Crtap - Musculoskeletal Research Center

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Excessive transforming growth factor-β signaling is a
common mechanism in osteogenesis imperfecta
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© 2014 Nature America, Inc. All rights reserved.
Ingo Grafe1, Tao Yang1, Stefanie Alexander1, Erica P Homan1, Caressa Lietman1, Ming Ming Jiang1,2,
Terry Bertin1, Elda Munivez1, Yuqing Chen1, Brian Dawson1,2, Yoshihiro Ishikawa3,4, Mary Ann Weis5,
T Kuber Sampath6, Catherine Ambrose7, David Eyre5, Hans Peter Bächinger3,4 & Brendan Lee1,2
Osteogenesis imperfecta (OI) is a heritable disorder, in both
a dominant and recessive manner, of connective tissue
characterized by brittle bones, fractures and extraskeletal
manifestations1. How structural mutations of type I collagen
(dominant OI) or of its post-translational modification
machinery (recessive OI) can cause abnormal quality and
quantity of bone is poorly understood. Notably, the clinical
overlap between dominant and recessive forms of OI suggests
common molecular pathomechanisms2. Here, we show that
excessive transforming growth factor-b (TGF-b) signaling
is a mechanism of OI in both recessive (Crtap−/−) and
dominant (Col1a2tm1.1Mcbr) OI mouse models. In the skeleton,
we find higher expression of TGF-b target genes, higher ratio
of phosphorylated Smad2 to total Smad2 protein and higher
in vivo Smad2 reporter activity. Moreover, the type I collagen of
Crtap−/− mice shows reduced binding to the small leucine-rich
proteoglycan decorin, a known regulator of TGF-b activity3,4.
Anti–TGF-b treatment using the neutralizing antibody 1D11
corrects the bone phenotype in both forms of OI and improves
the lung abnormalities in Crtap−/− mice. Hence, altered
TGF-b matrix-cell signaling is a primary mechanism in the
pathogenesis of OI and could be a promising target for the
treatment of OI.
Most cases of OI are caused by autosomal dominant mutations in the
genes encoding type I collagen (COL1A1 and COL1A2)1. In recent years,
mutations in additional genes encoding the proteins involved in posttranslational modifications of collagen have been identified as causing
recessive forms of OI. The first described was in cartilage-associated
protein (CRTAP), a member of the prolyl-3-hydroxylase complex that
is responsible for 3-hydroxylation of proline residue 986 of the α1 chain
of type I collagen2,5. Hypomorphic CRTAP mutations lead to partial
loss of 3-hydroxyproline (3Hyp) in fibrillar collagen and overmodification of other residues and result in recessive OI type VII, which
clinically overlaps with the dominant forms of OI2. The physiological
function of 3Hyp is incompletely understood, but biochemical and
genetic studies suggest that it is involved in collagen-protein inter
actions and required for normal bone mineralization6,7.
The extracellular matrix is an important reservoir for signaling
molecules and their regulators. In bone, TGF-β acts as a central
coordinator of bone remodeling by coupling the activity of boneresorbing osteoclasts and bone-forming osteoblasts8. TGF-β is produced by osteoblasts9, secreted predominantly in inactive, latent
forms10 and deposited into the bone matrix11, where it can be released
and activated during bone resorption by osteoclasts12. As an additional level of regulation, active TGF-β can be bound by proteoglycans13, which modulate its bioactivity4 in association with collagen
fibrils3. Because type I collagen is the most abundant component of
the extracellular matrix in bone, we hypothesized that alterations of
collagen observed in OI can affect the signaling-modulating function of the bone matrix. Consistent with this, Crtap−/− mice show
phenotypic overlap with animal models of upregulated TGF-β signaling14–16. For example, TGF-β overexpression results in low bone
mass14. In addition, Crtap−/− mice exhibit an enlargement in alveolar
airway space in lungs, similar to that observed in a model of Marfan’s
syndrome, where dysregulated TGF-β signaling contributes to lung
pathology15. Therefore, we first studied the status of TGF-β signaling
in the Crtap−/− mouse model of recessive OI.
Compared with wild-type (WT) samples, calvarial bone of Crtap−/−
mice showed a higher expression of the TGF-β targets Cdkn1a (cyclindependent kinase inhibitor 1a, P21) and Serpine1 (plasminogen activator inhibitor-1), consistent with elevated TGF-β activity (Fig. 1a).
To confirm activation of the intracellular TGF-β signaling pathway, we evaluated the status of Smad2, a second messenger protein,
which becomes phosphorylated after activation of TGF-β receptors.
Consistent with higher TGF-β signaling, immunoblot analyses demonstrated a greater ratio of phosphorylated Smad2 (pSmad2) to total
Smad2 in calvarial bone samples of Crtap−/− compared with WT mice
(Fig. 1b,c). To confirm higher TGF-β activity in vivo, we crossed
Crtap−/− mice with reporter mice expressing luciferase in response
1Department
of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA. 2Howard Hughes Medical Institute, Houston, Texas, USA.
Department, Shriners Hospital for Children, Portland, Oregon, USA. 4Department of Biochemistry and Molecular Biology, Oregon Health and Science
University, Portland, Oregon, USA. 5Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, Washington, USA. 6Genzyme Research
Center, Framingham, Massachusetts, USA. 7Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, Houston, Texas, USA.
Correspondence should be addressed to B.L. ([email protected]).
3Research
Received 13 October 2013; accepted 24 March 2014; published online 4 May 2014; doi:10.1038/nm.3544
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Figure 1 Excessive TGF-β signaling in Crtap−/− mice. (a) Quantitative RT-PCR of TGF-β target genes Cdkn1a
and Serpine1 in calvarial bone of WT and Crtap−/− mice at postnatal day 3 (P3). Results are shown as fold
change of the mean of WT group ± s.d.; n = 5 per group. (b) Western blot analysis of P3 calvarial protein
extracts showing amounts of activated Smad2 (pSmad2) relative to total Smad2 protein in Crtap−/− versus WT
mice; n = 3 per group. (c) Quantification of the western blot shown in b. Results are shown as fold change of
the mean of WT group ± s.d. (d) Bioluminescence in regions that overlap with skeletal structures in Crtap−/−
compared with WT mice that were intercrossed to TGF-β reporter mice (SBE-luc mice). Representative image
WT
Crtap–/–
of 3 litters at P10 is shown (scale bar, 1 cm). (e) TGF-β activity in conditioned medium from WT and Crtap−/−
bone marrow stromal cells cultured under osteogenic conditions. Results are shown as fold change of the mean of WT group ± s.d.; n = 6 per group.
(f) Immunostaining of lungs (P10) for pSmad2 (red) in WT and Crtap−/− mice, with DAPI (blue) staining of nuclei (40× magnification). Representative
images of n = 3 mice per group are shown (scale bars, 20 µm). *P < 0.05 WT versus Crtap−/−, determined by Student’s t-test.
to TGF-β (SBE-luc mice). Compared with WT SBE-luc littermates,
Crtap−/− SBE-luc mice showed more intense bioluminescence of areas
over skeletal structures, indicating higher TGF-β activity in vivo (Fig. 1d;
in three litters, Crtap−/− mice show a mean 2.86-fold higher (± 0.34 s.d.)
bioluminescence signal at the skull compared with WT mice).
To test whether higher TGF-β signaling is intrinsic to bone, i.e.,
tissue autonomous, we cultured bone marrow stromal cells (BMSCs)
under osteogenic conditions in vitro. By using a TGF-β reporter cell
line, we found that conditioned medium from Crtap−/− BMSCs exhibited greater TGF-β activity compared with medium from WT BMSCs
(Fig. 1e). Together, these findings indicate that loss of Crtap enhances
TGF-β signaling in bone in a tissue-autonomous fashion.
