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PTH-CALCITONIN-VITAMIN D-BONE |
Endocrine Unit, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Paola Divieti, M.D., Ph.D., Endocrine Unit, Wellman 5, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114. E-mail: divieti{at}helix.mgh.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The parathyroid glands are the main source of PTH, although small amounts of its mRNA were recently identified in hypothalamus and spleen (3). PTH synthesis and secretion are tightly controlled by calcium via a membrane-bound calcium-sensing receptor (4), although vitamin D (5, 6) and phosphate (7, 8) also play modulating roles. Under physiological conditions, a portion of the newly synthesized hormone undergoes intraglandular cleavage at a rate that also is regulated by extracellular calcium (9, 10). This cleavage results in the cosecretion of intact PTH and various C-terminal fragments, the predominant forms of which, identified to date, consist of peptides with N-termini located between residues 24 and 43 (11, 12, 13). Secreted intact PTH also undergoes endopeptidic cleavage(s) in peripheral tissues, mainly liver and kidney, by processes that degrade the resulting N-terminal fragments in situ but release additional C-fragments into the circulation (14, 15, 16). As a consequence of their obligatory renal clearance, the concentration of circulating C-terminal PTH (CPTH) fragments increases dramatically in patients with renal failure (17, 18, 19).
Recently, fragments of PTH lacking residues at the extreme N-terminus but otherwise large enough to cross-react with most commercially available intact PTH two-site immunoassays were detected after HPLC fractionation of normal plasma and, at much higher levels, in plasma of patients with advanced renal failure (19). Although their precise structure(s) has not been ascertained, these fragments exhibit chromatographic properties similar to those of synthetic PTH-(7-84) (18). Interestingly, human (h) PTH-(7-84) was recently shown to inhibit the calcemic actions of PTH-(1-84) and PTH-(1-34) in parathyroidectomized animals at doses much lower than would be predicted to effectively antagonize either hormonal form at the PTH1R (20, 21). Thus, these in vivo observations suggest that CPTH fragments might act upon bone cells via one or more mechanisms independent of the PTH1R per se.
The possibility that CPTH fragments (as well as intact PTH) might activate receptors distinct from the PTH1R was first postulated over 2 decades ago when Arber et al. (22) showed that a particular CPTH fragment, PTH-(53-84), possessed biological properties different from those of PTH-(1–34). Subsequent work from several different groups has produced direct evidence that CPTH fragments from within the sequence PTH-(35-84) bind specifically to bone and kidney cells and/or membranes and can exert direct actions on target cells in bone or cartilage. For example, CPTH fragments such as hPTH-(53-84) and hPTH-(60-84) increased alkaline phosphatase activity and expression of mRNAs for both alkaline phosphatase and osteocalcin in bone-derived cells and induced transient increases in cytosolic free calcium in chondrocytes (23, 24, 25, 26). Photoaffinity cross-linking studies to characterize the receptors for CPTH fragments (i.e. CPTHRs) expressed by ROS 17/2.8 osteosarcoma and rPT parathyroid cells were performed by Inomata et al. (27) using radioiodinated (Leu8,18,Tyr34)hPTH-(1-84) and (Tyr34)hPTH-(19-84), neither of which binds well, if at all, to the PTH1R. These studies showed that in ROS 17/2.8 cells, two proteins (80 and 30 kDa) interacted specifically with the radioligands used, whereas in rPT cells, only the 80-kDa protein was observed. Affinity labeling was inhibited by hPTH-(1-84), hPTH-(19-84), and, to a lesser extent, by CPTH fragments that were truncated even further at the N-terminus, whereas hPTH-(1-34) had no effect (27). Recently, hPTH-(7-84) was shown to bind to CPTHRs on ROS 17/2.8 cells with affinity comparable to that of hPTH-(1-84) (21).
Unequivocal evidence that such CPTHRs are distinct from the PTH1R was provided by our recent demonstration that specific [125I](Tyr34)hPTH-(19-84) binding is observed in clonal osteoblastic and osteocytic cell lines derived from mice in which the PTH1R gene had been eliminated by gene targeting (28). Further, CPTH fragments such as hPTH-(39-84) were shown to regulate cellular functions (i.e. connexin 43 expression and apoptosis) in clonal PTH1R-null osteocytes at concentrations shown to bind effectively to CPTHRs in these cells (28).
