Parathyroid hormone-related peptide interacts with bone morphogenetic protein 2 to increase...

Parathyroid Hormone-Related Peptide Interacts withBone Morphogenetic Protein 2 to IncreaseOsteoblastogenesis and Decrease Adipogenesis inPluripotent C3H10T1⁄2 Mesenchymal Cells

GEORGE K. CHAN, DENGSHUN MIAO, RON DECKELBAUM, ISABEL BOLIVAR,ANDREW KARAPLIS, AND DAVID GOLTZMAN

Calcium Research Laboratory, McGill University Health Center (G.K.C., D.M., I.B., D.G.), Lady Davis Research Institute-Jewish General Hospital (R.D., A.K.), and Department of Medicine, McGill University (G.K.C., D.M., R.D., I.B., A.K., D.G.),Montreal, Quebec, Canada H3A-1A1

We examined the effect of PTH-related peptide (PTHrP) onmodulating adipogenesis and osteoblastogenesis in the plu-ripotent mesenchymal cell line C3H10T1⁄2. These cells expressthe type 1 PTH/PTHrP receptor, thereby allowing PTHrP toinhibit bone morphogenetic protein 2 (BMP2) from enhancinggene expression of peroxisome proliferator-activated recep-tor � and the adipocyte-specific protein aP2 and from aug-menting the accumulation of lipid. In the presence of BMP2,PTHrP or a protein kinase C (PKC) stimulator (phorbol ester)increased the expression of indexes of the osteoblast pheno-type, including alkaline phosphatase, type I collagen, and os-teocalcin, whereas a PKC inhibitor (chelerythrin chloride)inhibited PTHrP action. PTHrP and a phorbol ester increased

gene expression of the BMP IA receptor, and both enhancedBMP2-dependent increases in promoter activity of the signal-ing molecule SMAD6. Overexpression of the BMP IA receptorfacilitated the capacity of BMP2 to increase osteoblastogen-esis in the absence of PTHrP and a dominant negative BMP IAreceptor variant inhibited this effect of BMP2. These resultsdemonstrate that PTHrP can direct osteoblastic, rather thenadipogenic, commitment of mesenchymal cells, implicate PKCsignaling in this activity, and show that PTHrP action in-volves enhanced gene expression of the BMP IA receptor,which facilitates BMP2 action in enhancing osteoblastogen-esis in pluripotent mesenchymal cells. (Endocrinology 144:5511–5520, 2003)

OSTEOBLASTS, THE PRINICIPAL bone-forming cells,and adipocytes, the main fat-storing cells, are be-

lieved to originate from the same pluripotent mesenchymalstem cells (1). Both cell types also contribute to the architec-tural structure of bone, and a decrease in bone volume isgenerally accompanied by an increase in adipocyte numberwithin the bone marrow (2). This reciprocal relationship isexemplified in the development of age-related osteopenia.Thus, bone marrow at a very early age is virtually devoid ofadipocytes; however as aging progresses, a decrease in bonevolume occurs with a reciprocal increase in fat depositswithin the marrow (2, 3). One hypothesis to explain thesephenomena is that the reduced number of osteoblasts and theincreased number of adipocytes are the result of increasingcommitment of pluripotent mesenchymal cells along the adi-pocytic, as opposed to the osteoblastic, lineage (4). Regula-tion of this process may involve bone morphogenetic pro-teins (BMPs). BMPs are expressed within the bone marrowstroma and are the only known morphogens that are able toinduce both adipogenesis and osteogenesis of pluripotentmesenchymal cells (5). BMP2, BMP4, and BMP7 have all beenshown to be effective inducers of both osteoblast and adi-

pocyte commitment in vitro; however, the cell lineage that isspecified is often determined by the concentration of appliedBMP. BMP family members may therefore serve to activatetranscription of distinct target genes in a dose-dependentmanner, leading to distinct cellular phenotypes. BMPs act bybinding to a type I and a type II receptor. BMP binding to thetype II receptor enhances the affinity for the type I receptor,and heterodimerization of the two receptors follows. Onceheterodimerization takes place, the type II receptor trans-phosphorylates and activates the type I receptor. It is the typeI receptor that contains the functional kinase domain that isessential for serine phosphorylation and activation of down-stream SMAD transcription factors (6).

PTH and PTH-related peptide (PTHrP) analogs have beenshown to effectively induce bone formation both in vivo andin vitro (7). Both peptides interact at a common G protein-coupled receptor, termed the type I PTH/PTHrP receptor(PTHR), which is linked to the adenylyl cyclase/protein ki-nase A (PKA) signaling system and the phospholipaseC/protein kinase C (PKC) systems (8). Several mechanismshave been suggested for the actions of PTH and PTHrP onincreasing bone formation. These include proliferation ofosteogenic progenitor cells (9), enhancing osteoblast differ-entiation (10), and inhibiting apoptosis of osteoblastic cells(11). PTH has also been shown to enhance the transcriptionalactivity of the osteoblast differentiation factor core bindingfactor A1 (CBFA1) via phosphorylation through an adenylylcyclase-PKA-dependent mechanism (12).

Abbreviations: ALP, Alkaline phosphatase; BMP2, bone morphoge-netic protein 2; CBFA1, core binding factor A1; Ihh, Indian hedgehog;N-Shh, N-terminal sonic hedgehog; PKA, protein kinase A; PKC, proteinkinase C; PPAR�, peroxisome proliferator-activated receptor �; PTHR,PTH receptor; PTHrP, PTH-related peptide; Shh, sonic hedgehog; TPA,12-O-tetradecanoylphorbol-13-acetate.

