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CELLULAR AND MOLECULAR
Departments of Orthopaedic Surgery (H.O., K.I., A.O., S.T.) and Pharmacology (K.-I.F., K.I., S.M.), Hirosaki University School of Medicine, Hirosaki, Japan; Hirosaki Memorial Hospital, Hirosaki, Japan (K.U.); and Aomori Central Hospital, Aomori, Japan (S.H.)
Received November 29, 2002; accepted February 3, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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).
Therefore, systemic factors as well as local factors have been considered to
play a role in the pathogenesis of this condition. The etiology of OPLL has
been extensively investigated from various standpoints, however, its mechanism
of development has not been clarified. The ossification process in OPLL is
thought to occur through the endochondral mechanism
(Hashizume, 1980;
Yamamoto et al., 2002), but
the involvement of multiple etiologic factors in the development of OPLL has
been suggested, including genetic factors
(Koga et al., 1998;
Numasawa et al., 1999;
Furushima et al., 2002),
dietary habits (Wang et al.,
1999), metabolic abnormalities
(Baba et al., 1997), and some
local factors. There are many studies on the progression of OPLL. OPLL often
progresses after posterior decompressive surgery of the cervical spine, such
as laminectomy or laminoplasty, which causes biological stimulation, defect of
the dorsal element, and cervical instability
(Matsunaga et al., 1994;
Nakamura, 1994;
Takatsu et al., 1999). These
pieces of clinical evidence support the hypothesis that the mechanical stress
that acts on the posterior ligaments plays an important role in the
progression of OPLL.
Differences between OPLL cells and non-OPLL cells have been recognized
histologically and morphologically (Goto et
al., 1998; Ishida,
1998). For example, OPLL cells have several different phenotypic
characteristics of osteoblasts. They are characterized by high alkaline
phosphatase (ALP) activity, an increase in cAMP in response to parathyroid
hormone (Ishida and Kawai,
1993b), and in vitro calcification. On the other hand, non-OPLL
cells have a typical uniform fibroblast-like morphology, which is spindle
shaped, and proliferate constantly (Goto et
al., 1998; Ishida,
1998). The ALP activity is not high, and no bone-like
calcification is observed in vitro (Ishida
and Kawai, 1993a). Thus, these cells do not show any osteoblastic
characters. Although differences have been noted between the two cell types,
there are few reports that have compared the two types of cells at the gene
expression level.
In this study, we compared OPLL cells with non-OPLL cells at the transcriptional level by differential display reverse transcription (RT)-polymerase chain reaction (PCR) and detected a difference in the expression of prostaglandin I2 (PGI2) synthase. Uniaxial cyclic stretch induced an increase in PGI2 synthase mRNA. Furthermore, OPLL cells had a hyper-responsiveness to PGI2 compared with non-OPLL cells. The possible role of PGI2 in the development of OPLL and the influence of mechanical stress are discussed.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Clinical Diagnosis and Spinal Ligament Samples. The diagnosis of
OPLL or non-OPLL (i.e., other cervical diseases with no relation to OPLL) was
confirmed on X-rays, computerized tomography, and magnetic resonance imaging
of the cervical spine preoperatively. Although the ligament samples were not
all from the same location in the cervical spine, we used all the samples for
the experiments, because the cells from OPLL patients showed similar
osteoprogenitor-like characteristics regardless of the location from which the
tissue was extirpated in the cervical spine
(Kon et al., 1997;
Ishida, 1998).
Cell Culture. The ligaments were harvested aseptically from patients during surgery, rinsed with phosphate-buffered saline, and the surrounding tissue was carefully removed under a dissecting microscope. In all cases, the ligaments were extirpated carefully from nonossified sites to avoid any possible contamination with osteogenic cells. The collected ligaments were minced into approximately 0.5-mm3 pieces, washed twice with phosphate-buffered saline, and then plated on 100-mm culture dishes and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 100 units/ml penicillin G sodium, and 100 µg/ml streptomycin sulfate in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The cells derived from the explants were harvested from the dishes with 0.02% EDTA/0.05% trypsin for further passages.
