MOLECULAR AND CELLULAR BIOLOGY, Oct. 2008, p. 6134–6147
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 28, No. 19
Posttranscriptional Regulation of Chicken ccn2 Gene Expression by
Nucleophosmin/B23 during Chondrocyte Differentiation?†
Yoshiki Mukudai,1Satoshi Kubota,2Harumi Kawaki,2Seiji Kondo,2Takanori Eguchi,2Kumi Sumiyoshi,2
Toshihiro Ohgawara,2,3Tsuyoshi Shimo,3and Masaharu Takigawa1,2*
Biodental Research Center, Okayama University Dental School,1and Department of Biochemistry and Molecular Dentistry2and
Department of Oral and Maxillofacial Surgery and Biopathological Science,3Okayama University Graduate School of Medicine,
Dentistry, and Pharmaceutical Sciences, Okayama 700-8525, Japan
Received 26 March 2008/Returned for modification 20 May 2008/Accepted 17 July 2008
CCN2/CTGF is a multifunctional factor that plays a crucial role in the growth and differentiation of
chondrocytes. The chicken ccn2 gene is regulated not only at the transcriptional level but also by the
interaction between a posttranscriptional element in the 3? untranslated region (3?-UTR) and a cofactor.
In the present study, we identified a nucleophosmin (NPM) (also called B23) as this cofactor. Binding of
NPM to the element was confirmed, and subsequent analysis revealed a significant correlation between the
decrease in cytosolic NPM and the increased stability of the ccn2 mRNA during chondrocyte differenti-
ation in vivo. Furthermore, recombinant chicken NPM enhanced the degradation of chimeric RNAs
containing the posttranscriptional cis elements in a chicken embryonic fibroblast extract in vitro. It is
noteworthy that the RNA destabilization effect by NPM was far more prominent in the cytosolic extract of
chondrocytes than in that of fibroblasts, representing a chondrocyte-specific action of NPM. Stimulation
by growth factors to promote differentiation changed the subcellular distribution of NPM in chondrocytes,
which followed the expected patterns from the resultant change in the ccn2 mRNA stability. Therefore, the
present study reveals a novel aspect of NPM as a key player in the posttranscriptional regulation of ccn2
mRNA during the differentiation of chondrocytes.
CCN2 (connective tissue growth factor [CTGF]/hypertro-
phic chondrocyte-specific gene product 24 [Hcs24]) is a cys-
teine-rich secretory protein of 36 to 38 kDa that has four
distinct modules, i.e., insulin-like growth factor-binding pro-
tein-likes, von Willebrand factor type C repeat, throm-
bospondin type 1 repeat, and C-terminal modules (4, 6, 54, 68,
69). CCN2 is a member of the CCN family (reviewed in ref-
erences 6, 38, 53, 54, 68, and 69), which comprises ccn1 (cef-
10/cyr61 [37, 66]), ccn2 (ctgf/hcs24/fisp12 [5, 46, 57]), ccn3 (nov
), ccn4 (elm-1/wisp-1 [20, 52]), ccn5 (ctgf-l/wisp-2/cop1 [52,
75]), and ccn6 (wisp-3 ). CCN2 was initially isolated from
angioendothelial cells as a growth factor related to platelet-
derived growth factor (PDGF) and was revealed to have
PDGF-like mitogenic and chemotactic activities toward fibro-
blasts (17, 23, 28, 33, 54). However, recent studies have re-
vealed that CCN2 is a multifunctional factor that regulates the
growth and/or differentiation, chemotaxis, adhesion of various
cells, and extracellular matrix formation by various cells, in-
cluding vascular endothelial cells (2, 29, 54, 64). Furthermore,
we showed earlier that CCN2 plays an important role in the
growth and differentiation of chondrocytes and osteoblasts
during endochondral ossification (46–48).
As to CCN2, it has been suggested that its gene expression
is regulated at multiple steps, such as transcriptional, post-
transcriptional, and translational stages (1), for playing its
multiple roles mentioned above. For instance, transforming
growth factor ? (TGF-?) induces the expression of CCN2
(28, 46), and a few TGF-? response elements have been
found in the ccn2 promoter region (11–13, 19, 39, 55). In
addition to such transcriptional regulation, we also reported
earlier that expression of the gene is regulated by its 3?
untranslated region (3?-UTR) at posttranscriptional stages
(30–32, 34–36, 44). Furthermore, we recently reported (45)
that a cis element in the 3?-UTR of chicken ccn2 mRNA and
its putative trans-factor counterpart collaboratively play an
important role in the posttranscriptional regulation by de-
termining the stability of ccn2 mRNA. In fact, the affinity of
binding between them is altered during chondrocytic differ-
entiation, thus suggesting the involvement of this RNA-
protein interaction in the precise posttranscriptional regu-
lation of ccn2 mRNA during endochondral ossification.
However, the identity of the putative trans factor and its
mechanism of action were not clarified at that time.
In the present study, we finally purified and identified this
trans-factor protein as nucleophosmin (NPM) (also called
B23), which actually bound to the minimal repressive cis
element in the 3?-UTR of ccn2 mRNA. With the recombi-
nant protein we produced, we could successfully reconstruct
the posttranscriptional regulatory events in vitro that were
observed during endochondral ossification in vivo. We also
obtained data showing that alteration of nucleocytoplasmic
shuttling of the trans factor is critical for the regulation of
chicken ccn2 expression during the differentiation of chon-
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Dentistry, Okayama University Graduate School
of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-
cho, Okayama 700-8525, Japan. Phone: 81-86-235-6645. Fax: 81-86-
235-6649. E-mail: email@example.com.
† Supplemental material for this article may be found at http://mcb
?Published ahead of print on 4 August 2008.
MATERIALS AND METHODS
Cell isolation and culture. Chicken embryonic fibroblasts (CEFs) were iso-
lated from a whole 10-day-old chicken embryo and maintained in Dulbecco’s
modified Eagle minimum essential medium supplemented with 10% fetal bovine
serum (FBS) in an atmosphere of humidified air containing 5% CO2at 37°C.
Chicken upper sternum chondrocytes (US cells) and lower sternum chondrocytes
(LS cells) were isolated from chicken embryo sternum cartilage on day 18 as
described previously (44).
For growth factor stimulation, the medium was exchanged for FBS-free me-
dium, and 24 h later, the cells were treated with 10 ng/ml of human TGF-?
(Biomedical Technologies Inc., Stoughton, MA), 200 ng/ml of recombinant hu-
man bone morphogenetic protein 2 (BMP 2) (R&D Systems, Minneapolis, MN),
10 ng/ml of human PDGF (Sigma Aldrich, St. Louis, MO), or 30 ng/ml of
recombinant human CCN2 (47) for a further 24 h in the absence of FBS.
Preparation of nuclear, cytoplasmic, and total cellular extracts. Nuclear and
cytoplasmic extracts were prepared by using a commercial kit, the CelLytic
NuCLEAR extraction kit (Sigma Aldrich), according to the manufacturer’s pro-
tocol. The protein concentrations of both fractions were determined by use of a
bicinchoninic acid protein assay kit (Pierce, Rockford, IL) (67). Total cell ex-
tracts were prepared with Cell Lytic-M lysis reagent (Sigma). In a few experi-
ments, the DNA concentrations in nuclear and total cell extracts were quantified
by measuring the fluorescence emitted from 5 ?l of the extract in the presence
of 100 ng/ml of bisbenzimide trihydrochloride (Hoechst 33258) at a wavelength
of 458 nm (excitation wavelength, 356 nm).
Isolation and purification of the trans-factor protein. The trans-factor protein
that bound to the 3?-UTR of chicken ccn2 mRNA was isolated by utilizing an
RNA affinity column according to a previous report (62) with slight modifica-
tions. All of the procedures were carried out at 4°C. In the first step, a heparin-
Sepharose column (HiTrap heparin HP [5 ml]; Amersham Bioscience, Piscat-
away, NJ) was prewashed with 50 ml of buffer B (50 mM Tris-HCl [pH 7.5]
containing 2 mM EDTA, 5% glycerol, 7 mM 2-mercaptoethanol [2-ME], and 100
mM NaCl). Two grams of the nuclear extract of CEFs was then applied to the
column. Then, the column was washed with 100 ml of buffer B containing 100
mM NaCl, and the bound proteins were eluted from the column by a step
gradient of NaCl concentrations (100 to 1,000 mM) in buffer B.
