In addi-tion, prior to the discovery of the human ortholog, murine CCN4⁄ WISP1 was identified as a novel gene with tumor-suppressor properties and that was initially Keywords cartilage; C
Trang 1secreted protein 1 mRNA splicing variants in chondrocytes Takeshi Yanagita1,2, Satoshi Kubota1, Harumi Kawaki1, Kazumi Kawata1, Seiji Kondo1,
Teruko Takano-Yamamoto3, Shinji Tanaka4and Masaharu Takigawa1
1 Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
2 Department of Orthodontics and Dentofacial Orthopedics, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
3 Division of Orthodontics and Dentofacial Orthopedics, Tohoku University Graduate School of Dentistry, Miyagi, Japan
4 Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, Japan
The Wnt1-induced secreted proteins (WISPs) were
identified together in a human study reported in 1998
and then later classified as members of the CCN
family, which also includes cysteine-rich 61 (Cyr61⁄
CCN1), connective tissue growth factor (CTGF⁄
CCN2) and nephroblastoma overexpressed (Nov⁄ CCN3) proteins as its classical members [1–5] In addi-tion, prior to the discovery of the human ortholog, murine CCN4⁄ WISP1 was identified as a novel gene with tumor-suppressor properties and that was initially
Keywords
cartilage; CCN family; chondrocyte
differentiation; splicing variant; Wnt-induced
secreted protein 1 (WISP1)
Correspondence
M Takigawa, Department of Biochemistry
and Molecular Dentistry, Okayama
University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences,
2-5-1 Shikata-cho, Okayama 700-8525,
Japan
Fax: +81 86 235 6649
Tel: +81 86 235 6645
E-mail: takigawa@md.okayama-u.ac.jp
Database
The nucleotide sequence data described
have been submitted to the GenBank
data-base under the accession numbers EF025921,
EF025922 and EF025923
(Received 20 November 2006, revised 15
January 2007, accepted 19 January 2007)
doi:10.1111/j.1742-4658.2007.05709.x
CCN4⁄ Wnt-induced secreted protein 1 (WISP1) is one of the CCN (CTGF⁄ Cyr61 ⁄ Nov) family proteins CCN members have typical structures composed of four conserved cysteine-rich modules and their variants lack-ing certain modules, generated by alternative spliclack-ing or gene mutations, have been described in various pathological conditions Several previous reports described a CCN4⁄ WISP1 variant (WISP1v) lacking the second module in a few malignancies, but no information concerning the produc-tion of WISP1 variants in normal tissue is currently available The expres-sion of CCN4⁄ WISP1 mRNA and its variants were analyzed in a human chondrosarcoma-derived chondrocytic cell line, HCS-2⁄ 8, and primary rab-bit growth cartilage (RGC) chondrocytes First, we found WISP1v and a novel variant of WISP1 (WISP1vx) to be expressed in HCS-2⁄ 8, as well as full-length WISP1 mRNA This new variant was lacking the coding regions for the second and third modules and a small part of the first module To monitor the expression of CCN4⁄ WISP1 mRNA along chondrocyte differ-entiation, RGC cells were cultured and sampled until they were mineral-ized As a result, we identified a WISP1v ortholog in normal RGC cells Interestingly, the WISP1v mRNA level increased dramatically along with terminal differentiation Furthermore, overexpression of WISP1v provoked expression of an alkaline phosphatase gene that is a marker of terminal dif-ferentiation in HCS-2⁄ 8 cells These findings indicate that WISP1v thus plays a critical role in chondrocyte differentiation toward endochondral ossification, whereas HCS-2⁄ 8-specific WISP1vx may be associated with the transformed phenotypes of chondrosarcomas
Abbreviations
ALP, alkaline phosphatase; CT, C-terminal cysteine knot; HUVEC, human umbilical vein endothelial cell; IGFBP, insulin-like growth factor binding protein-like; RGC, rabbit growth cartilage; TSP1, thrombospondin type 1; VWC, von Willebrand factor type C; WISP, Wnt-induced secreted protein.