0013-7227/03/$15.00/0 Endocrinology 144(12):5511–5520Printed in U.S.A. Copyright © 2003 by The Endocrine Society

doi: 10.1210/en.2003-0273

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We recently demonstrated that PTHrP is able in vitro toinhibit the terminal differentiation of committed preadipo-cytes by stimulating MAPK, which, in turn, can phosphory-late and down-regulate the adipogenic determining factorperoxisome proliferator-activated receptor � (PPAR�) (13).We also reported that in vivo PTHrP heterozygous null micethat are haplo-insufficient for PTHrP develop a prematureform of osteopenia, characterized by reduced trabecularbone volume and increased bone marrow adiposity (14). Inthe current studies we investigated whether we could dem-onstrate in vitro that PTHrP might play a role in directingmesenchymal cell differentiation toward the adipocytic orosteoblastic lineage. For these studies we employed the plu-ripotent mesenchymal cell line C3H10T1⁄2. These cells arederived from mouse embryo connective tissue and can beinduced to differentiate along several mesenchymal cell lin-eages (5, 15). When treated with low concentrations of BMP2,C3H10T1⁄2 cells differentiate along the adipocytic lineage;however, with higher concentrations of BMP2, osteogenesisis enhanced (16).

We therefore employed C3H10T1⁄2 cells to study the effectof PTHrP on inducing the osteoblast phenotype in these cells.Our studies show that in this system PTHrP can inhibit thecapacity of BMP to increase adipogenesis and can facilitatethe action of BMP to direct differentiation toward the osteo-blastic lineage. The PKC pathway is implicated as a signalingmechanism in this process, and the PTHrP effect involvesup-regulation of the BMP IA receptor, which enhances sen-sitivity to BMP and favors development of the osteoblastic,rather than the adipocytic, lineage.

Materials and MethodsCell culture and cell transfections

C3H10T1⁄2 clone 8 cells were obtained from American Type CultureCollection (Manassas, VA) and maintained in DMEM containing 10%heat-inactivated fetal calf serum. Fresh medium was applied every sec-ond day. To induce osteogenesis, cells were grown to confluence thenmaintained in �MEM supplemented with ascorbic acid (100 �g/ml),�-glycerophosphate (5 mm), and BMP2 (Research Diagnostics, Inc.,Flanders, NJ; 6 � 10�9 m) unless stated otherwise. Fresh medium wasapplied every 2 d. When required, PTHrP-(1–34) was added at a con-centration of 1 � 10�7 m unless otherwise stated. The PKC inhibitorchelerythrin chloride (17, 18) was assessed at concentrations of 1.0 �10�5, 5.0 � 10�6, 2.5 � 10�6, 1.25 � 10�6, and 5.0 � 10�7 m. The maximalconcentration permitting cell viability, as determined by trypan blueexclusion, was 1.25 � 10�6 m. This concentration was therefore em-ployed in all subsequent experiments.

C3H10T1⁄2 cells expressing the BMP IA receptor were generated aftercloning cDNA encoding the rat BMP IA receptor (19) (provided by Dr.Gideon Rodan, Merck & Co., West Point, PA) into the pcDNA3 expres-sion plasmid. The expression plasmid was then used for stable trans-fection as previously described (13). C3H10T1⁄2 cells expressing a BMPIA dominant negative receptor (DNBMP IA) lacking kinase activity weregenerated after cloning the cDNA (provided by Dr. Gideon Rodan) intothe pcDNA3 expression plasmid. Stably transfected cells were generatedin a similar manner, and control cells were generated by stable trans-fection with empty pcDNA3 plasmid. Selection was performed using400 �g geneticin/ml (Life Technologies, Inc., Gaithersburg, MD). Alltransfections were carried out using FUGENE6 reagent (Roche, India-napolis, IN). SMAD6 luciferase reporter transformants were generatedby cotransfection of C3H10T1⁄2 cells with 5 �g of a SMAD6 promoterfused to a luciferase reporter as previously described (20) and 0.5 �gpCDNA3, due to the lack of a selection marker for the SMAD6 luciferasereporter construct. Serial dilutions were performed, and selection ofindividual clones was carried out using 400 �g geneticin/ml medium.

Northern blot analysis

Total RNA was isolated using TRIzol reagent (Life Technologies,Inc.). For Northern blot analysis, 10 �g total RNA/sample were elec-trophoresed through a formaldehyde/agarose gel. The RNA was trans-ferred to a Bio-Trans nitrocellulose membrane (Amersham PharmaciaBiotech, Arlington Heights, IL). For detection of adipocytic genes, themembrane was probed with an internal EcoRI fragment of PPAR� mousecDNA (13) and the full-length cDNA of mouse aP2 (13). For detectionof osteoblastic genes, the membrane was probed with a 1135-bp frag-ment encoding the amino terminus of mouse alkaline phosphatase(ALP) cDNA, a 1600-bp fragment of rat �1R1 collagen 1 cDNA (21), andthe entire 467-bp coding region of rat osteocalcin cDNA. For detectionof BMP IA and DNBMP IA mRNA, a 233-bp fragment of the mouse BMPIA receptor cDNA was generated by PCR (5�-CTT GGA CCA GAA GAAGCC AG-3� and 5�-CTT TCG GTG AAT CCT TGC AT-3�). Northernanalysis detection was performed using a Cyclone Storage PhosphorSystem (Packard Instrument Co., Inc., Meriden, CT). Levels of mRNAwere quantified using Image (Scion Corp., Frederick, MD) and werestandardized by comparison with 18S RNA levels that were detectedsimultaneously, as previously described (13).