These fractions were assayed for binding to the minimal repressive cis region
of the 3?-UTR of chicken ccn2 mRNA (3?-100/50) (36) by using a UV cross-
linking method. The fractions that showed the maximal binding to the 3?-100/50
fragment were eluted at NaCl concentrations of 200 to 600 mM and then pooled.
The pooled fractions were subsequently loaded onto an RNA affinity chroma-
tography column that had been prepared by cross-linking N-hydroxysuccinimide
(NHS)-activated Sepharose in a column (HiTrap NHS-activated HP 1 ml; Am-
ersham Bioscience) and 5 mg of 3?-100/50 RNA fragment that had been synthe-
sized in vitro by utilizing a commercial kit (RiboMAX large-scale RNA produc-
tion systems; Promega, Madison, WI). The column was washed with 20 ml of
buffer B containing 100 mM NaCl, and the bound proteins were eluted from the
column by a step gradient of buffer B containing NaCl (100 to 1,000 mM). Each
fraction was assayed by the UV cross-linking assay, and the fractions that showed
maximum binding were combined and then concentrated by use of an Amicon
Ultra-4 centrifugal filter device (4 ml, 1,000 normal molecular weight limit;
Millipore, Bedford, MA).
Determination of internal amino acid sequence. The purified fraction was
further separated by two-dimensional electrophoresis with isoelectric focusing
and sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). The
RNA-binding proteins in the combined and concentrated fractions were verified
by Northwestern blotting analysis using 3?-100/50 RNA fragment as a radiola-
beled probe. Thereafter, the spot corresponding to the 40-kDa one stained by
Coomassie brilliant blue (CBB) was excised from a gel and subjected to internal
amino acid sequence analysis by Shimadzu Biotech (Tsukuba, Ibaraki, Japan).
REMSA and UV cross-linking assay. The RNA electromobility shift assay
(REMSA) and UV cross-linking assay were carried out as described previously
(45). The schemata of the probes used are shown in Fig. 3A. Extracted proteins,
recombinant nucleophosmin (described below), or bovine serum albumin (BSA)
was incubated at 25°C for 30 min with 5 ? 104cpm of radiolabeled RNA probes,
and then the binding mixture was incubated with 1 ?l of 1/100 diluted RNase
cocktail (Ambion, Austin, TX) for a further 10 min at 37°C. The RNA-protein
complex was subjected to 6% native PAGE in 0.5? Tris-borate-EDTA (TBE)
buffer (Invitrogen, Carlsbad, CA) for REMSA. The gels were subsequently dried
For the UV cross-linking assay, after RNase digestion, the protein-RNA
complexes were put on ice and UV irradiated for 10 min by a UV cross-linker
(Amersham Bioscience). Then, the samples were heated at 95°C for 5 min in an
SDS sample buffer (Sigma Aldrich) in the presence of 5% 2-ME and separated
by 12.5% or 4 to 20% gradient SDS-PAGE. The gels were subsequently dried
RNA immunoprecipitation assay. Ten micrograms of total RNA was incu-
bated with 2.5 ?g of nuclear extract of CEFs or recombinant NPM at 30°C for 1 h
in 250 ?l of a reaction buffer (20 mM HEPES [pH 7.9], 20% glycerol, 50 mM
KCl, 0.2 mM EDTA, 4.5 mM MgCl2, 0.5 mM ATP, 20 mM creatine phosphate,
2 units/mg RNAsin [Promega]), and then 200 ml of buffer N (50 mM Tris-HCl
[pH 8], 100 mM NaCl, 0.05% NP-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol [DTT]) containing 200 ng of antibody or preimmune rabbit immu-
noglobulin G (IgG) was added, and the mixture was further incubated at 4°C for
16 h. Protein A-Sepharose beads (Amersham Bioscience) (20 ?l) were washed
twice with 750 ?l of buffer K (100 mM Tris-HCl [pH 8], 12.5 mM EDTA, 150
mM NaCl, 1% SDS) and resuspended in the reaction mixture containing anti-
body-antigen-RNA complex. After incubation with shaking at 4°C for 1 h, the
reaction mixture beads were washed three times with buffer K. Thereafter, the
beads were suspended in 250 ?l of buffer K, heated at 80°C for 10 min, and
centrifuged. The RNA in the supernatant was purified by Isogen-LS (Nippon
Gene, Tokyo, Japan) and subjected to reverse-transcription-mediated-PCR (RT-
PCR) as described in our previous studies (44, 45).
Western blot analysis and Northwestern blot analysis. Western blot analysis
was carried out as described previously (45). The proteins in SDS sample buffer
with 2-ME (Amersham Bioscience) (10 ?g or protein amounts from the cells that
conferred 18 ?g of DNA) were heated, separated by 12.5% SDS-PAGE, and
transferred to a polyvinylidene difluoride (PVDF) membrane (Hybond P; Am-
ersham Bioscience). After the membrane was blocked with 5% skim milk, it was
incubated with a 1/1,000 dilution of polyclonal anti-CCN2 antibodies (ABcam,
Cambridge, United Kingdom), a 1/1,000 dilution of monoclonal anti-NPM an-
tibody (American Research Products, Inc., Belmont, MA), a 1/2,500 dilution of
monoclonal anti-?-tubulin antibody (Sigma Aldrich), or a 1/2,500 dilution of
monoclonal anti-lamin B1 antibody (Zymed, South San Francisco, CA). Then,
the membrane was incubated with a 1/20,000 dilution of peroxidase-conjugated
goat anti-mouse IgG antibody (American Qualex, La Mirada, CA). Subse-
quently, the blot was visualized by use of an ECL Western blotting analysis
system (Amersham Bioscience).
For Northwestern blotting, the protein was separated and transferred to a
PVDF membrane, as performed in Western blotting. The membrane was stained
with CBB prior to incubation with the probe. Then, the membrane was prein-
cubated for 16 h at 4°C in blocking buffer consisting of 10 mM Tris-HCl (pH 7.5),
50 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM DTT, 5% skim milk, 1 ?g/ml
yeast tRNA (Roche Applied Science, Penzberg, Germany), after which it was
incubated for 2 h at 25°C with the radiolabeled probe (1 ? 106cpm/ml) in
binding buffer comprising 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2,
1 mM EDTA, 1 mM DTT, 0.25% skim milk, and 1 ?g/ml yeast tRNA. There-
after, the membrane was washed three times with wash buffer consisting of 10
mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, and 1 mM DTT and was
subjected to autoradiography.
RNA degradation analysis in vivo. The cells in 3.5-cm and 10-cm tissue culture
dishes were grown until subconfluent, and then 10 ?g/ml of actinomycin D
(Sigma Aldrich) was added to the cultures in order to arrest de novo RNA
synthesis. In small interfering RNA (siRNA)-mediated knockdown experiments,
CEFs were transfected with synthetic siRNAs as described elsewhere and sub-
jected to actinomycin D treatment 24 h after transfection. After properly timed
intervals, total cellular RNA was isolated and subjected to Northern blotting or
real-time PCR analysis after reverse transcription in order to evaluate the ccn2
or gapdh mRNA level.
RNA preparation and Northern blot analysis. Total cellular RNA was isolated
by Isogen (Nippon Gene), according to the manufacturer’s protocol. Northern
blotting analysis was carried out as described previously (45). Total RNA was
denatured by glyoxysal, separated on 1% agarose gels, and then blotted onto
nylon membranes (Hybond N; Amersham Bioscience). After blotting, the mem-
brane was fixed with 5% acetic acid and stained with 0.02% methylene blue to
visualize rRNAs, hybridized, washed, and subsequently autoradiographed.
The probe for chicken ccn2 was prepared as described previously (45), and the
probe for chicken npm is described below (see “Preparation of recombinant
chicken NPM”). The plasmids were linearized by SpeI and transcribed in vitro by
bacteriophage T7 RNA polymerase (Promega) in the presence of 50 ?Ci of
[?-32P]UTP (3,000 Ci/mmol; Amersham Bioscience) for the preparation of ra-
diolabeled antisense RNA, followed by RQ1 DNase (Promega) digestion and
spin-column (ProbeQuant G-50; Amersham Bioscience) purification.
