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First published online 18 January 2006
doi: 10.1242/dev.02258
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1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, 240 Longwood Avenue, Boston, MA 02115, USA.
2 Endocrine Unit, Massachusetts General Hospital-Harvard Medical School, 50
Blossom Street, Boston, MA 02114, USA.
Author for correspondence (e-mail:
andrew_lassar{at}hms.harvard.edu)
Accepted 6 December 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Chondrogenesis, Runx2, Nkx3.2, Bapx1, PTHrP
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kronenberg,
2003). During this process, chondrocytes differentiate within an
aggregated mesenchyme, generating distinct cartilage primordia that begin
longitudinal growth. Cells lying within the central regions of the cartilage
undergo a further maturation process, withdrawing from the cell cycle,
enlarging in size and initiating synthesis of a new extracellular matrix
containing collagen X (Erlebacher et al.,
1995; Kronenberg,
2003). The resulting cells are termed hypertrophic chondrocytes,
which eventually die off to be replaced by bone and bone marrow. The
production and maturation of chondrocytes is eventually restricted to the
epiphysis of the bone in a structure termed the growth plate. The rate of
chondrocyte maturation in the growth plate is tightly regulated by a
PTHrP/Indian hedgehog (Ihh) signaling loop. PTHrP is normally produced by the
most distal perichondrium, at the articular surface, and its receptor is
synthesized in the proliferative and prehypertrophic zones of the growth plate
(Lanske et al., 1996;
Vortkamp et al., 1996).
Ectopic expression of either PTHrP or an activated form of the Parathyroid
Hormone (PTH)/PTHrP receptor results in a dramatic blockade of cartilage
maturation (Schipani et al.,
1997; Weir et al.,
1996). Conversely, mice lacking either PTHrP or its receptor,
exhibit dwarfism of their long bones due to premature chondrocyte maturation
(Karaplis et al., 1994;
Lanske et al., 1996). PTHrP
expression in turn is dependent upon the expression of Ihh, which is
transiently produced by chondrocytes as they initiate the maturation process
(Bitgood and McMahon, 1995;
St-Jacques et al., 1999;
Vortkamp et al., 1996).
The early steps of chondrogenesis, including mesenchymal condensation and
expression of chondrocyte-specific extracellular matrix proteins is crucially
dependent upon Sox-family transcription factors, including Sox9, Sox5 and Sox6
(de Crombrugghe et al., 2001;
Lefebvre, 2002). By contrast,
the latter steps of chondrogenesis appear to be regulated by Runx family
transcription factors. Runx2 is expressed in chondrocytes as they initiate
chondrocyte hypertrophy, and loss of this factor in genetically engineered
mice severely delays chondrocyte maturation in a number of developing bones
(Inada et al., 1999;
Kim et al., 1999).
Furthermore, forced expression of Runx2 in immature chondrocytes drives
premature maturation of chondrocytes by inducing expression of collagen X (col
X) and other hypertrophic markers (Enomoto
et al., 2000; Stricker et al.,
2002; Takeda et al.,
2001; Ueta et al.,
2001). It has recently been demonstrated that Runx3, which is also
expressed in maturing chondrocytes, works in combination with Runx2 to promote
chondrocyte maturation (Yoshida et al.,
2004), and that HDAC4 restricts ectopic and early onset
chondrocyte hypertrophy, and associates with Runx2
(Vega et al., 2004).
In the current study, we explore the role of the transcriptional repressor
Nkx3.2/Bapx1 in modulating the rate of chondrocyte maturation. Mice lacking
the Nkx3.2/Bapx1 gene exhibit major defects in the axial skeleton,
characterized by a lack of ventromedial elements in the vertebral column, and
by hypoplasia of the neural arches (Akazawa
et al., 2000; Lettice et al.,
2001; Lettice et al.,
1999; Tribioli and Lufkin,
1999). Consistent with this phenotype, we have found that signals
that induce somitic chondrogenesis, including sequential Shh and BMP signals,
induce the expression of Nkx3.2, and that forced expression of Nkx3.2 can
activate chondrogenesis in somites (Kim et
al., 2003; Murtaugh et al.,
2001; Zeng et al.,
2002). Nkx3.2 induces somitic chondrogenesis by acting as a
transcriptional repressor (Murtaugh et
al., 2001; Zeng et al.,
2002). In the course of characterizing the role of Nkx3.2 in
endochondral bone development, we observed that Nkx3.2 expression in the limb
skeleton was restricted to proliferative immature chondrocytes, and that its
expression in this region of the growth plate was dependent upon PTHrP
signals. On the basis of these results, we hypothesized that Nkx3.2/Bapx1
might negatively regulate chondrocyte maturation. However, analysis of the
long bones of mice lacking Nkx3.2/Bapx1 revealed that the limb bones
of these mice display relatively normal formation of hypertrophic chondrocytes
and bony tissue by Hematoxylin/Eosin staining (data not shown; S.P., A.B.L.
