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Collagen XXIV (Col24a1) Gene Expression is a SpecificMarker of Osteoblast Differentiation and Bone
Formation

Noritaka Matsuo ab; Shizuko Tanaka a; Hidekatsu Yoshioka b; Manuel Koch c;
Marion K. Gordon d; Francesco Ramirez eaResearch Division of the Hospital for Special Surgery, Laboratory of Genetics andOrganogenesis, New York, New York, USAb
Department of Anatomy, Biology, and Medicine, Oita University, Oita, Japanc
Institute of Biochemistry II, University of Cologne, Cologne, GermanydEnvironmental and Occupational Health Institute and Department of Pharmacology
and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway,
New Jersey, USAeChild Health Institute of New Jersey, Robert W. Johnson Medical School, New
Brunswick, New Jersey, USA
Online Publication Date: 01 March 2008
To cite this Article: Matsuo, Noritaka, Tanaka, Shizuko, Yoshioka, Hidekatsu, Koch, Manuel, Gordon, Marion K. andRamirez, Francesco (2008) 'Collagen XXIV (Col24a1) Gene Expression is a Specific Marker of Osteoblast Differentiation
and Bone Formation', Connective Tissue Research, 49:2, 68 - 75
To link to this article: DOI: 10.1080/03008200801913502
URL: http://dx.doi.org/10.1080/03008200801913502

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Connective Tissue Research, 49:68–75, 2008
c Informa Healthcare USA, Inc.
Copyright 
ISSN: 0300-8207 print / 1521-0456 online
DOI: 10.1080/03008200801913502

Collagen XXIV (Col24a1) Gene Expression is a SpecificMarker of Osteoblast Differentiation and Bone Formation
Noritaka Matsuo
Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery,
New York, New York, USA; and Department of Anatomy, Biology, and Medicine, Oita University,
Oita, Japan

Shizuko Tanaka
Laboratory of Genetics and Organogenesis, Research Division of the Hospital for Special Surgery,
New York, New York, USA

Hidekatsu Yoshioka
Department of Anatomy, Biology and Medicine, Oita University, Oita, Japan

Manuel Koch
Institute of Biochemistry II, University of Cologne, Cologne, Germany

Marion K. Gordon
Environmental and Occupational Health Institute and Department of Pharmacology and Toxicology,
Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey, USA

Francesco Ramirez
Child Health Institute of New Jersey, Robert W. Johnson Medical School, New Brunswick,
New Jersey, USA

INTRODUCTION
The collagens represent a very large family of extracellular
proteins that impart structural integrity to the connective tissueof virtually every organ system. The collagen family comprises
28 different α-chain trimers or types that give rise to alarge variety of suprastructures, such as fibrils, filaments, andnetworks [1, 2]. Fibril forming collagens are the most abundant
and ubiquitously distributed members of the collagen family.
They share a common primary structure, which consists ofa triple helical domain flanked at both ends by cysteine-richglobular peptides, and are widely expressed in soft and hardtissues (types I, III, and V) or predominantly in cartilage (types
II and XI).
Fibrillar collagens traditionally have been classified into
major (types I, II, and III) and minor (types V and XI) collagentypes based on their relative abundance in the connective tissues[1]. More recently, phylogenetic considerations have segregated
the fibrillar collagen sub units into a type A clade (α1(I),α2(I), α1(II), and α2(V) chains); a type B clade (α1(V), α3(V),α(XI), and α2(XI) chains); and a type C clade α1(XXIV) andα1(XXVII) chains) [3, 4]. Members of the last clade display
structural features of invertebrate collagens and are predicted

Collagen XXIV is an ill-characterized fibrillar collagen thatis predominantly expressed in the forming skeleton of the mouseembryo. Here we report that the Col24al gene is constitutivelytranscribed in the trabecular bone and periosteum of the newbornmouse as well. The bone specificity of Col24al was further
documented using three well-characterized cell culture models of
osteoblast differentiation. These in vitro analyses indicated that
Col24al transcription is activated at about the same time as that of
the osteocalcin gene, and gradually increases to eventually plateau
as osteoblasts begin to deposit a mineralizing matrix. These findings
lend further support to the hypothesis that collagen XXIV may be
implicated in the formation of a mineralization-competent bonematrix.
Keywords

Bone, Extracellular Matrix, Fibrillar Collagens,
Mineralization, Osteoblasts

Received 26 September 2007; accepted 13 November 2007; revised
12 November 2007.
Address correspondence to Francesco Ramirez, Department ofPharmacology and Systems Therapeutics, Mount Sinai School of
Medicine, One Gustave L. Levy Place, Box 1603, New York,NY 10029, USA. E-mail: francesco.ramirez@mssm.edu

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COLLAGEN XXIV GENE EXPRESSION IN BONE

FIG. 1. Col24al gene expression in the bone of newborn mice. (A) Dark-field micrographs of longitudinal sections of P4 tibias hybridized with indicated
35 S-labeled riboprobes. (B) Individual and merged images of longitudinal section of P4 tibias hybridized to Col24al and Col10al probes.

