ABSTRACT
Retinoic acid (RA) is known to play a role in various aspects of
skeletal development in vivo, including morphogenesis, growth
plate maturation, and apoptosis. In cell culture, RA treatment of
chondrocytes suppresses the differentiated phenotype characterized by
production of type II collagen and aggrecan. In an effort to discover
molecules involved in regulation of the chondrocyte phenotype or
related to developmental processes such as chondrogenesis, mRNAs from
bovine chondrocytes cultured with and without RA were amplified by
reverse transcription-polymerase chain reaction (PCR) and compared by
differential display. PCR products whose expression was inhibited by RA
treatment were cloned. One cDNA encodes a molecule we call
cartilage-derived retinoic acid-sensitive protein (CD-RAP), and its
properties are described here. The full-length bovine CD-RAP mRNA was
cloned after amplification by the rapid amplification of cDNA ends
procedure, and a part of the rat CD-RAP mRNA was amplified by reverse
transcription-PCR using sequence-specific primers. The bovine CD-RAP
mRNA contains an open reading frame of 130 amino acids. CD-RAP mRNA
expression, as determined by Northern blot analysis and in situ hybridization, was present only in cartilage primordia and
cartilage. The inhibition of CD-RAP mRNA expression by RA in vitro was time- and dose-dependent and was tested over concentrations
from 10
to 10
M.
Southern blot analysis of genomic DNA indicated that CD-RAP was encoded
by a single copy gene and that no other genes were closely related.
What appears to be the human homologue of CD-RAP was recently isolated
and cloned from a melanoma cell line and shown to function as a growth
inhibitory protein (Blesch, A., Boberhoff, A.-K., Apfel, R., Behl, C.,
Hessdoerfer, B., Schmitt, A., Jachimcza, P., Lottspeich, F., Buettner,
R., and Bogdahn, U.(1994) Cancer Res. 54, 5695-5701).
Neither CD-RAP nor this protein showed any homology to known proteins.
We speculate that, in vivo, CD-RAP functions during cartilage
development and maintenance.
Retinoic acid (RA) (
)is known to play a role in the
growth and differentiation of the central nervous system, skin, and
skeleton. Depending on the concentration, it can induce different
processes. At high doses, RA has teratogenic effects on pattern
formation, predominantly in the limb(1) , craniofacial
structures(2) , and the central nervous system(3) .
During normal skeletal development, RA may participate in induction and
morphogenesis of limbs (4) and in growth plate
maturation(5) . RA can repress chondrogenesis in vitro(6, 7) and induce osteogenesis in various cell
lines(8) . In terminally differentiated avian chondrocytes, RA
can stimulate proliferation and maturation to osteoblast-like cells as
well as matrix mineralization(9) .
RA-treated chondrocytes
have been used as a model system to investigate the mechanisms of
chondrocyte ``dedifferentiation,'' where expression of type
II collagen is repressed(10, 11) . In
chick(10) , rabbit(11) , and bovine (12) chondrocytes, the 
-chain of type I
collagen is increased, and in chick, both type I and III collagens are
increased(10, 13) . Expression of other matrix
molecules is also known to be altered by treatment of chondrocytes with
RA, including proteoglycans(14) , fibril-associated
collagens(12) , and matrix metalloproteinases(15) . In
culture, RA-treated chondrocytes rapidly change morphology from a
rounded, polygonal cell shape to a very distinct, flattened spindle
shape.
As an approach to better understanding the changes induced by
RA, we examined chondrocyte mRNA by the techniques of reverse
transcription-PCR and differential display. Bovine chondrocytes
cultured in the presence or absence of RA were compared for differences
in expression of mRNA. Products amplified from mRNA species that
appeared to be down-regulated by RA were selected for further analysis.
