Bone 46 (2010) 1100–1107

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Inositol hexakisphosphate inhibits mineralization of MC3T3-E1 osteoblast cultures

William N. Addison a, Marc D. McKee a,b,⁎
a
Faculty of Dentistry, McGill University, Montreal, Quebec, Canada
b
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

a r t i c l e i n f o a b s t r a c t

Article history: Inositol hexakisphosphate (IP6, phytic acid) is an endogenous compound present in mammalian cells and
Received 25 September 2009 tissues. Differentially phosphorylated forms of inositol are well-documented to have important roles in
Revised 4 January 2010 signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit
Accepted 8 January 2010
soft tissue calcification (specifically renal and vascular calcification) by binding extracellularly to calcium
Available online 14 January 2010
oxalate and calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely
Edited by: M. Noda unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on
osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent
Keywords: inhibition of mineralization without affecting cell viability, proliferation or collagen deposition. Osteoblast
Inositol hexakisphosphate differentiation markers, including tissue-nonspecific alkaline phosphatase activity, bone sialoprotein and
Phytic acid osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly
Osteopontin increased protein and mRNA levels of osteopontin, a potent inhibitor of crystal growth and matrix
Osteoblast mineralization. Inositol alone (without phosphate), as well as inositol hexakis-sulphate, a compound with a
Bone
high negative charge similar to IP6, had no effect on mineralization or osteopontin induction. Binding of IP6
to mineral crystals from the osteoblast cultures, as well as to synthetic hydroxyapatite crystals, was
confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by
binding to growing crystals through negatively charged phosphate groups and by induction of inhibitory
osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly
acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of osteopontin
gene expression.
© 2010 Elsevier Inc. All rights reserved.

Introduction inositol pyrophosphates (IP7-8) are all dephosphorylated and

Inositol hexakisphosphate (IP6, phytic acid) is the major form of
several highly phosphorylated inositol phosphates present in mam-
malian cells [1,2]. In IP6, all six hydroxyl groups of the inositol ring are
replaced by phosphate (Fig. 1A), resulting in a molecule with an
unusually high negative charge density. Although IP6 was first
identified as the major phosphate store in plant seeds (particularly
in grains and legumes) [3], IP6 was subsequently found to be
ubiquitous in mammalian cells and tissues [4,5]. In seeds, IP6 is
hydrolyzed during germination to maintain phosphate homeostasis
for plant cell metabolism [3]. In mammalian cells, intracellular
concentrations range from 10 to 100 μM [6–8], while urine and
plasma concentrations have been reported to be around 3 μM [9,10]
and 0.5 μM [11,12], respectively. IP6 has been implicated in a wide
variety of cellular functions such as cation transport, signal transduc-
tion, protein phosphatase inhibition, cell proliferation and DNA repair
(see [1,13,14] for reviews). IP6, lower inositol phosphates (IP1-5) and
⁎ Corresponding author. Faculty of Dentistry, Room M73, McGill University, 3640
University Street, Montreal, Quebec, Canada H3A 2B2. Fax: +1 514 398 8900.
E-mail address: marc.mckee@mcgill.ca (M.D. McKee).
phosphorylated by families of inositol phosphatases and kinases
thereby maintaining an intracellular pool of inositol phosphates with
distinct, and overlapping, cellular functions [1].
With respect to biomineralization, IP6 was demonstrated to inhibit
pericardial [15], vascular [16], tooth enamel [17] and renal calcifica-
tion [18,19]. Although the mechanism by which IP6 inhibits calcium
oxalate and calcium phosphate crystal growth in these systems is not
entirely known, IP6 is thought to adsorb onto growing crystal faces or
to stabilize nascent crystal nuclei, thus impeding further apposition of
mineral ions to the crystal. Like other small-molecule inhibitors (e.g.,
pyrophosphate and bisphosphonates) or protein inhibitors (e.g.,
osteopontin [20] and matrix gla protein [21]) of crystal growth, IP6
has an affinity for calcium ions by way of the negatively charged
phosphate residues [22,23]. Mineral-regulating proteins and peptides
are often post-translationally modified by phosphorylation of serine
and threonine residues [24,25]. Phosphates, in the form of phospho-
serine and phospho-threonine, provide a negatively charged side
group for binding to calcium. In addition, phosphorylations are often
ordered in clusters of three to five residues that seem to favor its
interaction with calcium within the hydroxyapatite crystal lattice
[26,27].