Real-time PCR analysis. After being transcribed to cDNA as described else-
where, the npm and ccn2 mRNA levels were quantitatively analyzed by a real-
time PCR method with LightCycler (Roche). Quantification was performed by
VOL. 28, 2008POSTTRANSCRIPTIONAL REGULATION OF ccn2 BY NPM 6135
an intercalator methodology with SYBR green real-time PCR master mix
(Toyobo, Osaka, Japan) as described previously (72). As a control, expression of
the glyceraldehyde 3?-phosphate dehydrogenase (GAPDH) gene was also ana-
lyzed. The nucleotide sequences of the specific primers used in the amplification
were 5?-GCA GAG AAG GAA TAT CAG TT-3? (sense) and 5?-CTC AAA
TCC ACC TAG TGA AAC-3? (antisense) for npm, 5?-CAC CAA CGA TAA
TGC TTT C3? (sense) and 5?-ACT TAG CTC TGT ACG TCT TCA-3? (anti-
sense) for ccn2, and 5?-AGG CTG TGG GGA AAG TCA-3? (sense) and 5?-
GAC AAC CTG GTC CTC TGT GTA T-3? (antisense) for gapdh. The integrity
of all amplicons was confirmed by melting curve analysis.
Preparation of recombinant chicken NPM. Chicken npm cDNA, including the
full-length open reading frame, was obtained by reverse-transcription-mediated-
PCR (RT-PCR), utilizing total cellular RNA from CEFs. The nucleotide se-
quences of sense and antisense primers were 5?-CAT ATG GAG GAC AGC
AGC GCC ATG GAC-3? and 5?-CTC GAG AGT CTG TCT CCA CTG CCA
G-3?, respectively. The amplicon was subcloned into pGEM-T Easy (Promega)
by the TA-cloning method, which was also utilized for the preparation of the
probe for Northern blotting. This plasmid was double digested with NdeI and
XhoI, and the npm cDNA was isolated and subcloned into the corresponding site
of pET21b(?) (EMD Biosciences, Madison, WI) in frame with the His6tag at
the C terminus. The resultant plasmid was designated pET21-cNPM. An Esch-
erichia coli strain, RosettaBlue(DE3)pLysS (EMD Biosciences), was trans-
formed with pET21-cNPM, and grown in Overnight Express instant TB (terrific
broth) medium (EMD Biosciences) containing ampicillin (50 ?g/ml), tetracy-
cline (12.5 ?g/ml), and kanamycin (34 ?g/ml) at 37°C for 16 h. Thereafter, the
cells were pelleted by centrifugation at 7,000 ? g for 10 min, suspended in
ice-cold binding buffer (20 mM phosphate [pH 7.4] containing 500 mM NaCl, 20
mM imidazole, 0.5% Tween 20, and 1 tablet/10 ml of Complete, Mini EDTA-
free protease inhibitor cocktail tablets [Roche Applied Science]), and disrupted
by sonication. The lysate was centrifuged at 12,000 ? g for 10 min at 4°C, and the
supernatant was filtered through a 0.45-?m filter. Chicken NPM-His6fusion
protein was purified with a HisTrap HP kit (Amersham Bioscience), according to
the manufacturer’s protocol. The filtered bacterial lysate was loaded onto the
column equilibrated with binding buffer, and after being washed with binding
buffer, the fusion protein was eluted with elution buffer (binding buffer contain-
ing 300 mM imidazole). The eluate was dialyzed and finally concentrated by an
Amicon Ultra-4 centrifugal filter device. The quantity of purified recombinant
chicken NPM-His6fusion protein was determined by a bicinchoninic acid protein
assay, and the quality was evaluated by CBB staining after SDS-PAGE and
Western blotting with anti-chicken NPM or anti-His (C-terminal) antibody (In-
vitrogen; data not shown). The purified protein was aliquoted, supplemented
with protease inhibitor cocktail (Sigma Aldrich), and stored at ?80°C until used.
Preparation of firefly luciferase-ccn2 fusion gene constructs. The reporter
plasmids containing firefly luciferase-chicken ccn2 fusion genes on a pGL3L(?)
backbone (34) were available from a previous study (45). In order to construct
additional plasmids for preparation of the reporter mRNAs in vitro, the parental
pGL3L(?) was double digested with HindIII and XbaI, and the isolated firefly
luciferase gene was subcloned between the corresponding sites in pGEM4 (Pro-
mega). This plasmid was designated Luc(?). The plasmids referred to as 3?-Full,
3?-100/50, and 3?-50, which contained the full-length 3?-UTR of chicken ccn2 or
ccn2 deletion mutations on a pGL3L(?) backbone (45), were double digested
with XbaI and EcoRI, and each 3?-UTR fragment was subcloned into the cor-
responding sites in Luc(?). As a result, the 3?-UTR fragments were located at
the 3? end of the firefly luciferase gene in these three resultant chimeric fusion
constructs, which were designated Luc-3?-Full, Luc-3?-100/50, and Luc-3?-50.
Proper construction of all plasmids was confirmed by nucleotide sequencing (58)
and restriction enzymatic digestion.
DNA transfection and luciferase assay. Twenty-four hours prior to transfec-
tion, 2 ? 105cells were seeded into each well in 35-mm tissue culture dishes.
Cationic liposome-mediated DNA transfection was carried out with 1 ?g of each
pGL derivative, which is described in the previous paragraph under “Preparation
of firefly luciferase-ccn2 fusion gene constructs,” in combination with 0.5 ?g of
pRL-TK, according to the manufacturer’s optimized methodology (FuGENE6;
Roche, Indianapolis, IN). Forty-eight hours after transfection, the cells were
lysed in 500 ?l of a passive lysis buffer (Promega), and the cell lysate was directly
used for the luciferase assay.
The dual luciferase assay system (Promega) was applied for the sequential
measurement of firefly (reporter) and Renilla luciferase (transfection efficiency
standard) activities with specific substrates. Quantification of both luciferase
activities and calculation of relative ratios were done as described previously
IVDA. An in vitro RNA degradation assay (IVDA) was carried out as de-
scribed previously (43) with slight modifications. Luc-3?-Full, Luc-3?-100/50, and
Luc-3?-50 were linearized by SpeI, transcribed, capped, and polyadenylated in
vitro in the presence of [?-32P]UTP by using a commercial kit (mMESSAGE
mMACHINE T7 Ultra; Ambion) according to the manufacturer’s protocol.
Schemata of the transcripts are shown in Fig. 5A. Fifty thousand cpm of each
radiolabeled RNA was incubated with 10 ?g of cytoplasmic extract in IVDA
reaction buffer consisting of 10 mM Tris-HCl (pH 7.5) containing 100 mM
potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 10 mM creatine
phosphate, 1 mM ATP, 0.4 mM GTP, and 0.1 mM spermine. After the appro-
priate incubation at 37°C, the RNA was purified by use of Isogen LS (Nippon
Gene) and subjected to 6% PAGE in the presence of 6 M urea in 1? TBE buffer,
followed by autoradiography. The optical density of each signal in the autora-
diograph was quantified by using a commercial computer software, Quantity One
(PDI Inc., New York, NY). In certain experiments, recombinant chicken NPM
(0.1 or 0.5 ?g) or the eluate from the RNA affinity column (1 ?g) was added
together with the cytosolic extract.
Synthesis and application of siRNA against chicken npm1. Two sets of siRNA
duplexes targeted to chicken npm1 mRNA were synthesized by iGene Thera-
peutics (Tsukuba, Japan). The nucleotide sequences of the sense strands of the
two double-stranded RNAs, si546 and si207, were 5?-UGA UGA GGA GGA
GAU UAA AAC ACC A-3? and 5?-CGA AGG CAA CCC AAC UAA AGU
AGU A-3?, respectively. For the knockdown experiments, 50 nM of each siRNA
was transfected into CEFs with the aid of a cationic liposomal transfection
reagent (RNAiFect; Qiagen, Hilden, Germany). The cells were harvested at 48 h
after transfection, and then total RNAs were extracted and subjected to reverse
transcription by avian myeloblastosis virus reverse transcriptase (Takara, Tokyo,
Japan), followed by real-time PCR analysis for quantification.
Statistical analysis. Unless otherwise specified, all experiments were repeated
at least twice, and similar results were obtained in the repeated experiments.
Statistical analysis was carried out by repeated measure analysis of variance,
Bonferroni/Dunn, or paired t test. Data were expressed as the means ? standard
Purification and identification of the protein(s) binding to
the 3?-UTR of ccn2 mRNA. Recently, we found that a 40-kDa
trans-factor protein bound specifically to an RNA cis element
(3?-100/50) in the chicken ccn2 3?-UTR, which posttranscrip-
tionally regulated gene expression during the course of chon-
drocyte differentiation (Fig. 1A) (45). First of all, we examined
whether the previous findings at the mRNA level correlated
with the resultant production of CCN2 protein during chon-
drocytic differentiation. Using CEF, LS, and US cells rep-
resenting immature mesenchymal cells, proliferating chon-
drocytes, and hypertrophic chondrocytes, Western blotting
analysis was performed with proteins from an equal number of
cells. As expected, increased production of CCN2 along with
chondrocytic differentiation was clearly observed (Fig. 1B).
