Adrenomedullin is a potent stimulator of osteoblastic activity
in vitro and in vivo
Jillian
Cornish1,
Karen E.
Callon1,
David H.
Coy3,
Ning-Yi
Jiang3,
Liqun
Xiao1,
Garth J. S.
Cooper1,2, and
Ian R.
Reid1
1 Department of Medicine and
2 School of Biological Sciences,
University of Auckland, Auckland, New Zealand; and
3 Peptide Research
Laboratories, Tulane University Medical Center, New Orleans,
Louisiana 70112
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ABSTRACT |
Adrenomedullin is a 52-amino acid vasodilator
peptide produced in many tissues, including bone. It has 20% sequence
identity with amylin, a regulator of osteoblast growth, and circulates in picomolar concentrations. The present study assesses whether adrenomedullin also acts on osteoblasts. At concentrations of 10
12 M and greater,
adrenomedullin produced a dose-dependent increase in cell number and
[3H]thymidine
incorporation in cultures of fetal rat osteoblasts. This effect was
also seen with adrenomedullin-(15
52), -(22
52), and -(27
52), but
adrenomedullin-(40
52) was inactive. These effects were lost in the
presence of amylin blockers, suggesting they were mediated by the
amylin receptor. Adrenomedullin also increased [3H]thymidine
incorporation into cultured neonatal mouse calvaria but, unlike amylin,
did not reduce bone resorption in this model. Adrenomedullin stimulated
phenylalanine incorporation into both isolated osteoblasts and
calvaria. When injected daily for 5 days over the calvariae of adult
mice, it increased indexes of bone formation two- to threefold
(P < 0.0001) and increased
mineralized bone area by 14% (P = 0.004). It is concluded that adrenomedullin regulates osteoblast
function and that it increases bone mass in vivo. The potential of this
family of peptides in the therapy of osteoporosis should be further
evaluated.
amylin; calvaria; bone resorption; peptide fragments
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INTRODUCTION |
ADRENOMEDULLIN is a 52-amino acid peptide first
described in 1993 by Kitamura et al. (20) and recently reviewed by
Kitamura and Eto (18). It was originally identified in a human
pheochromocytoma and has since been found to be present in normal
adrenal medulla and in many other tissues, including the atria,
ventricles, endothelial cells, lungs, brain, kidneys, and bone (14,
23). It circulates in picomolar concentrations in both rats and humans
(19). It is a potent vasodilator, acting directly on the renal,
cerebral, mesenteric, pulmonary, and systemic circulations, including
the vascular supply of the skeleton (17). Its hemodynamic effects are
probably mediated via receptors on vascular smooth muscle cells and
possibly endothelial cells. The kidney may also be a significant target
tissue. Binding to renal tubular membranes has been observed, and
sodium, potassium, and water excretion are increased by adrenomedullin.
It is a bronchodilator, and it modulates release of pituitary and
vasoactive hormones. The adrenomedullin receptor has recently been
cloned (16). It contains seven transmembrane domains and couples to
adenylyl cyclase. It has a structural resemblance to the other G
protein-linked receptors, and expression of the receptor mRNA is
widespread.
Adrenomedullin shows ~20% sequence identity with amylin and
calcitonin gene-related peptide and slightly less with calcitonin itself (24). All these peptides have an NH2-terminal ring
created by a disulfide bond and are amidated at their COOH terminals. Adrenomedullin differs from the others in that it has a linear NH2-terminal extension, consisting of 15 amino acids in the
human and 13 in the rat. The other members of this group inhibit bone resorption, probably via a direct action on the osteoclast calcitonin receptor (1, 36). We have recently shown that amylin also has direct
effects on osteoblast function, stimulating cell proliferation in vitro
and increasing indexes of bone formation in vivo (2, 3). In light of
these findings, we have assessed the actions of adrenomedullin in a
variety of models to determine whether it too is a modulator of bone
cell function.
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METHODS |
Osteoblast-like cell culture.
