The e�ect of bone morphogenetic protein-7 on theexpression of type I inositol 1,4,5-trisphosphate receptor inG-292 osteosarcoma cells and primary osteoblast cultures

Peter G. Bradforda,*, Jodi M. Maglicha, Alfred S. Ponticelli b,Keith L. Kirkwoodc

aDepartment of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, State University of New York at

Bu�alo, Bu�alo, NY 14214-3000, USAbDepartment of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Bu�alo, Bu�alo, NY

14214-3000, USAcDepartment of Periodontics, School of Dental Medicine, State University of New York at Bu�alo, Bu�alo, NY 14214-3000, USA

Received 6 April 1999; accepted 7 August 1999

Abstract

Bone morphogenetic protein-7 (BMP-7) a�ects di�erentiation of preosteoblasts enabling the resultant cells torespond optimally to acutely acting regulators. As the phosphoinositide cascade and, particularly, the calcium-

mobilizing inositol 1,4,5-trisphosphate (InsP3) receptor are integral to stimulus±secretion coupling in osteoblasts, thehypothesis that BMP-7 a�ects InsP3 receptor expression was examined in the G-292 human osteosarcoma cell lineand in primary cultures of human osteoblasts. G-292 osteosarcoma cells were found to be a valid experimental

model for primary human osteoblasts, expressing osteoblastic mRNAs encoding osteocalcin, bone sialoprotein,alkaline phosphatase, a1-collagen, epidermal growth-factor receptor, and BMP type II receptor. When cultured longterm in the presence of ascorbic acid and b-glycerophosphate, G-292 cells underwent further osteoblastic

di�erentiation, forming nodules and exhibiting restricted mineralization. G-292 cells responded to BMP-7 with anincrease in InsP3 receptor density. Ligand-binding studies established that BMP-7 (50 ng/ml) treatment of G-292cells increased InsP3 receptor density 2.4-fold with no apparent change in a�nity. Immunoblot analysis with

antibodies speci®c for type I, type II, and type III InsP3 receptors revealed that BMP-7 (50 ng/ml) treatmentresulted in a speci®c increase (20628%) in the type I receptor. Reverse transcription-polymerase chain reaction andNorthern blot analyses of G-292 and primary human osteoblasts con®rmed an increase in type I InsP3 receptormRNA upon BMP-7 treatment. These results demonstrate that G-292 cells respond to BMP-7 with an increase

InsP3 receptor density, consistent with the enhanced capacity of these cells to respond to Ca2+-mobilizing secretoryhormones during osteoblast di�erentiation. # 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Bone morphogenetic protein-7; Inositol 1,4,5-trisphosphate receptor; G-292 osteosarcoma; Bone morphogenetic protein

receptor; Osteocalcin

Archives of Oral Biology 45 (2000) 159±166

0003-9969/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PII: S0003-9969(99 )00122-3

www.elsevier.com/locate/archoralbio

* Corresponding author. Fax: +1-716-829-2801.

E-mail address: pgb@acsu.bu�alo.edu (P.G. Bradford).

Abbreviations: InsP3, inositol 1,4,5-trisphosphate; BMP-7, bone morphogenetic protein-7; TGF, transforming growth factor;

GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction; SDS±PAGE

sodium dodecyl sulphate±polyacrylamide gel electrophoresis.

1. Introduction

Inositol 1,4,5-trisphosphate mediates the calcium-

mobilizing e�ects of secretogogues such as hormones,

cytokines, and neurotransmitters (Berridge, 1997).

Agonist activation of cell-surface receptors stimulates

phosphatidylinositol-speci®c phospholipases C, with

resultant formation of inositol trisphosphate. Inositol

trisphosphate binds to speci®c receptors located pri-

marily on the endoplasmic reticulum, causing the

release of sequestered Ca2+ into the cytosol (Furuichi

and Mikoshiba, 1995). Elevated cytosolic Ca2+, in

turn, a�ects diverse cellular processes including, most

notably, secretion. There are three human genes encod-

ing distinct full-length inositol trisphosphate receptors,

termed types I, II, and III (Mignery et al., 1990;

SuÈ dhof et al., 1991; Blondel et al., 1993; Yamada et

al., 1994; Yamamoto-Hino et al., 1994).