Differential Display RT-PCR. After the cultures reached confluence, the total RNAs were extracted from the cell monolayers with an RNeasy kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. Each 1 µg of total RNA from OPLL and non-OPLL cells was reverse transcribed into cDNA using three 3'-anchored oligo(dT) primers. Three different arbitrary primers and three 3'-anchored oligo(dT) primers were used for the PCR amplification of the cDNA (1 µl). The PCR amplification was carried out in a volume of 20 µl using a Taq PCR Master Mix kit (QIAGEN), and the products were visualized by staining with SYBR Green-I (Amersham Biosciences Inc., Piscataway, NJ). The PCR cycling conditions were 95°C for 5 min, then 30 cycles of 95°C for 30 s, 42°C for 2 min, and 72°C for 30 s, and a final extension at 72°C for 5 min. The concrete pairs of primers were as shown in Tables 1 and 2. The reaction products were electrophoresed in a 6% SDS-polyacrylamide gel, and the differential bands were isolated from the gel. The cDNAs were eluted by boiling and then reamplified using the same primer pairs and PCR conditions.
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Uniaxial Cyclic Stretch. The cells (fifth passage) were placed on a
3.5 x 4.0-cm2 silicon chamber coated with 0.1% gelatin (IWAKI
GLASS, Tokyo, Japan) at a density of 10,000 cells/cm2. After the
cultures reached confluence, the cells were incubated in Dulbecco's modified
Eagle's medium supplemented with 1% fetal bovine serum for 24 h. The silicon
chamber was attached to a four-point bending and stretching apparatus that was
driven by a computer-controlled stepping motor (Scholertec Corp., Osaka,
Japan) (Naruse et al., 1998).
Uniaxial sinusoidal stretch (120% peak to peak, at 1 Hz) was applied in a
humidified atmosphere of 95% air and 5% CO2 at 37°C.
RNA Preparation and cDNA Synthesis. After different periods of cyclic stretch, the total RNAs were extracted simultaneously from the cell monolayers with an RNeasy kit (QIAGEN) according to the manufacturer's protocol. The total RNA was treated with RNase-free DNase I (Invitrogen Corp., Carlsbad, CA) and reverse transcribed into cDNA using Oligo(dT)12-18 primer (Invitrogen Corp.). One microgram of total RNA was heated at 70°C for 10 min in 10 µl of H2O supplemented with 0.5 µg of Oligo(dT)12-18 primer. The mixture was placed on ice, and cDNA synthesis was then performed by RT for 1 h at 37°C in a final volume of 20 µl of buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2) supplemented with 0.5 mM of dNTPs (Invitrogen Corp.), 2.5 mM of DTT (Invitrogen Corp.), 2 U of RNase inhibitor (TOYOBO, Osaka, Japan), and 200 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen Corp.). After the incubation, the cDNAs were heated to 72°C and then stored at -20°C until use for amplification by PCR.
PCR Analysis. For PCR amplification, specific oligonucleotide primers to human sequences were designed on the basis of the sequences in GenBank as follows: glycerol 3-phosphate dehydrogenase (G3PDH), 5'-TCCACCACCCTGTTGCTGTA-3' and 5'-ACCACAGTCCATGCCATCAC-3'; ALP, 5'-ATCGCCTACCAGCTCATGCAT-3' and 5'-GTTCAGCTCGTACTGCATGTC-3'; PGI2 synthase, 5'-GCCAAAAAAGAAGGTGCCGATTTC-3' and 5'-GAACTCCCGACCTCAAGTGATC-3'; and PGI2 receptor, 5'-GTCCATGCTCATCCTCTTTGCC-3' and 5'-GCGGAAAAGGATGAAGACCCA-3'.
