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J Biol Chem, Vol. 275, Issue 2, 999-1006, January 14, 2000
From the Endocrine Division, Lilly Research Laboratories,
Indianapolis, Indiana 46285
Osteocalcin is a major noncollagenous protein
component of bone extracellular matrix, synthesized and secreted
exclusively by osteoblastic cells in the late stage of maturation, and
is considered indicator of osteoblast differentiation. Osteocalcin expression is modulated by parathyroid hormone (PTH) and a variety of
other factors. The cAMP-dependent protein kinase pathway
has been shown previously to have an essential role in PTH signaling and regulation of osteocalcin expression. To determine the extent to
which other pathways may also participate in osteocalcin expression, we
used rat and human osteoblast-like cell lines to generate stably transfected clones in which the osteocalcin promoter was fused to a
luciferase reporter gene. These clones were examined for their
responsiveness to agents known to activate or interfere with protein
kinase A (PKA)- and protein kinase C (PKC)-dependent pathways. We have found that forskolin, cAMP, and PTH, as well as
insulin-like growth factor I (IGF-I) and basic fibroblast growth factor, all were effective in activating the osteocalcin promoter. Phorbol 12-myristate 13-acetate (PMA) was also a strong inducer of the
promoter, indicating that PKC plays a role in expression of
osteocalcin. In combination with PTH or forskolin, the effect of PMA
was additive to synergistic. Calphostin C, a selective inhibitor of
PKC, decreased the PMA-, PTH-, and IGF-I-induced luciferase activity in
a dose-dependent manner; a PKA inhibitor, H-89, also
blocked the induction by PTH and IGF-I but not by PMA. We conclude that
regulation of osteocalcin transcription is mediated by both
PKA-dependent and PKC-dependent mechanisms and
that the respective kinases reside on a linear or convergent pathway.
Extracellular matrix proteins play an important role in the
organization, architecture, and differentiated function of bone (1). A
major component of the matrix is osteocalcin, a 5-kDa Osteocalcin genes have been cloned from several species including rat,
mouse, and human (1, 4, 5). The expression of osteocalcin is regulated
by a variety of factors, including parathyroid hormone
(PTH)1 (6), 1,25-dihydroxy
vitamin D3 (4, 7), estrogens (8), glucocorticoids (9),
growth factors (10, 11), and cAMP (3, 6, 10). The osteocalcin promoter
region contains a number of potential regulatory sequences that may be
responsive to these factors. Also present are sequences that could
confer species and tissue specificity (12, 13).
Parathyroid hormone is a calcium-regulating peptide that plays a
significant role in the maintenance of bone function (14). A once-daily
administration of PTH-(1-34) results in increased bone mineral density
in normal and osteopenic bone in humans and animals (15). This increase
in bone density is correlated with enhanced osteoblast activity,
leading to increased bone formation (16). While the mechanism of
osteoblast activation by PTH is not fully understood, it is well
established that the hormone action on target cells is mediated by
binding of PTH to a G-protein-coupled receptor (17). Binding is
followed by activation of adenylyl cyclase, an increase in cAMP level,
and activation of cAMP-dependent protein kinase A (PKA). At
the same time, phospholipase C (PLC) may also be activated, leading to
the release of Ca2+ and induction of a protein kinase C
(PKC)-associated pathway (18, 19). Mutations causing inactivation or
constitutive activation of PTH receptors are associated with profound
genetic defects in humans (20, 21).
Our previous studies have shown that PTH activates osteocalcin
transcription, at least in part, via the cAMP-dependent PKA pathway (6). These results are consistent with the presence of cAMP
regulatory sequences within the osteocalcin gene promoter (6). However,
since this promoter contains additional potential response elements,
its activation by PTH may involve additional signal transduction
pathways. In this report, we present evidence that activation of
PTH-mediated osteocalcin transcription involves both PKA- and
PKC-dependent pathways.
Reagents and Media--
Rat PTH-(1-38); human PTH-(1-31),
PTH-(1-34), PTH-(13-34), PTH-(53-84), and PTH-(1-84); bovine
PTH-(3-34) and PTH-(7-34); and human PTH-related protein-(1-34) were
obtained from Bachem (Terrance, CA). Stocks of the polypeptides were
prepared in a PTH diluent (1 mM HCl, 0.15 M
NaCl containing 1 mg of bovine serum albumin/ml). Except where
specified, the rat PTH-(1-38) peptide was used in the experiments
described below. Calphostin C, okadaic acid, forskolin (FSK),
1,9-dideoxy-FSK, dibutyryl-3',5'-cAMP, phorbol 12-myristoyl 13-acetate
(PMA), and H-89 were obtained from Alexis Corp. (San Diego, CA). The
compounds were dissolved in Me2SO and diluted as needed
with PTH diluent. Human insulin-like growth factor I (IGF-I) and human
basic fibroblast growth factor (FGF) were from Sigma; both were
prepared in PTH diluent. Media components, Geneticin (G418), and fetal
bovine serum (FBS) used for cell cultivation were purchased from Life
Technologies, Inc.
