From the Department of Radiology and Radiation
Oncology, ¶ Department of Anatomy and Developmental Biology,
Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka,
Suita, Osaka 565-0871, Japan
Received for publication, August 29, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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We cloned a novel mouse cDNA,
CORS26 (collagenous
repeat-containing sequence of
26-kDa protein), encoding a secretory protein by
suppression subtractive hybridization between transforming growth
factor- In skeletal development in vertebrates, the formation of
chondrocytes from undifferentiated mesenchymal cells is one of the important processes, but the molecular mechanisms are not well understood. Identifying the genes underlying the induction of chondrocyte differentiation will provide powerful tools for
understanding skeletal development. The induction of chondrogenesis has
been extensively studied in vitro using primary cells and
clonal cell lines from a variety of sources (1-4). The mouse embryonic
fibroblast cell line, C3H10T1/2, are multipotential cells and have been
induced to undergo differentiation into myocytes, adipocytes,
osteoblasts, and chondrocytes under specific culture conditions and
treatments (5-8). The frequency of chondrogenic conversion in
C3H10T1/2 cells was much lower and irregular compared with other types
of conversion (5, 8), but it was recently reported that the induction
of chondrogenesis and the formation of spheroids in C3H10T1/2 cells
preferentially occurred when treated with transforming growth factor
(TGF)1- In the present study, to help clarify the mechanism for skeletal
development, mRNAs expressed in TGF- Cell Lines and Cell Culture--
The mouse cell line, C3H10T1/2,
was obtained from the RIKEN Cell Bank (Tsukuba, Japan), and micromass
cultures were performed as described previously (9). In brief,
trypsinized cells were suspended in Ham's F-12 medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS)
(Life Technologies, Inc.), penicillin (50 units/ml), and streptomycin
(50 µg/ml) at a concentration of 107 cells/ml, and then a
10-µl drop of this cell suspension was placed in the center of a
24-well dish at 37 °C in a humidified atmosphere containing 5%
CO2. The cells were allowed to adhere to the dish for
3 h, and the culture was flooded with 1 ml of medium. After 24-h
incubation, 1 ng/ml TGF- Construction of the Subtractive cDNA Library by
SSH--
Messenger RNAs obtained from C3H10T1/2 cells were isolated
using the FastTrack mRNA isolation kit (Invitrogen, San Diego, CA).
SSH was performed using the PCR-Select cDNA subtraction kit (CLONTECH, Palo Alto, CA) according to the
manufacturer's protocol. Tester cDNA was synthesized from
mRNAs of C3H10T1/2 cells treated with TGF- Dot-blot Differential Screening--
Cloned cDNA inserts
were initially evaluated for differential expression by DNA dot-blot
analysis. White colonies from a subtractive cDNA library were
picked randomly and boiled for 5 min in 20 µl of H2O and
then centrifuged. DNA in the supernatant was amplified by PCR using
universal vector primer M13 and M13R. The PCR products were purified by
QIAquick PCR purification kit (Qiagen, GmbH, Hilden, Germany) and
dot-blotted on Hybond-N+ nylon membranes (Amersham
Pharmacia Biotech, Buckinghamshire, United Kingdom). The
membranes were hybridized with [32P]dCTP-labeled tester
cDNA as positive and [32P]dCTP-labeled driver
cDNA as negative probes, respectively. The nucleotide sequences of
candidate positive clones were determined by sequencing, and cDNA
inserts were used as probes in Northern blot analysis confirming
differential gene expression.
DNA Sequencing and Sequence Analysis--
Sequencing of cDNA
inserts in the vector were performed by the ABI PRISM Dye Terminator
Cycle Sequencing Ready Reaction kit (PerkinElmer Life
Sciences) with the M13 vector primers on an ABI 373 automated
sequencer. For long cDNA inserts, synthetic oligonucleotides were
used as primers. The cDNA sequence was analyzed using sequence
analysis software, DNASIS (Hitachi Software Engineering, Yokohama, Japan). Data base homology searches for the cDNA sequence and deduced protein sequences were performed by BLAST programs with the
DNA and protein data bases at the National Center for Biotechnology
Information. Analysis of the primary structure of predicted proteins
were performed by the PROSITE motif search program.
