Induction of osteoblast differentiation indexes by PTHrP in
MG-63 cells involves multiple signaling pathways
Luisa
Carpio,
Julienne
Gladu,
David
Goltzman, and
Shafaat A.
Rabbani
Department of Medicine, McGill University Health Center, Montreal,
Quebec H3A 1A1, Canada
 |
ABSTRACT |
Parathyroid hormone
(PTH)-related peptide (PTHrP) can modulate the proliferation and
differentiation of a number of cell types including osteoblasts. PTHrP
can activate a G protein-coupled PTH/PTHrP receptor, which can
interface with several second-messenger systems. In the current study,
we have examined the signaling pathways involved in stimulated type I
collagen and alkaline phosphatase expression in the human
osteoblast-derived osteosarcoma cells, MG-63. By use of Northern
blotting and histochemical analysis, maximum induction of these two
markers of osteoblast differentiation occurred after 8 h of
treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen
and alkaline phosphatase stimulation in a dose-dependent manner. These
effects of PTHrP could also be blocked by inhibiting the
Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras
farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059.
Transient transfection of MG-63 cells with a mutant form of G
, which
can sequester 
-subunits, showed significant downregulation of
PTHrP-stimulated type I collagen expression, as did inhibition of
phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin.
Consequently, the 
-PI 3-kinase pathway may be involved in PTHrP
stimulation of Ras. Collectively, these results demonstrate that,
acting via its G protein-coupled receptor, PTHrP can induce indexes of
osteoblast differentiation by utilizing multiple, perhaps parallel,
signaling pathways.
parathyroid hormone-related peptide; osteoblast; cell
differentiation; hypercalcemia
 |
INTRODUCTION |
THE SEARCH for the
responsible pathogenetic factor in the development of hypercalcemia of
malignancy culminated in the discovery of parathyroid hormone
(PTH)-related peptide (PTHrP) a little more than a decade ago (6,
26, 32). Since then, PTHrP has been observed to be expressed by
a wide variety of normal adult and fetal tissues (12, 30).
Due to its wide tissue distribution and the degree to which it is
conserved across evolution, it was proposed that PTHrP may well have a
significant developmental role. It was subsequently discovered that
PTHrP plays a role in the proliferation and differentiation of a
variety of cell types, including chondrocytes, osteoblasts
(18), and keratinocytes (16, 17). Despite the
evidence that PTHrP plays an important role in cellular turnover and
maturation, little is currently known of the molecular mechanisms
involved in PTHrP-mediated cellular differentiation. Due to the
NH2-terminal sequence homology between PTH and PTHrP, these
two peptides can interact with a common receptor, PTH/PTHrP receptor
(1). The presence of this receptor in bone is well
established, and high receptor levels are seen in osteoblasts that are
actively differentiating (24), suggesting a role for the
receptor in osteoblast development. Active mineralization of bone
matrix involves the production of type I collagen and alkaline
phosphatase, which, among others, are established markers of osteoblast
differentiation (4).
The common PTH/PTHrP receptor is a member of the family of seven
transmembrane receptors that are coupled to heterotrimeric G proteins.
PTH and PTHrP are known to activate several second-messenger pathways
that are linked by distinct mediators to the PTH/PTHrP receptor
(27). For example, ligand binding is known to stimulate both intracellular cAMP and inositol trisphosphate through
G
s and G
q, respectively (1,
15). G protein-coupled receptors are also known to activate
mitogen-activated protein kinase (MAPK) activity in a manner that is
dependent on the profile of the involved G protein, the receptor to
which it is coupled, and the cell type in which they are found
(33). Previous studies have reported differential
activation of protein kinase A (PKA) or PKC pathways, and this may be
cell type specific and vary according to the differentiation stage and
exposure time to the ligand. Activation of MAPK has been shown to be
essential in the differentiation of several cell types, and it has been
shown that PKA and/or PKC activation can influence MAPK activity. G
protein activation will also involve the release of a 
-dimer
subunit (5, 21), and this subunit may regulate the
phosphorylation of the protein Shc, which can then lead to the
formation of a protein complex involving Shc-Grb2-Sos and the
subsequent activation of the protooncogene Ras (11). Ras
activation is known to result in activation of Raf and then in
activation of the enzymes MAPK kinase (MEK) and MAPK (22). Receptors coupled to trimeric G proteins may therefore activate one or
more of these possible pathways. Although it has been definitively established that PTH/PTHrP receptor signaling may occur via
G
s and/or G
q, the question of

-mediated signaling by this receptor is not yet so clearly proven.
The present study was undertaken to further examine the possible role
of multiple signal transduction pathways in the stimulation of
osteoblast differentiation by PTHrP. Through the use of chemical inhibitors of signal transduction and transient transfection of a

-sequestering mutant form of G
s, and employing a
human osteoblast-like osteosarcoma model, MG-63 cells, we show the
importance of activation of PKA, PKC, and Ras by PTHrP in inducing
osteoblast differentiation. Our results also demonstrate the
involvement of MAPK, which may be a point of convergence of these
activated signaling pathways in this system. These results, therefore,
emphasize the involvement of multiple pathways in PTHrP-induced indexes
of osteoblast differentiation.
 |
MATERIALS AND METHODS |
Cell culture.
MG-63 osteosarcoma cells were maintained in vitro in MEM [with
Earle's Salts (ES)] supplemented with 10% fetal bovine serum (FBS),
100 U/ml of penicillin-streptomycin sulfate (GIBCO-BRL). For transient
transfection assays, cells were plated at 1 × 105
cells/60-mm dish 24 h before transfection and growth in 5%
CO2 in MEM-ES. Cells were incubated with 10 µg/ml
lipofectin (GIBCO-BRL) and cultured overnight in serum-free MEM-ES
culture medium with 0.1, 1, or 10 µg of plasmid DNA. After overnight
incubation with lipofectin, fresh culture medium containing 10% FBS
was added. PTHrP treatment assays were performed within 48 h after transfection.
PD-98059 (Biomol), wortmannin (Sigma Canada), B1086 (Eisai Research
Institute, Andover MA), H-89 (Biomol), and chelerythrine chloride
(Biomol) were dissolved in dimethyl sulfoxide and stored at appropriate
stock concentrations and were diluted to the desired concentrations
immediately before use.