Osteoblasts were isolated by collagenase digestion from 20-day fetal
rat calvaria, as previously described (21). Calvaria were dissected
aseptically, and the frontal and parietal bones were stripped of their
periosteum. Only the central portions of the bones, free from suture
tissue, were collected. The calvaria were treated twice with
phosphate-buffered saline (PBS) containing 3 mM EDTA (pH 7.4) for 15 min at 37°C in a shaking water bath. After being washed once in
PBS, the calvaria were treated twice with 3 ml of 1 mg/ml collagenase
for 7 min at 37°C. After the supernatants were discarded from
digestions I and II, the calvaria were treated an additional two times
with 3 ml of 2 mg/ml collagenase (30 min, 37°C). The supernatants
of digestions III and IV were pooled and centrifuged, and the cells
were washed in Dulbecco's modified Eagle's medium (DMEM) with 10%
fetal calf serum (FCS), suspended in additional DMEM-10% FCS, and
placed in 75-cm2 flasks. The cells
were incubated under 5% CO2-95%
air at 37°C. Confluence was reached by 5-6 days, at which time
the cells were subcultured. After trypsinization using trypsin-EDTA
(0.05%:0.53 mM), the cells were rinsed in Eagle's minimum essential
medium (MEM) with 5% FCS, resuspended in fresh medium, and then seeded at 5 × 104 cells/ml in
24-well plates (0.5 ml cell suspension/well, i.e., 1.4 × 104
cells/cm2). The osteoblast-like
character of these cells has been established by demonstration of high
levels of alkaline phosphatase activity and osteocalcin production (10)
and a sensitive adenylate cyclase response to parathyroid hormone and
prostaglandins (12).
Proliferation studies (cell counts and thymidine incorporation) were
performed both in actively growing and nonactively growing cell
populations. To produce actively growing cells, subconfluent populations (24 h after subculturing) were changed to fresh MEM, which
contained 1% FCS and the experimental compounds. To produce nonactively growing cells, subconfluent populations were changed to
serum-free medium with 0.1% bovine serum albumin plus the experimental compounds. Cell numbers were analyzed at 6, 24, and 48 h after addition
of the peptide or vehicle. The cell numbers were determined after cells
were detached from the wells by exposure to trypsin-EDTA (0.05%/0.53
mM) for ~5 min at 37°C. Counting was performed in a hemocytometer
chamber. Results were expressed per well.
[3H]thymidine
incorporation into actively growing and nonactively growing cells was
assessed by pulsing the cells with
[3H]thymidine (1 µCi/well) 2 h before the end of the experimental incubation. The
experiment was terminated at 6, 24, or 48 h by washing the cells in MEM
containing cold thymidine followed by the addition of 10%
trichloroacetic acid. The precipitate was washed twice with
ethanol-ether (3:1), and the wells were desiccated at room temperature.
The residue was redissolved in 2 M KOH at 55°C for 30 min and
neutralized with 1 M HCl, and an aliquot was counted for radioactivity.
Results were expressed as disintegrations per minute (dpm) per well.
For both cell counts and thymidine incorporation, each experiment at
each time point was performed at least four different times using
experimental groups consisting of at least six wells.
[3H]phenylalanine
incorporation was assessed by pulsing the cells with
[3H]phenylalanine
(1µCi/well) for 4 h before the end of the experimental incubation.
Bone organ culture.
Bone resorption studies were carried out in neonatal mouse calvaria as
described previously (31). Mice were injected subcutaneously with 5 µCi 45Ca at 2 days of age, and
hemicalvaria were dissected out 4 days later. Hemicalvaria were
preincubated for 24 h in medium 199 with 0.1% bovine serum albumin and
then changed to fresh medium containing peptide or vehicle. Incubation
was continued for a further 48 h. To assess DNA synthesis,
[3H]thymidine (0.6 µCi/ml) was added in the last 4 h of the incubation, as described
previously (22). Similarly, in cultures in which protein synthesis was
determined,
[3H]phenylalanine (1 µCi/ml) was added. There were five to seven hemicalvaria in each
group.
In vivo studies.
The local effects of adrenomedullin on bone histology in vivo were
assessed in adult mice, using the model we have previously described
(4). Sexually mature male ARC Swiss Webster mice, aged between 40 and
50 days and weighing 25-35 g, were given injections (25 µl) over
the periosteum of the right hemicalvaria for 5 consecutive days.