In this report, we describe a cDNA derived from a small mRNA, the
protein product that we term cartilage-derived RA-sensitive protein
(CD-RAP). The CD-RAP mRNA contains an open reading frame of 130 amino
acids and was detected only in chondroprogenitor cells and
chondrocytes. In cultured chondrocytes, expression of CD-RAP mRNA was
inhibited by RA in a time- and dose-dependent manner. Insight into a
potential function for this molecule in cartilage may come from studies
of the human homologue, which was recently characterized in a melanoma
cell line as a growth inhibitory protein(16) .
EXPERIMENTAL PROCEDURES
Materials
General laboratory chemicals, RNase A,
and RNase T1 were from Sigma. Dulbecco's modified Eagle's
medium, restriction enzymes, and the random priming kit were from Life
Technologies, Inc. pGem11Zf
, reverse transcriptase,
and the poly(A) tract kit were from Promega. Hybond N paper was
purchased from Amersham Corp. The RACE kit was from CLONTECH (Palo
Alto, CA). Differential display PCR was performed using primers from
Gene Hunter Co. (Brookline, MA). [
-
P]dCTP
(3000 Ci/mmol) and [
-
P]ATP (5000 Ci/mmol)
were from Amersham Corp., and
S-UTP (800 Ci/mmol) was from
DuPont NEN. pBluescript II SK(+) cloning vector was from
Stratagene (La Jolla, CA). Pronase was from Calbiochem. Collagenase
(Class 2) was from Worthington. Fetal calf serum was from Hyclone
Laboratories (Logan, UT). dNTPs were from Pharmacia Biotech Inc.
Chondrocyte Cell Culture
Chondrocytes were
prepared from bovine articular cartilage as described by Kuettner et al.(17) . The isolated cartilage was digested for
1 h in 0.7% Pronase in Dulbecco's modified Eagle's medium
at 37 °C, followed by two washes with phosphate-buffered saline.
The cartilage was then digested by incubation with 0.15% collagenase
for 5 h at 37 °C in Dulbecco's modified Eagle's medium.
The released chondrocytes were filtered through nylon mesh and
harvested by centrifugation. The harvested cells were plated onto
100-mm cell culture dishes at a density of 1
10
cells/cm
. Cells were cultured in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum,
50 µg/ml ascorbic acid, and 50 µg/ml gentamycin at 37 °C in
a humidified atmosphere containing 5% CO
.
All-trans-RA was dissolved in 95% ethanol at a concentration
of 1 mg/ml and added to the prewarmed medium, which was then added to
the culture. After 4 days of culture, RA was added to the chondrocytes
at a concentration of 3
10
, 3
10
, or 3
10
M for up to 6 days. Control cultures were treated with equivalent
concentrations of ethanol vehicle. Medium was replaced every 2 days.
RNA Isolation
For isolation of total RNA from
chondrocytes, the cells were harvested by centrifugation and dissolved
in lysis buffer (4 M guanidinium SCN, 25 mM sodium
citrate, 0.5% sodium sarcosyl, and 0.7%
-mercaptoethanol). For
isolation of total RNA from different soft tissues, a 5-fold volume of
lysis buffer was added to the tissue pieces, followed by homogenization
of the tissues. Total cellular RNA was then isolated by CsCl
centrifugation according to the method of Chirgwin et al.(18) . Poly(A)
RNA was separated from
total RNA with magnetic beads after annealing to a biotin-labeled
oligo(dT) primer using the poly(A) tract kit. The reaction was
performed according to the manufacturer's protocol.
Northern Blot Analysis
RNA was electrophoresed in
an 1% agarose gel containing 6% formaldehyde and 1
MOPS buffer
(1
MOPS buffer = 20 mM MOPS, 5 mM sodium acetate, and 1 mM EDTA). Transfer of RNA to Hybond
N nylon membranes was carried out by capillary blotting with 20
SSC (1
SSC= 0.15 M NaCl and 0.015 M sodium citrate). After transfer, the nucleic acids were
cross-linked to the membrane in a Stratalinker (Stratagene). For
analysis of RNAs, cDNA probes were labeled with
[
P]dCTP by random priming and hybridized in 50%
formamide, 5
SSC, 5
Denhardt's
solution(19) , 0.5% SDS, and 100 µg/ml denatured salmon
sperm DNA at 42 °C for 16 h. After hybridization, filters were
washed twice in 2
SSC for 5 min at room temperature and twice
in 2
SSC and 0.1% SDS for 30 min each at 65 °C. The washed
filters were exposed to Hyperfilm-MP x-ray films.