8756-3282/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bone.2010.01.367
 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107 1101

Fig. 1. Effect of inositol hexakisphosphate (IP6) on mineralization of MC3T3-E1 osteoblast cultures. (A) Structure of IP6. The six inositol hydroxyl groups are replaced by phosphate.
(B) Osteoblast cultures were incubated with the indicated dose of IP6 for 12 days followed by von Kossa staining for mineral in the culture dishes (lower panel), and calcium content
determination from decalcified cell-matrix layer extracts is expressed as a percentage of untreated control (upper panel). (C) Osteoblast cultures were treated with either 4 μM IP6,
inositol (I), inositol hexakis-sulphate (IS), EDTA or 24 μM sodium phosphate (P) for 12 days followed by von Kossa staining for mineral in the culture dishes (lower panel), and
calcium content determination from decalcified cell-matrix layer extracts is expressed as a percentage of untreated control (upper panel). (D) von Kossa staining and calcium assays
after 12 days where cultures were treated with 4 μM IP6 for either the first, or the last, 6 days of the 12-day culture period. Inhibition of mineralization correlates with the
mineralization stage (days 6–12) and not with the cell differentiation and matrix assembly stage (days 0–6). Data are presented as means ± SE. ⁎p b 0.05; ⁎⁎p b 0.01 from Student's
t-test relative to the 0 μM control treatment.

Although mammalian cells synthesize IP6 de novo from phospha-
tidylinositol phosphate and lower inositol phosphates [1,28], there is
evidence that levels in tissue and body fluids can be modulated by IP6
from dietary sources. For example, rats fed an IP6-rich diet displayed
an elevation in plasma IP6 levels [12], and IP6 tissue levels correlate
with dietary IP6 content [29,30]. Hence, dietary IP6 has been
suggested as a therapeutic option for patients with kidney stones
[18], with a recent study showing that increased dietary IP6 is
associated with a reduced incidence of kidney stone formation [31].
Given that IP6 can adsorb to hydroxyapatite [32], and that
physiologic concentrations inhibit hydroxyapatite growth in a cell-
free physicochemical study [33], as well as in soft tissue calcification
in vivo and in vitro [15,16,18,31,34], it seems important to consider
the effects of IP6 on bone mineralization. Since the effects of IP6 on
osteoblast activity are largely unknown, the aim of this study was to
evaluate a possible role for IP6 in osteoblast mineralization using an
extracellular matrix-producing and mineralizing osteoblast cell
culture model. We report the first evidence that IP6 inhibits
mineralization of osteoblast cultures and up-regulates expression of
osteopontin—another potent mineralization inhibitor [20,35]. These
data suggest that IP6 may be a naturally occurring regulator of bone
mineralization as well as a specific signal for the regulation of
osteopontin gene expression.
Materials and methods
Cell culture
MC3T3-E1 (subclone 14) murine calvarial osteoblasts [36,37]
(courtesy of Dr. R. Franceschi, University of Michigan, Ann Arbor, MI)
were maintained in modified α-minimum essential media (Invitro-
gen) supplemented with 10% fetal bovine serum (Hyclone) and 1%
penicillin–streptomycin (Invitrogen) at 37 °C in a humidified
atmosphere of 5% CO2. All experiments were carried out at a plating
density of 50,000 cells/cm2. Cell differentiation and matrix mineral-
ization were initiated 24 h after plating by replacing the medium with
fresh medium supplemented with 50 μg/ml ascorbic acid (Sigma) and
10 mM β-glycerophosphate (Sigma). Cell culture medium, with or
1102 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107

without IP6, was changed every 48 h over a 12-day time period. For
mineralization inhibition experiments, D-myo-inositol 1,2,3,4,5,6-
hexakisphosphate (IP6) and D-myo-inositol 1,4,5-trisphosphate
(IP3) were purchased from Calbiochem. Myo-inositol, myo-inositol
hexakis-sulphate, ethylenediaminetetraacetic acid (EDTA), foscarnet
and sodium phosphate were purchased from Sigma. Signaling
pathway inhibitors U0126, DT3, KT5823, H89, calphostin C and
SB202190 were obtained from VWR; SP600125 from SuperArray;
deltamethrin, nifedipine, calmidazolium chloride, 2-APB, thapsigar-
gin, and S-Bay K8644 from Sigma; wortmannin, LY294002, RO318220,
and Akt inhibitor from Calbiochem; and PD98059 from Invitrogen
(see Table 1).
