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First published online 13 June 2007
doi: 10.1242/dev.000877
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1 Université Joseph Fourier, CNRS, UMR 5538, LEDAC, Institut Albert
Bonniot, La Tronche Cedex, F-38706, France.
2 INSERM, U823, Equipe DySAD, Institut Albert Bonniot, F-38042, France.
3 Max Planck Institut für Biochemie, Department of Molecular Medicine, Am
Klopferspitz 18a, 82152 Martinsried, Germany.
* Author for correspondence (e-mail: daniel.bouvard{at}ujf-grenoble.fr)
Accepted 4 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ICAP-1 (ITGB1BP1), Integrin, Cell differentiation, Cell adhesion, Osteoblast, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hynes, 1992;
Hynes, 2002). Integrins are
bi-directional signaling molecules, which switch between a low (inactive) and
a high affinity (active) state. The switch to the high affinity state is
controlled by intracellular signals that act on the cytoplasmic domain of
integrins and induce rapid and reversible changes in the conformation of the
integrin extracellular domains (inside-out signal)
(Calderwood, 2004). Following
activation, integrins bind their ligands, merge into large clusters, recruit a
multitude of proteins to form so called focal adhesions (FAs), and transmit
signals to various subcellular compartments (outside-in signal).
ICAP-1 [integrin cytoplasmic domain associated protein-1; also known as
integrin beta 1 binding protein 1 (ITGB1BP1)] is a ubiquitously expressed
protein identified in a yeast two-hybrid screen as a ß1
integrin cytoplasmic domain-interacting protein
(Chang et al., 1997). Human
cells express two ICAP-1 isoforms that are generated by alternative
splicing. The large isoform associates with the cytoplasmic tail of
ß1 integrin. The small isoform lacks a C-terminally located
integrin-binding site (Chang et al.,
1997) and is therefore unable to interact with ß1
integrin. Overexpression of ICAP-1 negatively regulates ß1
integrin function by diminishing the strength of adhesion to, and enhancing
cell migration on, fibronectin (FN)
(Bouvard and Block, 1998;
Bouvard et al., 2003;
Zhang and Hemler, 1999). How
ICAP-1 exerts its functional properties is still unclear. One study proposed
direct competition with talin for binding to ß1 integrin
(Bouvard et al., 2003). Talin
is a large cytoplasmic protein that binds and activates several integrins, and
links them to the actin cytoskeleton
(Calderwood et al., 2002;
Vinogradova et al., 2002).
Recruitment of ICAP-1 on ß1 integrin would dislodge talin and
thereby reduce the affinity state of ß1 integrins leading to
FA disassembly (Bouvard et al.,
2003). In line with this hypothesis is the finding that ICAP-1 is
absent from FAs. A second study suggests that ICAP-1 might act as a guanine
dissociation inhibitor (GDI) for the small GTPases RAC1 and CDC42
(Degani et al., 2002). A
reduced RAC1 and/or CDC42 activity could also explain the spreading defects of
cells overexpressing ICAP-1 (Bouvard et
al., 2003; Degani et al.,
2002).
Finally, the identification of additional binding partners such as KRIT1
and the nucleotide diphosphate kinase NM23-H2 (also known as NME2)
(Fournier et al., 2002;
Zawistowski et al., 2002;
Zhang et al., 2001) linked
ICAP-1 to additional signaling pathways. Loss-of-function mutations in
KRIT1 cause a human disease called cerebral cavernous malformation
type I (Laberge-le Couteulx et al.,
1999), which is characterized by abnormalities of the brain
vasculature. KRIT1 has been shown to bind microtubules and the small GTPase
RAP1A (Gunel et al., 2002;
Serebriiskii et al., 1997).
RAP1A can reverse the transformed phenotype of KRAS-overexpressing cells and
modulate integrin-mediated cell adhesion on FN
(Bos et al., 2001). NM23-H2 is
a protein with nucleoside diphosphate kinase activity that has been linked to
a variety of cellular activities including the suppression of metastasis and
cell motility of tumour cells in vitro. NM23-H2 can bind to the promoter
sequences of the PDGFA and c-Myc (MYC - Human Gene
Nomenclature Database) genes, modulate the activity of small GTPases such as
RAD (also known as RRAD) and RAC1, and localizes in cell ruffles upon integrin
ligation (Fournier et al.,
2002).