Thereafter, in order to identify this protein, we employed the
experimental strategy summarized in Fig. 1D. In the first step
of purification, a nuclear extract of CEFs was applied to a
heparin-Sepharose column equilibrated with buffer B. Frac-
tions that were eluted from the column were assayed for pro-
tein content and binding to the 3?-100/50 fragment, the mini-
mal repressive cis element in the 3?-UTR of chicken ccn2
mRNA (45), by conducting a UV cross-linking assay. Fractions
that were eluted at 200 to 600 mM NaCl concentrations
showed maximal binding to the radiolabeled 3?-100/50 frag-
ment (data not shown), so these fractions were combined,
pooled, and subjected to further purification. In the next step,
the pooled fractions (2 mg of protein) were subjected to RNA
affinity chromatography. The pooled fractions were loaded
onto the 3?-100/50 RNA affinity column, and after excessive
washing of the column, the bound protein was eluted by a
linear step gradient of 100 to 1,000 mM NaCl in buffer B. The
6136MUKUDAI ET AL.MOL. CELL. BIOL.
results of the UV cross-linking assay (Fig. 2A) conducted on
the resulting fractions revealed that the fractions eluting at 200
to 600 mM NaCl contained the protein bound to the RNA,
whereas no positive signal was observed in the flowthrough
fraction, suggesting successful purification by the RNA chro-
matography. Therefore, these fractions were combined and
pooled. For further verification of the binding between the
proteins and the 3?-100/50 RNA fragment, Northwestern blot-
ting was carried out (Fig. 2B). Five micrograms of the eluted
protein was subjected to 4 to 20% SDS-PAGE and stained with
CBB (Fig. 2B). A major band corresponding to an approxi-
mate molecular mass of 40 kDa and several minor bands were
observed. Thereafter, the membrane was incubated with ra-
diolabeled 3?-100/50 probe (Fig. 2B, NW lanes). A 40-kDa
protein, which corresponded to the major band seen by CBB
staining, strongly bound to the probe, whereas another major
band was also observed, one corresponding to an approximate
molecular mass of 90 kDa. Other minor bands stained with
CBB showed no binding to the probe. Furthermore, molecular
FIG. 1. (A) Chicken in vitro model of endochondral ossification
and posttranscriptional regulatory outcome of ccn2 expression therein.
CEF, LS, and US denote chicken embryonic fibroblasts representing
undifferentiated mesenchymal cells, lower sternum cartilage cells rep-
resenting immature/proliferating chondrocytes, and upper sternum
cartilage cells representing hypertrophic chondrocytes, respectively.
?, ?, ?, and ?? indicate approximate levels of each mRNA or
protein in each cell; 3 and 1 indicate equivalent and increased
mRNA stability, respectively. Note that ccn2 mRNA stability is in-
creased in US cells (arrow pointing up), where binding of a 40-kDa
protein to the 3?-100/50 posttranscriptional regulatory element is
nearly absent (?). (B) Western blotting analysis of the CCN2 proteins
in CEF, LS, and US cells. Total cell lysates comprising 18 ?g of DNA
were analyzed. (C) A core nucleotide sequence conserved among
mammalian species in the 3?-100/50 region and the corresponding
sequence in the ccn2 mRNA 3?-UTR. (D) Experimental strategy for
the identification of the 40-kDa protein. As summarized in panel A,
the protein (illustrated as spheres) binds specifically to the 3?-100/50
RNA fragment in the 3?-UTR, which has been supposed to destabilize
ccn2 mRNA. On the basis of this specific RNA-protein interaction,
affinity purification methodology was employed, as illustrated. ORF,
open reading frame.
FIG. 2. Purification and identification of chicken ccn2 mRNA 3?-
UTR-binding protein by affinity chromatography. (A) UV cross-linking
assay of eluate from a chicken ccn2 mRNA 3?-UTR affinity column.
NHS-activated Sepharose in a column was covalently conjugated to the
3?-100/50 fragment of chicken ccn2 mRNA 3?-UTR, which had been
transcribed in vitro (see Materials and Methods). The nuclear proteins
from CEFs that bound to the RNA were eluted by a linear step gradient
of NaCl concentrations (100 to 1,000 mM) and concentrated. The pro-
teins in the column flowthrough (FT) and fractions that bound to the
3?-100/50 RNA fragment were examined by conducting a UV cross-link-
ing assay with SDS-PAGE in a 4 to 20% gradient gel. The positions of
molecular mass standards (in kilodaltons) (Rainbow markers; Amersham
Bioscience) are shown at the left side of the panel. (B) Northwestern
blotting. The molecular mass standards (Marker) and 5 ?g of eluted
proteins (Extract) from the combined positive fractions (obtained with
200 to 600 mM NaCl in panel A) were subjected to SDS-PAGE in a 4 to
20% gradient gel and blotted onto a PVDF membrane. The membrane
was first stained with CBB and then incubated with radiolabeled 3?-100/50
probe for Northwestern analysis (NW). The prominent bands bound by
the probe are indicated by arrows at the right side of the panel. (C) Iden-
tification of the purified protein as nucleophosmin. A tryptic digestion
product of the 40-kDa protein was subjected to Edman degradation, and
the internal peptide sequences obtained were analyzed by the BLAST
program of the National Center of Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov). Amino acid sequences from five fractions
(fractions [Fr.] 14, 16, 18, 20, and 22; see the figure in supplemental
material for details) purified through a high-pressure liquid chromato-
graph column were found to be identical to those of chicken NPM
(GenBank accession number NM_205267), as indicated by the fraction
numbers and underlined sequences. The boldface superscript numbers to
the right of the sequences represent residue numbers of NPM counting
from the initiation methionine.
VOL. 28, 2008POSTTRANSCRIPTIONAL REGULATION OF ccn2 BY NPM 6137
mass markers did not bind to the probe, indicating that the
bands were probe-specific ones. Since the 40-kDa protein cor-
responded to the one we had determined to be a putative trans
factor of ccn2 mRNA, this protein was subjected to protein
sequencing for identification.
Tryptic digestion products of the approximately 40-kDa pro-
tein excised from the SDS-polyacrylamide gel were separated
by passage through a reverse-phase high-pressure liquid chro-
matograph column, and 11 specific peaks, which were not
found in the blank control gel, were selected (see figure in
supplemental material). These peaks were applied to an amino
acid sequencer, and the results were subjected to a BLAST
(National Center for Biotechnology Information; http://www
.ncbi.nlm.nih.gov) amino acid homology search. Of the se-
quences of the proteins in these peaks, five (Fig. 2C) showed
complete homology with the sequence of chicken NPM/B23
(GenBank accession number NM_205267), a protein known to
shuttle between the nucleus and cytoplasm (3).
NPM binds to the repressive cis element of the 3?-UTR of
chicken ccn2 mRNA. The ability of recombinant chicken NPM
(with His6at its C-terminal region) to bind to the 3?-UTR of
chicken ccn2 mRNA was determined by REMSA (Fig. 3B) and
the UV cross-linking assay (Fig. 3C). In these experiments, two
radiolabeled RNA probes were prepared (Fig. 3A) on the basis
of our recent study (45). One was the 3?-100/50 probe, the
minimal element in the 3?-UTR of chicken ccn2 mRNA for
RNA destabilization through binding to the 40-kDa putative
trans factor. The other was 3?-50, which was not capable of
binding to the putative trans factor. In REMSA (Fig. 3B), the
recombinant chicken NPM bound to 3?-100/50, resulting in a
shift of the position of the probe after electrophoresis. The
binding was enhanced by increasing the concentration of NPM.
In contrast, no shifted band was observed after incubation of
BSA with 3?-100/50. Furthermore, the results of the UV cross-
linking assay (Fig. 3C) clearly indicated the binding between
NPM and 3?-100/50. By incubating NPM with 3?-100/50, the
40-kDa band was observed, and its density increased with an
increase in the concentration of NPM used. Also in this case,
no specific band was observed when BSA was incubated with
3?-100/50. As expected, no signal was observed with the nega-
tive-control probe, 3?-50 (Fig. 3B). Finally, to further ascertain
the interaction of NPM and the ccn2 mRNA, we also carried
out an RNA immunoprecipitation analysis. As shown in Fig.