FIG. 2. Osteoblast-specific expression of Col24al. (A) RT-PCR screening of indicated transcripts in ROS17/2.8 (lane 1), ROS25 (lane 2), MC3T3-E1 (lane3), C3H10T1/2 (lane 4) osteoblastic cell lines, NIH-3T3 fibroblasts (lane 5), and primary MCC cultures (lane 6). Col24al expression was evaluated using 30
or 35 cycles of RT-PCR amplification, whereas 25 cycles were used for the other genes. (B) Semi quantitative estimates of Col24al transcripts in the indicatedosteoblastic cell lines performed using the indicated concentrations (fM) of competitor DNA.

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N. MATSUO ET AL.

[13, 14]. The present study therefore was designed to advanceknowledge of collagen XXIV function in bone by further
detailing the expression of the corresponding mouse gene(Col24al) in the postnatal bone and in established models ofin vitro osteoblastogenesis. The results of these analyses further
support our original hypothesis that collagen XXIV might be
implicated in the formation of a mineralization-competent bonematrix.

MATERIALS AND METHODS

FIG. 3. BMP-2 induction of Col24al transcription in differentiating
C3H10T1/2 cells. (A) RT-PCR analyses of indicated mRNAs in C3H10T1/2
cells stimulated to differentiate for 6 days with the indicated concentrations(ng/ml) of BMP-2. Transcripts were PCR-amplified for 35 cycles (Col24al)or 25 cycles (other genes) and resulting products were visualized by Southernblot hybridization or ethidium bromide staining, respectively. (B) The same
experimental conditions were used to visualized PCR-amplified products from
C3H10T1/2 cells that were incubated with 200 ng/ml of BMP-2 for the indicateddays.

to form homotrimes (collagens XXIV and XXVII) that are
predominantly expressed in the forming skeleton with mutually
exclusive patterns [5–8].
These findings were originally interpreted to indicate thatcollagens XXIV and XXVII might play comparable roles in cartilage and bone tissue, respectively, by regulating collagen I andII fibrillogenesis and/or matrix calcification and mineralization[6]. Indeed, recent studies have indicated that collagen XXVII
forms morphologically distinct assemblies that accumulate in
the pericellular matrix of the growth plate, concurrently withcartilage calcification during endochondral bone formation [9,10].
Osteoblasts play a central role in both intramembranousand endochondral ossification by depositing a mineralizationcompetent extracellular matrix that includes heterotypic collagens I, III, and V fibrils [11, 12]. Mutations in the subunitsof these major and minor collagen types have underscoredtheir importance in skeletal physiology by associating thestructurally impaired matrix with perturbed bone growth andintegrity [1, 2]. Primary osteoblasts or osteoblastic cell lines
have been used traditionally for the in vitro study of themolecular factors and cellular mechanisms responsible for or
associated with osteoblast differentiation and bone function

