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Endocrine Unit (P.D., K.C., R.S., H.J., F.R.B.), Massachusetts General Hospital, and Harvard Medical School, Boston Massachusetts 02114; and Chugai Pharmaceutical Company (N.I.), Ltd., Shizuoka, Japan
Address all correspondence and requests for reprints to: Paola Divieti M.D., Ph.D., Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: divieti{at}helix.mgh.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In rat osteoblastic cell lines, fragments from within the sequence
PTH(35–84), which cannot activate PTH1Rs, regulate expression of
alkaline phosphatase, osteocalcin, collagen 
1(I), and IGF binding
protein-5 (14, 15, 16). Direct effects of PTH C-fragments upon
osteoclasts and osteoclast progenitors (17) and upon
expression of collagen 1(I) and 1(X) expression in hypertrophic
chondrocytes (18) also have been observed. Several such
fragments induce cytosolic free calcium transients in human fetal
hypertrophic chondrocytes (19). Direct physical evidence
of a putative receptor (CPTHR) with binding specificity for C-terminal
PTH sequences was obtained by cross-linking of the peptide
125I-[Tyr34]hPTH(19–84)
(which does not bind to PTH1Rs) to 40-kDa and 90-kDa proteins in ROS
17/2.8 rat osteoblastic cells (6).
Collectively, these observations suggest that CPTHRs are expressed normally in bone and cartilage and that they may be involved in physiologic control of cell differentiation and function in these tissues. The problem of renal osteodystrophy is of particular interest in this regard, as PTH C-fragments normally are cleared mainly by the kidneys and thus accumulate to very high levels in blood during renal failure (7, 9, 12, 13). Also, hPTH (7–84) potently reduces the calcemic action of intact PTH(1–84) at concentrations much lower than those required for binding to the PTH1R (20).
To establish a model system in which to address the possible functions of CPTHRs in bone, we isolated, from cultures of enzymatically dispersed primary fetal murine calvarial cells, those that expressed CPTHRs in greatest abundance. To eliminate confounding effects of coexpressed PTH1Rs, these cells were derived from fetuses in which most exons encoding the PTH1R had been completely ablated by gene targeting (21, 22). These homozygous mice also were bred to ubiquitously express a transgene encoding a temperature-sensitive mutant SV40 large-T antigen (tsTAg), which enabled isolation of conditionally transformed clonal cell lines (21, 22). We report here that the subpopulation of calvarial-derived bone cells that expresses the highest levels of CPTHRs exhibits morphologic and molecular features characteristic of osteocytes, the most terminally differentiated cells of the osteoblast lineage (23, 24). Initial findings indicate that activation of CPTHRs may play a role in regulating osteocytic cell survival and intercellular communication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell isolation and culture
Cells were isolated by enzymatic digestion from calvarial bones
of 18.5 day-old tsA58(+)/PTH1R(-/-) fetuses, as previously described
(21). Animals were maintained in facilities operated by
the Massachusetts General Hospital Center for Comparative Resources in
accordance with the National Institute of Health Guide for the Care and
Use of Laboratory Animals and were employed using protocols approved by
the Institutional Animal Care and Use Committee.
The calvarial bones were dissected aseptically and sequentially
digested in 0.5 ml of -MEM containing 0.1% BSA, 1 mM
CaCl2 and 1 mg/ml of collagenase (type I and II;
ratio 1:3) (Worthington Biochemical Corp., Freehold, NJ).
Bones were sequentially digested six times for 20 min each at 37 C on a
rocking platform at 90 oscillations per minute under 5%
CO2 in air. Cells were cultured at 33 C in a
humidified atmosphere (95% air/5% CO2) using
growth medium [-MEM containing 10% FBS (lot no. 1011961 Life Technologies, Inc.) and 1% penicillin-streptomycin (PS)].
HeLa and BHK21 cells were obtained from Dr. Joel Habener (Molecular Endocrine Unit, Massachusetts General Hospital, Boston, MA) and NIH-3T3 cells were provided by Dr. Gino Segre (Endocrine Unit, Massachusetts General Hospital, Boston, MA). MS-1 cells are clonal conditionally immortalized murine bone marrow stromal cells previously isolated in our laboratory (25).