Patients with severe OI can exhibit lung abnormalities, and respiratory failure is one of the leading causes of death in these individuals17,18. Crtap−/− mice show an enlarged alveolar airway space
compared with WT mice, a feature associated with higher TGF-β
signaling in other models15,16. Accordingly, lungs of Crtap−/− mice
showed more intense staining for pSmad2 in alveolar cells compared
with lungs of WT mice, indicating that higher TGF-β activity is also
present in extraskeletal tissues (Fig. 1f).
To understand whether upregulated TGF-β signaling represents a
causal mechanism contributing to the bone and lung phenotypes in
Crtap−/− mice, we performed a rescue experiment with a pan–TGF-β
neutralizing antibody (1D11). We treated 8-week-old Crtap−/− mice
with 1D11 for 8 weeks; all control Crtap−/− and WT mice received a nonspecific control antibody (13C4). Treatment with 1D11 did not change
the body weight of Crtap−/− mice, indicating that TGF-β inhibition
did not affect their general nutritional status (Supplementary Fig. 1).
Mass spectrometric and collagen cross-links analyses showed that
1D11 did not considerably change the status of P986 3-hydroxylation
or collagen cross-links in Crtap−/− mice, suggesting that dysregulated
TGF-β signaling is a consequence of the altered collagen and not
directly involved in intracellular collagen processing or extracellular
fibril assembly (Supplementary Fig. 2).
As previously reported5, Crtap−/− mice exhibited reduced bone mass
and abnormal trabecular bone parameters (Fig. 2a,b). Microcomputed
tomography (microCT) imaging analysis of vertebrae and femurs
demonstrated that compared with control Crtap−/− mice, Crtap−/−
mice treated with 1D11 had significantly improved trabecular bone
nature medicine VOLUME 20 | NUMBER 6 | JUNE 2014
parameters, to near WT levels (Fig. 2a,b and Supplementary Tables 1
and 2). The effects of TGF-β inhibition with 1D11 on the skeleton have
been reported previously in WT and E-selectin ligand-1 knockout
(Esl1−/−) mice9, a model with higher TGF-β activity due to impaired
inhibition of TGF-β processing during its production. Whereas WT
mice had moderately elevated (33% improvement) bone volume/total
volume (BV/TV) after treatment with 1D11, Esl1−/− mice exhibited a
106% improvement after 1D11 treatment. This suggests that targeting
TGF-β in a pathophysiological situation where it is upregulated could
lead to a relatively more pronounced positive effect. In the present
study, 1D11 improved the BV/TV at the spine by 235% in Crtap−/−
mice, supporting the hypothesis that dysregulated TGF-β signaling
is a major contributor to low bone mass.
At the femur midshaft, the measures of cortical architecture (cortical
thickness, diameter, cross-sectional area and cross-sectional moments
of inertia) in Crtap−/− mice were lower compared to the same parameters in WT mice. Following 1D11 treatment, these parameters were no
longer significantly different from those in WT mice (Supplementary
Table 3). To test whether 1D11 treatment resulted in improved bone
strength, we performed biomechanical testing of the femurs. TGF-β
inhibition resulted in higher maximum load and ultimate strength in
treated compared with control Crtap−/− mice (Supplementary Table 4).
This indicates improved whole-bone and tissue strength and suggests improved resistance to fracture. However, 1D11 had no effects
on the higher brittleness of the OI bone, as post-yield displacement
in both control and 1D11-treated Crtap−/− mice remained reduced
(Supplementary Table 4). This probably reflects the abnormal mineralization associated with altered collagen structure and cross-linking.
Together, these findings indicate that upregulated TGF-β signaling
is a major contributor to the bone phenotype in OI resulting from
Crtap deficiency and that TGF-β inhibition restores bone mass and
microstructural parameters and improves whole-bone strength.
To understand the effects of TGF-β inhibition at the cellular level,
we performed histomorphometric analyses. In sections of vertebrae
in this study, we found higher osteoclast and osteoblast numbers per
bone surface in control Crtap−/− compared to WT mice, indicating a
higher rate of bone remodeling (Fig. 2c and Supplementary Table 5).
Consistently, the serum levels of the bone turnover markers osteocalcin and C-terminal cross-linked telopeptide of bone collagen (CTX)
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Figure 2 Phenotypic correction of Crtap−/− mice after treatment with the TGF-β neutralizing antibody 1D11.
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(a) MicroCT images of L4 vertebral bodies of 16-week-old WT, control antibody–treated Crtap−/− and 1D11-treated
−/−
Crtap mice after treatment for 8 weeks (scale bars, 500 µm). (b) MicroCT analysis results of L4 vertebral bodies
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for bone volume/total volume (BV/TV), trabecular number (Tb.N (per mm)) and trabecular thickness (Tb.Th) in
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WT, control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means ± s.d.; n = 8 per group.
(c) Histomorphometric analysis of L4 for osteoclast (N.Oc/BS (per mm bone surface)) and osteoblast (N.Ob/BS
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(per mm bone surface)) numbers per bone surface, and numbers of osteocytes per bone area (N.Ot/B.Ar (per mm 2
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−/−
bone area)) in WT, control Crtap and 1D11-treated Crtap mice. Results are shown as means ± s.d.; n = 6
0
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per group. (d) H&E staining of inflated lungs of 16-week-old WT, control Crtap−/− and 1D11-treated Crtap−/− mice
Crtap–/–
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after treatment for 8 weeks. Representative images of n = 8 mice per group are shown (scale bars, 100 µm).
(e) Quantification of the distance between alveolar structures by the mean linear intercept (MLI) method in lungs of WT,
control Crtap−/− and 1D11-treated Crtap−/− mice. Results are shown as means ± s.d.; n = 8 mice per group, 10 images analyzed per mouse. *P < 0.05.
NS, not significant, determined by one-way analysis of variance (ANOVA).
Mean linear intercept
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were elevated in 8-week-old (osteocalcin and CTX) and 16-weekold (CTX only) control Crtap−/− mice compared with levels in WT
mice (Supplementary Fig. 3). Similar changes have been described in
patients with OI, who show higher osteoclast and osteoblast numbers
consistent with elevated bone turnover19,20. Notably, mouse models
of upregulated TGF-β signaling also show higher bone resorption
and abnormal bone remodeling compared with WT mice8,14. Most
reports of the effects of TGF-β on bone cells are consistent with a
model where TGF-β stimulates the recruitment and initial differentiation of osteoclast and osteoblast precursors at the site of bone
repair8,21,22, followed by insulin-like growth factor-1–mediated osteo
blast differentiation23. However, at persistently high doses, TGF-β
can inhibit osteoblast differentiation by repressing Runx2 (ref. 24),
the key transcription factor that induces osteoblastic differentiation.
Therefore, fine tuning of TGF-β availability is crucial for the local
coupling of bone resorption with formation during bone remodeling,
and its imbalance can lead to bone pathology8,21.
In contrast to those from control Crtap−/− mice, bone sections of
1D11-treated Crtap−/− mice showed reduced osteoclast and osteoblast
numbers; these numbers were even lower than those observed in bone
sections from WT mice, indicating a supraphysiologic suppression
of bone remodeling resulting from TGF-β inhibition at the dose of
1D11 used (Fig. 2c). Our findings are different from those of previous
studies in WT mice, where 1D11 treatment resulted in lower osteoclast but higher osteoblast numbers25. This may reflect distinct effects
of TGF-β inhibition in a pathological situation, with higher TGF-β
signaling and bone remodeling compared with normal bone. TGF-β
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inhibits later differentiation of osteoblast precursors24, and upregulated TGF-β signaling could thereby lead to a higher proportion of
immature osteoblastic cells. In contrast, a higher number or proportion of immature osteoblasts could result in higher TGF-β secretion.