Thus, the expression of CPTHRs in bone offers a plausible mechanism by which circulating PTH fragments, truncated at their N-termini and including peptides as long as hPTH-(7-84) might exert biological actions, potentially different from those of intact PTH, by a means other than direct antagonism at the PTH1R. To determine whether the ability of hPTH-(7-84) to antagonize the calcemic response to PTH-(1-84) in vivo might reflect direct actions of this C-PTH fragment on bone, we studied its effects using in vitro assays of osteoclast formation and bone resorption.
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Materials and Methods |
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Animals
Animals were maintained in facilities operated by the
Massachusetts General Hospital Center for Comparative Resources in
accordance with the NIH Guide for the Care and Use of Laboratory
Animals and were employed using protocols approved by the institutional
animal care and use committee.
Bone resorption assay
Bone resorption was quantitated by the release of previously
incorporated 45Ca from newborn mouse calvarial
bones in vitro (31). Briefly, calvaria from 3-
to 4-d-old mice (CD-1 strain, Charles River Laboratories, Inc., Wilmington, MA) were obtained after maternal
administration of 50 µCi
45CaCl2 (NEN Life Science Products, Boston, MA), sc, on the 19th day of gestation.
The bones were divided in half and precultured in 1 ml DMEM containing
1 mM calcium, 2 mM
phosphate, 5% heat-inactivated horse serum, and 1%
antibiotic/antimycotic solution (Life Technologies, Inc.)
on a rocking platform at 90 oscillations/min in a 37 C incubator under
5% CO2 in air. After 24 h the medium was
replaced with 1 ml fresh medium containing the test substances (or
vehicle alone). After an additional 72 h, the bones were removed,
rinsed three times in PBS, placed in scintillation vials containing 0.4
ml 2 N HCl, and incubated for 2 h at room
temperature before addition of 5 ml scintillation fluid (Packard
Instruments, Downers Grove, IL). Aliquots of culture medium (0.5 ml)
were transferred to separate vials containing 5 ml scintillation fluid
for determination of released radioactivity. In some experiments
additional aliquots of culture medium were used for measurements of
cAMP as described below. Bone resorption was determined as the
percentage of total initial bone 45Ca
subsequently released into the medium during the 72-h treatment period.
Results are expressed as the mean ± SEM of
the percentage of 45Ca released for groups of
four bones and are representative of at least three independent
experiments.
Bone marrow culture
Bone marrow cells were isolated as previously described
(32). Briefly, 4- to 6-wk-old male mice (C57B/6 strain,
Charles River Laboratories, Inc.) were killed by carbon
dioxide asphyxiation, and tibias and femurs were aseptically removed
and dissected free of adhering tissue. The metaphyses were removed, and
the marrow cavity was flushed with 1 ml 
MEM to obtain marrow cells,
which were collected into 50-ml tubes and washed twice with MEM.
Cells were cultured in growth medium [MEM containing 10% FBS (lot
1011961 Life Technologies, Inc.) and 1%
penicillin-streptomycin] containing 100 nM dexamethasone
(Sigma) after plating at 1.5 x
106 cells/well in 24-well plates. Half of the
culture medium was replaced 3 times/wk with fresh medium containing a
2x concentration of the test substances (or vehicle). All cultures
were maintained in a 37 C incubator under 5% CO2
in air. After culture for 10 d, cells adherent to the surface of
each well were rinsed twice with PBS, fixed with 10% formalin in PBS
for 10 min at room temperature and with ethanol/acetone (50:50,
vol/vol) for 1 min before staining for tartrate-resistant acid
phosphatase (TRAP), as previously described (33).