Radioligand binding
The [Tyr34]hPTH(19–84) peptide was
radioiodinated with Na[125I] (2000 Ci/mmol) by
the chloramine-T method and purified by HPLC, as previously described
(6). For binding experiments, cells were plated in 24-well
dishes at 100,000 cells/ml and cultured at 33 C for 7–14 days.
Confluent monolayers then were washed with 0.5 ml binding buffer [100
mM NaCl, 5 mM KCl, 2 mM
CaCl2, 50 mM Tris-HCl (pH 7.8) plus
5% heat-inactivated horse serum] before incubation with
125I- [Tyr34]hPTH(19–84)
(100,000–200,000 cpm/well) in 0.5 ml binding buffer for 4 h at 15
C. Receptor number was ascertained by Scatchard analysis, using
[Tyr34]hPTH(19–84) or hPTH(1–84) as competing ligand.
Cellular protein was measured using the BCA protein assay kit
(Pierce Chemical Co., Rockford, IL) and was found to
average 0.33 mg/106 cells and 0.47
mg/106 cells for F cells and C cells,
respectively. In some experiments, cells were washed with acidic buffer
(50 mM glycine, 150 mM NaCl, pH 4.0) to remove
residual bound ligand, before addition of radioligand.
cAMP accumulation
Cells were rinsed twice with assay buffer (135 mM
NaCl, 6 mM KCl, 1 mM
MgCl2, 2.8 mM glucose, 1.2
mM CaCl2, and 20 mM
HEPES, pH 7.4) and then incubated for 15 min at 37 C with the same
buffer containing 0.1% heat inactivated BSA, 1 mM
isobutylmethylxanthine (IBMX), and agonist, conditions under which cAMP
accumulation was found to be linear with time for at least 15 min. The
buffer then was rapidly aspirated, the plates were frozen in liquid
nitrogen, and the frozen cells subsequently were thawed directly into
0.5 ml of 50 mM HCl. Cell-associated cAMP in the acid
extracts was measured using an RIA kit (NEN Life Science Products). Results were expressed as picomoles of cAMP produced
per well over 15 min.
Northern blot and RT-PCR analysis
Cells were plated in 10-cm dishes and cultured at the
appropriate temperature in growth medium. Total RNA was extracted using
the TRIreagent method (Sigma) and quantified by UV
absorbance. Fifteen micrograms of total RNA was separated by 1%
agarose-gel electrophoresis in the presence of formaldehyde and
transferred to a nylon membrane (Bio-Rad Laboratories, Inc. Hercules, CA) using standard procedures. Membranes were
hybridized with 32P-labeled cDNA probes labeled
by random primer extension, including the 1 subunit of human type I
collagen, mouse osteopontin, mouse alkaline phosphatase, rat
osteocalcin, mouse connexin 43 (Cx43), cbfa-1/osf-2 and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Rat osteocalcin was a
gift of Dr. Marie Demay, mouse Cx43 was a gift of Dr. David Paul and
cbfa-1/osf-2 was a gift of Dr. Gerard Karsenty.
RT-PCR was performed on total RNA using the SuperScript Preamplification System from Life Technologies, Inc. with a Peltier Thermal Cycler (MJ Research, Inc., Watertown, MA). The following pairs of primers were used for the cDNA amplification: CD44: 5'-CAAGTTTTGGTGGCACACAGC, 3'-GGTTAAGGAAGCTACCTGGC; mouse cbfa-1/osf-2; 5'-TGGAAGGGATGAAAGGCTGC, 3'-CGGTGTTTACCACAGCAGGT and alkaline phosphatase 5'-CCGTTCTTTCTCTATTC, 3'-ACAGATGGGTCCCACCG as previously reported (24, 25). The annealing temperature for CD44 and cbfa1/osf2 was 58 C, and the reaction was repeated for 30 cycles; for alkaline phosphatase the annealing temperature was 54 C, and the reaction was repeated for 35 cycles. PCR products were electrophoresed on 1.5% agarose gels and visualized using ethidium bromide staining.
Von Kossa staining
Cells, plated in six-well dishes, were maintained at 33 C for
3–4 days, until confluent, before refeeding with fresh medium
containing 10 mM ß-glycerophosphate and 50 µg/ml
ascorbic acid and transferring to nonpermissive conditions (39 C).