Previous studies with dye injections have shown that the injected
material spreads over much of the injected hemicalvaria but does not
cross the midline. The animals were maintained on a low-calcium diet
(0.1%) from 5 days before the first injection. Two groups of mice
(n = 12 in each) were injected daily
with adrenomedullin in doses of 4 × 10
10 and 4 × 10
9 mol,
respectively. These doses were chosen on the basis of our experience
with other bone-active peptides in this model. Animals in the control
group (n = 16) were injected with
vehicle (water) only. All animals were killed 1 wk after the last
injection. The study had the approval of the local institutional review
board.
The calvaria were dissected free of soft tissue, and gross morphology
was assessed by examination of the intact calvaria under a dissection
microscope. Bone tissue was fixed in 10% phosphate-buffered Formalin,
dehydrated in a graded series of ethanol solutions, and embedded
undecalcified in methyl methacrylate resin. Sections (4 µm thick)
were cut on a Leitz rotary microtome (Leica Instruments, Nussloch,
Germany) using a tungsten-carbide knife, mounted on gelatin-coated
slides, and air-dried. The sections were stained with a Goldner
trichrome stain and examined with the use of an Olympus BX 50 microscope (Olympus Optical, Tokyo, Japan), which was attached to an
Osteomeasure Image Analyzer (Osteometrics, Atlanta, GA).
Histomorphometric analyses were made of three adjacent fields (using a
×20 objective) in each hemicalvarium. This results in
measurements being made over ~90% of the length of each
hemicalvarium. The parameters assessed are as defined by the American
Society for Bone and Mineral Research (27) and are expressed per
millimeter of calvarial length. Osteoblasts were defined as cuboidal
cells immediately adjacent to osteoid. Osteoclast numbers included only multinucleated cells. The various surface estimates were based on
measurements of both periosteal and intramembranous surfaces: those
eroded by osteoclasts (eroded perimeter), those immediately adjacent to
osteoclasts (osteoclast perimeter), and those immediately adjacent to
osteoblasts (osteoblast perimeter). The precisions of these
histomorphometric measurements in our laboratory (expressed as
coefficients of variation of paired measurements) are as follows: mineralized bone area 1.3%, osteoid area 6.9%, osteoblast perimeter 6.8%, osteoblast number 1.7%, eroded perimeter 6.7%, osteoclast perimeter 7.9%, osteoclast number <1.0%, and calvarial length 0.2%. All measurements were made by one operator (J. Cornish) who was
blinded to the treatment group of each bone.
Materials.
Human adrenomedullin and its fragments were synthesized on
methylbenzhydrylamine resin, using standard solid-phase procedures, and
cleaved with hydrogen fluoride-anisole. Sequences containing a
disulfide bridge were cyclized by titration with
I2 in 90% acetic acid-water
solutions (26). Crude materials were purified by gel filtration on
Sephadex columns in 50% acetic acid followed by gradient elution on
C-18 silica using acetonitrile-0.1% trifluoroacetic acid eluants.
Homogeneity of final peptides was assessed by thin-layer chromatography, analytic high-performance liquid chromatography, amino
acid analysis, and matrix-assisted laser-desorption-ionization mass
spectroscopy. Purities were usually >98%.
EDTA and collagenase were obtained from Sigma Chemical, St. Louis, MO.
Trypsin-EDTA, MEM, DMEM, medium 199, and FCS were from GIBCO
Laboratories, Grand Island, NY.
[3H]thymidine,
[3H]phenylalanine, and
45Ca chloride were from Amersham,
England.
Statistical analysis.
Data are presented as means ± SE. The in vitro experiments were
analyzed using Student's t-tests for
pair-wise comparisons. Comparisons across more than two groups have
used analysis of variance (ANOVA), and subsequent pair-wise comparisons
were made using the Bonferroni inequality to adjust for multiple
comparisons, where appropriate. A value of
= 0.05 was accepted as
significant throughout. Where several experiments have been shown in
one figure, data are presented as treatment-to-control ratios, but the
P values shown were calculated using
the data from the individual experiments before the data were pooled.
