Distinct Phases of Coordinated Early and Late Gene Expression in Growth
Plate Chondrocytes in Relationship to Cell Proliferation, Matrix
Assembly, Remodeling, and Cell Differentiation
E TCHETINA,1F MWALE,1,2and AR POOLE1
Although much has been learned about growth plate development and chondrocyte gene expression during
cellular maturation and matrix remodeling in the mouse, there has been a limited study of the interrelation-
ships of gene expression between proteinases, growth factors, and other regulatory molecules in the mouse and
in other species. Here we use RT-PCR of sequential transverse sections to examine the expression profiles of
genes involved in chondrocyte growth, differentiation, matrix assembly, remodeling, and mineralization in the
bovine proximal tibial growth plate. Specifically, we studied the expression of genes encoding COL2A1 and
COL10A1, the latter a marker of cellular hypertrophy, the matrix metalloproteinases (MMP), MMP-13 and
MMP-9, as well as the transcriptional factors, Sox9 and Cbfa1, the growth factors basic fibroblast growth
factor (bFGF), parathyroid hormone–related peptide (PTHrP), transforming growth factor (TGF)?1, and ?2,
Indian hedgehog (Ihh), and the matrix protein osteocalcin. These were analyzed in relationship to cell division
defined by cyclin B2 expression. Two peaks of gene expression activity were observed. One was transient,
limited, and located immediately before and at the onset of cyclin B2 expression in the early proliferative zone.
The other was generally much more pronounced and was located in the early hypertrophic zone. The
upregulation of expression of COL2A1, its transcriptional activator Sox9, osteocalcin, MMP-13, and TGF?2
was observed immediately before and at the onset of cyclin B2 expression and also in the hypertrophic zones.
The upregulation of COL10A1, Cbfa1, MMP-9, TGF?-1, and Ihh gene expression was associated exclusively
with the terminal differentiation of chondrocytes at the time of mineral formation in the extracellular matrix.
In contrast, bFGF and PTHrP expression was observed in association with the onset of cyclin B2 expression
and hypertrophy. This initial cluster of gene expression associated predominantly with matrix assembly and
onset of cell proliferation is therefore characterized by expression of regulatory molecules distinct from those
involved at hypertrophy. Together these results identify separate phases of coordinated gene expression
associated with the development of the physis in endochondral bone formation. (J Bone Miner Res 2003;18:
Key words:growth plate, gene expression, chondrocyte differentiation, ECM remodeling, growth factors
assembling chondrocytes to growth-arrested hypertrophic
CENTRAL PROCESS in endochondral bone formation is
the progressive differentiation of proliferating matrix-
cells that direct the remodeling and mineralization of the
cartilage matrix that leads eventually to its subsequent re-
placement by bone in the growth plate (Fig. 1).
The maturational process of chondrocytes can be defined
by the expression of extracellular matrix genes as well as
chondrocyte morphology. Thus, the primary mammalian
growth plate physis can be divided into zones, namely in
order of development: resting, proliferative, and hypertro-
Dr Poole has served as a consultant for Amgen, Roche Labora-
tories, and Wyeth. All authors have no conflict of interest.
1Joint Diseases Laboratory, Shriners Hospitals for Children and Departments of Surgery and Medicine, McGill University, Montreal,
2Present Address: Orthopaedics Research Laboratory, Jewish General Hospital, Lady Davis Institute for Medical Research, Division
of Orthopedic Surgery, McGill University, Montreal, Quebec, Canada.
JOURNAL OF BONE AND MINERAL RESEARCH
Volume 18, Number 5, 2003
© 2003 American Society for Bone and Mineral Research
phic.(1)The resting chondrocytes show little or no cell
division but elaborate an extensive extracellular matrix like
the proliferating cells that express COL2A1, and the pro-
teoglycan aggrecan that constitute, together with other ma-
trix molecules, an extensive extracellular matrix (ECM).
After cessation of cell division, chondrocytes partly resorb
their ECM and enlarge (become hypertrophic) as they ex-
press the type X collagen gene (COL10A1).(1,2)
Proliferation and enlargement of chondrocytes in the
growth plate involves ECM remodeling whereby the triple
helix of type II collagen is cleaved by collagenase(s).(3–6)
This results in denaturation of the triple helix, which is then
susceptible to secondary cleavage by this collagenase and
by other metalloproteinases such as gelatinases A (MMP-2)
and B (MMP-9).(6)Our recent studies have revealed that
cleavage of type II collagen by the collagenase MMP-13
(collagenase 3) is an integral and required feature of chon-
drocyte hypertrophy and matrix remodeling in all zones of
The molecular mechanisms that govern the differentiation
of chondrocytes and remodeling of the ECM are now better
understood. In vitro studies indicate that differentiation of
these growth plate chondrocytes is an intrinsic process
given a permissive environment. However, signals gener-
ated by chondrocytes or perichondrium are likely to coor-
dinate the rate of long bone growth.(7–11)
Growth factors represent one group of such signals. Some
are responsible for the mutually exclusive processes of
chondrocyte proliferation and terminal differentiation.