BMP2-responsive luciferase assays

SMAD6 promoter-luciferase reporter assays were performed by amodification of a previously described method (20). Briefly, C3H10T1⁄2cells stably transfected with the SMAD6 promoter-luciferase reporterwere pretreated with BMP2 (6 � 10�9 m) alone for 5 d. To examine thePTHrP effects, in some culture plates PTHrP (10�7 m) was added withoutor with chelerythrin chloride (1.25 � 10�6 m) and with BMP2. To ex-amine the effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in some plates, TPA (1 �m) was added with or withoutchelerythrin chloride (1.25 � 10�6 m) and with BMP2 on the fourth dayfor 24 h. After an additional 24 h of serum deprivation, all cells werestimulated with PBS vehicle or BMP2 (6 � 10�9 m) for 24 h, and luciferaseactivities were measured.

PKA and PKC activity assay

C3H10T1⁄2 cells were incubated with BMP2 (6 � 10�9 m) for 4 d. The daybefore assays were to be performed, the cells were serum-deprived over-night. The following day, the PKA and PKC responses to PTHrP weremeasured intermittently after treatment of cells for 15 min with PTHrP-(1–34), followed by lysis. No readings were taken for d 1, as cells weretreated for a minimum of 24 h with BMP2 before serum deprivation. PKAand PKC activities were assessed with a PKA assay kit (Upstate Biotech-nology, Inc., Lake Placid, NY) and a PKC assay kit (Upstate Biotechnology,Inc.), respectively. For the PKA assay, kemptide (S6 kinase substrate;Upstate Biotechnology, Inc.) was used in place of the provided substrate.Six samples were prepared for each time point.

Oil Red O staining

After 14 d of incubation with BMP-2 (6 � 10�9 m), C3H10T1⁄2 cellswere washed twice with PBS, then fixed for 30 min with 10% formalin.Oil Red O stain (5% Oil Red O in 70% pyridine) was applied to the cellsfor 30 min, and then cells were washed three times with PBS.

ALP staining

Cytochemical staining for ALP was performed by incubating the cellsfor 15 min at room temperature in 100 mm Tris-maleate buffer containing0.2 mg/ml naphthol AS-MX phosphate (Sigma-Aldrich Corp., St. Louis,MO) dissolved in ethylene glycol monomethyl ether (Sigma-AldrichCorp.) as a substrate, and Fast Red TR (0.4 mg/ml; Sigma-Aldrich Corp.)as a stain for the reaction product.

Immunocytochemistry

Cultured cells were stained for type I collagen, osteopontin, andosteocalcin using the avidin-biotin-peroxidase complex technique asdescribed previously (9). The cells were first treated with 0.5% bovinetesticular hyaluronidase (Sigma-Aldrich Corp.) for 30 min at 37 C, fol-lowed by application of primary antibodies, affinity-purified goat an-

5512 Endocrinology, December 2003, 144(12):5511–5520 Chan et al. • PTHrP Stimulates Osteoblastogenesis

tihuman type I collagen antibody (Southern Biotechnology Associates,Inc., Birmingham, AL), goat antimouse osteocalcin (Biomedical Tech-nologies, Inc., Stoughton, MA), and PPAR� (Research Diagnostics Inc.,Flanders, NJ), overnight at room temperature. As a negative control, thepreimmune serum was substituted for the primary antibody. Afterwashing with high salt buffer (50 mm Tris-HCl, 2.5% NaCl, and 0.05%Tween 20, pH 7.6) for 10 min at room temperature, followed by two10-min washes with 50 mm Tris-HCl, 150 mm NaCl, and 0.01% Tween20, pH 7.6, the cells were incubated with a secondary antibody (biotin-ylated rabbit antigoat IgG, Sigma-Aldrich Corp.). Cells were thenwashed as before and incubated with the Vectastain ABC-AP kit (VectorLaboratories, Inc., Ontario, Canada) for 45 min. After washing as before,red pigmentation to identify regions of immunostaining was producedby a 10- to 15-min treatment with Fast Red TR/Naphthol AS-MX phos-phate (Sigma-Aldrich Corp.) containing 1 mm levamisole as endogenousALP inhibitor.

Quantification for both cytochemistry and immunocytochemistry byimage analysis was performed as previously described (9).

Computer-assisted image analysis

Computer-assisted image analysis was performed as described pre-viously (9). Briefly, images of stained culture dishes were photographedwith transmitted light over a light box. All images were processed usingNorthern Eclipse image analysis software (version 5.0, Empix Imaging,Inc., Mississauga, Canada). For determining the area of positive coloniesin cultured cells, thresholds were set using green and red channels. Thethresholds were determined interactively and empirically on the basisof three different images. Subsequently, this set threshold was used toautomatically analyze all recorded images of all sections that werestained in the same staining session under identical conditions.

Statistical analysis

Statistical analysis of cell cultures is based on three random fields.Statistical analysis was performed using a t test, Fisher’s test, orANOVA, followed by Bonferroni adjustment as appropriate. P � 0.05was taken as significant.

ResultsEvidence for functional type I PTH/PTHrP receptors(PTHR) in C3H10T1⁄2 cells

Northern analysis revealed PTHR mRNA expression inC3H10T1⁄2 cells, which remained constant throughout a 5-dincubation with BMP2 (Fig. 1, A and B). PTHrP is known tostimulate both PKA and PKC after binding to its G protein-coupled receptor (PTHR). To determine the functionality ofthe PTHR expressed in C3H10T1⁄2 cells, we determined basaland PTHrP-stimulated PKA and PKC activity over a 5-dincubation with BMP2. In the absence of PTHrP, PKA andPKC levels increased and then stabilized by d 4 (Fig. 1, C andE). In the presence of PTHrP, significant increases in bothPKA and PKC activity were observed at each time point (Fig.1, C and E). Furthermore, when assessed on d 4, PTHrPelicited a concentration-dependent increase in both PKA andPKC activities (Fig. 1, D and F).