The plasmid encoding the G
triple mutant was obtained from the
laboratory of Dr. H. R. Bourne (University of California, San
Francisco, CA) and has been previously described
(13).
Northern blot analysis.
Total cellular RNA was extracted by TRIzol extraction from control and
experimental cells after treatment with vehicle alone, PTHrP-(1-34) alone, or graded concentrations of the
chemical inhibitors. Ten micrograms of total cellular RNA were
electrophoresed on a 1.1% agarose-formaldehyde gel, transferred to a
nylon membrane (Nytran; S&S, Keene, NH) by capillary blotting, and then
fixed by drying and ultraviolet cross-linking for 10 min. The integrity of the RNA was assessed by ethidium bromide staining. Hybridization was
carried out with a 32P-labeled type I collagen cDNA and
with an 18S RNA probe with the use of a [32P]dCTP, as
previously described (2). After a 24-h incubation at 65°C,
filters were washed twice under low-stringency conditions [1×
standard sodium citrate (SSC) and 1% SDS at 60°C for 2 × 20 min] and under high-stringency conditions (0.1× SSC and 0.1% SDS at
60°C for 2 × 20 min). Autoradiography of filters was carried out at
70°C using XAR film (Eastman Kodak, Rochester, NY). The levels of type I collagen expression were quantified by densitometric scanning using the MAC BAS v1.01 alias program.
Alkaline phosphatase detection.
Alkaline phosphatase activity in control and PTHrP-treated MG-63 cells
was detected by a histochemical reaction. Cells were fixed in
citrate-buffered acetone. Slides were then immersed in alkaline-dye
solution containing diazonium salts and incubated for 30 min. Cells
were stained with Mayer's hematoxylin solution for 10 min to detect
the insoluble pigments formed as a result of alkaline phosphatase
activity. Slides were then evaluated as integrated densities of
staining by use of Scion Image, where total staining intensity was
measured (10).
Immune complex protein kinase assay.
Cells were washed twice with ice-cold PBS. Ice-cold RIPA lysis buffer
was added to cell monolayers and incubated on ice for 10 min. Cells
were scraped and transferred to Eppendorf tubes and further disrupted
by vortex. Cellular debris was pelletted, and the supernatant was
retained. Upon Bio-Rad Protein Assay quantitation of total protein
levels, 200-500 µg of total protein were coincubated with
0.2-2 µg of extracellular signal-regulated kinase (ERK)1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4°C. A 25-µl resuspended volume of protein G-agarose (Santa Cruz Biotechnology) was added and incubated at 4°C, rotating for 2-16 h. Immunoprecipitates were collected and rinsed with RIPA buffer.
Pellets were resuspended in 30 µl of appropriate kinase assay buffer
containing 10-1,000 ng of peptide substrate myelin basic protein
(MBP) and [
-32P]ATP (10 mCi/ml) and incubated
at 30°C for 30 min. Kinase reaction was terminated by addition of an
equal volume of 2× electrophoresis sample buffer and boiling for
2-3 min. Samples were analyzed by SDS-PAGE and autoradiography.
Statistical analysis.
All data are shown as means ± SE. Statistical analysis of results
was by Student's t-test or by analysis of variance.
Significant values were taken at P < 0.05. The mean,
SE, and P value measurements were performed using Excel
software (Microsoft, Port Redmond, WA).
 |
RESULTS |
Effect of PTHrP on indexes of osteoblast differentiation.
MG-63 osteoblast-like osteosarcoma cells, which exhibit characteristics
of early, immature osteoblasts, were incubated in the absence or
presence of 100 nM PTHrP-(1-34) for 0, 1, 3, 6, 8, 12, and 16 h. As shown in Fig.
1A, treatment with
PTHrP-(1-34) increased type I collagen mRNA
transcript levels within 6 h, and peak levels were reached after
8 h of treatment. Type I collagen levels were augmented
more than twofold at this time point compared with control, untreated
cells. Type I collagen levels decreased to basal by ~16 h. A second
marker of osteoblast differentiation, alkaline phosphatase, was
detected by histochemical reaction. After treatment of cells with 100 nM PTHrP-(1-34) for 6, 12, and 24 h, cells were
fixed, and alkaline phosphatase activity was detected by a
histochemical reaction. As seen in Fig. 1, B and C, PTHrP induced alkaline phosphatase activity, with highest
levels occurring at 24 h. Although staining density increased
slightly in untreated cells, the overall increase in staining in
treated cells was considerably greater, reaching levels as high as
sevenfold after 12 h of treatment. These results indicate that
PTHrP can induce indexes of differentiation in MG-63 osteoblastic
cells.

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of parathyroid hormone (PTH)-related peptide
(PTHrP) on MG-63 cell differentiation. Human osteoblast-like
osteosarcoma MG-63 cells were grown in 10% serum to 80% confluence
and then incubated in serum-free conditions. Cells were then treated
with vehicle or with 100 nM PTHrP-(1-34) for timed
intervals. A: total cellular RNA was extracted from MG-63
cells. Fifteen micrograms of total cellular RNA for each time point
(1-16 h) were electrophoresed onto a 1.1% agarose-formaldehyde
gel. Filters were probed with type I collagen and 18S cDNA to determine
the ratio of I collagen to 18S mRNA expression. B: alkaline
phosphatase activity in control and PTHrP-treated MG-63 cells was
detected by a histochemical reaction. Cells were fixed in
citrate-buffered acetone. Slides were then immersed in alkaline-dye
solution containing diazonium salts and incubated for 30 min. Cells
were stained with Mayer's hematoxylin solution for 10 min to detect
the insoluble pigments formed as a result of alkaline phosphate
activity. Slides were then evaluated microscopically. C:
alkaline phosphatase activity was quantified using the NIH Image-based
Scion Image analysis program. Quantification is presented as staining
density per field. Results represent means ± SE of 3 different
experiments. *Significant differences from control (P < 0.05).
|
|
Effects of inhibiting PKC on indexes of osteoblast differentiation.