Differential Display PCR
Differential display PCR
was performed using the RNAmap
kit from Gene Hunter Co.
following the manufacturer's instruction.
RACE
RACE was performed by using a commercially
available kit (Clontech). The reactions were carried out according to
the protocol given by the manufacturer. The CD-RAP mRNA was
reverse-transcribed using an oligonucleotide with the sequence
5`-TAGACTGAGCTCACTGGCAG-3` (primer 3; see Fig. 1) as a primer.
PCR amplification of the full-length CD-RAP cDNA was performed with the
oligonucleotides 5`-CGCGGATCCTCACTGGCAGTAGAAATCCCATA-3` (primer 4; see Fig. 1) and 5`-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATTCGATAG-3`
(primer 5; see Fig. 1). BamHI and EcoRI
cloning sites are underlined.
Figure 1:
Diagram
showing CD-RAP cDNA primers and products. The sequences of the primers
are shown under ``Experimental
Procedures.''
DNA Sequencing
DNA sequencing was performed
according to the dideoxy method (20) using a sequencing kit
from U. S. Biochemical Corp. following the manufacturer's
instructions or using the DyeDeoxy
Terminator Cycle
sequencing kit protocol with an Applied Biosystems Model 373 DNA
sequencer. All sequencing was performed on both strands of DNA.
Amplification of Rat and Mouse Homologues
5 µg
of total RNA isolated from rat fetal skeleton or 13.5-day mouse embryos
were reverse-transcribed using 30 pmol of a specific primer with the
sequence 5`-CGCGGATCCGAGCTCACTGGCAGTAGAAATCC-3` (primer 6; see Fig. 1). The reaction conditions were 50 mM Tris-HCl,
pH 8.3, 50 mM KCl, 5 mM MgCl
, 0.5 mM spermidine, 5 mM dithiothreitol, 0.5 mM dNTPs,
and 5 units of reverse transcriptase. PCR amplification was performed
with the primer used for the reverse transcription and an upstream
primer with the sequence 5`-CGCGAATTCATGCCCAAGCTGGCTGACCGGAA-3` (primer
7; see Fig. 1). The reaction mixture contained 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100, 0.2
mM dNTPs, 50 pmol of each primer, and 1 unit of Taq polymerase in a total volume of 100 µl. Amplification was
performed through 30 cycles in a Perkin-Elmer thermocycler. Each cycle
was for 30 s at 96 °C, 45 s at 50 °C, and 2 min at 72 °C.
The amplified fragment was digested with the restriction endonucleases BamHI and EcoRI. A recognition site for both of the
enzymes was created during the synthesis of the upstream primer and the
downstream primer, respectively. The digested fragment was cloned into
pGem11Zf
.