Cell Cultures
Rat osteosarcoma ROS17/2.8 and ROS25 cells, mouse preosteoblast MC3T3-E1 cells, mouse C3H10T1/2 mesenchymal
cells, mouse calvarial cells (MCCs), and mouse NIH-T3T
fibroblasts were used in this study. ROS17/2.8, ROS25, andNIH3T3 cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 10% heat-inactivated fetal bovine
serum in a humidified 5% CO2 and 95% air. MC3T3-E1
and C3H10T1/2 cells were cultured in Minimum Essential
Medium-α (α-MEM) supplemented with 10% heat-inactivatedfetal bovine serum in a humidified 5% CO2 and 95% air. MCC
were prepared from 4-day-old mice and cultured in α-MEM
supplemented with 10% heat-inactivated fetal bovine serum ina humidified 8% CO2 and 92% air [8]. Cells were seeded at thedensity of 1 × 106 cells/100 mm dish.
After reaching confluency, the growth medium was replacedby differentiation medium consisting of 5% FBS/α-MEM,
100 ng/ml bone morphogenetic protein (BM)-2, 10 mM βTglycerophosphate, and 50 µg/ml ascorbic acid (C3H10T1/2
differentiation medium), or 10% FBS/α-MEM, 10 mM βTglycerophosphate, and 50 µg/ml ascorbic acid (MC3T3-E1
and MCC differentiation medium) [15–17]. MCC cultures were
stained with Alizarin red or von Kossa to visualize calcium
deposits or phosphate deposits, respectively [17].
RNA Analyses
Total RNA was isolated from cultured cells using
a commercial kit (Invitrogen) according to the manufacturer’s recommendations. Reverse transcriptions werecarried out using Omniscript reverse transcriptase (Qiagen GmbH) with oligo(dT) primer, and the resulting single-stranded cDNA molecules were PCR amplified using primers specific for Col24al (forward primer,
5 -ATGCATTTAGGAGCCTACAG-3 ; reverse primer, 5 TCACGAGAGGTGACTCAATA 3 ), Colla2 (forward primer,
5 -TGGTCCTCTGGGCATCTCAGGC-3 ; reverse primer, 5 GGTGAACCTGCTGTTGCCCTCA-3 ), osteocalcin (Oc) (forward primer, 5 -ATGAGGACCCTCTCTCTGCT-3 ; reverse
primer, 5 -GGAGCTGCTGTGACATCCAT-3 ), or GAPDH(forward primer, 5 -ACCACAGTCCATGCCATCAC-3 ; reverse primer, 5 -TCCACCACCCTGTTGCTGTA-3 ).

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FIG. 4. Col24al accumulation in differentiating MC3T3-E1 cells. (A) RT-PCR analysis of the indicated mRNAs in cells cultured for the indicated days;
amplification was 35 cycles for Col24al and 25 cycles for the other genes. (B) Semi quantitative estimates of indicated transcripts performed using the indicatedconcentrations (fM or pM) of competitor DNA. (C) Bar graphs represent the quantification of the amplified products in (B) whose intensity was evaluated with theNIH ImageJ software. Results represent the means ± SE (n = 3).

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N. MATSUO ET AL.

Amplification conditions included a cycle at 94◦ GC for
2 min followed by 25 cycles (Colla2, Oc, and GAPDH) and35 cycles (Col24al) at 94◦ C for 30 sec, 56◦ C for 1 min, and72◦ C for 1.5 min, and a final elongation cycle at 72◦ C for 5 min.
For semiquantitative PCR analysis, competitor DNA was added
to each experimental sample at the indicated concentrations
and the resulting products were normalized against GAPDHlevels. PCR-amplified products were separated by 2% agarose
gel electrophoresis, and observed under ultraviolet illumination
after staining with ethidium bromide. To estimate the degree of
each transcript, relevant bands were quantified using the NIH
ImageJ software.
Three independent experiments were performed in duplicate
to estimate the relative amount of each gene product at
a given competitor concentration. The resulting data wereexpressed as the mean ± SE. In the case of C3H10T1/2
cells, Col24al transcripts could only be visualized by Southernblot hybridization of the PCR-amplified products to the same
32
P-labeled sequence.

In Situ Hybridizations
Mouse tibias from 4-day-old mice were fixed inparaformaldehyde and embedded in to prepare frozen sections

for in situ hybridizations to 35 S-labeled riboprobes [6]. Photoshop 7.0 imaging software (Adobe) was used for postprocessingof the in situ hybridization data.
RESULTS
We previously reported that onset of Col24al transcription in
the emerging skeletal elements of the developing mouse embryocommences at about E14.5 [6]. The study did not, however,address the possibility that Col24al expression may persist
post natally in bone tissue. In situ hybridizations therefore
were performed to examine Col24al expression in the growing
tibias of P4 mice. Other tissue-specific probes used in theanalysis included the extracellular matrix products of osteoblasts(α2(I) collagen [Colla2], and osteocalcin [Oc]), resting anddifferentiating chondrocytes (α1(II) collagen [Col2al]), andhyperthrophic chondrocytes (α1(X) collagen [Col10al]). The insitu hybridizations detected strong Col24al signals in trabecularbone and periosteum, colocalized with Colla2 and Oc transcripts(Figure 1A). Col24al signals were instead absent in cartilage,
which displayed the characteristic pattern of Col2al and Col10al
positive cells in distinct regions of the growth plate (Figure1A). In point of fact, the trabecular bone-specific expressionof Col24al was more evident in a merged image showing
no overlap with the Col10al-positive hyperthrophic cartilage