To directly test the function of ICAP-1 in vivo, we generated Icap-1-deficient mice. Most of the mutant mice are born and develop craniofacial dysmorphism and dwarfism caused by abnormal proliferation and differentiation of osteoblasts leading to a delayed closure of calvarial sutures. Furthermore, we show that ICAP-1 regulates ß1 integrin activity and the condensation of preosteoblastic cells, an absolute requirement for proper bone development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Mice were genotyped by Southern blot or by PCR.
Antibodies
Polyclonal anti-ICAP-1 antibodies were described previously
(Bouvard and Block, 1998).
Monoclonal antibodies against actin, vinculin (clone hVIN1) and talin (clone
8d4) were from Sigma-Aldrich (Germany). Polyclonal anti-ß1
integrin serum was a gift from Dr Johansson (The Biomedical Center, Uppsala
University, Sweden). Monoclonal ß1 antibodies 9EG7 and MB1.2
were from Pharmingen (France) and a gift from Dr Bosco (Robarts Research
Institute, Ontario, Canada), respectively. Polyclonal anti-collagen I, III and
osteonectin/BM-40 antibodies were from Dr R. Timpl (Max Planck Institute for
Biochemistry, Martinsried, Germany). Polyclonal antibodies against cyclin D1,
FGFR1 and FGFR3 were from SantaCruz (USA), against Ki67 from Novocastra (UK)
and against 5-bromo-2'-deoxyuridine (BrdU) from Roche (Germany).
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;
Otto et al., 1996). Cells were
grown in 
-MEM medium containing 10% FCS.
In vitro differentiation of isolated osteoblasts was performed essentially
as described (Globus et al.,
1998). 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 (-MEM, 10% FBS, 50 µg/ml ascorbic
acid, 10 mM ß-glycerophosphate) and changed every second day. The
differentiation process was visualized by alkaline phosphatase (AP) staining
for osteoblast activity and by Alizarin Red S staining for calcium
deposition.
For the adhesion assay, primary osteoblasts (passage 2) were seeded at 0.5x105 cells in a 96-well tray coated with various concentrations of FN or COL1. The cells were incubated for 1 hour at 37°C and then washed three times with PBS before staining with a Crystal Violet solution (0.1% Crystal Violet, 20% methanol) for 1 hour at room temperature. After three washes in water, cells were lysed in 0.1% SDS for 1 hour. The absorbance was read at 550 nm with a Beckman Coulter AD 340 absorbance detector.
Cell proliferation was estimated by the BrdU assay as previously described
(Fournier et al., 2005).
Immortalization of osteoblasts
Primary osteoblasts (passage 2) were infected with a retrovirus expressing
the large SV40 T antigen (Fässler et
al., 1995), cloned and tested for their ability to induce AP upon
differentiation (Mansukhani et al.,
2000). Clone SV2.1 from an Icap-1-deficient mouse and
clone SV6.5 from a wild-type animal were used. Rescue of ICAP-1 expression in
SV2.1 cells was via retroviral infection using the
pCLMFG-Icap-IRES-EGFP vector. A homogeneous cell population was
sorted based on EGFP fluorescence with a MoFlo cell sorter (Dako Cytomation).
ICAP-1 expression was checked by western blot, immunofluorescence and FACS
using EGFP as a marker. This non-clonal cell population is referred to as
SV2.1-Icap-1resc hereafter.
Compaction assay in hanging drops
Immortalized cells were harvested by trypsin digestion and washed twice in
DMEM medium. Drops of 10 µl of DMEM-SVF medium containing 25,000 cells were
spotted onto the cover lid of 10 cm Petri dishes, inverted and placed on a
Petri dish containing 8 ml of PBS. Spheroid compaction was then followed over
a 72-hour incubation period and images taken with a binocular microscope
equipped with a digital camera.