Trang 2described as ‘Expression in low-metastatic cells type 1
(Elm1)’ gene [6]
Currently, all six members tend to be referred to as
CCN1–6, based on the unified nomenclature [1,7]
Members have been described as being involved in a
variety of cell biological processes, such as apoptosis,
mitosis, cell adhesion, the production of extracellular
matrix and angiogenesis [2,8–12] It should be noted
that most have unfavorable roles in human fibrotic
dis-orders and several types of malignancy [13] As seen in
the other CCN family members, CCN4⁄ WISP1
posses-ses a typical structure composed of four conserved
cys-teine-rich modular domains encoded by separate exons
[14], which share sequence homologies with the
insu-lin-like growth factor binding protein (IGFBP: first),
von Willebrand factor type C repeat (VWC: second),
thrombospondin type 1 repeat (TSP1: third), and the
C-terminal cysteine knot (CT: fourth), respectively
The multimodular architecture of WISP1, along with
the production of its truncated isoforms in tumors,
rai-ses interesting questions regarding the participation of
each individual module in the various biological
func-tions of this factor [15]
The involvement of CCN4⁄ WISP1 in malignant
neo-plasms has been relatively well investigated [16] In
ear-lier studies, full-length WISP1 was also shown to restrain
tumor growth and metastasis, as represented by its initial
name, Elm1 [6] Recently, a novel variant of WISP1
(WISP1v), which lack the VWC module, was reported in
a few in human gastrointestinal cancer tissues, thus
sug-gesting the involvement of the variant in malignant phe-notypes of certain cancers [17–20] However, because of its initial discovery in mouse melanoma, no investigation was performed to clarify the distribution of WISP1 and its physiological roles in normal tissues
In this study, we analyzed the expression of full-length WISP1 mRNA and its splicing variants in nor-mal and nor-malignant-transformed chondrocytes For the first time, we describe the expression of WISP1v in nor-mal growth plate chondrocytes, which was regulated along with the terminal differentiation of those cells Furthermore, we also identified a novel WISP1 mRNA variant in malignant-transformed human chondrocytes The roles of these variants in chondrocyte biology have been analyzed and are now discussed
Results
CCN4⁄ WISP1 mRNA splicing variants in human chondrocytic HCS-2/8 cells
To characterize mRNAs from ccn4⁄ wisp1, RT-PCR was performed using total RNAs from HCS-2⁄ 8, human umbilical vein endothelial cells (HUVEC), HeLa, MDA231, SaOS2 and HEK293 cells with
speci-fic primers that recognize the IGFBP (sense) and CT (antisense) coding exons These primers were designed
to amplify the splicing variant lacking the VWC mod-ule area (WISP1v), as well as full-length WISP1 (Fig 1A) As a result, no ccn4⁄ wisp1 transcripts were
Fig 1 Analysis of CCN4 ⁄ WISP1 mRNA in several cell lines as evaluated by RT-PCR (A) The sense and antisense primers were designed to recognize the sequences in IGFBP module-encoding exons and CT module-encoding exons, respectively The length of the PCR products from the full-length CCN4 ⁄ WISP1 amplified by these primers was expected to be 606 bp (B) The results of the RT-PCR analysis CCN4 ⁄ WISP1 and WISP1v-cloned vectors were also amplified as controls According to the DNA size markers analyzed together, the size of the PCR product I was 600 bp, whereas those of II and III were 400 and 200 bp, respectively Experiments were repeated at least three times and representative results are shown Equal sample load was confirmed by the RT-PCR analysis of gapdh mRNA.
Trang 3detected with kidney-derived HEK293, HeLa cervical
cancer and MDA231 breast cancer cells Vascular
endothelial cells (HUVEC) and A371 melanoma cells
expressed only full-length mRNA (Fig 1B), whereas
osteoblastic SaOS2 expressed both CCN4⁄ WISP1 and
WISP1v Interestingly, we obtained three distinct
amplicons from the chondrocytic HCS-2⁄ 8 mRNA
Subsequent nucleotide sequence analysis revealed that
the longest and intermediate bands were full-length
CCN4⁄ WISP1 and WISP1v, respectively However,
the shortest band was a novel variant of WISP1
(WISP1vx) not previously described (Fig 2A) In
addi-tion, the presence of WISP1v in human osteogenic cell
lines was also revealed for the first time
Structure and predicted translation product of the novel CCN4/WISP1 mRNA splicing variant, WISP1vx
According to sequence analysis data, this novel vari-ant, WISP1vx, lacks VWC and TSP module-coding ex-ons and part of the IGFBP module-coding exon Because the alternative splicing site in IGFBP is located slightly upstream of the authentic site, the IG-FBP-coding exon in WISP1vx was 23 bp shorter than the full-length exon However, owing to the frame-shift, the IGFBP⁄ CT fusion coding frame is not trans-lated properly after the alternative splice site (Fig 2A) The deduced protein from WISP1vx is a single IGFBP module, in which eight C-terminal authentic amino acid residues are removed, and an extra 14 residues are added in their place
Importantly, the nucleotide sequences of exon– intron boundaries of WISP1vx strictly conserve the general rule of mRNA splicing sites in higher eukaryo-tes (Fig 2B) These findings indicate that WISP1vx is not an artifact of PCR amplification, but a splicing variant uniquely present in HCS-2⁄ 8 chondrocytic cells
Detection of CCN4⁄ WISP1 proteins in HCS-2/8 cells
In order to examine whether CCN4⁄ WISP1 and WISP1v mRNAs yielded corresponding proteins in HCS-2⁄ 8 cells, we analyzed the HCS-2 ⁄ 8 cell lysate using western blotting with an anti-(CCN4⁄ WISP1) serum targeting the CT module First, mammalian expression vectors for FLAG epitope-tagged CCN4⁄ WISP1, WISP1v and WISP1vx proteins were con-structed, and the proteins were overexpressed by DNA transfection of these expression vectors (Fig 3A) into HCS-2⁄ 8 cells The overexpressed CCN4⁄ WISP1, WISP1v and WISP1vx served as pos-itive controls for western blotting Because the FLAG epitope itself is a small octapeptide, the exogenesis CCN4⁄ WISP1 and its variants are indistinguishable from endogenous ones on SDS⁄ PAGE As shown in Fig 3B, analysis of transfectants with the anti-(CCN4⁄ WISP1) clearly showed strong enhancement
of the CCN4⁄ WISP1 and WISP1v signals that were also present in HCS-2⁄ 8 cells without the overexpres-sion As expected, no WISP1vx signal was detected, because it lacked the CT module in which the epitope
of H-55 antibody was located In contrast, when we used anti-FLAG IgG, we detected CCN4⁄ WISP1, WISP1v and WISP1vx only in overexpressed cells at the expected electromobility This indicates that
A
B
Fig 2 Exon–intron boundaries and the putative translation products
of the novel variant (WISP1vx) (A) Nucleotide sequences of the
amplicons I, II and III from HCS-2 ⁄ 8 cDNA were analyzed and the
structures of the corresponding mRNAs and deduced translational
products are illustrated Owing to the alternative splicing, a
frame-shift mutation was introduced, thus resulting in a premature
termin-ation of the transltermin-ation (III), as detailed in Fig 2B (B) Genomic,
mRNA and the deduced amino acid sequences are displayed at
upper, middle and lower, respectively Alternative and authentic
spli-cing sites are indicated with an emphasis of consensus sequences.