Primer Extension Analysis
For primer extension
analysis, a 35-mer oligonucleotide,
5`-CGGACTCTGAGCAAGACAGTGGCTAAGGCAGGAAT-3` (primer 8; see Fig. 1), was synthesized. The primer was end-labeled by
phosphorylation with [
-
P]ATP using T4
polynucleotide kinase. 10
cpm of the DNA primer were
hybridized with 1 µg of mRNA isolated from chondrocytes in 30
µl of hybridization buffer (40 mM PIPES, pH 6.4, 1 mM EDTA, pH 8.0, 0.4 M NaCl, and 80% formamide). The
hybridization mixture was first heated to 85 °C to denature the
nucleic acids following incubation at 30 °C for 16 h. The nucleic
acids were precipitated by addition of 170 µl of water and 400
µl of 100% ethanol at 0 °C for 1 h, collected by
centrifugation, and washed with 70% ethanol. For reverse transcription,
the primer/RNA hybrids were redissolved in 20 µl of reverse
transcription buffer (50 mM Tris-HCl, pH 8.3, 50 mM KCl, 10 mM MgCl
, 0.5 mM spermidine,
10 mM dithiothreitol, 1 unit/µl RNasin, 50 µg/ml
actinomycin D, and 5 units of avian myeloblastosis virus reverse
transcriptase). The reaction was incubated for 2 h at 42 °C. The
mRNA template was then removed by digestion with RNase A (5 µg/ml)
for 30 min at 37 °C. After extraction with phenol/chloroform, the
nucleic acids were precipitated by centrifugation and redissolved in 10
µl of formamide loading dye (80% formamide, 10 mM EDTA, 1
mg/ml xylene cyanol FF, and 1 mg/ml bromphenol blue). Half of the
reaction was heated for 5 min at 95 °C and analyzed by
electrophoresis through a 6% sequencing gel. The extended cDNA products
were detected by autoradiography.
Southern Blot Analysis
For Southern blot analysis,
genomic DNA was digested with restriction endonucleases and
electrophoresed through 0.7% agarose gel in 0.04 M Tris
acetate and 0.001 M EDTA, pH 8.0. After electrophoresis, the
nucleic acids were denatured by incubation of the gels in alkaline
solution (0.5 M NaOH and 1.5 M NaCl) for 30 min,
followed by neutralization for 30 min in 0.5 M Tris-HCl, pH
7.2, 1.5 M NaCl, and 0.001 M EDTA. The nucleic acids
were blotted and hybridized as described above.
In Situ Hybridization
Mouse tissue at 13.5 days
gestation was prepared for in situ hybridization as described
by Sandell et al.(21) . Tissue was hybridized with
antisense riboprobes of CD-RAP and type IIA procollagen mRNA. CD-RAP
antisense RNA was transcribed from a mouse cDNA fragment cloned into
pGem1Zf(+), which is described above. The plasmid was linearized
by restriction with EcoRI before in vitro transcription. For cloning of type IIA procollagen mRNA, cDNA from
a segment spanning exons 1 and 2 was amplified via PCR. The fragment
was designed with 5`-EcoRI and 3`-BamHI restriction
sites and inserted into the corresponding sites of pGem3Zf(+). The
total length of the cDNA insertion was 266 bp. It contained 59 bp of
exon 1 and 207 bp of exon 2. Antisense RNAs were transcribed in
vitro using Sp6 RNA polymerase in the presence of
S-UTP. Hybridizations were carried out at 40 °C
overnight with antisense riboprobes with a specific activity of
3-5
10
cpm/ml of tRNA hybridization solution
(50% formamide, 20% dextran sulfate, 20 mM dithiothreitol, 1
mg/ml tRNA, 300 mM NaCl, 10 mM Tris-Cl, pH 7.4, 10
mM Na
PO
, pH 6.4, 5 mM EDTA,
0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum
albumin). Slides were washed according to the procedure of Ausubel et al.(19) , followed by exposure to Beta-Max
autoradiographic film for 3 days and analysis with an MCID image
analysis system.
RESULTS
Identification of New Chondrocyte Molecules by
Differential Display
Chondrocytes were isolated from bovine
articular cartilage and plated on tissue culture dishes at a density of
10
cells/cm
. After 4 days of culture, all of
the cells were adhered as monolayers. They expressed mRNAs
characteristic for extracellular matrix molecules such as type II
collagen, aggrecan, and link protein, which were used as markers for
the normal chondrocyte phenotype(22) . The cells were then
treated with RA at a concentration of 3
10
M for 6 days to modulate the chondrocyte-specific gene
expression. At this time, synthesis of cartilage-characteristic type II
and XI collagens was not detectable (data not shown). Messenger RNA
from these cells was used for differential display by the method of
Liang and Pardee(23) . We isolated a total of 120 cDNA
fragments that appeared only in the display of mRNA from untreated
chondrocytes. Six of the 120 cDNA fragments were amplified from
independent mRNAs that were down-regulated by RA. One of them was
derived from an mRNA encoding a protein that was recently identified as
melanoma growth inhibitory activity (EMBL accession number X75450). The
expression of this molecule was detected only in a subset of melanoma
cell lines, but not in any normal tissue(16) . The sequences of
the two primers that amplified the molecule for the original display
were 5`-TTTTTTTTTTTTNG-3` for the downstream PCR primer and
5`-GCAATCGATG-3` for the upstream PCR primer (shown in Fig. 1).
