Differential Expressions of BMP Family Genes during

CELL STRUCTURE AND FUNCTION 25: 195–204 (2000)

© 2000 by Japan Society for Cell Biology

195

Differential Expressions of BMP Family Genes during Chondrogenic Differentiation of Mouse ATDC5 Cells

Haruhiko Akiyama1, Chisa Shukunami2, Takashi Nakamura1, and Yuji Hiraki2, �

1Department of Orthopedic Surgery, Graduate School of Medicine, and 2Department of Molecular

Interaction and Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-

8507, Japan

ABSTRACT. Clonal cell line ATDC5 enables the monitoring of the early- and late-phase chondrogenic

differentiation in a single culture. Undifferentiated ATDC5 cells differentiate into type II collagen expressing

chondrocytes through a cellular condensation stage (early-phase differentiation) and then to type X collagen-

expressing hypertrophic chondrocytes (late-phase differentiation). Progression of cellular differentiation was

accelerated by the activation of bone morphogenetic protein (BMP) signaling. ATDC5 cells expressed transcripts

for at least four members of the BMP family. The BMP-4 transcripts were expressed in all stages of differentiation,

as were transcripts for BMP type IA receptor (ALK-3) and BMP type II receptor. In contrast, transcripts for Growth/

Differentiation factor-5 (GDF-5) were induced during a cellular condensation, and those for BMP-6 were induced

during the formation of cartilage nodules, and declined as the differentiated ATDC5 cells became hypertrophic,

and BMP-7 transcripts were only detected after cells became calcified. Exogenously added BMP-4 indeed

promoted the early-phase differentiation. Late-phase differentiation of cells was also stimulated by BMP-4 and

BMP-6. Thus, the cumulative increase in BMP signaling promoted the sequential transitions of differentiation

steps of cells. These results indicate that the coordinated expressions of endogenous BMPs are involved in the

progression of chondrogenic differentiation in ATDC5 cells.

Key words: cartilage differentiation/BMP/ATDC5/endochondral bone formation

The bone morphogenetic protein (BMP) family plays a vital

role in skeletal development and are capable of inducing

ectopic bone formation through an endochondral pathway

which includes a cascade of events such as the recruitment

of progenitor cells, cellular condensation to form cartilagi-

nous bone precursors, vascular invasion, the replacement of

cartilage by bone, and marrow development (Rosen and

Thies, 1992). Several BMP family genes are expressed

at sites of bone formation with a unique spatiotemporal

pattern, suggesting that the coordinated expression of these

molecules is crucial for the cascade of events underlying

endochondral bone formation. Transcripts for BMP-2

and BMP-4 genes are localized to precartilaginous

mesenchymal condensation, and then the perichondrium

(Lyons, et al., 1990). Growth/differentiation factor (GDF)-5

is expressed predominantly in the precartilaginous

mesenchymal condensation and the cartilaginous cores of

the developing long bone (Storm, et al., 1994). In contrast,

BMP-6 and BMP-7 genes are expressed in mature

chondrocytes (Carey and Liu, 1995; Vortkamp, et al., 1996;

Vukicevic, et al., 1994).

In our previous studies (Shukunami, et al., 1997;

Shukunami, et al., 1996), we demonstrated that the clonal

cell line ATDC5 enables the monitoring of the multistep

chondrogenic differentiation in a single culture. In the

presence of insulin, chondrogenic differentiation is induced

to form cartilage nodules through cellular condensation

(early-phase differentiation). When the formation of carti-

lage nodules is completed, the cells are then converted

to type X collagen-expressing hypertrophic chondrocytes

followed by mineralization (late-phase differentiation).

However, insulin by itself induced only a cellular conden-

sation in the cultures of undifferentiated ATDC5 cells.

* To whom correspondence should be addressed.Tel and Fax: �81�75�751�4633E-mail: [email protected]: ActR, activin receptor; ALP, alkaline phosphatase; BMP,

bone morphogenetic protein; FBS, fetal bovine serum; GDF, growth/differ-entiation factor; G3PDH, glyceradehyde 3- phosphate dehydrogenase; OP,osteogenic protein; PTH, parathyroid hormone; RT-PCR, reverse transcrip-tion-polymerase chain reaction.