Reporter Vectors, Osteocalcin Probe, RNA Isolation, and RT-PCR
Analysis of Osteocalcin Gene Expression--
Plasmids containing the
rat or human osteocalcin gene promoter fragments were constructed using
the promoterless pGL-2 basic luciferase gene reporter vector (Promega
Corp., Madison, WI) as a backbone (6). The rat and the human promoter
sequences (1.154 and 0.844 kilobase pairs, respectively) were generated
by PCR on the basis of published information (rat promoter,
GenBankTM accession number M23637, nucleotides 1-1154;
human promoter, M34013, nucleotides 1-844). The PCR fragments were
recovered in pCR®II TA cloning vector (Invitrogen Corp.,
Carlsbad, CA), excised with KpnI and XhoI, and
subcloned into pGL-2 digested with KpnI and XhoI.
These plasmids (pRaOCN-luc and pHuOCN-luc) were used to establish
stable luciferase-expressing clones in ROS17/2.8 and SaOS-2 cell lines, respectively.
Northern Blot and RT-PCR Analysis of Osteocalcin Gene
Expression--
The osteocalcin probe was prepared by 32P
labeling of a full-length osteocalcin DNA obtained by PCR from a rat
cDNA library generated previously in this laboratory. The RNA for
Northern analysis was extracted from ~80% confluent ROS17/2.8 cells
using Ultraspec-IITM solution from Biotecx Laboratories
(Houston, TX) and enriched for the poly(A)+-containing
fraction by purification on an Oligotex column (Qiagen, Valencia, CA).
Manufacturers' instructions were followed in each case. The RNAs were
fractionated on a 1% agarose-formaldehyde gel, transferred to a nylon
membrane, and probed with the osteocalcin-specific probe. A 465-base
pair fragment of ubiquitin carrier protein DNA produced from a rat
cDNA library by PCR was used to normalize the signal. For the
RT-PCR experiments, the ROS17/2.8 cells were grown to confluence,
starved in serum-free medium containing 0.1% bovine serum albumin, and
treated with various forms of PTH. The RNAs were extracted using
Ultraspec-IITM, and 5 µg of total RNA were used for first
strand cDNA synthesis (SuperScriptTM; Life
Technologies, Inc.). The product (1-2 µl) was amplified by 10 cycles
of PCR using primers specific for osteocalcin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Aliquots of PCRs were
fractionated on a 1% agarose gel, transferred to a nylon membrane, and
probed with radiolabeled osteocalcin and GAPDH DNA. Quantitation was
carried out with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Quantitation of Secreted Osteocalcin--
A slightly modified
procedure of van Leeuwen et al. (22) was used to follow
osteocalcin production. ROS17/2.8 cells were seeded in six-well culture
dishes (4.5 × 105 cells/well) in Ham's F-12 medium
containing 10% FBS (Hyclone, Logan, UT) and 2 mM glutamine
and incubated at 37 °C under a 5% CO2, 95% air
atmosphere. After 24 h, the medium was aspirated and replaced with
the F-12 medium containing 2% serum. PTH-(1-38) was added at a
concentration of 10 Establishment of Stable Clones Expressing OCN-Luciferase
Activity--
Rat (ROS17/2.8) and human (SaOS-2) osteoblast-like cell
lines were used to generate stable transfectants. Initially, cells were
transiently transfected with either the OCN reporter vectors (see
below) or the promoterless pGL-2 vector control, which established that
FSK or PTH-(1-38) was effective in eliciting OCN-luciferase activation. The cells were maintained in Ham's F-12 or Dulbecco's modified Eagle's medium/F-12 medium (3:1), respectively, supplemented with 2 mM glutamine and 10% FBS. The cells were grown to
~90% confluence, trypsinized, and inoculated into T25 flasks at a
density of 5 × 105 cells/flask. After 24 h, the
attached cells were transfected with a mixture (1:1 molar ratio) of the
reporter vector (pRaOCN-luc or pHuOCN-luc) and pcDNA3.1 plasmid
(Invitrogen), carrying a neomycin resistance gene (a total of 3 µg of
DNA). Transfections utilizing the LipofectAMINE reagent (Life
Technologies, Inc.) were performed according to the manufacturer's
instructions. Geneticin-resistant clones capable of growth in the
presence of the antibiotic (1 mg/ml) were isolated over a period of
3-4 weeks, expanded, and assayed for luciferase activity as described below.
Transient Transfections--
SaOS-2 cells were transfected with
OCN-luc plasmids by the calcium phosphate method as described
previously (6).
Luciferase Assays--
Selected clones were maintained in T150
flasks in appropriate medium containing geneticin (1 mg/ml), grown to
80-90% confluence, harvested, and inoculated into 96-well plates at a
density of 5 × 104 cells/well. Each well contained a
total volume of 300 µl of medium. The plates were incubated for
24 h at 37 °C under a 5% CO2, 95% air atmosphere,
and the cells were synchronized by an additional period of 12-20 h of
incubation in 200 µl of the same medium containing 0.1% FBS.