Skeletal preparation, X-Gal staining and X-ray analysis
Staining of whole-mount embryos with Alcian Blue/Alizarin Red
(Aszodi et al., 1998) and X-Gal
(Sakai et al., 2001) was
carried out as described previously. X-ray images were obtained on a dual
energy setup developed at CEA/LETI (Grenoble, France).
Whole-mount in situ hybridization, histology, immunohistochemistry and in vivo cell proliferation
Whole-mount in situ hybridization was performed as described
(Rice et al., 2000).
Histochemistry and immunostaining on tissue sections were carried out as
described (Aszodi et al.,
1998). In vivo cell proliferation was analyzed using either the
BrdU incorporation assay (Aszodi et al.,
1998) or Ki67 immunohistochemistry. To detect cells with AP
activity, calvarial cryosections were fixed for 10 minutes in 3%
paraformaldehyde. After washing in PBS, the color reaction was developed in
the BCIP/NBT substrate solution (Roche, France). Immunofluorescence staining
and FACS analysis of primary osteoblasts were performed as previously
described (Bouvard et al.,
2003).
Activation index of ß1 integrin on primary osteoblasts
Activation index (AI) of ß1 integrin was estimated
essentially as previously described
(Calderwood et al., 2004).
Briefly, primary osteoblasts were isolated and passage-2 cells were aliquoted
into two pools containing either Tyrode's buffer alone or Tyrode's buffer
supplemented with 5 mM EDTA. After a 15-minute incubation at 4°C, cells
were incubated with or without the FITC labeled Fn7-10 fragment for 45 minutes
at 4°C in the presence or absence of 5 mM EDTA, washed in ice-cold
Tyrode's and analyzed on a FACScan (Becton Dickinson) flow cytometer. The
collected data were analyzed using CellQuest software (Becton Dickinson). In
parallel, cells were analyzed for ß1 expression using the
MB1.2 monoclonal antibody to detect the level of ß1 integrin
on the cell surface. The AI was calculated as follows: each specific mean
intensity fluorescence (MFI) was calculated by subtracting the background
obtained with Fn7-10 fragment incubation in the presence of EDTA or without
the primary antibody in the case of the MB1.2 labeling. AI=[(MFI Fn7-10)-(MFI
Fn7-10+EDTA)]/(MFI MB1.2)-(MFI MB1.2 control).
RNA and protein analyses
Total RNA was isolated from adult kidney using TRIzol reagent (Gibco BRL)
according to the manufacturer's recommendations. For northern analysis, 10
µg of total RNA was separated on a 1.2% agarose-2.2 M formaldehyde gel,
transferred to Hybond+ membrane (Amersham) and probed with a
32P-labeled Icap-1 cDNA.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corresponding
to the full-length protein (200 amino acids) and ICAP-1ß representing a
shorter, 150-amino acid protein. The short isoform results from alternative
splicing of exon 6, which contains the ß1 integrin-binding
site (Chang et al., 1997). In
mice, we were unable to detect the short isoform using RT-PCR amplification of
RNA isolated form different adult tissues (data not shown). Furthermore,
searches of EST and UCSC genome annotated databases also did not provide
evidence of a mouse Icap-1ß. By computer BLAST search, the gene
encoding ICAP-1 was localized on mouse chromosome 12 and on human chromosome
2. To study the in vivo function of ICAP-1, we generated an Icap-1-null allele by homologous recombination. The targeting strategy made use of a lacZ gene inserted in frame with the endogenous ATG and deleted exons 2 and 3 preventing the expression of a functional ICAP-1 protein (Fig. 1). Three correctly targeted embryonic stem (ES) cell clones were used to generate germline chimeric males. The null mutation was confirmed by Southern, northern and western blot analyses (Fig. 1B-D). Neither the Icap-1 mRNA nor the ICAP-1 protein were detected in tissues derived from homozygous mutant (Icap-1-/-) mice.
To determine the expression pattern of ICAP-1, heterozygous animals
(Icap-1+/-) were collected at various embryonic and adult
stages and subjected to ß-galactosidase (ß-gal) histochemistry
(Fig. 1E,F). At embryonic day
8.5 (E8.5), whole-mount staining demonstrated a faint ß-gal activity in
the developing heart and facial mesenchyme
(Fig. 1E). At later stages,
ß-gal activity became gradually visible all over the embryo with the
exception of the liver (Fig.