Cells were refed with fresh medium twice per week. After 3–4 weeks,
the presence of mineralized nodules was assessed by von Kossa staining,
as previously described (21). Briefly, after fixation in
95% ethanol for 15 min at 37 C, cells were gradually rehydrated with
water and stained with 5% silver-nitrate for 1 h at 37 C. Cells
then were exposed to incandescent light (100 W) for 15–30 min.
Western blot analysis and immunocytochemical staining
For Western blot analysis, confluent cell monolayers were washed
twice with PBS before lysis by incubation in ice-cold RIPA buffer (50
mM Tris-HCl, pH7.2, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate) for 5 min at 4 C. Lysates were centrifuged
at 14,000 rpm for 10 min, supernatants were collected and passed ten
times through a 22 gauge needle, and proteins were separated by
SDS-PAGE (12% acrylamide gel) and transblotted to Hybond
nitrocellulose membranes using standard procedures. Membranes were
first blocked for 30 min at room temperature in Tris-buffered saline,
pH 8.0, plus 0.05% TWEEN-20 (TBS-T) with 5% powdered milk solution,
incubated for 60 min with a 1:1000 dilution of first antibody specific
for murine Cx43 (Zymed Laboratories, Inc., San Francisco,
CA). The membranes were washed several times in TBS-T and then
incubated for 60 min with the second antibody/enzyme conjugate (HRP
antimouse, IgG diluted 1:5000). Immunoreactive bands were detected by
enhanced chemiluminesence assay (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) according to the
manufacturer’s instructions.
For immunocytochemical analysis, cells were plated on glass chamber slides at 72,000 cells/cm2, cultured for 2 days at 33 C and then incubated for an additional 4–6 days at 39 C. Cells were treated with vehicle alone (0.1% TFA) or with the appropriate hormone for the time indicated. The immunocytochemical staining was performed as previously described (24). Briefly, cells were fixed in 3% paraformaldehyde/2% sucrose in PBS and incubated for 5 min. in 0.05% Triton-X 100 diluted in PBS. The fixed slides were blocked in 5% BSA in TBS-T buffer for 2 h at room temperature. The cells then were incubated with a 1:125 dilution of anti-Cx43 monoclonal antibody for 30 min at room temperature. The bound antibody was detected using a Vectastain ABC kit, followed by staining with VIP substrate according to manufacturer’s instructions (Vector Laboratories, Inc. Burlingame, CA). Counterstaining was performed using 0.5% methyl green. In some cases, anti-Cx43 antibody was omitted to control for nonspecific staining.
Apoptosis
The pyknotic fragmentation of nuclei typical of apoptotic cells
was detected using Hoechst-33258 fluorescent dye (Sigma
Co., St. Louis, MO). Briefly, cells were cultured on glass coverslips
and maintained in culture at nonpermissive conditions for 4–6 days in
-MEM supplemented with 2.5% of FBS and 1% PS. After exposure to
the test hormones for 6 h, culture medium was aspirated and cells
were fixed in 4% paraformaldehyde in phosphate buffered solution and
then stained with Hoechst-33258 (2.5 mg/ml) for 5 min at room
temperature. Cells were washed and mounted with glycerol:PBS (9:1,
vol/vol). The stained nuclei were visualized by fluorescence microscopy
using a DAPI filter. In some experiments, apoptotic cells were detected
by the terminal deoxynucleotidyl transferase-mediated nick end labeling
(TUNEL) reaction, using the In situ Cell Death Detection Kit
from Roche Molecular Biochemicals (Indianapolis, IN)
following the manufacturer’s instructions.
Trypan blue staining was used for routine quantification of cell death,
as it previously was shown to correlate well with apoptosis in an
osteocytic cell line (26). Briefly, cells were plated at
50,000 cells/well in 24-well dishes at 33 C and shifted to
nonpermissive conditions (39 C) after 2 days. Cells were grown at 39 C
for an additional 4–6 days in -MEM supplemented with 2.5% of FBS
and 1% PS and then treated with different hormones for an additional
16–18 h. Nonadherent cells were combined with adherent cells that were
released from the cultures with trypsin-EDTA, centrifuged, and
resuspended in 0.1% trypan blue solution. The percentage of cells
exhibiting both nuclear and cytoplasmic staining was determined using a
hemocytometer.