H. Akiyama et al.

196

Instead, exogenously added BMP-2 directly induced

chondrogenesis by skipping out the condensation stage

(Shukunami, et al., 1998). In order to decipher mechanism

underlying the sequential differentiation of ATDC5 cells,

we studied the temporal patterns during differentiation of

cells in the presence of insulin and the actions of BMP

signaling molecules.

Materials and Methods

Materials

Human recombinant BMP-6 was a generous gift from Dr. J. M.

Wozney (Genetics Institute Inc., Cambridge, MA, USA). Xenopus

recombinant BMP-4 homodimer and BMP-4/7 heterodimer were

kindly provided by Takeda Chemical Industries Ltd. (Osaka,

Japan).

Cell culture

ATDC5 cells were maintained in a medium consisting of a 1:1

mixture of DME and Ham’s F-12 (DME/F12) medium (Flow

Laboratories, Irvine, U.K.) containing 5% fetal bovine serum

(FBS; JRH Biosciences, Lenexa, KS, USA), and ITS, i.e., 10 �g/

ml bovine insulin (I: Wako Pure Chemical, Osaka, Japan), 10 �g/

ml human transferrin (T: Boehringer Mannheim, Gaithersburg,

MD), and 3�10�8 M sodium selenite (S: Sigma, St. Louis,

MO, USA), as previously described (Shukunami, et al., 1997;

Shukunami, et al., 1996). In brief, the inoculum size of the cells

was 6�104 cells/well in 6-multiwell plates (Corning Glass,

Corning, NY, USA) at 37�C under 5% CO2 in air. On day 21, the

culture medium was switched to �MEM containing 5% FBS plus

ITS, and the CO2 concentration was shifted to 3% for the facili-

tation of cellular hypertrophy and mineralization in culture.

RNA extraction and hybridization analysis

Total RNA was prepared from the cultures by the single-step meth-

od of Chomczynski and Sacchi (Chomczynski and Sacchi, 1987).

For northern hybridization, 20 �g of total RNA was denatured with

6% formaldehyde, separated by 1% agarose gel (Seakem GTG,

FMC Bioproducts, Rockland, ME, USA) electrophoresis, and

transferred onto Nytran membranes with Turboblotter (Schleicher

& Schuell, Dassel, Germany). Hybridization was performed over-

night at 42�C with an appropriate cDNA probe (106 cpm/ml) in a

solution containing 50% formamide, 6�SSPE (0.9 M NaCl, 20

mM NaH2PO4, 6 mM EDTA at pH 7.4), 0.2% BSA (Calbiochem-

Novabiochem Co., La Jolla, CA, USA), 0.2% Ficoll 400

(Pharmacia, Uppsala, Sweden), 0.2% polyvinylpyrolidone (Wako

Pure Chemical, Osaka, Japan), 0.1% SDS, and 200 �g/ml dena-

tured salmon sperm DNA. Hybridization probes were prepared by

the random-primer method with a BcaBEST labeling kit (Takara,

Shiga, Japan) using the appropriate cDNA fragments: a 1.4 kb

EcoRI fragment of pKT1180 as a probe for �1(II) collagen mRNA

(Kimura et al., 1987); a 0.65 kb Hind III fragment of pSAm10h as

a probe for �1(X) collagen mRNA (Apte et al., 1992); a 2.5-Kb

EcoRI fragment of rAP54 (Noda et al., 1987) as a probe for

alkaline phosphatase (ALP) mRNA. The following cDNA frag-

ments were amplified by reverse transcription-polymerase chain

reaction (RT-PCR) with a specific primer set as described below,

and used as a probe for northern hybridization: a 0.48 kb fragment

of GDF-5 cDNA; a 0.6 kb fragment of BMP-4 cDNA; a 0.42 kb

fragment of BMP-6 cDNA; a 0.34 kb fragment of ALK-3 cDNA; a

0.93 kb fragment of BMP type II receptor cDNA. After hybridiza-

tion, the filters were washed for 30 min at 55�C in 2�SSPE and

0.1% SDS, then washed for 30 min at 55�C in 0.1�SSPE, 0.1%

SDS, and then exposed to X-OMAT film (Eastman Kodak Co.,

Rochester, NY, USA) at �80�C with a Cronex lightening plus

intensifying screen (DuPont, Boston, MA, USA).