Owing to the frame-shift mutation, a stop codon appears at the
fifteenth codon downstream of the alternatively splice site SD and
SA denote splicing donors and acceptors, respectively.
Trang 4CCN4⁄ WISP1 and WISP1v proteins were actually
translated from CCN4⁄ WISP1 and WISP1v mRNAs
in HCS-2⁄ 8 cells
A rabbit ortholog of the reported
CCN4/WISP1-splicing variant (WISP1v) that is associated with
human malignancies
HCS-2⁄ 8 was derived from a human chondrosarcoma
and it is one of the cell lines known to retain the
characters of chondrocytes, as represented by the
expression of type II and type X collagen genes To
assess whether WISP1v and WISP1vx are associated
with malignant phenotypes, or with normal
chondro-cytic phenotypes, we investigated whether CCN4⁄
WISP1 mRNA and its variants are also expressed in
normal primary chondrocytes Rabbit growth
carti-lage (RGC) cells were isolated, and chondrocytic
differentiation was induced in vitro Thereafter,
RT-PCR was performed to analyze the ccn4⁄ wisp1
mRNA expression pattern in RGC cells Interestingly,
WISP1v, as well full-length CCN4⁄ WISP1 mRNA,
was distinctly detected in normal RGC, whereas
WISP1vx was not As summarized in Fig 4,
nucleo-tide sequence analysis indicated that the splicing sites
used to generate rabbit WISP1v are at the same
posi-tion as those observed in the human gene, indicating
that it is exactly the same ortholog as that of human
WISP1v
Regulated expression of WISP1v along with the chondrocytic differentiation of RGC cells
In order to assess whether WISP1v is associated with the development of growth cartilage, primary growth cartilage cells collected from rabbits were cultured until they became confluent Afterwards, a long-term culture for the induction of terminal differentiation was car-ried out for over one month Fluctuation in type II collagen as a marker gene of chondrocyte ation showed that RGC cells were properly differenti-ated in vitro (Fig 5C) Alizarin red staining of RGC cells revealed increased mineralization by the cells from
2 weeks after reaching confluency (Fig 5A) Interest-ingly, the expression level of WISP1v increased gradu-ally along with the mineralization of chondrocytes (Fig 5D,E), thus suggesting that WISP1v plays a spe-cific role therein In contrast, full-length CCN4⁄ WISP1 was observed to be expressed almost constantly throughout all stages of chondrocyte differentiation (Fig 5D), thus implying a more basic biological role for full-length CCN4⁄ WISP1 mRNA in growth plate chondrocytes
Distribution of CCN4/WISP1 proteins in growth cartilage in vivo
In general, the distribution of a protein in a certain tis-sue is closely associated with its function in vivo
Fig 3 Production of the WISP1v proteins in HCS-2 ⁄ 8 cells and their overexpression (A) Molecular constructs for the overexpression of WISP1, WISP1v and WISP1vx A mammalian expression vector, pFLAG-CMV was employed as the parental vector The resultant constructs are illustrated These plasmids were designed to express WISP1, WISP1v and WISP1vx with the FLAG epitopes at the N-termini (B) West-ern blotting analysis probed by anti-(human CCN4 ⁄ WISP1) serum (left), or anti-FLAG serum (right) with the lysate of HCS-2 ⁄ 8 cells, together with those overexpressing WISP1, WISP1v, WISP1vx Apparent molecular masses of specific signals in SDS ⁄ PAGE are shown Positions of molecular mass markers are shown in the middle Specific signals are marked by arrows Experiments were repeated twice, yielding com-parable results.