TRAP-positive cells containing 3 or more nuclei were scored as
osteoclast-like multinucleated cells (TRAP+MNCs). Cells were counted at
x10 magnification in 20 contiguous fields along 2 orthogonal pathways
in each well, a method previously employed to account for the
nonuniform distribution of cells within wells (33). The
number of TRAP+MNCs contained in these 20 fields was expressed as the
number per well.
cAMP accumulation
Clonal rat osteosarcoma cell (ROS 17/2.8) were cultured in
48-well plates in Ham’s F-12 medium (Life Technologies, Inc.) supplemented with 10% FBS and 1%
penicillin-streptomycin. The cultures were maintained for 5–7 d after
reaching confluence by replacing the medium every other day. To assess
basal and agonist-induced cAMP accumulation, cells were rinsed twice
with assay buffer (DMEM containing 2 mM
isobutylmethylxanthine, 1 mg/ml heat-inactivated BSA, and 35
mM HEPES-NaOH, pH 7.4) and then incubated for 45 min at 23
C with the same buffer alone or in the presence of different peptides
(or with conditioned medium collected from resorption assays). The
buffer then was rapidly aspirated, the plates were frozen on powdered
dry ice, and the frozen cells were subsequently thawed directly into
0.25 ml 50 mM HCl. Cell-associated cAMP in the acid
extracts was measured as previously described (34).
Results were expressed as picomoles of cAMP produced per well over 45
min.
Statistical analysis
Results are expressed as the mean ± SEM or the
mean ± SD. The significance of differences between
treatment and control groups was assessed by the Mann-Whitney test.
Data were analyzed using the PRISM 3.0 software package for Macintosh
(GraphPad Software, Inc., San Diego, CA).
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Results |
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, addition of hPTH-(7-84) (300
nM) alone reduced basal
45Ca release by approximately 50% [control,
17.8 ± 5.7%; hPTH-(7-84), 9.6 ± 1.9%; P
< 0.001]. This effect was comparable to that of salmon calcitonin
(100 nM; 9.9 ± 1.1%; P <
0.001). In contrast, no inhibition of basal resorption was observed
with equimolar concentrations of much shorter N-truncated PTH analogs,
such as hPTH-(3–34) (300 nM; 18.7 ± 4.2%)
or PTHrP-(7-36) (300 nM; 15.4 ± 4.9%),
that bind as well or more effectively to the PTH1R as hPTH-(7-84).
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, hPTH-(7-84) (300 nM) also significantly inhibited (by 50%
or more) agonist-induced bone resorption caused by a variety of
osteotropic agents, including intact hPTH-(1-84) (3 nM),
hPTH-(1-34) (3 nM), VitD (10 nM),
PGE2 (100 nM), and IL-11 (10 ng/ml).
The antiresorptive effect of hPTH-(7-84) was dose dependent, with an
IC50 of approximately 200 nM (data
not shown). We considered the possibility that these inhibitory actions
of hPTH-(7-84) might reflect antagonism, at the PTH1R, of an effect of
locally secreted PTHrP to augment the responses to these other
agonists. As shown in Fig. 3, however,
neither hPTH-(3-34) (1 µM) nor PTHrP-(7-36) (1
µM), both of which act as PTH1R antagonists (see below)
at the concentrations used, inhibited resorption induced by VitD (10
nM). With respect to a possible nonspecific or irreversible
toxic effect of hPTH-(7-84), we observed that removal of the peptide
after 24 h of exposure to calvariae did not impair the resorptive
response of the bones to subsequently added VitD (data not shown).
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, we observed no inhibition of the cAMP
response to PTH-(1-34) by hPTH-(7-84) (0.1–1 µM),
whereas both PTHrP-(7-36) and PTH-(3-34), when present at 1
µM, inhibited the response by 50%. In related
experiments undiluted samples of conditioned medium from calvarial
resorption assays (described above) to which 300 nM
hPTH-(7-84), PTHrP-(7-36) or PTH-(3-34) had been added 72 h
earlier also were tested for inhibition of hPTH-(1-34)-induced cAMP
accumulation in ROS 17/2.8 cells. In none of these samples was
inhibition of the cAMP response to hPTH-(1-34) observed (data not
shown).