RT-PCR

The RT-PCR was performed as described previously (Shukunami,

et al., 1996). Briefly, first-strand cDNA was synthesized using

SuperScript II RNase H� reverse transcriptase (GIBCO BRL,

Gaithersburg, MD, USA) with total RNA extracted from ATDC5

cells. The PCR conditions were 94�C for 30 sec, 60�C for 1 min,

72�C for 1 min for 30 cycles, and final extension at 72�C for 5

min. The primers specific for mouse glyceraldehyde 3- phosphate

dehydrogenase (G3PDH) gene (Clontech, Palo Alto, CA, USA)

were used as a positive control for cDNA amplification. The fol-

lowing specific primers were used: 5'-CTCTTCAACCTCAG-

CAGCATCC-3' (sense primer) and 5'-TGGCAGTAGAAGGC-

CTGGTAGC-3' (antisense primer) for the detection of BMP-4

mRNA; 5'-TGACACGCAGACACATCTCC-3' (sense primer) and

5'-AGAAGGCACCTCCACAGAGC-3' (antisense primer) for

BMP-6 mRNA; 5'-GCTGAACTGCGGATCTTACG-3' (sense

primer) and 5'-AGCCTTGAGGTTCTTGCTGG-3' (antisense

primer) for GDF-5 mRNA; 5'-GTCGGACCTCTTCTTGCTGG-3'

(sense primer) and 5'-GACCGGATACTACGGAGATGG-3' (anti-

sense primer) for BMP-7 mRNA; 5'-GGCCTTGTCTGCT-

GCTAGTGG-3' (sense primer) and 5'-CTTCACGTCTTCCTG-

CACGC-3' (antisense primer) for type IB activin receptor (ActR-IB

or ALK-4) mRNA; 5'-GGAGACGATGTCACGAGGCC-3' (sense

primer) and 5'-CAGGTCCACATTGGTGACGG-3' (antisense

primer) for type IIB activin receptor (ActR-IIB) mRNA. For the

detection of mRNAs for the Smad family members, the following

primers were used: 5'- CCGTCCAACAATAAGAACCGC-3'

(sense primer) and 5'-ATCGTGGCTCCTTCGTCAGG-3' (anti-

sense primer) for the detection of Smad1 mRNA; 5'-ACCAG-

GTCTCTTGATGGCCG-3' (sense primer) and 5'-TGACGCATG-

GAAGGTCTCTCC-3' (antisense primer) for Madr2 mRNA; 5'-

AGACATTCCACGCCTCACAGC-3' (sense primer) and 5'-CA-

CACTGGAACAGCGGATGCT-3' (antisense primer) for Smad3

mRNA; 5'-CACCAGCACCACCAACTTCC-3' (sense primer) and

5'-CAACCTCGCTCTCTCAATCGC-3' (antisense primer) for

DPC4 mRNA; 5'-GCCGTGAAGCGATTGTTGG-3' (sense prim-

er) and 5'-GCGGTTCATTGTGGCTCAGG-3' (antisense primer)

for Smad5 mRNA; 5'-ACAAGCCACTGGATCTGTCCG-3' (sense

primer) and 5'-GGAGGTGATGAACTGTCGCG-3' (antisense

primer) for Smad6 mRNA; 5'-ATGCTGTACCTTCCTCCGCG-3'

Coordinated Bmp Expression during Chondrogenesis

197

(sense primer) and 5'-CACAGCCGATCTTGCTCCG-3' (antisense

primer) for Smad7 mRNA, and 5'-CAAGTTCCGAAGTGC-

CTCGCT-3' (sense primer) and 5'-GATCTTGCAGACAGTGGC-

CG-3' (antisense primer) for Smad8 mRNA. Aliquots (8 �l) of

each PCR reaction products were resolved on 3% NuSieve 3:1

agarose gels (FMC Bioproducts) alongside the markers. The

amplified products were stained with ethidium bromide. The

amplified PCR products were subcloned pCRII (Invitrogen, San

Diego, CA, USA), and the nucleotide sequences of all cDNA frag-

ments were verified by an ALP Express Sequencer (Amersham-

Pharmacia, Uppsala, Sweden). No products were observed when

the reverse transcriptase was omitted during the synthesis of

cDNA (data not shown).

Alizarin red staining

ATDC5 cells were cultured in DME/F12 medium containing 5%

FBS and ITS for 21 days. Then the culture medium was switched

to �MEM containing 5% FBS and ITS, and the cultures were con-

tinued for an additional 21 days. After a total of 42 days, the cul-

tures were incubated for an additional 8 days with or without 1,000

ng/ml of BMP-7 in �MEM containing 5% FBS and ITS. Cells

were rinsed three times with PBS and fixed with 95% methanol for

30 min. They were stained with 1% alizarin red S (Wako Pure

Chemicals) at pH 6.4 for 5 min and washed with distilled water, as

described (Shukunami, et al., 1997).