Thus, basic fibroblast growth factor (bFGF or FGF-2) and
parathyroid hormone–related peptide (PTHrP) stimulate
resting chondrocytes to proliferate and suppress terminal
differentiation of hypertrophic chondrocytes.(12–16)In addi-
tion it has been suggested that PTHrP, in combination with
another growth factor Indian hedgehog (Ihh), regulates
chondrocyte differentiation through the establishment of a
negative feedback mechanism, whereby Ihh and PTHrP can
together suppress hypertrophy.(10,17–19)
Transforming growth factor betas (TGF?s) are multifunc-
tional molecules regulating cellular proliferation, differen-
tiation, and extracellular matrix function.(20)TGF? trans-
ported from apoptotic chondrocytes to the region of cell
division would be expected to stimulate matrix production,
delay hypertrophic differentiation, and thus maintain
growth plate width.(21,22)TGF?1(23)and TGF?2
able to suppress chondrocyte hypertrophy partially because
of the regulation of PTHrP gene expression that exerts both
PTHrP-dependent and PTHrP-independent effects on endo-
chondral bone formation.(23–26)
Chondrocyte differentiation is controlled by specific
transcriptional mechanisms, which involve two key tran-
scription factors, Cbfa1 and Sox9. Sox9 has been shown
to regulate the rate of chondrocyte differentiation in
hypertrophy by controlling the expression of a series of
chondrocyte-specific genes including COL2A1, COL9A2,
COL11A1, and aggrecan.(27–29)Cbfa1 is essential for
osteoblast differentiation(30–32)and can also play a cru-
cial role in chondrocyte maturation during endochondral
ossification.(33–35)It acts as a transcription factor and can
induce MMP-13 expression.(36,37)
The complex sequential changes in gene expression dur-
ing chondrocyte differentiation have largely been studied
only in mice by in situ hybridization.(38)Whether these
findings extend to larger mammals is unclear. We have
previously used sequential in situ analyses of transverse
sections of the physis of the primary proximal tibial growth
plate of the bovine fetus, as we described earlier,(4,39)in-
corporating a reverse transcriptase-polymerase chain reac-
tion (RT-PCR) approach to examine the relationship be-
tween matrix remodeling and mineralization. In this study
we sought to define the coordinated expression of MMP-13,
MMP-9, and the regulatory genes described above in rela-
tionship to chondrocyte proliferation, ECM assembly, and
chondrocyte differentiation. We have identified two
peaks of expression of genes involved in ECM remodel-
ing immediately before and at the beginning of cell
proliferation and then at its cessation when cellular hy-
pertrophy occurs. We show that these are characterized
by the expression of distinct and sometimes mutually
exclusive regulatory molecules. These observations pro-
vide new insights into how matrix assembly proteolysis
and chondrocyte proliferation and differentiation may be
regulated in relationship to matrix assembly, remodeling,
Bovine primary growth plate. Modified and reproduced with
845GENE EXPRESSION IN THE GROWTH PLATE
MATERIALS AND METHODS
Bovine fetuses obtained from a local abattoir immediately
after the slaughter of pregnant cows were transported to the
laboratory. Fetal age was determined by measurement of
tibial length.(40)Fetuses ranged from 120 to 210 days old.
Tissue preparation was essentially as described.(4,39)Only
blocks of growth plate with a flat fracture surface were used.
Tissue blocks were trimmed to provide cross-sectional areas
of approximately 25 mm2. One hundred-micrometer-thick
transverse sections were cut parallel to the fracture face
(using a Vibratome; Ted Pella, Inc., Redding, CA, USA),
starting at the fracture face and extending through the hy-
pertrophic zone into the upper proliferative zone of the fetal
bovine growth plate. They represented tissue labeled as A,
B, C, and so on, from the fracture face. Their locations have
been previously characterized(4,39)and are shown in Fig. 1.
A series of sections was pooled (A with A, B with B, etc.)
to permit collection of a sufficient amount of tissue for the
analyses. Wet weights were determined immediately after
sectioning; the weights ranged from 10 to 15 mg, depending
on the sample. The weights of samples A and B were lower
because of some irregularity of the fracture face.
Total RNA isolation
Total RNA was isolated by a modification(6)of the
method of Chomczinski and Sacchi.(41)Fresh cartilage tis-
sue in solution D (4 M guanidine isothiocyanate, 25 mM
sodium citrate (pH 7.0), 0.5% lauroylsarcosine, and 0.1 M
2-mercaptoethanol) was vortexed vigorously for 30 min-
utes. The debris was removed by centrifugation at 5000g for
10 minutes at 4°C. Proteins and nucleic acids in the super-
natant were precipitated with 1 volume of isopropanol over-
night at ?20°C. The precipitate was removed at 10,000g for
20 minutes at 4°C and resuspended in digestion buffer (10
mM Tris-HCl, 5 mM EDTA, 1% SDS (pH 8.0), with 2
mg/ml of Proteinase K (Gibco), and incubated at 50°C until
the pellet disappeared. After extraction with 1 volume of
phenol, 0.2 volumes of chloroform and 0.1 volume of 2 M
sodium acetate (pH 4.0), the aqueous phase was recovered
by centrifugation (10,000g for 30 minutes at 4°C). Extrac-
tion was repeated until the interface disappeared. Then the
aqueous phase was precipitated with 2 volumes of 100%
ethanol and 0.1 volume of 3 M sodium acetate at ?20°C
overnight. RNA was pelleted at 16,000g for 30 minutes at
4°C, washed with 70% ethanol, dried, and resuspended in
diethyl pyrocarbonate treated (DEPC) water. The optical
density (OD) 260/280 nm was quantitated (1 OD 260 ? 40
?g of RNA) to assess the purity of the preparation require-
ment for (260/280 nM ? 1.8).(41)
We examined the expression of MMP-9 and MMP-13 in
relationship to osteocalcin, a hypertrophic chondrocyte and
osteoblast marker,(42)COL2A1, and its transcription factor
Sox9. These expressions were related to the chondrocyte
hypertrophic markers COL10A1, Cbfa1, and Ihh. These
were correlated with expression of regulatory molecules,
namely TGF?1TGF?2, bFGF, and PTHrP. Cell division
was investigated by examining the expression of cyclin B2,
which controls both mitotic entry and exit; it is expressed in
proliferating cells. The RT reaction was performed using
total RNA isolated from the cartilage in a total volume of 20
?l, containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl2, 10 mM DTT, 50 ?M each of dATP, dGTP,
dCTP, and dTTP, 0.5 ?g total RNA, and 200 U of Super-
Script TMII H-Reverse Transcriptase (as recommended by
Invitrogen, Burlington, Ontario, Canada).