To determine the involvement of PKC in PTHrP-mediated MG-63 cell
differentiation, cells were pretreated with chelerythrine chloride
(3, 7), a specific inhibitor of PKC, followed by coincubation with 100 nM PTHrP-(1-34) for 8 h,
the time of maximal induction of type I collagen transcript levels by
PTHrP treatment. As seen in Fig. 2,
inhibition of PKC by chelerythrine chloride treatment resulted in a
decrease in PTHrP-stimulated type I collagen levels in a dose-dependent
manner. Cell morphology (Fig. 3),
viability as determined by trypan blue dye exclusion (>95% viable),
and basal levels of type I collagen mRNA were unaffected by treatment of MG-63 cells with chelerythrine chloride alone. These results were
paralleled by a reduction in alkaline phosphatase levels. Pretreatment
with 5.0 µM chelerythrine chloride followed by coincubation with 100 nM PTHrP-(1-34) for 24 h resulted in alkaline
phosphatase levels that were comparable to levels in untreated cells
cultured for the same time period (Fig. 3). The ability of this
inhibitor to curtail the effects of PTHrP on MG-63 cell differentiation suggests that PKC activation is involved in this phenomenon.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of protein kinase C (PKC) inhibitor chelerythrine
chloride (Ch Cl) on type I collagen gene expression in MG-63 cells.
Top: MG-63 cells were grown in 10% serum to 80% confluence
and then incubated overnight in serum-free conditions (Ctl). Cells were
then pretreated with vehicle or Ch Cl for 1 h. Cells were then
treated with 100 nM PTHrP-(1-34) for 8 h. Ctl
represents the results of treatment with vehicle only for both the
pretreatment and treatment periods. Levels of type I collagen and
ratios of type I collagen to 18S mRNA were determined by Northern blot
analysis, as described in MATERIALS AND METHODS.
Bottom: results depict the ratios of type I collagen to 18S
mRNA and are expressed as %change relative to Ctl, which was assigned
a value of 100%. Bars represent means ± SE of 3 different
experiments. *Significant differences in the ratios from Ctl cells
(P < 0.05); **significant differences in the ratios
from PTHrP-only-treated cells (P < 0.05).
|
|

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of inhibitors of signal transduction on alkaline phosphatase
levels. A: MG-63 cells were grown in 10% serum to 80%
confluence on double-chamber slides and then incubated overnight in
serum-free conditions. Cells were pretreated with vehicle or PKA
inhibitor H-89 (30 µM) and PKC inhibitor Ch Cl (5.0 µM) for 1 h, phosphatidylinositol 3-kinase (PI 3K) inhibitor wortmannin (Wm; 100 nM) for 3 h, Ras inhibitor B1086 (5.0 µM), and mitogen-activated
protein kinase (MAPK) inhibitor PD-98059 (100 µM) overnight. The
cells were then treated with 100 nM PTHrP for 24 h. B:
alkaline phosphatase activity was quantified using the NIH Image-based
Scion Image analysis program. Quantification is presented as staining
density per field. *Significant differences in the ratios from Ctl
cells (P < 0.05); **significant differences in the
ratios from PTHrP-only-treated cells (P < 0.05).
|
|
Effects of inhibiting PKA on indexes of osteoblast differentiation.
In a number of cell types, PTHrP is known to be a strong activator of
adenylate cyclase via its stimulation of G
s. The cAMP generated then leads to activation of PKA. To determine whether activation of PKA is involved in PTHrP-induced MG-63 cell
differentiation, cells were pretreated with the PKA inhibitor H-89.
After incubation in serum-free medium for ~16 h, cells were
pretreated with H-89 at 15 and 30 µM concentrations for 1 h,
followed by coincubation with 100 nM PTHrP-(1-34).
Inhibition of PKA by H-89 treatment resulted in a dose-dependent
decrease in PTHrP-stimulated type I collagen mRNA levels (Fig.
4). The specificity of this response was
confirmed by the treatment of MG-63 cells with H-89 alone, which showed
little to no effect on basal type I collagen mRNA levels. Cell
viability by trypan blue dye exclusion (>94% viable) and morphology
(Fig. 3) were unaffected by H-89 at the doses indicated. These results
were paralleled by a reduction in alkaline phosphatase levels.
Pretreatment with 30 µM H-89 for 1 h, followed by coincubation with 100 nM PTHrP-(1-34) for 24 h, resulted in
levels of alkaline phosphatase that were comparable to those in
untreated cells (Fig. 3). The ability of this inhibitor to curtail the
effects of PTHrP on MG-63 cell differentiation suggests that PKA
activation is involved in this phenomenon.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of the PKA inhibitor H-89 on type I collagen gene
expression in MG-63 cells. Top: MG-63 cells were grown in
10% serum to 80% confluence and incubated overnight in serum-free
conditions. Cells were then pretreated with vehicle or H-89 for 1 h and then incubated with 100 nM PTHrP-(1-34) for
8 h. Ctl represents results of treatment with vehicle only for
both the pretreatment and treatment periods. Levels of type I collagen
and ratios of type I collagen to 18S mRNA were determined by Northern
blot analysis, as described in MATERIALS AND METHODS.
Bottom: results depict the ratios of type I collagen to 18S
mRNA and are expressed as %change relative to Ctl, which was assigned
a value of 100%. Bars represent means ± SE of 3 different
experiments. *Significant differences in the ratios from Ctl cells
(P < 0.05); **significant differences in the ratios
from PTHrP-only-treated cells (P < 0.05).
|
|
Effects of a G
s mutant on indexes of osteoblast
differentiation.
To confirm results obtained regarding PTHrP signaling via
G
s in PTHrP-mediated osteoblast differentiation, MG-63
cells were transiently transfected with a G
s triple
mutant. The G
s mutant used in our studies is designed to
stabilize a receptor-G
s-
-complex, effectively
blocking signaling from both G
s and 
. After
transient transfection, MG-63 cells were treated with 100 nM
PTHrP-(1-34) for 8 h. As seen in Fig.
5, in cells transfected with the
G
s mutant, PTHrP stimulation of type I collagen mRNA
levels was significantly reduced, confirming the involvement of
G
s in PTHrP-stimulated osteoblast differentiation and
suggesting the possible involvement of 
-subunits.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of expressing a dominant negative
G s triple mutant (G TM) on PTHrP-induced
type I collagen (1 collagen) gene expression in MG-63 cells.
Top: MG-63 cells were grown in 10% serum to semiconfluence.