The dynamic nature of inositol trisphosphate recep-

tor expression and its signi®cance to cellular physi-

ology have been recently revealed. Chronic regulators

of cell function a�ect the level of receptor expression,

which in turn determines the amplitude of the se-

cretory response to acute stimuli (Bradford et al.,

1992, 1993; Berridge, 1997). Treatment of human pro-

myelocytic HL-60 cells with retinoic acid or vitamin

D3 increases type I inositol trisphosphate receptor gene

expression, resulting in a greater density of cellular

inositol trisphosphate receptors, increased inositol

trisphosphate-dependent Ca2+ mobilization, and

enhanced secretory capacity in response to acute stimu-

lators. Similar hormonal regulation of inositol trispho-

sphate receptor expression has been shown in bone-

forming osteoblasts: exposure to 17b-oestradiol down-regulates type I inositol trisphosphate receptor gene ex-

pression and mRNA, and this is accompanied by a

reduced capacity of osteoblasts to secrete interleukin 6

(Kirkwood et al., 1996, 1997).

Bone morphogenetic proteins regulate cell growth

and di�erentiation and have speci®c a�ects on

osteoblasts (Hogan, 1996; Urist, 1997; Massague,

1998). Recombinant BMP-7 stimulates di�erentiation

of osteoblast progenitors and, in primary osteoblastic

cell cultures and in osteosarcoma cells, BMP-7 induces

the expression of osteoblastic markers including the

secretory proteins alkaline phosphatase and osteocalcin

(Sampath et al., 1992; Asahina et al., 1993, 1996; Li et

al., 1996). In considering the central role of the inositol

trisphosphate receptor in secretion, we have now

investigated the potential regulation by BMP-7 of

inositol trisphosphate receptor expression in G-292

human osteosarcoma cells and primary human osteo-

blasts.

2. Materials and methods

2.1. Cell culture

G-292 human osteosarcoma cells (ATCC CRL 1423)were cultured as described previously (Kirkwood et al.,

1996) using McCoy's 5A with 10% dialysed fetalbovine serum (Sigma Chemical Co., Lot No.96H9300), 2 mM glutamine, 100 U/ml penicillin, and100 mg/ml streptomycin at 378C in humidi®ed atmos-

phere of 5% CO2±95% air. For RNA extraction andmembrane preparation, cells were seeded at 2 � 104/cm2, cultured to approx. 80% con¯uence and then

treated with various agents as indicated before harvest-ing. For di�erentiation analyses, cells were maintainedat con¯uence in the presence of 50 mg/ml ascorbic acid

and 10 mM b-glycerophosphate for 2±8 weeks. Forassessment of mineralization, cells were stained with0.2% Alizarin Red S as described by Bodine et al.(1996). Primary cultures of human osteoblasts were

prepared by mechanical disruption and sequential col-lagenase digestion of bone explants retrieved fromimpacted maxillary third molar sites essentially as

described by Shlossman et al. (1982). The explantswere provided by Dr Richard Hall of the Departmentof Oral and Maxillofacial Surgery at SUNY Bu�alo

(IRB M1270-11). Primary osteoblastic cultures weremaintained and processed as described for G-292 cells.

2.2. Inositol 1,4,5-trisphosphate binding assays

Binding assays were performed as described by

Bradford et al. (1992). G-292 cells were harvested,washed in phosphate-bu�ered saline, and then hom-ogenized in ice-cold bu�er [100 mM KCl, 20 mMNaCl, 1 mM MgSO4, 5 mM HEPES (pH 7.2), 1 mM

phenyl methylsulphonyl ¯uoride, and protease inhibi-tor cocktail (P8340; Sigma)]. The homogenate was cen-trifuged (1000 g, 10 min) to remove intact cells and

nuclear debris. Membranes in the supernatant weresedimented by centrifugation (100,000 g, 30 min) andresuspended at 5±10 mg of protein/ml in 50 mM Tris,

pH 8.2, 1 mM EDTA bu�er and used directly in bind-ing assays, immunoblotting, or stored at ÿ808C.Steady-state [3H]inositol trisphosphate binding tomembranes was measured by vacuum ®ltration.