and Hans-Henning Arnold, unpublished)
(Akazawa et al., 2000;
Lettice et al., 2001;
Lettice et al., 1999;
Tribioli and Lufkin, 1999). To
explore the possibility that Nkx3.2/Bapx1 might negatively regulate
chondrocyte maturation, but that this role may not have been revealed in
Nkx3.2/Bapx1 deficient mice due to redundant/compensatory mechanisms that may
modulate chondrocyte maturation in the growth plate, we have employed a
gain-of-function approach in chick embryos to elucidate a potential role for
Nkx3.2 in the regulation of chondrocyte maturation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zeng et al., 2002), and
RCAS(A) retroviruses encoding deletion mutants of Nkx3.2 have been described
elsewhere (Kim et al., 2003;
Murtaugh et al., 2001).
RCAS(A)-Runx2 and RCAS(B)-Runx2 viruses, encoding mouse Runx2, were supplied
by Masahiro Iwamoto (Thomas Jefferson University, Philadelphia, USA), and have
been described previously (Enomoto et al.,
2000). RCAS(A)-chick Runx2
(Stricker et al., 2002) was
supplied by Stefan Mundlos (Max Planck Institute for Molecular Genetics,
Berlin, Germany). Details for the generation of RCAS(A)-PTHrP are available on
request.
In situ hybridization (ISH)
Details of the probes employed for ISH are available upon request. Frozen
sections of embryonic chick tissue, and paraffin sections of mouse, were
prepared exactly as described previously
(Murtaugh et al., 1999).
Non-radioactive section ISH, with digoxigenin (DIG)-labeled probes, was
performed as described previously
(Murtaugh et al., 2001). ISH
with 35S-labeled riboprobes and Hematoxylin and Eosin staining were
performed as described previously (Chung et
al., 1998).
Retroviral misexpression
Virus preparation was as described elsewhere
(Morgan and Fekete, 1996).
Concentrated viral supernatant was injected into the nascent wing buds of late
E3 [Hamburger-Hamilton (HH) stage 17-19] chick embryos. To ensure thorough
infection, each wing bud was injected in several anteroposterior regions along
its distal margin. Following injection, eggs were reincubated up to E8-E10,
and processed for Alcian Blue and Alizarin Red staining
(Murtaugh et al., 1999), or
ISH as described above.
Chick embryo explant culture, retroviral infection, and RT-PCR analysis
Dissection and culture of somitic or pre-somitic mesoderm (psm) explants
was previously described (Munsterberg et
al., 1995). Retrovirus infection was performed essentially as
described by Zeng et al. (Zeng et al.,
2002), with slight modifications (available upon request). Reverse
transcriptase (RT) reactions and polymerase chain reaction (PCR) analysis were
carried out as previously described
(Munsterberg et al., 1995;
Zeng et al., 2002). PCR
conditions and primer sequences have either been previously published or are
available on request.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), as
chondrocyte maturation begins, expression of these transcripts becomes
restricted to the distal portion of the developing cartilage elements
(Fig. 1). At Hamburger-Hamilton
(HH) stages 31 and 37 (Fig.
1C,D), Nkx3.2 expression appears to form a distal-to-proximal
gradient within the cartilage, which resembles the expression of Sox 9
(Fig. 1A,B), collagen IX (col
IX; Fig. 1E,F) and collagen II
(col II; Fig. 4C, parts a,c),
all markers of immature chondrocytes. Nkx3.2 expression declines in the
cartilage cells expressing Ihh, a marker of prehypertrophic chondrocytes
(Fig. 1G,H), and is expressed
at trace levels in mature, hypertrophic chondrocytes, marked by col X
expression (Fig. 1I,J).