Cells were then cultured in serum-free medium or transiently
transfected for 12 h with the empty vector (pcDNA1) or with vector
containing G TM. Cells were then cultured for 24 h
in serum-free medium. Untransfected and transfected cells were then
treated with 100 nM PTHrP-(1-34) or vehicle for
8 h. After stimulation with 100 nM PTHrP-(1-34),
total cellular RNA was extracted from untreated control and
experimental cells. Twenty micrograms of total cellular RNA were
electrophoresed onto a 1.1% agarose-formaldehyde gel. After transfer
of RNA, filters were probed with a 32P-labeled type I
collagen cDNA or with a 32P-labeled 18S RNA probe, as
described in MATERIALS AND METHODS. Bottom:
results depict the ratios of type I collagen to 18S mRNA and are
expressed as %change relative to Ctl, which was assigned a value of
100%. Bars represent means ± SE of 3 different experiments.
*Significant differences in ratios from vehicle-treated cells (Ctl)
(P < 0.05); **significant differences in ratios from
PTHrP-treated cells (P < 0.05).
|
|
Effects of inhibiting PI 3-kinase on indexes of osteoblast
differentiation.
To explore signal transduction components related to the 
-complex
that might be implicated in PTHrP-induced osteoblast differentiation, we examined the involvement of PI 3-kinase by using the chemical inhibitor wortmannin. In preliminary experiments, wortmannin was confirmed to have no effects on cellular viability by trypan blue dye
exclusion (>97%) or morphology (Fig. 3) at the doses indicated. Cells
were pretreated with wortmannin for 3 h, followed by coincubation with 100 nM PTHrP-(1-34). Type I collagen mRNA levels
were minimally affected in cells treated with wortmannin alone. The
ability of wortmannin to cause a dose-dependent decrease in
PTHrP-stimulated type I collagen levels (Fig.
6) suggests that PI 3-kinase is a component of the PTHrP-induced signaling involved in MG-63 cell differentiation. This was confirmed by the ability of wortmannin to
abrogate the PTHrP-mediated increase in alkaline phosphatase activity
after 24 h of coincubation (Fig. 3). Alkaline phosphatase activity
in the wortmannin-treated cells is comparable to basal, untreated cells
(Fig. 4). Taken together, these results suggest the involvement of PI
3-kinase in PTHrP-mediated osteoblast differentiation.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of the PI 3K inhibitor Wm on type I collagen gene
expression in MG-63 cells. Top: MG-63 cells were grown in
10% serum to 80% confluence and incubated overnight in serum-free
conditions. Cells were then pretreated with vehicle or Wm (50 nM, 100 nM) for 3 h. Cells were then treated with 100 nM
PTHrP-(1-34) for 8 h. Ctl represents results of
treatment with vehicle only for both the pretreatment and treatment
periods. Levels of type I collagen and ratios of type I collagen to 18S
mRNA were determined by Northern blot analysis, as described in
MATERIALS AND METHODS. Bottom: results depict
the ratios of type I collagen to 18S mRNA and are expressed as %change
relative to Ctl, which was assigned a value of 100%. Bars represent
means ± SE of 3 different experiments. *Significant differences
in the ratios from Ctl cells (P < 0.05). **significant
differences in the ratios from PTHrP-only-treated cells
(P < 0.05).
|
|
Effects of Ras inhibition on indexes of osteoblast differentiation.
Ras proteins are a major point of convergence of numerous signal
transduction pathways. Ras is required to be anchored to the plasma
membrane to function and must undergo the posttranslational addition of
a farnesyl group, which facilitates Ras insertion into the plasma
membrane (28, 36). We therefore examined the effects of
B1086, which is an inhibitor of the enzyme farnesyltransferase and
therefore inhibits Ras activity. In preliminary experiments, B1086 was
confirmed to have no effects of cellular viability as assessed by
trypan blue dye exclusion (>97%) or morphology (Fig. 3) at the doses
indicated. Overnight pretreatment of MG-63 cells with B1086 was
followed by 8 h of coincubation with 100 nM
PTHrP-(1-34). Total RNA was collected and subjected
to Northern blot analysis. Type I collagen mRNA levels were
significantly decreased in B1086-treated cells (Fig.
7), whereas type I collagen mRNA levels
were little affected in cells treated with B1086 alone. Histochemical
examination of alkaline phosphatase activity after identical treatment
conditions also revealed a significant decrease in this parameter in
B1086-treated cells (Fig. 3).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of the Ras inhibitor B1086 on type I collagen gene
expression MG-63 cells. Top: MG-63 cells were grown in 10%
serum to 80% confluence and then incubated overnight in serum-free
conditions. Cells were pretreated with vehicle or Ras inhibitor B1086
(5 µM, 10 µM) for 16 h. The cells were then treated with 100 nM PTHrP for 8 h. Ctl represents results of treatment with vehicle
only for both the pretreatment and treatment periods. Levels of type I
collagen and ratios of type I collagen to 18S mRNA were determined by
Northern blot analysis as described in MATERIALS AND
METHODS. Bottom: results depict the ratios of type I
collagen to 18S mRNA and are expressed as %change relative to Ctl,
which was assigned a value of 100%. Bars represent means ± SE of
3 different experiments. *Significant differences in the ratios from
Ctl cells (P < 0.05); **significant differences in the
ratios from PTHrP-only-treated cells (P < 0.05).
|
|
Effects of MAPK inhibition on indexes of osteoblast
differentiation.
The MAPK family of serine/threonine kinases includes extracellular
signal-regulated kinases (ERKs). Activation of the ERK group of MAP
kinases may occur via Ras and may involve stimulation of the enzyme
MEK, or MAPK kinase. MEK activates ERK MAPK directly and thus serves as
a point of control in that selective inhibition by the chemical
inhibitor of MEK, PD-98059 (2, 23), results in inhibition
of MAPK. To determine the involvement of MAPK in the PTHrP-mediated
induction of type I collagen expression, MG-63 cells were incubated
overnight with PD-98059. PD-98059 was confirmed to have no effects of
cellular viability by trypan blue dye exclusion (>97%) or morphology
(Fig. 3) at the doses indicated. After pretreatment, cells were
coincubated with 100 nM PTHrP for 8 h, and type I collagen levels
were determined by Northern blot analysis. A dose-dependent decrease
(Fig. 8) in PTHrP-stimulated type I
collagen gene expression was observed, implicating MAPK in
PTHrP-induced MG-63 differentiation. Cells treated with PD-98059 alone
showed negligible effects on type I collagen mRNA levels, confirming
the specificity of the response to PTHrP-induced increase of type I
collagen mRNA. These results were confirmed by measurement of alkaline
phosphatase activity in PD-98059-treated MG-63 cells (Fig. 3). Direct
measurement of the effects of PTHrP on MAPK activity in MG-63 cells was
also performed. PTHrP-(1-34) at 100 nM concentration
induced an increase in MAPK activity as measured by in vitro kinase
activity assay, as seen in Fig. 9. Peak
levels of MBP phosphorylation by MAPK were observed at 15 min, with
activity remaining significantly elevated at 20 and 25 min. By 30 min,
levels of MBP phosphorylation had returned to basal, untreated levels.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of the MAPK kinase (MEK) inhibitor PD-98059 on
type I collagen (a1 collagen) gene expression in MG-63 cells.