Fig. 1. Expression of the BMP family genes during the chondrogenic differentiation of ATDC5 cells. Cells were plated at 6�104 cells per well in 6-

multiwell plates, and maintained in DME/F12 medium containing 5% FBS and ITS. The culture medium was then changed to �MEM containing the same

supplements to facilitate cellular hypertrophy and mineralization on day 21. Total RNA was prepared on the indicated days of culture. In (a), the same mem-

brane filter was hybridized with the probes of mouse BMP-4, mouse GDF-5, mouse BMP-6, rat type II collagen and mouse type X collagen cDNAs. The

equivalent loading of RNA (20 �g/lane) was verified by ethidium bromide staining, as shown in the bottom panel. In (b), reverse-transcribed cDNAs were

amplified by PCR for the detection of BMP-7 mRNA in comparison with G3PDH mRNA. Aliquots (8 �l) of the PCR products were resolved on 3%

agarose gels alongside the markers.

H. Akiyama et al.

198

Results

Expression of BMP-4, BMP-6, BMP-7 and GDF-5 mRNAs during chondrogenic differentiation

The transcripts for the BMP family genes are expressed spa-

tially and temporally at the sites of skeletogenesis such as

limb buds and vertebrae during embryonic development

(Francis West, et al., 1995; Lyons, et al., 1990). ATDC5

cells express type I collagen in the undifferentiated stage.

However, the cells undergo differentiation to become type II

collagen-expressing chondrocytes in the presence of insulin

(Shukunami, et al., 1996). As shown in Fig. 1a, ATDC5

cells expressed transcripts for BMP-4 in the undifferentiated

(day 3) and condensation stages (day 5), prior to conversion

into chondrocytes. When the growth of differentiated cells

ceased by day 21 of culture, cells in cartilage nodules then

undergo late-phase differentiation to become hypertrophic

and calcifying chondrocytes (Shukunami, et al., 1997). The

BMP-4 mRNA level declined as the cartilage nodules

stopped growing after two weeks. The GDF-5 transcripts

were first detected in a cellular condensation stage on day 5

and continuously expressed in these cells, and declined as

differentiated cells began to calcify.

When the regions of cellular condensation are formed in

ATDC5 cell cultures from day 3 to day 6, type II collagen-

expressing chondrocytes are generated in the condensing

regions (Shukunami, et al., 1996). Chondrocytes thus

formed gradually proliferate to expand cartilage nodules in

the culture for another 10 days, as previously reported

(Shukunami, et al., 1996). As the number of chondrocytes

increased during this time period, the type II collagen

mRNA level increased and became maximal on day

14 or later. Type X collagen-expressing hypertrophic

chondrocytes then appeared in cartilage nodules, and mark-

edly increased in number from day 21 when the growth of

chondrocytes in the nodules completely ceased.

Interestingly, the BMP-6 and BMP-7 genes exhibited a

distinct temporal pattern of expression along with the

phenotypic transitions of ATDC5 cells. Transcripts for the

BMP-6 gene were absent until mature chondrocytes

appeared in the culture. The BMP-6 mRNA level increased

in parallel with the induction of type II collagen mRNA lev-

el in the culture, and peaked at around day 14 (Fig. 1a). The

level of BMP-6 mRNA was kept high during the maturation

of chondrocytes (from day 14 to day 21), and then declined

until the cells became calcified. In contrast, transcripts for

the BMP-7 gene were not detected until the cells became

calcified on day 42 or later (Fig. 1b). However, the level of

BMP-7 mRNA was so low as to be detected by northern

blotting.

Expression of BMP receptor mRNA during differenti-ation

As shown in Fig. 2a, ATDC5 cells expressed the transcripts

for the ALK-3 gene, encoding the BMP type IA receptor.

