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Department of Medicine (D.N., K.E.C., A.G., G.J.S.C., I.R.R., J.C.) and School of Biological Sciences (D.N., G.J.S.C.), University of Auckland, Auckland 1001, New Zealand
Address all correspondence and requests for reprints to: Associate Professor J. Cornish, Department of Medicine, University of Auckland, Private Bag 92 019, Auckland, New Zealand. E-mail: j.cornish{at}auckland.ac.nz
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several adrenomedullin receptors have been described. The first, L1, is a seven-transmembrane domain protein, which had been previously reported as an orphan G protein-coupled receptor (13, 14, 15). When transfected into COS cells, this receptor bound 125I-adrenomedullin with high affinity [dissociation constant (Kd) = 8.2 nM], and elevated cAMP levels in response to adrenomedullin. Two further adrenomedullin receptors were characterized after the cloning of receptor activity-modifying proteins (RAMPs) by McLatchie et al. (16). Coexpression of calcitonin receptor (CTR)-like receptor (CRLR), together with either RAMP2 or RAMP3, in Xenopus oocytes, produced new high-affinity binding sites for adrenomedullin. Adrenomedullin secretion, signal transduction, and biological activities have been recently summarized in a comprehensive review (17).
Bone undergoes remodeling, a continual coupled process of resorption and formation, that is essential for the maintenance of skeletal integrity. Bone remodeling is regulated by systemic hormones and by local factors, which affect cells of the osteoclast or osteoblast lineage by influencing rates of cell recruitment, replication, and differentiation. Recently, we demonstrated that adrenomedullin is mitogenic to fetal rat osteoblastic cells in vitro at periphysiological concentrations and promotes bone growth in vivo in adult mice when the peptide is administered either locally or systemically (18, 19). We have also shown that, unlike amylin and calcitonin, adrenomedullin does not suppress bone resorption. A study of the expression of adrenomedullin and the L1 adrenomedullin receptor, during mouse and rat embryogenesis, identified the expression of adrenomedullin in cartilage and in osteoblasts, and of L1 receptor in cartilage (3). However, a detailed analysis of expression of all the putative adrenomedullin receptors and of adrenomedullin itself in osteoblasts has not yet been undertaken.
In the current study, we demonstrate the expression of messenger RNA (mRNA) for adrenomedullin and its three putative receptors in rat osteoblasts. We show that treating primary osteoblasts for 16 h with PTH had no effect on adrenomedullin RNA levels, whereas transforming growth factor-ß (TGF-ß) and insulin-like growth factor-I (IGF-I) had a moderate inhibitory effect. We also demonstrate the production of adrenomedullin peptide in osteoblasts, and specific, high-affinity binding of adrenomedullin to these cells. Our results raise the possibility that adrenomedullin functions as a local regulator of osteoblast number and function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybond N+ nylon membrane, ProbeQuant G-50 micro columns,
-32P-deoxycytidine triphosphate, and
125I-adrenomedullin (rat) were from
Amersham Pharmacia Biotech (Little Chalfont, UK). PolyAT
tract mRNA Isolation System and RNase free deoxyribonuclease (DNase)
were from Promega Corp. (Madison, WI). Complete Protease
Inhibitor Cocktail, proteinase K, deoxyribonucleotide triphosphates
(dNTPs), and AmpliTaq DNA polymerase were from Roche Molecular Biochemicals (Mannheim, Germany). QIAquick gel extraction kit
and RNeasy RNA extraction kit were from QIAGEN (Valencia,
CA), and Random Primers DNA labeling system and PCR primers were
purchased from Life Technologies, Inc. (Gaithersburg, MD).
Rat-adrenomedullin was from American Peptide Co.
(Sunnyvale, CA), and rabbit anti-adrenomedullin (rat) polyclonal
antibody was from Peninsula Laboratories, Inc. (Belmont,
CA). Rabbit ExtrAvidin Peroxidase Staining Kit (Extra-3) and
Sigma Fast 3,3'-diaminobenzidine tablet sets (DAB) were
from Sigma-Aldrich Corp. (St. Louis, MO).
