Parathyroid Hormone and Parathyroid Hormone-related Protein
Exert Both Pro- and Anti-apoptotic Effects in Mesenchymal Cells*

Received for publication, September 14, 2001, and in revised form, February 26, 2002
Published, JBC Papers in Press, March 15, 2002, DOI 10.1074/jbc.M108913200

Hen-Li Chen‡, Burak Demiralp‡§, Abraham Schneider‡, Amy J. Koh‡, Caroline Silve¶,
Cun-Yu Wang�, and Laurie K. McCauley‡**‡‡

From the ‡Department of Periodontics, Prevention, and Geriatrics, the �Department of Biologic and Materials Sciences,
and the **Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, the §Department of
Periodontology, Faculty of Dentistry, Hacettepe University, 06100 Ankara, Turkey, and ¶INSERM U426, Faculty de
Medicine Xavier Bichat, 75870 Paris, France

During bone formation, multipotential mesenchymal
cells proliferate and differentiate into osteoblasts, and
subsequently many die because of apoptosis. Evi-
dence suggests that the receptor for parathyroid hor-
mone (PTH) and parathyroid hormone-related protein
(PTHrP), the PTH-1 receptor (PTH-1R), plays an impor-
tant role in this process. Multipotential mesenchymal
cells (C3H10T1/2) transfected with normal or mutant
PTH-1Rs and MC3T3-E1 osteoblastic cells were used to
explore the roles of PTH, PTHrP, and the PTH-1R in cell
viability relative to osteoblastic differentiation. Overex-
pression of wild-type PTH-1R increased cell numbers
and promoted osteocalcin gene expression versus inac-
tivated mutant receptors. Furthermore, the effects of
PTH and PTHrP on apoptosis were dramatically de-
pendent on cell status. In preconfluent C3H10T1/2 and
MC3T3-E1 cells, PTH and PTHrP protected against dex-
amethasone-induced reduction in cell viability, which
was dependent on cAMP activation. Conversely, PTH
and PTHrP resulted in reduced cell viability in postcon-
fluent cells, which was also dependent on cAMP activa-
tion. Further, the proapoptotic-like effects were associ-
ated with an inhibition of Akt phosphorylation. These
data suggest that parathyroid hormones accelerate
turnover of osteoblasts by promoting cell viability early
and promoting cell departure from the differentiation
program later in their developmental scheme. Both of
these actions occur at least in part via the protein ki-
nase A pathway.

The classical skeletal action of parathyroid hormone (PTH)1

is stimulation of osteoclastic bone resorption to maintain cal-
cium levels in blood. However, daily subcutaneous PTH injec-
tion increases bone mass and shows great potential in treating

osteoporosis (1). The mechanisms underlying the PTH anabolic
effect in bone are not fully understood. The increased bone
formation has been attributed to activation of growth factors (2,
3), osteoblast precursor cell proliferation (2, 4), and mature
osteoblast function (2). Recent studies suggest that suppression
of osteoblast apoptosis might play a major role in PTH anabolic
action (5); however, the impact of cell differentiation and the
pathways operating in this process have not been well
characterized.

The PTH-1 receptor (PTH/PTHrP receptor; PTH-1R) was the
first PTH receptor isolated, cloned, and sequenced and binds
both PTH and PTHrP with similar affinity (6, 7). The PTH-1R
responds to binding of PTH or PTHrP by activation of the
protein kinase A (PKA) and protein kinase C (PKC) pathways
(8) and clearly plays an important role in bone. Although other
PTH receptors have been identified (9, 10), the PTH-1R is the
major receptor responsible for skeletal actions of PTH and
PTHrP, as evidenced by similar phenotypes in the PTHrP and
PTH-1R null mouse models (11). Ablation of the PTH-1R gene
in mice results in a neonatal-lethal phenotype with severe
abnormalities in development of cartilage and bone (12).

To form bone, multipotential mesenchymal cells proliferate
and differentiate into osteoblasts, which secrete an extracellu-
lar matrix that becomes mineralized. Mature osteoblasts even-
tually become osteocytes or lining cells or vanish because of
apoptosis. Evidence suggests that the PTH-1R plays an impor-
tant role in proliferation, differentiation, and apoptosis that
occurs during bone formation. Expression of the PTH-1R is
considered a hallmark for osteoblastic differentiation, because
the PTH-1R is primarily associated with active collagen-pro-
ducing osteoblasts compared with osteoblasts in earlier or later
stages of differentiation (13). Further, osteocalcin mRNA, a
specific osteoblastic marker, is undetectable in osteoblasts
lacking the PTH-1R but becomes detectable after transfecting
PTH-1R null cells with wild-type PTH-1R (14). In addition,
transgenic mice overexpressing a constitutively active PTH-1R
(H223R) display increased osteoblastic proliferation and de-
creased apoptosis in trabecular bone leading to an increase in
trabecular bone volume (15).

In humans, genetic abnormalities of the PTH-1R also result
in profound skeletal defects. Constitutively active mutant
PTH-1 receptors H223R (16), T410P (17), and I458R (18) have
been identified as the cause for Jansen’s metaphyseal chondro-
dysplasia, a disease characterized by short limbed dwarfism as
a result of retarded chondrocyte differentiation. The expression
of H223R, T410P, and I458R in COS-7 cells results in a 4–8-
fold increase in basal cAMP accumulation when compared with
the wild-type PTH-1R control (16–18). In contrast, inactivated
mutant PTH-1 receptors �373–383 (19) and P132L (20) result

* This work was supported by National Institutes of Health Grants
DK53904 (to L. K. M.) and DE13788 (to C. W.) and The Scientific and
Technical Research Council of Turkey (NATO-A2) grant (to B. D.). The
costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.