FIG. 5. Col24al expression in differentiating MCC cultures. (A) RT-PCR analysis of the indicated mRNAs in cells cultured for the indicated days; amplification
was 35 cycles for Col24al and 25 cycles for the other genes. (B) Semi quantitative estimates of indicated transcripts performed using the indicated concentrations(fM or pM) of competitor DNA. (C) Bar graphs represent the quantification of the amplified products in (B) whose intensity was evaluated with the NIH ImageJsoftware. Results represent the means ± SE (n = 3). (D) Parallel determination of MCC terminal differentiation by assessing formation of mineral deposits using
Alizarin red or von Kossa staining; untreated MCC cultures are shown in day 12 (-) samples. (Continued)

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FIG. 5.

(Figure 1B). Similar data were obtained with adult bones (datanot shown), thus demonstrating that Col24al is continuously
expressed by osteoblasts during postnatal bone growth andremodeling.
The in vivo findings were re-examined in vitro by comparing
the dynamics of Col24al gene expression in several different
models of osteoblastogenesis. First, Col24al transcript levels
were evaluated by RT-PCR amplification of RNA purified
from undifferentiated cell lines representing different stagesof osteoblast differentiation (ROS17/2.8, ROS25, MC3T3-E1,
and C3H10T1/2 cells), in undifferentiated primary mouse
calvarial cells (MCC), and in NIH-3T3 fibroblasts as well.
Amplification of Colla2 and Oc transcripts also was included ineach experimental sample as markers of early and middle/late(Continued).

differentiation, respectively, while GAPDH was used as thenormalizing control.
The RT-PCR screen detected Col24al transcripts only in
ROS17/2.8, which correspond to mature osteoblasts, in MC3T3E1, which are preosteoblastic cells, and in MCC, which
represent an heterogeneous population of pre osteoblast cells[13, 16, 17] (Figure 2A). Semi quantitative RT-PCR analyses
determined that the concentration of Col24al transcripts is about
0.5 fM in ROS17/2.8 cells, and 0.01 fM in MC3T3-E1 cells(Figure 2B). MC3T3-E1 and MCC therefore were selected tomonitor Col24al mRNA accumulation during ostoblastogenesis; pluripotent C3H10T1/2 cells induced to differentiate along
the osteoblastic lineage were included in this analysis as well[18, 19].

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C3H10T1/2 cells were induced to differentiate into preosteoblasts by administration of increasing amounts of BMP-2
ranging from 62.5 to 500 ng/ml. In contrast to untreatedcells, BMP-2 slightly stimulated Col24al transcription at aconcentration of 250 ng/ml, and markedly increased it ata concentration of 500 ng/ml (Figure 3A). By contrast, Oc
induction was first noted at a lower BMP-2 concentration (62.5
ng/ml), a finding that probably reflects the higher levels of Oc
transcripts relative to those of Col24al (Figure 3A). Indeed
Col24al expression in this model of osteoblast differentiation
was so low that it could only be detected by Southern blot
hybridization of the PCR-amplified products, thus impedinga reliable quantification of the relative amounts of the twotranscripts. This last point notwithstanding, a time-course
analysis of BMP-2-induced differentiation of C3H10T1/2 cells(200 ng/ml) strongly suggested that transcription of Oc andCol24al is stimulated at about the same time (day 2), and that therespective transcripts accumulate thereafter at comparable rates(Figure 3B). These results therefore established that Col24al isa BMP-2 inducible gene in osteoblastogenic C3H10T1/2 cells.
Consistent with the above data, Col24al transcripts were
found to increase gradually in differentiating MC3T3-E1 cells
and concomitantly with Oc mRNA accumulation Figure 4A).
Semi-quantitative analyses revealed that Col24al and Oc display
distinct patterns of gene expression in differentiating MC3T3E1 cells, in that transcripts from the latter gene plateau earlier
than those of the former gene (day 3 versus 9, respectively; Figures 4B and 4C). They also confirmed that the absolute amountof Col24al transcripts between days 9 and 15 is very low, notably
about 1.5 × 104 and 103 -fold less than Col2al and Oc transcripts,
respectively (Figures 4B and 4C). This last finding reconciles
the difficulty of Col24al detection in differentiating C3H10T1/2,
as these cells are more heterogeneous and mostly correspond to
the earlier stage of progenitor osteoblast commitment (Figure 3).
The MC3T3-E1 data therefore indicated that Col24al is a low
expressed middle/late marker of osteoblast differentiation.
The last set of in vitro experiments employed MCC to validate
the above conclusions in a primary cell culture system that
replicates terminal differentiation of osteoblasts, as evidenced
by the formation of mineral nodules [17]. Accordingly, Col24al,Col1a2, and Oc mRNA levels in MCC cultures were evaluated inparallel with the emergence of calcium and phosphate deposits(by Alizarin red and von Kossa staining, respectively) (Figure 5).
The analyses revealed that, similar to Colla2 and Oc, Col24al
is continuously expressed through the terminal differentiation
process of primary MCC (Figures 5A–C). They also showedsome differences between differentiating MC3T3-E1 and MCC
cultures with respect to the accumulation patterns of Col24al andOc transcripts (Figures 4C and 5C). This apparent discrepancy
is likely to reflect intrinsic differences in the differentiation
properties of these two cellular models of osteoblastogenesis[16, 17]. However, the same-fold differences were noted in theabsolute amounts of Col24al, Oc, and Col2al transcripts between
late differentiating MC3T3-E1 and MCC cultures (Figure 4C).