Quantification of mineralization
After 12 days of culture, mineral was visualized by von Kossa
staining using a 5% silver nitrate solution (Sigma). For calcium
quantification of mineral deposited within the cell/matrix layer,
cultures were decalcified with 0.5 N HCl, and calcium in the
supernatant was determined spectrophotometrically (absorbance at
595 nm) using a calcium assay kit (Diagnostic Chemicals). The
calcium content of the cell/matrix layer was normalized to protein
content determined by the BCA protein assay kit (Pierce).
Assay for cell proliferation
Cell proliferation and viability in the presence of IP6 was measured
using the MTT assay [38]. Briefly, cells were incubated with 0.5 μg/μl
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bro-
mide) in medium for 3 h and solubilized with DMSO, and absorbance
was measured at 550 nm.
Assay for collagen deposition
To assess cell differentiation in terms of extracellular matrix
production and assembly, collagen matrix deposition was quantified
by picrosirius red staining followed by extraction with 0.1 N NaOH
and spectrophotometric measurement (absorbance at 562 nm) of
released stain as described previously [39]. Bovine calf skin collagen
type I (Sigma) was used as a standard.
Assay for alkaline phosphatase activity
To measure alkaline phosphatase activity as a marker of osteoblast
differentiation, cultures were washed three times with phosphate-
buffered saline and solubilized in 10 mM Tris, pH 7.4, 0.2% Igepal
(Sigma) and 2 mM phenylmethylsufonyl fluoride (Sigma). After
sonication and centrifugation, alkaline phosphatase activity in the
supernatant was determined spectrophotometrically (absorbance at
405 nm) using p-nitrophenylphosphate (Sigma) as a substrate and
bovine kidney alkaline phosphatase (Sigma) as a standard. One unit of
alkaline phosphatase hydrolyzes 1 μmol of p-nitrophenylphosphate/
min at 37 °C.
Western blotting
Cultures were scraped from the dishes and sonicated in buffer
containing 10 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X-100,
1% sodium deoxycholate, 0.5 M EDTA supplemented with a
protease inhibitor cocktail (Sigma) containing 4-(2-aminoethyl)
benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and
aprotinin. Protein concentration was determined by the BCA
protein assay (Pierce), and 5 μg of total protein was separated
by SDS-PAGE and transferred to nitrocellulose membranes (Pall
Life Sciences). The membranes were blocked with a 5% solution of
powdered skim milk in Tris-buffered saline/Tween 20, and then
immunoprobed using goat anti-osteopontin (R&D Systems), anti-
GAPDH-horseradish peroxidase conjugate (Abcam) and anti-goat-
horseradish peroxidase (Caltag). The blots were visualized by
chemiluminesence using ECL Plus (Amersham Biosciences). Band
densities on the Western blots were obtained using Quantity One
software (BioRad).
RNA isolation and RT-PCR
Total RNA was isolated using TRIzol reagent (Invitrogen) according
to the manufacturer's protocol. RNA was treated with DNase
(Invitrogen), and 1 μg was reverse-transcribed and amplified using
SuperScript one-step RT-PCR with Platinum Taq polymerase (Invitro-
gen). PCR products for Bsp (bone sialoprotein), Opn (Spp1, osteopon-
tin), Gapdh and Ocn (Bglap, osteocalcin) were analyzed by 2% agarose
gel electrophoresis. Primers (Invitrogen) used were as follows: Bsp,
5′-AACAATCCGTGCCACTCA-3′ and 5′-GGAGGGGGCTTCACTGAT-3′
[40]; Opn, 5′-CTGCTAGTACACAAGCAGACA-3′ and 5′-CATGA-
GAAATTCGGAATTTCAG-3′ [41]; Gapdh, 5′-CCACTCTTCCACCTTCG-3′
and 5′-GTGGTCCAGGGTTTCTTAC-3′ [42]; and Ocn, 5′-TGAACA-
GACTCCGGCG-3′ and 5′-GATACCGTAGATGCGTTTG-3′ [43]. Annealing
temperature and cycles performed were 45 °C/25 for Opn, 58 °C/24
for Ocn and Bsp and 55 °C/30 for Gapdh.