1F). On tissue sections, only a moderate ß-gal activity was
observed in liver, spleen, thymus and intestinal epithelial cells, whereas
other tissues expressed high ß-gal levels (data not shown). These results
are in agreement with previously published expression data
(Faisst and Gruss, 1998).
Icap-1+/- mice appeared normal. Southern blot genotyping of newborn mice from heterozygous intercrosses resulted in a normal number of homozygous mutants, suggesting that ICAP-1 has no rate-limiting function until birth. However, when the mendelian ratio of 4-week old litters from Icap-1+/- x Icap-1+/- and Icap-1+/- x Icap-1-/- intercrosses was evaluated, 20% of Icap-1-/- mice were missing (Table 1). The reason for this perinatal lethality is unknown.
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Delayed ossification in Icap-1-/- embryos
To analyze cartilaginous and bony tissues, the skeletons of homozygous
mutant and wild-type animals were stained at various developmental stages with
Alcian Blue (which stains cartilage) and Alizarin Red (which stains calcified
tissue). The Alcian Blue staining was indistinguishable between mutant and
control embryos throughout development, suggesting that cartilage formation is
not grossly affected in Icap-1-deficient mice
(Fig. 2E,F). The Alizarin Red
staining, however, was reduced in the skeleton of
Icap-1-/- mice as early as E14.5, indicating a defect in
ossification. The reduced Alizarin Red staining was most pronounced in the
parietal and frontal bones of the calvaria and in the maxillary and mandibular
components of the facial skeleton (not shown). At E16.5, the reduction in
Alizarin Red staining became even more prominent in the skull and in the bony
collar surrounding the long bones of the appendicular skeleton
(Fig. 2E).
At the newborn stage, Alizarin Red staining of long bones was similar between control and Icap-1-/- animals (Fig. 2F), suggesting that the ossification of the collar started late in Icap-1-/- mice, but was catching up at later stages. In the skull region, the postnatal development of the chondrocranium (the majority of the base of the skull) was normal in Icap-1-/- mice. This was shown by the normal ossification of the exoccipital, basioccipital, basisphenoid and presphenoid bones (Fig. 3A) and the normal formation and development of the synchondrosis of the skull base in newborn, P15, P21 and P60 control Icap-1-/- animals (Fig. 3A and data not shown). The ossification defect of calvarial bones, however, was obvious in newborn Icap-1-/- mice (Fig. 3B). The frontal, parietal and interparietal (supraoccipital) bones were reduced in size, giving rise to enlarged anterior and posterior fontanelles and to widened sagittal and metopic (interfrontal) sutures (Fig. 3B). At the age of 2 months, control mice had completed the ossification of the metopic sutures but still had patent lambdoid, sagittal and coronal sutures (Fig. 3C,F). In mutant mice of the same age, the posterior part of the metopic suture was still unossified (open) (Fig. 3D,E,G,H). In some Icap-1-/- mice, non-ossified, Alcian Blue-positive areas were observed extending from the posterior metopic suture to the frontal bones (Fig. 3E,H). Furthermore, the parietal bones were hypoplastic in mutant calvarias leading to shortened sagittal and V-shaped coronal sutures. The bone defect was also evident in other parts of the skeleton, such as in the vertebrae, where 15-day-old mutant mice showed non-fused vertebral arches (Fig. 3I) and in the pelvic bone, where mutant mice showed delayed fusion of the pubis and the ischial bone (Fig. 3J).
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Since the expression patterns of FGF-receptors correlate with the
osteogenic differentiation process (Iseki
et al., 1999; Rice et al.,
2000), we compared the expression of FGFR1 and FGFR3 in wild-type
and mutant newborn calvariae. In control tissue, both proteins were detected
in the osteoblasts of the calvarial bones and in cells of the osteogenic front
and weakly in the sutural mesenchyme (Fig.