PTHrP inhibits adipogenesis of pluripotent C3H10T1⁄2 cells

We incubated C3H10T1⁄2 cells over a 2-wk period eitherwith a concentration of BMP2 known to induce adipocytedifferentiation (16) or with the same concentration of BMP2in combination with PTHrP. BMP2-treated cultures demon-strated extensive adipogenesis as assessed by Oil Red Ostaining of the cells (Fig. 2, B and D). In contrast, markedlyreduced adipogenesis was observed in the presence of BMP2and PTHrP (Fig. 2, C and D).

To examine the mechanism of this effect, mRNA was iso-lated before cell treatment (d 0) and 7 d later from untreatedcells or cells treated with either BMP2 alone or BMP2 andPTHrP. Northern blots were performed to examine expres-sion of the adipogenic transcription factor PPAR� and thefat-specific gene aP2. BMP2 alone increased both PPAR� andaP2 expression by d 7, whereas with PTHrP treatment, areduction in both PPAR� and aP2 mRNA levels was ob-served (Fig. 2, E and F).

The expression of PPAR� was further confirmed by im-munocytochemistry of cells that were untreated (Fig. 2G) orwere incubated with either BMP2 (Fig. 2H) or BMP2 in com-bination with PTHrP (Fig. 2I). Untreated cells demonstratedsparse expression of PPAR� protein (Fig. 2, G and J), whereascells treated with BMP2 expressed abundant PPAR� protein(Fig. 2, H and J). Cells treated with both BMP2 and PTHrPshowed limited expression of PPAR� (Fig. 2, G and J). Con-sequently, PTHrP appeared capable of curtailing the adipo-genic program induced by BMP2 in these cells.

PTHrP enhances osteoblastogenesis inpluripotent C3H10T1⁄2

C3H10T1⁄2 cells were incubated over a 2-wk period withmedium alone or with either an adipogenic dose of BMP2

FIG. 1. Influence of BMP2 on PTHR expression and function inC3H10T1⁄2 cells. A, Northern analysis was performed to determine theexpression of PTHR over a 5-d incubation with BMP2 (6 � 10�9 M).18S RNA levels were assessed as a control. B, Computer-assistedquantification of Northern analysis was performed as described inMaterials and Methods. Relative levels of PTHR expression was stan-dardized vs. relative levels of 18S. C, PKA activity was measured asdescribed in Materials and Methods. Cells were incubated over 5 dwith BMP-2 (6 � 10�9 M), and enzyme activity was determined aftertreatment with BMP2 plus vehicle (control) or with BMP2 plusPTHrP (10�7 M). D, After 4 d of incubation with BMP-2, increasingconcentrations of PTHrP were added, and PKA activity was assessed.E, PKC activity was measured as described in Materials and Methodsand was determined under the same conditions as those described forPKA. F, The effect of increasing concentrations of PTHrP on PKCactivity was determined under the same conditions as those describedfor PKA. For C, D, E, and F, each bar represents the mean � SE of sixreplicates. *, P � 0.05 vs. control.

Chan et al. • PTHrP Stimulates Osteoblastogenesis Endocrinology, December 2003, 144(12):5511–5520 5513

alone or an adipogenic dose of BMP2 in combination withPTHrP. Indexes of osteoblastic differentiation were then ex-amined. Evidence of expression of ALP, type I collagen, andosteocalcin protein and mRNA were observed with PTHrPtreatment (Fig. 3, A–D), whereas only minimal expression ofthese markers was observed in cultures treated with BMP2alone (Fig. 3, A–D). Consequently, PTHrP appeared to stim-ulate osteoblastogenesis while inhibiting adipogenesis.

PTHrP acts by a PKC mechanism to increaseosteoblastogenesis and BMP IA receptor expressionand signaling

To assess whether the PKA pathway, the PKC pathway, orboth mediates the effects of PTHrP, C3H10T1⁄2 cells were

treated with low levels of BMP2 (Fig. 4). On d 4, vehicle alone(control), the PKA activator forskolin, the PKC activatorTPA, or both forskolin and TPA were added for 24 h. Afteran additional 10 d in culture, cells were examined for ex-pression of the osteoblast markers ALP, type I collagen, andosteocalcin. Treatment with forskolin had only a slight effecton the expression of these markers, whereas treatment withTPA produced dramatic increases (Fig. 4, A and B). Cellstreated with both forskolin and TPA did not show enhancedexpression as did those treated with TPA alone (Fig. 4A andB). Consequently, the PTHrP effect on osteoblastogenesisappeared to be predominantly due to a PKC-mediated re-sponse. Furthermore, there seemed to be no synergy betweenthe PKC and PKA pathways in mediating this action.