The finding that TGF-β inhibition reduces the elevated osteoblast
numbers in Crtap−/− mice suggests that the upregulated TGF-β signaling causally contributes to the higher number of osteoblasts.
We also observed higher osteocyte numbers per bone area in control Crtap−/− compared with WT mice; these numbers were reduced to
WT levels in 1D11-treated Crtap−/− mice (Fig. 2c and Supplementary
Table 5). In patients with OI, a higher osteocyte density has been
observed in individuals with more severe forms of the disease, probably reflecting the presence of immature primary bone owing to a
defect in physiological bone maturation26. Similarly, overexpression
of TGF-β in WT mice results in higher osteocyte density14. As a possible explanation, TGF-β has been shown to inhibit osteoblast apoptosis
during the transition of osteoblasts to osteocytes27 and could thereby
lead to a higher osteocyte density. Collectively, these findings indicate
that excessive TGF-β signaling contributes to a high bone turnover
status and impaired bone maturation in Crtap−/− mice and that inhibition of TGF-β signaling reverses these alterations.
We next investigated whether TGF-β inhibition affected the lung
abnormalities in Crtap−/− mice and found that 1D11 treatment
resulted in amelioration of this phenotype. Whereas lungs of control
antibody-treated Crtap−/− mice showed a 6.9-µm increase of the mean
distance between alveolar structures compared to WT mice, lungs of
Crtap−/− mice treated with 1D11 demonstrated only a 2.7-µm increase
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Hence, we hypothesize that decorin binding to collagen is critical
for TGF-β regulation and that this binding is disrupted with altered
collagen structure in OI. We found that although loss of Crtap did
not alter the expression of decorin and other SLRPs in calvarial bone
(Fig. 3a) or the qualitative abundance of decorin in trabecular bone
(Supplementary Fig. 4), it did reduce binding of decorin to type I
collagen (Fig. 3b; mean reductions of decorin binding to Crtap−/−
versus WT type I collagen at 3, 5 and 12 µM of decorin were 28.5%,
33.5% and 38.1%, respectively). On the basis of the requirement of
collagen binding for decorin to effectively reduce TGF-β bioactivity3,
it is possible that this altered binding in OI affects decorin’s ability to
sequester mature TGF-β in the matrix and modulate TGF-β function.
This could contribute to dysregulated TGF-β signaling, even if no
major changes in absolute TGF-β levels are present (Supplementary
Figs. 5 and 6). This notion is supported by the finding that COL1A1
and COL1A2 mutations in severe forms of dominant OI cluster in
regions that are known to bind proteoglycans33, further supporting
the relevance of proteoglycan-collagen interactions for normal bone
homeostasis. This implies that other proteoglycans that are competing
with decorin for the binding site on collagen34 may also contribute to
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compared to WT mice (Fig. 2d,e). This finding indicates that excessive TGF-β signaling is also a contributor to the lung abnormalities
in Crtap−/− mice. Upregulated TGF-β signaling has been linked to
developmental pulmonary abnormalities and disease in mature lungs.
For example, TGF-β overexpression in lungs results in impaired lung
development and enlarged airway space28, and upregulated TGF-β signaling is a pathomechanism of emphysema and bronchial asthma29,30.
Given the partial rescue of the lung phenotype with 1D11 in Crtap−/−
mice, it is possible that dysregulated TGF-β signaling affects lung
development when anatomic structures are established, in addition
to maintaining pulmonary tissue at later stages.
We next asked how collagen alterations due to loss of Crtap result
in dysregulated TGF-β signaling. Biochemical analyses have indicated
that collagen prolyl-3-hydroxylation does not fundamentally affect the
stability of collagen molecules and instead may affect collagen-protein
interactions6. An attractive hypothesis is that loss of 3Hyp and/or collagen overmodification could affect collagen interaction with small
leucine-rich proteoglycans (SLRPs). SLRPs bind to both type I collagen
and TGF-β and thereby modulate TGF-β activity3,4. For example, the
SLRP decorin inhibits distinct effects of TGF-β in osteosarcoma cells,
such as the upregulation of biglycan synthesis31, whereas it enhances
TGF-β activity in preosteoblastic cells4. The decorin-binding region
on type I collagen has been suggested to center at residues 961 and 962
of the triple helical domain32, which is located in close proximity to
the P986 residue. It is possible that the P986 3Hyp position marks an
interacting site for decorin–type I collagen binding, thereby mediating
the sequestration of TGF-β to collagen.
Cdkn1a (fold change)
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Figure 4 Inhibition of upregulated TGF-β signaling improves the bone phenotype in a
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mouse model of dominant OI (Col1a2tm1.1Mcbr). (a) Quantitative RT-PCR of TGF-β target
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genes Cdkn1a and Serpine1 in calvarial bone of P3 WT and Col1a2tm1.1Mcbr mice.
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Results are shown as fold change of the mean of WT group ± s.d.; n = 3 per group.
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(b) Western blot analysis showing activated Smad2 (pSmad2) relative to total Smad2
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protein in P3 calvaria of WT and Col1a2tm1.1Mcbr mice; n = 3 per group. (c) Quantification
of the western blotshown in b. Results are shown as fold change of the mean of
0
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WT group ± s.d. (d) MicroCT images of L4 vertebral bodies of 16-week-old WT, control
antibody–treated Col1a2tm1.1Mcbr and 1D11-treated Col1a2tm1.1Mcbr mice after treatment for 8 weeks (scale bars, 500 µm). (e) MicroCT analysis
results of L4 vertebral bodies for bone volume/total volume (BV/TV), trabecular number (Tb.N) and thickness (Tb.Th) in WT, control Col1a2tm1.1Mcbr
and 1D11-treated Col1a2tm1.1Mcbr mice. Results are shown as means ± s.d.; n = 6 per group. *P < 0.05. NS, not significant, determined by Student’s
t-test (a and c) and one-way ANOVA (e).
BV/TV (%)
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Figure 3 Reduced decorin binding to type I collagen of Crtap−/− mice.
(a) Quantitative RT-PCR of calvarial bone of P3 mice for the small
leucine-rich proteoglycans decorin (Dcn), biglycan (Bgn) and asporin (Aspn)
in WT and Crtap−/− mice. Results given as fold change of the mean of
WT group ± s.d.; n = 5 per group. NS, not significant, determined by
Student’s t-test. (b) Surface plasmon resonance analysis measuring the
binding of recombinant decorin core protein to type I collagen of WT
and Crtap−/− mice. Three technical replicates at each of the indicated
concentrations of decorin were performed in two independent biological
replicates (for each biological replicate, collagen was pooled from three
mice per genotype). Results are shown as the percentage of the mean of
WT (bars indicate mean per group). RU, response units.
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dysregulated TGF-β activity and that additional signaling pathways
could be altered35.
Because of the clinical overlap of some recessive and dominant
forms of OI, it is possible that dysregulation of TGF-β signaling is a
common disease mechanism. To address this hypothesis, we investigated the status of TGF-β signaling in a mouse model of dominant OI.
Knock-in mice carrying a G610C mutation in the Col1a2 gene
(Col1a2tm1.1Mcbr) phenocopy a dominantly inherited moderate form of
OI that was identified in an Amish population. Compared with bone
samples of WT mice, bone samples from Col1a2tm1.1Mcbr mice showed
higher expression of the TGF-β target genes Cdkn1a and Serpine1,
indicating upregulation of TGF-β signaling (Fig. 4a). Consistent with
these results, immunoblot analyses showed a greater ratio of pSmad2
to total Smad2 in bone of Col1a2tm1.1Mcbr compared with WT mice,
similar to our observation in Crtap−/− mice (Fig. 4b,c).