Top: MG-63 cells were grown in 10% serum to 80% confluence
and then incubated overnight in serum-free conditions. Cells were
pretreated with vehicle or PD-98059 (50 µM, 100 µM) for 16 h,
followed by PTHrP treatment for 8 h. Ctl represents results of
treatment with vehicle only for both the pretreatment and treatment
periods. Levels of type I collagen and ratios of type I collagen to 18S
mRNA were determined by Northern blot analysis, as described in
MATERIALS AND METHODS. Bottom: results depict
the ratios of type I collagen to 18S mRNA and are expressed as %change
relative to Ctl, which was assigned a value of 100%. Bars represent
means ± SE of 3 different experiments. *Significant differences
in the ratios from Ctl cells (P < 0.05); **significant
differences in the ratios from PTHrP-only-treated cells
(P < 0.05).
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of the PTHrP-(1-34) on MAPK
activity in MG-63 cells. Top: MG-63 cells were grown in 10%
serum to 80% confluence and then incubated overnight in serum-free
conditions. Cells were then treated with 100 nM
PTHrP-(1-34) for timed intervals. After stimulation
with PTHrP-(1-34), total cell lysates were collected
from untreated control and from experimental cells. Between 200 and 500 µg of total cellular protein were immunoprecipitated with
extracellular signal-regulated kinase-1 (ERK1) antibody.
Immunoprecipitates were incubated in reconstituted kinase reaction
buffer containing myelin basic protein (MBP) and
[32P]ATP, as described in MATERIALS AND
METHODS. Results represent means ± SE of 3 different
experiments. *Significant differences from time 0 (P < 0.05).
|
|
We next investigated effects of the various inhibitors of upstream
signaling on the ability of PTHrP to induce peak MAPK activity after 15 min of treatment (Fig. 10). All five
inhibitors inhibited the ability of PTHrP to induce MAPK activity,
thereby confirming the role of MAPK as a downstream target of these
signaling pathways.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 10.
Effect of inhibitors of signal transduction on MAPK
activity levels. Top: MG-63 cells were grown in 10% serum
to 80% confluence and then incubated overnight in serum-free
conditions. Cells pretreated with vehicle or PKA inhibitor H-89 (30 µM) and PKC inhibitor Ch Cl (5.0 µM) for 1 h, PI 3K inhibitor
Wm (100 nM) for 3 h, Ras inhibitor B1086 (5.0 µM), and MAPK
inhibitor PD-98059 (100 µM) overnight. The cells were then treated
with 100 nM PTHrP for 15 min. MAPK activity was then determined as
described in MATERIALS AND METHODS. Bottom:
results are expressed as %vehicle-only-treated cells (Ctl) and
represent means ± SE of 3 different experiments. *Significant
differences from Ctl (P < 0.05); **significant
differences from PTHrP-only-treated cells (P < 0.05).
|
|
 |
DISCUSSION |
Previous studies in our laboratory and others have demonstrated
the capacity of PTH and PTHrP to affect the differentiation of a number
of cell types, including osteoblasts. The current evidence indicates
that PTHrP can act as an autocrine or paracrine growth and/or
differentiation factor in a number of tissues (40-42). The existence of both PTHrP and the PTH/PTHrP receptor in bone and the
ability of a variety of bone-derived cells to produce PTHrP both in
culture and in vivo strongly suggest a local role in bone. Evidence for
such a role was also provided by the effects of targeted disruption of
the PTHrP gene in mice (19). Thus mice heterozygous for
the null mutation displayed haploinsufficiency and decreased bone
volume as the mice aged. Such observations, confirmed by targeted
overexpression of the same gene (38), suggest that PTHrP
may modulate the maturation and differentiation of osteoblasts.
Treatment of MG-63 cells with 100 nM PTHrP-(1-34) was
seen to induce increased expression of the osteoblast differentiation markers type I collagen and alkaline phosphatase. Although production of type I collagen is not exclusive to the differentiating osteoblast but is also produced by fibroblastic cells, type I collagen is considered a useful osteoblast differentiation marker when expressed in
an established sequence with other bone markers such as alkaline phosphatase (4). Both catabolic and anabolic effects of
PTHrP and PTH may be observed in bone via the PTH/PTHrP receptor
(8). Ishizuya et al. (14) have shown that
these discrepancies appear to be a function of exposure time, such that
intermittent exposure of cells to PTH-(1-34) of ~6
h will cause osteoblast differentiation. Treatment of MG-63
osteoblastic cells with PTHrP-(1-34) were consistent with these observations, in that levels of type I collagen mRNA were
seen to rise at ~6 h, peak at 8 h, and begin to decline with extended exposure time. These results suggest that there may exist an
inhibitory effect of prolonged exposure of osteoblasts to PTHrP. Subsequent experiments were therefore performed with 8-h incubation periods in keeping with these observations. The ability of the MG-63
cells to withstand prolonged exposure to each inhibitor was first
verified by trypan blue dye exclusion viability experiments as well as
by close observation for any adverse changes in cell morphology. It
seems possible that receptor desensitization due to internalization did
occur during this time interval, but this was not examined, and
signaling events leading to eventual differentiation would appear to
have been adequately initiated during the incubation times that were employed.
It is known that PTH/PTHrP receptor activation can lead to activation
of multiple G proteins, namely G
q and G
s,
with subsequent activation of phospholipase C (PLC) and adenylate
cyclase (25), respectively. Stimulation of PLC will result
in subsequent production of inositol 1,4,5-trisphosphate and
diacylglycerol (1, 2, 39), leading to mobilization
of calcium and PKC, respectively. Although there are varying reports
regarding the involvement of PLC in osteoblast differentiation, we have
seen that inhibition of PKC by treatment with chelerythrine chloride
can block induction of type I collagen and alkaline phosphatase.
