growth plate are thus most likely to reflect rapid Smad turnover
associated with active BMP signaling.
BMP canonical Smad signaling regulates the Ihh/PTHrP signaling loop
The Smad1/5CKO phenotype demonstrates that loss of BMP
receptor Smad signaling disrupts Ihh/PTHrP signaling in the growth plate.
Although Ihh levels are reduced, Ptch1 expression can be
detected, revealing that Ihh signaling does occur in mutants. Moreover,
Pthrp transcripts have been detected in Ihh-deficient mice
generated using a tamoxifen-inducible Col2-Cre allele
(Maeda et al., 2007). Our
findings, along with those of Maeda et al., raise the possibility that
although Ihh is required for induction of Pthrp expression,
Ihh-independent mechanisms might participate in the maintenance of
Pthrp expression.
Loss of Ihh leads to premature hypertrophy, growth plate
disorganization and a lack of osteoblasts in endochondral elements
(St Jacques et al., 1999).
Loss of PPR is also associated with accelerated differentiation
(Lanske et al., 1996).
Although Smad1/5 mutants exhibit diminished Ihh expression and
undetectable PPR, chondrocyte differentiation is impaired. We have shown
previously that loss of Bmpr1a and Bmpr1b leads to defects
in the transition of chondrocytes from a resting to a columnar proliferating
state, and to an inability of hypertrophic chondrocytes to complete terminal
differentiation (Yoon et al.,
2006). Hence, the diminished expression of PPR in Smad1/5
mutants might reflect a role for BMPs in commitment to differentiation. In
this scenario, the absence of PPR does not lead to accelerated
differentiation, owing to an earlier requirement for BMPs to permit
differentiation. Our results are also consistent with the possibility that
loss of PPR expression contributes to the growth plate defects in
Smad1/5 mutants. Although accelerated differentiation is seen in
PPR-/- mice at E18.5, chondrocytes in
PPR-/- mice become hypertrophic later than in WT mice
(Lanske et al., 1999),
consistent with the differentiation defect in Smad1/5 mutants at
midgestation stages. Moreover, in PPR-/- mice,
chondrocytes are replaced by invading blood vessels and osteoblasts more
slowly than in WT mice (Lanske et al.,
1999), but loss of PPR ultimately leads to excess bone formation
(Chung et al., 1998;
Lanske et al., 1996), similar
to the ossification phenotype in Smad1/5 mutants. A third possibility
is that BMPs have selective effects on signaling pathways independently of
PPR. It has been shown that phospholipase C (PLC)-dependent signaling
restrains chondrocyte proliferation and stimulates hypertrophic
differentiation; these actions are opposite to the responses mediated by the
cAMP/PKA (Prkaca) pathway downstream of PPR
(Guo et al., 2002). Hence,
BMPs may impact the balance between PLC and PKA pathway activation in
chondrocytes. Finally, we cannot rule out the possibility that there is a low
level of PPR expression in Smad1/5 mutant growth plates.
The greatly diminished expression of PPR in the growth plate raises the
possibility that canonical BMP pathways act directly on the PPR
promoter. Previous studies indicate that the highly conserved P2 promoter is
the major PPR promoter in skeletal tissues
(Bettoun et al., 1997;
McCuaig et al., 1995). This
promoter region contains multiple putative Smad-binding elements (data not
shown). Additional studies are needed to determine whether these sites are
active in chondrocytes.
Collectively, these studies demonstrate that canonical Smad signaling is the major BMP signal transduction pathway in chondrogenesis. If noncanonical BMP pathways play a role, they must do so in the context of active canonical signaling. Smads 1 and 5 are required to transduce canonical signals, are functionally redundant, required for all aspects of chondrogenesis, and do not require Smad4. Finally, our studies provide evidence that differential phosphorylation of canonical BMP receptor Smads might be an essential point of regulation of pathway activity in the proliferative zone of the growth plate.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/7/1093/DC1
|
Footnotes |
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|
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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0.05). All other
asterisks indicate significant differences from the no treatment control. NS,
not significant.