Trang 5Fig 4 The expression of WISP1v orthologue in normal RGC cells and its structure (A) The detection of WISP1v as well as CCN4 in RGC cells Total RNA from RGC cells (right) that had been cultured 3 weeks at confluence was analyzed The expected sizes of the amplicons were 550 bp from rabbit CCN4⁄ WISP1 and 289 bp from rabbit WISP1v, respectively, which were consistent with the electromobility seen here (left) A representative result of four independent series of experiments with similar results is displayed (B) Detailed structure of the exon–intron boundary of rabbit WISP1v in comparison with human WISPv.
Fig 5 Regulated expression of WISP1v along with chondrocytic differentiation RGC cells were differentiated in vitro, and the expression of
a marker gene and WISP1v was analyzed along with the time course (A) Alizarin red staining of RGC cells representing mineral deposition (B) Agarose gel electrophoresis analysis confirming the quality of the total RNA at each stage (C) Expression of the type II collagen gene as evaluated by quantitative real-time PCR (D) RT-PCR analysis with the primers recognizing WISP1v (E) A quantitative real-time PCR analysis performed with rabbit-specific WISP1v primers The sampling time points are represented as the numbers of weeks after the cell reached confluence Representative results of four independent series of experiments are displayed with error bars (SD of real-time PCR evaluation).
Trang 6Therefore following mRNA analysis, we examined the
distribution of CCN4⁄ WISP1 and WISP1v proteins in
developing mouse growth cartilage by
immunohisto-chemistry (Fig 6) Because no anti-(CCN4⁄ WISP1)
serum was able to detect the loss of VWC, which is
the structural determinant of WISP1v that
distingui-shes it from full-length WISP1 mRNA, we used an
antibody that recognized a common module between
the full-length CCN4⁄ WISP1 mRNA and WISP1v
proteins As a result, positive signals representing
full-length CCN4⁄ WISP1 mRNA and WISP1v were
observed, and were particularly strong in the hyper-trophic zone of the developing tibial sections of the mouse embryo Although these signals also include those from the full-length CCN4⁄ WISP1 mRNA, these
in vivo findings are consistent with the total outcome
of the constitutive expression of the full-length and the differentiation stage-dependent expression of WISP1v
in vitro
Effect of overexpression of CCN4/WISP1 and its variants on the phenotypes of human
chondrocytic HCS-2⁄ 8 cells Finally, the biological function of WISP1v and WISP1vx was evaluated using overexpression experi-ments FLAG epitope-tagged WISP1v and WISP1vx were overexpressed by DNA transfection of mamma-lian expression vectors into HCS-2⁄ 8 cells First, over-expression of WISP1v and WISP1vx was confirmed by western blotting with anti-FLAG IgG (Fig 3B) Thereafter, we collected total RNA and comparatively analyzed the expression levels of the marker genes of chondrocyte differentiation and mineralization by real-time quantitative RT-PCR No significant difference was observed in the mRNA level of type II collagen and aggrecan core protein genes; however, the alkaline phosphatase (ALP) mRNA level increased remarkably with WISP1v overexpression (Fig 7)
Discussion
In this study, we obtained some clues to clarify the physiological roles of WISP1v in normal cells In
par-Fig 6 Immunohistochemical staining of CCN4 ⁄ WISP1 and WISP1v
in the developing tibia of a mouse embryo The tibia at embryonic
day of 17 was probed using anti-(CCN4 ⁄ WISP1) serum recognizing
the CT module Dark brown signals indicate the presence of
CCN4 ⁄ WISP1 and ⁄ or WISP1v protein (right) A negative control
without primary antibody is also shown (left) Cartilaginous ECM
was counterstained by methyl green.
Fig 7 Effects of the overexpression of WISP1v and WISP1vx in HCS-2 ⁄ 8 on the differentiation marker genes of growth plate chondrocytes Quantitative real-time PCR analysis was performed with WISP1v and WISP1vx overexpressing HCS-2 ⁄ 8 cells The relative values against the expression of gapdh were computed and displayed with error bars (SD) Data are derived from three independent evaluations Statistical analysis was performed by Tukey–Kramer test *P < 0.05, significantly different as indicated by the bracket.