Stimulation of the PTH/PTHrP receptor also results in activation of
adenylate cyclase followed by a rapid increase in intracellular cAMP
and subsequent activation of PKA. We show that the inhibition of
adenylate cyclase, by treatment with H-89, results in an abrogation of
the PTHrP-(1-34)-stimulated increase in type I
collagen mRNA expression and also interrupts the production of the
later marker of MG-63 cell differentiation, alkaline phosphatase.
Although activation of PKA and PKC is known to occur in minutes, the
ability of these short-term signals to influence such a late-developing
cellular phenomenon as differentiation is well documented in the
literature. Previous studies by Tsai et al. (35) show that
100 nM PTHrP induces cAMP in UMR106 osteosarcoma cells in minutes, with
subsequent occurrence of differentiation at later time points. In
several studies investigating the effects of PTH on osteoblast
activity, the PKC and PKA signaling pathways appear to be activated
simultaneously and seem to cooperate in the anabolic effects of the
shared receptor in bone cells. Cross talk between these two signal
transduction systems may therefore occur in osteoblasts after PTH
stimulation. Further studies to elucidate this possibility were also
undertaken by combined inhibitor treatment of MG-63 cells, but these
show additive, and not synergistic, effects (data not shown).
Although it has been well established that the PTH/PTHrP receptor can
signal through both G
s and G
q
(1), the question of PTHrP signal transduction via

-subunit-dependent pathways remains to be definitively answered.
The ability of the transient transfection of a G
s mutant
(29), which sequesters 
-subunits, to abrogate the
effects of PTHrP treatment on osteoblast differentiation markers may
implicate 
-signaling. However, because the dominant negative
G
s (8) mutant utilized also inhibits
signaling via the endogenous G
s subunit, the results
obtained from our experiment may have simply confirmed results obtained
by inhibition of adenylate cyclase by H-89. Nevertheless, data obtained
by chemical inhibition of PI 3-kinase are consistent with a role for
the 
-subunits in mediating some of the effects of PTHrP. Further
studies in which 
-subunits are selectively inhibited will be
required, but such studies are currently technically challenging.
PI 3-kinase is a heterodimeric cytosolic protein composed of an 85-kDa
regulatory subunit and a 110-kDa catalytic subunit (9). PI
3-kinase is stimulated by both G protein-coupled receptors and receptor
tyrosine kinases. Stephens et al. (31) first discovered that PI 3-kinase is specifically activated by purified 
-subunits. The ability of the chemical inhibitor of PI 3-kinase, wortmannin, to
cause a significant reduction in the expression of both osteoblast differentiation markers in our studies suggests that PI 3-kinase is
involved in PTHrP-mediated MG-63 cell differentiation. Because PI
3-kinase inhibition is seen to decrease 
-dependent MAPK
activation, it is commonly presumed that 
-subunits activate PI
3-kinase and thus initiate a cascade of events that leads to the
phosphorylation of the protein Shc. Whether Rac or other GTP-exchange
proteins are involved in PI 3-kinase-mediated pathways was not tested. Nevertheless, the interaction of 
-subunits with SH2-containing proteins can be mediated by PI 3-kinase, such that Shc complexes to the
SH2-containing adaptor protein Grb2. Grb2 can then stably associate
with the Ras guanine nucleotide exchange factor Sos. Ras is anchored to
the plasma membrane, where Sos can stimulate the exchange of GDP for
GTP, thereby leading to Ras activation. We have found that chemical
inhibition of Ras by treatment with B1086, a farnesyltransferase
inhibitor, also diminished the effect of PTHrP-(1-34)
on induction of both type I collagen levels and alkaline phosphatase levels.
One of the best-characterized downstream targets of Ras is the family
of MAPKs (20). MAPKs are a family of serine/threonine kinases involved in the transduction of cellular signals to the nucleus. These proteins regulate a vast range of cellular processes including proliferation, differentiation, transformation, inflammation, apoptosis, and cytoskeletal rearrangement. Previous data also support the pivotal role of MAPKs in the induction of phenotypic indexes of osteoblast differentiation (34, 43). Upstream
of MAPK, the activation of Ras will lead to the sequential activation of Raf1, a serine/threonine kinase, and MEK, whose substrates are the
ERK1 and ERK2 of the MAPK family. It has also been shown that
activation of MAPK can occur independently of Ras activation, presumably via direct activation of Raf or MEK by PKA (37)
or PKC. Treatment of MG-63 cells with the MAPK inhibitor PD-98059 interrupted PTHrP stimulation of indexes of osteoblast differentiation, thereby suggesting that MAPK is involved in this process. The ability
of PTHrP to induce MAPK activity in MG-63 osteoblastic cells
complements the results of the studies on the effect of PD-98059 on
PTHrP induction of markers of differentiation. Furthermore, the
capacity of multiple inhibitors of upstream signaling to diminish MAPK
activity assay seems to suggest that these multiple signaling pathways
converge to some extent at MAPK. Whether other members of the MAPK
family, such as p38K or c-Jun NH2-terminal protein kinase,
are also involved in PTHrP signaling remains to be determined.
It is probable, then, that PTH/PTHrP activation in response to PTHrP
initiates parallel signaling via G
s leading to PKA
activation, via G
q leading to activation of PKC, and via

leading to activation of PI 3-kinase and Ras (Fig.
11). Convergence of these
signals may then occur via activation of MAPK. Further study is now
required to extend these observations to other osteoblastic cell models and to determine whether other kinases, such as the stress-activated kinases, play a role in PTHrP-mediated osteoblast differentiation. The
elucidation of the signaling pathways involved in the effects of PTHrP
on enhancing differentiation of osteoblastic cells clearly has
important implications with regard to our understanding of the actions
of this hormone in normal physiology and particularly with respect to
its anabolic actions in bone.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 11.