‡‡ To whom correspondence should be addressed: Dept. of Periodon-
tics/Prevention/Geriatrics, University of Michigan, 1011 N. University
Ave., Ann Arbor, MI 48109-1078. Tel.: 734-647-3206; Fax: 734-763-
5503; E-mail: mccauley@umich.edu.

1 The abbreviations used are: PTH, parathyroid hormone; PTHrP,
parathyroid hormone-related protein; PTH-1R, PTH-1 receptor; PKA,
protein kinase A; PKC, protein kinase C; CREB, cAMP response ele-
ment-binding protein; BMP, bone morphogenetic protein; PE, phyco-
erythrin; 7-AAD, 7-amino-actinomycin; ELISA, enzyme-linked immu-
nosorbent assay.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 22, Issue of May 31, pp. 19374–19381, 2002
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org19374

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in Blomstrand chondrodysplasia, which is a lethal genetic dis-
order with extremely advanced endochondral bone formation.
The mutant �373–383 expressed in COS-7 cells does not bind
PTH or PTHrP and fails to induce detectable stimulation of
either cAMP or inositol phosphate production (19). P132L, a
milder inactivated mutant receptor, results in severely reduced
PTH-induced cAMP accumulation and undetectable PTH-in-
duced inositol phosphate accumulation (20).

The role of the PTH-1R during bone formation has not been
thoroughly and mechanistically investigated. Therefore, this
study takes advantage of the different functional activities of
wild-type and mutant PTH-1 receptors to explore the impact of
the PTH-1R in a mesenchymal cell differentiation system.
Hence, the purpose of this study was to determine the effects of
overexpressing mutant and wild-type PTH-1 receptors on the
cellular growth program of mesenchymal cells.

EXPERIMENTAL PROCEDURES

Plasmid Constructs—The plasmid constructs utilized for the PTH-1R
transfection experiments included pEGFP-N2 (pE), (CLONTECH,
Palo Alto, CA) and pEGFP-N2 containing cDNA for the wild-type
PTH-1 receptor (WT) or mutant PTH-1 receptors �373–383 (�),
P132L (P), and H223R (H) (16, 19, 20). The pCMV-CREB (constitu-
tively expresses the wild-type CREB) (CLONTECH), pCMV-
CREB133 (a dominant-negative mutant vector preventing Ser133

phosphorylation of CREB) (CLONTECH), TAM-67 (a c-jun dominant-
negative vector) (21), and pcDNA (control plasmid for TAM-67) (In-
vitrogen) constructs were used to determine the role of CREB and
AP-1 complex in signal transduction of apoptosis pathway.

Cell Culture and Transfection—C3H10T1/2 is an undifferentiated
murine mesenchymal cell line (22) with the potential to become myo-
blasts, adipocytes, chondroblasts, and osteoblasts (23, 24). Their induc-
tion into the osteoblastic lineage can be stimulated with bone morpho-
genetic proteins (BMPs) (25, 26). C3H10T1/2 cells (American Type
Culture Collection, Manassas, VA) were cultured in basal medium
Eagle’s (Invitrogen) with 10% fetal bovine serum (Hyclone, Logan, UT)
and 100 units/ml of penicillin and streptomycin (Invitrogen). The cells
were passaged every 4–5 days. C3H10T1/2 cells were transfected with
different plasmid constructs (pE, WT, �, or P) using PerFect-2 (Invitro-
gen) or LipofectAMINETM Plus (Invitrogen). The transfected cells were
selected in 700 �g/ml Geneticin (Invitrogen) for 3 weeks to establish
stable cell lines. The subclones were generated by limiting dilution.
Geneticin (350 �g/ml) was added to cultures once a week to maintain
the stability of transfectants. For transient transfection experiments,
C3H10T1/2 cells were transfected with different plasmid constructs
(pE, �, WT, H, pCMV-CREB, CREB133, TAM-67, or pcDNA) using
LipofectAMINETM Plus.

MC3T3-E1 cells were obtained through Dr. Renny Franceschi from
Dr. M. Kumegawa (Meikai University, Sakado, Japan) and maintained
as described previously (27). Briefly, cells were grown in minimum
essential medium alpha medium (Invitrogen) with 10% fetal bovine
serum and 100 units/ml penicillin and streptomycin. The cells were
passaged every 4–5 days.

Adenylyl Cyclase Stimulation Assay—The adenylyl cyclase stimula-
tion and cAMP-binding protein assay were performed as described
previously with minor modification (28). The cells were plated in trip-
licate at 20,000 cells/cm2 into 24-well plates, and medium containing
ascorbic acid (Fisher) (50 �g/ml) was changed every other day. BMP-4
(R & D Systems Inc., Minneapolis, MN) (50 ng/ml) was added on day 3,
and the adenylyl cyclase stimulation assay was performed at day 8.
Briefly, the cells in triplicate wells were stimulated with 10�7 M hPTH
(1–34) (Bachem, Inc., Torrance, CA) or vehicle control (0.1% bovine
serum albumin with 4 mM HCl) in calcium- and magnesium-free Hanks’
balanced salt solution (Invitrogen) containing 0.1% bovine serum albu-
min and 1 mM isobutylmethylxanthine at 37 °C for 10 min. After aspi-
rating the medium, cAMP was extracted by adding 250 �l/well ice-cold
5% perchloric acid and incubating overnight at �20 °C. After thawing,
the pH was adjusted to 7.5 with 4 N KOH, and the neutralized extract
was then assayed for cAMP using a cAMP-binding protein assay. The
cAMP-binding protein assay was performed by incubating [3H]cAMP
(ICN, Irvine, CA) with standards or unknowns and a cAMP-binding
protein sufficient to bind �30% of radioactivity for 90 min on ice. The
samples were then incubated with dextran-coated charcoal for 20 min
and centrifuged to remove unbound from bound cAMP-binding protein-
[3H]cAMP complexes. The radioactivity of the supernatants was deter-

mined with a liquid scintillation spectrophotometer, and cAMP levels
were calculated by the log-logit method using the GraphPad Prism 3
program (GraphPad Software, San Diego, CA). Triplicate wells were
analyzed for DNA content by fluorometric analysis as described previ-
ously to standardize cAMP levels (13).