Osteoblast-like cell culture
The following protocol has been approved by the Animal Ethics
Committee, The University of Auckland, application N690. Osteoblasts
were isolated by collagenase digestion from 20-day fetal rat calvariae,
as previously described (20). Calvariae 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 calvariae were treated twice with PBS
containing 3 mM EDTA (pH 7.4) for 15 min at 37 C in a
shaking water bath. After washing once in PBS, the calvariae were
treated twice with 3 ml of 1 mg/ml collagenase for 7 min at 37 C. After
discarding the supernatants from digestions I and II, the calvariae
were treated twice more 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 DMEM with 10% FCS, suspended
in further 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 MEM with 5% FCS and resuspended in fresh medium,
then seeded according to the protocol of each experiment. The
osteoblast-like character of these cells has been established by
demonstration of high levels of alkaline phosphatase activity and
osteocalcin production (21), and a sensitive adenylate
cyclase response to PTH and PGs (22). UMR106–06 cells
were grown in DMEM/5% FCS in 75-cm2 flasks,
incubated under 5% CO2-95% air at 37 C.
Preparation of RNA
Total cellular RNA was purified from cells by a modified method
of single-step guanidinium thiocyanate-phenol-chloroform RNA extraction
(23). The protocol was followed up to the step of
isopropanol precipitation, then the RNA pellet was washed with ice-cold
70% ethanol, air-dried, and dissolved in water. The RNA was then
reprecipitated using 0.3 M sodium acetate (pH 5.2) and 2.5
vol of ethanol, incubated for 16 h at -20 C, centrifuged as
above, and dissolved in water. Poly A+ RNA was prepared from 1 mg total
RNA, using PolyAT tract mRNA Isolation System. For the analysis of
adrenomedullin transcription regulation, cells were seeded at 1.25
x 106 cells/75-cm2 flask,
one flask for each treatment, and incubated for 24 h in DMEM/0.1%
BSA. Media were replaced with fresh DMEM/0.1% BSA, and the cultures
were incubated for 16 h with the different factors at the
indicated concentrations. RNA for this experiment was extracted using
RNeasy mini kit. For all the RNA preparations, RNA concentration and
purity were determined by measuring the OD using a Gene Quant
spectrophotometer (Amersham Pharmacia Biotech), and the
quality was determined by electrophoresis on a 1% agarose gel.
Northern blot analysis
RNA was electrophoresed on a 1% agarose gel containing 1.2
M formaldehyde, 3 µg/ml ethidium bromide, and 1 x
MOPS buffer (20 mM 3-[N-morpholino] propanesulfonic acid,
pH 7.0, 5 mM sodium acetate, 1 mM EDTA). RNA
samples were denatured in RNA loading buffer (1 mM EDTA,
0.22 M formaldehyde, 7.7% formamide, 5% glycerol, and
bromophenol blue in 1x MOPS buffer) for 10 min at 65 C, chilled on
ice, and loaded onto the gel, which was run in 1.2 M
formaldehyde and 1x MOPS buffer. The gel was photographed under UV
light and the RNA transferred overnight to Hybond N+ nylon membrane by
capillary action in 20 x SSC (3 M NaCl and 0.3
M Na citrate, pH 7.0). RNA was immobilized on the membrane
by Stratalinker UV cross-linker (Stratagene, La Jolla,
CA). The probes used for hybridization were purified from agarose gels
containing amplification products of the relevant complementary DNA
(cDNA), using QIAquick gel extraction kit, and labeled with
-32P-deoxycytidine triphosphate using Random
Primers DNA labeling system. Unincorporated nucleotides were separated
from the labeled probe using ProbeQuant G-50 micro columns. The RNA
blots were prehybridized in 0.25 M
Na2HPO4, pH 7.5, 7% SDS,
and 1 mM EDTA for 2 h at 65 C. The probes were
denatured by adding NaOH to a final concentration of 0.2 M
and incubating for 10 min at room temperature. The blots were
transferred to fresh hybridization buffer, which contained the
denatured probe, and were hybridized for 20 h at 65 C. The
membranes were then washed in 0.2 x SSC and 0.5% SDS, first for
10 min at room temperature and then for 1 h at 65 C. All membranes
were later stripped and rehybridized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) probe to normalize mRNA levels. The GAPDH DNA
used for labeling was amplified by PCR from a construct of a 200-bp
fragment of rat GAPDH cDNA cloned into pBluescript vector. This
construct was a kind gift from the laboratory of Dr. Yoav Citri (The
Weizmann Institute of Science, Rehovot, Israel). The membranes were
exposed to x-ray films at -80 C with intensifying screens and were
also analyzed by a Phosphor Imager (BAS 2040; Fuji Photo Film Co., Ltd., Tokyo, Japan).