The level of these transcripts did not change significantly

during chondrogenic differentiation of ATDC5 cells. Simi-

larly, transcripts for the BMP type II receptor gene were

detected in all stages of differentiation of ATDC5 cells,

although the level of the transcripts upregulated during

maturation of differentiated chondrocytes from day 10 to

day 21 of culture. However, mRNA for these receptors were

detected in all stages. The transcripts for the BMP type IB

receptor (ALK-6) gene were not detected in the ATDC5

cells (data not shown). Transcripts for the type IB and type

IIB receptors for activin (ActR-IB and ActR-IIB) were also

found in all stages of differentiation of ATDC5 cells (Fig.

2b). ATDC5 cells similarly expressed transcripts for the

type I receptor for activin (ActR-I or ALK-2) (data not

shown).

The Smad family proteins play an important role in the

transduction of BMP signals from the cell-surface receptors

(Heldin, et al., 1996). As shown in Fig. 3, ATDC5 cells

clearly expressed transcripts for the Smad family genes in

all stages of differentiation.

Effect of the treatment with recombinant BMP-4 on the early-phase differentiation in ATDC5 cells

BMP-4 is expressed in undifferentiated ATDC5 cells,

indicating that BMP-4 participates in the early-phase differ-

entiation of these cells. When ATDC5 cells were cultured in

DME/F12 medium containing 5% FBS and ITS for 11 days,

cartilage nodules were apparently formed (Fig. 4a), as

reported previously (Shukunami, et al., 1996). Recombinant

BMP-4 (1,000 ng/ml) was added to the confluent monolayer

culture in the medium containing 5% FBS and ITS on day 5,

and the culture was maintained for additional 6 days. By

skipping out the condensation stage, almost all cells in cul-

ture underwent differentiation and formed a nearly continu-

ous sheet of chondrocytes (Fig. 4a) accompanying alcian

blue-positive matrix. As shown in Fig. 4b, recombinant

BMP-4 apparently induced the expression of type II

collagen gene within 24 hours in a dose dependent manner

in the confluent monolayer culture of ATDC5 cells.

Effect of the treatment with recombinant BMP-4 and BMP-6 on the late-phase differentiation

When type II collagen-expressing differentiated ATDC5

cells cease to grow in cartilage nodules, type X collagen-

expressing cells begin to appear in the nodules (Shukunami,

et al., 1997). We assessed by northern analyses the effects of

recombinant BMP-4 and BMP-6 on the late-phase differen-

tiation of ATDC5 cells. The cells were first cultured in

DME/F12 medium containing 5% FBS and ITS for 21 days


199

until the cells ceased to grow. Culture medium was then re-

placed with fresh medium containing various concentration

of recombinant BMP-4 or BMP-6. Cultures were incubated

for the indicated time periods (Fig. 5). Northern analyses

clearly indicated that both BMP-4 (Fig. 5a) and BMP-6

(Fig. 5b) stimulated the expression of type X collagen

mRNA in the cells in time- and dose-dependent manners.

Effect of the treatment with recombinant BMP-4/7

heterodimer on matrix mineralization

We previously reported that differentiated ATDC5 cells

underwent matrix mineralization in the cartilage nodules as

a consequence of the late-phase differentiation (Shukunami,

et al., 1997). In this stage on day 42 or later, the BMP-7

transcripts were expressed in these cells, indicating that

BMP-4/7 heterodimer may additionally participate in the

progression of the final stage of differentiation in culture.

We assessed the effects of recombinant BMP-4/7 on matrix

mineralization and the expression of ALP mRNA in these

Fig. 2.�Expression of the BMP receptor genes during the chondrogenic differentiation of ATDC5 cells. Cells were plated and grown as described in the

legend to Fig. 1. Total RNA was prepared on the indicated days of culture. In (a), the same membrane filter was hybridized with the probes of mouse ALK-

3, or mouse BMPR-II. The equivalent loading of RNA (20 �g/lane) was verified by ethidium bromide staining, as shown in the bottom panel. In (b), reverse-

transcribed cDNA was amplified by PCR for the detection of ActR-IB or ActR-IIB mRNA in comparison with G3PDH mRNA. Aliquots (8 �l) of the PCR

products were resolved on 3% agarose gels alongside the markers.

H. Akiyama et al.

200

cells. Differentiated cultures of ATDC5 cells were incu-

bated with BMP-4/7 (1,000 ng/ml) from day 42 to day 50.

As shown by alizarin red staining (Fig. 6a), mineralization

was clearly promoted by the treatment. The induction of a

high level of ALP activity in chondrocytes is closely associ-

ated with matrix mineralization (Shukunami, et al., 1997).