6F,G). In Icap-1-/- calvariae, FGFR1 and FGFR3
immunolabeling was fainter than in wild type, and this difference was
particularly pronounced at the osteogenic fronts
(Fig. 6F,G). In addition,
whole-mount in situ hybridization on E17.5 heads revealed that the expression
of both the early bone marker Runx2 (Cbfa1) and the later
marker bone sialic protein (Bsp; Ibsp - Mouse Genome
Informatics) were reduced in mutant mice
(Fig. 6H,I). Again, a weaker
signal was observed at the edge of the bony region, reflecting the marked
reduction of cells committed into the osteoblastic lineage within the
osteogenic front. Altogether, these results indicate that the osteogenic front
is not normally formed in mutant animals.
To test whether committed cells differentiate normally, primary osteoblasts were isolated from newborn wild-type and Icap-1-/- calvariae and incubated in medium supplemented with ascorbic acid and ß-glycerophosphate to induce osteoblast differentiation and the formation of mineralized bone nodules (Fig. 7A). After 2 weeks in the differentiation medium, AP activity was evident in almost all cells derived from control calvariae indicating their commitment to the osteoblast lineage. Icap-1-/- calvarial cells also started to express AP, albeit at lower levels (data not shown). After 4 weeks of culture, differentiating osteoblasts from Icap-1-/- calvariae contained fewer and smaller mineralized nodules as visualized by Alizarin Red (Fig. 7A) and von Kossa staining (data not shown). Similarly, immortalized Icap-1-/- cells (SV2.1-Icap-1-/-) showed markedly reduced AP staining and mineralized nodule formation relative to wild-type (SV6.5-Icap-1+/+) and rescued cells (SV2.1-Icap-1resc) (Fig. 7B and data not shown). These data show that ICAP-1 loss impairs osteogenesis and identifies a role of ICAP-1 as a cell-autonomous factor in osteoblast differentiation.
Since we routinely induced differentiation after cells derived from calvariae or immortalized osteoprogenitors had reached confluence in vitro, the numbers of neither the mutant (SV2.1-Icap-1-/-) nor the wild-type (SV6.5-Icap-1+/+) or rescued cells (SV2.1-Icap-1resc) significantly increased during the differentiation period (data not shown). Altogether, these findings suggest that the differentiation block occurs in addition to the cell proliferation defect.
ß1 integrin is highly expressed and activated in osteogenic front
ß1 integrins have been proposed to play a crucial role
during osteoblast proliferation and differentiation
(Moursi et al., 1997;
Zimmerman et al., 2000). Since
ICAP-1 interacts with the cytoplasmic tail of the ß1 integrin
chain and modulates integrin function in vitro
(Bouvard et al., 2003), we
analyzed ß1 integrin expression in wild-type and mutant
calvariae in vivo. At newborn stage, frontal sections through the sagittal
suture and the parietal bones were immunostained with an
anti-ß1 polyclonal antibody and with the monoclonal antibody
9EG7, which recognizes the ligand-bound form of ß1 integrins
(Fig. 8A and data not shown).
In sections from control mice, both antibodies strongly labeled the osteogenic
fronts and the bone surfaces, whereas the intervening mesenchyme was faintly
labeled. Conversely, sections from Icap-1-deficient mice showed clear
ß1 integrin staining of the bone surface but very faint
staining of cells at the osteogenic front. This apparently diminished
ß1 staining was mainly due to the reduced osteogenic front
population, rather than reduced expression on the cell surface of individual
cells, because FACS analysis showed only a slight reduction of
ß1 integrin expression on Icap-1-/-
osteoblasts (Fig. 8B, see
below).
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To investigate whether the loss of ICAP-1 expression interferes with
integrin activation, we estimated the ligand-binding affinity of the FN
receptor 5ß1 integrin both in wild-type and
Icap-1-/- primary osteoblasts. The cell-binding domain of
FN, corresponding to the type III repeats 7-10 (Fn7-10), was expressed,
purified and FITC-labeled. The capability of both mutant and wild-type primary
osteoblast cells to interact with Fn7-10 at a non-saturating concentration was
analyzed by FACS. As shown in Fig.
