Download ATP-DEPENDENT MINERALIZATION OF OSTEOBLASTIC CELLS

ATP-DEPENDENT MINERALIZATION OF OSTEOBLASTIC CELLS FROM "TIPTOE WALKING" (TTW) MICE IS
IMPAIRED IN VITRO.
+*Masuda, I; **Ikegawa, S; *Hirose, J; *Ryan, L
+*Medical College of Wisconsin, Div. of Rheumatology, Dept. of Medicine, Milwaukee, WI. 9200 W. Wisconsin Ave., Milwaukee, WI 53226, 414-456-8165, Fax:
414-456-6205, [email protected]
PC-1/NTPPPH has broad tissue distribution8 and is the dominant
NTPPPH in osteoblasts9. Biomineralization in ttw osteoblast MV was lower
than in w/t, suggesting that PC-1/NTPPPH is important for osteoblast
mineralization. Moreover, the promotive effect of extracellular ATP on MV
mineralization seen in w/t MV and osteoblasts was severely impaired in ttw
MV and osteoblasts. Regional decreases in density of trabecular bone in ttw
mice10 have been reported and ttw mice demonstrate increased bone turnover.
Their osteoblasts appear more PTH-responsive than w/t in vitro11. Although
extracellular ATP (or its degradation products such as adenosine) can strongly
potentiate the response of bone cells to PTH to stimulate bone cell growth and
differentiation in vitro12, ATP might not stimulate osteoblasts from ttw mice.
Failure of cellular responses to ATP would serve as an alternative explanation
for the decreased bone mineralization seen in ttw.
Proteins other than PC-1 have NTPPPH enzyme activity. Cartilage
intermediate layer protein (CILP)13, expressed specifically in cartilage, also
has a tentative functional domain encoding NTPPPH and has been identified
in porcine chondrocyte vesicles14,15. NTPPPH activity in ttw osteoblast
vesicles wasn't absent despite having non-functional PC-1/NTPPPH, and MV
still could enhance mineralization with ATP. These findings suggest that in
bone of ttw mice a form of NTPPPH other than PC-1/NTPPPH accounts for
residual NTPPPH activity and residual ATP-induced mineral formation.
MV
NTPPPH
PPi
(pmol/hr/mg protein)
(pmol/µg protein)
Wild type (w/t)
363.3 ± 69.4
0.150 ± 0.058
Tiptoe walking (ttw) 142.4 ± 22.2
0.111 ± 0.039
Table 1: NTPPPH activity and PPi in vesicles isolated from w/t and ttw
osteoblast cultured conditioned media. Assay were measured in triplicate and
values are representative of four separate experiments.
9000
45Ca (CPM/mg prote
Introduction: The "tiptoe walking" (ttw) mouse is an autosomal recessive
mutant manifesting multiple osteochondral lesions characterized by
pathologic calcium deposition including spinal and periarticular ligament
hyperostosis and cartilage calcification. It is a model of ossification of the
posterior longitudinal ligament (OPLL)1. A naturally occurring nonsense
truncation mutation was recently identified by genome mapping of ttw mouse.
The mutation produced a gene defect in plasma cell membrane glycoprotein1(PC-1), the inorganic pyrophosphate (PPi)-generating nucleoside
triphosphate pyrophosphohydrolase (NTPPPH)2. Forced expression of PC1/NTPPPH in murine osteoblastic MC3T3 cells resulted in more PC1/NTPPPH and PPi in their matrix vesicles and less mineral deposition than
forced expressed mutant PC-1 or empty plasmid. Therefore, PC-1/NTPPPH
was suspected of regulating bone mineralization by regulating PPi
production3. We studied osteoblastic cells derived from ttw mice that don't
express functional PC-1 and their littermate wild type (w/t) that do express
PC-1 to examine the mineralization capabilities of the cells and matrix
vesicles (MV).
Materials and Methods: Mice osteoblasts were isolated from explants of
joints from 14 week-old (elder age) ttw and w/t mice and cultured in
monolayer maintained in DMEM containing 10% fetal bovine serum and 1 %
antibiotics/antimycotics, at 37°C. They were characterized as being
osteoblasts by their expression of alkaline phosphatase and of osteoblastspecific protein, osteocalcin, as demonstrated by RT-PCR. Osteoblastic cells
of passage 2-4 were used for the experiments. To isolate MV, cells were left
in serum free DMEM for 72 hours, and culture-conditioned media were
collected. Media were initially centrifuged at 17,000g for 15 minuets at 4°C to
pellet cellular debris followed by ultracentrifugation at 200,000g for 100
minutes to isolate the MV fraction. NTPPPH and PPi assay were performed.