Determination of inositol hexakisphosphate binding to hydroxyapatite

Table 1 IP6 mineral-binding assays used hydroxyapatite from Berkeley

Inhibitors and pathways that had no effect on IP6-induced increase in osteopontin Advanced Biomaterials, and mineral crystals isolated from miner-
levels. alized, day-12 MC3T3-E1 osteoblast cultures as previously de-
Target/pathway Inhibitor Concentration scribed [44]. Briefly, to isolate mineral crystals from the cultures,
plates from 12-day mineralized cultures were washed three times
Akt Akt inhibitor 1 20 μM
Ca2+-ATPase Thapsigargin 1 μM
with phosphate-buffered saline, digested in trypsin and collagenase
Calcineurin Deltamethrin 20 nM before scraping and incubated in 0.5% sodium hypochlorite for
Calmodulin Calmidazolium chloride 10 μM 12 h at 4 °C (with rotation) to remove matrix components.
IP3 receptor 2-APB 150 μM Mineral was pelleted at 11,000g for 1 min and then washed five
JNK SP600125 10 μM
times in double-distilled water before a final wash in 98% ethanol
L-type calcium channel S-Bay K8644 0.5 μM
L-type calcium channel Nifedipine 5 μM followed by drying at 37 °C for 30 min. To determine binding of
MEK1 PD98059 25 μM IP6 to mineral, 3 mg of crystals was suspended in a 150 μl of
MEK1/2 U0126 10 μM 100 μM IP6 (pH adjusted to 7.2 with NaOH) for 15, 30, 45 or
P38 SB202190 15 μM
60 min followed by centrifugation at 2000g for 5 min. Experiments
Phosphatidylinositol 3-kinase LY294002 30 μM
Phosphatidylinositol 3-kinase Wortmannin 1 μM
were carried out in 500 μl polypropylene tubes. IP6 in the
Protein kinase A H89 10 μM supernatant was assayed as described by Magrill [32] by
Protein kinase C Calphostin C 0.4 μM precipitating the IP6 in an acidic Fe3+ 1.5 mg/ml solution, and
Protein kinase C RO318220 1 μM determining the decrease of Fe3+ spectrophotometrically as a
Protein kinase G DT3 0.25 μM
thiocyanate complex at 475 nm.
 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107
Fig. 2. Effect of inositol hexakisphosphate (IP6) on cell proliferation of MC3T3-E1
osteoblast cultures. Differentiating and mineralizing osteoblast cultures (DIFF, with
ascorbic acid and β-glycerophosphate) and undifferentiating osteoblast cultures
(UNDIFF, without ascorbic acid and β-glycerophosphate) were cultured in the presence
of 4 μM IP6 over 12 days during which the number of viable cells was assessed by the
MTT assay. Data are presented as means ± SE. ⁎⁎p b 0.01 from Student's t-test relative to
the untreated (no IP6) controls.
Statistical analysis of data
Statistical analyses were performed on triplicate samples using the
Student's t-test, and data are presented as standard error of the mean,
or as standard deviation, as indicated in the figure legends.
Results
Inositol hexakisphosphate inhibits mineralization of MC3T3-E1
osteoblast cultures
To examine the role of IP6 (Fig. 1A) on extracellular matrix
mineralization in osteoblast cultures, MC3T3-E1 cells were treated for
12 days with IP6, after which mineral was visualized by von Kossa
staining and quantified by a biochemical assay for calcium. As
demonstrated in Fig. 1B, IP6 dose-dependently inhibited mineraliza-
tion with maximal inhibition occurring at 4 μM. To further examine
the mechanism and relevance of IP6 mineral inhibition, the effect of
4 μM inositol alone (no phosphate groups), 4 μM inositol hexakis-
Fig. 3. Effect of inositol hexakisphosphate (IP6) on MC3T3-E1 osteoblast differentiation and collagen matrix assembly. Cultures were incubated with, or without, 4 μM peptide for
8 days, after which (A) collagen deposition was determined by quantification of picrosirius red staining, and (B) alkaline phosphatase activity was measured using p-
nitrophenylphosphate as a substrate. Data are presented as means ± SE. ⁎p b 0.05 from Student's t-test relative to the untreated control. (C) Osteoblast cultures were treated (or not
treated) for 12 days with 4 μM IP6 as indicated, after which RNA was extracted and RT-PCR analysis performed to examine gene expression of osteocalcin (Ocn) and bone sialoprotein
(Bsp) relative to the housekeeping gene Gapdh.