When the ATDC5 cell culture reached in the initial calcify-

ing stage on day 42, the culture was further incubated for

the indicated time periods in the presence of various

concentrations of recombinant BMP-4/7. As indicated by

northern blot analysis, the treatment with BMP-4/7 resulted

in an increase of the level of ALP mRNA in a dose-depend-

ent manner within 12 hours (Fig. 6b).

Discussion

ATDC5 cells retain the properties of chondroprogenitor

cells well (Shukunami, et al., 1996). Taking on a fibro-

blastic morphology, undifferentiated cells express the type

I collagen gene transcripts, and they actively proliferate

with a short doubling time of 16 hours. In the presence of

insulin, regions of cellular condensation appear transiently

in the culture, from which proliferating chondrocytes (with

a longer doubling time of 48 hours) are generated to form

cartilage nodules (Shukunami, et al., 1996). When the

formation of cartilage nodules is completed, the cells then

become hypertrophic and calcified as the late-phase differ-

entiation progresses (Shukunami, et al., 1997). These order-

ly transitions of cellular phenotype were positively and

negatively modulated by the exogenous growth and differ-

entiation factors added to the culture (Shukunami, et al.,

1998). Recombinant BMP-2 positively regulated the pheno-

typic transitions during early-phase and late-phase differen-

tiation. Parathyroid hormone (PTH) negatively regulated the

progression of differentiation (Shukunami, et al., 1997;

Shukunami, et al., 1996). These results strongly suggested

the presence of a complex network of endogenous growth/

differentiation signaling molecules underlying the orderly

transition of cellular phenotype in the ATDC5 cultures.

In the developing cartilaginous bone precursors, BMP-4

mRNA is expressed first in the perichondral mesenchymal

cells and late in perichondrium from which undifferentiated

mesenchymal cells are recruited (Wozney, 1993). The BMP-

4 transcripts were constitutively expressed in ATDC5 cells

irrespective of their differentiation stages. However, the

mRNA level slightly decreased prior to the induction of the

late-phase differentiation (day 21 or later). In contrast, the

GDF-5 mRNA were expressed after the onset of cellular

condensation. As shown in Fig. 2, ATDC5 cells also

expressed the BMP signaling counterparts, BMP type IA

receptor (ALK-3) and BMP type II receptor, in all stages of

differentiation. Intracellular signal transducing components

Smads were also expressed in the cells (Fig. 3). Thus,

ATDC5 cells retained their responsiveness to BMP-4

signaling.

Zou and coworkers demonstrated the distinct functions of

BMP receptors type IA and type IB (Zou, et al., 1997).

BMP type IB receptor (ALK-6) expression preceded the

early chondrogenic differentiation. Signaling from the

receptor is necessary for the early steps of mesenchymal

condensation and cell death. In contrast, BMP type IA

receptor (ALK-3) was essential for proper progression of

the cartilage differentiation program. In agreement with

these observations, ATDC5 cells properly kept track of the

early and late-phase differentiation program despite the lack

of BMP type IB receptor expression. ATDC5 cells are com-

mitted chondroprogenitor cells. In fact, when the confluent

cultures of undifferentiated ATDC5 cells were treated with

recombinant BMP-4, almost all of the cells were induced to

become chondrocyte-like without the formation of cellular

condensation (Fig. 4a). GDF-5 has also been shown to stim-

ulate chondrogenic differentiation (Tsumaki, et al., 1999).

ATDC5 cells expressed GDF-5 mRNA as well as mRNAs

for its type II receptors, BMP type II receptor and ActR-II

(Nishitoh et al., 1996). However, its type I receptor (ALK-6)

mRNA was not detected in these cells. Thus, it remained to

be elucidated the specific role of GDF-5 in differentiation of

ATDC5 cells.

Interestingly, BMP-6 and BMP-7 genes exhibited the

distinct and transient patterns of expression along with the

late-phase differentiation of ATDC5 cells. The expression

Fig. 3. Expression of the genes for the Smad family members during the

chondrogenic differentiation of ATDC5 cells. Cells were plated and grown

as described in the legend to Fig. 1. Total RNA was prepared on the indi-

cated days of culture. Reverse-transcribed cDNAs were amplified by PCR

for the detection of Smad1, Madr2, Smad3, DPC4, Smad5, Smad6, Smad7,

or Smad8 mRNA in comparison with G3PDH mRNA. Aliquots (8 �l) of

the PCR products were resolved on 3% agarose gels alongside the markers.