MV (40 µg of protein in 25 µl) were added in pentaplicate to 0.5 ml calcifying
medium3 with 45Ca, and incubated at 37°C for 48 hours to precipitate
minerals. The 45Ca in the pelleted mineral phase was solublized in HCl and
counted. Since ATP is a preferred substrate for NTPPPH and is known to
promote deposition of CPPD crystals and hydroxyapatite crystals in
chondrocyte vesicles4 and articular cartilage5, MV were incubated with or
without 1 mM ATP.
Results: NTPPPH activity in the MV from ttw was depressed significantly
but was not absent (Table 1). Values of PPi in these vesicles were very low
compared to the level that adult porcine chondrocytes produce (2-5 pmol/µg
protein). 45Ca precipitation in ttw MV without ATP was lower but not
significantly different from w/t MV; however, ttw MV ATP-induced
mineralization was significantly (p<0.01) inhibited by 70 % compared to w/t
(Figure 1). 45Ca precipitation with both ttw and w/t osteoblastic cells showed
similar trends to MV in that ttw osteoblasts mineralized poorly in the presence
of ATP compared to w/t.
Discussion: PPi is a potent inhibitor of apatite formation both in vitro and in
vivo6 and may have the physiological role of preventing calcium phosphate
crystal formation in articular cartilage. NTPPPH generates PPi on the outside
of joint cells and controls extracellular PPi levels. The non-functional ttw PC1/NTPPPH cannot hydrolyze ATP to generate PPi; therefore, extracellular PPi
concentrations might be insufficient to prevent the abnormal calcification seen
in soft tissues and cartilage of ttw mice. In osteoblasts, however, extracellular
PPi may promote mineral formation. Extracellular PPi is quickly hydrolyzed
to inorganic phosphate (Pi) by pyrophosphatases such as alkaline phosphatase,
thus providing anion for initial apatite nucleation and growth. As suggested by
studies in alkaline phsophatase knock-out mice which exhibit excess
extracellular PPi due to lack of hydrolysis of PPi, further growth of apatite is
impaired by supraphysiologic concentrations of PPi in calcifying cartilage7. It
is likely that high alkaline phosphatase activity is crucial to the ATPdependent enhancement of osteoblast MV mineralization by hydrolyzing
NTPPPH-generated PPi to Pi.
8000
7000
6000
5000
4000
3000
2000
1000
0
Figure1. Biomineralization assay of tiptoe (ttw) and wild type (w/t) mice MV
isolated from osteoblastic cells cultured conditioned media . Assay were
performed in pentaplicate and graph is representative of three separate
experiments.
References: 1Ohtsuki et al. (1998) Calcif Tissue Int 63:167-72. 2Okawa et al.
(1998) Nat Genet 19:271-73. 3Johnson et al. (1999) J Bone Miner Res 14:88392. 4Derfus et al. (1992) Arthritis Rheum 35:231-40. 5Ryan et al. (1992)
Arthritis Rheum 35:1520-5. 6Fleisch H. (1981) Metab Bone Dis Relat Res
3:279-87. 7Fedde et al. (1998) J Bone Miner Res 14:2015-26. 8Goding et al.
(1998) Immunol Rev 161:11-26. 9Lotz et al. (1995) Proc Natl Acad Sci USA
92:10364-68. 10Kobayashi et al. (1998) Calcif Tissue Int 62:426-36.
11
Terakado et al. (1995) Calcif Tissue Int 56:135-39. 12Bowler et al. (2000) J
Biol Chem 274:14315-324. 13Lorenzo et al. (1998) J Biol Chem 273:2346975. 14Masuda et al. (1995) J Clin Invest 95:699-704. 15Masuda et al. (1997)
Gene 197:277-87.
**RIKEN, Lab. for Bone and Joint Diseases, SNP Research Center, Tokyo,
JAPAN.
Session 7 - Bone Biology I - Esplanade Ballroom 303-305, Sun 11:30 AM - 1:00 PM
47th Annual Meeting, Orthopaedic Research Society, February 25 - 28, 2001, San Francisco, California
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