Following this starvation/synchronization period, the compounds under
study were added to triplicate wells, the PTH diluent serving as the
vehicle control. Unless stated otherwise, the length of each treatment
was 8 h. After the treatments, the solutions were aspirated, and
the cells were lysed for 15 min in 60 µl of the luciferase lysis
buffer (Roche Molecular Biochemicals). At this point, the luciferase
activity could be measured in lysates, or the plates, protected with
sealing film, could be stored at
The assays were performed using the Luciferase Reporter Gene Assay kit
from Roche Molecular Biochemicals Corp. Aliquots (20 µl) of cell
lysates were pipetted into wells of white opaque microtiter plates
(Dynex Technologies, Chantilly, VA) and placed in an automated injection MLX microtiter plate luminometer. The luciferase reaction mix
(100 µl) was injected sequentially into the wells. The light signals
generated in the reactions were integrated over an interval of 2 s, and the resulting luminescence values were used as a measure of
luciferase activity (relative units).
PTH Stimulates Osteocalcin mRNA Accumulation and Protein
Synthesis--
Initially, we evaluated the time course of OCN
induction in response to PTH treatment. This was evaluated by treating
ROS17/2.8 cells with PTH-(1-38) and then evaluating either the OCN
mRNA (Fig. 1A) or OCN
secreted into the medium. Northern blot analysis demonstrates that the
level of osteocalcin mRNA began to rise within 1 h of PTH
administration and continued to increase for at least 24 h (Fig
1A). The mRNA abundance increased over 4-fold in 24 h as compared with the untreated control.
The effect of PTH on osteocalcin secretion was also confirmed by
determination of protein secreted into the medium at various time
intervals as well as in response to various concentrations of
PTH-(1-38). As shown in Fig. 1 (B and C),
osteocalcin levels increased in a time- and dose-dependent
manner, with 72 h of PTH treatment showing the best response to
PTH-(1-38). Thus, PTH elevates both steady-state osteocalcin mRNA
level and the quantity of osteocalcin protein secreted by the cells.
Effects of Truncated Forms of PTH on OCN Expression--
We next
examined the effect of truncated PTH peptides on the level of OCN
expression (mRNA abundance and protein secretion). ROS17/2.8 cells
were treated with various truncated forms of PTH and the relative
abundance of OCN mRNA was measured by RT-PCR, using GAPDH mRNA
as an internal control. The results (Fig.
2A) demonstrate that only
those PTH fragments (PTH-(1-34), -(1-31), -(1-38), and -(1-84))
that are known to elicit cAMP accumulation (23) stimulated OCN
expression. Other peptides (PTH-(3-34), -(13-34), and -(53-84)) that
do not bind to PTH receptor and do not elevate cAMP level were
ineffective. These mRNA level changes were further confirmed by
quantitating the level of OCN secreted into the medium (Fig.
2B).
Establishment of Stable Geneticin-resistant OCN-Luciferase
Transfectant Clones Responsive to PTH--
To determine if PTH exerted
its effects at the transcriptional level, we analyzed OCN promoter
activity in stable transfectant cell lines generated in ROS17/2.8 and
SaOS-2 cells with OCN-luciferase plasmids. Examination of independently
and randomly selected geneticin-resistant clones revealed that these
clones exhibited a wide variation in both basal luciferase activity and
PTH-(1-38) responsiveness (Table I). For
subsequent experiments, we chose two rat clones (RG15 and RG24) and two
human clones (SG12 and SG40) that consistently maintained both basal
and PTH-inducible luciferase expression.
PTH Time and Dose Response of OCN-luc in Stable Transfectant
Lines--
Both ROS17/2.8 and SaOS-2 transfectants exhibited
time-dependent induction of reporter gene activity by
PTH-(1-38) (Fig. 3), with the highest
level of luciferase activity observed at about 8 h (4-30-fold
induction, depending on the clone). These results are in agreement with
our previous observations on transiently transfected SaOS-2 cells (6).
The PTH dose response was also examined in the four stable clones (Fig.
4). The four cell lines show a similar
pattern of activation by the hormone, being half-maximally stimulated
at PTH concentrations of 7.5 × 10 Activation of Osteocalcin Promoter-driven Luciferase Gene by
Various Truncated Forms of PTH--
We next confirmed that in stable
cell lines, distinct domains of PTH evoked specific OCN responses
(Refs. 6 and 23; Fig. 2). The PTH analogs PTH-(1-31) and PTH-(1-38),
which are known to raise intracellular cAMP concentration and stimulate
the PKA pathway (6, 23), were also able to stimulate reporter gene activity in the stable transfectant clones (Fig.
5). A truncated PTH-related protein,
PTHrP-(1-34), was nearly as active as PTH-(1-38). In contrast,
PTH-(3-34) and PTH-(7-34), the analogues incapable of elevating
intracellular cAMP level (23) were unable, at the concentration used,
to activate OCN-luc. These results are consistent with the known
involvement of PKA in PTH-induced osteocalcin expression. Both rat- and
human osteosarcoma-derived stable clones responded similarly in this
test.