3D, an anti-NPM antibody successfully coimmunoprecipitated
a ccn2 mRNA segment with native NPM in the nuclear extract,
further indicating the interaction between NPM and the ccn2
mRNA. These results together demonstrate the specific bind-
ing of NPM to 3?-100/50, thus strongly suggesting that NPM is
a trans factor of ccn2 mRNA.
Relevance of the ccn2 mRNA stability with subcellular dis-
tribution of NPM during differentiation of chondrocytes. Since
the 40-kDa trans-factor protein was anticipated to be an RNA-
destabilizing regulator during chondrocyte differentiation, the
intracellular fate of the ccn2 mRNA and the intracellular be-
havior of NPM were compared between proliferative LS and
hypertrophic US chondrocytes. As also described earlier, the
half-life (t1/2) of the ccn2 mRNA was prolonged by more than
twofold in US cells (20.4% remaining after 2 h of actinomycin
D treatment) compared to that in LS cells (3.8% remaining
under the same condition) (Fig. 4A), which was also consistent
with the results at the protein level (Fig. 1B). Using the same
conditions, we next analyzed NPM in chondrocytes. Since
NPM is known to be a shuttle protein moving between the
nucleus and cytoplasm, we were quite interested in the intra-
cellular distribution and the total content of NPM in these
FIG. 3. Specific binding of chicken NPM to the 3?-100/50 element
in the chicken ccn2 mRNA 3?-UTR. (A) Schematic representation of
the radiolabeled probes utilized in this study. The 3? half of the ccn2
mRNA, including the open reading frame (ORF) and the 3?-UTR, is
illustrated at the top. The sense-strand RNA fragments of the chicken
ccn2 mRNA 3?-UTR (3?-100/50 and 3?-50) were transcribed in vitro in
the presence of [?-32P]UTP. (B) REMSA of the RNA fragments of
ccn2 mRNA 3?-UTR. Two radiolabeled and folded RNA probes (3?-
100/50 and 3?-50) corresponding to the ones shown in panel A were
incubated with or without (?) 50 to 1,000 ng of recombinant chicken
NPM or BSA. After RNase digestion, the mixtures were analyzed by
electrophoresis through a 6% native polyacrylamide gel. (C) UV cross-
linking assay. The radiolabeled and folded RNA probes were incu-
bated with the protein and digested with RNase, as was performed in
REMSA. Then, the mixtures were irradiated by UV on ice and ana-
lyzed by SDS-PAGE using a 12.5% gel. The positions of molecular
mass standards (in kilodaltons) are shown at the left side of the panel.
The arrow (labeled NPM) indicates the position corresponding to the
molecular mass of the recombinant chicken NPM. (D) RNA immu-
noprecipitation analysis to confirm the specific binding of endogenous
NPM in the nucleus to ccn2 mRNA. Total CEF RNA was mixed with
CEF nuclear extract and was immunoprecipitated with the antibody
indicated, and the presence of ccn2 or actin (control) mRNA in the
immunocomplex was examined by RT-PCR after RNA extraction (Nu-
clear Extract). NPM, His, and Pre in the figure denote anti-chicken
NPM antibody, antipolyhistidine antibody, and a preimmune IgG,
respectively. Experiments were also repeated with recombinant NPM
only, instead of CEF nuclear extract, which detected the interaction of
NPM and ccn2 mRNA through the polyhistidine tag in the recombi-
nant NPM as well. The RT-PCR analysis of input RNA is shown in
lane Total. (?), negative-control antibody.
6138MUKUDAI ET AL.MOL. CELL. BIOL.
cells. Therefore, we carried out Western blotting to determine
the intracellular location of the trans factor (Fig. 4B). By an-
alyzing the proteins from an equal number of cells, total NPM
was found to be lower in US cells than in LS cells. Of more
interest, NPM was distributed dominantly in the cytoplasm in
LS cells, whereas most of the protein in the US cells had
accumulated in the nuclei, with quite low amounts in the cy-
toplasm. The results of Western blotting analysis for lamin B1
(a nuclear protein) and ?-tubulin (a cytoplasmic protein) con-
firmed the quality and quantity of each protein fraction (Fig.
4C). These findings indicate that cytoplasmic NPM can be an
RNA-binding destabilizer that regulates ccn2 expression dur-
ing chondrocyte differentiation.
In vitro reconstruction of the 5?-100/50-mediated posttran-
scriptional regulation by recombinant NPM. Specific binding
of NPM to 3?-100/50, the repressive cis element, and its neg-
ative correlation with ccn2 mRNA stability suggest that NPM
may regulate the level of ccn2 mRNA by acting as an RNA
destabilizer. In order to investigate this point, we established
an IVDA system. As shown in Fig. 5A, four chimeric fusion
probes [Luc(?), Luc-3?-Full, Luc-3?-100/50, and Luc-3?-50], in
which the full-length 3?-UTR of ccn2 mRNA and deletion
mutants of 3?-UTR of ccn2 mRNA were conjugated to the end
of the open reading frame of firefly luciferase, were prepared
to mimic RNA decay in vitro, according to previous studies
(43, 63). It has been widely known that regulated mRNA deg-
radation occurs in processing bodies (P bodies) in the cytosol
(51). The IVDA was carried out by using 10 ?g of the cytosolic
proteins of CEFs in the presence or absence of 0.5 ?g of
recombinant chicken NPM or 1 ?g of the eluate from the
chicken ccn2 mRNA affinity column (Fig. 5B). When incu-
bated with the cytosolic extract from CEFs alone, not only
Luc-3?-Full but also Luc-3?-100/50 underwent rapid degrada-
tion (t1/2? 0.5 h), whereas degradation of Luc(?) and Luc-
3?-50 was relatively slower (t1/2? 1 h) (Fig. 5B), suggesting that
this in vitro system could mimic the events that occurred in vivo
(Fig. 5A). Furthermore, in the presence of the recombinant
chicken NPM, the stability of Luc-3?-Full and Luc-3?-100/50
was drastically decreased (t1/2? 0.2 h), whereas that of Luc(?)
and Luc-3?-50 was only modestly decreased (t1/2? 0.7 h).
Thus, NPM was shown to be associated with both ccn2-specific
and nonspecific RNA degradation events in CEFs. Consistent
with these findings, results with the eluate from the RNA
affinity column, which contained endogenous NPM, were al-
most the same as those with recombinant chicken NPM.
Therefore, these data indicate that NPM is the trans-regulatory
factor of chicken ccn2 mRNA acting via RNA destabilization.
Destabilization effect of NPM on ccn2 mRNA is more spe-
cific and robust in chondrocytes. Next, IVDA was conducted
by using cytosolic extracts from both LS and US cells (Fig. 6)
as well as from CEFs. Each of the three radiolabeled probes
(shown in Fig. 5A) was incubated with 10 ?g of cytosolic
protein from LS or US cells. With LS proteins, the half-life of
Luc-3?-50 was as long as that of Luc(?) (t1/2? 0.7 h), and the
stability of Luc-3?-Full and Luc-3?-100/50 was decreased (t1/2?
0.25 h). Interestingly, the addition of NPM to Luc-3?-Full or
Luc-3?-100/50 in the LS cytosolic extract accelerated mRNA
decay far more drastically (Fig. 6) (t1/2? 0.1 h) than that in the
CEF extract (Fig. 5B), whereas the effect of NPM was minimal
on Luc(?) and Luc-3?-50 (t1/2? 0.52 h). These findings indi-
cate that NPM acts as a more specific and efficient regulator of
ccn2 mRNA degradation in the cytosolic extract of chondro-
cytes. Similar increased potential and functional specificity of
NPM were also observed in the case of the US cell cytosolic
extract. However, all of the RNA probes were more stable in
the presence of the US cell extract than in the presence of the
LS cell extract, and the half-lives of Luc(?) and Luc-3?-50 were
longer than those of Luc-3?-Full and Luc-3?-100/50 [t1/2? 1.6 h
for Luc(?), t1/2? 0.9 h for Luc-3?-Full, t1/2? 0.55 h for
Luc-3?-100/50, and t1/2? 1.4 h for Luc-3?-50]. The addition of
NPM to the US cell extract also resulted in drastically quick
degradation of Luc-3?-Full and Luc-3?-100/50 (t1/2? 0.1 h),
whereas the nonspecific effect on Luc(?) and Luc-3?-50 was
quite small (t1/2? 1 h for Luc(?) and t1/2? 0.8 h for Luc-3?-
50). These findings indicate that NPM obtains the ability to
exert highly specific action to regulate ccn2 mRNA stability in
chondrocytes, which is supported by the composition of the
cytosolic molecules that are specific to chondrocytes.