Northern Blot Analysis--
Total RNA was isolated from
C3H10T1/2 cells before and after 6-h treatment with TGF- Rapid Amplification of cDNA Ends--
Rapid amplification of
cDNA ends (RACE) was performed in both directions using the SMART
cDNA amplification kit (CLONTECH) from
mRNA of C3H10T1/2 cells treated with TGF- Amplification of Full-length CORS26--
The full-length coding
sequence of CORS26 was amplified by LA Taq
polymerase (TaKaRa) using primers
5'-CTGTCAAGCTTCCCTGCGAGACTCTT-3' and
5'-GCAAGCCAGATGGGAGAAAAGTTTAT-3' from TGF- In Vitro Transcription and Translation--
The TNT T7 quick
coupled transcription/translation system (Promega) was used to
transcribe and translate the full-length CORS26 cDNA
construct in the presence of [35S]methionine (Amersham
Pharmacia Biotech) according to the manufacturer's instructions. Five
microliters of the products were electrophoresed on a 12.5%
SDS-polyacrylamide gel. The gel was treated with Enlightning (PerkinElmer Life Sciences), dried and autoradiographed.
RT-PCR Analysis--
Total RNAs isolated from various cell lines
were digested with RNase-free DNase I (Promega) at 37 °C for 15 min.
Then the denatured 1 µg of total RNA was reverse-transcribed in 20 µl of a reaction mixture containing 5× RT buffer, 10 mM
dithiothreitol, 0.5 mM amount of each dNTP, 500 ng
of oligo(dT)15 primers, 100 units of RNase inhibitor, and
200 units of Moloney murine leukemia virus reverse transcriptase
(Wako Pure Chemicals, Osaka, Japan) at 37 °C for 1 h and
subsequently heated at 70 °C for 10 min. After the RT reaction, PCR
was carried out in a total volume of 25 ml of a mixture containing 1 µl of RT samples, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.2 mM amount of each dNTP, 0.4 µM amount
of each primer, and 1.0 unit of AmpliTaq Gold DNA polymerase
(PerkinElmer Life Sciences). Twenty-five cycles of denaturation
(94 °C, 30 s), annealing (54 °C, 30 s), and extension (72 °C, 45 s) were carried out in a DNA thermal cycler
(PerkinElmer Life Sciences). The primer sequences for
CORS26 were 5'-GAAACAATGGGAACAATGGAG-3' and
5'-TGCTGAAGGTGAAGAAATACA-3'.
Aliquots (10 µl) of the PCR products were electrophoresed in 1.5%
agarose gel, and the gel was stained with ethidium bromide and
photographed under UV light. The amplified 300-bp PCR products were
subcloned into pGEM-T Easy vector.
In Situ Hybridization--
One microgram of recombinant pGEM-T
Easy vector containing the 300-bp fragment located at the coding
region (nucleotides 305-604) of CORS26 was used as a
template. Plasmids were linearized with SpeI to prepare the
antisense riboprobe and NcoI to prepare the sense riboprobe.
In vitro transcription was performed with
35S-UTP (Amersham Pharmacia Biotech) using T7 and
SP6 RNA polymerase (Promega). Unincorporated labels were removed by
ethanol precipitation, and the counts/min were determined on a
scintillation counter.
Embryos were obtained from ICR mice. In situ hybridizations
were carried out as described previously (14) with modifications. In
brief, mouse embryos were fixed with 4% paraformaldehyde and embedded
in paraffin for sectioning. The 7-µm-thick sections were pretreated
with proteinase K and HCl and acetylated. Hybridization was performed
with riboprobes (1 × 105 cpm per slide) at 53 °C
overnight. After hybridization, the sections were washed with 5× SSC
containing 10 mM dithiothreitol at 50 °C for 30 min, SF
solution (2× SSC, 50% formamide, and 20 mM
dithiothreitol) at 65 °C for 30 min, NTE buffer (0.5 M
NaCl, 10 mM Tris-Cl, and 1 mM EDTA) at 37 °C
for 30 min, treated with 20 µg/ml RNase A at 37 °C for 30 min and then washed with 2× SSC and 0.1× SSC at room temperature. The
sections were dehydrated in a graded series of ethanol, air-dried,
coated with NTB-2 emulsion (Eastman Kodak Co.) and exposed for 2-3
weeks. Microphotographs were taken using both light- and dark-field optics.