RT-PCR
Before using RNA as a template for RT-PCR, it was treated with
DNase, to remove any DNA contaminants. One hundred micrograms of RNA
were incubated for 30 min at 37 C with 4 U of RQ-1 RNase-free DNase, 40
U RNaseOUT, 2 mM 1,4-dithiothreithol, 40 mM
Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM
MgCl2, and 10 mM
CaCl2 in a final vol of 100 µl. The RNA samples
were then incubated for 20 min at 37 C with 50 µg/ml proteinase K, in
a buffer containing 10 mM Tris-HCl (pH 8.0), 5
mM EDTA, and 0.5% SDS, in a final vol of 500 µl. Samples
were extracted with equal volumes of water-saturated phenol, and then
with equal volumes of chloroform, and precipitated with 0.3
M Na acetate (pH 5.2) and 2.5 vol of ethanol.
All RT-PCR amplifications were carried out using aerosol barrier
pipette tips. cDNA was synthesized from 1 µg DNase-treated RNA in 1x
PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl)
and 2 mM MgCl2, using 0.1U random
hexamer primer, 1 mM of each dNTP, 5 mM
1,4-dithiothreithol, 40 U RNaseOUT, and 200 U of M-MLV-reverse
transcriptase, in a final vol of 20 µl. The reaction was incubated
for 60 min at 37 C, and then heat inactivated at 95 C for 5 min. The RT
reaction was used directly for subsequent PCR amplification. The PCR
was carried out in a final vol of 50 µl containing 1x PCR buffer, 2
mM MgCl2, 1 µM of each
primer, 0.4 mM of each dNTP, and 1U of AmpliTaq DNA
polymerase. PCR was carried out in an automatic DNA thermal cycler
(Mastercycler Personal; Eppendorf, Hamburg, Germany).
After an initial denaturation step of 2 min at 94 C, 35 PCR cycles were
performed. Denaturation was carried out at 94 C for 30 sec, annealing
at 55 C for 30 sec, and primer extension at 72 C for 30 sec for the
first cycle, adding 5 sec per cycle after that. The primer pairs used
to amplify the specific cDNAs are listed in Table 1
. PCR products were purified from
agarose gels using QIAquick gel extraction kit, and their sequences
were determined on an ABI 377 XL DNA Sequencer (PE Applied Biosystems, Foster City, CA).