Comparison of Nkx3.2 expression with BrdU uptake, which marks active DNA
synthesis (Fig. 1K,L), suggests
that Nkx3.2 expression is restricted to the proliferative zone within the
cartilage. It should be noted, however, that the greatest amount of cellular
proliferation occurs in the most distal chondrocytes of the developing
cartilage element, which do not express Nkx3.2 (arrows in
Fig. 1A-F). Thus, not all
proliferating chondrocytes express Nkx3.2. Its expression is restricted to the
immature, proliferative chondrocytes within the developing cartilage element,
and is specifically downregulated as cells begin to express the
prehypertrophic marker Ihh, and to withdraw from the cell cycle. This
expression pattern is consistent with that very recently described by
Francis-West and colleagues, who noted that this gene is also expressed in
soft tissues, such as developing tendons, muscle sheaths and surrounding
mesenchyme, in developing chicken limbs
(Church et al., 2005).
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)
programmed to express Nkx3.2 (RCAS-Nkx3.2)
(Murtaugh et al., 2001), and
the embryos were allowed to develop through embryonic day 9-10 (E9-E10), at
which point cartilage maturation should normally be well underway.
Nkx3.2-infected wings exhibited considerably shorter and thicker cartilage
elements than did their contralateral control counterparts, and displayed a
lack of mineralized bone, as detected by Alizarin Red staining
(Fig. 2A). This phenotype was
observed in 98% of RCAS-Nkx3.2-infected wings (n>80); in some of
these wings the bones were sometimes severely misshapen, but we never observed
any patterning defects. To further characterize this phenotype, we sectioned RCAS-Nkx3.2-infected and control wings and subjected them to in situ hybidization (ISH) analysis. The contralateral control wings exhibited the ordinary pattern of chondrocyte maturation, as evidenced by sequential expression domains of collagen IX (col IX), col II and Sox9 in immature chondrocytes, Ihh in prehypertrophic chondrocytes, and col X in hypertrophic chondrocytes (Fig. 2B, parts a,c,e,g, and 2C, parts a,c). By contrast, RCAS-Nkx3.2-infected wings displayed uniform expression of Sox9 throughout the entire cartilage, with greatly reduced Ihh expression and barely, if any, detectable expression of col X (Fig. 2B, parts b,d,f,h, and 2C, parts b,d). Moreover, the usual separation between the two col X expression domains, comprising late hypertrophic chondrocytes and trabecular bone (Fig. 2B, part g), is completely absent from RCAS-Nkx3.2-infected limbs (Fig. 2B, part h). Thus, forced expression of Nkx3.2 inhibits the expression of both prehypertrophic and hypertrophic chondrocyte marker genes. The expression of osteopontin (OP), which marks late hypertrophic chondrocytes, was barely detectable in RCAS-Nkx3.2-infected wings (Fig. 7A, compare parts m and n), indicating that the Nkx3.2-induced depletion of Ihh- and col X-expressing cells is not due to an accelerated maturation of such cells into late hypertrophic chondrocytes, but rather reflects a blockade in the maturation of immature chondrocytes. Consistent with this result, BrdU labeling of RCAS-Nkx3.2-infected wings indicated that the normal boundary between proliferative and quiescent chondrocytes present in the control wing was absent in the infected cartilage; instead, proliferative cells were distributed uniformly throughout the infected skeletal elements (Fig. 2C, parts e,f). These results are consistent with the normal expression pattern of Nkx3.2, as described above, and strongly suggest that downregulation of this gene is required for chondrocyte maturation to proceed.
Both DNA binding and transcriptional repression by Nkx3.2 are required to inhibit chondrocyte maturation
To further clarify how Nkx3.2 expression blocks chondrocyte maturation, we
investigated whether DNA binding or transcriptional repression activity was
necessary for this effect. Nkx3.2-NQ contains a glutamine substitution of a
conserved asparagine (amino acid 200) located in the homeodomain; this mutant
is defective in DNA-binding activity but still retains an intrinsic
transcriptional repressor activity (Kim et
al., 2003). Viral misexpression of Nkx3.2-NQ, unlike wild-type
Nkx3.2, failed to alter the morphology of the limb skeleton (zero abnormal
wings, n=11; Fig.
3A,D). Furthermore, analysis of col II and col X expression
revealed a normal distribution of mature chondrocytes in RCAS-Nkx3.2-NQ
infected limbs, in spite of an efficient viral infection throughout the limb
(Fig. 3G,J, and data not show).