Treatments That Increase Intracellular Accumulation of cAMP Enhance
OCN Transcription--
To further evaluate the involvement of the PKA
pathway in PTH-induced OCN-luc expression in stably transfected cells,
we examined the effect of various concentrations of FSK, a well known
activator of adenylyl cyclase (6, 24), and dibutyryl cAMP. As shown in
Fig. 6A, SaOS-2 cells exposed
to dibutyryl cAMP (10 Phorbol 12-Myristate 13-Acetate Is a Potent Activator of
Osteocalcin Promoter--
Because the PTH receptor can signal through
phospholipase C and thus, indirectly, through the PKC pathway (18, 19),
we asked whether activators of PKC could stimulate OCN transcription. As shown in Fig. 8, when SG12 cells were
treated with PMA, the expression of OCN-luc was significantly
stimulated (28-fold increase over control level). A combination of PTH
and PMA or of FSK and PMA was at least additive, if not synergistic
(136- and 166-fold increase, respectively, as compared with PTH alone
(63-fold) and FSK alone (45-fold)). Very similar results were obtained
with SaOS-2 cells transiently transfected with OCN-luc plasmid (not shown).
Independent Activation of OCN Transcription by PTH and PMA--
To
determine whether PTH and PMA can act through independent pathways to
activate osteocalcin transcription, we asked if cells pretreated with
one activator are able to respond to a subsequent challenge with the
same or the other agent. SG12 cells were incubated with either PTH or
PMA for 12 h and then challenged with either PMA or PTH for
additional 8 h. For comparison, the standard 8-h treatment and
continuous 20-h exposure were also evaluated. The results (Fig.
9) show that cells exposed to PTH or PMA
remained responsive to subsequent challenge with PMA or PTH,
respectively, although the amplitude of response was different. While
the long term exposure to PMA significantly diminished response to
either agent, the desensitization or down-regulation of the
PKC-dependent pathway reduced but, importantly, did not
abrogate the PTH-dependent signaling. This indicates that
desensitization of one pathway still allows the other pathway to
mediate OCN transcription.
Involvement of Protein Kinase C in PTH Activation of OCN
Promoter--
To obtain further confirmation of the involvement of
protein kinases in PTH signaling, we have analyzed the SG12 clone
responses to a selective protein kinase C inhibitor, calphostin C (25). As shown in Fig. 10A,
calphostin C inhibited the PTH-inducible OCN-luc expression in a
dose-dependent fashion. The compound was also effective in
abolishing PMA-stimulated OCN-luc expression, indicating a major role
for the PKC pathway in regulating osteocalcin transcription. The known
PKA inhibitor, H-89 (26), which inhibited the PTH-induced expression
(6), had no effect on PMA-induced OCN-luc expression (Fig.
10B).
In order to further define whether or not both the PKA and PKC pathways
are activated independently, we evaluated the effects of H-89 or
calphostin C on OCN mRNA levels after stimulation with either PTH
or cAMP. The results (Fig. 11) show
that a 24-h PTH treatment (lane 2) resulted in a
4.9-fold stimulation of OCN mRNA levels, in comparison with control
(lane 1). Treatments with PTH plus H-89
(lane 3) or PTH plus calphostin C
(lane 4) resulted in at least a partial reduction
in OCN mRNA levels (2.9- and 2.4-fold, respectively, in comparison
with control). A treatment with cAMP resulted in a 3.5-fold increase in
OCN mRNA level (lane 5), while H-89
co-treatment with cAMP reduced the OCN mRNA levels to 1.6-fold (lane 6), which was comparable with the reduction
obtained with H-89 alone (lane 8). More
importantly, a calphostin C co-treatment with cAMP was ineffective
(lane 7). These results establish that (a) PTH stimulation of OCN transcription is mediated by
either PKA or PKC pathways, (b) cAMP-mediated activation of
OCN involves primarily, if not exclusively, PKA pathway, and
(c) both of the pathways act independently of each
other.
Insulin-like Growth Factor I Stimulates OCN-luc
Transcription--
IGF-I, an important effector of bone remodeling,
has been shown to up-regulate osteocalcin synthesis in osteosarcoma
cells (27). Furthermore, its activity is enhanced by PTH and cAMP and
is down-regulated by estrogen (28). Therefore, we determined if IGF-I
stimulated OCN-luc expression and if this growth factor acted through
PKA- or PKC-dependent pathways or both in the SG12 clone.
Cells were treated with IGF-1 and/or PTH in the absence or presence of
H-89 or calphostin C. The results (Fig.
12) demonstrate that IGF-I
significantly stimulated OCN-luc expression and that PTH had an
additive effect on this stimulation. Both H-89 and calphostin C were
highly effective in inhibiting IGF-1 activation of OCN-luc. These
results further establish that IGF-1 activation of OCN-luc also is
likely to utilize both PKA and PKC signaling cascades. Similar results
(additivity with PTH activation and inhibition by H-89) were obtained
with FGF (data not shown).
