β1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis
β1 integrins mediate the BMP2 dependent transcriptional control of osteoblast differentiation and osteogenesis
Molly Brunner 0 1
NoeÂ mie Mandier 0 1
Thierry Gautier 0 1
Genevieve Chevalier 0 1
Anne- Sophie Ribba 0 1
Philippe Guardiola 1
Marc R. Block 0 1
Daniel Bouvard 0 1
0 Centre de Recherche INSERM 1209, CNRS 5309, Institute for Advanced Bioscience; Universit eÂ Grenoble Alpes, Grenoble, France, 2 Centre Hospitalier Universitaire and University of Angers, SNP Plateform, Institute for Biological Health , Transcriptome and Epigenomic, Angers , France
1 Editor: Dominique Heymann, Universite de Nantes , FRANCE
Osteoblast differentiation is a highly regulated process that requires coordinated information from both soluble factors and the extracellular matrix. Among these extracellular stimuli, chemical and physical properties of the matrix are sensed through cell surface receptors such as integrins and transmitted into the nucleus to drive specific gene expression. Here, we showed that the conditional deletion of β1 integrins in the osteo-precursor population severely impacts bone formation and homeostasis both in vivo and in vitro. Mutant mice displayed a severe bone deficit characterized by bone fragility and reduced bone mass. We showed that β1 integrins are required for proper BMP2 dependent signaling at the pre-osteoblastic stage, by positively modulating Smad1/5-dependent transcriptional activity at the nuclear level. The lack of β1 integrins results in a transcription modulation that relies on a cooperative defect with other transcription factors rather than a plain blunted BMP2 response. Our results point to a nuclear modulation of Smad1/5 transcriptional activity by β1 integrins, allowing a tight control of osteoblast differentiation.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This work was supported by Institut
National du Cancer (INCa) to DB, Association pour
la Recherche sur le Cancer to DB, and Societe
Francaise contre les cancer de l'enfant (SFCE),
SFCE-CRAUFESD16, to DB. MB was supported by
a fellowship from the French Research Ministry
(MRT). The funders had no role in study design,
Proliferation, differentiation and survival of adherent cells are all tightly dependent on cell
interaction with the extracellular matrix (ECM) or neighboring cells. Therefore, the nature
and the physical proprieties of the extracellular matrix are important players during
mammalian development and tissue integrity. During bone formation and homeostasis, osteoblasts are
responsible for the deposition of novel bone matrix at sites of osteoclast dependent bone
]. New bone matrix synthesis is a direct response to changes that occur within the
surrounding environment. While not totally understood, an emerging hypothesis is that the
remodeled bone matrix impacts on both the physical quality (stiffness) and composition of the
bone with the release of ECM fragments and growth factors [
]. Matrix stiffness and resulting
cellular tension have emerged to be critical for mesenchymal stem cell fate and proliferation
]. Recently, it has been shown that mesenchymal stem cells are able to integrate the
data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing interests: The authors have declared
that no competing interests exist.
extracellular environment stiffness to control YAP/TAZ signaling and thereby cell
proliferation and differentiation [
]. Additionally, soluble growth factors such as BMPs, WNTs or
PTHrP also play a pivotal role to control proper osteoblast proliferation and differentiation
]. Among those soluble factors, BMP2 is of prime interest in this latter process. Indeed,
BMP2 deletion during development leads to a severe defect in post-natal bone formation [
Likewise, BMP receptor 1A (BMPR1A) or SMAD-4 (a BMP effector) deletions in mature
osteoblasts, as well as the overexpression of the BMP-inhibitor Noggin, led all to a significant
reduction in osteoblast activity [
These mechanical and matrix associated cues are likely very important for controlling
osteoblast/osteoclast coupling as mentioned above as well as developmental and
pathological processes. During bone development, pre-osteoblasts are located both within the
connective tissue (periosteous) and at mineralized surfaces, while differentiated osteoblasts are
exclusively found in close contact to mineralized bone matrix [
]. These findings favor the
view that matrix rigidity may provide a signal necessary for pre-osteoblast to osteoblast
Integrins as the main receptors for cell adhesion to the ECM are likely important
mechano-transducers in these processes. Although lacking any enzymatic activity, they are
crucial for bidirectional signaling. In one hand, they are involved in matrix organization and
deposition [9±11] thereby creating a signaling niche that facilitate outside-in signaling from
cognate receptors such as growth factors receptors. On the other hand, integrin affinity and
clustering are regulated from an inside-out signaling pathway. Fibronectin (FN) through its
interaction with α5β1 integrins, as well as type I collagen, was shown to be necessary for
osteoblast differentiation and survival [12±14], while the deletion of β1 integrin specific inhibitor
such as ICAP-1 was reported to affect bone formation in mice [
]. Mechanistically, ICAP-1,
by preventing an excessive talin and kindlin-2 recruitment on β1 integrin cytoplasmic tails,
favors β1 integrin cycling between low and high affinity that is required for fibrillogenesis
and collagen deposition [
]. Finally, it was shown that α5β1 activation by an agonist
peptide also promotes osteoblast differentiation through the activation of a FAK and MAPK/
ERK-dependent pathway [
]. Unexpectedly, the conditional deletion of β1 integrins in
mature osteoblasts using the type I collagen (Col 2.3) promoter driving Cre recombinase
expression led to a relatively slight phenotype in mice [
]. Since, the expression of a
dominant negative (DN) form of β1 integrins under the control of the osteocalcin (OCN)
promoter that targets mature osteoblasts demonstrated a limited role for these integrins in this
cell type [
], the absence of a severe bone phenotype in the former mouse model may be
explained by the relatively late deletion of β1 integrins. This hypothesis was further
confirmed by the analysis of conditional deletions of β1 integrins at different developmental
stage during osteogenesis [
In this study, we analyzed the role of β1 integrins at an early stage of osteoblast
differentiation using the Osterix (Osx) promoter to drive Cre recombinase expression (β1Ost-ko mice).