Reverse transcription-PCR using these primers amplified a cDNA fragment
of 100 bp in length. Northern blot analysis confirmed that the mRNA,
which was the original template for the reaction, could be
down-regulated by RA in vitro (Fig. 2A).
Because of the sensitivity of the expression of this mRNA to RA, it was
called CD-RAP.
Figure 2:
A, detection of CD-RAP mRNA by Northern
blot analysis. 5 µg of total RNA isolated from chondrocytes grown
for 4 days in monolayer (lane 1) or treated for an additional
6 days with RA (lane 2) were electrophoresed through a 1%
agarose gel and analyzed by Northern blot hybridization with a
radiolabeled CD-RAP cDNA probe, followed by autoradiography. B, RACE of the full-length CD-RAP cDNA. mRNA from chondrocytes
was reverse-transcribed with primer 3 (see Fig. 1). After
ligation of the anchor primer to the 3`-end of the cDNA, PCR
amplification was performed with primers 4 and 5 (see Fig. 1).
An aliquot of the reaction was analyzed through a 2% agarose gel. Lane 1, molecular weight marker
X HaeIII
fragments; lane 2, PCR products after amplification of one-one
hundredth of the anchor ligation reaction; lane 3, PCR
products after amplification of one-tenth of the anchor ligation
reaction. The size of DNA fragments is indicated in
kilobases.
Full-length Cloning by RACE
From the results of
the Northern blot hybridizations, it was determined that the
full-length mRNA is a small molecule
500 bases in length (Fig. 2A). RACE was used to amplify the full-length
mRNA to its 5`-end (Fig. 2B). For RACE, we created a
specific primer based on DNA sequence information derived from the
100-bp cDNA fragment we had cloned after differential display. This
primer was used for reverse transcription of the CD-RAP mRNA. After
synthesis of the cDNA, the mRNA template was hydrolyzed with alkaline
solution, and the 3`-end of the cDNA, which represents the 5`-end of
the mRNA, was ligated to an anchor oligonucleotide using T4 RNA ligase.
A primer complementary to the anchor oligonucleotide and a nested
downstream primer were used for PCR. The oligonucleotides used for
reverse transcription and RACE are shown in Fig. 1. After 35
cycles in a thermocycler, an aliquot of the reaction was analyzed on a
2% agarose gel. Three distinct bands could be detected after agarose
gel electrophoresis. Two of them migrated close together and were
500 bp long. This coincided with the expected size of the
full-length CD-RAP mRNA (Fig. 2B).The cDNA
fragments in the 500-bp range were purified from the agarose gel and
cloned into pBluescript II SK(+). Nine individual clones were
analyzed by dideoxy sequencing. All of them represented cDNA fragments
amplified from the CD-RAP mRNA. The clone with the longest cDNA insert
was used for further analysis. According to the sequence information
from this clone, the full-length CD-RAP mRNA would be 548 bp long.
Primer Extension Analysis
The 5`-end of the CD-RAP
mRNA was confirmed by primer extension analysis. A new primer with a
length of 35 nucleotides was synthesized for the primer extension
experiments (see Fig. 1). This primer started 88 nucleotides
downstream of the sequence that appeared to be the 5`-end of the mRNA
based upon the analysis of the RACE product. The primer was end-labeled
with [
-
P]ATP and annealed to 1 µg of
mRNA. The mRNA was then reverse-transcribed in the presence of
actinomycin D to avoid self-priming. The products of the primer
extension reaction were analyzed by electrophoresis through a 6%
sequencing gel and detected by autoradiography (Fig. 3). For
determination of the length of the extended products, a dideoxy
sequencing reaction was run next to the primer extension reaction.