Transient Transfection with FLAG-tagged CORS26 and
Immunoblotting--
A CORS26 cDNA was tagged
with a FLAG epitope at the COOH terminus by PCR using oligonucleotides
5'-TTTGCCGAGCCATGCTCGGGAGGC-3' and
5'-TCACTTGTCATCGTCGTCCTTGTAGTCCTTAGTTTCAAAGAGCAGAAA-3'.
The sequence for a FLAG tag is underlined. The amplification
conditions were 80 °C for 5 min, followed by 25 cycles at 94 °C
for 30 s, 60 °C for 30 s, and 72 °C for 1 min and a
final incubation at 72 °C for 5 min. The PCR product was cloned into
pGEM-T Easy vector, and the insert was excised with
EcoRI and recloned into the EcoRI site of
pcDNA3.1(+) vector (Invitrogen). The orientation and nucleotide sequence of pcDNA3.1/CORS26-FLAG was confirmed by an
automated ABI 373 sequencer. The pcDNA3.1/CORS26-FLAG construct was
transiently transfected into COS-7 cells (RIKEN Cell Bank) using the
SuperFect Transfection Reagent (Qiagen) according to the
manufacturer's instruction. A pcDNA3.1(+) vector was transfected
in parallel as a negative control. Transfections were performed on
cells seeded into six-well tissue culture plates in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% FBS 24 h
before use at a density of 3 × 105 cells/well. Cells
were typically transfected with 2 µg of DNA, incubated with the DNA
suspension for 3 h, and replenished with fresh medium. Then,
48 h after transfection, conditioned medium was collected, and
cells were lysed with TNE buffer (10 mM Tris-HCl (pH 7.8),
1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40).
Protein from the conditioned medium was concentrated for 1 h with
a Centricon-10 concentrator (Millipore, Bedford, MA). The proteins from
the medium and cell extracts were separated on a 12.5%
SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane in a
Trans-Blot semidry electrophoretic transfer cell (Bio-Rad) and
immunoblotted with anti-FLAG M2 monoclonal antibody (Sigma) diluted
1:500. Immunocomplexes were detected with a secondary antibody
conjugated to peroxidase (Dako, Glostrup, Denmark) and
visualized with ECL reagent (Amersham Pharmacia Biotech).
Cell Growth Analysis Using CORS26 Stably Transfected
Cells--
A DNA fragment containing a coding region of
CORS26 was inserted into the
EcoRI-EcoRV sites of pcDNA3.1(+) vector.
C3H10T1/2 cells were seeded into 35-mm culture dishes in DMEM
supplemented with 10% FBS at a density of 3 × 104
cells per dish. Twenty-four hours later, the cells were transfected with 2.5 µg of either control pcDNA3.1 vector or
pcDNA3.1/CORS26 using the SuperFect Transfection Reagent
(Qiagen) according to the manufacturer's instruction. The cells were
incubated for 48 h and then trypsinized and seeded at a 1:20 ratio
in 100-mm culture dishes in DMEM containing 10% FBS. Sixteen hours
later, the cells were switched to a selective medium containing 500 µg/ml G418 (Promega). After 4 weeks of culture in the selective
medium, clonal isolates were expanded, and expression of
CORS26 mRNA was verified by RT-PCR as described above.
To analyze the growth of C3H10T1/2 cells transfected with
pcDNA3.1/CORS26, the transfectants were seeded in 24-well culture
plates at a density of 1 × 104 cells/well and
cultured with DMEM supplemented with 10% FBS. The number of cells were
counted with a hemocytometer at the indicated days.
Molecular Cloning and Sequence Analysis of the CORS26
cDNA--
To identify genes specifically expressed during skeletal
development, the SSH technique was utilized using the mRNA
extracted from C3H10T1/2 cells with or without TGF-
We determined the sequence of the isolated partial cDNA clones and
carried out homology searches in the GenBankTM using
BLAST2. Among all clones that had no significant homology with any
known genes in the nucleotide sequence data bases, a 380-bp cDNA
clone (clone 129) showed a marked induction by TGF- Predicted Protein Product of CORS26--
The open reading frame
predicted a polypeptide sequence of 246 amino acids corresponding to a
calculated molecular mass of 26.8 kDa with an isoelectric point
of 6.2. Indeed, a protein of ~26 kDa was generated by coupled
in vitro transcription and translation of the
CORS26 cDNA in rabbit reticulocyte lysates (Fig.