Viable Cell Enumeration Assay—The cells were plated in triplicate in
24-well plates and treated with experimental reagents at designated
times, and viable cell numbers were determined by the trypan blue dye
exclusion method using a hemacytometer. To determine the effects of
PTH/PTHrP on apoptosis, caspase inhibitors 2 � 10�7 M YVAD-cmk
(inhibits caspase-1) and DEVD-fmk (inhibits caspase-3 and caspase-8)
(CLONTECH) were used to inhibit apoptosis (29) and dexamethasone
(10�7 M) was used to induce apoptosis (5) as described previously.

Flow Cytometry Assay—During apoptosis, the cell membrane phos-
pholipid phosphatidylserine is translocated from the inner to the outer
leaflet of the plasma membrane. Annexin V binds with high affinity to
cells with exposed phosphatidylserine. Annexin V conjugated to fluoro-
chromes such as phycoerythrin (PE) can be used in flow cytometry for
apoptosis analysis (30, 31). Annexin V-PE staining is used in conjunc-
tion with a vital dye, 7-amino-actinomycin (7-AAD), for detection of
early apoptotic cells (Annexin V-PE-positive and 7-AAD-negative).
C3H10T1/2 cells were plated in duplicate at 20,000 cells/cm2 in 12-well
plates and transfected with plasmid DNA using LipofectAMINETM Plus
on the second day. Treatment with or without serum deprivation was
performed by incubation in basal medium Eagle’s with or without 10%
fetal bovine serum for 16 h after washing cells. The floating cells were
collected, and the adherent cells were trypsinized with trypsin/EDTA
(Invitrogen). Floating and adherent cells were combined, rinsed with
cold phosphate-buffered saline, and then resuspended in 1� annexin V
binding buffer. Annexin V-PE and 7-AAD (PharMingen, San Diego, CA)
staining were performed by adding 5 �l of both agents to all treatment
groups. The controls included annexin V-PE only, 7-AAD only, and no
staining. The cells were incubated in the dark for 15 min, and flow
cytometry was performed within 1 h at the University of Michigan Flow
Cytometry Core. Annexin V-PE-positive and 7-AAD-negative cells were
defined as apoptotic cells.

Cell Death ELISA—Apoptosis cleaves cellular DNA into histone-
associated fragments. The cell death detection ELISA Plus (Roche Mo-
lecular Biochemicals) is a quantitative sandwich enzyme immunoassay
utilized to measure nucleosomal particles in cytoplasmic fractions. To
measure apoptosis in postconfluent cells, WT subclone cells were plated
at 5,000 cells/cm2 at day 0. On day 7, after a 3-h treatment with caspase
inhibitors (2 � 10�7 M YVAD-cmk � 2 � 10�7 M DEVD-fmk) or vehicle
(0.04% Me2SO), the cells were exposed to PTH (1–34) (10�8 M) or vehicle
treatment, and apoptosis ELISA was performed at day 8 as described
previously (32). The cells were lysed, and 20 �l of supernatant was
added to each well in a streptavidin-coated microtiter plate. Subse-
quently, a mixture of anti-histone-biotin and anti-DNA-peroxidase
monoclonal antibodies was added and incubated with shaking for 2 h.
Following several washing steps, the nucleosomes were quantified by
adding 2,2�-azino-di-[3-ethylbenzthiazolin sulfonate] diammonium salt
to the peroxidase retained in the immunocomplex. The subsequent color
reaction was measured in an ELISA plate reader at 405 nm against a
2,2�-azino-di-[3-ethylbenzthiazolin sulfonate] diammonium salt blank
(reference wavelength at 490 nm).

Northern Blot Analysis—The cells were plated in duplicate at
50,000 cells/cm2 in 60-mm dishes with medium containing ascorbic
acid (50 �g/ml) for each cell type. After the cells reached confluence,
the culture medium was changed into medium containing BMP-4 (50
ng/ml) and ascorbic acid (50 �g/ml) for 2 days, and then the total RNA
was isolated 4 days after confluence. The steady state expression of
osteocalcin was determined by Northern blot analysis. RNA isolation
and Northern blot analysis were performed as described (33). Briefly,
total RNA (10 �g) was electrophoresed on 1.2% agarose-formaldehyde
gels. The RNA was transferred to nylon membranes (Duralon UV)
(Stratagene, La Jolla, CA) via passive transfer and cross-linked with
UV transillumination (Stratalinker 2400, Stratagene). The mem-
branes were hybridized with a cDNA probe for osteocalcin labeled
with [�-32P]dCTP (Amersham Biosciences) using the Rediprime la-
beling system (Amersham Biosciences). After hybridization and
washing, radioactivity counts were measured using an Instant Im-
ager (Packard Instrument Co., San Diego, CA) and blots exposed to
Biomax MR film (Eastman Kodak Co.) at �70 °C for 24–72 h. The
blots were stripped and probed with an 18 S ribosomal RNA cDNA to
control for RNA loading.

Western Blot Analysis—MC3T3-E1 cells were plated at 5,000 cells/
cm2 in 60-mm dishes. The culture medium was changed at days 1, 3, 5,
and 7. At day 8, the cells received PTHrP (1–34) (10�7 M) (0–120 min),

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and the proteins were isolated for Western blot analysis as described
(34). After resolving by 10% SDS-PAGE, the proteins were transferred
onto polyvinylidene difluoride membranes (Bio-Rad) and blocked in
TBST (10 mM Tris, pH 8.0, 0.85% NaCl, 0.1% Tween 20) with 5% nonfat
dry milk for 1 h. The membranes were then incubated overnight at 4 °C
in a 1:500 dilution of Akt antibody (Cell Signaling, Beverly, MA) or
1:1,000 dilution of phosphorylated Akt antibody (Cell Signaling) in
TBST. After three washes with TBST and incubation for 1 h with
secondary antibody, the membranes were washed five times with TBST
and developed by chemiluminescent detection according to protocols
supplied by the manufacturer (ECL; Amersham Biosciences).