Schematic diagram of PTHrP receptor signaling involved in
regulating differentiation in MG-63 osteoblastic cells. PLC,
phospholipase C; IP3, inositol triphosphate; DAG, diacylglycerol; PIP2,
phosphatidylinosotol phosphate-2.
|
|
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. A. Rabbani, Calcium Research Laboratory, Royal Victoria Hospital, 687 Pine Ave. West, Rm. H4.72, Montreal, QC H3A 1A1 Canada
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 January 2001; accepted in final form 26 April 2001.
 |
REFERENCES |
1.
Abou-Samra, AB,
Juppner H,
Force T,
Freeman MW,
Kong XF,
Schipani E,
Urena P,
Richards J,
Bonventre JV,
Potts JT, Jr,
Kronenberg HM,
and
Segre GV.
Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related-peptide from rat osteoblast-like cells: a single receptor stimulates intracellular accumulation of both cAMP and inositol trisphosphates and increases intracellular free calcium.
Proc Natl Acad Sci USA
89:
2732-2736,
1992[Abstract].
2.
Aklilu, F,
Gladu J,
Goltzman D,
and
Rabbani SA.
Role of mitogen-activated protein kinases in the induction of parathyroid hormone-related protein.
Cancer Res
60:
1753-1760,
2000[Abstract/Free Full Text].
3.
Araujo dos Santos, A,
and
Giestal de Araujo E.
The effect of PKC activation on the survival of rat retinal ganglion cells in culture.
Brain Res
853:
338-343,
2000[ISI][Medline].
4.
Aubin, JE,
Liu F,
Malaval L,
and
Gupta AK.
Osteoblast and chondroblast differentiation.
Bone
17:
77-83,
1995.
5.
Birnbaumer, L.
Which G protein subunits are the active mediators of signal transduction?
Trends Pharmacol Sci
8:
209-211,
1987[ISI].
6.
Burtis, WJ,
Brady TG,
Orloff JJ,
Ersbak JB,
Warrell RP, Jr,
Olson BR,
Wu TL,
Mittnick ME,
Broadus AE,
and
Stewart AF.
Immunochemical characterization of circulating parathyroid hormone-related protein in patients with humoral hypercalcemia.
N Engl J Med
322:
1106-1112,
1990[Abstract].
7.
Cai, YC,
Ma L,
Fan GH,
Zhao J,
Jiang LZ,
and
Pei G.
Activation of N-methyl-D-aspartate receptor attenuates acute responsiveness of
-opioid receptors.
Mol Pharmacol
51:
583-587,
1997[Abstract/Free Full Text].
8.
Canalis, E,
McCarthy TL,
and
Centrella M.
Differential effects of continuous and transient treatment with parathyroid hormone-related peptide (PTHrP) on bone collagen synthesis.
Endocrinology
126:
1806-1812,
1990[Abstract].
9.
Carter, AN,
and
Downes CP.
Phosphatidylinositol 3-kinase.
Proc Natl Acad Sci USA
88:
7908-7912,
1992[Abstract].
10.
Choi, HJ,
Hyun MS,
Jung GJ,
Kim SS,
and
Hong SH.
Tumor angiogenesis as a prognostic predictor in colorectal carcinoma with special reference to mode of metastasis and recurrence.
Oncology
55:
575-581,
1998[ISI][Medline].
11.
Clapham, DEG
Protein 
subunits.
Annu Rev Pharmacol Toxicol
37:
167-203,
1997[ISI][Medline].
12.
Guise, TA,
Yoneda T,
Yates AJ,
and
Mundy GR.
The combined effect of tumor-produced parathyroid hormone-related protein in patients with humoral hypercalcemia of cancer.
N Engl J Med
322:
1106-1112,
1993[Abstract].
13.
Iiri, T,
Bell SM,
Baranski TJ,
Fujita T,
and
Bourne HR.
A Gsa mutant designed to inhibit receptor signaling through Gs.
Proc Natl Acad Sci USA
96:
499-504,
1999[Abstract/Free Full Text].
14.
Ishizuya, T,
Yokose S,
Hori M,
Noda T,
Suda T,
Yoshiki S,
and
Yamaguchi A.
Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells.
J Clin Invest
99:
2961-2970,
1997[Abstract/Free Full Text].
15.
Juppner, H,
Abou-Samra AB,
Freeman M,
Kong XF,
Schipani E,
Richards J,
Kolakowski LF, Jr,
Hock J,
Potts JT, Jr,
Kronenberg HM,
and
Segre GV.
A G protein-linked receptor for parathyroid hormone, and parathyroid hormone-related peptide.
Science
254:
1024-1026,
1991[ISI][Medline].
16.
Kaiser, SM,
Laneuville P,
Bernier SM,
Rhim JS,
Kremer R,
and
Goltzman D.
Enhanced growth of a human keratinocyte cell line induced by antisense RNA for parathyroid hormone-related peptide.
J Biol Chem
267:
13623-13628,
1992[Abstract/Free Full Text].
17.
Kaiser, SM,
Sebag M,
Rhim JS,
Kremer R,
and
Goltzman D.
Antisense-mediated inhibition of parathyroid hormone-related peptide production in a keratinocyte cell line impedes differentiation.
Mol Endocrinol
8:
139-147,
1994[Abstract].
18.
Kano, J,
Sugimoto T,
Fukase M,
and
Chihara K.
The direct involvement of cAMP-dependent protein kinase in the regulation of collagen synthesis by parathyroid hormone (PTH) and parathyroid hormone-related peptide in osteoblast-like osteosarcoma cells (UMR-106).
Biochem Biophys Res Commun
184:
525-529,
1992[ISI][Medline].
19.
Karaplis, AC,
Luz A,
Glowacki J,
Bronson RT,
Tybulewicz VL,
Kronenberg HM,
and
Mulligan RC.
Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene.
Genes Dev
8:
277-289,
1994[Abstract].
20.
Khosravi-Far, R,
White MA,
Westwick JK,
Solski PA,
Chrzanowska-Wodnicka M,
Van Aelst L,
Wigler MH,
and
Der CK.
Oncogenic Ras activation of Raf/mitogen-activated protein kinase-independent pathways is sufficient to cause tumorigenic transformation.
Mol Cell Biol
16:
3923-3933,
1996[Abstract].
21.
Logothetis, DE,
Kurachi Y,
Galper J,
Neer EJ,
and
Clapman DE.
The 
subunits of GTP-binding proteins activate the muscarinic K+ channel in heart.
Nature
325:
321-326,
1987[ISI][Medline].
22.