3A), suggesting that the
predicted initiation sequence serves as the start site of the
translation. Hydropathy analysis (16) revealed that the predicted
protein was predominantly hydrophilic, and the signal peptide of 22 amino acid residues and the cleavage site were also predicted at the
amino-terminal end on the basis of the rules of von Heijne (17) (Fig.
3B). No transmembrane spanning region was predicted. To
investigate whether CORS26 protein was secreted from cells, FLAG-tagged
CORS26 was transiently transfected into COS-7 cells and
analyzed by Western blotting using an anti-FLAG antibody. These
findings demonstrated that COS-7 cells synthesized the CORS26 protein,
and the protein was secreted into the medium (Fig.
4). Amino acid sequence analysis of the
predicted CORS26 protein using the BLASTp search program revealed that
the COOH-terminal globular region of CORS26 was 34, 32, and 30%
identical to precerebellin (18), complement protein C1q-related factor
(CRF) (19), and adipocyte complement-related protein of 30 kDa (Acrp30,
equivalent to AdipoQ) (20, 21), respectively. A unique feature of this protein is that the NH2-terminal part contained
uninterrupted collagen-like Gly-X-Y repeats (23 repeats) immediately downstream of a short noncollagenous sequence (at
amino acids positions 45-113). NH2-terminal
Gly-X-Y repeats are known to be found in the
complement protein C1q A, B, and C chains (22-24), Acrp30, chipmunks
hibernation-specific proteins (HP-20, HP-25, and HP-27) (25) and CRF
(19). In the COOH-terminal half, CORS26 shares homology with the
globular domain of the C1q subunits, precerebellin, Acrp30, CRF, and
collagens VIII and X (26) (Fig. 5).
It was also notable that there was a proline-rich region in the
NH2-terminal part (at amino acid positions 55-62). The
predicted protein also contained other putative functional sites,
phosphorylation sites for protein kinase C (at amino acid positions
152-154) and casein kinase II (at amino acid positions 77-80 and
138-141) and several N-myristoylation sites.
Expression of CORS26 mRNA in the Adult Mouse Tissues--
We
examined the expression of CORS26 mRNA in various adult
mouse tissues by Northern blot analysis (Fig.
6). CORS26 cDNA hybridized to double transcripts of 2.3 and 2.0 kb. The CORS26 mRNA
was expressed in rib growth plate cartilage and kidney.
CORS26 mRNA was not detected in other tissues. In growth
plate cartilage, the hybridization signal at 2.0 kb was stronger than
the signal at 2.3 kb, while, in kidney, the signal at 2.3 kb was
stronger than the signal at 2.0 kb. The 2.0-kb size of the transcripts
agrees approximately with the length of the cloned CORS26
cDNA.
Expression of CORS26 mRNA in Various Fibroblastic or
Osteoblastic Cell Lines--
By RT-PCR analysis, the expression of
CORS26 mRNA was not detectable in NIH3T3 cells,
BALB/3T3 cells, C3H10T1/2 cells, and MC3T3-E1 cells (Fig.
7).
In Situ Hybridization of CORS26 in the Embryonal Mouse
Tissues--
To examine the expression of CORS26 during
embryonic development in vivo, we performed in
situ hybridization using embryonal mouse serial sections at 13-15
days p.c. CORS26 transcripts were localized in regions of
cartilage primordium of occipital bone and that of the vertebral body
of a 13-day-p.c. embryo (Fig. 8, A-C and F-H). In Meckel's cartilage, high
levels of CORS26 mRNAs were seen in a 15-day-p.c. embryo
(Fig. 8, D and I). In the cartilage primordium of
digital bone of a 14-day-p.c. embryo, CORS26 was expressed
in prechondrocytes, but not in mature chondrocytes (Fig. 8,
E and J). These experiments showed that
CORS26 mRNA was present at relatively high
concentrations in precartilaginous primordia and developing cartilages.