Membranes (0.2±0.5 mg protein) were added to bu�ercontaining 3±60 nM [3H]inositol trisphosphate (21 Ci/mmol; Dupont NEN) in a ®nal volume of 500 ml.Mixtures were incubated with shaking at 48C for40 min, diluted rapidly with 4 ml of ice-cold Tris±EDTA bu�er, and then ®ltered under vacuum through

glass-®bre ®lters (Whatman GF/B). Analysis of thisprocedure has shown that membranes are quantitat-ively retained (>95%) on these ®lters. The amount of

P.G. Bradford et al. / Archives of Oral Biology 45 (2000) 159±166160

[3H]inositol trisphosphate retained on the ®lters wasdetermined using a Wallac 1409 liquid scintillation

counter. Non-speci®c binding was determined in thepresence of 10 mM inositol trisphosphate (Calbiochem-Novabiochem Corp.). Data were analysed by

Scatchard plots to determine the KD and Bmax for[3H]inositol trisphosphate binding.

2.3. Immunoblotting

Type I, II, and III inositol trisphosphate receptor ex-pression in G-292 cells was analysed by immunoblot-

ting using type-speci®c antibodies. G-292 membraneproteins (20±50 mg) were separated by SDS±PAGE(6%), transferred to nitrocellulose membranes, and

probed with speci®c antibodies raised against C-term-inal tail peptides of the rat types I, II, or III inositoltrisphosphate receptor. The antibodies are type speci®c

and cross-react with human inositol trisphosphatereceptors (Wojcikiewicz, 1995). The antibodies weregraciously provided by Dr Richard J. Wojcikiewiczof SUNY Health Sciences Center. Antibody±protein

complexes were detected using peroxidase-linked anti-rabbit immunoglobulin and ECL reagent (AmershamLife Science). Reactions were quanti®ed using

Chemiluminescence Imaging System (BioRad) or scan-ning densitometry.

2.4. RNA isolation, RT-PCR, and Northern analysis

Isolation of total cellular RNA by guanidine thio-

cyanate extraction and CsCl ultracentrifugation andthe RT-PCR procedures were performed as described

by Kirkwood et al. (1996). Speci®c primers were syn-thesized by Bio-Synthesis, Inc (Lewisville, TX) andGenosys Biotechnologies, Inc. (The Woodlands, TX).

PCR products were analysed by agarose gel electro-phoreses and their authenticity was assessed bySouthern blot analysis and by DNA sequencing after

cloning into pGEM-11Zf(+) (Promega, Madison, WI).Total RNA (15±30 mg) was used for Northern blotanalysis as described by Kirkwood et al. (1996). Blots

were reacted with 32P-labelled probes speci®c for inosi-tol trisphosphate receptor types, a1-collagen, bone sia-loprotein, or GAPDH. Quanti®cations of hybridizedprobe were made using Bio-Rad Phosphor Imaging

and Molecular Imager Systems, version 1.4.

3. Results

3.1. G-292 cells provide an experimental model systemfor human osteoblasts

3.1.1. Extracellular matrix deposition and nodule

formation in G-292 cell culturesThe G-292 cell line was derived from a human

osteosarcoma. To determine whether the cell line was a

valid model for human osteoblasts, we have performedseveral cellular and molecular analyses. G-292 cellsproliferate readily but can be induced to di�erentiate

under speci®c culture conditions. Fig. 1 shows photo-micrographs of human G-292 cells and cultures of pri-mary osteoblasts. In Fig. 1 (upper left), G-292 cells

were cultured for 21 days at con¯uence in McCoy'smedia containing 10% dialysed fetal bovine serum.These cells exhibited noticeable deposition and accre-tion of osteoid and extracellular matrix protein, typi-

®ed by the ®brillar nature of secreted proteins. In Fig.1 (lower panels), cells were cultured for 8 weeks butsupplemented with 50 mg/ml ascorbic acid and 10 mM

b-glycerophosphate. Ascorbic acid and b-glycero-phosphate are required for osteoblast nodule forma-tion and mineralization (Stein and Lian, 1993).