After autoradiography, two major extension products could be detected:
a predominant band at 88 nucleotides and a minor band at 117
nucleotides (Fig. 3). The smaller band matched the size we
expected for the full-length CD-RAP mRNA. The larger extended product
indicates the potential presence of an alternative transcription start
site, 29 bases farther upstream. Additional bands that yielded very
weak signals were most likely premature termination products.
Figure 3:
Definition of the 5`-end of the CD-RAP
mRNA by primer extension analysis. Primer 8 (see Fig. 1) was
radiolabeled with [
-
P]ATP and used for
reverse transcription of 1 µg of chondrocyte mRNA. The extended
products were analyzed through a 6% sequencing gel and detected by
autoradiography. In lane 1, the radiolabeled oligonucleotide
alone was loaded. Lane 2 shows the cDNA products extended from
the radiolabeled primer. For the determination of the length of the
synthesized cDNAs, a dideoxy sequencing reaction was loaded next to the
primer extension reaction.
Fig. 4shows the complete cDNA sequence of the CD-RAP clone.
The bovine sequence spans the complete mRNA molecule from the 5`-start
site of transcription to the poly(A) tail. A cDNA encoding a protein
responsible for melanoma inhibitory activity (16) appears to be
the human homologue of CD-RAP, and its sequence is shown in Fig. 4. No other homologous proteins were found in the data
bank. The CD-RAP cDNA sequence revealed an open reading frame of 130
amino acids encoding a protein with a typical hydrophobic leader
sequence followed by a unique domain protein. No linkage sites for N-linked carbohydrate (Asn-Gly-Ser/Thr) or O-linked
carbohydrate (Ser-Gly) were found. The protein included four cysteine
residues.
Figure 4:
Full-length cDNA and protein sequences of
bovine CD-RAP compared with the human and rat sequences. The potential
cleavage site of a hydrophobic signal sequence is indicated by the arrow.
To screen for tissue specificity, a rat cDNA clone was
prepared using the primers shown in Fig. 1. This cDNA probe was
amplified by reverse transcription-PCR from total RNA isolated from
skeletal cartilage of a fetal rat. The primers for reverse
transcription and PCR were chosen from identical sequences in bovine
and human CD-RAP cDNAs. Fig. 4compares this 322-bp rat cDNA
fragment with bovine sequence. The DNA conservation between these
species is 85.5% for human and bovine, 90% for human and rat, and 87%
for rat and bovine.
Southern Blot Analysis
Southern blot analysis was
used to assess copy number of the CD-RAP gene and whether closely
related genes could be found. 5 µg of bovine genomic DNA were
digested with the restriction endonuclease EcoRI or PstI. The digested DNAs were separated by electrophoresis
through a 0.7% agarose gel and blotted onto nylon membranes. After
hybridization and autoradiography, a single band was detected,
indicating that CD-RAP is present as a unique gene (Fig. 5).
When the blot was probed under low stringency conditions, no additional
bands were observed, indicating that there are no other closely related
genes in the genome. Similar results were obtained when the rat probe
was tested on rat genomic DNA (data not shown).
Figure 5:
Southern blot analysis of CD-RAP. 10
µg of bovine genomic DNA were digested with the restriction
endonucleases EcoRI (lane 2) and PstI (lane 3) and separated through a 0.7% agarose gel. The DNA
fragments were blotted onto a nylon membrane and hybridized with a
radiolabeled CD-RAP cDNA fragment. Lanes 4 and 5 show the
hybridization products with the digested DNAs in lanes 2 and
3. Lane 1: molecular size marker (
HindIII
fragments). The size of DNA fragments is indicated in
kilobases.