Osteocalcin is a small protein synthesized exclusively by mature
osteoblastic cells and secreted into the extracellular space, where it
becomes a major component of the mineral-binding extracellular matrix
(1). We have been interested in cellular mechanisms governing the
uniquely tissue-specific and temporally restricted expression of this
abundant protein. Previously, we showed that PTH-(1-34), a peptide
that stimulates bone formation, also enhanced osteocalcin
transcription. This effect of PTH required activation of a
cAMP-dependent PKA pathway (6). Since PTH acts through a
receptor that signals via both PKA and PLC pathways, we asked if OCN
could be additionally or synergistically activated by agents that
stimulate PKC.
We have demonstrated PTH induction of osteocalcin by (a)
quantitation of secreted protein, (b) mRNA analysis by
RT-PCR and Northern blot, and (c) analysis of OCN promoter
activity in rat and human osteoblast-like osteosarcoma cell lines that
were stably transfected with OCN promoter coupled to a luciferase
reporter gene. For the most part, there was considerable agreement
among all of these approaches, leading to the conclusion that
osteocalcin expression is subject to regulation utilizing both PKA and
PKC pathways and that PTH-(1-34) activation of OCN also is mediated via both of the pathways. A previous study has shown that cAMP and PTH
increase OCN mRNA levels by increasing mRNA stability (29). Our
results establish that PTH activation of osteocalcin also involves
transcriptional activation of the OCN promoter (this study and Ref.
6)
Most of the studies were done in stable transfectants that contain
either rat OCN-luc or human OCN-luc promoters. While a range of basal
and PTH-inducible activities was observed among the various clones
(Table I), two isolates of each species were chosen for their robust
responsiveness to PTH. Both the rat and human stable transfectants
exhibited a time- and dose-dependent OCN activation in
response to PTH-(1-34) treatment. These responses (Figs. 3 and 4) are
consistent with the kinetics of endogenous osteocalcin gene expression
in ROS17/2.8 cells (Fig. 1), although some differences were observed
between the clones with respect to the maximal level of response to the
hormone. In general, ROS17/2.8 transfectants appeared to exhibit a
somewhat higher basal level of OCN-luc expression.
While PTH treatment of untransfected cells results in
time-dependent accumulation of the osteocalcin mRNA
(Fig. 1), some of the stable transfectants we have generated do not
follow the same kinetics when PTH-induced OCN-luciferase expression is
measured (Fig. 3, clones RG24 and SG12). In these clones, the level of luciferase activity declines with prolonged incubation. These results
are similar to our previous study using transient transfection (6). We
interpret this behavior as resulting from clonal variation among the
isolates (e.g. a decreased stability of the luciferase protein in some but not other clones). Irrespective of the differences in the level of response, all isolates responded to PTH, FSK, cAMP, and
PMA.
The results presented here reinforce previous observations that
cAMP-dependent PKA is required for PTH activation of
osteocalcin expression (6). This was shown by RT-PCR analysis of
ROS17/2.8 cells treated with various truncated forms of PTH,
determination of OCN secretion in ROS17/2.8 cells, and OCN-luc
activation in stable transfectant lines (Figs. 2 and 5). In each case,
only the N-terminally intact PTH peptides that elicit cAMP accumulation (PTH-(1-31), -(1-34), -(1-38), and -(1-84)) could induce OCN-luc response. Moreover, dibutyryl cAMP added directly to culture medium stimulated the OCN-luc expression, OCN secretion (Fig. 6, A
and B), and OCN mRNA levels (Fig. 11). Finally,
forskolin, a nonspecific activator of adenylyl cyclase, stimulated
OCN-luc expression in both ROS17/2.8 and SaOS-2-derived clones (RG15
and SG12, respectively). A forskolin analog, 1,9-dideoxyforskolin, that
does not activate adenylyl cyclase had no effect on OCN-luciferase
expression (results not shown).
In osteoblasts, PTH acts through a cognate G-protein-coupled
seven-transmembrane domain receptor (PTHR1) that recognizes both PTH
and PTHrP (30). The signal from the receptor is transmitted through the
participation of both PKA and PLC pathways. PKA activation has been
demonstrated in both normal and osteoblast-like cells, while PLC
activation, as measured by calcium release and inositol 1,4,5-trisphosphate production, could only be shown in cells stably transfected to express a high copy number of the PTH receptor gene
(31-33). These observations leave uncertain the relative contribution of the PLC and phosphoinositol pathway to PTH signaling, but they do
suggest a possible role. Importantly, calcium release could be
demonstrated in the absence of cAMP production (31).
PLC activation leads to generation of inositol 1,4,5-trisphosphate and
diacylglycerol, in turn leading to Ca2+ mobilization and
activation of PKC. We evaluated activators of PKC (PMA, IGF-I, and FGF)
for their ability to modulate OCN-luciferase expression in osteosarcoma
cells. The data indicate that PMA was able to activate OCN
independently of the PKA-mediated mechanism. Several pieces of evidence
point to the importance of PKC in effecting osteocalcin expression.