Examination of the these cells revealed clear indices ofosteoblastic di�erentiation including nodule formationand limited but positive staining for mineralizationwith Alizalin Red S, a marker of matrix calcium depo-

sition (not shown). Cultures of primary human man-dibular cells used in this study are shown in Fig. 1(upper right).

3.1.2. Osteoblast selective RNA expression pattern byG-292 cells

Molecular and biochemical assessments of G-292cells further supported their characterization as osteo-blastic. G-292 cells expressed osteoblast selective

Fig. 1. Photomicrographs of G-292 osteosarcoma cells and

primary cultures of human mandibular osteoblasts. (Upper

left) G-292 cells cultured at con¯uence for 3 weeks in

McCoy's medium plus 10% fetal calf serum. (Upper right)

Passaged cultures of human mandibular bone cells. (Lower

panels) G-292 cells maintained at con¯uence in the presence

of 50 mg/ml ascorbic acid and 10 mM b-glycerophosphate for

8 weeks. Magni®cations are as indicated.

P.G. Bradford et al. / Archives of Oral Biology 45 (2000) 159±166 161

mRNAs, including osteocalcin, BMP type II receptor,

bone sialoprotein, and a1-collagen. Fig. 2 shows theresults of RT-PCR analyses using primer pairs speci®c

for BMP type II receptor and osteocalcin mRNAs.The major PCR products were of sizes predicted by

the choice of ampli®cation primers: 282 bp for BMPtype II receptor and 171 bp for osteocalcin. The DNA

sequences obtained from multiple clones of the BMPtype II receptor PCR product were identical to the

cDNA sequence encoding part of the kinase domaindownstream of the activating serine phosphorylation

site (Liu et al., 1995; Kawabata et al., 1995). The

sequenced osteocalcin cDNAs matched that of theoriginally reported human osteocalcin mRNA (Celeste

et al., 1986), except for six extra nucleotides whichwere found uniformly in the G-292 osteocalcin

sequence. The inclusion of these extra nucleotides(GTGCAG) adds two extra codons (gly-ala) to the 3 'end of the second exon of the osteocalcin cDNA. Thesigni®cance of this change is unknown.

Northern blot analysis demonstrated that G-292

cells expressed bone sialoprotein and a1-collagenmRNAs (Fig. 3). Bone sialoprotein, a major structural

protein of bone matrix, is speci®cally expressed by os-teoblasts during bone formation and remodelling (Kim

et al., 1994; Lekic et al., 1996). a1-Collagen, the majorinterstitial collagen of bone and a requirement for

proper mineralization, is expressed by osteoblasts andtheir precursors (Zohar et al., 1998). Hybridization

with speci®c probes for BSP and a1-collagen was

readily observed with RNAs of 3.0 kb and 5.7 kb, re-spectively, derived from G-292 cells grown to 90% cell

con¯uence (days 3±5 post-plating). In addition, immu-noblot analysis demonstrated that G-292 cells express

proteins typical of osteoblasts, including the epidermalgrowth-factor receptor, oestrogen receptor b, NF-kBp65, and AP-2 (data not shown). Taken together, theseresults support the validity of using the G-292 osteo-sarcoma cell as a model for human osteoblasts.