Interestingly, in pre-osteoblasts, the β1 integrin deletion led to a severe bone mass reduction in
young mice as previously reported [
]. However, it was not reported how integrins affect
osteoblast differentiation at the molecular level. We showed that, mechanistically, this
phenotype was associated in vivo with a clear decrease in type I collagen deposition and Smad1/5
phosphorylation. Using in vitro osteoblast cultures, we demonstrated that β1 integrins deletion
neither affects Smad1/5 phosphorylation ex vivo nor their nuclear localization, but rather acts
downstream into the BMP signaling pathway, likely by regulating the establishment of a
proper network of transcription factors involved in the transition between pre-osteoblast to
2 / 17
Fig 1. β1 integrin deletion in β1Ost-ko mice. A. Immunofluorescence analysis of β1 integrin expression on wild-type and β1Ost-ko P4 mice sections (cb:
cortical bone; bm: bone marrow; dash line represents cortical the bone surface; arrows show osteoblasts deleted of β1 integrins; scale bar represents
10μm). Note that β1 integrin deletion is not total in β1Ost-ko mice. B and C. Western-blot analysis and quantification of β1 integrin expression in long
bones lysates from wt and β1Ost-ko P30 mice. β1 integrin expression was normalized with actin ( : p<0.05).
Efficiency of β1 integrin deletion by Osx-Cre deletor mice
Since, β1 integrins have a limited role in mature osteoblasts, we hypothesize that deletion
using the Col1-Cre deletor mice might occur at a stage too late to impair the osteoblast
differentiation and reveal any important function of β1 integrins in this process. To specifically
address this question, we crossed the Osterix-Cre (Osx-Cre) deletor mice with mice bearing β1
integrins floxed alleles [
]. To characterize the β1 integrin deletion using the Osx-Cre deletor
mice, we performed immunofluorescence staining on femur from 4-days old WT and β1Ost-ko
mice. As expected, a clear reduction in the number of β1 integrin-positive osteoblasts at the
surface of cortical bone (Fig 1A, white arrow heads) was observed. This was also accompanied
by an increase in β1 integrin-negative osteocytes embedded into cortical bone. β1 integrin
deletion was then quantified by Western-blotting of long bones lysates from wild-type and
β1Ost-ko mice (Fig 1B). As expected, we observed a clear reduction in β1 integrin expression in
β1Ost-ko mice compared to wild-type. Western-blotting quantification and normalization with
actin allowed us to estimate a 2.78-fold reduction in β1 integrin expression in β1Ost-ko mice
compared to WT (Fig 1B and 1C). This incomplete deletion of β1 integrins in β1Ost-ko mice
can be explained by the presence of progenitors that still do not express the Osterix
transcription factor or by the partial recombinase deletion of the β1 integrin gene. Nevertheless, the
deletion was sufficient to induce a drastic phenotype in β1Ost-ko mice, highlighting the
important role of this integrin class in bone formation and homeostasis.
β1 integrins are important regulators of bone formation at the
Mutant mice deleted for β1 integrins (β1Ost-ko mice) as well as heterozygous mice (Ost-Cre+;
β1+/f) were significantly smaller upon aging than wild-type mice (Fig 2A and 2B). Interestingly,
3 / 17
Fig 2. β1 integrin deletion at pre-osteoblastic stage leads to severe bone phenotype. A. Growth curve of wild-type, heterozygous and β1 integrin
deleted (β1Ost-ko) mice. B. Picture of wild-type and β1Ost-ko mice showing the growth defect due to β1 integrin deletion at pre-osteoblastic stage. C.
Total bone mineral density (BMD) of wild-type, heterozygous and mutant mice measured using micro Computed Tomography ( : p<0.005). D and E.
Histomorphometric analysis of osteoblast number (Ob. N.) D. and surface (Ob. S.) E. at P30 depending on mice genotype ( : p<0.05). F and G.
Alizarin Red and Alcian Blue staining of skeleton from 3-days or 15-days old wild-type and β1Ost-ko mice. Note the increased porosity of skull, jaw and
long bones, as well as bending and hypertrophic callus resulting from former fractures in β1Ost-ko mice.
this growth defect was more visible after weaning (post-natal day 21, P21). When looking
specifically at the bone tissue, mutant and heterozygous mice displayed a significant reduction in
bone mineral density measured by micro Computed Tomography (Fig 2C).
Histomorphometric analyses of β1Ost-ko mice showed a reduced number of osteoblasts and a reduced bone
4 / 17
surface covered by osteoblasts compared to their wild-type littermates (Fig 2D and 2E). This
was accompanied by a strong mineralization defect, increased porosity and fragility of long
bones as well as flat bones such as calvaria and scapula at P3 (Fig 2F). This occasionally leads
to fractures, bending, and hypertrophic callus resulting from prior fractures in β1Ost-ko mice
long bones such a ribs and tibia (Fig 2G, upper and lower panel respectively). All together
these data highlighted the importance of β1 integrins at early stages of osteoblast
differentiation for proper bone formation as previously reported (21).
β1 integrin deletion leads to reduced Type I Collagen deposition and
We previously reported the role of β1 integrin activation/de-activation cycle in fibronectin and
collagen matrix deposition, a prerequisite for mineralization (11). Hence, we analyzed matrix
deposition capability of osteoblasts deleted for β1 integrins. Immunofluorescence staining for
Type I Collagen on femur sections of E14.5 WT or β1Ost-ko embryos were carried out and
revealed that β1 integrin deletion strongly impaired Type I collagen deposition at the
periosteum surface of forming bones (Fig 3A). The collagen deposition defect was also confirmed ex
vivo using deleted (β1-/-) or control immortalized osteoblasts induced to differentiate in a
conditioned medium. At confluence, wild-type osteoblasts showed an important Type I Collagen
deposition that increased after 4 days of differentiation. In contrast, β1-/- osteoblasts exhibited
a reduced collagen deposition at confluence that did not increase significantly during
differentiation (Fig 3B). We previously demonstrated that mineralization requires a proper matrix
deposition by osteoblasts. Accordingly, differentiation in a conditioned medium for 4, 10 or
15 days revealed a complete absence of mineralization of β1-/- osteoblasts visualized by Alizarin
red staining. These results were validated with 3 independent clones to rule out any clonal
effect on differentiation capability and demonstrated the importance of β1 integrins for proper
Type I Collagen matrix deposition and subsequent mineralization by osteoblasts. Moreover,
mineralization capability of osteoblasts isolated from wild-type (Ost-Cre-; β1f/f) or mutant
(Ost-Cre+; β1f/f) mice confirmed that β1 integrins deletion during osteoblast differentiation
impairs osteoblast mineralization and that blocking Cre expression with doxycycline rescued
the mineralization phenotype in Ost-Cre+; β1f/f (Fig 3D).