All other data are from individual, representative experiments.
In the in vivo experiment, the primary end-point for each
histomorphometric index was the determination of whether it was different in the injected right hemicalvaria from the contralateral, uninjected bone of the same animal. Ratios of each index in these two
bone regions were derived and compared between groups by ANOVA. These
data and the associated statistical significances are presented in
Figs. 7 and 8. The absolute values of each histomorphometric index are
also presented for the injected and uninjected hemicalvaria by
treatment group (Table 1).
 |
RESULTS |
Effect of adrenomedullin on isolated osteoblasts.
The effect of adrenomedullin on proliferation of fetal rat osteoblasts
was assessed by the measurement of cell numbers. Treatment with
adrenomedullin for 24 h, in cultures grown in medium containing 1%
FCS, produced a dose-dependent increase in the numbers of actively growing osteoblasts (Fig. 1). A significant
increase was observed at adrenomedullin concentrations of
10
12 M and greater. This
stimulation was maintained for 48 h (Fig. 2A). To
determine whether proliferation in response to adrenomedullin was
dependent on the basal growth rate of the cells, these experiments were
repeated in nonactively growing osteoblastic preparations. The time
course of the increase in cell number in response to adrenomedullin
(10
10 M) in these cells was
similar to that seen in actively growing cells (Fig.
2B).

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Fig. 1.
Dose response of adrenomedullin (ADM) treatment for 24 h on cell
no./well in cultures of actively growing fetal rat osteoblasts. Data
have been pooled from 2 separate experiments, are expressed as ratios
of treatment to control, and are means ± SE. * Significantly
different from control.
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Fig. 2.
Time course of effect of ADM
(10 10 M) on cell no./well
in actively (A) and nonactively
(B) growing fetal rat osteoblasts.
Data are means ± SE. Significantly different from control:
* P < 0.003 and
** P < 0.01.
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The effect of adrenomedullin on DNA synthesis in osteoblasts was
assessed by the measurement of
[3H]thymidine
incorporation into isolated fetal rat osteoblasts. Treatment with
adrenomedullin for 24 h produced a dose-dependent increase in
[3H]thymidine
incorporation (Fig.
3A).
This stimulation was maintained for 48 h (Fig.
3B).

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Fig. 3.
A: dose response of ADM treatment on
[3H]thymidine
incorporation into nonactively growing fetal rat osteoblasts at 24 h.
There is a significant treatment effect when assessed by analysis of
variance (ANOVA; P = 0.01).
B: time course of effect of ADM on
[3H]thymidine
incorporation into nonactively growing fetal rat osteoblasts. Data are
means ± SE. * Significantly different from control.
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To determine in what part of the adrenomedullin molecule its anabolic
activity resides, we investigated the effects of various synthetic
fragments of the peptide on the proliferation of fetal rat osteoblasts
(Fig. 4). Treatment for 24 h with the
(15
52), (22
52), or (27
52) peptides produced a similar stimulation
of proliferation to that from the intact adrenomedullin molecule. However, adrenomedullin-(40
52) did not produce any increase in cell
number. When the disulfide bond of adrenomedullin was broken, the
resultant reduced peptide had diminished agonist activity, which was
detectable only at concentrations
10
10 M, with a maximal
effect one-half of that seen with the intact molecule (data not shown).

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Fig. 4.
Effect of indicated fragments of ADM on cell no./well in cultures of
actively growing fetal rat osteoblasts cultured for 24 h. Data from 1 representative experiment for each fragment have been pooled and are
expressed as ratios of treatment to control. Data are means ± SE.
Significantly different from control:
* P < 0.01.
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In view of the similarity of the present findings to those of amylin in
these model systems, we assessed the effects of putative amylin
receptor blockers on these actions of adrenomedullin. Amylin-(8
37) is
a specific blocker at the amylin receptor in skeletal muscle (7), which
we have recently shown to block the proliferative effect of amylin in
fetal rat osteoblasts (J. Cornish, unpublished observations). Amylin in
which the disulfide bond has been broken (reduced amylin) is also an
effective amylin blocker in bone (J. Cornish, unpublished
observations). These peptides completely blocked the proliferative
effects of both intact adrenomedullin (Fig.