1103
sulphate (similar negative charge array as IP6), 4 μM EDTA (a cation
chelator, like IP6) and 24 μM phosphate alone on mineralization were
also examined. Treatment with inositol alone and phosphate alone
control for the possibility that IP6 is hydrolyzed by phosphatases,
thereby raising the level of inositol or phosphate in the medium. IP6,
like EDTA, is also a chelator of divalent metal cations, and the EDTA
control thus examines the possibility that IP6 inhibits mineralization
by altering free Ca2+ levels. As shown in Fig. 1C, whereas 4 μM IP6
inhibited mineralization, these controls had no effect on mineral
deposition. This suggests that inhibition of mineralization by IP6 is not
attributable simply to a change in inositol or phosphate levels in the
environment of the cells as a result of IP6 hydrolysis, nor is it
attributable to nonspecific cation chelation. Lack of inhibition by
inositol hexakis-sulphate underscores the importance of the phos-
phate groups in mediating IP6 action. To further understand the
mechanism by which IP6 inhibits mineralization, cultures were
treated with 4 μM IP6 either during days 0–6 (the matrix deposition
and assembly stage), during days 6–12 (the matrix mineralization
stage), or for the entire 12-day duration of the culture period. IP6
inhibited mineralization only when present during days 6–12,
suggesting that IP6 blocks culture mineralization mainly by direct
binding to hydroxyapatite crystals during the mineralization stage
and not by adversely affecting osteoblast differentiation and matrix
deposition during the early matrix assembly stage.
Absence of an effect of inositol hexakisphosphate on osteoblast
differentiation
Cell proliferation under differentiating and mineralizing condi-
tions, as measured by MTT assay, was normal after IP6 treatment (Fig.
2). Proliferation of undifferentiating osteoblasts (cultured without
ascorbic acid or β-glycerophosphate) was slightly decreased by IP6
treatment. To determine whether the mineralization-inhibiting effect
of IP6 was attributable to a disruption of osteoblast matrix assembly
and differentiation, we examined the effect of IP6 on collagen
deposition and alkaline phosphatase activity—two prominent mar-
kers of osteoblast differentiation and function. Collagen deposition
(Fig. 3A) remained abundant after treatment with 4 μM IP6. Alkaline
phosphatase activity (Fig. 3B), although slightly elevated, was not
adversely affected by treatment with 4 μM IP6. Later markers of
osteoblast differentiation were also examined by RT-PCR and, as
shown in Fig. 3C, mRNA levels of osteocalcin and bone sialoprotein
showed no changes. Taken together, these data indicate that IP6 does
not impair the ability of osteoblasts to synthesize a collagenous
1104 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107
matrix, express alkaline phosphatase and differentiate to produce
specific bone matrix proteins.
Inositol hexakisphosphate up-regulates osteopontin
An important feature of osteoblast mineralization is the secretion
of the mineralization inhibitor osteopontin into the extracellular
matrix. Recent data from both in vitro and in vivo studies have shown
that osteopontin expression is sensitive to the local levels of anions
such as phosphate and pyrophosphate [45,46]. To determine whether
IP6 could elicit a similar response, we examined levels of osteopontin
in IP6-treated osteoblast cultures. RT-PCR showed that osteopontin
mRNA was up-regulated in the presence of IP6 (Fig. 4A). To determine
whether the effect of IP6 was reflected at the protein level, we
examined the IP6-treated cultures by Western blotting and observed
that IP6 used at a 4 μM concentration led to a marked increase in
osteopontin levels (Fig. 4B) that was detectable as early as 24 h after
treatment and which peaked at 48 h (data not shown). We next
examined whether this up-regulation was directly attributable to IP6
Fig. 4. Induction of osteopontin (OPN) by inositol hexakisphosphate (IP6) in MC3T3-E1
osteoblast cultures. (A) Cultures were treated with, or without, 4 μM IP6 for 12 days
after which RNA was extracted and RT-PCR performed for gene expression of Opn and
the housekeeping gene Gapdh. (B) Cultures were treated with 4 μM IP6 for 48 h and the
cell lysate was analyzed by Western blotting using anti-OPN and anti-GAPDH antibody.