No specific signal was detected above background levels by sense
riboprobes as controls.
Growth of CORS26 Transfected Cells in Vitro--
We generated
C3H10T1/2 cells stably transfected with the mammalian expression vector
containing CORS26. The expression of CORS26
mRNA in the transfectant was verified by RT-PCR analysis (data not
shown). The growth of CORS26 transfectants was significantly enhanced when compared with that of control cells. Moreover, saturation densities of CORS26 transfectants were higher than that of
control cells (Fig. 9).
During skeletal development, condensation of multipotential
mesenchymal cells to differentiate toward the various cell types is an
important process. One such process is the formation of chondrocytes
from undifferentiated mesenchymal cells. It was recently demonstrated
that the mouse embryonic mesenchymal cell line, C3H10T1/2, when
cultured at high density, is induced to undergo chondrogenic differentiation by TGF- In the present study, we demonstrated isolation of a novel gene,
CORS26, encoding a secretory protein of 246 amino acids
using a suppression subtractive hybridization technique between
TGF- Recently, it was reported that the crystal structure of Acrp30, a
member of the C1q family of proteins, showed homology with that of the
tumor necrosis factor (TNF) family proteins. Moreover, TNFs (28) and
C1q proteins have similar gene structure in the globular domains (29).
These similarities suggest an evolutionary link between the C1q-like
proteins and TNFs and establish a C1q/TNF molecular superfamily. It has
been reported that TNFs have monospecific receptors (TNFRs) (30). Since
CORS26 and C1q share significant similarities in the structure of the
collagenous domain, which has been shown to be important for
ligand-receptor interaction (13), it is possible that CORS26
might signal through the TNFRs or TNFR-like receptors.
Both Northern blot analysis and RT-PCR analysis showed a unique pattern
of gene expression of CORS26 in various tissues and cell
lines. CORS26 mRNA expression was found in rib growth
plate cartilage and early stages of chondrogenic differentiation of C3H10T1/2 cells from which the CORS26 cDNA was isolated.
Although the expression level was lower than that in cartilage,
CORS26 was also expressed in kidney of adult mice. Indeed,
DNA sequence homology searches in the GenBankTM Mouse EST
data bases using the BLAST search program revealed that several mouse
EST sequences isolated from whole embryo or adult kidney libraries were
homologous or identical to the 5' and 3' ends of the CORS26
cDNA. These findings from the EST data base are consistent with the
tissue distribution of the CORS26 gene. However,
CORS26 mRNA was not detected in fibroblastic cell lines
and osteoblastic cell lines. The expression of CORS26 in kidney raises the question about its possible role in this tissue, but
at present, details are unclear.
The basic form of the skeleton are first recognizable when mesenchymal
cells aggregate into regions of high cell density called condensations.
They subsequently differentiate into cartilage and bone and continue to
grow by cell proliferation, maturation, and matrix deposition. In
situ hybridization of mouse embryos 13-15 days p.c. showed that
CORS26 was highly expressed in precartilaginous primordia
and Meckel's cartilage. In each case, CORS26 transcripts appeared to be localized in condensed prechondrocytic mesenchymal cells
or in less mature chondrocytes, whereas the levels of CORS26 mRNA were lower or undetectable in the mature chondrocytes.
Furthermore, to test the role of the CORS26 gene in
vitro, we generated C3H10T1/2 cells stably transfected with
pcDNA3.1/CORS26. Since CORS26 mRNA is expressed in
condensed prechondrocytic mesenchymal cells in vivo, we
examined the effect of the CORS26 gene on cell
proliferation. Overexpression of CORS26 enhanced growth of
C3H10T1/2 cells and increased saturation densities in vitro.
This finding suggested that secreted CORS26 protein acted as a growth
factor based on its mitogenic activity on C3H10T1/2 cells.
Although the function of CORS26 in vivo is not clear, one
possible function is the local regulation of mesenchymal condensation as secreted autocrine/paracrine factors during an early stage of
skeletal development. Further analysis of CORS26 will lead to a better understanding of the molecular mechanism in skeletal development.