Statistical Analysis—All of the assays were repeated at least three
times with similar results unless otherwise noted. The data were ana-
lyzed using either analysis of variance or a Student’s t test with the
Instat 2.1 biostatistics program (GraphPad Software).

RESULTS

Cyclic AMP Response to PTH Stimulation Confirms the Bio-
logic Activity of the Transfected PTH-1Rs—PTH binds to the
PTH-1R and activates adenylyl cyclase, which leads to in-
creased cAMP. To verify the existence and function of the
transfected PTH-1Rs, adenylyl cyclase stimulation assays were
performed after cells were induced to differentiate with the
addition of BMP-4 and standardized to DNA levels in all sam-
ples. BMPs are widely used to induce osteoblastic differentia-
tion in C3H10T1/2 cells (25, 26). In stable mixed populations
(Fig. 1A), all groups had elevated cAMP after PTH (1–34)
stimulation reflecting the biologic activity of the PTH-1R. WT
transfectants had more biologically active PTH-1Rs, as demon-
strated by the greatest cAMP response. In contrast, the groups
with mild (P132L) and severely (�373) inactivated mutant
PTH-1Rs showed a lower cAMP response and the least cAMP
response, respectively. The cAMP response of the plasmid-only
group (pEGFP) indicated the existence of functional endoge-
nous wild-type PTH-1Rs in C3H10T1/2 cells. Representative
subclones were also utilized in experiments to get homogene-
ous populations of each type of stable transfectant. The result
of adenylyl cyclase stimulation of selected subclones (Fig. 1B)

demonstrated significant cAMP responses in all groups except
�373. The cAMP response after PTH stimulation was the high-
est in the WT subclone and the lowest in �373 similar to their
respective mixed populations. Interestingly, we noted that WT
mixed populations and subclones demonstrated a 65 and 60%
increase in DNA content when compared with the respective
�373 groups, suggesting that the expression of functional PTH-
1Rs resulted in increased numbers of cells. The assays were
also performed in subclones without BMP induction of differ-
entiation. Basal cAMP levels were not altered in the absence of
BMP induction (P132L, 2.24 � 1.26 pmol cAMP/�g DNA;
�373–383, 1.27 � 1.07 pmol cAMP/�g DNA; pEGFP, 0.95 �
0.66 pmol cAMP/�g DNA; and WT, 1.02 � 0.81 pmol cAMP/�g
DNA). Without BMP treatment, no PTH-induced cAMP levels
were found in P132L and �373 subclones. WT and pEGFP had
2.7- and 3.3-fold elevated cAMP levels, which demonstrated a
PTH response, but significantly lower than when BMP-in-
duced. This suggests that BMP treatment may induce PTH
receptor expression as previously demonstrated (25, 26, 35).

PTH and PTHrP Inhibit Apoptosis of Preconfluent Cells
through the PKA Pathway—The anabolic action of PTH has been
attributed to its antiapoptotic effects. Thus, viable cell enumer-
ation assays were performed to determine the effect of PTH on
dexamethasone-induced apoptosis in preconfluent WT subclone
cells (Fig. 2). PTH (1–34) demonstrated an antiapoptotic-like
effect by blocking the dexamethasone-induced decrease in viable
cell number. In this assay system, dexamethasone has previously
been reported to stimulate apoptosis, and PTH was found to
prevent the dexamethasone-induced effect (5).

Cyclic AMP is an important intracellular secondary messen-
ger in the PTH-1R signal transduction pathway. To explore the
mechanism of PTH on apoptosis in preconfluent cells, flow
cytometry detection of apoptosis with parallel adenylyl cyclase
stimulation assays were performed on transient transfectants
of pEGFP, WT, and H223R. WT had a higher PTH-induced
cAMP response than H223R as reported in COS-7 cells (17). In
addition, H223R demonstrated the highest basal cAMP levels
among all groups indicative of its constitutive activity (Fig. 3A).
The H223R transfected cells had a significantly reduced per-
centage of apoptotic cells (annexin V-PE-positive and 7-AAD-
negative cells) after serum withdrawal (Fig. 3B). Likewise,
treatment with forskolin (10�6 M), an activator of cAMP, pre-
vented the dexamethasone-induced reduction in viable cell
numbers in preconfluent C3H10T1/2 cells (data not shown).
These data suggest that cAMP is responsible for the antiapo-
ptotic effect of PTH in preconfluent undifferentiated cells.

FIG. 1. Determination of biologic activity of PTH-1R in trans-
fectants of stable mixed populations (A) and stable subclones
(B). The cells were plated at 20,000 cells/cm2 and induced to differen-
tiate with the addition of ascorbic acid (50 �g/ml) and BMP-4 (50 ng/ml).
The cells in triplicate wells were stimulated with PTH (1–34) (10�7 M)
or vehicle. The cAMP levels were determined by cAMP-binding protein
assay, standardized to DNA levels, and expressed as the means � S.E.
*, p � 0.005 versus respective control groups.

FIG. 2. PTH blocks the dexamethasone-induced cell number
reduction in preconfluent cells. WT subclone cells were plated
overnight in triplicate, treated for 1 h with PTH (1–34) (10�8 M) or
vehicle, and then treated with dexamethasone (DEX; 10�7 M) or vehicle
for 6 h. Viable cell numbers were determined by trypan blue dye
exclusion and hemacytometer enumeration. The results are expressed
as the means � S.E. *, p � 0.05 versus PTH(�)Dex(�). **, p � 0.01
versus PTH(�)Dex(�).