First, PMA actions were at least additive or synergistic with those of
PTH or FSK (Fig. 8). Second, H-89 treatment did not abolish stimulation
by PMA (Fig. 10B), showing that activation of PKC can
activate osteocalcin promoter activity independently of the PKA
pathway. Third, cells desensitized to PTH-(1-38) by an extended
treatment with the hormone were still able to respond robustly to PMA
stimulation and vice versa (Fig. 8). Finally, the growth factors (IGF-I
and FGF), known for their actions through both PKA- and
PKC-dependent pathways, strongly stimulated OCN-luc
expression in a manner additive with PTH (Fig. 12 and data not shown).
The experiments with protein kinase inhibitors (H-89 and calphostin C)
revealed that each of the inhibitors could block activation of the OCN
promoter. Thus, calphostin C, a highly selective inhibitor of protein kinase C (34), was able to abolish OCN-luc transcription elicited by either PTH or PMA (Figs. 10 and 11)
or IGF-I (Fig. 12) but was ineffective in blocking
cAMP-stimulated OCN expression (Fig. 11). On the other hand, a PKA
inhibitor, H-89, while strongly diminishing OCN-luc expression when
PTH, cAMP, FSK, or IGF-I (Figs. 7, 11, and 12) was the inducing agent,
had no effect on PMA-induced OCN-luc expression (Fig. 9). Collectively,
these results suggest that both PKA and PKC activation pathways are
involved in PTH signaling. However, it is not clear whether these
activation steps are independent of or interdependent on each other.
Most of the evidence suggests that they act independently. First, H-89
blocks cAMP- or PTH-stimulated, but not PMA-stimulated, OCN
transcription. Second, calphostin C blocks PMA- or PTH-stimulated, but
not cAMP-stimulated, OCN transcription. Thus, the simplest
interpretation of these data is that activation of either of the
pathways leads to OCN transcription, and PTH activation of OCN utilizes
both of the pathways. However, an additional possibility is that
although the initial signals generated by PTH and PMA differ, the
PTH-dependent and PKC-dependent transduction
pathways converge onto an element common to both (e.g.
adenylyl cyclase) that serves to transmit the signals necessary to
promote expression of the osteocalcin gene. This notion is consistent
with the well documented ability of G-proteins, PKA, and PKC to
activate differentially various isoforms of adenylyl cyclase (35). The
synergy between PTH and PMA and between PMA and FSK (Fig. 8) is also
consistent with such an interpretation.
The extent to which PTH signals via PKC remains an open question.
However, it is quite possible that factors that elicit PKC signaling
(such as IGF-1 and FGF-2) may work in concert with PTH to enhance OCN
transcription. Taken together, our data point to the existence of
multiple regulatory circuits involved in transcriptional control of
osteocalcin expression. Which of these circuits is also responsible for
the temporal and tissue-specific pattern of osteocalcin biosynthesis
remains to be elucidated.
The role of OCN in bone mineralization is not clear. OCN is produced by
osteoblasts during a late stage of maturation. Recent study suggests
that OCN knockout animals may develop higher bone density, suggesting
that it may serve to inhibit mineralization (36). PTH promotes
osteoblast differentiation in vivo and also causes an
increase in OCN mRNA levels (16). Bone formation and maturation are
complex processes and clearly involve several steps in tandem (12).
They could involve an initial expansion of osteoblasts, followed by
formation of early osteoid matrix. Once an appropriate amount of matrix
is laid down, it is certainly possible that mineralization could
proceed utilizing enzymes such as alkaline phosphatase or matrix
proteins such as bone sialoproteins. If OCN is indeed a true inhibitor
of mineralization, it may serve as a termination signal for bone
formation once the bone matrix is fully matured (37). PTH may play a
role in all aspects of the process, including OCN production.
*
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.
The abbreviations used are:
PTH, parathyroid
hormone;
PTHrP, parathyroid hormone-related protein;
OCN, osteocalcin;
OCN-luc, osteocalcin promoter fused to luciferase reporter gene;
FSK, forskolin;
PMA, phorbol 12-myristate 13-acetate;
H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide;
FBS, fetal bovine serum;
PKA, cAMP-dependent protein
kinase;
PKC, protein kinase C;
PLC, phospholipase C;
FGF, human basic
fibroblast growth factor;
IGF-I, human insulin-like growth factor I;
PCR, polymerase chain reaction;
RT-PCR, reverse transcriptase-PCR;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Activation of Osteocalcin Transcription Involves Interaction of
Protein Kinase A- and Protein Kinase C-dependent
Pathways*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carboxyglutamic acid-containing noncollagenous protein of uncertain function; one of its possible roles may be to control nucleation of
hydroxyapatite crystals (2). Osteocalcin is synthesized and secreted
exclusively by mature osteoblasts during the late stage differentiation
and mineralization (1, 3) and, thus, may serve both as a marker of bone
formation and as an indicator of the maturation stage of osteoblastic
cell populations.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 to 109 M,
and the incubation continued for an additional 72-h period, unless
otherwise indicated. The control well received vehicle diluent.