β1 integrins are essential for Smad1/5 phosphorylation in vivo
Next we wondered whether the collagen deposition defect alone accounted for the bone
phenotype. A major osteogenic factor regulating Alkaline Phosphatase (ALP), Osterix (Osx)
expression, and involved in bone development is the Bone Morphogenetic Protein 2 (BMP2)
]. Some ex vivo studies demonstrated a link between BMP2 and integrin signaling [
9, 13, 23
Moreover, the expression of a dominant negative BMP2 receptor in maturing osteoblasts
partly phenocopied β1 integrin deletion in pre-osteoblasts [
]. However, it is still not clear
whether the crosstalk between BMP2 and integrins is relevant in vivo, and which mechanisms
are implicated. Thus, we asked whether the drastic bone phenotype induced upon β1 integrin
deletion at the pre-osteoblastic stage in vivo could be linked to any BMP2 signaling defect. To
address this important question, we compared Smad1/5 phosphorylation in vivo in wild-type
and β1Ost-ko mice. As revealed by immunofluorescence, wild-type osteoblasts at the cortical
bone surface were strongly positive for p-Smad1/5 staining, while on β1Ost-ko mice sections,
this staining was almost absent (Fig 4A). These results were confirmed by Western-blotting of
bone lysates showing a 2-fold reduction in Smad1/5 phosphorylation in β1Ost-ko compared to
WT mice (Fig 4B and 4C). Therefore, these data demonstrated the positive control of β1
integrins on Smad1/5-dependent BMP2 signaling in vivo.
5 / 17
Fig 3. β1 integrins are necessary for Type I Collagen deposition and mineralization. A. Immunofluorescence analysis of Type I Collagen
deposition on wild-type and β1Ost-ko E14.5 embryos femur sections (h.c.: hypertrophic cartilage; scale bar represents 10μm). Note that Type I
Collagen deposition onto β1Ost-ko developing bones surface was almost absent. B. Immunofluorescence staining of Type I Collagen deposition by
wild-type and β1 deleted (β1-/-) immortalized osteoblasts induced to differentiate for 4 days (day 4) or not (day 0)(Scale bar represent 10μm). C.
and D. Mineralization capability, visualized by Alizarin red staining, of WT vs β1-/-, and Ost-Cre expressing β1f/f vs control (Ost-Cre negative)
immortalized osteoblasts induced to differentiate for 4, 10 or 15 days (C.) or 0, 6 and 14 days (D.). Doxycycline was added to the medium (+dox)
to block Cre expression. Note the lack of mineralization of β1-/- and Ost-Cre+;β1f/f osteoblasts cultures and the mineralization rescued in presence
β1 integrins are necessary for the expression of BMP2 targeted genes
independently on Smad1/5 phosphorylation
As mentioned above, previous ex vivo studies demonstrated a link between integrins and
BMP2 receptor activation [
]. However, it is not clear in bone at which level β1 integrins act
6 / 17
Fig 4. β1 integrins are essential for Smad1/5 phosphorylation in vivo, but controls the expression of BMP2 targeted genes regardless of Smad1/5
phosphorylation ex vivo. A. Immunofluorescence analysis of β1 integrins as well as Smad1/5 phosphorylation (P-Smad1/5) on wild-type and β1Ost-ko
P4 mice sections (c.b.: cortical bone; b.m.: bone marrow; scale bar represents 10μm). Note the almost absence of Smad1/5 phosphorylation in the lack of
β1 integrins. B and C. Western-blot analysis and quantification of Smad1/5 phosphorylation in long bones lysates from wt and β1Ost-ko P30 mice.
Smad1/5 phosphorylation was normalized with actin ( : p<0.05). D. Western-blot analysis of Smad1/5, p38, ERK1/2 and AKT phosphorylation in
immortalized WT and β1-/- osteoblasts cultures treated or not with BMP2. E. Quantitative PCR analyses of BMP2-independent Runx2, or
BMP2-dependent Alkaline Phosphatase (ALP), Osteocalcin (OCN) and Osterix (Osx) mRNA expression levels in WT or β1-/- osteoblasts cultures
treated or not with BMP2 ( : p<0.005). Note the absence of induction by BMP2 of target genes in β1-/- osteoblasts.
7 / 17
to favor BMP2-dependent signaling. In order to further characterize the input of β1 integrins in
the BMP2/Smads pathway, we studied BMP2 downstream signaling in immortalized
osteoblasts cultures deleted or not for β1 integrins. Unexpectedly, when analyzing the
phosphorylation of key proteins of main signaling pathways, such as p38, Smad1/5, ERK1/2 or AKT, in
response to BMP2, no major differences were observed between WT and β1-/- cells (Fig 4D).
Interestingly, the previously observed Smad1/5 phosphorylation defect upon β1-/- osteoblasts
removal in vivo was no longer reproduced ex vivo after stimulation with BMP2. Indeed,
treatment with BMP2 led to a significant Smad1/5 phosphorylation in both WT and β1 deleted cells
(Fig 4D, upper panel). Likewise, p38 phosphorylation in response to BMP2 was not affected by
β1 integrin loss, while ERK1/2 phosphorylation remained unchanged regardless of the
expression or β1 integrins. Conversely, we noted a slight reduction in AKT phosphorylation after
BMP2 treatment that was not reproduced in β1-/- cells. To further investigate β1-/- osteoblasts
response to BMP2 treatment ex vivo, we analyzed the final events of BMP2-dependent pathway
activation, i.e. the expression of BMP2 target genes. Unexpectedly, quantitative PCR analyses
revealed that the expression of BMP2 target genes (ALP, OCN and Osx) was clearly reduced in
response to BMP2 treatment in β1 deleted compared to WT osteoblasts (Fig 4E). One
explanation for the apparent discrepancy between efficient Smad1/5 phosphorylation and the absence
of target genes expression in the absence of β1 integrins may be the lack of efficient nuclear
translocation of Smads in β1-/- osteoblasts. Indeed, it was previously reported that integrins can
regulate the nuclear translocation of signaling proteins such as ERK or α-NAC [
9, 25, 26
test this hypothesis, we performed immunofluorescence analyses of p-Smad1/5 subcellular
localization under basal conditions or after BMP2 treatment. Importantly, WT as well as
β1-/osteoblasts exhibited a clear nuclear translocation of Smad1/5 in response to BMP2 (Fig 5).