FIG. 2. Influence of BMP2 and PTHrP on adipogenesis in C3H10T1⁄2 cells. A, C3H10T1⁄2 cells were cultured for 14 d and then stained with OilRed O. B, Cells were incubated with BMP2 (6 � 10�9 M) alone for 14 d and then stained with Oil Red O as described in Materials and Methods.C, Cells were incubated with BMP2 (6 � 10�9 M) and PTHrP (10�7 M) for 14 d and then processed as described in A. Each photomicrographin A–C is representative of three random fields from three different cultures. D, Quantification of Oil Red O staining per field was performedwith Northern Eclipse software as described in Materials and Methods. Cells were untreated, treated with BMP2 alone (6 � 10�9 M; left bar),or treated with BMP2 and PTHrP (10�7 M right bar). Each bar represents the mean � SE of triplicate determinations using three random fieldsfrom three different cultures. *, P � 0.05. E, Northern blots of PPAR� and aP2 were performed as described in Materials and Methods and areshown in the upper panel. RNA was extracted from cells before treatment (d 0) and on d 7 with and without treatment. Cells were treated withBMP2 (6 � 10�9 M) alone (control) or with BMP2 plus PTHrP (10�7 M PTHrP). Numbers represent two different experiments. Levels of mRNAencoding 18S were determined as a loading control (lower panel). F, Computer-assisted quantification of Northern analysis was performed,whereby relative levels of the adipocytic markers PPAR� and aP2 were standardized vs. relative levels of 18S. G, Untreated C3H10T1⁄2 culturedcells after a 14-d incubation following analysis for PPAR� expression. H, Cells were treated with BMP2 (6 � 10�9 M) alone for 14 d and thenanalyzed for PPAR� protein expression as described in Materials and Methods. I, Cells were incubated with BMP2 (6 � 10�9 M) and PTHrP(10�7 M) for 14 d, followed by analysis for PPAR� protein expression. Photomicrographs in G–I are each representative of three random fieldsfrom three different cultures. J, Quantitation of PPAR� protein expression was performed using Northern Eclipse software as described inMaterials and Methods. C3H10T1⁄2 cells were untreated (left bar), treated with BMP2 (6 � 10�9 M) alone (center bar), or treated with BMP2and PTHrP (10�7 M right bar). Each bar represents the mean � SE of triplicate determinations using three random fields from three differentcultures. *, P � 0.05.


To further determine whether PTHrP enhances osteoblas-togenesis as a result of enhanced PKC signaling, C3H10T1⁄2cells were induced to differentiate with BMP2 alone or withBMP2 and either PTHrP alone or PTHrP and the PKC in-hibitor chelerythrin chloride. After culturing, cells werestained for the osteoblast-specific protein osteocalcin. Cellstreated with PTHrP and chelerythrin demonstrated signifi-cantly lower levels of osteocalcin expression compared withcells treated with PTHrP alone (Fig. 4, C and D). Conse-quently, the PTHrP effect on osteoblastogenesis appeared tobe due at least in part to a PKC-mediated response.

In view of the fact that even in the absence of PTHrP,

higher concentrations of BMP2 have been reported to en-hance differentiation along the osteoblast lineage (16), wenext assessed whether PTHrP might augment the sensitivityof the cells to BMP2 by amplifying its signaling. We thereforefirst examined the effect of PTHrP on expression of the BMPI receptors that transduce the BMP signal via its serine/threonine kinase activity. Cells were incubated with low(adipogenic) concentrations of BMP2 alone, with BMP2 plusPTHrP, or with BMP2 plus forskolin or TPA (added on d 4).On d 5 RNA was isolated and examined for expression of theBMP I receptors. The BMP IB receptor was undetectable byNorthern blot or RT-PCR (data not shown). The BMP IA

FIG. 3. Effects of BMP2 and of PTHrP on the osteoblastphenotype. A, C3H10T1⁄2 cells were either cultured un-treated or were incubated with BMP2 (6 � 10�9 M) aloneor with BMP2 plus PTHrP (10�7 M) and after 14 d wereexamined for the expression of ALP, type I collagen (ColI), and osteocalcin (OCN) protein as described in Mate-rials and Methods. Photomicrographs are representativeof three different fields from four different cultures. B,Expression of ALP, type I collagen, and osteocalcin ofuntreated cells or cells after treatment with BMP2 (6 �10�9 M) alone or with BMP2 and PTHrP was quantifiedusing Northern Eclipse imaging software as described inMaterials and Methods and is represented as the per-centage of the field that stained positively for each os-teoblastic marker (% staining/field). Each bar representsthe mean � SE of three random fields per culture from fourdifferent cultures. *, P � 0.05 vs. control (BMP2 alone).C, Expression of mRNA encoding ALP, type I collagen,and osteocalcin was determined in untreated cells, cellstreated with BMP2 alone, or cells treated with BMP2 andPTHrP, as described in Materials and Methods. mRNAloading was assessed by probing with 18S. D, Quantifi-cation of Northern analysis was performed to determinethe expression of osteoblastic markers in untreated cells,cells treated with BMP2 (6 � 10�9 M), and cells treatedwith BMP2 and PTHrP. Computer-assisted quantifica-tion of Northern analysis was performed as described inMaterials and Methods. Relative levels of ALP, type Icollagen, and osteocalcin expression were standardizedvs. relative levels of 18S.


receptor was detected in all four samples, and expressionwas standardized against the 18S levels of each respectivelane. Equivalent expression was observed in control cells andforskolin-treated cells. BMP IA receptor expression wasmarkedly increased, however, in both PTHrP-treated andTPA-treated cells (Fig. 5, A and B).

To determine whether the increased BMP IA receptor ex-pression was associated with increased postreceptor signal-

ing, we stably transfected C3H10T1⁄2 cells with a SMAD6promoter-luciferase reporter construct that has been dem-onstrated to be responsive to BMP2-induced signaling (20).Cells were pretreated with an adipogenic dose of BMP-2alone (control) or with BMP-2 plus PTHrP for 5 d. A third setwas pretreated with an adipogenic dose of BMP-2 alone for5 d, with TPA added to the pretreatment mixture for 24 h ond 4 to mimic PTHrP stimulation of PKC. Pretreatment with