To test whether excessive TGF-β signaling also represents a causal
mechanism in dominant OI, we treated 8-week-old Col1a2tm1.1Mcbr
mice with the TGF-β neutralizing antibody 1D11 and control
Col1a2tm1.1Mcbr and WT mice with the control antibody 13C4 for
8 weeks. Similar to 1D11 treatment in Crtap−/− mice, 1D11 treatment
in Col1a2tm1.1Mcbr mice restored the trabecular bone parameters at the
spine compared to control-treated Col1a2tm1.1Mcbr mice to WT levels,
including bone volume, trabecular number and trabecular thickness
(Fig. 4d,e and Supplementary Table 6). Together, these findings indicate that dysregulated TGF-β signaling is also a key contributor to the
pathogenesis of dominant OI and that anti–TGF-β therapy corrects
the bone phenotype in dominant OI.
From a clinical-translational perspective, potential negative effects
of systemic TGF-β inhibition in patients with OI have to be considered.
Whereas Tgfb1−/− mice develop a severe dysregulation of the immune
system with inflammatory disease within the first weeks of life36,
we did not observe obvious negative effects on general health, behavior
or growth in either Crtap−/− or Col1a2tm1.1Mcbr mice treated with 1D11,
suggesting that the effects of partial pharmacological inhibition of TGF-β
in adult mice are different from those of a complete loss of TGF-β1
during development. In humans, fresolimumab, which is similar to
1D11 in its affinity and specificity to the three isoforms of TGF-β, has
been tested in phase 1 clinical studies in patients with primary focal
segmental glomerulosclerosis37, idiopathic pulmonary fibrosis38 and
cancer39. In these studies, fresolimumab was, in general, well tolerated, with possible dose-related adverse events including skin rashes
or lesions, epistaxis, gingival bleeding and fatigue.
The molecular mechanisms of OI are incompletely understood.
As a result, current treatment options for patients with OI are mainly
limited to antiosteoporosis therapies with antiresorptive drugs. Of
note, a recent randomized controlled trial of the anabolic agent teriparatide showed that adult patients with severe OI responded differently than those with mild OI40. This suggests that there are genotypic
differences in response to therapies targeted at modifying cell signaling and that TGF-β inhibition may be a promising target in severe
OI owing to mutations in the genes encoding collagen and proteins
involved in its post-translational modification. Overall, our data support the concept of dysregulated matrix-cell signaling as a mechanism
in the pathogenesis of different forms of brittle bone disease and point
to a disease-specific mechanism–based strategy for the treatment of
OI by neutralizing overactive TGF-β activity.
Methods
Methods and any associated references are available in the online
version of the paper.
674
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank D. Spencer and his lab (Baylor College of Medicine) for providing the
IVIS camera system and training, M. Starbuck and F. Gannon for consultation and
advice in bone histomorphometry, M. Warman and C. Jacobsen (Boston Children’s
Hospital) for providing the Col1a2tm1.1Mcbr mice and helpful information. We also
thank L. Fisher (US National Institute of Dental and Craniofacial Research) for
providing the decorin antibody LF-113. We thank M. Bagos for help with
microCT analyses, A. Abraham for help with the biomechanical testing, W. Song
and S. Liu for their help with serum bone turnover analyses, the US National
Institutes of Health (NIH) for providing the ImageJ software and A. Choi and
H. Lam (Harvard Medical School) for providing the ImageJ modification for lung
morphometry, which was generated by P. Thompson. Also, we thank D. Rifkin
(New York University Medical Center) for providing PAI-luciferase reporter mink
lung epithelial cells. In addition, we thank R. Morello, G. Sule, D. Baldridge and
G. Ghosal for their helpful discussions and P. Fonseca for editorial assistance.
This work was supported by a research fellowship from the German Research
Foundation/Deutsche Forschungsgemeinschaft (I.G.), a Michael Geisman
Fellowship from the Osteogenesis Imperfecta Foundation (I.G.), grant support
from Shriners Hospitals for Children (H.P.B.), NIH grants 5F31DE020954
(E.P.H.), 5F31DE022483 (C.L.), R37 AR037318 and R01 AR036794 (D.E.), and P01
HD70394 (B.L. and D.E.) and the Howard Hughes Medical Institute Foundation
(B.L.). This work was also supported by the Baylor College of Medicine Intellectual
and Developmental Disabilities Research Center (HD024064), the Eunice Kennedy
Shriver US National Institute of Child Health & Human Development and the
Rolanette and Berdon Lawrence Bone Disease Program of Texas.
AUTHOR CONTRIBUTIONS
I.G. and B.L. conceptualized the study. T.Y., T.K.S., C.A., D.E. and H.P.B.
contributed to the design of the study and experiments. I.G., T.Y., S.A., E.P.H.,
C.L., M.M.J., T.B., E.M., Y.C., B.D., Y.I., M.A.W. and C.A. performed and analyzed
experiments. I.G., T.Y. and B.L. wrote the manuscript with contributions from all
authors. B.L. supervised the project.
COMPETING FINANCIAL INTERESTS
The authors declare competing financial interests: details are available in the online
version of the paper.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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ONLINE METHODS
Animals, anti–TGF-b treatment and tissue collection. We generated Crtap−/−
mice as previously described5 and maintained them on a mixed C57BL/6129Sv genetic background. Mice harboring a G610C mutation in the Col1a2
gene (Col1a2tm1.1Mcbr)41 were a gift from M. Warman and were bred to WT
C57BL/6 mice. We used mice that were heterozygous for the Col1a2tm1.1Mcbr
allele for experiments. TGF-β reporter mice that express luciferase in response
to the Smad2/3-dependent TGF-β signaling pathway (SBE-luc mice), were
obtained from Jackson Laboratory (B6.Cg-Tg(SBE/TK-luc)7Twc)42 and
bred to Crtap+/− mice for two generations to generate Crtap−/− mice and
WT littermates expressing the reporter transgene. All mice were housed in
the Baylor College of Medicine Animal Vivarium, and animal experiments
were performed following the approved protocol of the Animal Care and Use
Committee at the Baylor College of Medicine.
For protein and RNA analyses, we isolated calvaria of P3 mice, cleaned them
of extraskeletal tissue and snap-froze them in liquid nitrogen. For immuno
staining of Crtap−/− P10 mice lungs, we equally inflated the lungs of each
mouse immediately after euthanasia by gravity with 4% paraformaldehyde at a
constant pressure of 25 cm H2O and then suture-closed them at the trachea.
Lungs were then gently dissected from the thorax and fixed in 4% paraformaldehyde overnight.
We treated 8-week-old female Crtap−/− and Col1a2tm1.1Mcbr mice with the pan–
TGF-β neutralizing antibody 1D11 (provided by Genzyme/Sanofi; clone 1D11,
diluted 1:10 for injections) for 8 weeks (10 mg kg−1 body weight, intraperitoneal
(i.p.) injections three times each week). Control Crtap−/−, Col1a2tm1.1Mcbr and
WT mice received a control antibody (13C4 provided by Genzyme/Sanofi; clone
13C4, diluted 1:10 for injections) of the same IgG1 isotype. Mutant female mice
(Crtap−/− and Col1a2tm1.1Mcbr) of each litter were randomly assigned to treatment
(1D11) or control group. After treatment, mice were euthanized, and we collected
lumbar spines and femurs and fixed them in 10% formalin for microCT and bone
histomorphometry. We stored contralateral femurs of Crtap−/− mice in –20 °C
wrapped in saline-soaked gauze until biomechanical testing was performed. We
equally inflated lungs of Crtap−/− mice, and collected and fixed them as described
for P10 mice. No blinding was possible during treatment because 1D11 or control
antibody was injected according to group allocation. In all subsequent analyses,
the investigators were blinded to genotype and treatment group.