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, hPTH-(7-84) alone exerted no effect
on the formation of TRAP+MNCs, although basal osteoclast formation in
this system is low, and an inhibitory effect therefore might not be
easily detectable. On the other hand, when osteoclast formation was
stimulated by VitD (10 nM), hPTH-(7-84) (300
nM) caused a striking (70%) reduction in the formation of
TRAP+MNCs relative to the effect of VitD alone [VitD, 153 ± 38
cells; VitD + hPTH-(7-84), 53 ± 14 cells]. In contrast,
PTHrP-(7-36) did not inhibit TRAP+MNC formation induced by VitD [VitD
+ PTHrP-(7-36), 127 ± 22 cells; Fig. 5A]. To determine whether
shorter C-terminal PTH fragments could regulate osteoclast formation,
we tested the effects of hPTH-(39-84), alone or in combination with
VitD (10 nM). As shown in Fig. 5B, hPTH-(39-84) alone, at
3000 nM, slightly stimulated osteoclast formation (41
± 11 cells), as reported previously for short CPTH fragments
(24, 33). Like hPTH-(7-84), however, hPTH-(39-84)
dramatically inhibited osteoclast formation promoted by VitD [VitD,
207 ± 31 cells; VitD + hPTH-(39-84), 46 ± 10 cells].
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One possible mechanism for such an effect could involve direct antagonism by hPTH-(7-84) to prevent binding of hPTH-(1-34) or hPTH-(1-84) to PTH1Rs expressed on osteoblasts or marrow stromal cells. Because the antagonism in vivo is observed at much lower doses of hPTH-(7-84), relative to intact PTH, than that predicted to be necessary for direct antagonism at the PTH1R, however, we also considered the alternative possibility that hPTH-(7-84) may exert unique PTH1R-independent antiresorptive effects by activating CPTHRs expressed in bone cells. Our results are fully consistent with this latter hypothesis. Thus, we observed concentration-dependent inhibition of bone resorption in ex vivo calvarial organ cultures that was not mimicked by shorter, N-truncated PTH fragments that 1) are more effective PTH1R antagonists than is hPTH-(7-84) and 2) do not bind detectably to CPTHRs expressed on bone cells (28). Similar results were obtained in studies of osteoclastogenesis using whole bone marrow cultures, which further suggests that CPTHRs may be involved in the regulation of osteoclast formation. Because the number of mononuclear TRAP-positive cells formed in the marrow cultures also was reduced by hPTH-(7-84), the predominant action in osteoclastogenesis may be to inhibit formation of osteoclast precursors. The rapidity (1–2 h) of the hPTH-(7-84) effect observed in vivo, however, suggests that interference with the activity of mature osteoclasts also may be involved.
The antiresorptive effect of hPTH-(7-84) observed in the
calvarial assay system contrasted sharply with the inability of
hPTH-(3-34) or PTHrP-(7-36), introduced at similar concentrations, to
inhibit resorption. Because both of these shorter, N-truncated peptides
are effective in vitro PTH1R antagonists, whereas
hPTH-(7-84) is not (as shown in Fig. 4
), these results argue strongly
against a mechanism involving direct antagonism by hPTH-(7-84) at the
PTH1R of either endogenous PTHrP present within the cultured bones or
exogenously added PTH. Moreover, the antiresorptive effect of
hPTH-(7-84) in vitro was not restricted to resorption
induced by added PTH, but was encountered in both control cultures and
cultures treated with a variety of unrelated bone-resorbing agonists,
including VitD, PGE2 and IL-11. These findings
point to a more generalized antiresorptive mechanism by which
PTH-(7-84), presumably acting via CPTHRs, may limit the formation and,
possibly, the activity of mature osteoclasts. This could reflect
interference with the up-regulation of RANKL or macrophage
colony-stimulating factor, the down-regulation of OPG, or both, that
normally are triggered in marrow stromal cells and osteoblasts by these
diverse resorbing agents (35). In this regard, we observed
expression of CPTHRs by PTH1R-null osteoblasts and osteocytes
(28) and by clonal marrow stromal cells that are capable
of supporting PTH- or vitamin D-dependent osteoclast formation from
hemopoietic progenitors in vitro (our unpublished
observations). We also cannot yet exclude that the inhibition of
resorption was mediated partly by a proapoptotic effect of hPTH-(7-84)
on bone cells via activation of the CPTHR, as we previously reported in
osteocytic cells (28). Moreover hPTH-(7-84) could act
directly on mature osteoclasts, their hemopoietic precursors, or both
to dampen cellular responsiveness to activation of RANK or
c-Fms by their respective stromal cell or osteoblast-derived
ligands. Indeed, evidence that shorter CPTH fragments alone can
modestly induce osteoclast formation, as seen in the present study with
hPTH-(39-84) and reported previously (24, 33), in contrast
to the inhibitory effects of the same fragment upon osteoclast
formation induced by vitamin D, points to a complexity in CPTHR action
that is not readily explained at present, but that could involve
disparate effects on distinct cell types involved in osteoclast
formation. Direct analysis of CPTHR expression in such cells would
be needed to address this possibility.