These results led us to conclude that β1 integrins are important regulators of BMP2/Smads
signaling ex vivo, but not at the level of BMP receptor activation or Smad phosphorylation but
rather downstream in the signaling pathway.
β1 integrins regulate a subset of genes involved in bone mineralization
Next, we performed an unbiased transcriptomic analysis from wild-type and β1 deficient cells
upon BMP2 stimulation to disclose a broader view of how the loss of β1 integrins impacts the
BMP2 response. In good agreement with the reported phosphorylations above and Smads
nuclear localization, we noticed that the BMP2 response was not completely obliterated (Fig 6,
S1 and S2 Tables). Indeed, we observed in control cells that out of the 897 genes that were
regulated by BMP2, 572 were upregulated and 325 downregulated (S1 Table). In β1 deficient cells
714 genes were regulated by BMP2 with 447 genes upregulated and 267 genes downregulated
(S2 Table). In the BMP2 induced gene response, there was a significant overlap with 233
common genes upregulated and 78 genes downregulated in both WT and β1 deficient genotypes
(Fig 6A). While most genes that were modulated by BMP2 in both genotypes did not display
any significant differences, few of them exhibited more than a two-fold in between control
and β1 deficient cells difference (S3 Table). It is noteworthy, that some of these genes were
described to be involved in osteoblast function or differentiation such as FGFR2, FN1 [
Additionally, the analysis of modified biological processes, upon BMP2 treatment in both
control and mutant cells using the online Amigo2 algorithm, further supported the idea that
BMP2 induced osteoblastic signature was specifically affected by β1 integrin deficiency (Fig
6B). Indeed, gene signature related to bone development that was enriched in control cells
upon BMP2 stimulation was no longer found in β1 deficient cells, and the overall signature
corresponding to the osteoblastic differentiation was significantly reduced (enriched 4.67 fold
in control versus 3.85 fold in β1 deficient cells).
8 / 17
Fig 5. Efficient Smad1/5 nuclear translocation in absence of β1 integrins. Smad1/5 nuclear translocation was analyzed using
immunofluorescence under basal conditions (untreated) or after BMP2 treatment (+BMP2; PY: phospho-tyrosine; scale bar represents
10μm). Note the clear P-Smad1/5 nuclear translocation after BMP2 treatment in WT, as well as β1 deleted (β1-/-) osteoblasts.
Although the response to BMP2 appeared to be somehow preserved in β1 deficient cells,
some important genes involved during osteoblastic differentiation were not induced or
dramatically reduced such as osterix (SP7), type I and XII collagens (two collagens upregulated
during osteogenesis) and Itga11 [
]. In sharp contrast, Zfp521, a negative regulator of
Runx2 was upregulated in β1 deficient cells . Thus, this analysis further confirmed that β1
integrins play a pivotal role in the control of gene expression required for supporting a full
osteoblastic differentiation, but likely not directly by regulating BMP2 response.
Herein, we provided evidence and confirmed that β1 integrins are required at the early stages
of osteoblast differentiation in vivo and consequently for proper bone formation. We further
demonstrated that β1 integrins influence the expression of BMP2 target genes by acting not at
the BMP receptor level as suggested by earlier studies [
9, 13, 23, 31
] but rather downstream the
BMP2 signaling pathway and SMADs activation/nuclear translocation, probably at the
transcriptional level. These data underline the complexity of the regulation of osteoblast
differentiation and exemplify the necessity for a cell to be at the right place at the right time in order to
differentiate properly and become functional.
Integrins involvement in growth factor signaling has long been recognized [
Surprisingly, in vivo and ex vivo results showed an apparent contradiction. Indeed, in vivo BMP2
9 / 17
Fig 6. Transcriptomic analyses of BMP2 response. A. Diagrams representing the number of up and down regulated genes in control and
β1 deficient cells in response to BMP2 stimulation, respectively. B. Amigo2 online software analysis of GO biological process modified upon
BMP2 treatment in control (blue) and β1 deficient cells (red). Biological processes were selected based upon biological interest (bone related
signature). FDR = False Discovery Rate.
10 / 17
signaling is affected at the level of SMAD1/5 phosphorylation whereas ex vivo, SMAD1/5
phosphorylation and nuclear translocation were not affected. Despite this, the expression of some
of the BMP2 target genes were still blunted in the absence of β1 integrins. We can explain this
apparent discrepancy by the fact that BMP2 expression is regulated by the BMP/SMAD
pathway itself . Thus, a defective response to BMP2 (at the transcriptional level) due to β1
integrin loss would lead to a reduced secretion of BMP2 in the microenvironment in vivo,
consequently reducing BMPR and SMAD1/5 activation in osteoblasts. Hence, β1 integrins by
regulating BMP-2 and -4 expression and secretion might indirectly regulate SMAD1/5
phosphorylation level in vivo. Supplementation of exogenous BMP2 ex vivo and consequently the
independence of osteoblasts to their own BMP2 production may explain why p-SMAD1/5
defect was not observed under ex vivo experimental conditions. Alternatively, the nature of the
extracellular matrix on which the osteoblasts adhere onto may also conciliate in vivo and ex
vivo data. Indeed, osteoblasts express β1 integrin subunits that can associate with α1 and α5
and form functional receptor dimers for type I collagen and fibronectin (FN), respectively. On
the other hand, they also express αv integrin subunit that associates to β3 or β5 to form FN or
Bone Sialoprotein (BSP) receptors. Therefore, one can speculate that in vivo the ECM located
close to differentiating osteoblasts does not allow the engagement of αv containing integrins
by osteoblasts. In this case, the absence of integrin activation upon β1 integrin deletion leads to
an impairment of BMP2 signaling at the receptor or SMAD1/5 level. In contrast ex vivo αv
integrins may compensate the β1 integrin loss and rescues SMAD1/5 phosphorylation
downstream BMPR activation. Indeed, our results suggest that integrins in general are necessary for
proper BMPR activation and SMAD1/5 phosphorylation but that β1 integrins in particular are
required for SMAD1/5-dependent transcriptional regulation of BMP2 target genes. Further
experiments would be needed to address the role of αv vs β1 integrins and respective
complementary matrices at different levels of BMP signaling.