Thus, the DNA-binding activity of Nkx3.2 is required to block chondrocyte
maturation. As the Nkx3.2-NQ protein is as stable as wild-type Nkx3.2
(Nkx3.2-WT; D.-W.K. and A.B.L., unpublished), this result suggests that the
blockade of chondrocyte maturation observed with wild-type Nkx3.2 was not
simply due to the titration (by protein-protein interaction) of important
factors necessary for chondrocyte hypertrophy, as a consequence of Nkx3.2
overexpression. Instead, it appears that Nkx3.2 represses chondrocyte
maturation by binding to the regulatory element of a gene(s) involved in this
process.
Prior work has indicated that Nkx3.2 functions as a transcriptional
repressor, and that regions of the protein C-terminal to the homeodomain are
critical for this activity (Kim and
Lassar, 2003; Murtaugh et al.,
2001). While an Nkx3.2 mutant lacking this C-terminal domain,
Nkx3.2
C, fails to repress transcription, fusion of the repression
domain of the Drosophila Engrailed protein onto this protein, to
generate Nkx3.2C-En, restores transcriptional repressor activity to
this fragment of Nkx3.2 (Murtaugh et al.,
2001). Viral misexpression of Nkx3.2C had no
morphologically detectable effect on the limb skeleton (zero abnormal wings,
n=23; Fig. 3B,E). When
the infected cartilage was examined by ISH, chondrocyte maturation appeared
normal, in spite of a thorough viral infection of the cartilage
(Fig. 3H.K, and data not
shown). By contrast, misexpression of Nkx3.2C-En yielded shortened and
thickened limb cartilage elements (80% abnormal wings, n=20;
Fig. 3C,F) similar to those
observed following infection with RCAS-Nkx3.2-WT. As expected, these cartilage
elements failed to undergo hypertrophy
(Fig. 3I,L). We conclude that
the ability of ectopic Nkx3.2 to block chondrocyte maturation correlates with
its ability to repress transcription of a gene(s) normally required to promote
this process.
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C-VP16, by appending the strong transcriptional activation domain
of the Herpes Simplex Virus VP16 protein onto the C terminus of
Nkx3.2C. This derivative of Nkx3.2 no longer represses reporter gene
activity, but instead acts as a strong activator of gene expression in vitro
(Kim and Lassar, 2003). Viral
misexpression of Nkx3.2C-VP16 resulted in an extended zone of
mineralization (Alizarin Red staining in
Fig. 4A), which is likely to be
the result of accelerated chondrocyte maturation. To confirm that
Nkx3.2C-VP16 accelerates chondrocyte maturation, we analyzed by ISH the
distribution of chondrogenic markers at two different stages after viral
infection. At E8.5 (Fig. 4B),
the Nkx3.2C-VP16-infected wing seems slightly longer than the control
wing (Fig. 4B, parts a,b).
Although col X is just beginning to be expressed within the core of the
control wing skeletal element (Fig.
4B, part c), col X expression is considerably stronger and more
broadly expressed within the core of the RCAS-Nkx3.2C-VP16-infected
skeletal element (Fig. 4B, part
d). At E10 (Fig. 4C), in
RCAS-Nkx3.2C-VP16-infected wings, col X expression is upregulated and
the distance between the peripheral-most extent of the two col X expression
domains is expanded relative to the control wing
(Fig. 4C, parts e,f). In
addition, the distance between the two col II expression domains, which
consist of both early and late hypertrophic chondrocytes, is broader than in
the control uninfected wing (Fig.
4C, parts c,d). Thus, Nkx3.2C-VP16 accelerates chondrocyte
maturation in vivo. This finding is consistent with the notion that Nkx3.2
inhibits chondrocyte maturation by transcriptionally repressing a gene(s) that
directly or indirectly promotes chondrocyte maturation, and that a reverse
function form of Nkx3.2 (i.e. Nkx3.2C-VP16) should activate the
expression of such a gene(s) and therefore accelerate chondrocyte
maturation.