8D, we consistently observed an increase in Fn7-10 binding to
Icap-1-/- osteoblasts compared with wild-type cells. The
activation index, which normalizes the specific binding of FITC-Fn7-10 to the
total ß1 surface expression level, was elevated by
approximately three-fold in Icap-1-/- as compared with
wild-type cells (Fig. 8E).
During in vitro differentiation of both primary and immortalized
osteoblasts, we consistently observed that the bone nodules formed by
Icap-1-/- cells were fewer, smaller and less compact than
those formed by wild-type cells (Fig.
9A). As differentiation of bone cells requires an initial step of
cell condensation (Globus et al.,
1998; Ornitz and Marie,
2002), a defect in this step might lead to altered or delayed
differentiation. To address this question, we cultured immortalized
osteoprogenitors in suspension using the hanging drop technique. Under these
conditions, wild-type or rescued cells aggregated and formed compact spheroids
within 48 hours, whereas spheroids formed by Icap-1-/-
cells were less compacted (Fig.
9B and data not shown).
Since ICAP-1 loss increases integrin affinity (Fig. 8), we investigated whether blocking of ß1 integrins in their activated state would mimic ICAP-1 deficiency. To this end, we complemented the culture medium with the integrin-activating monoclonal antibody 9EG7 and then formed spheroids (Fig. 9B). Whereas spheroidtriggered compaction of Icap-1-/- cells was not affected by treatment with the 9EG7 antibody, compaction of control or rescued cells was consistently delayed. These findings indicate that modulation of integrin affinity by ICAP-1 is required for proper compaction of osteoblastic cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
first mechanism is responsible for the generation of the flat bones of the
skull vault as well as for the formation of the bony collar around the
diaphysis of the long bones. In Icap-1-/- mice,
intramembranous ossification is affected, as both periosteal and calvarial
osteogenesis are delayed. Alcian Blue staining revealed that the formation of
cartilaginous templates of endochondral bones is grossly undisturbed in mutant
mice. Since ICAP-1 is also expressed in chondrocytes (data not shown), we
cannot fully exclude mild differentiation and/or proliferation defects of
mutant chondrocytes, which might also contribute to the small stature of
Icap-1-/- mice. We focused our investigations on
intramembranous ossification and show for the first time the important role of
ICAP-1 during osteoblast differentiation. The ossification of calvarial bones
(frontal, parietal and interfrontal) was dramatically delayed during embryonic
development, leading to open fontanels and wide sutures at birth. These
symptoms mirror the calvarial abnormalities of mice carrying mutations for the
transcription factors Runx2 (Otto
et al., 1997) and Atf4
(Yang et al., 2004), which
have been identified as key regulators of osteoblast differentiation. The
remarkable phenotypic similarities between the Icap-1-/-
mice and these mouse models suggest that ICAP-1 is a newly recognized and
important regulator of osteoblast development. Cranial sutures regulate bone
expansion during postnatal life and remain non-ossified as long as the brain
is growing (Opperman, 2000).
Osteogenesis at the suture involves the differentiation of mesenchymal cells
into preosteoblasts and into osteoblasts at the sutural margins and the
subsequent deposition of a collagenous matrix along the bony plates. Increased
growth of the calvarial bones leads to premature closure of the sutures
(craniosynostoses), whereas delayed bone growth results in suture latency.
ICAP-1 is expressed at the osteogenic fronts and its deficiency leads to a
dramatic reduction in the osteoblast population at the osteogenic fronts,
which in turn prevents correct temporal fusion of cranial sutures. The reduced
osteoblast number at the osteogenic fronts is due to reduced proliferation and
impaired osteoblast differentiation.
ICAP-1 regulates osteoblast proliferation
We have previously reported that ICAP-1 has a dual localization and is
found in the cytoplasm/membrane and in the nucleus
(Fournier et al., 2005). Loss
of ICAP-1 expression in immortalized osteoblasts considerably reduced cell
proliferation and cyclin D1 expression. Therefore, the reduced proliferation
in the osteogenic front is likely to result from a lack of ICAP-1 in the
nucleus, which would lead to reduced cyclin D1 expression. This might explain
the severe decrease in the cell population committed to the osteoblast lineage
in the sutural region that we observed in the Icap-1-deficient
animals.