FIG. 4. Effects of PKA and PKC signal-ing pathways on the osteoblast pheno-type. A, C3H10T1⁄2 cells were incubatedfor 14 d with BMP2 (6 � 10�9 M). On d4 of this incubation, vehicle (control),forskolin (100 �M), TPA (1 �M), or bothforskolin (100 �M) and TPA (1 �M) wereadded for 24 h. After the 14-d incuba-tion period, cells were examined for theexpression of ALP, type I collagen (Col1), and osteocalcin (OCN) as describedin Materials and Methods. Photomicro-graphs are representative of three ran-dom fields from three different experi-ments. B, Expression of the markersshown in A was quantitated usingNorthern Eclipse imaging software asdescribed in Materials and Methods.The results are represented as the per-centage of the field that stained posi-tively for each osteoblastic marker (%staining/field). Each bar represents themean � SE of triplicate fields from threedifferent experiments. *, P � 0.05 vs.control (BMP2 alone); **, P � 0.05 vs.BMP2 and forskolin treatment. C,C3H10T1⁄2 cells were incubated for 14 dwith vehicle only (Control) or withBMP2 (6 � 10�9 M) alone or in combi-nation with PTHrP (10�7 M) or PTHrPand chelerythrin chloride (1.25 � 10�6

M). After the 14-d incubation period, theexpression of osteocalcin was deter-mined by immunocytochemistry. Pho-tomicrographs are representative ofthree random fields from three differentexperiments. D, Quantitation of osteo-calcin staining was performed as de-scribed in Materials and Methods. Eachbar represents the mean � SE of threefields per culture from three separatecultures. * and **, P � 0.05 vs. BMPalone (control) and vs. BMP plusPTHrP, respectively.


PTHrP or TPA in combination with BMP-2 significantly in-creased BMP-2-stimulated SMAD6 promoter-reporter activ-ity (Fig. 5C) compared with the control. Furthermore, theeffects of PTHrP or TPA in combination BMP2 were abol-ished with the addition of the PKC inhibitor chelerythrinchloride (Fig. 5C). Consequently, BMP IA receptor signalingappeared to be enhanced by PTHrP and PKC activation.

BMP IA receptor mediates BMP2-induced osteoblastogenesis

To determine whether increased BMP IA receptor expres-sion can induce differentiation along the osteoblast lineage,

we stably transfected pluripotent C3H10T1⁄2 cells with thecDNA encoding the BMP IA receptor or with the emptyvector (pcDNA3) as a control (Fig. 6, A and B). When cellsoverexpressing the BMP IA receptor were treated with BMP2(6 � 10�9 m) in the absence of PTHrP, these cells expressedconsiderably higher levels of ALP, collagen type I, and os-teocalcin than the pcDNA3-transfected cells (Fig. 6A). Tofurther confirm the role of the BMP IA receptor in this pro-cess, we also stably transfected wild-type C3H10T1⁄2 cellswith a dominant negative form of the BMP IA receptor(DNBMP IA). These cells, BMP IA-overexpressing cells, andempty-vector transfected cells (pcDNA3) were then treatedwith a higher dose (1 � 10�8 m) of BMP2 alone or with PTHrP(Fig. 6C). Control cells readily expressed ALP when treatedwith the higher dose of BMP2 even in the absence of PTHrP,but PTHrP produced a further increase in activity (Fig. 6, Cand E). Cells overexpressing the functional BMP IA receptorwhen treated with the higher concentration of BMP2 dem-onstrated even greater expression of the osteoblastic marker,but PTHrP added no further enhancement (Fig. 6, C and E).In contrast, cells transfected with DNBMP IA failed to ex-press the osteoblast marker ALP even in the presence of thehigher dose of BMP2 or of BMP2 plus PTHrP (Fig. 6, C andE), confirming the critical role of the BMP IA receptor inmodulating differentiation of C3H10T1⁄2 cells along the os-teoblastic lineage. Stable expression of the BMP IA andDNBMP IA mRNA was confirmed by Northern analysis(Fig. 6D).

Discussion

Several in vitro systems have been employed to enhancethe regulation of osteoblastic cell differentiation by auto-crine/paracrine or endocrine factors. These systems includeprimary cultures derived from fetal rat calvaria or long bone,or established clonal multipotential and osteoblastic celllines. Each system presents both advantages and disadvan-tages. Isolated clonal lines may exhibit heterogeneity of ex-pressed genes and of regulation by hormones and growthfactors. This may reflect true functional heterogeneity, that is,in vivo heterogeneity, or may reflect aberrant behavior orinstability resulting from immortalization or culture manip-ulations. Primary cultures, on the other hand, are limited bythe fact that they contain a diverse mixture of osteoblasticcells at different stages of differentiation and cells of otherlineages, including fibroblastic cells. Among the most com-monly used multipotential cell lines are 2T3 cells derivedfrom the calvaria of a transgenic mouse expressing simianvirus-40 T antigen driven by the BMP2 promoter (22), C2C12cells, a subclone of a mouse myoblastic cell line (23) andC3H10T1⁄2 cells. In our studies we employed C3H10T1⁄2 clone8 cells isolated from a line of C3H mouse embryo cells (24).C3H10T1⁄2 clone 8 cells are an established mesenchymal stemcell line that can be induced in both the parent line andsubclones to differentiate into multiple mesenchymal phe-notypes (15). Other than the PTHrP receptor, which can beexpressed in multiple cell types, markers of the osteoblastphenotype were not observed in our studies before the ad-dition of BMP2 and PTHrP and have not previously beenreported in C3H10T1⁄2 cells before the addition of exogenous

FIG. 5. Effects of PTHrP and of PKA and PKC signaling pathways onthe BMP IA receptor. A, Northern blot of the BMP IA receptor (upperpanel). RNA was extracted from cells treated with BMP2 (6 � 10�9