5) and its (27
52) fragment (data not
shown) in these cells. In contrast, reduced adrenomedullin was a less
effective antagonist, diminishing stimulation of cell numbers by
one-half when present in a 10-fold excess with the intact peptide (data
not shown).

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Fig. 5.
Effect of amylin receptor blockers, amylin-(8 37)
(10 9 M) and reduced amylin
(10 9 M), on ADM
(10 10 M) action on cell
number and thymidine incorporation in actively growing fetal rat
osteoblasts. Data are means ± SE. There is a significant treatment
effect in each experiment by ANOVA (P < 0.004), and pair-wise comparisons indicate that only ADM is
significantly different from control.
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The stimulation of osteoblast proliferation by adrenomedullin was
accompanied by similar changes in
[3H]phenylalanine
incorporation into these cells, implying similar changes in protein
synthesis. Thus the incorporation of this amino acid was 3,534 ± 44 dpm/well in control cells, 3,815 ± 68 dpm/well in cells exposed to
adrenomedullin 10
10 M, and
3,818 ± 100 dpm/well in cells exposed to adrenomedullin-(27
52) 10
10 M
(P < 0.05 for both compared with
control).
Effect of adrenomedullin in bone organ culture.
The effects of adrenomedullin were also assessed in cultured neonatal
mouse calvaria. There was a dose-dependent increase in
[3H]thymidine
incorporation (Fig.
6A)
consistent with the findings in isolated cells. However, there was no
significant change in 45Ca release
from prelabeled calvaria treated for 48 h with adrenomedullin at
concentrations of 10
8 to
10
11 M (Fig.
6B), and
45Ca release stimulated by
parathyroid hormone (10
8 M)
was also unaffected by adrenomedullin (data not shown). In contrast,
amylin typically reduces basal resorption by 30% in this model (2).
Incorporation of
[3H]phenylalanine was
increased from 27.3 ± 0.7 dpm/µg in control bones to 30.1 ± 0.6 and 30.0 ± 0.6 in bones exposed to adrenomedullin (10
9 M) and
adrenomedullin-(27
52)
(10
9 M), respectively
(P < 0.05 for both
compared with control).

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Fig. 6.
A: dose-response relationship for
[3H]thymidine
incorporation into neonatal mouse calvaria after 48 h of treatment with
ADM. Data are means ± SE. There is a significant treatment effect
(P = 0.0002). * Significantly
different from control. B: percentage
of 45Ca-release/bone from neonatal
mouse calvaria after 48 h of treatment with ADM. Data are means ± SE. There was no significant change from control.
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Effect of adrenomedullin in vivo.
Table 1 sets out the histomorphometric indexes in the uninjected and
injected hemicalvaria from each of the three groups. The statistical
analysis has been performed on the ratios of each index in the injected
to the uninjected halves of each calvaria, and these results are shown
in Figs. 7 and
8. There were two- to threefold increases
in the indexes of osteoblast activity in those bones exposed to either
dose of adrenomedullin. Resorption indexes showed slight upward trends
in the presence of adrenomedullin, but only for eroded perimeter was
this significant. Mineralized bone area was increased by 13.6% with
the higher dose of adrenomedullin. Periosteal area was not changed
(P = 0.54).

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Fig. 7.
Effect of ADM on indexes of osteoblast
(top) and osteoclast
(bottom) activity in adult male mice
injected daily over right hemicalvaria with either peptide or vehicle
for 5 days. Data are ratios of each histomorphometric parameter in
injected hemicalvaria to that in contralateral bone in same animal.
Data are presented as means and SE. When analyzed by ANOVA, there was a
significant treatment effect on each of the osteoblast indexes
(P < 0.0001) and on eroded perimeter
(P = 0.03).
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Fig. 8.
Effect of ADM on mineralized bone area in adult male mice injected
daily over right hemicalvaria with either peptide or vehicle for 5 days. Presentation of data is as in Fig. 7. There was a significant
treatment effect (P = 0.004).