3.2. E�ect of BMP-7 on inositol 1,4,5-trisphosphate

receptor in G-292 cells

3.2.1. Binding studies

To investigate the potential regulation by BMP-7 ofinositol trisphosphate receptor expression in G-292cells, we determined the extent of [3H]inositol trispho-sphate binding to membranes isolated from BMP-7-

treated or untreated cells. Scatchard analysis demon-strated that 72 h of treatment with 50 ng/ml BMP-7increased inositol trisphosphate receptor number with-

out a change in binding a�nity (Table 1). In four inde-pendent experiments, BMP-7 increased the bindingBmax 2.4-fold (239231% of control, 21.0±50.2 fmol/

mg), whereas the calculated KD remained essentiallyunchanged (4 vs 5 nM). These results suggest thatBMP-7 treatment of G-292 cells increases inositol

trisphosphate receptor density.

3.2.2. Immunoblot analyses with inositol trisphosphatereceptor type-speci®c antibodies

Three distinct inositol trisphosphate receptors (typesI, II, and III) have been described and shown by RT-PCR to be expressed in osteoblasts and osteosarcoma

cell lines (Kirkwood et al., 1996). To determine whichof the receptor types are increased upon BMP-7 treat-ment, immunoblot analysis was performed utilizing

membrane protein preparations and speci®c antibodiesagainst types I, II, and III inositol trisphosphate recep-

Fig. 2. G-292 osteosarcoma cells expressing BMP type II

receptor and osteocalcin mRNAs. RT-PCR products speci®c

for BMP type II receptor (lane 2, 282 bp) and osteocalcin

(lane 3, 176 bp) were analysed by agarose gel electrophoresis

alongside standard-sized marker DNAs (lane 1). Sizes of the

standard DNAs are shown in bp on the margin.

Fig. 3. Northern blot analyses showing expression of a1-col-lagen, bone sialoprotein, and GAPDH by G-292 cells. Shown

are hybridizations of G-292 RNAs of 5.7, 3.0, and 1.8 kb

with probes for a1-collagen, bone sialoprotein, and GAPDH,

respectively.

P.G. Bradford et al. / Archives of Oral Biology 45 (2000) 159±166162

tors. As shown in Fig. 4 and summarized in Table 2,treatment of G-292 cells with 50 ng/ml BMP-7 for 72 hresulted in an approximate 2-fold increase (2.06 � ,

P < 0.001) in type I inositol trisphosphate receptorprotein, with no signi®cant changes in types II and III.

3.3. E�ect of BMP-7 on type I inositol trisphosphatereceptor mRNA in G-292 cells and human osteoblasts

3.3.1. Type I inositol trisphosphate receptor mRNAanalysis by RT-PCR and Northern blotHaving demonstrated an increase in type I inositol

trisphosphate receptor protein upon BMP-7 treatment,we utilized RT-PCR and Northern blot to determinethe steady-state amounts of type I inositol trispho-sphate receptor mRNA in G-292 cells as well as in pri-

mary cultures of human osteoblasts in the presenceand absence of BMP-7. By semiquantitative RT-PCRwith normalization to ampli®ed GAPDH, BMP-7

caused an approx. 5-fold increase in type I inositoltrisphosphate receptor mRNA (data not shown).Northern blot analyses con®rmed these results.

Treatment with 50 ng/ml BMP-7 for 48 h increasedtype I inositol trisphosphate receptor mRNA 3-fold inG-292 cells (2.820.5, mean2SEM, n= 3) and in

human primary osteoblasts (2.8±3.1, range of n = 2)when normalized to GAPDH concentrations (Fig. 5).

4. Discussion

BMP-7 induces osteogenesis in vivo (Sampath et al.,1992) and osteoblastic di�erentiation of uncommitted,

clonal, embryonic mesenchymal progenitor cells in cell

culture (Asahina et al., 1996). Depending on theirstage of di�erentiation, BMP-7 has multiple e�ects on

osteoprogenitor cells (Asahina et al., 1993) and com-

mitted preosteoblastic cell lines, such as MC3T3-E1cells, including the upregulation of the secretory pro-

ducts osteocalcin, bone sialoprotein, and alkaline phos-

phatase (Li et al., 1996). The present studies were

conducted to determine whether BMP-7 could alsoregulate the expression of the inositol trisphosphate

receptor as a potential mechanism of enhancing the se-

cretory capacity in G-292 osteosarcoma cells and os-teoblasts.