M) alone (control), BMP2 plus PTHrP (10�7 M PTHrP), BMP2 plusforskolin (100 �M Forskolin), or BMP2 plus TPA (1 �M; TPA) underconditions preparing them for a luciferase reporter assay as describedin Materials and Methods. Levels of 18S mRNA were concomitantlydetermined as a loading control (lower panel). Incubations and blotswere performed as described in Materials and Methods. B, Quanti-fication of BMP IA expression from three independent Northern blots.Analysis was performed using Scion image, with values obtained bydetermination of relative BMP IA expression divided by their respec-tive 18S mRNA levels. Fsk, Forskolin treatment. C, Luciferase re-porter activity in C3H10T1⁄2 cells stably transfected with a SMAD6promoter-luciferase reporter construct as described in Materials andMethods. Cells were pretreated with vehicle, BMP2 (6 � 10�9 M)alone, BMP2 plus PTHrP (10�7 M), BMP2 plus PTHrP and chel-erythrin chloride (1.25 � 10�6 M), BMP2 plus TPA (1 �M), or BMP2,TPA, and chelerythrin chloride as described in Materials and Meth-ods. Cells were then treated with vehicle (PBS) or BMP2 (6 � 10�9

M) alone. Each bar represents the mean � SE of quadruplicate de-terminations. *, P � 0.05 vs. treatment with vehicle; **, P � 0.05 vs.pretreatment with BMP2 alone, followed by treatment with BMP2;***, P � 0.05 vs. pretreatment with BMP2 plus PTHrP, followed bytreatment with BMP2; ****, P � 0.05 vs. pretreatment with BMP2plus TPA, followed by treatment with BMP2.


regulatory factors. Nevertheless, as limiting dilutions to pre-pare isolated subclones were not performed in our studies,it is quite possible that committed early mesenchymal pro-genitors, including osteoprogenitors, existed in our culturesin addition to multipotential mesenchymal stem cells.

Previous studies involving pluripotent mesenchymal celllines have shown that BMP2 can induce the adipocytic phe-

notype (16, 25–31). We showed increased activity of PKA andPKC as a result of PTHrP stimulation of the PTHR in themesenchymal cell line C3H10T1⁄2 cells and that this respon-siveness to PTHrP appeared to manifest itself in part byinhibiting BMP2-induced adipogenesis. The inhibition byPTHrP involved a reduction in BMP2-induced increases inmRNA encoding PPAR� and aP2 as well as a decrease in

FIG. 6. Effect of overexpression and inhibition of the BMP IA receptor on PTHrP effects on the osteoblastic phenotype. A, C3H10T1⁄2 cells weretransfected with the empty pcDNA3 vector (top three panels) or with the vector expressing the BMP IA receptor (bottom three panels), incubatedwith a low concentration of BMP2 (6 � 10�9 M), and then stained for ALP, type I collagen, and osteocalcin as described in Materials and Methods.Photomicrographs are representative of four random fields from four different experiments. B, Quantitation of ALP, collagen type I (Col I), andosteocalcin (OCN) staining as described in Materials and Methods after treatment with BMP2 (6 � 10�9 M) in cells transfected with the emptypcDNA3 vector or with the vector expressing the BMP IA receptor. Each bar represents the mean � SE of four random fields per culture fromfour separate cultures. *, P � 0.05. C, C3H10T1⁄2 cells were transfected as described in Materials and Methods with the empty pcDNA3 vector(left panels), with the vector expressing the BMP IA receptor, BMP IA (middle panels), or with the vector expressing a dominant negative BMPIA receptor, DNBMP IA (right panels). Cells were then incubated with a higher concentration of BMP2 (1 � 10�8 M) or with BMP2 and PTHrP(10�7 M) and stained for ALP. Photomicrographs are representative of four random fields from four different experiments. D, Gene expressionof endogenous BMP IA receptor (pcDNA3), the expressed BMP IA receptor (BMP IA), and the dominant negative BMP IA receptor (DNBMPIA). RNA was extracted from cells, and Northern blots were performed as described in Materials and Methods. The blot was probed with thecDNA encoding the amino terminus of the BMP IA receptor. Levels of the 18S mRNA were concomitantly determined as a loading control. E,Quantitation of ALP staining, as described in Materials and Methods, after treatment with BMP2 (1 � 10�8 M) or with the combination of BMP2(1 � 10�8 M) and PTHrP (10�7 M) of cells transfected with the empty pcDNA3 vector, the vector expressing the BMP IA receptor, or the vectorexpressing the DNBMP IA receptor. Each bar represents the mean � SE of measurements of four random fields per culture from four separatecultures. * and **, P � 0.05 vs. pcDNA3 and BMP IA, respectively.


cytological staining of lipid. We also found that in the pres-ence of PTHrP, concentrations of BMP2 that normally inducean adipocytic phenotype now induced markers of the os-teoblast lineage. Consequently, the effect may involve thespecification of multipotent precursor cells to the osteoblastlineage, trans-differentiation of adipocytes to osteoblasticcells, or the simultaneous inhibition of committed adipocyteprogenitors and stimulation of early committed osteopro-genitor cells within the C3H10T1⁄2 cell cultures.

To explore the signaling pathway involved in the PTHrP-induced effect, we employed activators of both the PKA andPKC pathways and found that the PKC pathway predomi-nantly mediated commitment to the osteoblast phenotype.To further support the role of PTHrP stimulation of PKC asthe mechanism for enhanced osteogenesis, the PKC inhibitorchelerythrin chloride was found to limit the osteogenic po-tential of PTHrP. Our finding that PTHrP enhances the de-velopment of the osteoblastic lineage by a PKC-dependentmechanism differs from a recent report that the effect ofPTH-(1–34) to increase ALP positivity in C3H10T1⁄2 cellstransfected with both BMP2 and PTHR appeared to be me-diated by forskolin rather than TPA (32). However, thosestudies employed C3H10T1⁄2 cells constitutively expressingBMP2, making comparison of the two systems difficult.

