1 Departamento de Biología Celular y Fisiología, Instituto de
Investigaciones Biomédicas, Universidad Nacional Autónoma de
México, Mexico City, Mexico
2 Departamento de Inmunología, Instituto de Investigaciones
Biomédicas, Universidad Nacional Autónoma de México,
Mexico City, Mexico
3 Genetics and Cell Biology Section, National Cancer Center Research Institute,
Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
* Author for correspondence (e-mail: jchimal{at}servidor.unam.mx)
Accepted 9 July 2004
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SUMMARY |
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Key words: Joint formation, Integrin, Hedgehog, BMP, Wnt14, Chondrogenesis, Chondrocyte differentiation, Mouse
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Introduction |
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The important structural function of the extracellular matrix (ECM) in the
mature cartilage suggests that the components of the ECM and integrins
(receptors for ECM proteins) (Bokel and
Brown, 2002; Hynes,
2002
) exert a major role in cartilage differentiation. However,
experimental evidence supporting this idea is scarce. Alterations in ECM
components such as collagens (Li et al.,
1995
; Barbieri et al.,
2003
), link protein (Wai et
al., 1998
) or aggrecan
(Watanabe and Yamada, 1999
)
are responsible for some structural changes in cartilage. Also chondrocyte
differentiation occurs in the absence of ß1 integrins, but these cells
are unable to form columns in growth plates. These chondrocytes show reduction
of proliferation (Aszodi et al.,
2003
; Fässler and Meyer,
1995
). In addition, mutant mice lacking
1,
2,
6,
v, ß3, or ß5 integrins do not show skeletal
malformations (Bouvard et al.,
2001
), suggesting that other integrins control skeletal
development or that functional redundancy may occur.
Since the role of integrins on skeletal development is poorly understood,
and much less for joint formation, we investigated the effects of
5ß1 integrin inhibitors and BMP on joint formation in the
embryonic mouse limb culture system. Blocking ß1 and
5 integrins
with specific antibodies or RGD peptides (arginine-glycine-aspartatic acid) in
the developing mouse zeugopod led to inhibition of pre-hypertrophic
chondrocyte differentiation and ectopic joint formation between proliferating
chondrocytes and hypertrophic chondrocytes. Moreover, ectopic joint resulted
in expression of Wnt14, Gdf5, chordin, autotaxin and type I collagen
and CD44, while expression of IHH and type II collagen was downregulated.
Also, misexpression of
5ß1 integrin in chick embryos resulted in
joint fusion and formation of pre-hypertrophic chondrocytes. In addition, we
investigated the effects of BMPs on ectopic joint formation induced by the
inhibition of
5ß1 integrin. This is the first report on the
involvement of
5ß1 integrin in the control point by which
chondrocytes take the decision between the cartilage differentiation program
and the joint formation program.
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Materials and methods |
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Microinjections and mouse limb organ cultures
Complete embryos at embryonic day (E) 14.5 were injected at the wrist with
2 µl of antibody solution or just with PBS using a pneumatic microinjector
model PV830 (World Precision Instruments) equipped with glass microneedles.
Then embryonic forelimb organ cultures were prepared as described by Vortkamp
et al. (1996) and Lanske et
al. (1996
). Briefly, limbs
were placed on a Nucleopore filter. Filter and limb were then placed on top of
a stainless steel grid (Plain weave mesh 0.38 mm; Goodfellow) inside a well of
a 24-well tissue culture plate containing BGJb medium with 1% bovine serum
albumin. The grid keeps the limb at the liquid/air interface. Limb cultures
were then kept for 4 days at 37°C in a 5% CO2 incubator. For
some experiments, cultures were supplemented with 1 µg/ml BMP7 or 400 ng/ml
SHH.
Skeletal preparation and tissue staining
Cultured limbs were fixed overnight in 96% ethanol and stained as
previously described (Otto et al.,
1997).