One prediction of our results might be that PTH-(1-84), which binds to CPTHRs with affinity comparable to that of PTH-(7-84) (21), should elicit less bone resorption than PTH-(1-34), which does not interact effectively with CPTHRs (28). Although few direct in vitro comparisons have been performed (24, 36), the available data do not indicate substantial or consistent differences in resorptive responses to these two peptides. Indeed, we observed in the calvarial resorption assay that the intact hormone reproducibly induced less 45Ca release than did PTH-(1-34) at equimolar concentrations, although this difference was never statistically significant. This could indicate that when PTH1Rs and CPTHRs are exposed simultaneously to equimolar concentrations of a common ligand, the PTH1R-mediated resorptive response strongly predominates. Alternatively, it is possible that despite comparable binding affinity, intact PTH cannot activate CPTHRs as effectively as N-truncated peptides (by analogy with the disparate activation of PTH1Rs observed with PTH-(1-34) vs. PTH-(3-34)). On the other hand, because CPTH fragments normally circulate in plasma at concentrations at least 5- to 10-fold higher than those of intact PTH, a requirement for higher molar concentrations of CPTH ligands to activate CPTHRs might be expected. This concept is consistent with our finding that a 10- to 100-fold molar excess of CPTH ligand is needed to elicit functional antagonism of PTH1R-mediated resorption in vitro. The observation that hPTH-(7-84) could antagonize the calcemic response to hPTH-(1-84) at equimolar doses in vivo might be related to differential bioavailability or metabolism of the two peptides after their ip or iv administration (20, 21).
Secretion of CPTH fragments by the parathyroid glands is positively regulated by blood calcium (37). Thus, one possible physiological role of the antiresorptive action of N-truncated PTH fragments in vivo could be to modulate the extent of bone resorption induced by intact PTH in a manner responsive to the extracellular calcium concentration. Such a mechanism, for example, might allow for maximal release of calcium from bone only during severe hypocalcemia to supplement the ongoing renal and (indirect) intestinal actions of PTH. It is important to note that the chemical identities of all circulating CPTH fragments have not yet been completely defined. In particular, the existence in blood of PTH-(7-84) per se has not been directly demonstrated. On the other hand, the recent immunochemical characterization of nonintact PTH peptides, which are especially abundant in renal failure, is consistent with the presence of extended CPTH fragments longer than those previously inferred from analyses of secreted or peripherally generated cleavage products, the N-termini of which ranged between positions 24 and 43 of the intact PTH sequence (19, 38). Thus, the possibility that PTH fragments similar or identical to PTH-(7-84) may be present in blood, especially in renal failure, at concentrations high enough to activate CPTHRs and thereby exert direct effects on bone resorption must be considered.
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Footnotes |
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1 P.D. and M.R.J. contributed equally to this work. 
Abbreviations: CPTH, C-terminal PTH; hPTH, human PTH; PTH1R, type 1 PTH/PTHrP receptor; TRAP, tartrate-resistant acid phosphatase; TRAP+MNC, TRAP-positive cells containing three or more nuclei (osteoclast-like multinucleated cells); VitD, 1,25-dihydroxyvitamin D3.
Received May 24, 2001.
Accepted for publication September 17, 2001.
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), osteotropic agents alone
(
), or osteotropic agents in combination with 300 nM
hPTH-(7-84) (
). Osteotropic agents were employed at the following
concentrations: hPTH-(1-84), 3 nM; hPTH-(1-34), 3
nM; VitD, 10 nM; PGE2, 100
nM; and IL-11, 10 ng/ml. Results are expressed as the
mean ± SEM of quadruplicate determinations. Each
experiment was repeated three times. *, P < 0.05
for difference between osteotropic agent alone vs.
osteotropic agent plus hPTH-(7-84).
,
dashed line), or PTHrP-(7-36) (