Other mechanisms for enhanced osteogenesis have also beenascribed to the capacity of PTHrP to stimulate PKA. The os-teoblast differentiation transcription factor CBFA1 is essentialfor osteogenesis in vivo (33–35) and has been shown to be atarget of PKA. Posttranslational phosphorylation of CBFA1 viaPTHrP signaling enhances the transcriptional activity of CBFA1(12). It is possible that in our system enhanced osteoblasticcommitment by PTHrP is independent of CBFA1 activation byPKA, or that PKC may also activate CBFA1. Alternatively,CBFA1 function may occur downstream of PTHrP action. Thus,BMP2-induced osteoblastic differentiation of C3H10T1⁄2 cellsappears to involve CBFA1 mRNA and protein expression (26,36, 37). CBFA1 can enhance the transcriptional activation ofSmad proteins (38, 39), and our studies show that PTHrP-stimulated osteoblastic commitment requires a BMP2-depen-dent response. Consequently, CBFA1 stimulation may still con-verge on the pathway of PTHrP-induced osteogenesis,although further downstream in the signaling pathway.

The mechanism of PTHrP action in the C3H10T1⁄2 cellsappears to involve PKC-induced gene expression of the BMPIA receptor. This differs from results obtained in the T an-tigen immortalized clonal cell line 2T3, in which the expres-sion of a constitutively active BMP IA receptor inducedadipocyte differentiation, whereas the expression of a con-stitutively active BMP IB receptor induced formation of min-eralized bone matrix. However, both receptor subtypes areexpressed in developing bone, and targeted deletion of theBMP IB receptor gene has revealed no significant change inosteoblast differentiation (40). Additionally, gene array anal-ysis of differentiating osteoprogenitor cells demonstratesthat increased BMP IA expression correlates with the termi-nal differentiation of an osteoprogenitor cell (41). Conse-quently, an increase in BMP IA receptor expression may wellregulate specification of osteoblast development.

The receptor-regulated SMADs, SMAD1, SMAD5, andSMAD8, are directly activated by the BMP type I receptor,

and both SMAD1 (42) and SMAD5 (43) have been implicatedin BMP2-induced osteoblastic differentiation. SMAD4 maythen associate with the activated SMADs, forming an acti-vated SMAD complex that can translocate to the nucleus andparticipate in the regulation of target genes. SMAD com-plexes can also increase transcriptional regulation of inhib-itory SMADs that include SMAD6 (20). To examine the func-tional integrity of the BMP IA receptor that was increased byPTHrP, we assessed the capacity of BMP2 to increase SMAD6promoter activity after pretreatment of target cells with ei-ther PTHrP or a PKC agonist. The results demonstrate theenhanced promoter activity induced by BMP2 after pretreat-ment with PTHrP or a PKC activator and illustrate the func-tional capacity of the receptor.

We demonstrated that ectopic overexpression of BMP IAreceptors in C3H10T1⁄2 cells enhances osteoblastic differen-tiation in the presence of concentrations of BMP2 that aregenerally ineffective in the absence of PTHrP. Furthermore,the higher concentrations of BMP2 that are effective in in-ducing osteoblast commitment even in the absence of PTHrPbecame ineffective in the presence of a dominant negativeform of the BMP IA receptor, and PTHrP was ineffective inthe presence of the dominant negative BMP IA receptor.These findings support the view that PTHrP acts to increasesensitivity to BMP2 by enhancing the expression of func-tional BMP IA receptors, which then signals the initiation orprogression of a genetic osteogenic program.

Sonic hedgehog (Shh) is a member of the hedgehog family ofmorphogens that includes Indian hedgehog (Ihh), an importantregulator of skeletal development. Both Shh and Ihh sharesubstantial amino acid sequence homology, and Shh has beenemployed in vitro to mimic the effects of Ihh. During endo-chondral bone formation, Ihh can stimulate PTHrP production,which then mediates the inhibitory effect of Ihh on chondrocyticdifferentiation. Shh has recently been shown to inhibit BMP2-induced adipogenesis in C3H10T1⁄2 cells (44, 45). Furthermore,recombinant N-terminal Shh (N-Shh) has also been reported toenhance osteoblastic commitment in the presence of BMP2. Thissynergistic effect was mediated at least in part by BMP-stim-ulated SMAD signaling to increase gene transcription. Al-though it would be tempting to hypothesize that the effects ofhedgehog analogs on enhancing BMP2-induced osteoblasticcommitment are, in fact, mediated by PTHrP, it appears thatN-Shh has no effect on PTHrP or PTHR expression in C3H10T1⁄2cells (45), nor does exogenous PTHrP appear to affect N-Shh-induced endochondral bone formation (46). Consequently,these effects may independently converge on the BMPpathway.

Our studies therefore demonstrate that PTHrP plays a criticalrole in regulating an inverse relationship between adipocytesand osteoblasts by inhibiting cell differentiation toward theadipocytic lineage and synergizing with BMP2 to enhance dif-ferentiation toward the osteogenic lineage. This supports a rolefor PTHrP in cell fate determination that may prove to be animportant component of its anabolic effect on the skeleton.

Acknowledgments

We gratefully acknowledge Dr. Jean Jacques Lebrun for his invalu-able assistance. We also thank Dr. Wataru Ishida for providing theSMAD6 promoter luciferase construct.


Received March 3, 2003. Accepted August 20, 2003.Address all correspondence and requests for reprints to: Dr. David

Goltzman, Calcium Research Laboratory, Room H 4.67, Royal VictoriaHospital/McGill University Health Center, 687 Pine Avenue, Montreal,Quebec, Canada H3A 1A1. E-mail: [email protected].

This work was supported by grants (to D.G. and A.K.) from theCanadian Institute for Health Research and the National Cancer Instituteof Canada and a scientist award (to A.K.) from the Canadian Institutefor Health Research.

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