Modulation of the intracellular distribution of NPM by
growth factors. In addition to CCN2, several growth factors,
such as TGF-? (26, 49), BMPs (14, 18, 42, 78), and PDGF (27),
FIG. 4. Relationship between intracellular ccn2 mRNA stability
and subcellular distribution of NPM in chondrocytes. (A) Degradation
profiles of ccn2 mRNA in LS and US cells. RNA synthesis in LS or US
cells was arrested by actinomycin D, and a time-course study of the fate
of the remaining ccn2 mRNA was analyzed by Northern blotting anal-
ysis (top panels). The level of 28S rRNA remained unchanged through
the experimental time course (data not shown). The numbers (hours)
indicate the time periods after transcriptional arrest was initiated. The
signal intensities of the autoradiograms were quantitatively analyzed
and plotted compared to the values at time zero (bottom panel). The
broken line with open circles and solid line with closed circles denote
the data obtained with LS and US cells, respectively. (B) Nucleocyto-
plasmic distribution of NPM in LS and US cells. Proteins from the
nuclear extract (N) or cytoplasmic extract (C) from cells comprising
the same amount of DNA were subjected to 12.5% SDS-PAGE and
blotted onto a PVDF membrane. The blot was then incubated with
anti-NPM antibody and then with secondary antibodies for visualiza-
tion of signals, as described in Materials and Methods. (C) Successful
fractionation confirmed by Western blotting of each fraction (10 ?g
protein) with anti-lamin B1 (nuclear marker) or anti-?-tubulin (a cy-
toplasmic marker) antibody.
VOL. 28, 2008POSTTRANSCRIPTIONAL REGULATION OF ccn2 BY NPM 6139
have been reported to play important roles in growth and
differentiation of chondrocytes. Moreover, these growth fac-
tors not only stimulate the expression of ccn2 (46) but also
alter the stability of ccn2 mRNA in vivo (45). In fact, these
growth factors repressed the degradation of ccn2 mRNA in LS
cells, whereas conversely, they accelerated it in US cells.
Therefore, suspecting the involvement of NPM in the regula-
tion of ccn2 by such growth factors, we investigated npm gene
expression and the subcellular distribution of NPM protein in
LS and US cells stimulated by these growth factors. Northern
blotting (Fig. 7A) revealed that all of the growth factors en-
hanced the expression of npm mRNA in LS cells. However, the
level of NPM protein was found to be rather decreased by
growth factor treatment, as revealed by Western blotting anal-
ysis (Fig. 7B). These data suggest a possible regulation of NPM
production at a translation level. More importantly, Western
blotting also revealed that stimulation of LS cells by all of the
growth factors resulted in remarkable decreases in cytoplasmic
NPM (Fig. 7B). This effect was most prominent by stimulation
with TGF-? and BMP 2 and was modest with PDGF and
CCN2. To our surprise, the effect of the growth factors on the
US cells was the opposite. In US cells, NPM was found mostly
in the nuclei without stimulation. However, stimulation by any
of the growth factors resulted in drastic accumulation of NPM
in the cytoplasm but also in an increased amount of NPM in
the nuclei. The results of Western blotting analyses of marker
FIG. 5. Effect of NPM on the degradation of target RNA with or without a chicken ccn2 3?-UTR segment in a reconstituted system with CEF
cytosolic extract in vitro. (A) Schematic representation of the structures of the RNAs utilized in this study. The 3? half of the ccn2 mRNA including
the 3? half of the open reading frame (ORF) and the 3?-UTR are illustrated at the top, and the full-length and deletion mutant fusion mRNAs
are schematically displayed. The full-length ccn2 3?-UTR and two short segments of ccn2 3?-UTR were connected in the sense orientation to the
3? end of firefly luciferase genes (F. Luciferase) derived from pGL3L(?). These three fusion RNAs (Luc-2?-Full, Luc-3?-100/50, and Luc-3?-50)
and firefly luciferase-only RNA [Luc(?) as a negative control] were capped (hatched box) and polyadenylated (AAA) in the presence of
[?-32P]UTP by using a commercial kit (see Materials and Methods). The results of in vivo expression of the same mRNAs are also summarized.
Arrows pointing down and right indicate lower and equivalent gene expression levels relative to that of Luc(?) in chicken cells (44). (B) IVDA
of the fusion RNAs. The radiolabeled RNAs shown in panel A were incubated with cytoplasmic extracts (10 ?g) from CEFs in the absence (?)
or presence of recombinant chicken NPM (0.5 ?g) or eluate from the chicken ccn2 mRNA affinity column (1 ?g). After timed intervals, RNA was
purified and subjected to urea-denatured 6% PAGE. The graphs at the bottom of the panel show the relative remaining values and approximate
fitted degradation curves of the RNA standardized against each RNA at time zero. The values and curves for the negative control (closed ovals
and solid line), NPM (closed rectangles and dotted line), and eluate (closed triangles and broken line) are depicted. The values are shown as
percentages on semilogarithmic graphs (value at time zero ? 100). Statistically significant differences from the values for the control without NPM
or eluate are indicated by asterisks as follows:*, P ? 0.05;**, P ? 0.001.
6140 MUKUDAI ET AL.MOL. CELL. BIOL.
proteins confirmed the quality and quantity of each protein
fraction. The different redistribution patterns of NPM that
resulted from stimulation by the growth factors are consistent
with the differentiation-dependent effects of these growth fac-
tors on the resultant ccn2 gene expression between LS and US
cells (45), suggesting the role of NPM as an RNA destabilizer
acting in the cytoplasm in the ccn2 regulation by the growth
Reproduction of the effect of growth factors on ccn2 mRNA
stability in IVDA, which was saturable by excess NPM. As
stated above (see “Modulation of the intracellular distribution
of NPM by growth factors”), certain factors had the opposite
effect on the stabilization of ccn2 mRNA between LS and US
cells. In the present study, IVDA (Fig. 8), in which Luc-3?-
100/50 was incubated with cytoplasmic proteins, yielded the
same results, which again indicates the reliability of IVDA as
an in vitro assay system. In LS cells, the RNA was more stable
in the presence of cytosolic proteins from cells stimulated with
TGF-? (t1/2? 1.7 h), BMP 2 (t1/2? 1.2 h), PDGF (t1/2? 0.6 h),
or CCN2 (t1/2? 0.9 h), than in the presence of control cell
extract (t1/2? 0.25 h). However, of importance, the RNA
stabilization effect of the growth factors was totally abolished
by the addition of 0.1 ?g NPM. Indeed, in the presence of
excess NPM, ccn2 mRNA decayed in the growth factor-treated
cell lysates as rapidly as in the control cell lysate. On the other
hand, in US cells, stimulation by the growth factors rather
decreased the RNA stability (t1/2? 0.6 h for the control, t1/2?
0.15 h for TGF-?, t1/2? 0.35 h for BMB 2, t1/2? 0.15 h for
FIG. 6. Robust effect of NPM on selective degradation of target RNA in a reconstituted system with chondrocyte cytosolic extract in vitro. The
radiolabeled RNAs shown in Fig. 5A were incubated with cytoplasmic extracts (10 ?g) from LS or US cells in the absence (?) or presence of
recombinant chicken NPM (0.5 ?g). After timed intervals, the RNA was purified and subjected to urea-denaturing 6% PAGE. In the graphs at
the bottom of the panel, the relative remaining amounts and fitted degradation curves of RNA standardized against each RNA at time zero
(negative control [closed ovals and solid line] and NPM [closed rectangles and dotted line]) are shown on a semilogarithmic graph (value at time
zero ? 100). Statistically significant differences between the two groups are indicated by asterisks as follows:*, P ? 0.05;**, P ? 0.001. A clear-cut
and selective effect of NPM on the degradation of the 3?-100/50-containing RNA was observed.