The human G-292 osteosarcoma cells selected for

these studies exhibit an osteoblastic phenotype and

provide a good model system for signal-transduction

Fig. 4. Immunoblot analyses of G-292 proteins with anti-

bodies speci®c for types I, II, and III inositol trisphosphate

(InsP3) receptor protein. Membrane proteins (35 mg) from G-

292 cells untreated or treated for 72 h with 50 ng/ml BMP-7

were separated by 6% SDS±PAGE, transferred to nitrocellu-

lose, and reacted with type-speci®c antibodies to the InsP3

receptor. Rat brain membrane protein was included in the

analyses as a control.

Table 2

E�ect of BMP-7 treatment on inositol trisphosphate (InsP3) receptor protein concentrations in G-292 cellsa

InsP3 receptor protein

(Percent of untreated G-292 Cells)

Treatment Type I Type II Type III

BMP-7 206.428.5b 110.324.7 96.823.1

(50 ng/ml) (n= 7) (n= 3) (n= 3)

a Binding of antibodies speci®c for either type I, II, or III InsP3 receptor protein was detected with horseradish peroxidase-

coupled secondary antibody and the ECL reagent. Values are InsP3 receptor protein concentrations in BMP-7 treated cells

expressed as a percent of concentrations in untreated cells. Values are means2SEMs for the indicated number of di�erent mem-

brane preparations.b P< 0.001 vs untreated.

Table 1

E�ect of BMP-7 treatment on KD and Bmax of [3H]inositol

trisphosphate binding to G-292 membranesa

G-292 cell treatment KD (nM) Bmax (fmol/mg)

Control 422 21.023.6

BMP-7 (50 ng/ml) 522 50.226.5b

a Values are means2SEMs from four independent exper-

iments.b P< 0.001 vs control.

P.G. Bradford et al. / Archives of Oral Biology 45 (2000) 159±166 163

analysis. Under long-term continuous culture con-

ditions with ascorbic acid and b-glycerophosphate, G-

292 cells elaborated extracellular matrix and underwent

limited nodule formation and mineralization. Despitethe apparently low frequency and long time course of

nodule formation in these cultures, the fact that these

structures formed at all supports the characterizationof G-292 cells as osteoblastic. Under proliferative con-

ditions, G-292 cells expressed type I collagen, bone sia-

loprotein, and osteocalcin. These cells also expressedBMP type II receptors and responded to low physio-

logical doses of BMP-7. In regard to the expression

and secretion of bone matrix proteins, similar morpho-

logical characterizations and responsiveness to BMP-7have been described for fetal rat calvarial cells (Li et

al., 1996).

Our previous studies demonstrated that the inositol

trisphosphate receptor expression during cellular di�er-entiation controls the extent of secretion in response to

acute stimuli (Bradford et al., 1992, 1993) and that

chronic regulators of osteoblast biology a�ect inositol

trisphosphate receptor gene expression (Kirkwood etal., 1996, 1997). In light of the fact that BMP-7 a�ects

the extent of bone matrix protein deposition by osteo-

blasts, it was reasonable to hypothesize that inositoltrisphosphate receptor expression may be regulated by

BMP-7.

Using speci®c antisera reactive against each of the

three principal types of inositol trisphosphate receptor

protein, we demonstrated that G-292 cells express allthree receptor types, con®rming our previous RT-PCR

analyses (Kirkwood et al., 1996). In addition to the

major immunoreactive inositol trisphosphate receptorspecies that are also readily detectable in rat brain, G-

292 cells express minor species of all three receptor

types for inositol trisphosphate. The inositol trispho-sphate receptor is a post-translationally modifed, and

these minor, higher molecular-weight species may rep-

resent glycosylated receptor forms. Using the rat brain

membranes as a standard, the relative amounts oftypes I, II, and III inositol trisphosphate receptor pro-tein in G-292 cells were approx. 5%, 60%, and 16-

fold, respectively, that of rat brain. Type I inositoltrisphosphate receptor is heavily expressed in brain, es-pecially in the Purkinje cells of the cerebellum, where it

may constitute 1% of the cellular protein (Furuichi etal., 1989). The amount of inositol trisphosphate recep-

tor in rat brain is 18 ng/10 mg microsomal protein andthe relative abundance of the three types is 9621%,421%, and 020%, respectively (Wojcikiewicz, 1995).