Oligo sequences used for PCR are shown in Table 1. PCR
was performed in a total volume of 25 ?l containing 10 mM
Tris-HCl (pH 8.3), 1.5 mM MgCl2, 0.4 mM each of dATP,
dGTP, dCTP, and dTTP, 0.8 ?M of each primer, 1 ?1 of RT
mixture, and 2.5 U of AmpliTaq DNA polymerase (Perkin
Elmer, Woodbridge, Ontario, Canada). The 30 cycles of
PCR included denaturation (95°C, 1 minute), annealing
(50°C, 1 minute), and extension (72°C, 5 minutes). After
agarose (1.6%) gel electrophoresis, PCR products were vi-
TABLE 1. OLIGO SEQUENCES FOR PCR
Matrix metalloproteinase 13 (MMP-13): MMP-13-D (1241–1259)
GATAAAGACTATCCGAGAC, MMP-13-R (1369–1386)
Matrix metalloproteinase 9 (MMP-9): MMP-9-D (139–156)
GCAGAGGAATACCTGTAC, MMP-9-R (361–377)
Transforming growth factor beta1 (TGF?1): TGFb1-D (126–143)
GGCAACAAAATCTATGAC, TGFb1-R (471–490)
Transforming growth factor beta 2 (TGF?2): TGFb2-D (943–
962) GATTTGACGTCTCAGCAATG, TGFb2 (1324–1343)
Indian hedgehog (Ihh): IHH-D (476–494)
CAGGTCATCGAGACTCAGG, IHH-R (773–789)
Basic fibroblast growth factor (bFGF): bFGF-D (975–994)
GGCACACATTAATCTACATG, bFGF-R (1262–1282)
Parathyroid hormone related protein (PTHrP): PTHrP-D (233–
253) GAAATCAGAGCTACCTCGGAG; PTHrP-R (317–336)
Core binding factor (Cbfa1): Cbfa1-D (365–385)
GGTTCAACGATCTGAGATTTG, Cbfa1-R (573–594)
Sox-9: Sox9-D (21–39) CATGAAGATGACCGACGAG, Sox9-R (283–
Procollagen type II (COL2A1): COL2A1-D (949–970)
GAACCCAGAAACAACACAATCC, COL2A1-R (1075–1095)
Procollagen type X (COL10A1): COL10A1-D (213–232)
CTGAGCGATACCAAACACC, COL10A1-R (297–319)
Osteocalcin: OC-D (123–141) CTTTGTGTCCAAGCAGGAG, OC-R
Cyclin B2: CB2-D (932–949) GTTGACTATGACATGGTG; CB2-R
Glyceraldehyde 3-phosphate dehydrogenase: G3PDH-D (605–628)
GCTCTCCAGAACATCATCCCTGCC, G3PDH-R (927–950)
846TCHETINA ET AL.
sualized by ethidium bromide staining. GAPDH was used as
reference for gel loading. The band intensities were deter-
mined to be below saturation by dilution analyses. Each
analysis was performed at least three times at different
dilutions of each sample of the original cDNA. The result of
the single dilution for all the samples in a given set that
showed most clearly differences in expression (e.g.,
COL2A1) is presented in Figs. 2 and 3. Results were ana-
lyzed using National Institutes of Health 1.60 software to
determine the pixel intensity for each band and autoback-
ground subtraction was used to control for background
signal (Fig. 3). These results were reproducible for at least
two growth plates each from a different fetus.
The isolated clones of each amplified cDNA fragment
were sequenced (Sheldon Center, McGill University) to
verify the identity of each cDNA product. To confirm the
lack of chromosomal DNA contamination of RNA samples,
PCR was also performed with RNA aliquots. To avoid
variation in efficiency between experiments, all sections
were simultaneously subjected to RT, and all samples of
cDNA were simultaneously amplified in PCR.
RESULTS AND DISCUSSION
Using a microanalytical method, involving RT-PCR anal-
yses of sequential transverse sections of the primary prox-
imal tibial growth plate of the bovine fetus, 12 samples of
transverse sections representing hypertrophic (A–C), matu-
ration (D–F), and lower to upper proliferative (G–L) zones
of bovine growth plate (Fig. 1) were obtained as identified
previously.(4,39)The analyses we show are for a single fetus.
Repeat studies of other fetuses have revealed essentially the
Gene expression profile of collagens, MMPs, and their
Original RT-PCR analyses are shown in Fig. 2. They are
also presented in relationship to GAPDH expression in Fig.
3. Expression of COL10A1, the definitive marker of hyper-
trophic chondrocytes, was detected suddenly in sample C
(Figs. 2 and 3), thereby defining the early hypertrophic
zone. Expression was increased in samples B and A in
relationship to GAPDH. The increase in COL10A1 mRNA
in sample A was accompanied by the onset of osteocalcin
expression, which is also expressed by the most mature
hypertrophic chondrocytes(43)as well as by osteoblasts in
zones of the growth plate.
A representative RT-PCR analyses of gene expression in the
to GAPDH expression determined using NIH1.60 software.
Relative RT-PCR analysis of gene expression in relationship
847 GENE EXPRESSION IN THE GROWTH PLATE
the primary spongiosa.(44)Thus, the hypertrophic zone was
defined as being represented by samples A, B, and C.
Osteocalcin was also clearly expressed by chondrocytes in
sample L before onset of expression of cyclin B2. This was
not seen in association with hypertrophy because there was
no detectable COL10A1 expression.