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; Weir et al.,
1996), we initially investigated whether PTHrP might be
upregulated by Nkx3.2 misexpression. As shown in
Fig. 5A, expression of PTHrP is
in fact downregulated in articular chondrocytes following infection with
RCAS-Nkx3.2 (Fig. 5A, compare
parts a and b). This is not surprising, given that ectopic Nkx3.2 both
diminishes the level of Ihh expression and blocks the progression of Ihh
expression into regions of the cartilage that lie proximal to the articular
perichondrium (Fig. 2B, parts
e,f; Fig. 7A, parts g,h), where
it is normally required to maintain expression of PTHrP
(St-Jacques et al., 1999). We
then considered the possibility that Nkx3.2 might actually be functionally
downstream of PTHrP, and therefore mediate at least some of the effects of
PTHrP on chondrocytes.
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To confirm that expression of Nkx3.2 in growth plate cartilage depends on PTHrP signaling, we analyzed its expression under conditions where PTHrP signaling had been either artificially activated or suppressed. To examine the first case, we infected wing buds of chicken embryos with an RCAS construct encoding PTHrP. As expected, viral PTHrP misexpression led to a phenotype similar to that of Nkx3.2 misexpression; cartilage elements within the limb skeleton were shorter and displayed a decrease in Alizarin Red staining (Fig. 5B, parts a,b, see humerus and ulna marked by arrows). We analyzed sections of RCAS-PTHrP infected limbs by in situ hybridization and observed that chondrocyte maturation was severely delayed. Although expression of both Sox9 and col II was extinguished in the central region of the control uninfected ulna (Fig. 5B, parts c,g; between arrowheads), expression of both of these genes was maintained within the central region of the contralateral RCAS-PTHrP-infected ulna (Fig. 5B, parts d,h; see arrows). In addition, in contrast to the control wing, the RCAS-PTHrP-infected ulna displayed a single Ihh expression domain in the middle of this cartilage element and a loss of col X expression (Fig. 5B, parts i-l). Interestingly, although Nkx3.2 expression was diminished in the central region of the control uninfected ulna (Fig. 5B, part e; between arrowheads), viral misexpression of PTHrP maintained the expression of Nkx3.2 in the central region of the contralateral infected ulna (Fig. 5B, part f; see arrows). Thus, increased levels of PTHrP signaling prolong the expression of Nkx3.2 throughout the entire cartilage template, concomitant with a delay in chondrocyte maturation.
To evaluate Nkx3.2 expression in the absence of PTHrP signaling, we
examined mice embryos deficient for either the PTHrP (PTHrP-/-) or
the PTH/PTHrP-receptor (PTHrP-receptor-/-) gene. Mice lacking PTHrP
or its receptor exhibit accelerated chondrocyte maturation
(Karaplis et al., 1994;
Lanske et al., 1996),
essentially the opposite phenotype to that seen following Nkx3.2
misexpression. In growth plates of wild-type E18.5 mouse embryos, we found
that Nkx3.2/Bapx1 is expressed in the columnar layer of proliferative
chondrocytes, but not in pre-hypertrophic and hypertrophic cells
(Fig. 5C, parts a-c; histone H4
mRNA marks proliferating cells). This expression pattern is consistent with
the fact that the PTHrP-receptor is expressed in such proliferating cells
(Lee et al., 1996), and would
therefore be expected to activate target gene expression in this cellular
compartment. In contrast to what we found in wild-type animals, Nkx3.2/Bapx1
expression is greatly reduced or undetectable in growth plates of both
PTHrP-/- and PTHrP-receptor-/- mutant mice
(Fig. 5C, parts d-g). Thus,
PTHrP signaling is either directly required to maintain Nkx3.2/Bapx1
expression in chondrocytes, or indirectly required to maintain expression of
this gene by augmenting either the proliferation or survival of the columnar
chondrocytes that express this gene. To distinguish between these two
possibilities we examined Nkx3.2/Bapx1 expression in mice embryos engineered
to lack expression of both PTHrP and the cyclin-dependent kinase inhibitor
p57.
Whereas PTHrP knockout mice embryos lack columnar proliferative
chondrocytes within their growth plates, this cell population is restored in
mice deficient for both PTHrP and p57 expression
(MacLean et al., 2004).
Bapx1/Nkx3.2 expression is confined to the immature chondrocytes in the growth
plates of E17.5 PTHrP+/-; P57+/+ mice
(Fig. 5D, parts a,b). These
Bapx1/Nkx3.2-expressing chondrocytes, which also express collagen II, are
specifically deleted from the growth plate in PTHrP-/-;
P57+/+ mice (Fig.