Thus, it may be approximated that G-292 cells primar-ily express type I inositol trisphosphate receptor pro-

tein, as had been determined for RNA (Kirkwood etal., 1996).It is interesting that the e�ect of BMP-7 is to

increase type I inositol trisphosphate receptors selec-tively. The approximate doubling of the steady-stateprotein level is consistent with the 2.8-fold increase in

type I inositol trisphosphate receptor mRNA observedafter BMP-7 treatment. The physiological importance

of a 2-fold increase in type I inositol trisphosphatereceptor protein is at present unknown. Importantly,however, similar hormone-dependent increases (3-fold)

in inositol trisphosphate receptors levels in HL-60 cellscorrelate with a 2- to 3-fold increase in inositol

trisphosphate-stimulated intracellular Ca2+ mobiliz-ation, and a 5- to 10-fold increase in secretory responseto acute stimulators (Bradford et al., 1992). The signi®-

cance of such regulation has also been highlighted bythe observation that small groups of inositol trispho-sphate-regulated Ca2+-release channels work as el-

emental units to adjust the ``amplitude'' of the Ca2+

response in a given cell (Berridge, 1997; Bootman et

al., 1997). Depending on the concentration of Ca2+

release channels and the resultant amplitude of theCa2+ signal, gradations in cellular responses are eli-

cited, including gradations in secretion (Dolmetsch etal., 1997; Tse et al.,1997).

The regulation by BMP-7 of inositol trisphosphatereceptor expression is signi®cant to the biology of os-teoblasts. The receptor is essential in transducing phos-

phoinositide turnover, in dictating the extent ofintracellular calcium mobilization, and in accommodat-ing secretion (Berridge, 1997). Many acute stimulators

of osteoblast secretion, including parathyroid hor-mone, thrombin, endothelins, bradykinin, and some

prostaglandins signal through the inositol trispho-sphate pathway. Our data show that chronic ex-pression of the type I inositol trisphosphate receptor

gene is hormonally regulated in osteoblasts. This regu-lation results in a corresponding changes in the densityof microsomal inositol trisphosphate receptors, in ino-

sitol trisphosphate-dependent Ca2+ mobilization, and,perhaps most signi®cantly, in secretory capacity in re-

Fig. 5. Northern blot analyses showing increase in steady-

state type I inositol trisphosphate (InsP3) receptor mRNA in

G-292 cells and human primary osteoblasts (HOST) after 48 h

treatment with 50 ng/ml BMP-7. Blots of 20 mg total RNA

from cells treated (+) or not treated (ÿ) with BMP-7 were

hybridized with 32P-labelled type I inositol trisphosphate

receptor cDNA probe and exposed to phosphorimaging. Blots

were also reacted with probe for GAPDH to control for

RNA loading. Results are representative of two (HOST) and

three (G-292) independent experiments.

P.G. Bradford et al. / Archives of Oral Biology 45 (2000) 159±166164

sponse to acute cell stimulators. Thus, in preosteoblas-tic cells and osteoblasts, BMP-7 sensitizes the cellular

signal-transduction capacity and allows for enhancedsecretion of matrix proteins essential for bone for-mation. This information points to new targets for the

design and development of osteoprotective drugs.

Acknowledgements

BMP-7, also known as osteogenic protein-1, wasgenerously provided by Dr David Rueger of Creative

Biomolecules, Inc., Hopkinton, MA 01748, USA.Assistance with the handling of BMP-7 was providedby Dr Dennis Higgins of the State University of New

York at Bu�alo.

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