Osteocalcin concentrations in the culture supernatants were determined
using a rat osteocalcin radioimmunoassay kit (Biomedical Technologies,
Stoughton, MA).
80 °C for assay at a later time.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PTH stimulates osteocalcin production in
ROS17/2.8 cells. A, time course of PTH-induced
osteocalcin mRNA synthesis. The cells were grown to 80% confluence
in T225 flasks, washed with phosphate-buffered saline, and incubated in
0.1% FBS-containing medium for 8 h before the addition of PTH
(5 × 10 8 M). At the indicated times
(h), the cells were harvested, and RNA was extracted as described under
"Experimental Procedures."
Poly(A)+ RNAs (2 µg/lane) from PTH-treated cells were
fractionated on an agarose gel, transferred to a nylon membrane, and
probed with a 32P-labeled osteocalcin cDNA probe. The
relative increases (-fold induction over the 0-h control, normalized
for ubiquitin carrier protein mRNA) in mRNA abundance following
PTH administration were as follows: 1 h, 1.25×; 6 h, 2.38×;
24 h, 4.13×. B, time course of induction of
osteocalcin protein production and secretion. ROS17/2.8 cells were
grown as described under "Experimental Procedures," treated with or
without PTH-(1-38) (108 M). The accumulated
osteocalcin protein secreted into the culture supernatants was assayed
at the indicated time intervals by radioimmunoassay. During the time of
treatment, there was very little difference in cell density between
control and PTH-treated cultures (2.86 versus 3. 17 × 105 cells/dish). C, osteocalcin secretion at
various PTH Concentrations. ROS17/2.8 cells were treated with indicated
concentrations of PTH-(1-38) and OCN secretion was assayed after a
72-h treatment.
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Fig. 2.
Effects of various analogues of PTH on
osteocalcin expression. A, RT-PCR analysis of OCN
mRNA. ROS17/2.8 cells were inoculated into six-well plates (5 × 105 cells/well) and grown to confluence. The medium was
replaced with serum-free medium containing 0.1% bovine serum albumin,
and the cells were incubated for 24 h. The various analogues of
PTH were added (each at 10 8 M), and
incubations continued for an additional 24-h period. The RNAs were
extracted and processed for RT-PCR as described under
"Experimental Procedures." The PCR products
were separated by gel electrophoresis, transferred to a nylon membrane,
and probed with 32P-labeled rat cDNA probes for
osteocalcin and GAPDH. The autoradiogram (inset) was scanned
in a densitometer, and the osteocalcin/GAPDH ratios were determined.
With the exception of the control in the graph, the
numbering in the gel lanes
(inset) corresponds to the PTH analogues indicated in the
graph (lane 2, PTH-(1-34);
lane 3, PTH-(1-31), etc.). Untreated control is
not shown in the inset and was similar to lane
5, and for the purpose of quantitation, this was considered
as 1. B, effect of PTH analogues on OCN secretion. All
fragments were added to ROS17/2.8 cells at 108
M, and the OCN levels were determined after 72 h of
treatment. There were no discernible differences in cell number during
the duration of the treatment.
OCN-luciferase activity and inducibility in stable transfectant clones
9 to 5 × 1010 M and reaching a maximum level at 5 × 107 to 5 × 105 M. The
activity in clone RG15 showed no leveling off even at the highest dose
(5 × 105 M) of PTH used in this
experiment.
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Fig. 3.
Time course of induction of OCN-luc activity
by PTH-(1-38). The selected ROS17/2.8 and SaOS-2-derived clones
(RG and SG, respectively) in microtiter wells
were treated with PTH (5 × 10 8 M) for
the indicated length of time. The additions of the hormone were
staggered in such a way that all cells were incubated for the total of
24 h. The medium was washed, the cells were extracted with
luciferase lysis buffer, and the enzyme activity was measured as
described under "Experimental
Procedures."
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Fig. 4.
Effect of various concentrations of
PTH-(1-38) on OCN-luc activity. Cells in microtiter dishes were
treated with indicated amounts of PTH for 8 h, lysed, and assayed
for expression of luciferase activity as described above.
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Fig. 5.
Effect of different analogues of PTH on
OCN-luc activity. Each analogue was added at 5 × 10 8 M concentration, and the cells (RG15 and
SG12) were exposed to the peptides for 8 h.
6 to 103
M), or FSK (107 to 105
M), demonstrated a concentration-dependent
increase in OCN-luc expression. We also evaluated whether treatment
with these agents resulted in enhanced OCN protein secretion. The
results (Fig. 6B) show that PTH, FSK, and cAMP treatment of
ROS 17/2.8 cells resulted in a 5-7-fold stimulation of OCN secretion
during the 72-h treatment period. We also evaluated OCN-luc expression
in the presence of H-89, a selective inhibitor of the PKA pathway. The
results (Fig. 7) of a transient
transfection experiment using a single FSK concentration
(105 M) and various concentrations of H-89
(0.1-10 µM) indicate a concentration-dependent inhibition of FSK-stimulated
OCN-luc expression by H-89. Collectively, these results further confirm
that activation of the PKA pathway leads to activation of OCN
expression.