Trang 7ticular, expression of WISP1v among normal
chondro-cytes and osteoblastic cells was confirmed, which has
heretofore not been elucidated Expression of WISP1v
increased during the course of chondrocytic terminal
differentiation, thus suggesting a role for WISP1v
therein This hypothesis was supported by the fact that
overexpression of WISP1v enhanced the gene
expres-sion of one of the mineralization markers in
chondro-cytic cells Moreover, the presence of a novel splicing
variant not described previously was discovered in
HCS-2⁄ 8 cells These findings are summarized in
Fig 8
To date, WISP1v has been investigated only from
the point of view of tumorigenesis and malignant
phe-notypes of tumors [16] Indeed, expression of WISP1v
has been reported only in malignant tumor cells [7,17]
Scirrhous carcinoma of the stomach is known to be
associated with WISP1v and it is characterized by
rapid growth with a vast fibrous stroma, high
invasive-ness and a substantially poor prognosis [20] Because
little is known about the molecular pathogenesis of the
disease, a pathological role for WISP1v should be
investigated further It should be noted that most
CCN family members are associated with certain types
of malignancy
Regarding physiological functions, some CCN
fam-ily members play an important role in skeletal growth,
as typically represented by CCN2⁄ CTGF [1,4,5]
CCN2⁄ CTGF was found to be highly expressed in
hypertrophic chondrocytes, as shown by in situ
hybrid-ization of both whole-mount neonates and their
longi-tudinal sections [21] A series of in vitro evaluations
revealed that CCN2⁄ CTGF plays an important role in
cartilage formation and endochondral ossification
RGC and rabbit articular cartilage cells transfected
with recombinant adenoviruses generating mRNA for
CCN2⁄ CTGF, thus result in increased proteoglycan
synthesis [22,23] Moreover, rCCN2⁄ CTGF effectively
increased both the ALPase activity and the matrix cal-cification of RGC cells [24,25] In the developing skel-etal system, CCN1⁄ Cyr61 is expressed in the cartilage
of a number of skeletal elements Regarding these ele-ments, CCN1⁄ Cyr61 is first expressed in condensing mesencymes, with expression persisting throughout the stages of chondrogenesis [26] Strong expression of CCN1⁄ Cyr61 in the developing cardiovascular and skeletal systems suggests that it might play an import-ant role in angiogenesis and⁄ or chondrogenesis [27– 31] In contrast, no report has described the role of CCN4⁄ WISP1 in cartilage development and endochon-dral ossification Here, we showed for the first time the expression and possible function of the CCN4⁄ WISP1 variant at a late stage of endochondral ossification In addition to the effect on ALP gene expression, we also evaluated the effect of WISPv overexpression on type X collagen gene expression, but no significant effect was observed (data not shown) Together with the fact that osteoblastic cells also expressed WISP1v, this variant is thought to play a role in mineralization rather than hypertrophy of chondrocytes
In comparison with CCN1⁄ Cyr61 and CCN2 ⁄ CTGF, the role of CCN4⁄ WISP1 in chondrocyte dif-ferentiation appears more specific to the mineralizing stage of endochondral ossification This fundamental difference may be interpreted from an evolutionary point of view Genome-wide prediction of CCN ortho-logs in several species indicated that CCN4⁄ WISP1 or-thologs were found only in animals with a calcified skeleton, whereas CCN1⁄ Cyr61 and CCN2 ⁄ CTGF could be identified in invertebrates such as Ciona intes-tinalis Thus, CCN4⁄ WISP1 is thought to be required for the establishment of a calcified endoskeleton This hypothesis is consistent with our findings on WISP1v However, it may not apply exactly to full-length CCN4⁄ WISP1, because no clear change was observed according to the chondrocytic differentiation
Fig 8 Formation and function of
CCN4 ⁄ WISP1 variants Structure,
expres-sion and function of the CCN4⁄ WISP1
vari-ants are summarized All of the findings
were obtained in this study except for that
of gastrointestinal cancers reported by
Tan-aka et al [17,20] ++, enhanced expression
evaluated by real-time RT-PCR; ±, modest
expression evaluated by real-time RT-PCR;
–, expression undetectable.
Trang 8In addition, no substantial induction of ALPase
activ-ity was seen in the overexpression experiment (data
not shown) We suspect that full-length CCN4⁄ WISP1
does not have such a close relationship with
endochon-dral mineralization, but rather another basic cell
biolo-gical role in chondrogenesis may exist In fact, our
preliminary data indicate the increasing expression of
CCN4⁄ WISP1 end ⁄ or its variant along with
chondro-genesis of mesenchymal stem cells (Kawaki et al.,