5D, parts c,d). To examine if Bapx1/Nkx3.2 expression is regained
under conditions that restore the presence of immature columnar chondrocytes
despite the absence of PTHrP signaling, we examined the expression of
Bapx1/Nkx3.2 in mice lacking expression of both PTHrP and p57. Because only
the maternally inherited allele of p57 is expressed
(Hatada and Mukai, 1995),
P57+/-m mice (which contain only a paternal wild-type p57 allele)
lack expression of this gene. Interestingly, although the population of
immature chondrocytes expressing col II is expanded in PTHrP-/-;
P57+/-m mice, expression of Bapx1/Nkx3.2 is not restored in these
cells (Fig. 5D, parts e,f).
Thus PTHrP signals are required to maintain the expression of Bapx1/Nkx3.2 in
immature chondrocytes of the growth plate.
To evaluate if PTHrP signals are sufficient to induce the expression of Nkx3.2 in chondrocytes, we treated cultures of hypertrophic upper sternal chondrocytes derived from 15-day-old chick embryos with PTHrP in vitro. After 3 days of treatment with PTHrP, we noted that both Ihh and collagen X expression was extinguished in these cultures, whereas Nkx3.2 expression was significantly induced (Fig. 5E). Taken together, these data indicate that expression of Nkx3.2/Bapx1 in the growth plate lies downstream of PTHrP signaling, and suggest that Nkx3.2/Bapx1 may mediate at least some of the effects of this signaling pathway on chondrocyte maturation.
Nkx3.2 represses Runx2 expression
We wondered whether Nkx3.2 blocks chondrocyte hypertrophy by blocking the
expression of Runx2, which is a positive regulator of this process. To
evaluate this possibility, we first compared the expression of endogenous
Nkx3.2 and Runx2 during cartilage maturation in the chick limb. We found that
Nkx3.2 and Runx2 are expressed in a reciprocal pattern within the developing
cartilage element; Runx2 expression is most intense in the central region of
the cartilage element where Nkx3.2 expression is most diminished (see
arrowheads in Fig. 6A, parts
a,b). To test whether Nkx3.2 might directly or indirectly modulate Runx2
expression, we infected chicken wing buds with RCAS viruses encoding either
wild-type Nkx3.2 or the `reverse function' Nkx3.2-C-VP16, and examined
Runx2 expression by ISH. RCAS-Nkx3.2 infection reduced Runx2 expression
specifically in the central region of the developing cartilage that would
ordinarily mature into hypertrophic chondrocytes and express col X (outlined
by orange boxes in Fig. 6B,
parts a,b). By contrast, infection with RCAS-Nkx3.2-C-VP16 increased
Runx2 transcript levels principally in the peripheral regions of the cartilage
(outlined by blue boxes in Fig.
6B, parts e,f), while accelerating col X expression in the central
region of the cartilage (Fig.
6B, parts g,h). Quantification of Runx2 expression indicated that
misexpression of Nkx3.2 led to a twofold decrease of Runx2 expression in the
central region of the skeletal element that would normally become hypertrophic
(Fig. 6C, left panel, orange
bars). Conversely, forced expression of Nkx3.2-C-VP16 increased Runx2
expression approximately twofold in the peripheral regions of the developing
cartilage (Fig. 6C, right
panel, blue bars).
In addition to assaying the effects of Nkx3.2 on Runx2 expression in vivo,
we also examined these issues in a chondrogenic culture system in vitro.
Presomitic mesoderm (psm) dissected from HH stage 10 chicken embryos can
initiate a chondrogenic program following either sequential treatment with Shh
and Bmp4 (Murtaugh et al.,
1999), or following infection with RCAS-Nkx3.2 in the presence of
Bmp4 signals (Murtaugh et al.,
2001). Psm explants cultured with Shh for 2 days and Bmp4 for an
additional 6 days begin to differentiate into mature chondrocytes, as
indicated by the expression of both Runx2 and col X
(Fig. 6D, lane 1). Culture of
such explants in Bmp4 for 12 days increased the expression of both Runx2 and
col X (Fig. 6D, lane 3). In
striking contrast, psm infected with RCAS-Nkx3.2 and exposed to Bmp4 signals
for either 6 or 12 days expressed very robust levels of the immature
chondrocyte marker epiphycan, but only trace levels of Runx2 and col X
(Fig. 6D, lanes 2 and 4). Thus,
retroviral-encoded Nkx3.2 induced chondrogenesis in explants of presomitic
mesoderm while blocking chondrocyte maturation in these cultures by either
directly or indirectly repressing the expression of both Runx2 and col X.