Immunoblotting. We extracted protein from snap-frozen P3 calvaria samples
by transferring them to 300 µl lysis buffer (0.0625 M Tris-HCl, pH 7.5, 2% SDS,
5 mM NaF, 2 mM Na3VO4 and Roche cOmplete proteinase inhibitor) and
homogenized for 1 min, followed by incubation at 95 °C for 40 min. We transferred the supernatant to Centrifugal Filter Units/Amicon Ultra 3K (Millipore)
and centrifuged to concentrate the protein. We measured the total protein
concentration of the lysate using the Micro BCA reagent (Pierce) following the
manufacturer’s directions. 40 µg of calvaria protein extracts were suspended
in Laemmli buffer containing 5% β-mercaptoethanol and separated on Mini
Protean TGX SDS-PAGE gels (gradient 4–20%; Bio-Rad) and transferred onto
polyvinylidene difluoride membranes for western blot analyses. We incubated
PVDF membranes with pSmad2 monoclonal antibody (Cell Signaling #3108,
1:750 in TBST containing 5% BSA overnight), followed by secondary horseradish peroxidase–linked anti-rabbit antibody (GE, 1:5,000 in TBST containing
5% BSA for 2 h), treated them with ECL Plus Western Blotting Detection
System (GE) and exposed them to X-ray films. Subsequently, we stripped
antibodies from membranes using ReBlot Plus reagent (Millipore) and incubated them with Smad2 monoclonal antibody (Cell Signaling #5339, 1:2,000 in
TBST containing 5% BSA overnight), followed by similar secondary antibody
incubation and ECL-mediated visualization. X-ray films were scanned, and the
density of each band was quantified using ImageJ software (NIH).
Quantitative real-time RT-PCR. We extracted total RNA from snap-frozen
P3 mouse calvaria using Trizol reagent (Invitrogen). The Superscript III RT
system (Invitrogen) was used to synthesize cDNA from total RNA according to the manufacturer’s protocol. We performed quantitative RT-PCR on
a LightCycler v.1.5 (Roche) using gene-specific primers and SYBR Green I
reagent (Roche). The gene encoding β2 microglobulin was used as the reference gene for normalizing cDNA concentrations.
nature medicine
In vivo bioluminescence imaging. We injected P10 Ctrap−/− mice and WT
littermates that expressed the TGF-β reporter transgene (SBE-luc mice) with
d-luciferin (Goldbio, 150 mg kg−1, i.p.), anesthetized them with isoflurane and
performed imaging 10 min after injection using a bioluminescence imaging
system (Xenogen).
Primary osteoblast culture and TGF-β reporter cells. We isolated bone marrow cells from tibias and femurs of approximately 2-month-old Crtap−/− and
WT mice and cultured them in α-MEM supplied with 10% FBS, 100 U mL−1
penicillin and 100 ug mL−1 streptomycin. We changed the medium every
second day and discarded unattached cells. After 7 d, we reseeded the attached
cells, defined as bone marrow stromal cells (BMSCs), to 24-well plates at 2.5 ×
104 cells per cm2 and cultured them in osteogenic medium (α-MEM, 10%
FBS, 500 µM ascorbic acid and 10 mM β-glycerophosphate) for 3 d. We collected conditioned medium and incubated this with plasminogen activator
inhibitor-1–luciferase reporter mink lung epithelial cells43 (these cells were
obtained from the laboratory of D. Rifkin; they were not recently further profiled or tested for mycoplasma contamination). After 24 h, we collected the
cell lysates for luciferase activity assays, which were measured using the DualLuciferase Reporter System (Promega). The results were normalized to the
total protein amount quantified using the Micro BCA reagent (Pierce).
MicroCT and bone histomorphometry. We scanned lumbar vertebrae and
femurs using a Scanco µCT-40 microCT for quantification of trabecular and
cortical bone parameters. We analyzed vertebral and femoral trabecular bone
parameters using the Scanco analysis software by manually contouring the
trabecular bone of vertebral body L4 as well as the distal metaphyseal section of
the femur. The cortical bone parameters at the center of the femoral midshaft
were quantified using the automated thresholding algorithm included in the software. Scanned undecalcified Crtap−/− mouse spine samples were then embedded
in plastic for sectioning. We performed toluidine blue staining and TRAP staining using standard protocols for visualization and quantification of osteoblasts
and osteoclasts, respectively, using the Bioquant Osteo Image Analysis System.
Immunostaining and histology. For immunohistochemistry, we collected
hind limbs of P5 mice, fixed them overnight in 4% paraformaldehyde and
embedded them in paraffin. After deparaffinization and rehydration, we performed heat-induced antigen retrieval (Dako, S1700) followed by treatment
with hyaluronidase for 30 min (2 mg mL−1; Sigma). Endogenous peroxidase
was blocked using 3% hydrogen peroxide for 10 min. After incubation with
blocking solution (3% normal goat serum, 0.1% BSA, 0.1% Triton X-100
in PBS), we incubated sections in antibodies specific for TGF-β1 (G1221,
Promega) and decorin (LF-113, provided by L. Fisher) for 60 min (1:25 dilution each in PBS, control samples were incubated in PBS only) at 37 °C and
subsequently incubated them with secondary antibody (SuperPicTure Ploymer
Detection kit, Invitrogen, 87-9663, undiluted following the manufacturer’s
protocol). We added substrate DAB according to the manufacturer’s recommendations and dehydrated and mounted samples using Cytoseal XYL xylenebased mounting medium (Thermo Scientific). We processed sections of WT
and mutant littermates at the same time. Images of the trabecular bone were
taken with a light microscope (Axioplan 2, Zeiss) using identical exposure
times for WT and mutant littermates.
We equally inflated lungs of P10 and 16-week-old Crtap−/− mice during tissue
collection, fixed them in 4% paraformaldehyde and performed paraffin embedding. We used lungs of P10 Crtap−/− and WT mice for immunostaining for
pSmad2. Briefly, we treated paraffin sections with xylene, and rehydrated and
heated them for 20 min for antigen retrieval (pH 6; Dako). We then incubated
sections in blocking solution (3% normal donkey serum, 0.1% BSA, 0.1% Triton
X-100 in PBS) and subsequently incubated them with rabbit anti-pSmad2 antibody (1:500) (Cell signaling, #3101), donkey anti-rabbit secondary antibody
conjugated to Alexa Flour 594 (1:600) (Invitrogen, A-21207), and mounted them
with Prolong Gold antifade reagent with DAPI (Invitrogen). Fluorescent images
from these sections were taken using a Zeiss microscope (Axiovision Software)
using identical exposure times.
For lung histology and morphometry of 16-week-old mice, we stained para
sagittal sections using a standard protocol for H&E staining. We used the mean
doi:10.1038/nm.3544
npg
linear intercept (MLI) method to quantify the distance between alveolar structures as previously described44. Briefly, we captured ten histological fields per
mouse at 20× magnification from all lobes of both lungs using a light microscope
(Axioplan 2, Zeiss) and measured the MLI using modified ImageJ software
(NIH, modified by P. Thompson). After manual removal of blood vessels, large
airways and other nonalveolar structures, the software automatically thresholds
the alveolar tissue in each image and overlays a line grid comprised of 1,353 lines
with each line measuring 21 pixels over the image. The number of lines that
intercepted alveolar structures was used to calculate the MLI.