manuscript in preparation)
The functional significance of WISP1vx remains to be
investigated Expression of WISP1vx was detected only
in chondrosarcoma-derived HCS-2⁄ 8 cells We are
cur-rently collecting clinical samples of chondrosarcomas to
verify the significant relationship between WISP1v
expression and phenotypes of particular type of
malig-nancy Further functional evaluations are also required
to elucidate the pathological roles of this variant
Experimental procedures
Cell culture
A human chondrosarcoma-derived chondrocytic cell line,
HCS-2⁄ 8 [32], human cervical carcinoma-derived HeLa,
human melanoma cell line A371, human osteoblastic SaOs2
and human embryonic kidney-derived HEK293 [33] cells
were grown in DMEM supplemented with 10% fetal
bovine serum HUVECs derived from human umbilical
cords were purchased from Biowhittaker (Walkersville,
MD) and used between passages three and seven These
cells were cultured in EBM-2 complete medium (Clonetics,
San Diego, CA) RGC cells were isolated from growth
car-tilage in ribs of 5-week-old rabbits, as described previously
[25] The primary RGC cells were cultured in
a-modifica-tion of minimum essential medium (Sigma, St Louis, MO)
containing 10% fetal bovine serum Cell cultures were
maintained in a humidified 5% CO2 atmosphere at 37C
without any passage until analysis
Alizarin red staining
RGC cells in six-well plates were washed with NaCl⁄ Piand
fixed using 4% paraformaldehyde⁄ NaCl ⁄ Pi Following
fix-ation cells were washed again in NaCl⁄ Piand stained with
a 1% alizarin red solution, as described previously [34]
Macroscopic images were captured after 15 extensive
washes with NaCl⁄ Pi
DNA transfection
HCS-2⁄ 8 cells were seeded onto six-well plates at 500 000
cells per well Cells were transfected with 2 lg of plasmid
DNA using 8 lL of FuGENE6 transfection reagent (Roche
Applied Science, Penzberg, Germany) according to the manufacturer’s instructions Cells were lysed in 50 lL of 1· passive lysis buffer (Promega, Madison, WI), and collected for western blotting at 48 h after transfection RNA was also extracted from cells for RT-PCR at 48 h after transfec-tion
RNA extraction and RT-PCR analysis
Total RNA was extracted from the cells using the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Valencia, CA) Total RNA (500 ng) was reverse-transcribed
to cDNA by using an oligo(dT) with avian myeloblastosis virus reverse transcriptase (Takara, Tokyo, Japan) For semiquantitative PCR analysis, the cDNAs were amplified with Blend-Taq Plus (Toyobo, Osaka, Japan) with a regular thermal cycler
The sets of synthetic primers used for amplification are described below, where the numbers in parentheses indicate the expected sizes of the PCR products: human glyceralde-hyde-3-phosphate dehyrogenase gene (gapdh), sense 5¢-GCC AAAAGGGTCATCATCTC-3¢ and antisense 5¢-GTCTTC TGGGTGGCAGTGAT-3¢ (215 bp); human ccn4 ⁄ wisp1, sense 5¢-CTCAGCAGCTTGGGGACAAC-3¢ and antisense
CAG-3¢ and antisense 5¢-TGGCTGGTACACAGCCAGA CACTTC-3¢ (550 bp); human wisp1v, sense 5¢-GCAATAG GAGTGTGTGCACAGGTGG-3¢ and antisense 5¢-GAT GCCTCTGGCTGGTACAC-3¢ (345 bp) The amplification conditions were as follows: for human ccn4⁄ wisp1 and human wisp1v cDNA, 94C (5 min) for 1 cycle, followed
by 94C (30 s), 65 C (30 s), 72 C (45 min) for 35 cycles, and a final incubation at 72C for 5 min; for rabbit ccn4⁄ wisp1 and rabbit wisp1v cDNAs, 94 C (5 min) for
1 cycle, followed by 94C (30 s), 60 C (30 s), 72 C (30 s) for 35 cycles, and a final incubation at 72C for 5 min PCR products were electrophoresed in a 1% agarose gel containing ethidium bromide and visualized under UV light Photographs of the stained gels were taken and analyzed quantitatively with transilluminator fasiii (Toyobo)
Antibodies and molecular clones
An anti-(CCN4⁄ WISP1 H-55) serum recognizing the CT module (Santa Cruz Biotechnologies, Santa Cruz, CA) and
an anti-(FLAG M2) mAb (Sigma) were utilized in western blotting or immunohistochemistry
For molecular cloning of wisp1-related cDNA fragments, PCR products were fractionated by agarose gel electrophor-esis and were extracted from the gels using the Gel Extrac-tion kit (Qiagen) Next, purified PCR products were inserted into pGEM T-easy vector according to the manu-facturer’s instructions (Promega)
Trang 9The original human CCN4⁄ WISP1 and WISP1v
mam-malian expression vectors were given by Shinji Tanaka
(Tokyo Medical and Dental University), in which human
cDNAs were inserted in pCR3.1 (Invitrogen, San Diego,
CA) [17] Three FLAG epitope-tagged expression vectors
were newly constructed
ccn4⁄ wisp1 and wisp1v cDNAs were excised from the
pCR3.1-based plasmids by digestion with HindIII and