The reaction was performed using a Taq PCR Master Mix kit as follows: 1 µl of cDNA was used as the template in a 20-µl amplification mixture containing 1 U of Taq DNA polymerase, 0.5 µM each of the 5' and 3' primers, and distilled water. All the products were assayed in the exponential phase of the amplification curve, and the PCR cycles were determined for each primer pair. PCR was performed in a PerkinElmer 9600 thermal cycler. The cycling conditions for G3PDH were 94°C for 20 s, 60°C for 30 s, 72°C for 90 s for 17 cycles, and a final extension at 72°C for 10 min. The cycling conditions for ALP were 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min for 23 cycles, and a final extension at 72°C for 10 min. The cycling conditions for PGI2S and PGI2 receptor were 94°C for 20 s, 61°C for 30 s, 72°C for 90 s for 24 and 36 cycles, respectively, and a final extension at 72°C for 10 min. The amplified products were resolved by electrophoresis in a 2.5% w/v agarose gel and visualized by staining with SYBR Green-I. The SYBR Green-I fluorescence was converted into a TIFF image by a charge-coupled device camera (C-900 ZOOM; OLYMPUS, Japan), and the intensity was quantified by QuantiScan software (BIOSOFT, Ferguson, MO). All the products were corrected for the G3PDH mRNA levels.
Enzyme-Linked Immunosorbent Assay of PGI2.
PGI2 concentration in the medium after loading mechanical stress on
OPLL cells was evaluated by measuring its stable metabolite
6-keto-PGF1
by enzyme-linked immunosorbent assay
kit (Assay Designs, Inc., Ann Arbor, MI) according to the manufacturer's
protocol.
Drugs. ONO-8713 and ONO-AE-248ONO were kindly provided by Ono Pharmaceutical Co., Ltd. (Osaka, Japan). All other chemicals used in this study were of high-quality analytical grade.
Statistical Analysis. All data are expressed as the mean ± S.E.M. The Friedman test for a control was used in all experiments. P < 0.05 was considered significant. n means the number of ligament cell preparations obtained from different spinal ligament samples.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Effect of Mechanical Stress on PGI2 Synthase Expression.
It has been reported that mechanical stress plays an important role in the
progression of OPLL (Kitajima et al.,
2001). Furthermore, mechanical stress produces PGI2 in
osteoblasts (Rawlinson et al.,
1993; Zaman et al.,
1997). To investigate the relationship between PGI2
signaling and mechanical stress, we performed RT-PCR with primers for
PGI2 synthase. The mRNA expression of PGI2 synthase in
OPLL cells was higher than that in non-OPLL cells maintained in the resting
state (Fig. 3). In OPLL cells,
cyclic stretch significantly increased the mRNA expression of PGI2
synthase about 145 and 170% (P < 0.05) after stimulation for 6 and
9 h, respectively, compared with the cells maintained in the resting state (0
h) (Fig. 4). On the other hand,
the expression level of PGI2 synthase did not change in non-OPLL
cells.
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Effect of Mechanical Stress on PGI2 Production.
PGI2 production during the mechanical loading was examined by
enzyme-linked immunosorbent assay method. After loading the cyclic stretch on
OPLL cells, medium was collected, and the concentration of
6-keto-PGF1, the stable metabolite of
PGI2, was evaluated. PGI2 released into the medium was
significantly increased by cyclic stretch for 9 h (P < 0.05, 3128
± 132 pg/ml, n = 4) compared with the medium of OPLL cells
maintained in the resting state for 9 h (1091 ± 54 pg/ml, n =
4).
Effects of Beraprost and Dibutyryl-cAMP on ALP Expression. It has
been reported that PGI2 stimulates cAMP synthesis in osteoblasts
(Partridge et al., 1982;
Rawlinson et al., 1991;
Khanin et al., 1999). To
investigate the role of PGI2 and cAMP in the osteogenic
differentiation of OPLL cells, beraprost, a stable analog of PGI2,
and dibutyryl cAMP, a membrane-permeable cAMP analog, were added to the
culture medium at final concentrations of 1 and 100 µM, respectively, and
then the ALP mRNA expression level was assessed as a marker gene for
osteogenic differentiation. Beraprost increased the mRNA expression of ALP
about 150 and 240% (P < 0.05) after addition for 6 and 9 h,
respectively, and dibutyryl cAMP increased it about 150 and 200% (P
< 0.05) after addition for 6 and 9 h, respectively, compared with the cells
with no drugs (Figs. 5 and
6). No change was observed in
non-OPLL cells.