Biomechanical testing by three-point bending. We tested Crtap−/− and
WT femurs by three-point bending using a span of 6 mm with an Instron
5848 device (Instron Inc., Norwood MA). All the femurs were tested wet at
room temperature. We preloaded femurs to 1 N at a rate of 0.05 N s−1 for
5 s. Following the preloading, we loaded the femurs to failure at a rate of
0.1 mm s−1. Load and displacement data was captured at rate of 40 Hz by using
BLUEHILL Software (Instron 5848). To determine the yield point, a region
was identified after the preload and before the maximum load on the loaddisplacement curve. We separated this region into five segments, from which
the fitted line of the segment with greatest slope was taken. Next, a 0.012-mm
offset was implemented on the line. The point of intersection between the
offset line and the load-displacement curve was the 0.012 offset yield point.
This yield point corresponded more closely to a 0.2% offset strain, which
is commonly chosen in the literature. The elastic region was identified as
the region from the completion of the preload to the yield point. Postyield
region was identified as the region from the yield point until the point at
which the change in load exceeded –1 N, indicating failure. Elastic displacement was the displacement during which the specimen remained in the elastic
region. Postyield displacement was the displacement during which specimen
remained in the postyield region. Total displacement was calculated as the
sum of elastic displacement and postyield displacement. Using the trapezoidal numerical integration method, we calculated energy to failure as the area
under the load-displacement curve. We determined maximum load by finding
the highest load value recorded by BLUEHILL before the specimen failed.
To calculate stiffness, we applied the least-square fit method to the steepest
segment of the elastic region of the load-displacement curve. Stiffness was
the slope of least-square fit line. Geometric data (diameter and moment of
inertia) obtained from microCT analysis of the femoral midshaft were used
to calculate the intrinsic material properties: ultimate strength, toughness to
failure and elastic modulus.
Serum bone turnover markers. We quantified serum osteocalcin using the
Mouse Osteocalcin EIA Kit from Biomedical Technologies Inc. and C-terminal
cross-linked telopeptide of bone collagen (CTX) using the RatLaps EIA Kit
from Immunodiagnostic Systems Ltd. Both analyses were performed according to the manufacturer’s protocols.
Collagen SDS-PAGE, mass spectrometry and cross-links analyses. For mass
spectrometry, we prepared type I collagen from Crtap−/− and WT tibias. We
defatted bone with chloroform/methanol (3:1 v/v) and demineralized it in
0.5 M EDTA, 0.05 M Tris-HCl, pH 7.5, all steps at 4 °C. We finely minced
the bone samples and solubilized collagen by heat denaturation (90 °C) in
SDS-PAGE sample buffer. Collagen α-chains were cut from SDS-PAGE gels
and subjected to in-gel trypsin digestion. We performed electrospray mass
spectrometry on the tryptic peptides using an LCQ Deca XP ion-trap mass
spectrometer equipped with in-line liquid chromatography (ThermoFinnigan)
doi:10.1038/nm.3544
using a C8 capillary column (300 µm × 150 mm; Grace Vydac 208 MS5.315)
eluted at 4.5 µl per min. Sequest search software (ThermoFinnigan) was used
for peptide identification using the NCBI protein database.
We quantified pyridinoline cross-links (hydroxylysyl pyridinoline and
lysyl pyridinoline) by HPLC after hydrolyzing demineralized bone in 6 N HCl
as described45.
Surface plasmon resonance analysis. We performed surface plasmon
resonance experiments using a BIACore X instrument (GE Healthcare
Bio-Sciences). We immobilized purified native mouse tendon type I collagen
from three WT and three Crtap−/− mice (pooled for each genotype) on CM5
sensor chips (GE Healthcare Bio-Sciences AB, BR100399) by amide coupling
at a concentration of about 0.5 ng mm−2 (500 RU) and 0.8 ng mm−2
(800 RU), respectively. The experiments were conducted at a flow rate of 10 µl
min−1 and 20 °C in HBS-P buffer (10 mM HEPES buffer, pH 7.4, containing
150 mM NaCl and 0.005% surfactant P20). We then injected recombinant
human decorin core protein (R&D systems) onto both type I CM5 chips.
We determined the concentration of the stock solution of human decorin by
amino acid analysis. The binding response of decorin to WT and Crtap−/−
mouse type I collagen was normalized by the amounts of immobilized type I
collagen on the CM5 sensor chips. We used three concentrations of decorin
(3, 5 and 12 µM), and for each concentration the analysis was repeated three
times. We performed this experiment twice with collagen isolated from
different mice each time.
Statistical analyses. For comparisons between two groups, we used unpaired
two-tailed Student’s t-tests. For comparisons between three groups, we performed
one-way ANOVA if equal variance and normal distribution of groups were
confirmed, followed by all pairwise multiple comparison using the Holm-Sidak
method. If the equal variance or normal distribution test failed, we performed
Kruskal-Wallis one-way ANOVA on ranks, followed by all pairwise multiple comparison using the Tukey’s test. A P value less than 0.05 was considered statistically significant for Student’s t-test, ANOVA and Kruskal-Wallis
one-way ANOVA on ranks. For post hoc pairwise multiple comparisons, each
P value was compared to a critical level depending on the rank of the P value
and the total number of comparisons made to determine whether differences
between groups are significant. We used Sigma Plot V11.0 (Systat Software
Inc.) for statistical analyses. The effects of 1D11 on bone and lungs of OI mice
were unknown at study start. To determine the initial sample size per group of
mice, we calculated that to detect a minimal difference of 20% in bone mass
(BV/TV) by MicroCT between 1D11- and control-treated OI mice with a 90%
power, a group size of eight mice is required.
41.Daley, E. et al. Variable bone fragility associated with an Amish COL1A2 variant
and a knock-in mouse model. J. Bone Miner. Res. 25, 247–261 (2010).
42.Lin, A.H. et al. Global analysis of Smad2/3-dependent TGF-β signaling in
living mice reveals prominent tissue-specific responses to injury. J. Immunol. 175,
547–554 (2005).
43.Abe, M. et al. An assay for transforming growth factor-β using cells transfected
with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal.
Biochem. 216, 276–284 (1994).
44.Chen, Z.H. et al. Autophagy protein microtubule-associated protein 1 light chain-3B
(LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema.
Proc. Natl. Acad. Sci. USA 107, 18880–18885 (2010).
45.Eyre, D. Collagen cross-linking amino acids. Methods Enzymol. 144, 115–139
(1987).
nature medicine
Supplementary Information
Excessive TGFβ signaling is a common mechanism in Osteogenesis Imperfecta
Ingo Grafe, Tao Yang, Stefanie Alexander, Erica Homan, Caressa Lietman, Ming Ming Jiang,
Terry Bertin, Elda Munivez, Yuqing Chen, Brian Dawson, Yoshihiro Ishikawa, Mary Ann Weis,
T. Kuber Sampath, Catherine Ambrose, David Eyre, Hans Peter Bächinger, Brendan Lee
Correspondence to Brendan Lee: [email protected]
Nature Medicine: doi:10.1038/nm.3544
Weight (grams±SEM)
Supplementary Figure 1
26
25
24
23
22
21
20
19
18
17
16
15
14
13
WT
WT
–/– control
Crtap-/Crtap
control
1D11
Crtap–/– 1D11
Crtap-/-
8
9
10
11
12
13
14
15
16
Age (weeks)
Supplementary Figure 1. Weight curves showing weight of WT, control Crtap –/– mice and 1D11-treated Crtap –/– mice during the study
period. N=8 per group.