Effects of PTH-1 Receptor Expression on Mesenchymal Cells19376

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CREB and the AP-1 complex are two downstream mediators
of the PKA pathway (36, 37). To further characterize the PTH/
PTHrP antiapoptotic effect in preconfluent cells, WT subclone
cells were transiently transfected with specific constructs that
inhibit either CREB (CREB133) or the AP-1 complex (TAM-67)
and evaluated in viable cell enumeration assays (Fig. 4).
PTHrP prevented the apoptosis induced by dexamethasone in
the plasmid-only group but not in the CREB133 or TAM-67
transfection groups. This indicates that the PKA downstream
mediators, CREB and AP-1 complex, play important roles in
mediating the antiapoptotic effect of PTH/PTHrP in preconflu-
ent mesenchymal cells.

Overexpression of Functional PTH-1R Increases Viable Cell
Numbers—Viable cell enumeration assays were performed on
stable mixed populations (Fig. 5A) and stable subclones (Fig.
5B) to determine the effects of the PTH-1R transfection on cell
numbers over time. Viable cell numbers after an 8-day culture
in both assays were the lowest in �373 and increased respec-
tively in pEGFP, P132L, and WT. The WT cells demonstrated
a more than 2-fold increase in viable cell numbers compared
with �373 cells in both mixed populations and representative
subclones at day 8. This suggests that overexpression of func-
tional wild-type PTH-1 receptor leads to an increase in cell
numbers over time but does not distinguish between effects on
proliferation versus cell death.

PTH and PTHrP Reduce Numbers of Postconfluent Cells—
The adenylyl cyclase stimulation assays indicated differential
responses to PTH among groups. Therefore, viable cell enumer-
ation assays were performed to explore the effect of PTH on
stable subclones during an 8-day culture (Fig. 6A). Interest-
ingly, PTH treatment significantly decreased WT cell numbers
at day 8 but not earlier. The cells becoming confluent at day 4
or 5 in the assay suggested that the PTH effect was restricted

to postconfluent cells. In addition, no significant reduction in
cell numbers was observed in other groups, suggesting that
this phenomenon was associated with biologically active PTH-
1Rs. Furthermore, viable cell enumeration assays were per-
formed to determine whether the PKA pathway was responsi-
ble for the effect on viable cell numbers of WT subclones at day
8 (Fig. 6B). When cells were cultured with activators of PKA,
PTH (1–34), and forskolin, there was a reduction in viable cell
numbers at day 8. In contrast, the antagonist, PTH (7–34),
which binds the PTH-1R without activation of cAMP, had no
effect. These data suggest that the PKA pathway mediates the
PTH-induced reduction in viable cell numbers in postconfluent
cells.

The reduction in numbers of postconfluent WT subclone cells
by PTH or forskolin might be due to either a decrease in
proliferation or an increase in apoptosis and was contrary to
the PTH protection against apoptosis found in preconfluent
cells. Therefore, the effect of PTH on apoptosis of day 8 WT
subclone was explored using additional viable cell enumeration
assays (Fig. 7A). Caspase inhibitors, which prevent apoptosis,
significantly inhibited the PTH-induced decrease in viable cell
numbers. This suggests that the reduction in cell numbers in
response to PTH in more mature cells is due to a proapoptotic
mechanism. To further confirm this hypothesis, cell death
ELISAs were performed on day 8 WT subclone cells (Fig. 7B).
PTH treatment increased DNA fragmentation, a characteristic
of apoptotic cell death, whereas caspase inhibitors prevented it.

These data suggest a biphasic response where early during a

FIG. 3. High basal cAMP levels reduce the apoptosis induced
by serum withdrawal in preconfluent cells. C3H10T1/2 cells were
plated in triplicate, transfected the next day, serum-deprived for 16 h,
and then stained with annexin V-PE and 7-AAD. Apoptosis was deter-
mined by flow cytometry. Parallel adenylyl cyclase stimulation assays
were performed. A, H223R transient transfected cells demonstrated
higher basal cAMP levels than other transfectants. *, p � 0.0001 versus
other vehicle-treated groups. B, H223R transient transfectants had
reduced apoptosis induced by serum withdrawal. *, p � 0.05 versus
other groups. The results are expressed as the means � S.E.

FIG. 4. PTHrP protection from apoptosis via CREB and AP-1
complex in preconfluent cells. WT subclone cells were plated over-
night in triplicate, transfected with different plasmids for 3 h, treated
for 1 h with PTHrP (1–34) (10�7 M) or vehicle, and then treated with
dexamethasone (Dex; 10�7 M) or vehicle for 6 h. Viable cell numbers
were determined by trypan blue dye exclusion and hemacytometer
enumeration. A, cells transfected with CREB or CREB133 (a dominant-
negative mutant vector preventing Ser133 phosphorylation of CREB). *,
p � 0.05 versus control and PTHrP(�)Dex(�). **, p � 0.05 versus
control. ***, p � 0.01 versus control. B, cells transfected with pcDNA or
TAM-67 (a c-jun dominant-negative vector). *, p � 0.05 versus control
and PTHrP(�)Dex(�). **, p � 0.05 versus control. Transient transfec-
tion of CREB133 or TAM-67 abolished the PTHrP protective effect on
dexamethasone-induced reduction in cell numbers. The representative
results from two assays are expressed as the means � S.E.

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proliferative phase PTH protects against apoptosis, whereas
later after confluence it stimulates apoptosis. These effects may
be associated with the differentiation program of the cell.
Hence, the impact of the PTH-1R expression on a marker of
osteoblast differentiation was further evaluated.