XbaI, and then inserted between the corresponding
restric-tion enzymatic sites of the Mammalian Amino-Terminal
FLAG vector (Sigma) The resultant expression plasmids
contain the ccn4⁄ wisp1 and its variant cDNAs fused
in-frame to the FLAG epitope tag We designated these
CMV-FLAG–WISP1vx, respectively, and utilized them in
the overexpression experiments
DNA sequence analysis
The nucleotide sequences of the cloned PCR products and
newly constructed expression plasmids were determined by
automated fluorescence DNA sequencing using the ABI
Prism Dye Terminator Cycle Sequencing Kit (PE Applied
Biosystems, Foster City, CA), following the
manufac-turer’s recommendations The primer recognizing the
bac-teriophage T7 promoter was employed in the sequencing
reactions for the TA-cloned cDNAs At least three
inde-pendent clones were analyzed for each mRNA variant
and identical results were obtained To confirm the
nucleotide sequence of the expression vectors,
CMV-FLAG–WISP1, CMV-FLAG–WISP1v and CMV-FLAG–
WISP1vx, ccn4⁄ wisp1-specific primers used in RT-PCR
analysis were utilized Products of the sequencing
reac-tions were then purified using SIGMA SPIN columns
(Sigma), and the samples were dissolved in 20 lL of
Applied Biosystems)
Quantitative real-time PCR
Optimal primer concentrations were determined based on
the protocols of Toyobo SYBR Green PCR Master Mix
Manual (Toyobo) Reactions were performed in 20 lL of a
reaction mixture (Toyobo) containing 2 lL cDNA, 5 lm
each primer and 1· SYBR Green Master Mix The primer
sets used for the evaluation of ccn4⁄ wisp1 and its variants
were the same as the regular RT-PCR The nucleotide
sequences of the primer sets for the quantitative evaluation
of human alkaline phosphatase (ALP), type II collagen and
AGCTTCAGAAGCTCAACACCA-3¢ and antisense 5¢-AT
type II collagen, sense 5¢-GAGGGCAATAGCAGGTTCA
CGTA-3¢ and antisense 5¢-TGGGTGCAATGTCAATGA
TGG-3¢ (133 bp); human aggrecan, sense 5¢-TCTTCA GTCCCGTTCTCCAC-3¢ and antisense 5¢-AACATCACT GAGGGCGAAGC-3¢ (93 bp), The amplification condi-tions were as follows: for human ccn4⁄ wisp1 and human alp cDNA, 95C (1 min) for 1 cycle, followed by 95 C (1 s), 59C (1 s), 72 C (30 s) for 50 cycles, and a melting process from 50 to 95C for 5 min, for rabbit ccn4 ⁄ wisp1 and rabbit ccn4⁄ wisp1v cDNAs, 94 C (5 min) for 1 cycle, followed by 94C (5 s), 60 C (5 s), 72 C (30 s) for 45 cycles, and melting process from 50 to 95C for 5 min, human aggrecan, 94C (30min) for 1 cycle, followed by
94C (5 s), 60 C (0 s), 72 C (30 s) for 45 cycles, and melting process from 50 to 95C for 5 min Similarly, rab-bit gapdh cDNA fragment (283 bp) was analyzed with sense
(5¢-CACAATGCCGAAGTGGTCGT-3¢) primers under the following conditions: 94C (30 s) for 1 cycle, followed by
94C (5 s), 65 C (10 s), 72 C (15 s) for 45 cycles, and melting process from 50C to 95 C for 5 min Rabbit type II collagen cDNA fragment (370 bp) was analyzed
antisense (5¢-AGCCGCCATTGATGGTCTCC-3¢) primers under the conditions of; 94C (30 s) for 1 cycle, followed
by 94C (5 s), 65 C (0 s), 72 C (60 s) for 45 cycles, and melting process from 50C to 95 C for 5 min Each ampli-fication reaction was performed and checked for absence of nonspecific PCR products by the melting curve analysis in
a LightCyclerTM system (Roche) Relative cDNA copy numbers were computed based on the data with a serial dilution of a representative sample for each target gene
Immunohistochemistry
Paraffin-embedded sections of normal mouse tibia were deparaffinized with xylene, rehydrated and washed with NaCl⁄ Pi After blocked in a blocking agent (Histofine: Nichirei, Tokyo, Japan), the samples were incubated over-night at 4C in an anti-(CCN4 ⁄ WISP1 H-55) serum (Santa Cruz Biotechnologies) at a concentration of 1 : 50
After washing several times in NaCl⁄ Pi, the sections were immersed in methanol, containing 0.3% H2O2 for 30 min
to block endogenous peroxidase activity To reduce nonspe-cific binding, 10% goat serum (Vector Laboratories, Burlin-game, CA) in NaCl⁄ Pi (GS-NaCl⁄ Pi) was applied to the specimens for 30 min Sections were then incubated with a specific antibody against CCN4⁄ WISP1 (1 : 50) diluted in GS-NaCl⁄ Piat 4C overnight
Specific signals were probed by a horseradish peroxy-dase-conjugated secondary antibody (Histofine: Nichirei), and visualized using 3,3-diaminobenzidine tetrachloride (Sigma) Finally, the samples were counterstained with methyl green solution (Wako Pure Chemical Industries, Osaka, Japan)
For negative controls, we skipped the primary antibody reaction step
Trang 10Western blotting
The cell lysate was prepared in a SDS sample buffer
con-taining 2.5% b-mercaptoethanol Each sample (25 lL) was
heated for 5 min at 95C and then was separated by 15%