Relative Abunda
#*
*
DGLNGLPGPIGPPGPR
100
60
40
20
750
crtap-/1D11 Ab
WT
100
100
80
80
774.02+ 2+
782.0
α1(I)
810
820
b
*
#*
DGLNGLPGPIGPPGPR
60
60
40
40
740
740
Relative
Abundance
Relative
Abundance
100
100
100
80
80
80
60
60
m/z
P986 peptide
m/z
crtap-/- control
1D11 Ab
crtap-/Ab
WT
α1(I)
α1(I)
α1(I)
2+
774.02+
774.0
800
800
bone
810
810
820
820
*
*
DGLNGLPGPIGPPGPR
782.02+ DGLNGLPGPIGPPGPR
#*
*
60
40
40
40
20
20
20
00
740
0740
740
100
100
80
Relative Abundance
750 crtap-/760 1D11
770 Ab
780treated
790
750
760
770
780
790
750
750
750
760
760
760
crtap-/Ab
crtap-/- control
1D11 Ab
α1(I)
α1(I)
770
770
770
780
780
780
m/z
m/z
m/z
774.02+
774.02+
80
60
60
40
40
20
20
0
0740
740
750
750
760
760
crtap-/- control Ab
770
770
780
780
m/z
m/z
774.02+
790
790
790
800
800
800
810
810
810
820
820
820
*
*
DGLNGLPGPIGPPGPR
*
*
DGLNGLPGPIGPPGPR
790
790
800
800
810
810
820
820
WT
Crtap–/–
control
Crtap–/–
1D11
P986
alpha 1(I)
96.6%
(2.1)
5.8%
(3.3)
2.0%
(1.2)
c
Hydroxylysyl pyridinoline
(HP res/collagen)
20
20
0
0
3Hyp
0.5
0.4
0.05
NS
NS
NS
0.3
0.2
0.1
0.0
0.04
NS
* NS
0.03
0.02
0.01
0.00
WT
25
HP / LP (ratio)
740
Lysyl pyridinoline
(HP res/collagen)
crtap-/1D11
Ab
treated
bone
760
770
780
790
800
P986 peptide
m/z
0
Relative
Abundance
Relative
Abundance
Relative
Abundance
Relative Abundance
Abundance
Relative
a
80
20
*
* NS
15
10
5
0
Crtap–/– 1D11
α1(I)
*
Supplementary
Figure 2. No effect
of TGFβ* inhibition
on the abnormal type I collagen post-translational modification in Crtap –/– mice.
DGLNGLPGPIGPPGPR
80
(a) Tandem
mass
spectra
of
extracted
type
I
collagen
from
tibia of WT, control Crtap –/–, and 1D11- treated Crtap –/– mice (16 week old mice,
60
after40treatment for 8 weeks). (b) Status of collagen residue Pro986 alpha 1(I) 3-hydroxylation in bone samples of bone samples of WT, control
20 –/– and 1D11- treated Crtap –/– mice assessed by tandem mass spectra analyses. Mean of percentage of 3-hydroxylated residues (±SD) is
Crtap
0
shown,
per group.
(c). Hydroxylysyl
740 n=5
750
760
770
780
790
800pyridinoline
810
820 (HP), lysyl pyridinoline crosslinks (LP) levels and HP/LP ratio of bone type I collagen of
m/z treated Crtap –/– mice. Results are given as means±SDs, n=4 mice per group, *P<0.05 for Crtap –/– vs. WT.
WT, control Crtap –/– and 1D11NS, not significant.
100
b
200
150
100
50
0
WT
Crtap-/Crtap–/–
*
40
30
20
10
0
WT
Crtap-/Crtap–/–
Serum OCN (ng/ml)
*
50
Serum CTX (ng/ml)
Serum OCN (ng/ml)
250
140
35
NS
NS
120
NS
100
80
60
40
20
0
WT
Crtap–/– Crtap-/Crtap–/–
Crtap-/control 1D11
Serum CTX (ng/ml)
a
NS
30
*
25
#
20
15
10
5
0
WT
Crtap-/Crtap–/–
Crtap–/– Crtap-/-
control 1D11
Supplementary Figure 3. Serum bone-turnover markers osteocalcin (OCN) and C-terminal cross-linked telopeptide of bone collagen (CTX) at
start (a. 8 weeks of age) and end of the treatment study (b. 16 weeks of age). (a) Results are given as means±SDs, n=8 for WT, n=14 for Crtap –/–
mice. (b) Results are given as means±SDs, n=8 for WT, n=7 per Crtap –/– group. *P<0.05 for Crtap –/– vs. WT, #P<0.05 for Crtap –/– 1D11 vs.
Crtap –/– control. NS, not significant.
Crtap–/–
WT
control
a
b
c
d
e
f
Supplementary Figure 4. Immunostaining for decorin in the distal femur metaphysis of WT and Crtap –/– mice is shown in 20X (a–c) and 40X
magnification (d–f). Control femurs were incubated in secondary antibody only. (n=3 per genotype; scale bars=100μm (a–c), 50μm (d–f)).
Crtap–/–
WT
control
a
b
c
d
e
f
Supplementary Figure 5. Immunostaining for TGFβ1 in the distal femur metaphysis of WT and Crtap –/– mice is shown in 20X (a–c) and 40X
magnification (d–f). Control femurs were incubated in secondary antibody only (n=3 per genotype; scale bars=100μm (a–c), 50μm (d–f)).
Col1a2tm1.1Mcbr
WT
control
a
b
c
d
e
f
Supplementary Figure 6. Immunostaining for TGFβ1 in the distal femur metaphysis of WT and Col1a2tm1.1Mcbr mice is shown in 20X (a–c)
and 40X magnification (d–f). Control femurs were incubated in secondary antibody only. (n=3 per genotype; scale bars=100μm (a–c), 50μm (d–f)).
Supplementary Table 1
BV/TV
(%)
Tb.N
(1/mm)
Tb.Th
(µm)
Tb.Sp
(mm)
BMD BV
(mg HA/ccm)
Wild type
SD
31.173
6.543
4.845
0.583
63.988
9.482
0.145
0.034
703.189
19.014
Crtap-/- control
SD
8.354
2.045
2.130
0.409
38.888
2.931
0.450
0.118
677.416
16.138
Crtap-/- 1D11
SD
27.953
5.645
4.290
0.435
64.650
7.407
0.171
0.030
746.862
26.564
ANOVA P value
<0.001
<0.001
<0.001+
<0.001+
<0.001
Pairwise P values
Wild type vs. Crtap-/- control
Wild type vs. Crtap-/- 1D11
Crtap-/- control vs. Crtap-/- 1D11
<0.001
n.s.
<0.001
<0.001
0.031
<0.001
<0.05
n.s.
<0.05
<0.05
n.s.
<0.05
0.023
<0.001
<0.001
Supplementary Table 1. MicroCT analyses of vertebral body L4 of WT, control Crtap –/– and 1D11 treated Crtap –/– mice (16 week old mice, after
treatment for 8 weeks). Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th),
trabecular separation (Tb.Sp), and bone mineral density of bone volume (BMD BV); n=8 per group, + indicates Kruskal-Wallis one-way ANOVA on
ranks where the equal variance test failed. n.s.=not statistically significant.
BV/TV
(%)
Tb.N
(1/mm)
Tb.Th
(µm)
Tb.Sp
(mm)
BMD BV
(mg HA/ccm)
Wild type
SD
10.698
3.558
2.808
0.684
37.413
5.071
0.341
0.108
733.864
25.997
Crtap-/- control
SD
2.873
1.082
0.861
0.237
32.863
3.428
1.209
0.345
727.151
28.765
Crtap-/- 1D11
SD
12.343
4.523
2.953
0.767
40.663
5.842
0.323
0.121
756.539
36.913
ANOVA P value
<0.001+
<0.001+
0.015
<0.001
0.162
<0.05
n.s.