ICAP-1 regulates integrin activity
Isolated Icap-1-null primary osteoblasts displayed an increased
cell adhesion to FN and COL1. We have previously shown that overexpression of
ICAP-1 in HeLa cells disrupts focal adhesion, probably by inhibiting the
association of the cytoplasmic tail of ß1 integrin with talin
(Bouvard et al., 2003). It has
been shown that talin binding to the ß integrin domain is a key step in
the regulation of integrin activation
(Calderwood et al., 2004;
Calderwood et al., 2002). In
good agreement with this, we found an increase in Fn7-10 binding to integrin
in mutant cells that was not accompanied by any upregulation of
ß1 integrin expression. This confirms that ICAP-1 regulates
ß1 integrin affinity.
ICAP-1 regulates osteoblast differentiation
Osteoblast differentiation is a multistep process that first required an
initial condensation of the mesenchymal cells to form the osteogenic front
(Fig. 10)
(Hall and Miyake, 2000). The
cell population at the osteogenic front is visible during calvaria bone
development at the edge of the expanding bone within the intervening
mesenchyme. This condensed cell population further differentiates and
expresses different osteoblastic markers including RUNX2, AP and BSP. Our
knowledge of how integrins or cell adhesion is implicated into this process is
poorly documented, but recent reports suggested an unexpected role for matrix
stiffness in controlling early osteoblast differentiation
(Engler et al., 2006;
McBeath et al., 2004).
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2ß1 integrin regulates
osteoblast-specific gene expression and osteoblast differentiation
(Takeuchi et al., 1996;
Xiao et al., 1998). Similarly,
5ß1 integrin interaction with FN promotes
differentiation (Moursi et al.,
1997) and survival (Globus et
al., 1998) of cultured calvarial osteoblasts. Finally, transgenic
overexpression of a dominant-negative form of ß1 integrin in
osteocytes blocks ß1 integrin function, reduces adhesion to
collagen and FN, and diminishes bone mass in cortical and flat bones
(Zimmerman et al., 2000).
Although these studies identify an important role for ß1
integrins for osteogenesis, they do not address the role of integrin affinity
modulation in bone development. Our data demonstrate for the first time that
not only ß1 integrin affinity increases, but also affinity
decreases, are of paramount importance for proper bone development both in
vivo and in vitro. We consistently observed a marked reduction of osteoblast cells in Icap-1-deficient animals. Even the expression of the very early marker RUNX2 at the osteogenic front region was significantly reduced. Furthermore, the in vitro differentiation assays revealed that the bone nodules formed by Icap-1-null immortalized osteoprogenitors were smaller and less compacted as compared with nodules formed from wild-type or rescued cells. This reduced compaction of Icap-1-deficient cells was further confirmed by the hanging drop technique, suggesting that ICAP-1 is required for osteoblast condensation, which is a crucial and early step during osteoblast differentiation.
Our data suggest that the proliferation and the differentiation defects
occur independently and that they both contribute to the abnormal
osteogenesis. In support for this notion, we observed that limited ICAP-1
expression in Icap-1-deficient preosteoblast cell lines fully
restored their proliferation rate, but only partially their potential to
differentiate and form nodules in vitro. Furthermore, in our differentiation
assay, we routinely use confluent cells to rule out the possibility that the
proliferation defect is influencing the formation of mineralized nodules.
Indeed, we observed only a slight increase in cells during the differentiation
period and this increase was similar in control and Icap-1-deficient
cultures. Our data indicate that the condensation defect of the
Icap-1-deficient preosteoblasts further limits the number of
progenitors that will finally differentiate into mature osteoblasts
(Fig. 9). The functional
importance of the condensation of progenitors for osteoblast development has
also been observed in connexin 43 (GJA1)-deficient mice
(Lecanda et al., 2000).
Previous work implicated ß1 integrins in cell compaction
(Robinson et al., 2004;
Robinson et al., 2003). Our
data suggest that ICAP-1 might control ß1 integrin function
during this process by regulating activation/deactivation cycles. To our
knowledge this is the first direct evidence reporting that integrin affinity
is important for cell cohesion and differentiation.
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ACKNOWLEDGMENTS |
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