201

of BMP-6 mRNA increased in parallel with the increase of

type II collagen mRNA in the ATDC5 cells, and then

declined (Fig. 1a), suggesting that the BMP-6 expression

was induced in association with nodule formation. When

the cultures of differentiated ATDC5 cells were treated with

recombinant BMP-6, it facilitated the phenotypic conver-

sion of proliferating cells to hypertrophic cells, as evidenced

by the upregulation of type X collagen expression (Fig. 5b).

The stimulatory effects of recombinant BMP-6 on the late-

phase differentiation of ATDC5 cells is similar to those of

BMP-4 (Fig. 5a). Differentiated ATDC5 cells express ALK-

3, but do not express ALK-6. Thus, the stimulatory effects of

BMP-6 on differentiation of ATDC5 cells may possibly be

mediated by ALK-3 in ATDC5 cells.

We previously showed that the ATDC5 cultures

underwent matrix mineralization in the differentiated carti-

lage nodules without �-glycerophosphate supplement

(Shukunami, et al., 1997). BMP-7 mRNA was expressed in

a matrix mineralization stage in these cells, and recombi-

nant BMP-4/7 actually promoted a marked upregulation of

ALP mRNA and matrix mineralization in these cells (Fig.

6), suggesting that BMP-7 plays a role in the progression of

Fig. 4. Effects of recombinant BMP-4 on the early-phase chondrogenic differentiation of ATDC5 cells. Cells were plated at 6�104 cells per well in a

6-multiwell plate and grown to confluency on day 5 in DME/F12 medium containing 5% FBS and ITS. Cells were then incubated in the absence or the pres-

ence of recombinant BMP-4 (1,000 �g/ml) for another 6 days. Cells were then assessed for cartilaginous nodule formation under phase-contrast microscopy

(a) (Bar denotes 100 µm). (b) After 5 days, medium was replaced with the fresh medium containing various doses of BMP-4. For the time-dependence, cells

were treated with 1,000 ng/ml of BMP-4 for the various time periods indicated. For the dose-dependence, cells were treated with 100 or 1,000 ng/ml of

BMP-4 for a total of 48 hours. Total RNA was prepared and subjected for northern analysis of type II collagen mRNA. The equivalent loading of RNA (20

�g/lane) was verified by ethidium bromide staining, as shown in the bottom panel. Three independent experiments were performed and gave similar results.

H. Akiyama et al.

202

mineralization in ATDC5 cells.

Chondrogenic differentiation includes a series of cellular

events. which enable cells to undergo orderly transitions of

the cartilage phenotype, as characterized by the sequential

expression of unique cartilage matrix. The present results

indicated that, in addition to the basal expression of BMP-4,

the differentiation stage-specific expression of BMP-6 and

BMP-7 contributed to the progressive multistep differentia-

tion after induction of cellular condensation by insulin in

ATDC5 cells. Thus, the mouse clonal cell line ATDC5 is a

useful in vitro model for molecular analysis of the multistep

chondrogenic differentiation during endochondral bone for-

mation.

Acknowledgements. We thank Dr. J. M. Wozney (Genetics Institute Inc.)

for his kind provision of human recombinant BMP-6. This work was partly

Fig. 5. Effects of recombinant BMP-4 or BMP-6 on the late-phase chondrogenic differentiation of ATDC5 cells. Cells were plated at 6�104 cells per well

in a 6-multiwell plate and cultured for 21 days in DME/F12 medium containing 5 %FBS and ITS. After 21 days, medium was replaced with the fresh medi-

um containing various doses of recombinant BMP-4 (a) or BMP-6 (b). For the time-dependence, cells were treated with 1,000 ng/ml of BMP-4 or BMP-6

for the various time periods indicated. For the dose-dependence, cells were treated with 100 or 1,000 ng/ml of BMP-4 or BMP-6 for a total of 48 hours. Total

RNA was prepared and subjected for northern analysis of the expression of type X collagen mRNA. The equivalent loading of RNA (20 �g/lane) was veri-

fied by ethidium bromide staining, as shown in the bottom panel. Three independent experiments were performed and gave similar results.


203

supported by The Naito Foundation and Terumo Life Science Foundation

to YH.

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(Received for publication, June 20, 2000

and accepted, June 30, 2000)

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Differential Expressions of BMP Family Genes during