<0.05
n.s.
n.s.
n.s.
<0.001
n.s.
<0.05
<0.05
0,004
<0.001
Pairwise P values
Supplementary Table 2. MicroCT analyses of trabecular bone in proximal femurs for WT, control Crtap –/–, and 1D11- treated Crtap –/– mice (16
week old mice, after treatment for 8 weeks). Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular
thickness (Tb.Th), trabecular separation (Tb.Sp) and bone mineral density of bone volume (BMD BV); n=8 per group. + indicates Kruskal-Wallis
one-way ANOVA on ranks where the equal variance test failed. n.s.=not statistically significant.
Cortical thickness
BMD BV
Diameter a.p.
CSA
2
CSMI m.l.
4
CSMI a.p.
(mm)
(mg HA/ccm)
(mm)
(mm )
(mm )
(mm4)
Wild type
SD
0.242
0.014
1084.726
28.375
1.239
0.063
0.905
0.079
0.134
0.023
0.221
0.042
Crtap-/- control
SD
0.203
0.020
1084.885
34.256
1.142
0.065
0.731
0.082
0.101
0.021
0.162
0.034
Crtap-/- 1D11
SD
0.221
0.026
1096.127
39.754
1.186
0.080
0.808
0.118
0.111
0.028
0.186
0.038
ANOVA P value
0.003
0.753
0.039
0.005
0.032
0.021
0.012
n.s.
n.s.
0.001
n.s.
n.s.
0.011
n.s.
n.s.
0.006
n.s.
n.s.
Pairwise P values
<0.001
n.s.
n.s.
Supplementary Table 3. MicroCT analysis of cortical bone at the femur midshaft for WT, control Crtap –/–, and 1D11-treated Crtap –/– mice (16 week old
mice, after treatment for 8 weeks). Means±SDs are shown for cortical thickness, bone mineral density of bone volume (BMD BV), anterior-posterior (a.p.)
diameter, cross-sectional area (CSA), and cross-sectional moments of inertia (CSMI) for medio-lateral (m.l.) and anterior-posterior (a.p.) axis; n=8 per group.
n.s.=not statistically significant.
Maximum
load
(N)
Stiffness
(N/mm)
Energy
to failure
(N*mm)
Ultimate
strength
(MPa)
Toughness
to failure
(MPa)
Elastic
modulus
(GPa)
Total
displacement
(mm)
Elastic
displacement
(mm)
Post-yield
displacement
(mm)
Wild type
SD
22.831
2.860
230.578
37.053
7.846
3.985
154.704
7.478
11.912
5.695
6.960
0.731
0.444
0.207
0.068
0.014
0.376
0.203
Crtap-/- control
12.943
151.689
1.228
114.496
2.208
6.663
0.127
0.079
0.048
SD
2.402
27.384
0.913
18.240
1.677
1.154
0.057
0.006
0.058
Crtap-/- 1D11
SD
18.818
2.337
200.804
15.644
1.991
0.834
145.633
19.074
3.408
1.851
7.248
0.271
0.156
0.055
0.073
0.013
0.083
0.055
ANOVA P value
<0.001
0.009
0.009
0.004
0.009
0.658
0.015
0.360
0.012
<0.001
0.003
0.005
0.001
0.005
0.009
0.007
n.s.
n.s.
0.017
n.s.
0.017
0.023
0.019
0.015
n.s.
n.s.
0.016
n.s.
n.s.
n.s.
Pairwise P values
-/-
Wild type vs. Crtap 1D11
Supplementary Table 4. Results of biomechanical testing of femurs from WT, control Crtap –/– and 1D11 treated Crtap –/– mice by 3-point bending (16 week
old mice, after treatment for 8 weeks). N=6 for WT, n=4 for control Crtap –/– and n=3 for 1D11 treated Crtap –/– mice. Results are shown as means±SDs.
n.s.=not statistically significant.
BV/TV
(%)
Tb.N
(1/mm)
Tb.Th
(µm)
Tb.Sp
(µm)
N.Oc/BS
(1/mm)
Oc.S/BS
(%)
N.Ob/BS
(1/mm)
Ob.S/BS
(%)
N.Ot/B.Ar
(1/mm2)
Wild type
SD
11.956
3.067
4.003
0.656
29.554
3.536
226.736
50.077
3.226
0.812
15.051
3.089
18.142
2.368
20.827
2.754
549.002
83.665
Crtap-/- control
SD
3.817
1.656
1.909
0.692
19.502
2.560
587.008
289.835
4.768
0.914
19.885
3.407
24.487
5.421
26.888
6.983
735.561
72.617
Crtap-/- 1D11
10.203
3.411
30.182
277.886
1.772
6.829
8.638
9.748
565.044
SD
3.097
0.878
6.982
69.118
0.673
2.235
2.855
4.718
51.014
ANOVA P value
<0.001
<0.001
0.002
0.003+
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.002
<0.05
0.005
0.012
0.011
n.s.
<0.001
n.s.
n.s.
n.s.
n.s.
0.007
<0.001
<0.001
0.002
n.s.
<0.001
0.003
0.001
<0.05
<0.001
<0.001
<0.001
<0.001
<0.001
Pairwise P values
-/-
Wild type vs. Crtap 1D11
-/-
-/-
Crtap control vs. Crtap 1D11
Supplementary Table 5. Histomorphometry analyses of L4 vertebral bodies of WT, control Crtap –/–, and 1D11- treated Crtap –/– mice (16 week old mice,
after treatment for 8 weeks). Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th),
trabecular separation (Tb.Sp), number of osteoclasts/bone surface (N.Oc/BS), osteoclast surface/bone surface (Oc.S/BS), number of osteoblasts/bone surface
(N.Ob/BS), osteoblast surface/bone surface (Oc.S/BS), and number of osteocytes/bone area (N.Ot/B.Ar); n=6 per group. + indicates Kruskal-Wallis one way
ANOVA on ranks where equal variance test failed. n.s.=not statistically significant.
BV/TV
(%)
Tb.N
(1/mm)
Tb.Th
(µm)
Tb.Sp
(mm)
BMD BV
(mg HA/ccm)
Wild type
SD
24.708
3.282
3.939
0.330
62.533
3.497
0.193
0.025
716.669
7.626
Col1α2tm1.1Mcbr control
SD
10.608
1.487
2.071
0.195
51.067
2.999
0.435
0.047
731.436
11.792
Col1α2tm1.1Mcbr 1D11
SD
29.715
1.984
4.244
0.199
69.983
2.329
0.166
0.013
754.115
7.844
ANOVA P value
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
0.015
0.002
<0.001
n.s.
<0.001
<0.001
<0.001
n.s.
<0.001
<0.001
<0.001
Pairwise P values
Wild type vs. Col1α2tm1.1Mcbr control
Wild type vs. Col1α2tm1.1Mcbr 1D11
Col1α2tm1.1Mcbr control vs. Col1α2tm1.1Mcbr 1D11
Supplementary Table 6. MicroCT analyses of vertebral body L4 of WT, control Col1a2tm1.1Mcbr and 1D11 treated Col1a2tm1.1Mcbr mice (16 week old mice,
after treatment for 8 weeks). Means±SDs are shown for bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th),
trabecular separation (Tb.Sp), and bone mineral density of bone volume (BMD BV); n=6 per group, n.s.=not statistically significant.

Crtap - Musculoskeletal Research Center

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