It was nicely shown that osteoblast differentiation is finely regulated in vivo. The less
differentiated cells being in a softer environment while mature osteoblasts being located on the rigid
bony surface [
]. β1 integrins being one of the major osteoblast cell surface receptors involved
in the matrix sensing, we can speculate that β1 removal might interfere with the osteoblast
capacity to sense their extracellular environment. Indeed, our in vivo data highlight a critical
role for β1 integrins in osteoblast function, while in vitro data supports that β1 integrins are
required for a selective transcriptional regulation of BMP2 target genes. This appears to be
downstream of Smad1/5 phosphorylation and nuclear translocation. How exactly this network
is regulated by β1 integrins remains speculative, but we anticipate that β1 integrins regulate the
formation of molecular complexes promoting an osteogenic specific transcription program.
Supporting the hypothesis of a missing co-factor to cooperate with a BMP2 dependent gene
expression, is the observation that β1 deficient cells are still responsive to a BMP2 stimulation.
Since we recently reported that β1 is a key regulator of YAP nuclear localization, [
] we can
speculate that YAP/TAZ signaling might be involved. Indeed, is was recently shown that YAP/
TAZ co signal with Smads to commit cells into the bone lineage in a fish model [
Therefore, one can hypothesize that β1 integrins might control Smad dependent transcription
through the regulation of the Smad co-transcription factor YAP or TAZ. Whatever the
cofactor involved, it is noteworthy that under BMP2 stimulation the expression of the key
osteogenic transcription factor osterix (a Runx2 target gene) was compromised in β1 deficient cells.
As RUNX2 and osterix cooperate with Smad1/5 to activate the expression of BMP2 target
genes, one can imagine that the defect observed in β1 deleted cells might be due to a RUNX2
or osterix transcriptional defect. This would explain the selective defect in the BMP2 response
in β1 deficient cells. Clearly, further investigations will be needed to precisely decipher how β1
integrins influence BMP2-dependent transcription of osteogenic genes and whether YAP
11 / 17
nuclear translocation might be involved in the establishment of this network, but our data
clearly support that β1 integrin signaling is involved in the fine coordination of different inputs
that led to the regulation of a specific gene expression pattern.
Mouse strain with floxed alleles of β1 integrin subunit (Itgb1tm1Ref) was kindly provided by Pr
R. FaÈssler (Max Planck Institute, Martinsried, Germany). The Osx1-GFP:Cre deletor mouse
was from Dr A. McMahon. Mice were generated in a mixed background (Sv129/C57B6). Mice
were kept at Grenoble University Animal house facility under regular conditions of husbandry
accordingly to the European rules and the project have been approved by the University
Ethical committee (National ethical committee number 11, project number: APAfiS 10218). Mice
were euthanized using CO2 asphyxia methods accordingly to the French legislation on animal
Antibodies plasmids and reagents
Antibodies: Anti-phospho-ERK (#4370), -phosphop38 (#4831), phosphor-AKT (#4058),
andÐphospho-Smad1/5 (#9511) were from Cell Signaling (Ozyme, St Quentin en Yvelines
France). Anti-β1 integrin (MB1.2) was from BD Biosciences (Le Pont de Claix, France). For
IHC, β1 integrin clone 4B7R antibody (Abcam, Paris, France) was used. The
anti-phosphotyrosine (PY) monoclonal antibody 4G10 used as hybridoma supernatant was produced in our
laboratory. Anti-Actin (clone AC-40), anti -Type I Collagen and doxycycline were from
Sigma-Aldrich (L'Isle d'Abeau, France). BMP2 was from Shenandoah Biotechnology Inc. and
was used at a 200μg/mL concentration. pEl-BRE4xLuc plasmid was a gift from J. MassagueÂ
Cell cultures and cell lines
Osteoblast cell lines were generated from newborn calvaria as previously described [
were immortalized with a retrovirus expressing the large SV40 T antigen, and were maintained
at 37ÊC with 5% CO2 in DMEM 10% FCS supplemented with penicillin and streptomycin.
Immortalized β1f/f cells were infected with an adenoviral supernatant encoding the Cre
recombinase for 1h in PBS supplemented with 2% FCS and 1mM MgCl2. Osteoblast were cloned and
β1 integrin deletion was checked by immunofluorescence.
Histomorphometry and bone density measurements
Tibiae from 2-month old animals were fixed and embedded in methylmethacrylate. Sections
were deplasticized and stained for Masson-Goldner with hematoxylin (Gill II), acid fuchsin/
ponceau xylidine, and phosphomolybdic acid/orange G to stain the cells and osteoid, and light
green to stain the mineralized matrix [
]. Primary cancellous bone was defined as the 120 μm
band below the growth plate. Cancellous bone was defined as the remaining trabecular area
that extends down 2 mm. The absolute osteoblast number in the cancellous bone was
evaluated and reported. pQCT of the distal femur was performed with XCT Research SA (StraTec
Medizintechnik). Trabecular BMD was measured at 9% of the bone length below the growth
plate using peel-mode 20 [
12 / 17
Mice skeletons were fixed in 70% ethanol during 2 days, transferred to acetone 2 more days,
then stained 2 days in staining solution (Alizarin Red 0.05g/l, Alcian Blue 0.15g/l, acetic acid
5% v/v). Finally, remaining soft tissues were digested in 1% KOH.
Immunofluorescence and TRAP staining
Cells were fixed with 4% paraformaldehyde-PBS for 15min. Following permeabilization and
blocking with goat serum, cells were incubated with primary antibodies during 1 hour.