1-treated and untreated C3H10T1/2 cells. The deduced amino
acid sequence of CORS26 consists of 246 amino acids with a
secretory signal peptide and contains a collagenous region
(Gly-X-Y repeats) at the NH2
terminus and a complement factor C1q globular domain at the COOH
terminus. CORS26 is structurally similar to C1q and to
adipocyte-specific protein Acrp30. Transfection analysis suggested that
CORS26 is a secretory protein. Northern blot analysis revealed that
CORS26 mRNA was present at high levels in rib growth plate cartilage and at moderate levels in kidney of adult mice. CORS26 mRNA was not detected in NIH3T3 cells,
BALB/3T3 cells, C3H10T1/2 cells, or osteoblastic MC3T3-E1 cells
by reverse transcription-polymerase chain reaction analysis. In
situ hybridization of mouse embryos between 13 and 15 days
postcoitus revealed relatively high levels of CORS26
mRNA in condensed prechondrocytic cells of cartilage primordia and
developing cartilages. However, CORS26 mRNA were undetectable in mature chondrocytes. Furthermore, overexpression of
CORS26 enhanced the growth of C3H10T1/2 cells in
vitro. The present findings suggest that the CORS26
gene may play an important role in skeletal development.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (9), bone
morphogenetic protein-2 (10), or a combination of osteoinductive
bone proteins (11) in high-density micromass cultures. The formation of
the spheroids resembled the condensation of mesenchymal cells seen in
precartilage. Thus, C3H10T1/2 cells in high-density micromass cultures
are suited for studying the molecular mechanisms involved in skeletal development.
1-treated C3H10T1/2 cells
were subtracted with those in untreated C3H10T1/2 cells using the
suppression subtractive hybridization (SSH) technique (12), and we
isolated a novel gene, CORS26 (collagenous
repeat-containing sequence of 26-kDa protein).
Sequence analysis revealed that CORS26 possesses a collagenous
structure at the NH2 terminus and a complement factor C1q
globular domain at the COOH terminus. Due to the structural similarity
between CORS26 and subunits of complement factor C1q, this novel
protein is thought to be a member of the C1q-related protein family.
The presence of the signal peptide, plus the hydrophilic nature of
CORS26, suggests that CORS26 is a secretory protein. Indeed, the CORS26
protein was secreted from COS-7 cells by transient transfection
analysis. CORS26 mRNA is specifically expressed in cartilage and kidney in the adult mouse. Moreover, the expression of
this novel gene was observed in cartilage primordium or developing cartilage in embryonal mouse tissues in vivo. Thus, a
possible secretory protein encoded by the CORS26 gene may be
one of the important signaling molecules produced by prechondrocytic
mesenchymal cells or early chondrocytes during skeletal development.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 was added to the culture medium. Human TGF-
1 was purchased from R & D Systems (Minneapolis, MN). The mouse cell lines, NIH3T3 and MC3T3-E1, were also obtained from the
RIKEN Cell Bank, and BALB/3T3 cells were from Cancer Cell
Repository of Tohoku University (Sendai, Japan).
1 for 6 h, and
driver cDNA was from mRNAs of untreated C3H10T1/2 cells.
Products from the secondary PCR were cloned into pT7Blue T vector
(Invitrogen), and transformed into competent Escherichia
coli JM109 cells.
1 or mouse
rib growth plate cartilage using a RNeasy kit (Qiagen). Rib
growth plate cartilages were obtained from 10-day-old mice. Twenty
micrograms each of total RNA was denatured using glyoxal and
electrophoresed in 1% agarose gel, then transferred to
Hybond-N+ nylon membranes (Amersham Pharmacia Biotech) and
UV-cross-linked onto the membrane. For analysis of CORS26
expression in various normal adult mouse tissues, the Multiple Tissue
Northern blot (CLONTECH) was purchased. The
blotting membrane was incubated in prehybridization solution of 50%
formamide, 5× standard saline citrate (SSC), 5× Denhardt's solution,
0.1% SDS, and 50 µg/ml salmon sperm DNA at 43 °C for 3 h,
and then hybridization was performed using a 1.6-kb cDNA fragment
of CORS26 labeled with [32P]dCTP by random
priming (TaKaRa, Shiga, Japan) at 43 °C for 16 h. The membrane
was washed in 2× SSC containing 0.1% SDS at room temperature for 15 min, 0.5× SSC containing 0.1% SDS at 55 °C for 60 min, and 0.1×
SSC at room temperature for 10 min, respectively. The membranes were
autoradiographed with Hyper-film (Amersham Pharmacia Biotech) at
80 °C. After autoradiography, the membrane was stripped and
rehybridized with mouse glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) probe for internal loading control.