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DISCUSSION |
The present data establish that adrenomedullin acts on osteoblasts,
whether they be of fetal rat origin cultured as disaggregated cells,
derived from neonatal mice and cultured as an intact tissue, or studied
in vivo in adult mice. Stimulation of cell proliferation occurs whether
assessed by cell numbers or by thymidine incorporation into DNA,
indicating that the result is not dependent on the measurement technique and it is also independent of the basal growth rate of the
osteoblasts. The degree of stimulation of osteoblast proliferation by
adrenomedullin is comparable to that produced by recognized osteoblast
growth factors such as transforming growth factor-
and insulin-like
growth factor I, which each increase cell numbers by ~20% in our
laboratory (based on data from at least 5 experiments with each
peptide; J. Cornish, unpublished observations). Adrenomedullin is not a
nonspecific mitogen, however, because it inhibits proliferation of its
principal target, the vascular smooth muscle cell (15). The explanation
for such opposing effects in these tissues is unknown, but two of the
second messenger systems linked to G protein-coupled receptors
[adenosine 3',5'-cyclic monophosphate (cAMP) and
diacylglycerol] can have opposing effects on cell proliferation
(29). Thus differential activation of these systems could account for
such an effect. Testing of this hypothesis must await an assessment of
the second messenger systems involved in the action of adrenomedullin
on osteoblasts.
Adrenomedullin also increases protein synthesis in vitro and the area
of mineralized and unmineralized bone in vivo. The changes in bone area
are comparable to those produced by amylin (3) and insulin (6) in this
model and are greater than those associated with calcitonin use (3).
The histomorphometric changes are unlikely to represent a nonspecific
effect of the injected peptide, since other peptides, such as
calcitonin gene-related peptide (3) and COOH-terminal parathyroid
hormone-related peptide (5), have no effect on osteoblast activity in
this model. How the concentrations of adrenomedullin used here compare
with those occurring in vivo is uncertain, since the initial high level
will be rapidly reduced as a result of dilution by tissue fluid,
diffusion of peptide away from the injection site, and enzymatic
degradation. Thus there may be no peptide present for much of each 24-h
period, and the average peptide concentration may well be in the
physiological range. It is quite clear, however, that the concentration
of adrenomedullin required to produce a half-maximal effect on
osteoblast proliferation in vitro is comparable to the circulating
concentrations of this hormone found in vivo. This, together with the
present studies in adult mice, suggests that adrenomedullin is likely
to be a physiological influence on osteoblast proliferation in vivo.
Although the physiological role of this hormone is still being defined,
the present data suggest that it might extend beyond vasodilation and
salt and water metabolism to include regulation of bone metabolism.
This is supported by recently published evidence for the expression of
adrenomedullin and its receptor in high concentrations in osteoblasts
during the later stages of rodent embryogenesis and also in the
maturing chondrocytes of fetal mice (23). Taken together with the
present data, this suggests that adrenomedullin might be an important
paracrine regulator of skeletal growth throughout life. An increasing
number of conditions are being identified in which there are major
perturbations of circulating adrenomedullin concentrations, including
acute sepsis (13), hyperthyroidism (34), and pregnancy (8), and high
peptide concentrations have been reported in umbilical cord plasma and amniotic fluid (8). The increase in bone turnover seen in these conditions and in the fetus might be contributed to by adrenomedullin. The recognition of adrenomedullin's effects on bone is also important in the anticipation of possible skeletal effects, should this peptide
be used therapeutically as a vasodilator.
It is of interest to compare the effects of adrenomedullin on bone with
those of amylin. The effects of both agents on osteoblast proliferation
are similar in terms of maximal effect, time dependence, and the
stimulation of growth of both quiescent and actively growing cells (3).
Adrenomedullin produces detectable growth stimulation at lower
concentrations than amylin, although the substantial loss of amylin
onto plastic surfaces may have contributed to this apparent difference.