In contrast to COL10A1, COL2A1 expression was de-
tected throughout the growth plate. But when PCR was
performed using equally diluted samples, two peaks of
COL2A1 expression were observed in samples K, D, and A.
The latter occurs in D immediately before upregulation of
COL10A1 expression. This upregulation in the early hyper-
trophic zone was originally reported.(43)The same distribu-
tion pattern of these collagens was shown for the rat growth
plate.(45)The upregulation of COL2A1 expression was seen
in the early proliferative as well as the hypertrophic zones.
This was accompanied in both sites by peaks of expression
of the COL2A1 transcriptional activator Sox9.(26–29)Coor-
dinated expression patterns for COL2A1 and Sox9 have
been shown in the mouse growth plate but previously not in
the hypertrophic zone.(26,27,46)
The peak of expression of Cbfa1 was only seen in the
hypertrophic zone, coinciding with that of Sox9, COL2A1
and COL10A1, Ihh, and MMP-13. The same type of expres-
sion for Cbfa1 was seen in the hypertrophic zone in mouse
growth plate chondrocytes, although its expression was re-
ported to be elevated before differentiation to the hypertrophic
was shared with MMP-9 in sample E before hypertrophy. A
peak of expression of Cbfa1 was found also in terminal hy-
pertrophic chondrocytes by in situ hybridization method,(49)
although Cbfa1 expression is not always an obligate require-
ment for differentiation in mouse chondrocytes.(31,32)
The collagenase MMP-13 was weakly expressed in the
proliferative and maturation zone, although, as in COL2A1
and Sox9, it showed a small transient peak in the upper
proliferative region (sample K). Its expression was progres-
sively upregulated in the hypertrophic zone where marked
upregulation of COL10A1, COL2A1, and Cbfa1 occurred.
Our results thus confirm and add to previously obtained in
situ hybridization data of prenatal development of mice(50,51)
and humans(52,53)in which MMP-13 expression was found
only in hypertrophic chondrocytes. Although in situ hybrid-
ization is probably not always sensitive enough to detect the
expression level of MMP-13 in the proliferative zone be-
cause of the lower level of its expression, MMP-13 was
associated with proliferation also in the rat growth plate.(45)
In contrast, the RT-PCR approach allowed us to identify this
additional peak of MMP-13 expression in early proliferative
chondrocytes associated with cartilage matrix remodeling
earlier in chondrocyte development and proliferation. We
also clearly detected expression throughout the growth plate
albeit usually at low levels. These results on expression of
MMP-13 are also supported by our previous studies show-
ing the existence of an earlier peak of collagenase activity in
the samples representative of the proliferative zone in ad-
dition to the pronounced increase of collagen cleavage
activity in the hypertrophic zone.(4)
MMP-9 (gelatinase B) expression was not found in the
proliferative zone but it also was expressed before hyper-
trophy and in the hypertrophic zone. As noted above, the
transient earlier upregulation before hypertrophy corre-
sponded to that seen for Cbfa1. The expression of MMP-9
has previously been reported to be restricted to the hyper-
trophic zone in rabbit and mouse growth plates.(54–56)
Cyclin B2 showed several peaks of expression in the
bovine growth plate, namely in samples J, H, D, and A.
Because cyclin B2 is not only expressed by chondrocytes,
the expression maximum shows the cumulative data for all
dividing cells present in the sample. These can include other
types of cells, for instance, endothelial cells of cartilage
blood vessels. Because blood vessel ingrowth occurs from
the bone area it may result in upregulation of cyclin B2
expression in samples A, C, and D. In contrast, the upregu-
lation of cyclin B2 expression in samples J and H is likely
primarily caused by chondrocyte proliferation. The reduc-
tion in expression of COL2A1 and its transcription factor
Sox9 in samples H and I occur at a time of cyclin B2
expression in samples J and H. Thus, it seems to reflect a
reduction in collagen assembly that favors cell division.
Upregulation of differentiation-related growth factors
The process of physeal growth and remodeling is known
to be controlled by growth factors. The expression patterns
of genes encoding some of these regulatory molecules are
shown in Figs. 2 and 3. Transient peaks of expression of
bFGF, PTHrP, and TGF?2were found in sample K.
Whereas the expression of bFGF and PTHrP was reduced
elsewhere in the growth plate, that of TGF?2again tran-
siently increased in sample G and then in sample C in the
hypertrophic zone. It also again peaked in sample A at the
junction with the primary spongiosa, a site where osteocal-
cin expression was selectively observed.
PTHrP expression was similar to that in the resting and
proliferative zones of mouse growth plate.(15)PTHrP has
been detected (in conjunction with PTH/PTHrP receptors)
at the junction of the proliferative and hypertrophic zones of
mice,(58)but we did not observe this. PTHrP expression has
been reported to be downregulated in the mouse when
hypertrophy occurs.(13,15,16)However, here we observed this
downregulation in association with cyclin B2 expression
(the onset of proliferation) and in close relationship to
TGF?2and bFGF expression. Others have also observed
higher expression of PTHrP in epiphyseal chondrocytes
than in growth plate chondrocytes.(58)Our data point to
expression at the interface between these two zones.