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|
Misexpression of Runx2 does not rescue a PTHrP-induced blockade of chondrocyte maturation
Ectopic expression of either PTHrP or Nkx3.2 in limb cartilage blocks
chondrocyte hypertrophy (Figs 2
and 5) and simultaneously
represses the expression of Runx2 (Fig.
6) (Guo et al.,
2006). In light of the inability of Nkx3.2 to block chondrocyte
maturation in the presence of exogenous Runx2, we wondered if this would also
be the case for PTHrP signals. To evaluate this issue, we analyzed col X and
Ihh expression in psm explants infected with RCAS(A)-Runx2, in either the
absence or presence of increasing levels of PTHrP. Although 10-8 M
PTHrP repressed col X expression and only slightly dampened the expression of
Ihh, 10-7 M PTHrP significantly decreased the expression of both
col X and Ihh in psm cultures infected with RCAS(A)-Runx2
(Fig. 7C, lanes 1-4), despite
the continued expression of retrovirally encoded Runx2 RNA in these
cultures. Thus, PTHrP signals can blunt the ability of exogenous Runx2 to
promote chondrocyte maturation. As PTHrP signals are thought to block
chondrocyte maturation by increasing cAMP levels and protein kinase A (PKA)
activity in the cell (Guo et al.,
2002), we evaluated whether the elevation of cAMP levels by
forskolin would similarly block the ability of retroviral Runx2 to induce
chondrocyte hypertrophy. Administration of increasing levels of forskolin
phenocopied the effects of PTHrP administration and blocked the expression of
col X (but not Ihh) at lower concentrations, and extinguished the expression
of both col X and Ihh at higher concentrations
(Fig. 7C, lanes 5-7). These
findings indicate that elevated cAMP levels are sufficient to block
chondrocyte maturation downstream of Runx2 gene expression. Together
with our observation that Nkx3.2 expression requires PTHrP signals in the
growth plate, these results indicate that PTHrP signaling can repress
chondrocyte hypertrophy both by affecting Runx2 transcription (via Nkx3.2
induction), and by another mechanism that blocks chondrocyte maturation by
acting either downstream or in parallel to Runx2 mRNA expression.
|
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Retroviral misexpression of Nkx3.2 in chicken wings resulted in a severe delay
of chondrocyte maturation, indicating that Nkx3.2 expression needs to be
downregulated to allow chondrocyte maturation to proceed. Both the DNA binding
and the transcriptional repressor activities of Nkx3.2 were required to
inhibit chondrocyte hypertrophy, suggesting that Nkx3.2 represses the
transcription of a gene(s) necessary for chondrocyte maturation. Consistent
with this notion, we found that ectopic expression of a `reverse function'
mutant of Nkx3.2 that has been converted into a transcriptional activator
hastens chondrocyte maturation, presumably by activating the expression of a
Nkx3.2 target gene(s) that is necessary to promote chondrocyte hypertrophy.
Taken together, these findings strongly suggest that Nkx3.2 either directly or
indirectly represses the expression of a gene(s) that is normally required for
chondrocyte maturation to proceed.
Nkx3.2/Bapx1 delays chondrocyte maturation at least in part by repressing Runx2 expression
We observed that Nkx3.2 and Runx2 are expressed in reciprocal patterns
during chondrocyte maturation, such that Runx2 expression is most intense in
the central region of the cartilage element where Nkx3.2 expression is most
diminished. Forced expression of Nkx3.2 represses Runx2 expression in the
regions of the chick limb cartilage that would normally become hypertrophic in
vivo, and dramatically represses Runx2 expression in cultured somite explants
in vitro. Most significantly, we found that infection of limbs or somite
cultures with RCAS-Runx2 restored chondrocyte maturation in tissues infected
with RCAS-Nkx3.2. Thus, forced expression of Runx2, which promotes chondrocyte
maturation, is epistatic to the effects of forced Nkx3.2 expression.