Tissue-specific Expression of CD-RAP mRNA
To
investigate the tissue distribution of CD-RAP mRNA, total RNAs from
different rat tissues such as heart, lung, liver, kidney, skeletal
muscle, and spleen were used for Northern blot hybridization. As a
positive control, we included total RNA from skeletal cartilage of a
fetal rat. After exposure to x-ray films for up to 2 weeks, a
hybridization signal was detected only in total RNA from the rat fetal
skeletal cartilage (Fig. 6A). To determine whether the
absence of CD-RAP mRNA was related to a developmental stage, we tested
for CD-RAP mRNA expression in a similar spectrum of fetal bovine
tissues. In addition, RNA from different cartilages was analyzed,
including the vertebral column, knee, and shoulder. Northern blot
analysis showed the presence of CD-RAP mRNA in total RNA isolated from
all of the cartilaginous tissues, but from none of the other tissues
that were tested (Fig. 6B).
Figure 6:
Tissue-specific expression of CD-RAP mRNA. A, expression in adult rat tissues. 5 µg of total RNA from
adult rat tissues (lanes 1-6) and rat fetal skeleton (RFS; lane 7) were used for Northern blot
hybridization. Exposure time was 1 week. B, expression in
fetal bovine tissues. 5 µg of total RNA from different cartilages
derived from the vertebral column (vert.col; lane 1),
knee (lane 2), and shoulder (lane 3) and from heart,
kidney, liver, lung, skeletal muscle, and spleen (lanes
4-9, respectively) were analyzed by Northern blot
hybridization with a radiolabeled CD-RAP cDNA probe. Exposure times
were 1 week. As a control for equal loading of total RNA, the Northern
blots shown in A and B were hybridized with a cDNA
probe specific for ribosomal elongation factor 1 (ELF1)
mRNA(22) .
To confirm the tissue
distribution of CD-RAP mRNA, in situ hybridization was
performed in a mouse embryo. Sections of tissues from a 13.5-day embryo
were used. A representative set of hybridizations is shown in Fig. 7. A probe for the mRNA of type IIA procollagen was used to
localize cartilage primordia and to confirm the developmental stage of
the mouse embryo. Comparison of the two hybridizations shows that
CD-RAP mRNA is colocalized with the type II procollagen mRNA in the
cartilage primordia of the developing vertebral column, ribs, cranium,
and nasal septum. Interestingly, no expression of CD-RAP mRNA occurred
in the non-cartilaginous tissues of the otic vesicle, where type II
collagen expression is abundant.
Figure 7:
Autoradiographs of in situ hybridization with type IIA procollagen (A) and CD-RAP (B). Tissue sections were from a 13.5-day mouse embryo.
Tissues labeled are the primordia of the cranium (CR),
vetebral column (VC), and ribs (R).
Regulation of Gene Expression by RA
Since the
concentration of RA originally used for the modulation of chondrocytes
was much higher than physiological concentrations, we examined the
effect on gene expression of lower RA concentrations (Fig. 8A). The effects of three different RA
concentrations (3
10
, 3
10
, and 3
10
M)
were tested. This represents a range from more physiological
concentrations (3
10
M) to
concentrations routinely used for dedifferentiation of chondrocytes in vitro (3
10
M). During
culturing with RA, the morphology of the chondrocytes changed from a
polygonal to a fibroblast-like cell shape as described
previously(11) . Total RNA was isolated after 2, 4, and 6 days
of RA treatment. Under these conditions, CD-RAP mRNA expression showed
dose- and time-related down-regulation by RA at all concentrations
tested (Fig. 8A). In cells treated for 2, 4, and 6 days
with the ethanol carrier only, expression of CD-RAP mRNA did not
significantly change. The weaker signals after 4 and 6 days of ethanol
treatment in Fig. 8B (lanes 3 and 4)
are due to different amounts of total RNA loaded onto the gel. This is
shown by the levels of elongation factor 1 mRNA detected after
hybridization of the same blots with an elongation factor 1-specific
cDNA probe (Fig. 8B, ELF1).