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Fig. 6.
Osteocalcin induction by forskolin and cyclic
AMP. A, concentration-dependent activation
of OCN-luc promoter. The SG12 cells were treated with FSK
(10 5 M) for 4 h, while PTH-(1-38)
(5 × 108 M) and dibutyryl cAMP
(2.5 × 104 M) were present for 8 h. B, osteocalcin secretion stimulated by forskolin and
cAMP. Osteocalcin secretion was measured after 72 h of treatments
with PTH 1-38 (5 × 108 M), FSK
(105 M), or dibutyryl cAMP (2.5 × 104 M).
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Fig. 7.
Effect of various doses of H-89 on
FSK-stimulated OCN promoter activity. SaOS-2 cells were
transiently transfected with OCN-luc promoter, as described (6), using
the calcium phosphate precipitation method, and after 24 h, they
were further treated with FSK (10 5 M) with
the indicated concentrations of H-89. The enzyme activity was
determined and normalized to cellular protein, and it is expressed as
-fold increase versus control.
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Fig. 8.
Synergistic effects of PTH, FSK, and PMA on
osteocalcin promoter activity. Stable transfectant clone SG12 was
treated with 5 × 10 9 M PTH,
105 M FSK, or 107 M
PMA or with pairwise combinations of each for 8 h. Cell lysates
were analyzed for luciferase activity as described under
"Experimental Procedures."
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Fig. 9.
Desensitization of SG12 clone to PTH and
PMA. SG12 cultures (5 × 104 cells/well) in
medium containing 0.1% FBS were either untreated (control) or
subjected to treatments diagrammed as A, B, or
C. In A, the cells were exposed to PTH (5 × 10 8 M, 28-fold induction), PMA
(106 M, 26-fold), or both (56-fold), for
8 h (indicated as the standard treatment). In B, the
cultures were exposed to PTH or PMA for 12 h and were then
challenged with an additional dose of either PTH or PMA for 8 h.
There was no change of medium at any of the time points; thus, the
originally present test compounds had not been removed upon additional
treatments, and the total length of exposure to the first agent was
20 h (12 + 8 h). The procedure shown in C involved
continuous 20-h exposure to PTH and/or PMA. Note that even after
prolonged treatment with PMA, the cells could be stimulated by PTH
(6-fold induction over control level).
View larger version (20K):
[in a new window]
Fig. 10.
Dose-dependent inhibition of
PTH- or PMA-induced OCN-luc expression by calphostin C. SG12 cells
were grown in a microtiter plate and starved in 0.1% FBS as described
under "Experimental Procedures." Calphostin C was added to the
cells at the concentrations shown (A), and the incubation
continued for 2 h at 37 °C. During this time, the plate was
illuminated with an incandescent light, which is necessary to achieve
full inhibitory potency (36). After 2 h, 5 × 10 8 M PTH or 106 M
PMA was added, and the treatment continued for a further 8-h period. In
B, the inhibitor H-89 (105 M) is
shown to be ineffective against PMA-induced OCN-luc expression (compare
with Fig. 11).
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Fig. 11.
Effect of H-89 and calphostin C on
cAMP-stimulated osteocalcin mRNA levels. The ROS17/2.8 cells
were incubated with PTH-(1-38) (10 8 M PTH)
or dibutyryl cAMP (2.5 × 104 M) with or
without H-89 (105 M) or calphostin C
(106 M) for 6 h. The total cellular RNA
was analyzed by Northern blot, using 32P-labeled rat probes
for OCN or GAPDH. The signal intensity was quantitated by densitometric
scanning, and the ratios of OCN/GAPDH levels were determined. The
values are expressed as -fold induction relative to control.
Lane 1, Dulbecco's modified Eagle's medium
control; lane 2, PTH; lane
3, PTH plus H-89; lane 4, PTH plus
calphostin C; lane 5, dibutyryl cAMP;
lane 6, cAMP plus H-89; lane
7, dibutyryl cAMP plus calphostin C; lane
8, H-89 alone; lane 9, calphostin C
alone.
View larger version (20K):
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Fig. 12.
Effect of insulin-like growth factor I on
osteocalcin promoter activity. The SG12 cells were incubated with
human IGF-I for 8 h in the presence or absence of PTH (5 × 10 10 M), and luciferase activity was
measured. Where indicated, H-89 (105 M) or
calphostin C (107 M) was added 2 h
before PTH and the growth factor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
To whom correspondence should be addressed: Endocrine Research,
Lilly Research Laboratories, Corporate Center, Eli Lilly and Company,
Indianapolis, IN 46285. Tel.: 317-276-6929; E-mail:
Chandra@lilly.com.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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