VOL. 28, 2008 POSTTRANSCRIPTIONAL REGULATION OF ccn2 BY NPM 6141
PDGF, and t1/2? 0.2 h for CCN2). More importantly, the
addition of NPM resulted in rapid and comparable degrada-
tion of the RNA in all cell extracts. These results further
confirm that NPM plays an important role in the regulated
degradation of ccn2 mRNA and that NPM is a ccn2-specific
RNA-destabilizing molecule, and this effect can be precisely
controlled by its subcellular redistribution during chondrocytic
Effect of npm silencing on ccn2 gene expression by siRNAs in
vivo. Finally, in order to confirm the requirement of NPM in
the regulation of ccn2 in living cells, we employed an RNA
interference-mediated knockdown strategy with CEFs. Two
independent siRNAs were synthesized. One siRNA duplex,
designated si546, was predicted to exert a maximal knockdown
effect against the npm mRNA, whereas the other one, si207,
was predicted to be less effective in silico. Consistent with this
prediction, transfection of si546 into CEFs showed striking
gene silencing effect on npm, while a modest repressive effect
was observed with si207 (Fig. 9). The expression level of gapdh
was not significantly altered by the siRNAs (Fig. 10), ruling out
the possibility of off-target effects, and thus, it was utilized as
an internal control to compute the relative gene expression
levels. Also, the resultant NPM protein levels produced by the
transfectants corresponded well to the mRNA levels observed
above (Fig. 9B). Next, under the same conditions, the steady-
state ccn2 mRNA level was evaluated. Exactly as expected,
strong or modest silencing of npm resulted in a remarkable or
subtle increase in the steady-state ccn2 mRNA level. These
results clearly indicate that NPM is acting as a specific negative
regulator of ccn2 gene expression in vivo, confirming the utility
and reliability of our in vitro system.
Increased production of CCN2 by npm silencing and the
mechanism of action. Subsequently, the effects of npm silenc-
ing on CCN2 production were analyzed by Western blotting
analysis in order to estimate the biological significance of the
upregulated ccn2 expression. As clearly seen in Fig. 10, CCN2
production levels showed changes comparable to those in
mRNA levels (Fig. 9), indicating that NPM acts as a regulator
of CCN2 production. Then, to further examine whether NPM
regulated ccn2 expression by altering the stability of ccn2
mRNA in vivo, which was clearly indicated by the in vitro data,
mRNA degradation analysis was carried out in vivo. After the
pretreatment by the siRNAs for 24 h, nascent mRNA synthesis
was arrested by actinomycin D, and the fate of remaining
mRNAs was chased following a time course. As a result, deg-
radation of ccn2 mRNA was found to be remarkably slower in
si546-treated cells than in the cells with control siRNA. Addi-
tionally, the degradation profile of another mRNA, gapdh, was
not altered by si546. These data represent two critical aspects
concerning target specificity; one is the specificity of the
siRNAs to npm mRNA, and the other is the specificity of NPM to
ccn2 mRNA. Collectively, in vivo knockdown of npm resulted
in a prolonged half-life of ccn2 mRNA, whereas in vitro addi-
tion of NPM caused accelerated degradation of ccn2 mRNA.
Thus, the consistency of in vivo and in vitro data strongly
indicate that our IVDA system indeed reflects the biological
reality taking place in living cells.
Our previous study (44) revealed that the 3?-UTR of chicken
ccn2 mRNA contained a repressive cis element of gene expres-
FIG. 7. Effects of growth factors on the expression and subcellular distribution of NPM in LS and US chondrocytes. (A) Northern blot analysis
of LS and US cells stimulated with growth factors. LS and US cells were stimulated with TGF-? (10 ng/ml), BMP 2 (200 ng/ml), PDGF (10 ng/ml),
or CCN2 (30 ng/ml) for 24 h in the absence of FBS or not stimulated (?) as a negative control, and total RNA was then extracted, denatured by
glyoxysal, separated on 1% agarose gel, and blotted onto a nylon membrane. Prior to hybridization with the npm probe, the membrane was stained
by methylene blue (28S) in order to show the same amount of total RNA. The signals were quantified, standardized against the level of 28S RNA,
and presented as relative values versus control (1.00) at the bottom of the panel. (B) Subcellular fractionation and Western blot analysis. LS and
US cells were stimulated with growth factors or not stimulated as described above for panel A, and 10-?g amounts of proteins from the nuclear
extract (N) or cytoplasmic extract (C) were subjected to 12.5% SDS-PAGE and blotted onto a PVDF membrane. The blot was incubated with
anti-NPM, anti-lamin B1 (nuclear control), or anti-?-tubulin (cytosolic control) antibody. The NPM signals from the nuclei (N) and cytosol
(C) were standardized against the signals of lamin B1 (NPM/L) and ?-tubulin (NPM/T), respectively. Normalized signals relative to the control
(1.00) are displayed.
6142 MUKUDAI ET AL.MOL. CELL. BIOL.
sion, as does that of mammalian species (30–32, 34–36). Fur-
thermore, our recent study (45) indicated that the stability of
chicken ccn2 mRNA is regulated in a differentiation stage-
dependent manner in chondrocytes and that the stability of
chicken ccn2 mRNA negatively correlates with the interaction
between a putative 40-kDa trans factor and the repressive cis
element of the 3?-UTR of ccn2 mRNA. Following these stud-
ies, we sought to identify the putative 40-kDa trans-factor by
employing an RNA affinity chromatography technique with a
3?-100/50 fragment of ccn2 mRNA 3?-UTR. The results of a
UV cross-linking assay of the 3?-100/50 fragment with the
purified proteins (Fig. 2A) showed a few bands representing
their specific binding. Also, upon Northwestern blotting (Fig.
2B), two specific bands were observed. One was approximately
40 kDa in molecular mass, and the other was approximately 90
kDa. On the basis of our previous study (45), we chose and
further investigated the 40-kDa protein in the present study.
However, the 90-kDa protein may not be disregarded, since it
also bound to the probe rather more strongly than the 40-kDa
protein did (Fig. 2B). Therefore, in the future, we will inves-
tigate the properties of this protein as well.
The protein was processed by an endopeptidase, and puri-
fied fragments were subjected to Edman analysis. The five
internal peptide sequences obtained (Fig. 2C) indicated that
NPM (40, 41, 50) was the 40-kDa binding protein for the
3?-100/50 fragment. NPM has been given other names, such as
FIG. 8. Differentiation-dependent regulation of the stability of the target RNA containing 3?-100/50 by growth factors via NPM in vitro. LS and
US cells were stimulated with TGF-? (10 ng/ml), BMP 2 (200 ng/ml), PDGF (10 ng/ml), or CCN2 (30 ng/ml) for 24 h in the absence of FBS or
left alone (?) as a negative control, and the cytoplasmic extract was obtained. Radiolabeled RNA (Luc-3?-100/50) was incubated with the
cytoplasmic extracts (10 ?g) in the absence (?) or presence of recombinant chicken NPM (0.1 ?g). After timed intervals, the RNA was purified
and subjected to urea-denaturing 6% PAGE. In the graphs at the bottom of the panel, the relative remaining amounts and fitted degradation
curves of RNA standardized against each RNA at time zero (negative control [closed ovals and solid line] and NPM [closed rectangles and dotted
line]) are shown on semilogarithmic graphs (value at time zero ? 100). Note the opposite effects of growth factors on the reporter RNA stability
between the two, both of which were abolished when excess NPM was added. Statistically significant differences in the absence of NPM between
the values of growth factor-treated groups and the control group are indicated by asterisks as follows:*, P ? 0.05.
VOL. 28, 2008 POSTTRANSCRIPTIONAL REGULATION OF ccn2 BY NPM6143
B23 (7, 8), NO38 (60, 61), and numatrin (16). NPM was first
found to be located mainly in a granular component of nuclei
(56, 74) and to play a role in the assembly of preribosomal
particles (60). Later, it was shown to shuttle between the nu-
cleus and cytoplasm (3). Furthermore, a number of recent
reports revealed that NPM interacts with proteins, such as the
transcriptional factor YY1 (24), Rev protein of human immu-
nodeficiency virus type 1 (15), and tumor suppressor p53 (10),
and with single- or double-stranded DNA (9, 10, 76, 77) and
RNA (3, 70, 73). Since these findings suggested multifunc-
tional roles of NPM as a nucleocytoplasmic shuttle protein, we
investigated whether or not NPM is a trans factor critical for
the posttranscriptional regulation of ccn2 mRNA.