We therefore concluded that together the in vivo and in vitro data
indicated that Col24al is constitutively expressed by osteoblasts
during bone formation.
DISCUSSION
Collagen XXIV is a new member of the fibrillar collagengroup that exhibits structural features of invertebrate fibrillar
collagens [6]. Unlike collagen XXVII, the other member of
the C clade of invertebrate-like fibrillar collagens, collagen
XXIV is predominantly, if not exclusively, expressed in theforming bone elements of the mouse embryo [6]. Furthermore,cell type-specific transcription of Col24al is driven by nuclear
factors implicated in the regulation of osteoblastogenesis [8].
The present study provides additional evidence for a potential
role of collagen XXIV in bone physiology. The evidence
includes in vitro experiments showing that Col24al expressionin pluripotent C3H10T1/2 cells is induced by BMP-2, and that
Col24al transcripts accumulate progressively and concomitantlywith those of Oc in differentiating MC3T3-E1 and MCC cultures. We also showed that Col24al is constitutively expressedin mineralizing primary osteoblast cultures and in the bone ofadult mice.
Furthermore, the analyses enabled us to estimate thatcollagen XXIV is the lowest represented collagen moleculein bone. We argue from these correlative lines of evidence
that deposition of collagen XXIV into the bony matrix may
participate in establishing the structural requirements for tissue
mineralization. Our contention is supported by preliminary data
indicating that osteopenia characterizes the otherwise-normal
Col24al null mice.
The extracellular matrix, and fibrillar collagen assemblies
in particular, play a key role in regulating bone mineralization
and bone integrity [20]. Mutations in the subunits of collagens
I and V or in collagen I-modifying enzymes have been
associated with significant bone abnormalities and increased
risk of fractures (2, 21, 22). Moreover there is emerging
evidence from the oim/oim mouse that abnormal collagenI assembly also may perturb the activity of resident cells
through an ill-defined mechanism, thus exacerbating the lossof bone integrity [23]. Additional studies from geneticallytargeted mice have implicated nonstructural components of theextracellular matrix (biglycan, osteocalcin, and osteonectin) in
the control of bone mass during physiological growth and adultremodeling through the control of local and systemic signals of
osteoregulation [24–28]. In spite of its low abundance, collagen
XXIV may represent yet another matrix regulator of bone mass
maintenance.
There are numerous examples of severe pathologies associated with loss of function of minor collagen types [1, 2]. A
case in point is collagen XIX, the least abundant of all collagentypes, whose loss in mice impairs muscle differentiation andesophageal function, and leads to perinatal lethality [29].
Ongoing investigations are aimed at elucidating the precise roleof collagen XXIV in bone physiology by characterizing cellularDownloaded By: [Ramirez, Francesco] At: 00:41 7 April 2008

COLLAGEN XXIV GENE EXPRESSION IN BONE

and/or structural mechanisms responsible for reduced bone massin Col24al-deficient mice.

ACKNOWLEDGMENTS
We thank Bayan Sudan for excellent technical support, andKaren Johnson for organizing the manuscript. We dedicate
the study to the memory of Shizuko Tanaka. This work was
supported by NIH grant AR-38648.
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