TGF?2has previously been localized (as protein) in the
zone of calcified cartilage,(57)but TGF?2has also been
reported to be expressed in chondrocytes of proliferative
and hypertrophic zones of rat growth plate.(59)These obser-
vations are in general agreement with our results, although
we observed transient expression in these sites. In contrast,
TGF?1expression peaked only in the hypertrophic zone in
sample C in agreement with the finding that this growth
factor is an abundant product of hypertrophic chondro-
cytes.(60,61)The expression of Ihh was also restricted to the
hypertrophic zone in samples C and B where COL10A1 is
expressed. This confirms previous results showing that ex-
848TCHETINA ET AL.
pression of Ihh parallels that of COL10A1 mRNA in mouse
EC cells (ATDC5),(62)early postnatal mouse ribs,(63)and in
the zones of cartilage calcification in Cbfa1-deficient
Upregulation of genes during terminal chondrocyte
Terminal chondrocyte differentiation marked by cessa-
tion of cell division and onset of hypertrophy, characterized
by the expression of COL10A1, was closely associated with
the expression of MMP-13 as we and others have shown.(1,5)
MMP activity has been shown to be required for ECM
remodeling(5)and is maximal at this time.(4,5)The upregu-
lation of MMP-13 is accompanied by and preceded by
co-expression of MMP-9 another matrix metalloproteinase
that is involved in the remodeling of the hypertrophic
zone.(56)Cbfa1, a transcription factor for MMP-13,(37,64)is
co-expressed with MMP-13. However, it was also co-
expressed with MMP-9 immediately before hypertrophy.
The significance of this is unclear but may relate to early
initiation of collagen resorption.(4)The upregulation of
COL2A1 that was originally described(44)accompanies the
peak of MMP-13 and Sox9 expression. These observations
clearly show that genes for both matrix synthesis and deg-
radation are co-upregulated at this time.
In relationship to the regulation of these events, it is
interesting to note the increased expression for the growth
factors Ihh, TGF?1, and TGF?2is seen when the genes
COL10A1, MMP-13, and COL2A1 are first upregulated with
onset of hypertrophy. It has been clearly shown that TGF?1
and TGF?2 can suppress hypertrophy.(24,28)However,
TGF?1 and Ihh may also positively affect chondrocyte
hypertrophy because TGF?1has been reported to upregu-
late MMP-13,(65)and Ihh is able to upregulate the expres-
sion of COL10AL mRNA.(66)They are all co-expressed in
the hypertrophic zone as in other growth plates.(48,66)
Upregulation of genes during early chondrocyte
proliferation and matrix assembly
An unexpected but reproducible finding was the transient
peaks of expression of MMP-13, Sox9, and COL2A1 early
in the assembly of the physis in relationship to onset of
cyclin B2 expression in the proliferative zone. Their expres-
sion was accompanied by a transient and marked increase in
expression of TGF?2bFGF and PTHrP. It has been shown
that TGF? can stimulate PTHrP expression.(25)These ob-
servations therefore implicate TGF?2in this stimulation.
PTHrP can induce bFGF expression,(14)which would favor
the coincidence of expression of the two genes seen here.
This occurs at a time of matrix assembly and limited re-
modeling before proliferation with less collagenase activity
and expression than is seen later in hypertrophy.(4,5)bFGF is
known to induce chondrocyte proliferation.(67,68)Cyclin B2
expression is seen subsequent to onset of bFGF expression.
PTHrP,(55)like TGF?2and bFGF,(54,69,70)has been reported
to stimulate MMP-13 expression in rodents. Whether these
growth factors/hormones play a similar role here is unclear.
The TGF? family are known to provide potent stimuli for
matrix assembly.(71,72)The significance of the very transient
and isolated peaks of expression of TGF?2between the
upper proliferative and the hypertrophic zone remains to be
established. Because Sox9 is also a target of signaling by
PTHrP,(46)the co-expression of PTHrP and Sox9 at the time
of onset of cyclin B2 expression seems logical.
In conclusion, these results provide a unique insight into
the interrelationships of gene expression in chondrocyte
proliferation and differentiation, matrix assembly, degrada-
tion, mineralization, and vascularization in the bovine with
the enlarged physis. They offer an opportunity to identify
distinct phases of gene expression in early and late physeal
growth and development and provide a means of carefully
comparing this with gene expression of regulatory and
matrix molecules within the same tissue. By examining the
growth plate of the much larger bovine fetus, it is apparent
that, although there are similarities to the mouse, there are
also recognizable differences in the relative expression of
some of these genes revealed by this semiquantitative ap-
proach compared with in situ hybridization used in studies
of the murine growth plate.
1. Poole AR 1991 The growth plate: Cellular physiology, cartilage
assembly and mineralization. In: Hall B, Newman S (eds.) Carti-
lage: Molecular Aspects. CRC Press, Boca Raton, FL, USA, pp.
2. Cancedda R, Descalzi-Cancedda F, Castagnola P 1995 Chondro-
cyte differentiation. Int Rev Cytol 159:265–358.
3. Mwale F, Billinghurst C, Wu W, Alini M, Webber C, Reiner A,
Ionescu M, Poole J, Poole AR 2000 Selective assembly and
remodelling of collagens II and IX associated with expression of
the chondrocyte hypertrophic phenotype. Dev Dyn 218:648–662.
4. Mwale F, Tchetina E, Wu W, Poole AR 2002 The assembly and
remodelling of the extracellular matrix in the growth plate in
relationship to mineral deposition and cellular hypertrophy: An in
situ study of collagens II and IV and proteoglycan. J Bone Miner
5. Wu W, Tchetina E, Mwale F, Hasty K, Pidoux I, Reiner A, Chen
J, van Wart HE, Poole AR 2002 Proteolysis involving MMP-13
(collagenase-3) and the expression of the chondrocyte hypertrophic
phenotype. J Bone Miner Res 17:639–651.
6. Poole AR, Howell DS 2001 Etiopathogenesis of osteoarthritis. In:
Moskowitz RW, Howell DS, Goldberg VM, Mankin HJ (eds.)
Osteoarthritis: Diagnosis and Management, 3rd ed. WB Saunders
Co, Philadelphia, PA, USA, pp. 29–47.
7. Bohme K, Winterhalter KH, Bruckner P 1995 Terminal differen-
tiation of chondrocytes is a spontaneous process and is arrested by
transforming growth factor-beta-2 and basic fibroblast growth fac-
tor in synergy. Exp Cell Res 216:191–198.