For tissue staining, limbs were fixed in 4% paraformaldehyde at 4°C overnight, dehydrated and embedded in Paraplast by standard techniques. Tissue sections were then stained with Weigert's acid iron chloride haematoxilin and then stained with Fast Green. Subsequently, sections were stained with Safranin O.
cDNA probes and in situ hybridization
For in-situ hybridizations the following cDNA probes were used: mouse
Gdf5 (Storm and Kingsley,
1999); Wnt14 (Katoh,
2002
); chordin (Bachiller et
al., 2003
). For chicken embryos Wnt14, Gdf5 and
Ihh cDNA probes were used. Fragments of autotaxin (accession number
NM015744), Cd44 (accession number XM283773) and aggrecan
(accession number L07049) mouse genes were obtained by RT-PCR. First-strand
cDNA was synthesized with a First-strand cDNA Kit (Roche Applied Science) and
1 µg of RNA of mouse embryo at E12. The following primers (5' to
3') were used: autotaxin 5' primer,
5'-CAGCAAGTCGAATTAAGAGG-3' and 3' primer
5'-GGCCAGCGTATACAGATTAG-3' (corresponding to region 127-692);
Cd44 5' primer 5'-AAGACTTGAACAGGACAGGA-3' and
3' primer 5'-AGAGATGCCAAGATGATGAG-3' (corresponding to
region 1747-2248); aggrecan 5' primer
5'-GAGGAGCCATACACATCTTC-3' and 3' primer
5'-CACTGAGGTCCTCTACTCCA-3' (corresponding to region 2506-3040).
PCRs were performed in a total volume of 25 µl using Taq DNA polymerase
(Invitrogen). The cycling conditions were 15 seconds at 94°C for
denaturation, 30 seconds at 55°C for annealing, 1 minute at 72°C for
elongation, and then 30 minutes at 72°C after the last cycle (35 cycles).
The PCR products were cloned into pGEM T-easy (Promega). The authenticity of
the fragments was confirmed by dideoxy sequencing.
Digoxigenin 11 UTP-labeled single-stranded RNA probes were prepared using a DIG RNA labeling kit (Roche Applied Science) according to the manufacturer's instructions. Tissue sections on slides were treated with 1 µg/ml proteinase K (preincubated at 37°C) for 5 minutes at room temperature, then washed with PBT and incubated with hybridization buffer at 65°C for 15 minutes, after which the corresponding Digoxigenin-labeled probe was added and incubated overnight at 65°C. Next morning, sections were washed and incubated with FITC-labeled anti-digoxigenin antibodies at 4°C overnight. Next day, sections were washed and observed under a Nikon Fluorescence Microscope Eclipse E600, and photographed with a Nikon digital camera Coolpix 995 (Nikon Inc).
Immunofluorescence and TUNEL
For immunohistochemical analysis, slides were incubated overnight with
antibodies at 4°C. Next morning, slides were incubated with the
corresponding FITC-labeled secondary antibody for 2 hours at 37°C and
observed under a Nikon Fluorescence Microscope Eclipse E600, and photographed
with a digital camera Nikon Coolpix 995 (Nikon Inc.). Apoptosis was detected
by the TUNEL assay using an in-situ cell death detection Kit, TMR (Roche
Applied Science) according to the manufacturer's instructions.
Electroporation of 5ß1 integrin cDNA into chick embryos.
Fertilized White Leghorn chicken eggs (ALPES, Puebla Mexico) were windowed
and staged according to Hamburger and Hamilton
(1951). Non-linearized
full-length human
5 chain of integrins cDNA clone (6 µg/µl;
pECE+
5) and non-linearized full-length human ß1 chain of integrins
cDNA clone (6 µg/µl; pECE+ß) were injected into the autopod of the
hindlimb at HH stage 27 (Hamburger and
Hamilton, 1951
) with a microneedle. DNA was mixed with Chinese ink
and mineral oil (1.5 µl of pECE+
5 plus 1.5 µl of pECE+ß1
plus 0.5 µl of Chinese ink and 0.5 µl of mineral oil) and approximately
0.5-1.0 µl of mix were microinjected. Electroporation was performed at 28
volts with 10 pulses of 70 mseconds. After electroporation, chick embryos were
returned to the incubator and analyzed by skeletal staining, histology,
immunofluorescence and in-situ hybridization.