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Effects of Adenylate Cyclase Inhibition on the Gene Expression Induced
by Beraprost. To investigate the involvement of adenylate cyclase in the
signal transduction in OPLL cells stimulated by PGI2, the cells
were incubated with 100 µM SQ22536
(Schilling et al., 1998), a
potent inhibitor of adenylate cyclase, for 30 min and then incubated with
beraprost in the presence of SQ22536 for 9 h. The increase in ALP mRNA
expression induced by beraprost was diminished by the addition of 100 µM
SQ22536 (Fig. 7).
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Effects of Adenylate Cyclase Inhibition on the Gene Expression Induced by Mechanical Stress. To investigate the involvement of adenylate cyclase in the signal transduction in OPLL cells stimulated by mechanical stress, the cells were incubated with 100 µM SQ22536 for 30 min and then subjected to uniaxial cyclic stretch in the presence of SQ22536 for 9 h. The expressions of ALP were significantly increased by stretch (P < 0.05, ALP/G3PDH = 0.99 ± 0.45, n = 4) compared with the group without stretch (ALP/G3PDH = 0.52 ± 0.26, n = 4), and these stretch-induced expressions of ALP were almost completely suppressed by SQ22536 (ALP/G3PDH = 0.54 ± 0.27, n = 4).
The Presence of PGI2 Receptor. The presence of PGI2 receptor was confirmed in OPLL and non-OPLL cells by RT-PCR using specific primers for PGI2 receptor (Fig. 8). There was no significant difference in the expression of PGI2 receptor between the two types of cells. This result suggests that the difference in sensitivity to PGI2 between the two types of cells may be due to differences in the intracellular signal transduction.
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Effects of Prostaglandin E2 Receptor Agonist and Antagonists on the ALP Expression. To investigate the involvement of prostaglandin E2 receptors in the signal transduction in OPLL cells stimulated by PGI2, the cells were incubated with 1 µM ONO-8713 (an EP1 antagonist), 100 µM AH6809 (an EP1 and EP2 antagonist), 10 µM ONO-AE-248 (an EP3 agonist), and 100 µM AH23848B (an EP4 antagonist) for 30 min and were followed by the incubation with 1 µM beraprost for 9 h except for the case of the EP3 agonist. The expressions of ALP were increased by beraprost about 199% (P < 0.05, ALP/G3PDH = 1.06 ± 0.15, n = 4) compared with the group without beraprost (ALP/G3PDH = 0.59 ± 0.18, n = 4). The expressions of ALP in the presence of prostaglandin E2 receptor antagonists were 150% (P < 0.05, ALP/G3PDH = 0.86 ± 0.16, n = 4), 171% (P < 0.05, ALP/G3PDH = 0.94 ± 0.08, n = 4), and 145% (P < 0.05, ALP/G3PDH = 0.76 ± 0.11, n = 4), respectively. On the other hand, the EP3 agonist (ONO-AE-248) failed to increase the ALP expression (ALP/G3PDH = 0.61 ± 0.08, n = 4).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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).
PGI2 is converted from prostaglandin H2 by
PGI2 synthase, the key enzyme in PGI2 production.
Differential display RT-PCR revealed higher expression of this enzyme in OPLL
cells compared with non-OPLL cells. This result suggests that OPLL cells have
an osteoblastic phenotype rather than a fibroblastic phenotype and that the
PGI2-signaling system plays some role in the progression of OPLL.
Several lines of evidence support the view that OPLL cells have an
osteoblastic phenotype and the metaplasia of OPLL cells into osteoprogenitor
cells had already occurred in OPLL (Ishida
and Kawai, 1993a; Kon et al.,
1997).