Statistical analysis
All results were expressed as the mean ± SD.
Each experiment was repeated at least twice. Significance of
differences between treatment and control groups was assessed by
Student’s t test using Bonferroni correction.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify cells with high expression of CPTHRs, single colonies were
isolated from the mixed F1 population by limiting dilution and then
screened for specific binding of
125I-[Tyr34]
hPTH(19–84). Among twenty subclones isolated, three clones, initially
designated C1, C14, and C59 (i.e. C cells) were selected for
further characterization on the basis of their distinctly high specific
binding of the
125I-[Tyr34] hPTH(19–84)
radioligand. Like the previously described osteoblastic clonal cell
lines (21), these three C cells did not express any other
species of Gs-linked PTH receptors, as demonstrated by the absence of a
detectable cAMP response to either hPTH(1–34) or hPTH(1–84) (not
shown). Comparison of the CPTHR binding of these C cells with those of
the previously described osteoblastic (F) cells, which had been
selected for high alkaline phosphatase activity rather than for CPTHR
binding (21), is shown in Fig. 1
. The amount of total radioactivity
bound averaged 14.4 ± 3.2% for the three C cell clones,
vs. 2.98 ± 0.51% for the three previously reported F
cell lines. Nonspecific binding, assessed in the presence of
10-6
M hPTH(1–84), was 2.8 ± 1.2% and
1.04 ± 0.15% for the C and F cell clones, respectively. As
assessed by Scatchard analysis, CPTHR expression by C cells ranged from
1,900,000 to 3,400,000 sites/cell on the C cells but was less than
600,000 sites/cell on the F cells (ranging from 200,000 to 600,000)
(Fig. 1, inset).
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, hPTH(1–84) displaced the
125I-[Tyr34] hPTH(19–84)
radioligand as effectively as [Tyr34]
hPTH(19–84) (IC50 = 20–50 nM for
both). Binding by the fragment hPTH(24–84) also was equivalent to that
of hPTH(1–84), whereas the shorter peptide hPTH(39–84) was much less
potent, with an IC50 in the range of 500–700
nM. Human PTH(1–34) displaced the radioligand slightly
(24 ± 11.5%; n = 3 experiments) at 10,000 nM
but was ineffective at 1000 nM.
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). Binding was reduced 19 ± 5%
and 66 ± 7% by preincubation with hPTH(1–84) at concentrations
of 200 nM and 1000 nM, respectively.
Pretreatment with hPTH(39–84) reduced CPTHR binding by less than 20%
when added at 10,000 nM but not at all at 1000
nM (not shown). To assure that this change was due to a
reduction in total binding sites and not simply to persistent receptor
occupancy by preadministered hPTH(1–84), cells were rinsed, before
radioligand addition, with acidic buffer that was found in preliminary
experiments to completely and reversibly remove previously bound
radioligand. CPTHR binding also was down-regulated following
preincubation for 16 h in the presence of calcium ionophore
(ionomycin, 1 µM) or active phorbol ester (TPA, 10
nM), which reduced specific binding by 71 ± 3% and
48 ± 12%, respectively. The effects of these drugs were at least
partly reversible, as radioligand binding returned to 100% and 63% of
controls, respectively, 24 h after their removal (data not shown).
No down-regulation occurred when cells were pretreated with inactive
phorbol ester (4- phorbol) at concentrations as high as 100
nM (not shown). Also, the effect of ionomycin (1
µM) was not blocked by prior incubation of the cells with
the intracellular calcium chelator BAPTA-AM (25 µM) (not
shown). Finally, no down-regulation of CPTHR binding occurred following
exposure for 24 h to hPTH(1–34) (1 µM),
1,25(OH)2D3 (10
nM), 8-bromo cAMP (1 mM), forskolin (10
µM), insulin (100 ng/ml), IGF-1 (100 nM) or
dexamethasone (100 nM) (data not shown).
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, A–C). This
stellate appearance was seen in all three C cell lines and was distinct
from the more cuboidal shape of the osteoblastic (F) cells that
previously had been isolated from the same calvarial digest on the
basis of high alkaline phosphatase activity (21) (Fig. 4D). Because of their osteocytic morphology, the three C cell lines
were renamed OC cells.