Lopez-Ilasca, M.
Signaling from G-protein-coupled receptors to mitogen-activated protein (MAP)-kinase cascades.
Biochem Pharmacol
56:
269-277,
1998[ISI][Medline].
23.
Mayer, AM,
Brenic S,
and
Glaser KB.
Pharmacological targeting of signaling pathways in protein kinase C-stimulated superoxide generation in neutrophil-like HL-60 cells: effect of phorbol ester, arachidonic acid and inhibitors of kinase(s), phosphatase(s) and phospholipase A2.
J Pharmacol Exp Ther
279:
633-644,
1996[Abstract].
24.
McCauley, LK,
Koh AJ,
Beecher CA,
Cui Y,
Rosol TJ,
and
Franceschi RT.
PTH/PTHrP receptor is temporally regulated during osteoblast differentiation and is associated with collagen synthesis.
J Cell Biochem
61:
638-647,
1996[ISI][Medline].
25.
Morris, AJ,
and
Malbon CC.
Physiological regulation of G protein-linked signaling.
Physiol Rev
79:
1373-1430,
1999[Abstract/Free Full Text].
26.
Moseley, JM,
Kubota M,
Diefenbach-Jagger H,
Wettenhall RE,
Kemp BE,
Suva LJ,
Rodda CP,
Ebeling PR,
Hudson PJ,
Zajac JD,
and
Martin TJ.
Parathyroid hormone-related protein purified from a lung cancer cell line.
Proc Natl Acad Sci USA
84:
5048-5052,
1987[Abstract].
27.
Partridge, NC,
Bloch SR,
and
Pearman AT.
Signal transduction pathways mediating parathyroid hormone regulation of osteoblast gene expression.
J Cell Biochem
55:
321-327,
1994[ISI][Medline].
28.
Schlessinger, J,
and
Ullrich A.
Growth factor signaling by receptor tyrosine kinases.
Neuron
9:
383-391,
1992[ISI][Medline].
29.
Skoglund, G,
Mehboob HA,
and
Holz GG.
Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin gene cAMP response element.
Diabetes
49:
1156-1164,
2000[Abstract].
30.
Soifer, NE,
Van Why SK,
Ganz MB,
Kashgarian M,
Siegel NJ,
and
Stewart AF.
Expression of parathyroid hormone-related protein in the rat glomerulus and tubule during recovery from renal ischemia.
J Clin Invest
92:
2850-2857,
1993[ISI][Medline].
31.
Stephens, L,
Smrcka A,
Cooke FT,
Jackson TR,
Sternweis PC,
and
Hawkins PT.
A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein beta gamma subunits.
Cell
77:
83-93,
1994[ISI][Medline].
32.
Strewler, GJ,
Stern PH,
Jacobs JW,
Eveloff J,
Klein RF,
Leung SC,
Rosenblatt M,
and
Nissenson RA.
Parathyroid hormone-like protein from human renal carcinoma cells: structural and functional homology with parathyroid hormone.
J Clin Invest
80:
1803-1807,
1987[ISI][Medline].
33.
Sugden, PH,
and
Clerk A.
Regulation of the ERK subgroup of MAP kinase cascades through G protein-coupled receptors.
Cell Signal
9:
337-351,
1997[ISI][Medline].
34.
Takeuchi, Y,
Suzawa M,
Kikuchi T,
Nishida E,
Fujita T,
and
Matsumoto T.
Differentiation and transforming growth factor-1
receptor down-regulation by collagen 11
211
integrin interaction is mediated by focal adhesion kinase and its downstream murine osteoblastic cells.
J Biol Chem
272:
29309-29316,
1997[Abstract/Free Full Text].
35.
Tsai, JA,
Bucht E,
Stark A,
Sjostedt U,
and
Torring O.
Parathyroid hormone-related protein (1-37) induces cAMP response in human osteoblast-like cells.
Calcif Tissue Int
62:
250-254,
1998[ISI][Medline].
36.
Ullrich, A,
and
Schlessinger J.
Signal transduction by receptors with tyrosine kinase activity.
Cell
61:
203-212,
1990[ISI][Medline].
37.
Verheijen, MHG,
and
Defize LHK
Parathyroid hormone activates mitogen-activated protein kinase via a cAMP-mediated pathway independent of Ras.
J Biol Chem
272:
3423-3429,
1997[Abstract/Free Full Text].
38.
Weir, EC,
Philbrick WM,
Amling M,
Neff LA,
Baron R,
and
Broadus AE.
Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation.
Proc Natl Acad Sci USA
93:
10240-10245,
1996[Abstract/Free Full Text].
39.
Wu, S,
Pirola CJ,
Green J,
Yamaguchi DT,
Okano K,
Juppner H,
Forrester JS,
Fagin JA,
and
Clemons TL.
Effects of N-terminal, midregion, and C-terminal parathyroid hormone-related peptides on adenosine 3',5'-monophosphate and cytoplasmic free calcium in rat aortic smooth muscle cells and UMR-106 osteoblast-like cells.
Endocrinology
133:
2437-2444,
1993[Abstract].
40.
Wysolmerski, JJ,
Broadus AE,
Zhou J,
Fuchs E,
Milstone LM,
and
Philbrick WM.
Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.
Proc Natl Acad Sci USA
91:
1133-1137,
1994[Abstract].
41.
Wysolmerski, JJ,
McCaughern-Carucci JF,
Daifotis AG,
Broadus AE,
and
Philbrick WM.
Overexpression of parathyroid hormone-related protein or parathyroid hormone in transgenic mice impairs branching morphogenesis during mammary gland development.
Development
121:
3539-3547,
1995[Abstract/Free Full Text].
42.
Wysolmerski, JJ,
and
Stewart AF.
The physiology of parathyroid hormone-related protein: an emerging role as a developmental factor.
Annu Rev Physiol
60:
431-460,
1998[ISI][Medline].
43.
Xiao, G,
Jiang D,
Thomas P,
Benson MD,
Guan K,
Karsenty G,
and
Franceschi RT.
MAPK pathways activate and phosphorylate the osteoblast-specific transcription factor, Cbfa1.
J Biol Chem
275:
4453-4459,
2000[Abstract/Free Full Text].
Am J Physiol Endocrinol Metab 281(3):E489-E499
0193-1849/01 $5.00
Copyright © 2001 the American Physiological Society