Mechanical stress is known as a regulator of bone remodeling, which
increases not only the osteoblast cell number but also the expressions of
various osteogenic marker genes, such as alkaline phosphatase, type I
collagen, osteopontin, and osteocalcin
(Harter et al., 1995). OPLL
often progresses after posterior decompressive surgery of the cervical spine,
such as laminectomy or laminoplasty, which causes cervical instability
(Matsunaga et al., 1994;
Nakamura, 1994;
Takatsu et al., 1999). These
clinical observations suggest that the mechanical stress that acts on the
posterior ligaments is an important factor in the progression of OPLL. In
fact, uniaxial cyclic stretch increases the expressions of osteogenic marker
genes in both OPLL cells (Tanno et al., unpublished observations) and ligament
tissues (Iwasaki et al., unpublished observations). On the other hand,
mechanical loading induces PGI2 production in osteocytes and
osteoblasts, resulting in an induction of bone remodeling
(Rawlinson et al., 1993;
Zaman et al., 1997).
Furthermore, exogenous PGI2 stimulates an increase in ALP activity
in osteocytes and osteoblasts (Rawlinson
et al., 1993). The present study revealed that uniaxial cyclic
stretch enhanced the expression of PGI2 synthase and also
PGI2 production. Beraprost, a stable PGI2 analog,
induced an increase in ALP mRNA in OPLL cells but not in non-OPLL cells. These
observations suggest that mechanical stress affects the progression of OPLL
through the activation of the PGI2-signaling system.
PGI2 interacts with a specific receptor, IP, which is a G
protein-coupled cell surface receptor. The IP receptor was detected in fetal
bone and osteoblasts (Fortier et al.,
2001). Although the IP receptor was expressed in both OPLL cells
and non-OPLL cells, beraprost only increased the ALP mRNA expression in OPLL
cells. Ligament cells have been reported to express other prostagrandin
receptors (i.e., EPs). However, antagonists for EP receptors, including EP1,
EP2, and EP4, did not affect on the ALP expression induced by beraprost, and
an agonist for the EP3 receptor by itself failed to induce the ALP expression.
These results allow us to speculate that the cellular effects of
PGI2 are mediated through the PGI2 receptor but there
are differences between OPLL cells and non-OPLL cells with regard to the
response to mechanical stress and intracellular signal transduction.
PGI2 enhances cAMP synthesis in the osteoblastic-like cell line
UMR-106 (Khanin et al., 1999)
and osteoblasts (Partridge et al.,
1981), and this is mediated by adenylate cyclase. Dibutyryl cAMP
mimics the effect of PGI2 in osteoblasts
(Partridge et al., 1982;
Rawlinson et al., 1991;
Khanin et al., 1999). The
present study demonstrated that beraprost and dibutyryl cAMP only increased
the ALP mRNA expression in OPLL cells and that SQ22536, a potent adenylate
cyclase inhibitor, diminished the stimulatory effect of beraprost. These
results suggest that the PGI2/cAMP system plays a pivotal role in
the osteogenic differentiation of OPLL cells.
In conclusion, PGI2 synthase was expressed more highly in OPLL cells than in non-OPLL cells. Uniaxial cyclic stretch, as a mechanical stress, further increased PGI2 synthase expression only in OPLL cells, resulting in the stimulation of ALP expression via an increase in the intracellular cAMP level. We propose that the increase in PGI2 synthase induced by mechanical stress plays a key role in the progression of OPLL, at least in part through the induction of osteogenic differentiation in spinal ligament cells through the PGI2/cAMP system.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: OPLL, ossification of the posterior longitudinal ligament of the spine; PGI2, prostaglandin I2; RT, reverse transcription; PCR, polymerase chain reaction; G3PDH, glycerol 3-phosphate dehydrogenase; ALP, alkaline phosphatase; BLAST, Basic Local Alignment Search Tool.
Address correspondence to: Ken-Ichi Furukawa, Department of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan. E-mail: furukawa{at}cc.hirosaki-u.ac.jp
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, significantly different from 0 h, P < 0.05.
PGI2 synthase/G3PDH at 0 h was 1.40 ± 0.68 in OPLL cells and
0.97 ± 0.30 in non-OPLL cells.