(C) MC3T3-E1 cells were treated either with 4 μM IP6, inositol (I), inositol hexakis-
sulphate (IS), EDTA or 24 μM sodium phosphate (P) for 48 h and analyzed by Western
blotting for OPN. (D) Cultures were pretreated with 300 μM foscarnet for 30 min, and
then treated with 10 mM phosphate (P) or 4 μM IP6 for 48 h and analyzed by Western
blotting for OPN.
and not to phosphate or inositol. Phosphate and inositol alone were
unable to increase osteopontin levels (Fig 4C). Inositol hexakis-
sulphate and EDTA also had no effect on osteopontin levels,
suggesting that induction of osteopontin is IP6-specific and not
attributable to negative charge or ion chelation. The phosphate
transporters Pit-1 and Pit-2 have previously been shown to mediate
phosphate regulation of osteopontin gene expression [47]. To further
confirm that IP6 and phosphate regulate osteopontin by different
mechanisms, and to examine the involvement of the phosphate
transporters, we treated cultures with 4 μM IP6 in the presence of the
phosphate uptake inhibitor, foscarnet (phosphonoformic acid).
Phosphate (10 mM; a dose previously shown to up-regulate
osteopontin [47]) was used as a positive control for osteopontin up-
regulation by phosphate. Western blotting confirmed that phosphate-
mediated osteopontin induction was blocked by foscarnet, whereas
IP6-mediated osteopontin induction was insensitive to this inhibitor
(Fig 4D). Phosphate uptake is therefore not necessary for IP6
regulation of osteopontin. Osteopontin gene expression has previ-
ously been shown to be regulated by the mitogen-activated protein
kinase (MAPK) pathways [45,46], whereas IP6 is known to activate
protein kinase C [48]. Interestingly, inhibition of MAPK and protein
kinase C pathways had no effect on IP6-induced osteopontin
expression (data not shown). Table 1 contains a list of related
pathways and inhibitors that were examined but which showed no
effect on IP6 induction of osteopontin expression.
Inositol hexakisphosphate binds to hydroxyapatite
To determine whether IP6 adsorbs to hydroxyapatite crystals, IP6
was first incubated with mineral crystals and then centrifuged to
separate bound and unbound IP6. IP6 in the supernatant was then
measured spectrophotometrically. Binding of IP6 to mineral derived
from osteoblast cultures (Fig. 5A) and to the synthetic hydroxyapatite
standard (Fig. 5B) was confirmed. IP6 did not degrade or bind to the
plastic vessel walls over the same time period (Fig. 5C).
Specificity of inositol hexakisphosphate action
To ascertain the specificity of IP6 action, and to determine whether
other related inositol phosphates could have the same mineralization-
inhibiting effect, we similarly examined mineralization and osteo-
pontin expression after inositol-1,4,5-trisphosphate (IP3) treatment.
The 1,4,5-trisphosphate configuration is the dominant inositol tri-
sphosphate isomer in mammalian cells [49]. IP3 inhibited minerali-
zation in osteoblast cultures with significantly less potency than IP6
(Fig. 6A). Whereas IP6 at the 4 μM concentration completely inhibited
mineralization, 4 μM IP3 displayed no effect on mineral inhibition. In
addition, IP3 failed to increase osteopontin protein levels at 0.4–40 μM
unlike IP6 which displayed a concentration-dependent stimulation of
osteopontin protein at similar doses (Fig. 6B). The fact that the
decreased number of phosphate groups in IP3 (3 phosphates) as
compared to IP6 (6 phosphates) reduced its mineralization-inhibition
potency and eliminated its ability to regulate osteopontin levels
indicates that although the observed effects of IP6 appear to be
specific, other related inositol phosphates could potentially play a
lesser role in mineral regulation.
Discussion
IP6 is a highly phosphorylated, negatively charged inositol
phosphate ubiquitous to cells, tissues and body fluids [4,5,29].
Among other widespread functions, it has been proposed to inhibit
soft-tissue calcification in vivo [16,18,31,50]. IP6 inhibits kidney stone
[18] and pericardial calcification [15] and has also been shown to
inhibit hydroxyapatite formation in cell-free crystal growth study [33]
and in enamel mineralization [17]. However, prior to the present
 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107
Fig. 5. Inositol hexakisphosphate (IP6) binding to synthetic hydroxyapatite and mineral isolated from MC3T3-E1 osteoblast cultures. Graphs show depletion of IP6 from the
supernatant after incubation of 100 μM IP6 with (A) 3 mg osteoblast culture-isolated mineral, (B) synthetic hydroxyapatite or (C) without any mineral. IP6 concentration was
determined by Fe3+ precipitation as described in Materials and methods. Data are presented as the means ± SD. ⁎p b 0.05; ⁎⁎p b 0.01 from Student's t-test relative to the 0-min data
point.
study no reports existed on the effect of IP6 on osteoblast culture
mineralization. In this paper we provide new insights into the role of
IP6 on skeletal tissues by examining its effects on matrix-producing
and mineralizing MC3T3-E1 osteoblast cell cultures. We show that IP6
inhibits osteoblast culture mineralization, adsorbs to osteoblast
culture mineral and up-regulates levels of osteopontin. Taken
together, these results suggest that IP6 may be a physiologic regulator
of bone mineralization by activating multiple inhibitory mechanisms
that include direct inhibition of crystal growth and indirect changes in
osteoblast gene expression.