First, in order to confirm the binding of NPM to the 3?-UTR
of ccn2 mRNA, we prepared two radiolabeled probes, 3?-
100/50 and 3?-50 (Fig. 3A). The former probe was the target
RNA segment, whereas the latter was a negative control. In-
deed, REMSA and the UV cross-linking assay (Fig. 3B and C)
indicated the specific binding of recombinant chicken NPM to
3?-100/50. Moreover, this specific interaction of NPM and the
3?-100/50 probe was further confirmed by RNA immunopre-
cipitation analysis. With this protein, we could eventually re-
constitute the 3?-100/50-mediated regulation of ccn2 expres-
FIG. 9. RNA interference-mediated knockdown of npm and its
effect on ccn2 gene expression. (A) Steady-state mRNA levels of npm
in CEFs at 48 h after the transfection of siRNAs as evaluated by
real-time RT-PCR analysis. Two different siRNAs targeted to npm
were synthesized and applied. The gapdh mRNA level, which was not
significantly altered by those siRNAs (data not shown), was used as a
standard. The mRNA levels are represented as relative values against
that obtained with a control RNA duplex. (B) Western blotting anal-
ysis of NPM in CEFs transfected with the corresponding siRNAs
under the same conditions as those shown in panel A. The same
amount of total proteins (10 ?g) was loaded for each sample. (C) Ef-
fects of npm knockdown by the siRNAs on the steady-state mRNA
levels of ccn2 in CEFs. The ccn2 mRNA levels were standardized
against gapdh mRNA levels and are shown as relative values against
those with a control RNA duplex.
FIG. 10. Outcome and mechanism of enhanced ccn2 gene expres-
sion by npm gene silencing. (A) Cellular CCN2 levels in CEFs as
evaluated by Western blotting after the introduction of the siRNAs
targeting npm. The same set of siRNAs was employed as in Fig. 9. The
same amount of total proteins (10 ?g) was loaded for each sample.
(B) No significant effect of npm gene silencing on the degradation of
gapdh mRNA in vivo. Nascent mRNA synthesis was arrested by acti-
nomycin D treatment, and the fate of the mRNA in CEFs with a
control RNA duplex (solid line) or si546 (dotted line) was pursued by
real-time RT-PCR analysis. Relative values were computed against
those at time zero. (C) Effect of npm gene silencing on the degradation
of ccn2 mRNA in CEFs transfected with a control RNA duplex (solid
line) or si546 (dotted line). Standardized values of ccn2 mRNA levels
(ccn2/gapdh) were further normalized against those at time zero and
6144 MUKUDAI ET AL.MOL. CELL. BIOL.
sion in vitro, which was actually observed during chondrocyte
differentiation in vivo.
Prior to establishing an IVDA system, we comparatively
evaluated the gene expression and subcellular distribution of
NPM in LS and US cells. Northern blotting analysis showed
comparable ccn2 gene expression levels in LS and US cells
(data not shown). However, interestingly, in US cells, most of
the NPM protein had accumulated in the nuclei, and its dis-
tribution in the cytoplasm was very low (Fig. 4B). Considering
the increased ccn2 mRNA stability in US cells (Fig. 4A), NPM
was thus suspected to be the critical determinant to accelerate
the selective degradation of ccn2 mRNA in the cytoplasm.
Involvement of the shuttling property of NPM in ccn2 regula-
tion was also indicated herein.
For IVDA, four chimeric reporter RNAs were prepared,
and their stability was assessed in cytosolic extract from CEFs.
Unexpectedly, the stabilities of all of the reporter RNAs were
significantly decreased by the addition of NPM to the CEF
extract. Several earlier studies (21, 22, 59) showed that NPM
possesses an intrinsic RNase activity that preferentially cleaves
single-stranded poly(A), poly(U), and poly(C). Hence, the de-
creased RNA stability in the presence of NPM might involve
the effect of the nonspecific RNase activity of NPM in part.
However, the degree of decreased stability caused by NPM was
greater for the mRNAs with the binding target (Luc?-3?-Full
and Luc-3?-100/50) than for those of the controls [Luc(?) and
Luc-3?-50]. These results indicate the sequence-specific en-
hancing effect of NPM on RNA degradation. Therefore,
together with the results of the RNA-binding analysis, we
showed that NPM is an mRNA destabilizer that binds to the
repressive cis element of 3?-UTR of ccn2 mRNA and accel-
erates the degradation with significant specificity in CEF
extracts. Furthermore, this NPM function as a negative
posttranscriptional regulator of ccn2 expression indicated in
vitro was clearly confirmed in vivo through an siRNA-me-
diated gene silencing approach.
As stated above, the effect of NPM on mRNA degradation
did not appear to be highly specific in CEF extracts. Neverthe-
less, subsequent analysis with chondrocyte cytosolic extracts
revealed a quite interesting aspect of NPM function. Surpris-
ingly, IVDA with cytosolic proteins from LS and US cells (Fig.
6) showed much stronger and more specific effects of NPM on
the stability of ccn2 mRNA than the effects obtained with
cytosolic proteins from CEFs. The molecular background of
this cell-type-dependent enhancement of functional specificity
is unknown. However, since CCN2 plays a critical role in both
the growth and differentiation of chondrocytes, precise regu-
lation of ccn2 mRNA would be especially required in this
particular type of cells. Therefore, the enhanced regulatory
potential of NPM in LS and US cells suggests that the NPM-
mediated posttranscriptional regulation is one of the precise
control systems that have been developed specifically in chon-
TGF-?, BMP 2, PDGF, and CCN2 are known to promote
the differentiation of growing cartilage cells. Therefore, upon
stimulation, these factors increase ccn2 expression in LS cells,
whereas they rather decrease it in US cells by driving the cells
to terminal hypertrophy (45). It is of interest that although
stimulation with these factors hardly changed the expression
level of the npm gene in LS and US cells (Fig. 7A), the cellular
distribution of NPM protein was drastically changed depend-
ing on the differentiation stage of the chondrocytes (Fig. 7B).
In LS cells, incubation with the growth factors resulted in the
accumulation of NPM in the nuclei. In contrast, in growth
factor-treated US cells, NPM was observed in the cytosolic
fraction, in which it was hardly observed without stimulation;
thus, opposite results were obtained with LS and US cells. It is
noteworthy that the results of IVDA (Fig. 8) indicated that the
stimulation by growth factors stabilized the target RNA in LS
cell extract, whereas it rather destabilized the target in US cell
extracts, which is in agreement with our previous study (45).
Here again, the higher the NPM content in the cytosol, the
more fragile was the ccn2 mRNA. Total ablation of such an
effect of these growth factors by excess NPM in vitro indicates
that NPM acts as a major regulator of ccn2 mRNA stability
upon growth factor stimulation. These results further indicate
that the stability of ccn2 mRNA in chondrocytes was critically
controlled by the intracellular distribution of shuttling NPM.
In a number of previous studies, important physiological or
pathological roles of CCN2 were reported, such as roles in cell
growth and differentiation in development (46, 47, 71), angio-
genesis (2, 64, 68), and wound healing (23). In addition, CCN2
is known to play a critical role in chondrocytes during endo-
chondral ossification (46–48, 65, 69), with its gene expression
being precisely controlled during differentiation. Indeed, ex-
pression of the ccn2 gene is controlled not only at the tran-
scriptional level (11–13, 19) but also by posttranscriptional
events (30–32, 34–36, 44, 45). In the present study, we first
identified the posttranscriptional regulatory protein as NPM
binding to the repressive cis element to enhance RNA degra-
dation with pronounced efficacy in chondrocytes. Moreover,
we demonstrated that the intracellular distribution of NPM
was a critical parameter of ccn2 regulation during the differ-
entiation of chondrocytes. To our knowledge, no study con-
cerning the role of NPM in skeletal development has previ-
ously appeared in the literature; thus, our present findings
represent the first report of a novel NPM-mediated gene reg-
ulation system critical for proper chondrogenesis and endo-
chondral ossification. Generally, mRNAs are regulated post-
transcriptionally by multiple complexes composed of an RNA
cis element and trans-factor protein(s). Hence, our next goal is
to uncover the precise regulatory mechanism of degradation of
ccn2 mRNA by the interaction among 3?-UTR, NPM, and any
other factor(s) that needs to be identified. Further investiga-
tion is now ongoing.
This work was supported by the programs Grants-in-Aid for Scien-
tific Research (grant S to M.T.) and (grant C to S. Kubota) and
Grants-in-Aid for Exploratory Research (to M.T.) of the Ministry of
Education, Culture, Sports, Science, and Technology of Japan, and by
a grant from the Foundation of Sanyo Broadcasting (to S. Kubota).
We thank Takako Hattori, Takashi Nishida, Masanao Minato,
Tsuyoshi Yanagita, Kazumi Kawata, Chisa Kuroda, Kyouji Nakao, and
Toshihiro Ogawara for helpful suggestions; Kazumi Ohyama for tech-
nical assistance; and Yuki Nonami for secretarial assistance.
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