PAGE, then the separated proteins were transferred to a
poly(vinylidene difluoride) membrane (Immobilon,
Milli-pore, Bedford, MA) Following the established protocol for
western blotting, FLAG-tagged proteins were detected
using a mouse anti-(FLAG M2) mAb (Sigma) Horseradish
peroxidase-conjugated goat anti-(mouse IgG) (Chemicon,
Temecula, CA) was used as a secondary antibody These
antibodies were used at dilutions of 1 : 1000 and 1 : 5000,
respectively Similarly, CCN4⁄ WISP1 proteins were
detec-ted, using anti-(CCN4⁄ WISP1 H-55) serum (Santa Cruz
Biotechnologies) Horseradish peroxidase-conjugated
anti-(rabbit IgG) (DAKO-Japan, Tokyo, Japan) was used as a
secondary antibody These antibodies were used at dilutions
of 1 : 200 and 1 : 1000, respectively Throughout the
proce-dure, 3% BSA–NaCl⁄ Pi was used as a blocking solution
Signals were detected using ECLTMwestern blotting
detec-tion regaments (Amersham Biosciences, Uppsala, Sweden)
Autoluminograms were obtained with an ECL mini-camera
(Amersham Biosciences)
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research (S) (to MT) and (C) (to SK) from
Japanese Society for Promotion of Science (JSPS) and
for Exploratory Research (to MT) from the Ministry
of Education, Culture, Sports, Science, and
Technol-ogy of Japan The authors thank Drs Takako Hattori
and Takashi Nishida for their helpful suggestions and
Ms Yuki Nonami for valuable secretarial assistance
A371 cell was obtained by Dr Akira Sasaki
References
1 Perbal B & Takigawa M (2005) CCN Proteins: A New
Family of Cell Growth and Differentiation Regulators
Imperial College Press, London
2 Brigstock DR (1999) The connective tissue growth
fac-tor⁄ cysteine-rich 61 ⁄ nephroblastoma overexpressed
(CCN) family Endocrin Rev 20, 189–206
3 Perbal B (2001) NOV (nephroblastoma overexpressed)
and the CCN family of genes: structural and functional
issues Mol Pathol 54, 57–79
4 Takigawa M (2003) CTGF⁄ Hcs24 as a multifunctional
growth factor for fibroblasts, chondrocytes and vascular
endothelial cells Drug News Perspect 16, 11–21
5 Takigawa M, Nakanishi T, Kubota S & Nishida T
(2003) Role of CTGF⁄ HCS24 ⁄ ecogenin in skeletal
growth control J Cell Physiol 194, 256–266
6 Blacketer MJ, Koehler CM, Coats SG, Myers AM & Madaule P (1993) Regulation of dimorphism in Sac-charomyces cerevisiae: involvement of the novel protein kinase homolog Elm1p and protein phosphatase 2A Mol Cell Biol 13, 5567–5581
7 Brigstock DR, Goldschmeding R, Katsube KI, Lam
SC, Lau LF, Lyons K, Naus C, Perbal B, Riser B, Takigawa M et al (2003) Proposal for a unified CCN nomenclature Mol Pathol 56, 127–128
8 Pennica D, Swanson TA, Welsh JW, Roy MA, Lawrence DA, Lee J, Brush J, Taneyhill LA, Deuel B, Lew M et al (1998) WISP genes are members of the connective tissue growth factor family that are up-regulated in wnt-1-transformed cells and aberrantly expressed in human colon tumors Proc Natl Acad Sci USA 95, 14717–14722
9 Perbal B (2001) NOV (nephroblastoma overexpressed) and the CCN family of genes: structural and functional issues Mol Pathol 54, 57–79
10 Zhang Y, Pan Q, Zhong H, Merajver SD & Kleer CG (2005) Inhibition of CCN6 (WISP3) expression pro-motes neoplastic progression and enhances the effects of insulin-like growth factor-1 on breast epithelial cells Breast Cancer Res 7, 1080–1089
11 Luo X, Ding L & Chegini N (2006) CCNs, fibulin-1C and S100A4 expression in leiomyoma and myometrium: inverse association with TGF-beta and regulation by TGF-beta in leiomyoma and myometrial smooth muscle cells Mol Hum Reprod 12, 245–256
12 Zoubine MN, Banerjee S, Saxena NK, Campbell DR & Banerjee SK (2001) WISP-2: a serum-inducible gene dif-ferentially expressed in human normal breast epithelial cells and in MCF-7 breast tumor cells Biochem Biophys Res Commun 30, 421–425
13 Perbal B, Brigstock DR & Lau LF (2003) Report on the second international workshop on the CCN family
of genes Mol Pathol 56, 80–85
14 Kubota S & Takigawa M (2006) CCN family genes in the development and differentiation of cartilage tissues Clin Calcium 16, 486–492
15 Desnoyers L, Arnott D & Pennica D (2001) WISP-1 binds to decorin and biglycan J Biol Chem 276, 47599–47607
16 Cervello M, Giannitrapani L, Labbozzetta M, Notarbartolo M, D’Alessandro N, Lampiasi N, Azzolina
A & Montalto G (2004) Expression of WISPs and of their novel alternative variants in human hepatocellular carci-noma cells Ann NY Acad Sci 1028, 432–439
17 Tanaka S, Sugimachi K, Kameyama T, Maehara S, Shirabe K, Shimada M, Wands JR & Maehara Y (2003) Human WISP1v, a member of the CCN family,
is associated with invasive cholangiocarcinoma
Hepatology 37, 1122–1129
18 YuC, Le AT, Yeger H, Perbal B & Alman BA (2003) NOV (CCN3) regulation in the growth plate and CCN