Previous studies have shown that IP6 inhibited hydroxyapatite
crystallization from solution [33]. In the present study, we demon-
strate that IP6 inhibits hydroxyapatite formation within the abundant
collagenous extracellular matrix produced by the well-characterized
MC3T3-E1 osteoblast cell line. Considering that cellular, urinary and
plasma concentrations of IP6 are in the range of 15–100 μM [6–8],
3 μM [9,10] and 0.5 μM [11,12], respectively, our observations that
4 μM IP6 inhibits osteoblast mineralization would suggest that IP6 is a
physiologic inhibitor of mineralization. This micromolar concentra-
tion used in our study is consistent with the observed inhibition of
hydroxyapatite crystallization from urine at 12 μM [33], and the
inhibition of bovine pericardium calcification by 0.39 μM IP6 [15]. IP6
has previously been shown to prevent calcification in other soft
tissues; subcutaneously injected IP6 inhibited calcium phosphate
formation in a rat model of aortic calcification [16], and orally
administered IP6 prevented calcium oxalate formation in a rat kidney
stone animal model [18].
Crystal growth inhibitors such as pyrophosphate, bisphosphonates
and osteopontin act by adsorbing to growing crystal surfaces. IP6, like
the above-mentioned inhibitors, is also negatively charged and
possesses a high affinity for divalent metal cations [22,23]. This
affinity for Ca2+, and also Ca2+-containing crystal surfaces, may be the
mechanism for inhibition of calcium oxalate and calcium phosphate
mineral formation. IP6 adsorption to synthetic hydroxyapatite
reduces the rate of mineral dissolution [51,52] presumably by similar
mechanisms.
The ability of IP6 to interact with divalent metal ions in solution
could suggest ion chelation as a mechanism for the observed
inhibition of mineralization since in principal, calcium chelation
would decrease ion supersaturation and thus reduce the amount of free
calcium for crystal incorporation. However, our observations suggest
that inhibition of mineralization was not attributable to calcium ion
chelation from the culture medium because the experiments were
carried out in medium containing physiologic Ca2+concentration
(1.8 mM) whereas inhibition was observed at 4 μM IP6. IP6 binds
1105
to Ca2+ with a stoichiometry of 1:1 [22,23] and thus the effect of IP6
on free Ca2+ levels would be negligible. Furthermore, the effects of
IP6 could not be imitated by EDTA, a well-known calcium ion
chelator.
High concentrations (mM) of exogenous IP6 have been shown to
inhibit cell growth in a number of carcinogenesis models [53]. In our
observations, using micromolar concentrations, IP6 showed little
effect on osteoblast viability, proliferation and differentiation. Thus,
IP6 did not affect the ability of osteoblasts to proliferate, to assemble a
collagenous matrix and to subsequently express alkaline phosphatase
and other late markers of osteoblast differentiation. However, our
results demonstrated that IP6 acted to increase levels of osteopontin, a
potent proteinaceous inhibitor of mineralization. In vivo, osteopontin
is expressed and accumulates both in bone and at sites of pathologic
mineralization where it is believed to regulate and limit, respectively,
tissue mineralization [20]. Osteopontin levels in osteoblast cells are
known to be regulated by high levels of phosphate (10 mM) and
pyrophosphate (0.5 mM) [45,46]. In the present study, osteopontin
levels were increased by 4 μM IP6 whereas controls using equimolar
concentrations of phosphate (24 μM) failed to increase osteopontin.
This result suggests that osteopontin up-regulation by IP6 occurs via a
mechanism different from phosphate and does not release phosphate
by IP6 dephosphorylation.
The mechanism by which IP6 induces osteopontin expression
remains unknown. IP6 has been shown to activate the protein kinase
C pathway in insulin-secreting cells [48] and the protein kinase C
pathway is upstream of osteopontin gene expression [45]. Despite
this, protein kinase C inhibitors failed to inhibit osteopontin induction
by IP6 suggesting an alternative mechanism of action.