Interestingly, infection of chick wings with RCAS-Nkx3.2 leads to a profound
blockade of chondrocyte maturation but only a partial loss of Runx2 expression
in the developing cartilage, resulting in an approximately 50% decreased
expression of Runx2 in cells located in the position of normally hypertrophic
chondrocytes. These findings suggest either that chondrocyte hypertrophy
requires a threshold level of Runx2 or that Nkx3.2 represses other factors
necessary for the activity of this residual level of Runx2. Indeed chondrocyte
maturation is completely absent in mice lacking both Runx2 and Runx3, but is
also considerably delayed in mice that lack Runx3 but have only one intact
allele of Runx2 (Yoshida et al.,
2004), suggesting that a threshold level of Runx factors are
indeed necessary to promote chondrocyte hypertrophy. Although these findings
are consistent with the notion that Nkx3.2 blocks chondrocyte maturation by
dropping Runx2 levels below a threshold that is required for this process to
take place, it is also plausible that Nkx3.2 blocks the expression of
co-factors required for Runx2 activity, and that overexpression of Runx2
somehow bypasses the need for such co-factors. Nkx3.2 has recently been shown
to bind to a Runx2 promoter in gel shift assays, and can repress transcription
driven by this regulatory element in vitro
(Lengner et al., 2005),
suggesting that Nkx3.2 may directly repress Runx2 expression.
Nkx3.2/Bapx1 acts downstream of the PTHrP signaling pathway
Two crucial and well-characterized paracrine regulators in endochondral
bone development are Indian hedgehog and PTHrP, which form a feedback loop to
control the rate at which chondrocytes undergo maturation
(Lanske et al., 1996;
Vortkamp et al., 1996). Here,
we provide several lines of evidence that the Nkx3.2/Bapx1 gene is a
target of PTHrP signaling that regulates chondrocyte maturation in the growth
plate. Chicken Nkx3.2 and mouse Bapx1 are expressed in proliferative
chondrocytes, the site of active PTHrP signaling. Nkx3.2 expression is
significantly expanded throughout the cartilage following the misexpression of
PTHrP, and is lost in mouse limbs deficient for both PTHrP and PTHrP-R
expression. Because the proliferating columnar chondrocytes that express
Nkx3.2 are specifically lost in the absence of PTHrP signals, we evaluated
whether these signals were necessary for the expression of Nkx3.2 or only for
the maintenance of this cell population. Although loss of expression of the
cdk inhibitor p57 restored col II expressing columnar chondrocytes in PTHrP
deficient mice, these cells failed to express Bapx1/Nkx3.2 in the absence of
PTHrP signals, suggesting that PTHrP signals are indeed necessary to maintain
the expression of Bapx1/Nkx3.2 in the growth plate. Consistent with this idea,
we have found that application of PTHrP to cultures of hypertrophic chicken
chondrocytes could induce the expression of Nkx3.2.
PTHrP signals block chondrocyte hypertrophy by at least two parallel pathways
In addition to maintaining the expression of Bapx1/Nkx3.2 in immature
chondrocytes, our results indicate that PTHrP signals can also block
chondrocyte maturation by a mechanism(s) that is independent of the
suppression of Runx2 transcription. This conclusion stems from the observation
that PTHrP or forskolin can block the ability of retroviral-encoded Runx2 to
induce either Ihh or col X gene expression in somitic explants (this work), or
mineralization in cultured chondrocytes
(Iwamoto et al., 2003).
Repression of chondrocyte maturation in RCAS-Runx2 infected somites by either
PTHrP or forskolin administration occurs without an affect on viral Runx2
transcript levels. This observation is consistent with recent findings that
PTH/PTHrP signals can block the ability of transgenic Runx2, driven by the col
II regulatory regions, to induce chondrocyte hypertrophy
(Guo et al., 2006). Thus, we
think it is most likely that PTHrP signals block chondrocyte maturation in the
growth plate by at least two parallel pathways, one transcriptional, involving
the induction of Nkx3.2/Bapx1, which in turn blocks the transcription of Runx2
and/or a co-factor necessary for chondrocyte maturation, and another distinct
pathway that blocks chondrocyte maturation downstream of Runx2 mRNA
expression (outlined in Fig.
8). According to this scenario, PTHrP signals would continue to
repress chondrocyte maturation (by a Nkx3.2/Bapx1-independent pathway) in mice
lacking Nkx3.2/Bapx1, thus explaining the apparently normal
development of the limb skeleton in such mice.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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