Secondary antibodies used were conjugated with Alexa 488 and Alexa 555 from Jackson
Immunoresearch (Interchim, MontlucËon, France). Samples were mounted using Mowiol 4±88
reagent (Sigma Aldrich) supplemented with DAPI (Life technologies, St Aubin, France) and
were analyzed using an upright Axioimager M2 microscope (Carl Zeiss SAS, Le Pecq,
For paraffin-embedded tissues, sections were prepared and immunostained following
deparaffinization and hydration. TRAP stainings were performed using the TRAP kit from
Sigma Aldrich and according to the manufacturer's instructions.
Bone and cell lysates and Western-blotting
Protein lysates from bones were obtained from mice long bones carefully cleaned of any
muscle and bone marrow. Bone from 3 mice were pooled and frozen at -80ÊC then crushed by
shaking in presence of steel ball (Retsch MM400; 3 cycles of 1 minute at 30 movements/s) and
lysed in RIPA lysis buffer containing proteases and phosphatases inhibitors. Cells were lysed
using RIPA lysis buffer containing proteases and phosphatases inhibitors. Lysates from both
bone and cell culture were centrifuged at 15000rpm for 30 min at 4ÊC, and supernatants were
used for immunoblotting using standard protocol. Experiments were repeated two times.
In vitro differentiation of isolated osteoblasts was performed essentially as previously described
]. Briefly, 60,000 cells per well were plated in a 24-well tray. After 3 days of culture, when
cells were confluent, the medium was switched to differentiation medium (α-MEM, 10% FCS,
50 μg/ml ascorbic acid, 10 mM β-glycerophosphate) and changed every other day. The
differentiation process was visualized by Alizarin Red S staining for calcium deposition as described
RNA extraction, Reverse transcription, Real-Time PCR and transcriptomic
RNA samples were prepared and analyzed as previously described (Brunner et al., 2011).
Mouse primers were the following: RUNX2 forward, 5’- CCGCACGACAACCGCACCAT-3’;
and reverse 5’- CGCTCCGGCCCACAAATCTC-3’; ALP forward, 5’- GCCCTCTCCAAGA
CATATA-3’ and reverse 5’- CCATGATCACGTCGATATCC-3’; OCN forward, 5’- AAG
CAGGAGGGCAATAAGGT-3’ and reverse 5’- AGCTGCTGTGACATCCATAC-3’; Osx
forward, 5’- CCTAGCAGACACCATGAG-3’ and reverse 5’-
Total RNA was isolated from β1f/f and β1-/- immortalized osteoblast and purified using
TRIzol reagent (Thermo Fisher Scientific, Waltham, USA) and RNeasy Kit (Qiagen,
Courtaboeuf, France) following the manufacturer's instructions. Total RNA quantification was
performed using the Nanodrop ND- 1000 spectrophotometer (Thermo Fisher Scientific,
Waltham, USA). RNA was reverse-transcribed with the iScript Reverse Transcription Supermix
13 / 17
(Biorad, Hercules, USA). Real-time qPCR analysis was performed using iTaq Universal
SybrGreen Supermix (Biorad, Hercules, USA) on Biorad CFX96.
The integrity of the extracted RNAs was assessed with the Bioanalyzer 2100 and the
RNA6000 Nano kit (Agilent Technologies Incorporation, Santa Clara, USA). A RNA integrity
number (RIN) greater or equal to 7.00 was achieved for all samples. No sign of DNA
contamination was detected in any of the samples analyzed. The starting amount of total RNA used for
the reactions was 400 nanograms per sample, for all samples. The Illumina Total Prep RNA
Amplification Kit (Applied Biosystems / Ambion, Austin, USA) was used to generate
biotinylated, amplified cRNA according to the manufacturer recommendations. Hybridization,
staining and detection of cRNAs on Illumina Mouse WG-6 v2 Expression BeadChips were
performed according to the manufacturer's protocol. The MouseWG-6 v2.0 BeadChip profiles
more than 45,200 transcripts derived from the National Center for Biotechnology Information
Reference Sequence (NCBI RefSeq) database (Build 36, Release 22), the Mouse Exonic
Evidence Based Oligonucleotide (MEEBO) set as well as from exemplar protein-coding sequences
described in the RIKEN FANTOM2 database. The Illumina I-Scan system was used to scan all
Expression BeadChips, according to Illumina recommendations.
Using the Gene Expression Module 1.9.0 of GenomeStudio V2011.1 software (IlluminaÐ
USA), the Quantile normalization method was applied to the primary probe data. Processed
probe data were then filtered according to the following criteria: minimal signal intensity fold
change of 1.50 across all samples, minimal probe signal intensity absolute change of 150 across
all samples. Filtered data were then log2-transformed, and the expression values compared
between the β1-/-cells and wild-type β1f/f samples using Omics Explorer 3.2(42) (Qlucore,
Sweden). Genes were considered differentially expressed when their expression level satisfied two
criteria: the adjusted p-value (q-value) was < 0.01(which corresponded to a |R|> 0.96 ii) the
absolute fold change between the mean expression value in the samples from mutant cells
compared to that in controls was > 1.5.
Gene ontology analysis was performed using Amigo2 online software (http://amigo2.
berkeleybop.org/amigo/landing) on selected genes. BMP2 regulated genes in control (897) and
β1 integrin deficient cells (714) were submitted to the Amigo2 Term Enrichment Process.
Biological process answers were analyzed according to web site specifications. Relevant processes
were collected based on implication in BMP2 driven processes and a cut-off value of 1.6 fold
enrichment was chosen as filter. Raw data analysis is provided as S4 Table.
S1 Table. Gene list up- and downregulated upon BMP2 in β1f/f osteoblasts.
S2 Table. Gene list up- and downregulated upon BMP2 in β1-/- osteoblasts.
S3 Table. BMP2 induced shared gene list in β1f/f with β1-/- osteoblasts.
S4 Table. Raw data analysis of Amigo2 on β1f/f with β1-/- osteoblasts upon BMP2.