The effects of both hormones on osteoblasts are blocked by the amylin
antagonists, amylin-(8
37) and reduced amylin, suggesting involvement
of the same receptor in these actions of amylin and adrenomedullin. The
partial sequence identity between the peptides and the similar spectrum
of actions in other tissues are consistent with this possibility (25).
A significant difference between amylin and adrenomedullin exists with
respect to their effects on bone resorption. Amylin is consistently
found to inhibit basal and stimulated resorption in organ culture (2,
28, 33) and to reduce the activity of isolated osteoclasts (1). We have
confirmed this action of amylin in both the neonatal mouse calvarial
model (30) and in vivo in the adult mouse (3) but could detect no
suppression of resorption by adrenomedullin in these models. Consistent
with this is the failure of adrenomedullin to produce hypocalcemia in
the rat (35). In fact, the present in vivo studies show the opposite
trend, probably because of the coupling of resorption to formation,
which comes into play over the longer time course of this experiment.
The dissociation of the osteoblast and osteoclast effects of amylin and
adrenomedullin suggests that these actions are mediated by different
receptors. One possibility would be that amylin produces its inhibition
of bone resorption via the osteoclast's calcitonin receptor, whereas
the anabolic effects on bone of both amylin and adrenomedullin are
mediated by a distinct receptor on the osteoblast. These possibilities
will need to be tested by binding studies.
The present data include a detailed study of the structure-activity
relationships of adrenomedullin. Previously, such data have only been
available for its effects on vascular smooth muscle. In that tissue,
removal of up to 15 NH2-terminal amino acids has little
effect on the peptide's vasodilator activity (9, 11, 32). However,
loss of the ring structure either by more extensive NH2-terminal truncation or by formation of a linear analog
not containing the disulfide bond eliminates specific binding and cAMP
formation in rat vascular smooth muscle cells (9, 32). Removal of the
COOH-terminal amide also results in loss of agonist activity (9), and
NH2-terminal peptides are inactive (9, 11). In contrast,
activity in osteoblasts is preserved in peptides as short as
adrenomedullin-(27
52), suggesting that the disulfide bond and the
ring structure created by it are not necessary. This suggests that a
different receptor mediates the effects of adrenomedullin in
osteoblasts from that in vascular smooth muscle. This dissociation of
the vascular and osteotropic effects of adrenomedullin may be very
important if its proliferative effects on bone were to be used
therapeutically. The diminution in activity of the adrenomedullin molecule after reduction of the disulfide bond is not surprising, since
this will change the conformation of the peptide and interfere with its
receptor binding even if the sequence with affinity for the receptor is
intact.
The activity of these short adrenomedullin fragments is also surprising
when comparison is made with amylin. When the ring is removed from
amylin, the resulting peptide [amylin-(8
37)] has no
agonist activity on osteoblasts and is in fact an antagonist. This
suggests that activation of the osteoblast amylin receptor is
dependent, in part, on the COOH-terminal sequence of the peptide and
that modification in this region can restore activity lost as a result
of NH2-terminal truncation of the molecule. This
observation may be relevant to the design of amylin analogs for use in
bone and other tissues.
In conclusion, the present studies demonstrate that adrenomedullin is a
potent stimulator of osteoblast proliferation and protein synthesis
that increases bone mass and is active at periphysiological concentrations. This raises the possibility that it may have a role in
the regulation of bone metabolism both in health and disease. One of
the major challenges in osteoporosis research at present is the
development of a practical therapy that increases bone mass by
stimulating osteoblast activity. The further definition of the bone
actions of the amylin-adrenomedullin class of peptides is a promising
new research direction in this important area. The present data suggest
that the development of a relatively short peptide from this family,
which has a selective anabolic action on bone, is a real possibility.
 |
ACKNOWLEDGEMENTS |
We thank Qixia Cindy Lin, Usha Bava, and Tom Mulvey for technical
help.
 |
FOOTNOTES |
This research was supported by the Health Research Council of New
Zealand.
Address for reprint requests: I. R. Reid, Dept. of Medicine, Univ. of
Auckland, Private Bag 92019, Auckland, New Zealand.
Received 10 April 1997; accepted in final form 19 August 1997.
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REFERENCES |
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