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, OC cell expression of
osteocalcin and Cx43 mRNAs was higher than in F1–14 osteoblastic cells
or in early cultures of normal murine calvarial osteoblasts, whereas
osteopontin mRNA was barely detectable in the OC cells. Transcripts for
alkaline phosphatase or cbfa-1/osf-2 could not be detected by Northern
blot analysis in these cells but were present at low levels, as
revealed by RT-PCR (Fig. 5B). Type 1 collagen was well expressed in all
of the cell lines. None of the mRNAs measured by Northern blot analysis
increased significantly in cells incubated for 5 days at 39 C
vs. 33 C. Cx43 expression, as detected by direct
immunostaining of fixed cells, was observed in cytoplasm and along the
dendritic processes (Figs. 4
and 6).
After incubation for 2 h with hPTH(1–84) (100
nM) or hPTH(39–84) (1000
nM), Cx43 staining was more prominent, especially
in a perinuclear location (Fig. 6). This represented mainly a
redistribution of Cx43, as it was not accompanied by an increase in
total cellular Cx43 protein, as assessed by Western blotting (Fig. 6D).
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). Mineralization (Fig. 7B
vs. A) was increased when cells were maintained at 39 C,
conditions under which the transforming T antigen is inactive.
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, CPTHR
specific binding was much greater in the osteocytic cells than in the
unfractionated original calvarial digest (F1) or the previously
isolated osteoblastic (F1–14) or bone marrow stromal cells (MS-1)
(25). Minimal specific binding was detected on HeLa, BHK1,
NIH3T3 cells or, as previously reported, on LLC-PK1 porcine kidney
cells (6).
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(A and B) when OC cells, which lack
functional PTH1R genes, were incubated for 6 h with 100
nM hPTH(1–84), increased nuclear pyknosis and
chromatin condensation appeared, as revealed by DNA staining with
Hoechst dye 33258. Increased apoptosis also was observed using a TUNEL
immunocytochemical assay (Fig. 8, C and D). Similarly, when cell death
was monitored using trypan blue staining of combined adherent and
nonadherent cells from these cultures (26), hPTH(1–84)
induced a doubling in trypan blue-stained cells after 16 h (Fig. 9A). This effect of hPTH(1–84) upon cell
death was concentration dependent and maximal at 10
nM, with an EC50 in the
range of 0.1 nM (Fig. 9B). The effect was
mimicked by 100 nM hPTH (24–84) and
1000 nM hPTH(39–84), which produced,
respectively, 1.8- and 1.5-fold increases over basal. (Fig. 9, C and
D). As expected, hPTH(1–34) (1000 nM) was
inactive, consistent with its inability to effectively bind to CPTHR
sites on these cells (Fig. 2). In these experiments, the percentage of
trypan blue-stained cells in controls was substantial (20–30%),
perhaps because of prolonged incubation in reduced serum at the
nonpermissive temperature. A similar proapoptotic effect of hPTH(1–84)
was observed in analogous experiments performed at 37 C, however,
where basal rates of cell death were lower (Fig. 9A). Cell death
triggered by hPTH(1–84) was blocked completely by prior addition of
the caspase-3 inhibitor DEVD (50 µg/ml) (Fig. 9E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We report here that clonal murine calvarial-derived cell lines with
many characteristics of osteocytes also express unusually large numbers
of receptors that specifically recognize the C-terminal portion of
intact PTH. In fact, these cells express considerably higher levels of
CPTHRs (up to 10-fold more per cell) than do other bone cell subtypes,
including mature osteoblasts (21). It is difficult to
identify the osteocytic phenotype with complete confidence in
vitro, absent the normal surrounding lacunar environment, but the
three conditionally immortalized, clonal cell lines that we selected
exclusively on the basis of high CPTHR expression share many
morphological features and a distinctive profile of gene expression
with normal osteocytes (23) and with a previously
characterized osteocytic cell line (24). These include a
stellate shape, with numerous dendritic processes, and abundant
expression of mRNAs for osteocalcin, Cx43, and CD44 but not for
alkaline phosphatase or cbfa-1/osf-2, which are more characteristic of
early or mature osteoblasts. The osteocytic phenotype of these cells is
most intense when they are maintained at a temperature (i.e.