Expression of Functional PTH-1R Increases Osteocalcin Gene
Expression—Osteocalcin is a specific osteoblastic marker.
Thus, the expression of osteocalcin was used to determine the
effects of the PTH-1R on osteoblastic differentiation in mesen-

chymal cells. Northern blot analyses were performed on total
RNA isolated from differentiated stable mixed populations and
subclones. WT had the highest and �373 had the lowest ex-
pression levels of osteocalcin in both stable mixed populations
(data not shown) and subclones (Fig. 8). These data suggest
that the expression of functional PTH-1R promotes mesenchy-
mal cell differentiation toward the osteoblastic lineage. In ad-
dition, PTH reduced the numbers of cells after 8 days of culture
in WT, but not in other transfectants (Fig. 7A), further sup-
porting the role of PTH in inducing apoptosis in more differen-
tiated mesenchymal cell types.

The PTH/PTHrP Bi-directional Effects on Apoptosis Are De-
pendent on the Differentiation State in MC3T3-E1 Cells—In
C3H10T1/2 transfectants, PTH (1–34) was antiapoptotic in
preconfluent cells and proapoptotic in more differentiated post-
confluent cells. This was further tested in a well characterized
MC3T3-E1 osteoblastic differentiation system. Caspase inhib-
itors prevented the dexamethasone-induced reduction in viable
cell numbers in preconfluent cells (Fig. 9A). This suggests that
dexamethasone decreased the viable cell numbers via apoptosis
similar to other reports (5). As with the C3H10T1/2 cells,
PTHrP prevented the reduction in cell viability induced by
dexamethasone, and forskolin mimicked the antiapoptotic ef-
fect of PTHrP in preconfluent cells (Fig. 9B). Forskolin alone
slightly but not significantly reduced cell numbers in precon-
fluent cells. Conversely, PTH (1–34) and PTHrP (1–34) both
significantly decreased the viable cell numbers in differenti-

FIG. 5. Overexpression of functional PTH-1R increases viable
cell numbers over time. The cells were plated at 5,000 cells/cm2 into
triplicate wells, and viable cell numbers were determined at days 4, 6,
and 8 using the trypan blue dye exclusion method. A, stable mixed
populations. *, p � 0.05 versus WT and �373. B, stable subclones. *, p �
0.01 versus all other groups. Representative viable cell numbers from
two assays are expressed as the means � S.E. Viable cell numbers at
day 8 were the highest in WT and the lowest in �373.

FIG. 6. PTH reduces viable cell numbers in postconfluent cells.
A, stable subclones were plated at 5,000 cells/cm2 into triplicate wells.
PTH (1–34) (10�8 M) or vehicle was added at days 1, 3, 5, and 7. Viable
cell numbers were determined at days 4, 6, and 8. PTH decreased viable
cell numbers in WT group at day 8. *, p � 0.05 versus day 8 WT vehicle
group. B, WT subclone cells were plated at 5,000 cells/cm2 into triplicate
wells. PTH (7–34) (10�8 M), PTH (1–34) (10�8 M), forskolin (10�6 M), or
vehicle were added at days 1, 3, 5, and 7, and viable cell numbers were
determined at day 8. PTH (1–34) and forskolin (FSK), but not PTH
(7–34), decreased viable cell numbers. *, p � 0.05 versus control. **, p �
0.01 versus control. The results are expressed as the means � S.E.

FIG. 7. PTH induces apoptosis in postconfluent cells. WT sub-
clone cells were plated at 5,000 cells/cm2 into triplicate wells. A, PTH
(1–34) (10�8 M) or vehicle was added at days 1, 3, 5, and 7. In addition,
caspase inhibitors (combination of 2 � 10�7 M YVAD-cmk and DEVD-
fmk) or vehicle 3-h pretreatment were performed at days 5 and 7. Viable
cell numbers were determined at day 8 using the trypan blue dye
exclusion method. Caspase inhibitors (Casp Inh) reversed the PTH-
induced reduction in viable cell numbers at day 8. *, p � 0.005 versus
other three groups. B, cells were treated with PTH (1–34) (10�7 M) or
vehicle control after a 3-h pretreatment with caspase inhibitors (com-
bination of 2 � 10�7 M YVAD-cmk and DEVD-fmk) or vehicle control at
day 7. Cell death ELISA was performed at day 8. *, p � 0.005 versus
caspase inhibitor(�)PTH(�). **, p � 0.005 versus caspase
inhibitor(�)PTH(�). All of the data are expressed as the means � S.E.

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ated MC3T3-E1 cells (Fig. 9C). These data confirm that the
effects of PTH/PTHrP on apoptosis are dependent on the dif-
ferentiation state of the cells.

PTH Prevents Akt Phosphorylation in Postconfluent Cells—
Akt is an antiapoptotic signaling molecule in multiple cell types
when challenged with cell death inducers (38, 39). Phosphoryl-
ation is critical for its activation by upstream kinases and for
the maintenance of its activity. To explore the possible mech-
anism of the PTH proapoptotic effect in more mature cells,
postconfluent differentiated MC3T3-E1 cells were used in time
course Western blot analyses for Akt and phosphorylated Akt
(Fig. 10). PTHrP (1–34) decreased the Akt phosphorylation
within 30 min. This suggests that the reduction in phosphoryl-
ated Akt is a mechanism by which PTH exerts its proapoptotic
effect on more mature cells. In contrast, preconfluent cells
express Akt protein, but phosphorylation was not detected at
steady state nor with PTHrP nor with dexamethasone treat-
ment for 1 or 2 h (data not shown). Because the untreated
groups did not have phosphorylated Akt, the inhibition of
apoptosis in preconfluent osteoblast precursors is unlikely via
mechanisms relative to Akt phosphorylation.

DISCUSSION

To form bone, multipotential mesenchymal cells proliferate
and differentiate into osteoblasts. Once mature osteoblasts
have finished bone formation, the majority (50–70%) originally
located at the remodeling site die because of apoptosis, and the
rest become osteocytes or lining cells (40). In this study we
report that PTH and its receptor play important roles in this
process. Expression of functional PTH receptors increases via-
ble cell numbers and promotes osteoblastic differentiation.
Furthermore, PTH treatment protects against apoptosis in less
mature cells and promotes apoptosis in more mature cells.