1. For the 3'-RACE method, 5'-GGGCCAAAGGTGAGAAAGGA-3' and 5'-GCCCCCGTATCAGGTGTGTA-3' were
designed as gene-specific primers, and for the 5'-RACE method, 5'-GGCCCCAAATCTCCCAGTCAT-3' and 5'-GGTCGCCTTTGTCTCCTTTCT-3' were designed as gene-specific primers. The nested PCR products were subcloned into the pT7Blue T vector and sequenced.
1-treated
C3H10T1/2 cDNA and cloned directly into pGEM-T Easy (Promega,
Madison, WI). The amplification conditions were 80 °C for 5 min,
followed by 30 cycles at 94 °C for 30 s, 65 °C for 40 s, and 72 °C for 2.5 min and a final incubation at 72 °C for 5 min. This construct was sequenced for accuracy and was used for
in vitro transcription and translation.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 treatment, and
we selected several clones specifically up-regulated by TGF-
1 treatment.
1 treatment (Fig.
1). We, therefore, focused further
analysis on this clone. To obtain a full-length sequence, we carried
out the 5'- and 3'-RACE using a TGF-
1-treated C3H10T1/2-derived
cDNA. The full nucleic acid sequence obtained (1,879 bp) and the
deduced amino acid sequence are shown in Fig.
2. The full length of 1,879 bp obtained
was in good agreement with the transcript size of ~2.0 kb estimated
on Northern blots. An open reading frame extends from nucleotide
positions 100-837 and encodes a protein of 246 amino acids with an
estimated molecular mass of 26.8 kDa. The first ATG codon
(nucleotides 100-102) lies in the context of the Kozak consensus
initiation site of the eukaryotic mRNA translation (15). The
CORS26 cDNA contains two consensus polyadenylation signals at nucleotide positions 1,462 and 1,837. Comparison of a
CORS26 cDNA sequence to the GenBankTM data
base using the BLAST program revealed that there was no significant
homology with any known genes. The CORS26 cDNA sequence was submitted to the GenBankTM as a novel gene with
accession number AF246265.
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Fig. 1.
Northern blot analysis confirming
differential gene expression for clone 129. Total RNA obtained
from C3H10T1/2 cells without or with TGF- 1 treatment was
electrophoresed. The blot was stripped and reprobed with a GAPDH probe
as the loading control.
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Fig. 2.
Nucleotide sequence and the predicted amino
acid sequence of CORS26. The triangle
indicates the putative signal peptide cleavage site. The position of
collagen-like domains are circled. Two separate consensus
polyadenylation signals are underlined.
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Fig. 3.
A, in vitro transcription and
translation of CORS26 cDNA. The translation products were separated
on 12.5% SDS-polyacrylamide gel and visualized by autoradiography. An
~26-kDa protein was generated. The protein molecular mass is
indicated by kilodaltons on the left side. B,
hydrophobicity plot of CORS26 protein. Positive values indicate
hydrophobic, and negative values indicate hydrophilic regions.
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Fig. 4.
Detection of FLAG-tagged CORS26 protein in
conditioned medium. Western blot analysis of proteins from
conditioned medium (CM) and cell lysates of COS-7 cells
transiently transfected with pcDNA3.1/CORS26-FLAG (lanes
1 and 3) or pcDNA3.1 vector (lanes 2 and
4). The proteins (20 µg) were separated on 12.5%
SDS-polyacrylamide gel and immunoblotted with an anti-FLAG M2
antibody.
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Fig. 5.
Comparison of the amino acid sequence of the
COOH-terminal globular domain of CORS26 with those of other
proteins. The references were CRF, precerebellin, AdipoQ/Acrp30,
and C1qC. Conserved amino acids are boxed. The numbers on
the left indicate amino acid residues for each
protein.
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Fig. 6.