39 C) that is nonpermissive for expression of functional transforming
tsTAg. For example, mineralization (Fig. 7) was much more intense in
cells cultured at 39 C than at 33 C, as we previously observed in other
clonal osteoblastic cells isolated from these mice
(21).
The OC cells were isolated intentionally from animals that genetically lack functional PTH1Rs to eliminate possible confounding effects of coexpressed PTH1Rs and to allow us to focus directly upon CPTHR-dependent actions of intact PTH. The results of radioreceptor binding assays in OC cells, using the 125I-[Tyr34]hPTH(19–84) radioligand, were remarkably similar to those reported previously with ROS17/2.8 cells (6, 16), which suggests that the CPTHRs on these different cell types probably are similar or identical. Thus, like Inomata et al., we found that hPTH(1–84) and the hPTH(19–84) peptide bound equivalently to CPTHRs and that hPTH(39–84) bound more weakly, with an IC50 roughly 15-fold higher. We have further narrowed the ligand domain responsible for this higher affinity interaction, moreover, by studying hPTH (24–84), which was found to bind at least as well as hPTH(1–84). This indicates that CPTHRs require sequences within the region hPTH(24–38) for high affinity binding, although other, more C-terminal domains also must be involved to account for the residual affinity of hPTH(39–84). The small amount of binding observed at high concentrations of hPTH(1–34) is consistent with the presence of a binding determinant within hPTH(24–38), most of which is contained within hPTH(1–34). It seems that this domain probably cannot function well independently of the cooperative effects of more C-terminal sequences, however. The manner in which of these independent domains contribute to the formation of an active conformation of the ligand remains obscure, especially as available spectroscopic studies do not suggest a highly ordered secondary structure for the C-terminal sequence of PTH (32, 33, 34).
Enumeration of CPTHR sites on the OC cells led to estimates of 1,900,000 to 3,400,000 per cell, which was 6- to 10-fold higher than on the osteoblastic (F) cells obtained from the same bones and at least 5-fold higher than on ROS 17/2.8 cells (6). A survey of other available cell lines indicated that, with the exception of a bone marrow stromal cell line (MS-1) (25), little or no binding was detectable on NIH-3T3, HeLa or BHK21 fibroblast cells, as was reported previously for OK and LLC-PK1 kidney cells, YCC cells, and SaOS-2, MG63 and UMR106–01 osteosarcoma cells (6). Thus, the very high abundance of expressed CPTHRs on osteocytic OC cells is unique, even among other bone cells tested, and suggests that these receptors may play an important role in osteocyte regulation. The possibility that bone is an important C-PTH target tissue in vivo is further supported by our recent observation that 125I-[Tyr34]hPTH(19–84) radioligand binds specifically to frozen sections of 18.5-day normal mouse fetal calvarial bone (unpublished results) and that hPTH (7–84) reduces the calcemic effects of intact PTH(1–84) in vivo at concentrations much lower than those required for effective antagonism of the PTH1R (20).
We observed homologous down-regulation of CPTHR binding following preincubation with hPTH(1–84). This response required relatively high concentrations of ligand (>100 nM), in contrast to the effects upon apoptosis, and was mimicked by addition of active phorbol ester or ionomycin. The failure of BAPTA to block the ionomycin effect suggests mediation by a sustained influx of calcium, presumably from the extracellular compartment, as opposed to a rapid burst of calcium released from intracellular stores. This is consistent also with our previous report of CPTH-stimulated calcium uptake in SaOS-2 human osteoarcoma cells (35). Also, in C-PTH-treated human fetal chondrocytes, others have reported increased cytosolic free calcium due to activation of calcium influx via nickel-insensitive channels (18). Our down-regulation data are most compatible with involvement of a calcium-dependent PKC, although further study will be needed to pursue this possibility and to clarify the role and source of the calcium signal implicated in this response.