8. Long F, Linsenmayer TF 1998 Regulation of growth region carti-
lage proliferation and differentiation by perichondrium. Develop-
9. Luan Y, Praul CA, Gay CV, Leach RM Jr 1996 Basic fibroblast
growth factor: An autocrine growth factor for epiphyseal growth
plate chondrocytes. J Cell Biochem 62:372–382.
10. Vortkamp A, Lee K, Lanske B, Serge GV, Kronenberg HM, Tabin
CJ 1996 Regulation of the rate of cartilage differentiation by Indian
Hedgehog and PTH-related protein. Science 273:615–622.
11. Shida JL, Jingushi S, Izumi T, Ikenoue T, Iwamoto Y 2001 Basic
fibroblast growth factor regulates expression of growth factors in
rat epiphyseal chondrocytes. J Orthop Res 19:259–264.
12. Kato Y, Iwamoto M 1990 Fibroblast growth factor is an inhibitor
of chondrocyte terminal differentiation. J Biol Chem 265:5903–
13. Karaplis AC, Luz J, Gowacki J, Bronson RT, Tybulewicz VL,
Kronenberg HM, Mulligan RC 1994 Lethal skeletal dysplasia from
targeted disruption of the parathyroid hormone-related peptide
gene. Genes Dev 8:277–289.
849 GENE EXPRESSION IN THE GROWTH PLATE
14. Iwamoto M, Shimazu A, Pacifici M 1995 Regulation of chondro-
cyte maturation by fibroblast growth factor-2 and parathyroid
hormone. J Orthop Res 13:838–845.
15. Amizuka N, Warshawsky H, Henderson JD, Goltzman D, Karaplis
AC 1994 Parathyroid hormone-related peptide-depleted mice show
abnormal epiphyseal cartilage development and altered endochon-
dral bone formation. J Cell Biol 126:1611–1623.
16. Weir EC, Philbrick WM, Amling LA, Neff LA, Baron R, Broadus
AE 1996 Targeted overexpression of parathyroid hormone-related
peptide in chondrocytes causes chondrodysplasia and delayed en-
docondral bone formation. Proc Natl Acad Sci USA 93:10240–
17. Lanske B, Karaplis AC, Lee K, Luz A, Vortkamp A, Pirro A,
Karperien M, Defize LHK, Ho C, Mulligan RC, Abou-Samra AB,
Juppner H, Segre GV, Kronenberg HM 1996 PTH/PTHrP receptor
in early development and Indian hedgehog-regulated bone growth.
18. Yoshida E, Noshiro M, Kawamoto T, Tsutsumi S, Kuruta Y, Kato
Y 2001 Direct inhibition of Indian hedgehog expression by para-
thyroid hormone (PTH)/PTH-related peptide and upregulation of
retinoic acid in growth plate chondrocyte cultures. Exp Cell Res
19. St-Jacques B, Hammerschmidt M, McMahon AP 1999 Indian
hedgehog signaling regulates proliferation and differentiation of
chondrocytes and is essential for bone formation. Genes Dev
20. Roberts AB, Sporn MB 1993 Physiological actions and clinical
applications of transforming growth factor-? (TGF?). Growth
21. Gibson G 1998 Active role of chondrocyte apoptosis in endochon-
dral ossification. Microsc Res Tech 43:191–204.
22. Yang X, Chen L, Xu X, Li C, Huang C, Deng CX 2001 TGF-
beta-Smad3 signals repress chondrocyte hypertrophic differentia-
tion and are required for maintaining articular cartilage. J Cell Biol
23. Serra R, Karaplis A, Sohn P 1999 Parathyroid hormone-related
peptide (PTHrP)-dependent and independent effects of transform-
ing growth factor ? (TGF?) on endochondral bone formation.
J Cell Biol 145:783–794.
24. Terkeltaub RA, Johnson K, Rohnow D, Goomer R, Burton D,
Deftos LJ 1998 Bone morphogenetic proteins and bFGF exert
opposing regulatory effects on PTHrP expression and inorganic
pyrophosphate elaboration in immortalized murine endochondral
hypertrophic chondrocytes (MCT cells). J Bone Miner Res 13:
25. Pateder DB, Rosier RN, Schwartz EM, Reynolds PR, Puzas JE,
D’Souza M, O’Keefe RJ 2000 PTHrP expression in chondrocytes,
regulation by TGF-?, and interaction between epiphyseal and
growth plate chondrocytes. Exp Cell Res 256:555–562.
26. Szuts V, Mollers U, Bittner K, Schurmann G, Muratoglu S, Deak
F, Kiss I, Bruckner P 1998 Terminal differentiation of chondro-
cytes is arrested at distinct stages identified by their expression
repertoire of marker genes. Matrix Biol 17:435–448.
27. Zhao Q, Eberspaecher H, Lefebvre V, deCrombrugghe B 1997
Parallel expression of Sox9 and COL2A1 in cells undergoing
chondrogenesis. Dev Dyn 209:377–386.
28. DeCrombrugghe B, LefebvreV, Behringer RR, Bi W, Murakami S,
Huang W 2000 Transcriptional mechanisms of chondrocyte dif-
ferentiation. Matrix Biol 19:389–394.
29. Bi W, Huang W, Whitworth DJ, Deng JM, Zhang Z, Behringer
RR, de Crombrugghe B 2001 Haploinsufficiency of Sox9 results in
defective cartilage primarodia and premature skeletal mineraliza-
tion. Proc Natl Acad Sci USA 98:6698–6703.