Considering the highly-charged nature of IP6, it is unlikely that IP6
can passively diffuse across the cell membrane. Despite this, rapid
uptake of exogenous IP6 by various cancer cell lines has been
demonstrated in vitro [54]. The mechanisms of this uptake are not
entirely clear, although pinocytosis and endocytosis have been
proposed in these studies [54,55]. A related inositol phosphate, IP3,
has also been shown to travel across cells via gap junctions [56]. The
effects of IP6 could potentially also be mediated by an as yet
unidentified receptor.
IP6 has intriguing chemical properties in terms of charge,
modulation of charge by dephosphorylation and chelation ability,
and it has been implicated in a wide variety of biologic processes.
Therefore, the use of appropriate controls [14] is important to assess
the physiologic relevance of any observed effects. In this study, we
show that IP6, in the presence of physiological calcium concentra-
tions, inhibits mineralization and up-regulates osteopontin. The ion
1106 W.N. Addison, M.D. McKee / Bone 46 (2010) 1100–1107
Fig. 6. Specificity of inositol hexakisphosphate (IP6) action in MC3T3-E1 osteoblast
cultures. (A) von Kossa staining for mineral (bottom panel) and calcium quantification
after 12 days of treatment with the indicated concentrations of either IP6 or inositol
trisphosphate (IP3). (B) Cultures were treated with the indicated dose of either IP6 or
IP3 for 48 h, and protein was extracted and analyzed by Western blotting using anti-
osteopontin (OPN) and anti-GAPDH antibody. Band quantification is expressed as the
means of the integrated densities obtained using Quantity One software normalized to
the GAPDH bands. Data are presented as the means ± SE. ⁎p b 0.05 from Student's t-test
relative to the 0 μM treatment.
chelator EDTA, and the similarly highly charged inositol hexakis-
sulphate, did not imitate the effect of IP6 on mineralization and
osteopontin levels. Inositol and phosphate alone also had no effect.
IP3, a lower inositol phosphate, was less potent than IP6 at inhibiting
mineralization and did not up-regulate osteopontin at similar doses.
These data collectively suggest that the action of IP6 is specific and not
simply the result of its ion-chelating or high charge density. The
observation that IP3 is a less potent mineralization inhibitor than IP6,
however, indicates that the phosphate residues are indeed important
to the mechanism by which extracellular matrix mineralization is
inhibited.
The fact that inositol hexakis-sulphate had no effect on mineral-
ization in our bone cell cultures differs from observations where
inositol hexakis-sulphate impaired enamel formation [57] and
inhibited casein kinase I and II phosphorylation of dentin proteins
[58] in a tooth organ culture system. However, the 4 μM concentration
used in our work is much less than the 100 μM concentration used in
the tooth organ experiments. The discrepancy could also simply be
attributable to a difference in experimental systems used (i.e.,
osteoblast cell culture versus tooth germ cultures). Finally, kinases
other than casein kinase I and II present in osteoblasts, such as Golgi
kinase [59] and casein kinase-like ectokinases [60], could be
compensating for any casein kinase inhibition by inositol hexakis-
sulphate.
In undifferentiating osteoblasts IP6 caused a slight reduction in cell
proliferation. These observations could be related to the anti-
proliferative effects of IP6 observed in certain cancer cell lines [61]
which serve as the basis for a therapeutic role of IP6 in cancer therapy
[62].
In summary, our in vitro observations on the effects of IP6 on
osteoblast cultures indicate that IP6 may regulate physiologic bone
mineralization by binding to hydroxyapatite crystals through nega-
tively charged phosphate groups, and/or by serving as a specific signal
at the cellular level for the regulation of osteopontin gene expression—
another potent mineralization inhibitor. With the list of ubiquitous,
physiologic inhibitors of bone mineralization rapidly expanding,
additional information is required about their interactions and relative
contributions to the mineralization process. Furthermore, if indeed
systemic levels of IP6 may be safely and adequately manipulated by
dietary intake, then further investigation is needed to determine
potential therapeutic benefits of this approach for the management of
mineralization disorders.
Acknowledgments
These studies were funded by a grant from the Canadian Institutes
of Health Research (CIHR; MT11360 to M.D.M.). M.D.M. is a member
of the Jamson T.M. Wong Laboratories for Calcified Tissue Research of
the Centre for Bone and Periodontal Research. W.N.A. was the
recipient of a studentship from the CIHR Training Grant in Skeletal
Health Research.
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