Functional studies of CPTHRs on OC cells have pointed so far to involvement in at least two general types of cellular processes: cell survival and intercellular communication. We observed a rapid CPTHR-dependent change in cellular expression of Cx43, a gap-junction protein important in mediating intercellular communication and previously shown to be expressed by both osteoblasts and osteocytes (36, 37). The possible physiologic significance of this action is not yet clear. The response of osteocytes to mechanical stress in vivo is promoted by PTH(1–34) and blocked by parathyroidectomy (38). It is interesting to speculate that activation of CPTHRs by C-PTH fragments, preferentially secreted by the parathyroids during hypercalcemia (9), might trigger communication from osteocytes to other bone cells so as to limit osteoclast formation or activity and thereby exert negative feedback on bone resorption. Further understanding of CPTHR function and ligand activity, facilitated by availability of these osteocytic cell lines, will be needed to pursue such hypotheses.
We also observed that CPTHR-interacting ligands increased the rate of osteocyte cell death in vitro. As this action was accompanied by evidence of increased nuclear pyknosis, chromatin condensation and TUNEL staining and was completely blocked by the caspase-3 inhibitor DEVD, we conclude that apoptosis, mediated by activation of the caspase cascade, is the likely mechanism. Importantly, this proapoptotic effect was observed also with the C-terminal fragment hPTH(39–84) (at an appropriately higher concentration), which indicates that the ligand domain required for CPTHR activation of this response likely is located C-terminal to the above-mentioned hPTH(24–38) region needed for high-affinity binding. In contrast, others (26) have observed an opposite, anti-apoptotic action of hPTH(1–34), via the PTH1R activation, in dexamethasone-stimulated osteoblastic and osteocytic cells. In OC cells, which lack endogenous PTH1Rs, hPTH(1–84) exerts only a pro-apoptotic effect (via CPTHRs), as does hPTH(39–84). This suggests the possibility that PTH1Rs and CPTHRs may exert opposite effects upon cell survival in osteocytes and that these cells may be able to integrate signals arising from these two types of PTH receptors in response to discoordinate changes in circulating intact hormone and C-PTH fragments. In renal insufficiency, for example, excessive CPTHR activation by high levels of circulating CPTH fragments might lead to exaggerated, pathological loss of osteocytes and contribute to some forms of renal osteodystrophy (39).
Expression of the PTH1R by osteocytes has been documented in vivo (40), and it is of interest to consider the potential for interactions between PTH1Rs and CPTHRs in these cells. It is possible that the genetic absence of PTH1R in our cells may have modified in some way the expression or function of CPTHRs. Almost certainly, the response of these PTH1R-null cells to hPTH(1–84) in vitro differs from that of authentic osteocytes in vivo, in which both receptor types presumably can be activated concurrently by the intact hormone. Preliminary evidence, in ROS 17/2.8 cells, indicates that C-PTH fragments may modify the function of hPTH(1–84) [but not hPTH(1–34)] in cells that express both types of PTH receptors (41). The possible involvement of PTH1Rs in modulating CPTHR function, or vice versa, now can be addressed directly in our OC cells by reconstituting them through transfection with cDNA encoding PTH1Rs; efforts in this direction are underway in our laboratory.
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Acknowledgments |
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Footnotes |
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Received July 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. Jolette, C. E. Wilker, S. Y. Smith, N. Doyle, J. F. Hardisty, A. J. Metcalfe, T. B. Marriott, J. Fox, and D. S. Wells Defining a Noncarcinogenic Dose of Recombinant Human Parathyroid Hormone 1-84 in a 2-Year Study in Fischer 344 Rats Toxicol Pathol, December 1, 2006; 34(7): 929 - 940. [Abstract] [Full Text] [PDF]
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 A. A. Selim, M. Mahon, H. Juppner, F. R. Bringhurst, and P. Divieti Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling Am J Physiol Cell Physiol, July 1, 2006; 291(1): C114 - C121. [Abstract] [Full Text] [PDF]