30. Rodan G, Harada S 1997 The missing bone. Cell 89:677–680.
31. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Daguchi
K, Shimuzu Y, Bronson RT, Gao YH, Inada M, Sato M, Koamoto
R, Kitamura Y, Ytoshiki S, Kishimoto T 1997 Targeted disruption
of Cbfa1 results in a complete lack of bone formation owing to
maturation arrest of osteoblasts. Cell 89:775–764.
32. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G 1997
Osf2/Cbfa1: A transcriptional activator of osteoblast differentia-
tion. Cell 80:371–378.
33. Komori T 2000 A fundamental transcription factor for bone and
cartilage. Biochem Biophys Res Commun 276:813–816.
34. Takeda S, Bonnamy J-P, Owen MJ, Ducy P, Karsenty G 2001
Continuous expression of Cbfa2 in non-hypertrophic chondrocytes
uncovers its ability to induce hypertrophic chondrocyte differenti-
ation and partially rescues Cbfa1-deficient mice. Genes Dev 15:
35. Ueta C, Iwamoto M, Katanami N, Yoshida C, Liu Y, Enomoto-
Iwamoto M, Ohmori T, Enomoto H, Nakata K, Takada K, Kurisu
K, Komori T 2001 Skeletal malformations caused by overexpres-
sion of Cbfa1 or its dominant negative form in chondrocytes. J Cell
36. Porte D, Tuckermann J, Becker M, Baumann B, Teurich S, Hig-
gins T, Owen MJ, Schorpp-Kistner M, Angel P 1999 Both AP-1
and Cbfa1-like factors are required for the induction of interstitial
collagenase by parathyroid hormone. Oncogene 18:667–678.
37. Jimenez MJG, Balbin M, Lopez JM, Alvarez J, Komori T, Lopez-
Otin C 1999 Collagenase-3 is a target of Cbfa1, a transcription
factor of runt gene family involved in bone formation. Mol Cell
38. Ng LJ, Wheatley S, Muscat GEO, Conway-Campbell J, Bowles J,
Wright E, Bell DM, Tam PPI, Cheah KSE, Koopman P 1997 Sox9
binds DNA, activates transcription, and coexpresses with type II
collagen during chondrogenesis in the mouse. Dev Biol 183:108–
39. Alini M, Matsui Y, Dodge GR, Poole AR 1992 The extracellular
matrix of cartilage in the growth plate before and during calcifi-
cation: Changes in the composition and degradation of type II
collagen. Calcif Tissue Int 50:327–335.
40. Pal S, Tang LH, Choi H, Haberman LC, Roughley PJ, Poole AR
1981 Structural changes during development in bovine fetal epi-
physeal cartilage. Collagen Relat Res 1:151–176.
41. Chomczynski P, Sacchi N 1987 Single step method of RNA
isolation by acid guanidiumthiocyanate-phenol-chloroform extrac-
tion. Anal Biochem 162:156–159.
42. Lian JB, McKee MD, Todd AM, Gerstenfeld LC 1993 Induction of
bone-related proteins, osteocalcin and osteopontin and their matrix
ultrastructural localization with development of chondrocyte hy-
pertrophy in vitro. J Cell Biochem 52:206–219.
43. Sandberg M, Vuorio E 1987 Localization of types I. II and III
collagen mRNAs in developing human skeletal tissue by in situ
hybridization. J Cell Biol 104:1077–1084.
44. Pullig O, Weseloh G, Ronneberger D-L, Kakonen SM, Swoboda B
2000 Chondrocyte differentiation in human osteoarthritis: Expres-
sion of osteocalcin in normal and osteoarthritic cartilage and bone.
Calcif Tissue Int 67:230–240.
45. Alvarez J, Balbin M, Santos F, Fernandez M, Ferrando S, Lopez
JM 2000 Different bone growth rates are associated with changes
in the expression pattern of types II and X collagens and collage-
nase 3 in proximal growth plates of the rat tibia. J Bone Miner Res
46. Huang W, Chung U, Kronenberg HM, Crombrugghe B 2001 The
chondrogenic transcription factor Sox9 is a target of signalling by
the parathyroid hormone-related peptide in the growth plate of
endochondral bones. Proc Natl Acad Sci USA 98:160–165.
47. Kim IS, Otto F, Zabel B, Mundlos S 1999 Regulation of chondro-
cyte differentiation by Cbfa1. Mech Dev 80:159–170.
48. Inada M, Yasui T, Nomura S, Miyake S, Deguchi K, Himeno M,
Sato M, Yamagiwa H, Kimura T, Yasui N, Ochi T, Endo N,
Kitamura Y, Kishimoto T, Komori T 1999 Maturational distur-
bance of chondrocytes in Cbfa1-deficient mice. Dev Dyn 214:
49. Enomoto H, Enomoto-Ivomoto M, Iwamoto M, Nomura S, Him-
eno M, Kitamura Y, Kishimoto T, Komori T 2000 Cbfa1 is a
positive regulatory factor in chondrocyte maturation. J Biol Chem
50. Gack S, Vallon R, Schmidt J, Grigoriadis A, Tuckermann J,
Shenkel J, Weiher H, Wagner E, Angel P 1995 Expression of
interstitial collagenase during skeletal development of the mouse is
restricted to osteoblast-like cells and hypertrophic chondrocytes.
Cell Growth Differ 6:759–767.
51. Mattot V, Raes MB, Henriel P, Eeckhout Y, Stehelin D, Vanden-
bunder B, Desblends X 1995 Expression of interstitial collagenase
is restricted to skeletal tissue during mouse embryogenesis. J Cell
52. Johansson N, Saarialho-Kere Y, Airiola C, Herva R, Nissinen L,
Collagenase-3 (MMP-13) is expressed by hypertrophic chondro-
cytes, periosteal cells, and osteoblast during human fetal bone
development. Dev Dyn 208:387–397.