C3H10T1/2 cells stably transfected with various PTH recep-
tors had different levels of osteocalcin and altered cell num-
bers. Both PTH (1–34) and PTHrP (1–34) bind to the PTH-1
receptors with similar affinity (7), activate the same signal
transduction pathway with similar potency (41, 42), and exert
anabolic actions in bone (43). In our study, overexpression of
wild-type PTH receptors increased cell number after 8 days in

culture, whereas overexpression of inactivated receptors re-
sulted in lower cell numbers compared with controls. Our data
suggest that functional PTH receptors, likely through affecting

FIG. 8. Expression of functional PTH-1R increases osteocalcin
gene expression. A, autoradiograph of Northern blot analysis of os-
teocalcin mRNA and 18 S rRNA. B, plot of osteocalcin expression after
adjusted to 18 S rRNA expression. The results are expressed as the
means � S.E. from duplicate samples. WT had the highest and �373
had the lowest, level of osteocalcin (OCN) expression. *, p � 0.05 versus
pEGFP. **, p � 0.001 versus pEGFP.

FIG. 9. PTH/PTHrP effects on apoptosis are dependent on the
differentiation state of the MC3T3-E1 cells. A, cells were plated
overnight in triplicate at 10,000 cells/cm2, after a 3-h pretreatment with
caspase inhibitors (Casp Inh; combination of 2 � 10�7 M YVAD-cmk and
DEVD-fmk) or vehicle, dexamethasone (Dex; 10�7 M), or vehicle was
added, and viable cells were enumerated 6 h later. *, p � 0.05 versus
other three groups. B, cells were plated overnight in triplicate at 30,000
cells/cm2, treated for 1 h with PTHrP (1–34) (10�7 M), forskolin (FSK;
10�7 M) or vehicle, and then treated with dexamethasone (10�7 M) or
vehicle for 6 h. *, p � 0.05 versus control. **, p � 0.01 versus dexam-
ethasone only group. C, cells were plated in triplicate at 5,000 cells/cm2

and cultured in differentiation medium (50 �g/ml ascorbic acid) for
either 6 or 8 days before treatment. Viable cell numbers were deter-
mined after 1 h of PTH (1–34) (10�8 M) (6-d group), PTHrP (1–34) (10�7

M) (8-d group), or vehicle treatment followed by 6 h of dexamethasone
vehicle treatment (to simulate similar conditions as A, B) by trypan
blue dye exclusion and hemacytometer enumeration. *, p � 0.05 versus
control. In preconfluent cells, caspase inhibitors, PTHrP, and forskolin
prevented the dexamethasone-induced reduction in viable cell num-
bers. In contrast, PTH and PTHrP treatment decreased viable cell
numbers in the postconfluent differentiated cells. The results are ex-
pressed as the means � S.E. of triplicate samples.

FIG. 10. PTHrP-mediated inhibition of Akt phosphorylation in
postconfluent differentiated cells. MC3T3-E1 cells were cultured in
differentiation medium (50 �g/ml ascorbic acid) for 8 days and treated
with PTHrP (1–34) (10�7 M) for 0–120 min followed by protein isolation
and Western blot analysis. Akt and phosphorylated Akt were analyzed.
PTHrP inhibited Akt phosphorylation but did not alter Akt levels.

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proliferation or apoptosis, contribute to the increase of mesen-
chymal cell numbers. These results agree with studies report-
ing that PTH stimulates osteoblast proliferation (2, 4) and
increases osteoblast number (44) and that a constitutively ac-
tive PTH-1R increases osteoblast number in trabecular bone in
transgenic mice (15). Additionally, Watson et al. (45) suggest
that the PTH-1R localizes to the nucleus and may be associated
with ligand-independent proliferation. Further, our data may
explain the disparity in the results from many other cell sys-
tems (46, 47), because the effect of PTH, PTHrP, and the
PTH-1R likely depends on the differentiation state of the cell.

The effect of the PTH receptor expression on osteoblastic
differentiation of mesenchymal cells was reflected by osteo-
calcin expression levels. After BMP-4 treatment, overexpres-
sion of functional PTH receptors resulted in the highest level
of osteocalcin expression, whereas overexpression of two
types of inactivated mutant PTH receptors resulted in lower
levels of osteocalcin expression. Our results agree with pre-
vious reports indicating that the presence of the PTH recep-
tor is essential for detection of osteocalcin mRNA expression
by Northern blot analysis (14). BMPs are well known to
induce the differentiation of C3H10T1/2 cells into PTH-
responsive osteoblasts (25). Still, C3H10T1/2 cells produce
endogenous BMP-1, BMP-2, and BMP-4 (48, 49); thus they
may become more differentiated after their confluence even
without exogenous BMP. It is likely that the postconfluent
WT cells used in our study represent a more differentiated
cell stage in contrast to preconfluent cells. The specific de-
tails of the interactions between BMP and PTH signaling
pathway are as yet unclear. However, BMP may play a role in
the anabolic effects of intermittent PTH treatment in vivo
through the following mechanisms: 1) The biologically active
MH2 domain of Smad1 leads to the immediate up-regulation
of the PTH-1R gene in mesenchymal progenitors C3H10T1/2
(35). Thus, BMP induces osteoblastic differentiation, which
leads to expression of PTH-1R and potentially enhances PTH
anabolic effects. 2) Runx2 is a transcription factor regulating
both differentiation and function of osteoblasts (50). BMPs
increase the expression of Runx2, and PTH induces PKA-
mediated post-transcriptional modification of Runx2 (26, 51,
52). Runx2 and c-Fos�c-Jun physically interact and coopera-
tively bind the AP-1- and runt domain-binding sites in the
collagenase-3 promoter (53). This process may also be crucial
for activation of other osteoblastic genes, including osteocal-
cin and COL1A1 and COL1A2, which also contain AP-1- and
runt domain-binding sites in their promoters (54, 55). Thus,
BMP may aid in PTH anabolic actions in vivo through Runx2.