14 / 17
We are extremely grateful to Pr. Reinhard FaÈssler for providing us with β1 integrin derived
tools. We thank Alexei Grichine for microscopy assistance. M. Brunner was supported by a
fellowship from the French Research Ministry (MRT). We thank I. Nakchbandi for the
histomorphometric analysis. This work was supported by INCa and SFCE grants.
Conceptualization: Molly Brunner, Daniel Bouvard.
Data curation: Philippe Guardiola, Daniel Bouvard.
Formal analysis: Molly Brunner, Thierry Gautier, Marc R. Block, Daniel Bouvard.
Funding acquisition: Daniel Bouvard.
Investigation: Molly Brunner, NoeÂmie Mandier, Genevieve Chevalier, Philippe Guardiola,
Marc R. Block, Daniel Bouvard.
Methodology: Daniel Bouvard.
Project administration: Daniel Bouvard.
Resources: Anne-Sophie Ribba, Daniel Bouvard.
Supervision: Daniel Bouvard.
Validation: Molly Brunner, NoeÂmie Mandier, Philippe Guardiola, Marc R. Block, Daniel
Writing ± original draft: Marc R. Block, Daniel Bouvard.
Writing ± review & editing: Molly Brunner, Thierry Gautier, Marc R. Block, Daniel Bouvard.
15 / 17
16 / 17
1. Martin TJ , Sims NA . Osteoclast-derived activity in the coupling of bone formation to resorption . Trends Mol Med . 2005 ; 11 ( 2 ): 76 ± 81 . https://doi.org/10.1016/j.molmed. 2004 . 12 .004 PMID: 15694870
2. Nistala H , Lee-Arteaga S , Smaldone S , Siciliano G , Carta L , Ono RN , et al. Fibrillin-1 and -2 differentially modulate endogenous TGF-beta and BMP bioavailability during bone formation . J Cell Biol . 2010 ; 190 ( 6 ): 1107 ± 21 . https://doi.org/10.1083/jcb.201003089 PMID: 20855508
3. Engler AJ , Sen S , Sweeney HL , Discher DE . Matrix elasticity directs stem cell lineage specification . Cell . 2006 ; 126 ( 4 ): 677 ± 89 . https://doi.org/10.1016/j.cell. 2006 . 06 .044 PMID: 16923388
4. Dupont S , Morsut L , Aragona M , Enzo E , Giulitti S , Cordenonsi M , et al. Role of YAP/TAZ in mechanotransduction . Nature . 2011 ; 474 ( 7350 ): 179 ± 83 . https://doi.org/10.1038/nature10137 PMID: 21654799
5. Long F , Ornitz DM . Development of the endochondral skeleton . Cold Spring Harb Perspect Biol . 2013 ; 5 ( 1 ):a008334. https://doi.org/10.1101/cshperspect.a008334 PMID: 23284041
6. Bandyopadhyay A , Tsuji K , Cox K , Harfe BD , Rosen V , Tabin CJ . Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis . PLoS Genet . 2006 ; 2 ( 12 ):e216. https:// doi.org/10.1371/journal.pgen. 0020216 PMID: 17194222
7. Mishina Y , Starbuck MW , Gentile MA , Fukuda T , Kasparcova V , Seedor JG , et al. Bone morphogenetic protein type IA receptor signaling regulates postnatal osteoblast function and bone remodeling . J Biol Chem . 2004 ; 279 ( 26 ): 27560 ±6. https://doi.org/10.1074/jbc.M404222200 PMID: 15090551
8. Devlin RD , Du Z , Pereira RC , Kimble RB , Economides AN , Jorgetti V , et al. Skeletal overexpression of noggin results in osteopenia and reduced bone formation . Endocrinology . 2003 ; 144 ( 5 ): 1972 ± 8 . https:// doi.org/10.1210/en.2002-220918 PMID: 12697704
9. Maes C , Kobayashi T , Selig MK , Torrekens S , Roth SI , Mackem S , et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels . Dev Cell . 2010 ; 19 ( 2 ): 329 ± 44 . https://doi.org/10.1016/j.devcel. 2010 . 07 .010 PMID: 20708594
10. Hynes RO . The extracellular matrix: not just pretty fibrils . Science . 2009 ; 326 ( 5957 ): 1216 ±9. https://doi. org/10.1126/science.1176009 PMID: 19965464
11. Brunner M , Millon-Fremillon A , Chevalier G , Nakchbandi IA , Mosher D , Block MR , et al. Osteoblast mineralization requires beta1 integrin/ICAP-1-dependent fibronectin deposition . J Cell Biol . 2011 ; 194 ( 2 ): 307 ± 22 . https://doi.org/10.1083/jcb.201007108 PMID: 21768292
12. Globus RK , Doty SB , Lull JC , Holmuhamedov E , Humphries MJ , Damsky CH . Fibronectin is a survival factor for differentiated osteoblasts . J Cell Sci . 1998 ; 111 (Pt 10): 1385 ± 93 .
13. Jikko A , Harris SE , Chen D , Mendrick DL , Damsky CH . Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2 . J Bone Miner Res . 1999 ; 14 ( 7 ): 1075 ± 83 . https://doi.org/10. 1359/jbmr. 1999 . 14 .7.1075 PMID: 10404007
14. Moursi AM , Globus RK , Damsky CH . Interactions between integrin receptors and fibronectin are required for calvarial osteoblast differentiation in vitro . J Cell Sci . 1997 ; 110 (Pt 18): 2187 ± 96 .