53. Stahle-Backdahl M, Sandstedt B, Bruce K, Lindahl A, Jimenez
MJ, Lopez-Otin C 1997 Collagenase-3 (MMP-13) is expressed
Heino J, Kahari VM1997
850TCHETINA ET AL.
during human fetal ossification and re-expressed in postnatal bone Download full-text
remodelling and rheumatoid arthritis. Lab Invest 76:717–728.
54. Sakiyama H, Inaba N, Toyoguchi T, Okada Y, Matsumoto M,
Moriya Y, Ohtsu H 1994 Immunolocalization of complement C1s
and matrix metalloproteinase 9 (92 kDa gelatinase/type IV colla-
genase) in the primary ossification of centre of human femur. Cell
Tissue Res 277:239–245.
55. Kawashima-Ohya Y, Satakeda H, Kuruta Y, Satakeda H, Kuruta
Y, Yan W, Akagawa Y, Hawakawa T, Noshiro M, Okada Y,
Nakamuro S, Kato Y 1998 Effects of parathyroid hormone
(PTH) and PTH-related peptide on expression of matrix metall-
proteinase-2, -3, and –9 in growth plate chondrocyte cultures.
56. Vu TH, Shipley JM, Bergers G, Berger JE, Helms JA, Hanahan D,
Shapiro SD, Senior RM, Werb Z 1998 MMP-9/gelatinase B is a
key regulator of growth plate angiogenesis and apoptosis of hy-
pertrophic chondrocytes. Cell 93:411–422.
57. Zerath E, Holy X, Mouillon JM, Farbox B, Machwate M, Andre C,
Renault S, Marie PJ 1997 TGF?2prevent impaired chondrocyte
proliferation induced by unloading in growth plates of young rats.
Life Sci 24:2397–2406.
58. Farquharson C, Jeffries D, Seawright T, Houston B 2001 Regula-
tion of chondrocyte terminal differentiation in the postembryonic
growth plate: The role of the PTHrP-Indian hedgehog axis. Endo-
59. Zhang R, Simmon DJ 1996 Transforming growth factor-?2mRNA
level in unloaded bone analyzed by quantitative in situ hybridiza-
tion. J Bone Miner Res 11:S322.
60. D’Angelo M, Pacifici M 1997 Articular chondrocytes produce
factors that inhibit maturation of sternal chondrocytes in serum-
free agarose cultures: A TGF? independent process. J Bone Miner
61. Horner A, Kemp P, Summers C, Bord S, Bishop NJ, Kelsaall AW,
Coleman N, Compston JE 1998 Expression and distribution of
transforming growth factor-? isoforms and their signalling recep-
tors in growing human bone. Bone 23:95–102.
62. Akiyama H, Shigeno C, Hiraki Y, Shikunami C, Kohno H, Akagi
M, Konishi J, Nakamura T 1997 Cloning of a mouse smoothened
cDNA and expression patterns of hedgehog signalling molecule
during chondrogenesis and cartilage differentiation in clonal
mouse EC cells ATDC5. Biochem Biophys Res Commun 235:
63. Iwasaki M, Jikko A, Le AX 1999 Age-dependent effects of hedge-
hog protein on chondrocytes. J Bone Joint Surg Br 81:1076–1082.
64. Selvamurugan N, Chou WY, Pearman AR, Palumanti MR, Par-
tridge N 1998 Parathyroid hormone regulates the rat collagenase-3
promoter in osteoblastic cells through the cooperative interaction
of the activator protein-1 site and the runt domain binding se-
quence. J Biol Chem 273:10647–10657.
65. Uria JA, Jimenez MG, Balbin M, Freije JMP, Lopez-Otin C 1998
Differential affects of TGF? on the expression of collagenase-1
and collagenase-3 in human fibroblasts. J Biol Chem 273:9769–
66. Akiyama H, Shigeno C, Iyama K, Ito H, Hiraki Y, Konishi J,
Nakamuro T 1999 Indian hedgehog in the late-phase differentia-
tion in mouse chondrogenic EC cells ATDC5: Upregulation of
type X collagen and osteoprotegerin ligand mRNAs. Biochem
Biophys Res Commun 257:239–245.
67. Wroblewski J, Edwall-Arvidsson C 1995 Inhibitory effects of
basic fibroblast growth factor on chondrocyte differentiation.
J Bone Miner Res 10:735–742.
68. Mancilla EE, DeLuca JA, Uyeda JA, Czerwiec FS, Baron J 1998
Effects of fibroblast growth factor-2 on longitudinal bone growth.
69. Uria JA, Balbin M, Lopez JM, Alvarez J, Vizoso F, Takigawa M,
Lopez-Otin C 1998 Collagenase-3 (MMP-13) expression in chon-
drosarcoma cells and its regulation by basic fibroblast growth
factor. Am J Pathol 153:91–101.
70. Borden P, Solymar D, Sucharczuk A, Lindman B, Cannon P,
Heller RA 1996 Cytokine control of interstitial collagenase and
collagenase-3 gene expression in human chondrocytes. J Biol
71. Glansbeek HL, van Beuningen HM, Vitters EL, van der Kraan P,
van den Berg WB 1998 Stimulation of articular cartilage repair in
established arthritis by local administration of transforming growth
factor-? into murine knee joint. Lab Invest 78:133–142.
72. Morales TI, Roberts AB 1988 Transforming growth factor ?
regulates the metabolism of proteoglycans in bovine cartilage
organ cultures. J Biol Chem 263:12828–12831.
Address reprint requests to:
Elena Tchetina, PhD
Joint Diseases Laboratory
Shriners Hospitals for Children
1529 Cedar Avenue
Quebec H3G 1A6, Canada
Received in original form April 29, 2002; in revised form October
23, 2002; accepted November 15, 2002.
851 GENE EXPRESSION IN THE GROWTH PLATE