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Results |
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Inhibition of 5ß1 integrins induces molecular changes characteristic of joint formation
Because one of the main integrins that bind RGD ligands is 5ß1,
we explore whether inhibition of integrin function by blocking the
5
subunit would have the same effect as the anti-ß1 antibodies or the RGD
peptide. Microinjection of the anti-
5 mAb HM
5 induced the
formation of an ectopic joint (Fig.
2A). Another anti-mAb, MFR5, also gave origin to an ectopic joint
(data not shown). Together, these data support the idea that
5ß1
integrin binds to an RGD-contained ligand in the ECM of a developing limb in
order to prevent the appearance of joints.
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5ß1 integrin is downregulated during joint formation
Because our data suggested that 5ß1 integrin ligation to an RGD
ligand in the developing limb contributed to prevent the appearance of a new
joint, we explored the pattern of expression of
5ß1 during joint
formation. The whole autopod of a mouse embryo at E14.5 was stained for the
5 integrin subunit. The
5 integrin was found in the all-forming
digits of the hand (Fig. 3A).
At the zone where the joints of the fingers are being formed,
5
integrin expression was not observed (Fig.
3A). Closer examination of the interzone confirmed that
5
integrin was not present in the forming joint
(Fig. 3B). In addition,
Wnt14 was expressed in the interzone where
5 integrin was not
expressed (Fig. 3C). In the
more advanced skeletal elements such the ulna,
5 integrin expression
was evident in pre-hypertrophic chondrocytes and colocalized with IHH but not
with type X collagen (Fig.
3D-F). In addition,
5 integrin was seen in less intensity
in proliferating chondrocytes, and was evident in perichondrium and joints
formed (Fig. 3D-F).
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5ß1 integrin induces joint fusion and differentiation of pre-hypertrophic cells
Our results showed that inhibition of integrin 5ß1 induces
ectopic joints, so we evaluated whether misexpression of human
5ß1
integrin inhibits joint formation. Full-length cDNA clones for human integrin
5 and ß1 chain were electroporated into the autopod of embryonic
chick legs at HH stage 27. After incubating chick embryos for 5 days, we found
that misexpression of
5ß1 integrin inhibited joint formation
(Fig. 5A-B). Histological
analysis of fingers confirmed continuity in the cartilage tissue between the
phalanges (Fig. 5C-D). To
determine that misexpression of
5ß1 integrin occurred in the chick
leg, we evaluated it by immunofluorescence with a monoclonal antibody specific
for human
5ß1 integrin, which does not crossreact with chick. The
results showed expression of human
5ß1 integrin
(Fig. 5E-F). Since ectopic
joint formation by inhibition of
5ß1 integrin coincides with
inhibition of pre-hypertrophic chondrocytes, we evaluated whether
misexpression of
5ß1 integrin up-regulates IHH, molecular marker
of pre-hypertrophic chondrocytes. Results showed that expression of human
5ß1 integrin in the fingers colocalized with IHH
(Fig. 5E-H). Wnt14 was
not expressed under these conditions (Fig.
5I-J). In conclusion, misexpression of
5ß1 integrin
induces joint fusion and this phenotype correlates with differentiation of
pre-hypertrophic chondrocytes.
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Discussion |
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A remarkable finding of our study, of interest to the understanding of the
joint formation mechanisms, is the structural differences between normal and
ectopic joints induced by integrin blockage. Normal joints are flanked by
growth plates with proliferating chondrocytes closest to the articular ends,
and hypertrophic chondrocytes further from the joints. By contrast, ectopic
joints are induced between proliferating chondrocytes and hypertrophic
chondrocytes. Possibly, when the cartilage condensations, which are entirely
made up of proliferating chondrocytes, reach a particular size, for a reason
that is still totally unknown, the cells in the center of the element exit the
cell cycle (Karaplis et al.,
1994; Schipani et al.,
1997
; Rossi et al.,
2002
). When they do, there are two alternative choices they can
make. When the skeletal pattern is first being established, the condensations
that reach this critical size initiate joint formation in their center. Later,
once the skeletal pattern is established, when the skeletal elements again
reach a critical size, the cells in the center, instead of becoming pre-joint
cells, adopt the alternative fate of becoming pre-hypertrophic cells, leading
down the path to hypertrophy and eventual ossification. So, the decision of
which cell fate to adopt when exiting the cell cycle, pre-joint or
pre-hypertrophic, could be explained on the basis of the presence or absence
of
5ß1 integrin in the perichondrial cells and/or postmitotic
chondrocytes.