15. Bouvard D , Aszodi A , Kostka G , Block MR , Albiges-Rizo C , Fassler R . Defective osteoblast function in ICAP-1-deficient mice . Development . 2007 ; 134 ( 14 ): 2615 ± 25 . https://doi.org/10.1242/dev.000877 PMID: 17567669
16. Millon-Fremillon A , Bouvard D , Grichine A , Manet-Dupe S , Block MR , Albiges-Rizo C . Cell adaptive response to extracellular matrix density is controlled by ICAP-1-dependent beta1-integrin affinity . J Cell Biol . 2008 ; 180 ( 2 ): 427 ± 41 . https://doi.org/10.1083/jcb.200707142 PMID: 18227284
17. FromigueÂ O , Brun J , Marty C , Da Nascimento S , Sonnet P , Marie PJ . Peptide-based activation of alpha5 integrin for promoting osteogenesis . J Cell Biochem . 2012 ; 113 ( 9 ): 3029 ± 38 . https://doi.org/10. 1002/jcb.24181 PMID: 22566152
18. Hamidouche Z , Fromigue O , Ringe J , Haupl T , Vaudin P , Pages JC , et al. Priming integrin alpha5 promotes human mesenchymal stromal cell osteoblast differentiation and osteogenesis . Proc Natl Acad Sci U S A . 2009 ; 106 ( 44 ): 18587 ± 91 . https://doi.org/10.1073/pnas.0812334106 PMID: 19843692
19. Phillips JA , Almeida EA , Hill EL , Aguirre JI , Rivera MF , Nachbandi I , et al. Role for beta1 integrins in cortical osteocytes during acute musculoskeletal disuse . Matrix Biol . 2008 ; 27 ( 7 ): 609 ± 18 . https://doi.org/ 10.1016/j.matbio. 2008 . 05 .003 PMID: 18619537
20. Zimmerman D , Jin F , Leboy P , Hardy S , Damsky C . Impaired bone formation in transgenic mice resulting from altered integrin function in osteoblasts . Dev Biol . 2000 ; 220 ( 1 ):2± 15 . https://doi.org/10.1006/ dbio. 2000 .9633 PMID: 10720426
21. Shekaran A , Shoemaker JT , Kavanaugh TE , Lin AS , LaPlaca MC , Fan Y , et al. The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype . Bone . 2014 ; 68 : 131 ± 41 . https://doi.org/10.1016/j.bone. 2014 . 08 .008 PMID: 25183373
22. Rodda SJ , McMahon AP . Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors . Development . 2006 ; 133 ( 16 ): 3231 ± 44 . https:// doi.org/10.1242/dev.02480 PMID: 16854976
23. Chang SF , Chang CA , Lee DY , Lee PL , Yeh YM , Yeh CR , et al. Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad . Proc Natl Acad Sci U S A . 2008 ; 105 ( 10 ): 3927 ± 32 . https:// doi.org/10.1073/pnas.0712353105 PMID: 18310319
24. Zhao M , Harris SE , Horn D , Geng Z , Nishimura R , Mundy GR , et al. Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation . J Cell Biol . 2002 ; 157 ( 6 ): 1049 ± 60 . https://doi.org/10.1083/jcb.200109012 PMID: 12058020
25. Aplin AE , Stewart SA , Assoian RK , Juliano RL . Integrin-mediated adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1 . J Cell Biol . 2001 ; 153 ( 2 ): 273 ± 82 . PMID: 11309409
26. Quelo I , Gauthier C , Hannigan GE , Dedhar S , St-Arnaud R . Integrin-linked kinase regulates the nuclear entry of the c-Jun coactivator alpha-NAC and its coactivation potency . J Biol Chem . 2004 ; 279 ( 42 ): 43893 ±9. https://doi.org/10.1074/jbc.M406310200 PMID: 15299025
27. Mansukhani A , Bellosta P , Sahni M , Basilico C . Signaling by fibroblast growth factors (FGF) and fibroblast growth factor receptor 2 (FGFR2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts . J Cell Biol . 2000 ; 149 ( 6 ): 1297 ± 308 . PMID: 10851026
28. Brunner M , Jurdic P , Tuckerman JP , Block MR , Bouvard D , Jeon K . New Insights into Adhesion Signaling in Bone Formation . International Review of Cell and Molecular Biology , Vol 305 . 2013 ; 305 :1± 68 . https://doi.org/10.1016/B978-0 -12-407695-2 . 00001 -9 PMID: 23890379
29. Nakashima K , Zhou X , Kunkel G , Zhang Z , Deng JM , Behringer RR , et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation . Cell . 2002 ; 108 ( 1 ): 17 ± 29 . PMID: 11792318
30. Hesse E , Saito H , Kiviranta R , Correa D , Yamana K , Neff L , et al. Zfp521 controls bone mass by HDAC3-dependent attenuation of Runx2 activity . J Cell Biol . 2010 ; 191 ( 7 ): 1271 ± 83 . https://doi.org/10. 1083/jcb.201009107 PMID: 21173110
31. Lai CF , Cheng SL . Alphavbeta integrins play an essential role in BMP-2 induction of osteoblast differentiation . J Bone Miner Res . 2005 ; 20 ( 2 ): 330 ± 40 . https://doi.org/10.1359/JBMR.041013 PMID: 15647827
32. Ghosh-Choudhury N , Choudhury GG , Harris MA , Wozney J , Mundy GR , Abboud SL , et al. Autoregulation of mouse BMP-2 gene transcription is directed by the proximal promoter element . Biochem Biophys Res Commun . 2001 ; 286 ( 1 ): 101 ±8. https://doi.org/10.1006/bbrc. 2001 .5351 PMID: 11485314
33. Sabra H , Brunner M , Mandati V , Wehrle Haller B , Lallemand D , Ribba AS , et al. β1 integrin dependent Rac/group I PAK signaling mediates YAP activation of Yes associated protein 1 (YAP1) via NF2/merlin . J Biol Chem . 2017 .
34. Uemura M , Nagasawa A , Terai K. Yap/Taz transcriptional activity in endothelial cells promotes intramembranous ossification via the BMP pathway . Sci Rep . 2016 ; 6 : 27473 . https://doi.org/10.1038/ srep27473 PMID: 27273480
35. Bentmann A , Kawelke N , Moss D , Zentgraf H , Bala Y , Berger I , et al. Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function . J Bone Miner Res . 2010 ; 25 ( 4 ): 706 ± 15 . https://doi.org/10.1359/jbmr.091011 PMID: 19821765
36. Kawelke N , Bentmann A , Hackl N , Hager HD , Feick P , Geursen A , et al. Isoform of fibronectin mediates bone loss in patients with primary biliary cirrhosis by suppressing bone formation . J Bone Miner Res . 2008 ; 23 ( 8 ): 1278 ± 86 . https://doi.org/10.1359/jbmr.080313 PMID: 18348696