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 J. Huan, K. Olgaard, L. B. Nielsen, and E. Lewin Parathyroid Hormone 7-84 Induces Hypocalcemia and Inhibits the Parathyroid Hormone 1-84 Secretory Response to Hypocalcemia in Rats with Intact Parathyroid Glands J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1923 - 1930. [Abstract] [Full Text] [PDF]
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 P. A. Friedman and W. G. Goodman PTH(1-84)/PTH(7-84): a balance of power Am J Physiol Renal Physiol, May 1, 2006; 290(5): F975 - F984. [Abstract] [Full Text] [PDF]
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 S. P Shrader and K. R Ragucci Parathyroid Hormone (1-84) and Treatment of Osteoporosis Ann. Pharmacother., September 1, 2005; 39(9): 1511 - 1516. [Abstract] [Full Text] [PDF]
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 A. B. Hodsman, D. C. Bauer, D. W. Dempster, L. Dian, D. A. Hanley, S. T. Harris, D. L. Kendler, M. R. McClung, P. D. Miller, W. P. Olszynski, et al. Parathyroid Hormone and Teriparatide for the Treatment of Osteoporosis: A Review of the Evidence and Suggested Guidelines for Its Use Endocr. Rev., August 1, 2005; 26(5): 688 - 703. [Abstract] [Full Text] [PDF]
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 P. Divieti, A. I. Geller, G. Suliman, H. Juppner, and F. R. Bringhurst Receptors Specific for the Carboxyl-Terminal Region of Parathyroid Hormone on Bone-Derived Cells: Determinants of Ligand Binding and Bioactivity Endocrinology, April 1, 2005; 146(4): 1863 - 1870. [Abstract] [Full Text] [PDF]
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T. M. Murray, L. G. Rao, P. Divieti, and F. R. Bringhurst Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands Endocr. Rev., February 1, 2005; 26(1): 78 - 113. [Abstract] [Full Text] [PDF]
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 P. D'Amour, J.-H. Brossard, A. Rakel, L. Rousseau, C. Albert, and T. Cantor Evidence That the Amino-Terminal Composition of Non-(1-84) Parathyroid Hormone Fragments Starts before Position 19 Clin. Chem., January 1, 2005; 51(1): 169 - 176. [Abstract] [Full Text] [PDF]
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D. Miao, B. He, B. Lanske, X.-Y. Bai, X.-K. Tong, G. N. Hendy, D. Goltzman, and A. C. Karaplis Skeletal Abnormalities in Pth-Null Mice Are Influenced by Dietary Calcium Endocrinology, April 1, 2004; 145(4): 2046 - 2053. [Abstract] [Full Text] [PDF]
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 R. J. Midura, X. Su, J. A. Morcuende, M. Tammi, and R. Tammi Parathyroid Hormone Rapidly Stimulates Hyaluronan Synthesis by Periosteal Osteoblasts in the Tibial Diaphysis of the Growing Rat J. Biol. Chem., December 19, 2003; 278(51): 51462 - 51468. [Abstract] [Full Text] [PDF]
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F. R. Bringhurst Circulating Forms of Parathyroid Hormone: Peeling Back the Onion Clin. Chem., December 1, 2003; 49(12): 1973 - 1975. [Full Text] [PDF]
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 A. B. Hodsman, D. A. Hanley, M. P. Ettinger, M. A. Bolognese, J. Fox, A. J. Metcalfe, and R. Lindsay Efficacy and Safety of Human Parathyroid Hormone-(1-84) in Increasing Bone Mineral Density in Postmenopausal Osteoporosis J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5212 - 5220. [Abstract] [Full Text] [PDF]
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S. J. Silverberg, P. Gao, I. Brown, P. LoGerfo, T. L. Cantor, and J. P. Bilezikian Clinical Utility of an Immunoradiometric Assay for Parathyroid Hormone (1-84) in Primary Hyperparathyroidism J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4725 - 4730. [Abstract] [Full Text] [PDF]
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 H. Reichel, A. Esser, H.-J. Roth, and H. Schmidt-Gayk Influence of PTH assay methodology on differential diagnosis of renal bone disease Nephrol. Dial. Transplant., April 1, 2003; 18(4): 759 - 768. [Abstract] [Full Text] [PDF]
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W. G. Goodman, I. B. Salusky, and H. Juppner New lessons from old assays: parathyroid hormone (PTH), its receptors, and the potential biological relevance of PTH fragments Nephrol. Dial. Transplant., October 1, 2002; 17(10): 1731 - 1736. [Full Text] [PDF]
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P. Divieti, M. R. John, H. Juppner, and F. R. Bringhurst Human PTH-(7-84) Inhibits Bone Resorption in Vitro Via Actions Independent of the Type 1 PTH/PTHrP Receptor Endocrinology, January 1, 2002; 143(1): 171 - 176. [Abstract] [Full Text] [PDF]
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