Findings of this study suggest that the perichondrium may also exert an
important role in joint formation. Supporting this interpretation, Aszodi et
al. (Aszodi et al., 2003) have
observed that joints develop normally in ß1 integrin-deficient mice under
the control of the type II collagen promoter. It must be taken into account
that type II collagen is expressed in all chondrocytes but not in
perichondrium. So, the strong expression of
5ß1 integrin that we
observed in the perichondrium of skeletal elements beginning pre-hypertrophic
differentiation is not affected by the absence of ß1 integrin in type II
collagen-expressing cells in ß1 integrin-deficient mice. On this basis,
the effect of
5ß1 integrin on ectopic joint formation and
inhibition of pre-hypertrophic cells might be mediated by the perichondrium.
Moreover, rescue experiments of this study with SHH protein revealed that
hedgehog signaling inhibits ectopic joint formation. Since the perichondrium
expressed Patched, which is considered a major target of IHH
signaling, it is likely that the effect of ectopic SHH was mediated by the
perichondrium. Also, a direct effect of SHH on chondrocytes may explain
inhibition of the ectopic joint (Long et
al., 2001
).
In accordance with the above-described hypothesis, the ectopic joint
induced by blocking 5ß1 integrin displayed the morphology of a gap
perpendicular to the long axis of the bone. This joint pattern differs from
that observed by Hartmann and Tabin
(2001
) in experiments
misexpressing Wnt14. The joint induced by misexpression of
Wnt14 is extended through the whole cartilage rudiment and is most
probably due to instructive signals delivered by Wnt14 that induce
downstream genes of the joint pathway in all cartilage cells misexpressing
this gene. It is well established that the same integrin is capable of
inducing very different cell responses depending on the cell type that
expresses them. For example,
5ß1 integrin binding to fibronectin
in fibroblasts leads to cell survival
(Aplin et al., 1999
); in
mammary cells, it leads to milk production
(Schmidhauser et al., 1990
);
and in leukocytes, it leads to endothelial cell migration or cytokine
production (Rosales and Juliano,
1995
). Thus, although the pattern of
5ß1 integrin
expression in the cartilage of the developing limb is rather dispersed, we do
not necessarily expect as diffuse an effect as the one described after
Wnt14 misexpression. The presence of
5ß1 integrin in the
perichondrium might influence the decision of which cell fate is adopted,
either pre-joint or pre-hypertrophic, by proliferating chondrocytes when they
exit the cell cycle in a local horizontal population of cells differentiating
in concert, since blocking integrins produces joints perpendicular to the long
axis of the skeletal element. In this sense, integrins modulate the response
to other signals as suggested by Aplin et al.
(Aplin et al., 1998
).
An additional finding of our study is that the response of chondrocytes to
BMP signaling is regulated by integrin. In normal developing limbs, BMP
treatments cause intense chondrogenesis and inhibition of joint formation
(Duprez et al., 1996;
Macias et al., 1997
;
Brunet et al., 1998
;
Merino et al., 1999
). By
contrast, in our experiments, addition of BMP to the culture medium inhibited
differentiation of pre-hypertrophic cells and enhanced formation of ectopic
joints. This finding is in agreement with the normal expression of several
bmp genes in the developing joints
(Macias et al., 1997
), as it
would be difficult to explain why bmp genes are expressed in the
joints if they block joint formation
(Duprez et al., 1996
;
Brunet et al., 1998
;
Merino et al., 1999
;
Storm and Kingsley, 1999
).
According to our results, in the absence of
5ß1 integrin BMP
signaling can direct proliferating chondrocytes to the joint program. On the
contrary, in the presence of
5ß1 integrin, BMP signaling directs
proliferating chondrocytes to the pre-hypertrophic program expressing
Ihh (Macias et al.,
1997
). Whether this modulation of BMP response is mediated through
the perichondrium, or whether it is a direct effect of BMPs on the
chondrocytes, remains to be clarified.
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ACKNOWLEDGMENTS |
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