Figure 8:
Time course and dose response of CD-RAP
mRNA to RA. A, total RNAs from chondrocytes treated with RA
for 2, 4, and 6 days at a concentration of 3
10
M (lanes 1-3), 3
10
M (lanes 4-6), or 3
10
M (lanes 7-9) were
analyzed. Total RNA from chondrocytes grown for 4 days in monolayer
without RA was used to show CD-RAP mRNA levels before the RA treatment (lane 10). B, total RNA from chondrocytes grown for 4
days in monolayer (lane 1) and cultured for an additional 2,
4, and 6 days with the same amount of EtOH, which was used as the RA
carrier (lanes 2-4), was analyzed for CD-RAP mRNA
expression. In both A and B, 5 µg of each RNA
were analyzed by Northern blot hybridization with a radiolabeled bovine
CD-RAP cDNA probe. As a control for RNA loading, hybridization was
performed with a cDNA probe for ribosomal elongation factor 1 (ELF1).
DISCUSSION
An mRNA encoding a cartilage protein, CD-RAP, has been cloned
from cultured chondrocytes. It is down-regulated by RA in vitro in a time- and dose-dependent manner. The CD-RAP mRNA encodes a
unique 130-amino acid protein containing a potential signal peptide,
four cysteine residues, no obvious processing sites, and no sites of N- or O-linked glycosylation. We have characterized
the full-length mRNA, demonstrated the start site of transcription, and
showed that this gene is unique in the genome. In vivo, CD-RAP
mRNA appears to be synthesized only by chondrocytes and
chondroprogenitor cells. No expression was detected in RNA from various
other adult rat or fetal bovine tissues; however, abundant expression
was observed in rat fetal skeletal cartilage and in all bovine fetal
cartilages tested. In embyonic tissue, CD-RAP mRNA is expressed in the
cartilage primordia of 13.5-day mouse embryos. At this time, the
cartilage primordia of the skeleton begins to form. Our results
indicate that CD-RAP mRNA is synthesized by cells in the cartilage
primordia and in differentiated cartilage. In the mouse embryo,
expression of CD-RAP mRNA is similar to that of type II procollagen
mRNA(21, 24) . Current studies are underway to
determine the temporal sequence of expression of type II procollagen
splice forms and CD-RAP.
Previous studies have indicated that RA is
potentially involved in cartilage differentiation. Although not itself
a morphogen in vivo, RA is a capable of initiating pattern
formation in chick limbs (25, 26) possibly through the
induction of hox genes (27) in mesenchymal cells. RA
can further stimulate the differentiation of chondrocytes to
hypertrophic chondrocytes during growth plate maturation(28) .
In the growth plate, one role of RA may be to suppress the expression
of typical chondrocyte extracellular matrix molecules such as type II
collagen and aggrecan. RA-induced stimulation of type X collagen,
alkaline phosphatase, and osteocalcin synthesis followed by
mineralization and apoptosis may be important in vivo for
development of the hypertrophic chondrocyte phenotype(29) . The
RA-dependent differences of CD-RAP mRNA levels in vitro may
provide information relevant to understanding cartilage
differentiation.
The cDNA we have characterized is almost certainly
the bovine version of a molecule recently characterized in a melanoma
cell line (16) . Blesch et al.(16) showed
that the expressed protein has growth inhibitory activity on certain
melanoma cell lines. They found its expression limited to these
melanoma cells and a neuroepithelial cell line. Addition of melanoma
growth regulatory protein to the culture medium caused melanoma cells
to round up and to decrease their incorporation of
[
H]thymidine. It is not clear how these
activities could relate to chondrogenesis; however, a change in cell
shape and decrease in proliferation are characteristics of the
condensation phase of chondrogenesis. The relationship between the
expression of CD-RAP in malignant cells and normal physiological
expression by chondrosarcomas remains to be examined. Preliminary
studies indicate the presence of CD-RAP mRNA in rat and human
chondrosarcomas. Further studies will be needed to clarify the role of
CD-RAP in the development and maintenance of cartilage.