Expression of the CORS26
mRNA in adult mouse tissues. A Northern blot of several
tissues was hybridized with the CORS26 cDNA. Double
transcripts of 2.3 and 2.0 kb are detected in kidney and rib growth
plate cartilage, respectively. Size markers are indicated on
the left. The blot was rehybridized with a GAPDH
probe.
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Fig. 7.
RT-PCR analysis of CORS26
expression in mouse cell lines. Aliquots of the PCR products
were electrophoresed in 2% agarose gel, and the gel was stained with
ethidium bromide. A 300-bp CORS26-specific band was not seen
in the mouse cell lines examined. PCR for GAPDH expression was also
performed as a quality control. The molecular mass marker is
X174/HaeIII.
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Fig. 8.
In situ hybridization analysis of
CORS26 expression in embryonal mouse tissues.
Bright-field (A-E) and dark-field (F-J)
microphotographs of the sections hybridized to CORS26
antisense riboprobes are shown. The CORS26 signal is
shown in the cartilage primordium of occipital bone (A and
F), of lumbar vertebral body (B and
G), and sacral vertebral body (C and
H) of a 13-day-p.c. embryo. D and I
show the signal in Meckel's cartilage of a 15-day-p.c. embryo.
CORS26 signals were detected in the prechondrocytes of
developing digits of a 14-day-p.c. embryo (E and
J). Bar, 1,500 µm in A, 200 µm in
B, C and 100 µm in D and E.
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Fig. 9.
Growth of CORS26
transfectants in vitro. C3H10T1/2-CORS26
(closed circle) or C3H10T1/2-vector (open circle)
were seeded into 24-well culture plates at a density of 1 × 104 cells/well. The number of cells were counted in
triplicate at the indicated days. Results are shown as mean ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. C3H10T1/2 cells cultured under this condition form a three-dimensional spheroid structure, and the morphology of the cells in the spheroid resembled that of the cells
seen in precartilage condensations (9). The formation of the
spheroid in vitro mimic the condensation event of
chondrogenesis in vivo.
1-treated and untreated C3H10T1/2 cells cultured at high
density. Sequence analysis reveals that CORS26 protein has a
hydrophobic signal peptide at the NH2 terminus and lacks a
putative transmembrane domain. The presence of a putative signal
peptide suggests that CORS26 enters the secretory pathway. Actually,
CORS26 was secreted from COS-7 cells after transient transfection of a
FLAG-tagged CORS26 expression vector. The deduced amino acid
sequence of CORS26 displays structural similarity to several
C1q related proteins, such as C1q A, B, and C chains, Acrp30, and CRF,
containing the collagenous repeats (Gly-X-Y) at
the NH2 terminus and the globular domain at the COOH
terminus. This suggested that CORS26 may belong to the C1q family
proteins. C1q family proteins are known to homo- or hetero-oligomerize
via the collagenous structures, suggesting that CORS26 might form
oligomers with itself or other proteins. The COOH-terminal region of
the protein contains three potential phosphorylation sites.
Consequently, this molecule is a potential target for protein
phosphorylation via protein kinase C and casein kinase II. In addition,
a cysteine residue in the globular region of the C1q B and C
chains plays an important role in the formation of disulfide bonds with
IgG (27) and the stabilization of the triplex strands in the
collagenous domain (24). These cysteine residues are replaced by other
residues in CORS26 similar to Acrp30 and CRF.
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FOOTNOTES |
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* This work was supported by a Grant-in-aid from the Ministry of Education, Culture and Science of Japan (No. 11470396).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF246265.
§ To whom correspondence should be addressed: Dept. of Radiology and Radiation Oncology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-2967; Fax: 81-6-6879-2970; E-mail: tmaeda@radiol.dent.osaka-u.ac.jp.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M007898200
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ABBREVIATIONS |
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The abbreviations used are: TGF, transforming growth factor; SSH, suppression subtractive hybridization; FBS, fetal bovine serum; PCR, polymerase chain reaction; RT-PCR, reverse transcription-PCR; bp, base pair(s); kb, kilobase pair(s); p.c., post coitus; RACE, rapid amplification of cDNA end(s); DMEM, Dulbecco's modified Eagle's medium; CRF, complement protein C1q-related factor; Acrp, adipocyte complement-related protein; TNF, tumor necrosis factor; TNFR, TNF receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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