The reported effects of PTH on apoptosis appear to be de-
pendent on the cell culture system. PTH is antiapoptotic in
osteoblasts (5) and chick embryo hypertrophic chondrocytes
(56), whereas it promotes apoptosis in 293 cells, a transformed
primary embryonal kidney cell line (29). Interestingly, our
findings of a bi-directional effect of PTH on cells of differing
maturity stage are similar to those reported for another impor-
tant factor in skeletal development, fibroblast growth factor
(57–59). Fibroblast growth factor treatment increases prolifer-
ation in immature osteoblasts but promotes apoptosis in differ-
entiating osteoblasts (59). Our study indicates that the effects
of PTH on apoptosis are similarly dependent on cell status.
PTH is antiapoptotic in preconfluent cells and proapoptotic in
more differentiated postconfluent cells. The underlying mech-
anisms of this dual effect are not totally clear at present but
appear to be dependent on the PKA pathway. This is in con-
trast to the proapoptotic effects of PTH reported in kidney cells
that were found to be dependent on the PKC pathway (29).

The PTH-1R responds to binding of PTH or PTHrP by acti-

vation of PKA and PKC pathways. The PKA pathway is medi-
ated by the Gs protein that activates adenylyl cyclase and leads
to cAMP production and protein kinase A activation. The PKC
pathway is mediated by the Gq protein that activates phospho-
lipase C, resulting in an increase in intracellular Ca2� levels
and activation of protein kinase C. In previous work, PTH
induced apoptosis in 293 cells through PKC pathway rather
than PKA pathway (29). However, other studies indicate that
cAMP suppresses apoptosis in rat periosteal cells (60) and
human osteoblasts (61). Transgenic mice overexpressing a con-
stitutively active PTH-1R (H223R), which results in higher
basal cAMP levels, also had decreased apoptosis in trabecular
bone leading to an increase in osteoblast number in trabecular
bone (15). In our study, the transient transfection of H223R
resulted in high basal cAMP levels and suppressed apoptosis as
determined by flow cytometry assays in preconfluent mesen-
chymal cells. In addition, forskolin, an activator of cAMP, mim-
icked the antiapoptotic effect of PTHrP in preconfluent
MC3T3-E1 cells. Furthermore, CREB and AP-1 complex are
two mediators downstream of cAMP. We found that inhibition
of either the CREB or the AP-1 complex abolished the PTHrP
protective effect on viable cell numbers in preconfluent
C3H10T1/2 transfectants. CREB has been found to be anti-
apoptotic in other cell types (62, 63), whereas AP-1 family
members have been found to be both anti- and proapoptotic (64,
65). The c-jun N-terminal kinase can be activated by stimuli
such as mitogenic signals and growth factors (66) and is re-
sponsible for phosphorylating transcription factors such as c-
Jun and the activating transcription factor-2, which may be
important in the PTHrP signaling cascade. Reports indicate
that early expression of the c-jun N-terminal kinase is associ-
ated with a protection from apoptosis, whereas later expression
is associated with a stimulation of apoptosis (67, 68). Dissecting
out the specific AP-1 family members involved in the regulation
of apoptosis is complex as evidenced by findings that JunB is
pro-apoptotic and c-Jun is anti-apoptotic (65). Interestingly, in
contrast to the early protective effect, cAMP likely promotes
apoptosis in postconfluent cells as evidenced by reduction of
viable cell numbers after induction of cAMP production. In
addition, in the more differentiated cells, the proapoptotic ef-
fect is likely due to the loss of Akt phosphorylation that has a
well known protective role in cell survival (69, 70). Akt was not
found to be a mediator of the protective effects of PTH in the
less differentiated cells because there was no alteration in Akt
or its phosphorylation with PTH treatment of undifferentiated
cells. Interestingly, reports of cAMP inhibition of Akt activity
in 3T3, COS, and HEK293 cells corroborate our data and indi-
cate that the inhibition is mediated through an upstream reg-
ulation of Akt phosphoinositide-dependent kinase (71).

In summary, we show that PTH, PTHrP, and the PTH-1
receptor play important roles at multiple stages during bone
formation through their effects on cell survival. PTH prevents
the apoptosis of more immature preconfluent mesenchymal
cells, increases their numbers and promotes their differentia-
tion into osteoblasts for bone formation. In more mature post-
confluent cells, PTH induces apoptosis of osteoblasts. Finally,
one may speculate that this promotion of turnover of more
mature cells may help to clear the way for the less differenti-
ated cells to produce extracellular matrix and hence contribute
to the anabolic actions of PTH in bone.

Acknowledgments—We gratefully acknowledge Dr. Ernestina
Schipani (Endocrine Unit, Massachusetts General Hospital and
Harvard Medical School, Boston, MA) for providing us with plasmids
encoding mutant PTH-1 receptors. In addition, we thank Mark A.
Kukuruga in the University of Michigan Flow Cytometry Core for
advice in flow cytometry techniques and the Center for Biorestoration of
Oral Health for scientific critique and support.

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Effects of PTH-1 Receptor Expression on Mesenchymal Cells 19381

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Wang and Laurie K. McCauley
Hen-Li Chen, Burak Demiralp, Abraham Schneider, Amy J. Koh, Caroline Silve, Cun-Yu

and Anti-apoptotic Effects in Mesenchymal Cells
Parathyroid Hormone and Parathyroid Hormone-related Protein Exert Both Pro-

doi: 10.1074/jbc.M108913200 originally published online March 15, 2002
2002, 277:19374-19381.J. Biol. Chem. 

  
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