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Receptor binding assay
All the tubes used for binding assays were silanized with a
solution of 2% dichlorodimethylsilane in acetone, to minimize peptide
adherence to plastic. For the membrane binding assay, the membranes
were thawed, aliquoted into 0.5-ml tubes at 20 µg protein/tube, and
centrifuged at 13,000 rpm for 10 min at 4 C in a microfuge. The
membrane pellets were resuspended in 250 µl of binding buffer (100
mM HEPES, pH 8, 118 mM NaCl, 5 mM
KCl, 1.2 mM MgSO4, 8.8 mM
dextrose, and 0.2% BSA), containing the
125I-adrenomedullin and unlabeled rat
adrenomedullin competitor, as described for each experiment. After
incubation for 2 h at room temperature, binding reactions were
terminated by centrifugation as above and one wash with 250 µl of
ice-cold binding buffer. The radioactivity of the pellets was
determined in a 1480 Wizard-3 Automatic 
-counter (Wallac, Inc. Oy, Turku, Finland). A similar procedure was followed for
binding to intact cells. Cells were grown overnight in 12-well plates,
incubated for 30 min in serum-free medium supplemented with 0.1% BSA,
and washed twice with 0.5 ml binding buffer. The cells were then
overlaid with 0.5 ml binding buffer containing
125I-adrenomedullin and the unlabeled rat
adrenomedullin competitor, and incubated at room temperature for 2
h. At the end of the incubation, the cells were washed twice with 0.5
ml ice-cold binding buffer, and then lysed by incubation with 1 ml/well
of 0.5 M NaOH solution for 30 min at 55 C. The cell lysate
was collected into counting vials, wells were washed again with 1 ml
0.5 M NaOH, which was then pooled with the lysate from each
well. The radioactivity was counted in a -counter as above. The
results shown are representative of at least three experiments
performed in triplicate. All binding data analyses were performed by
nonlinear regression, using GraphPad Software, Inc. 3.1
(GraphPad Software, Inc., San Diego, CA).
Immunocytochemistry
Cells were seeded into eight-chamber slides at a density of
5 x 104 cells/chamber, containing
5%FBS/MEM and incubated for 24 h at 37 C in
5%CO2-95% air. Cells were subsequently fixed in
2% paraformaldehyde, incubated with 5% Triton X-100 for 10 min, and
washed three times with PBS. For blocking of nonspecific signal, cells
were incubated for 1 h at room temperature with normal goat serum,
diluted 1:50, then washed three times with PBS. Primary antibody
diluted (1:1000) in PBS/1% BSA was added to the cells, and PBS/1% BSA
alone or normal rabbit serum diluted (1:1000) was added to the negative
controls. Slides were incubated for 1 h at room temperature and
then washed three times with PBS. Detection of the primary antibody was
achieved using a Rabbit ExtrAvidin Peroxidase Staining Kit (Extra-3).
Sigma Fast 3,3'-diaminobenzidine tablet sets were used as
substrate for the peroxidase. Slides were washed three times with PBS,
counterstained with Mayer’s hematoxylin, rinsed 3 times in distilled
water, and coverslipped using Gurr Aquamount mountant (BDH Laboratory,
Poole, England).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, both
primary osteoblasts and UMR106–06 cells express the genes for
adrenomedullin, L1, RAMP1, RAMP2, RAMP3, and CRLR. CTR, however, is
expressed in the UMR106–06 cell line but not in primary osteoblastic
cells. As negative controls for the RT-PCR, the same reactions were
carried out without RNA or in the absence of the reverse-transcriptase
enzyme. All these controls were negative (data not shown).
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). A very high expression of RAMP2 was
detected in both primary osteoblasts and UMR106–06 cells, and a
moderate expression level of RAMP3 in primary osteoblasts only. These
two RAMPs are the ones which have been shown in the past to form
specific adrenomedullin receptors when coexpressed with CRLR. Our
Northern analysis also showed a low level of expression of RAMP1 in
primary osteoblasts, and a very low level in UMR106–06. Quantitative
analysis of the bands’ intensity is shown in Fig. 2B. All values are
presented as a ratio between the RAMP and the GAPDH signals.
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). We
determined, by immunocytochemistry, the expression of adrenomedullin
peptide in the cells. Figure 3B shows strong staining of cells
incubated in the presence of primary anti-adrenomedullin antibody (I)
and no staining in the control (II), which was incubated without the
primary antibody. An additional control, using normal rabbit serum
instead of the primary antibody, was also negative. These experiments
demonstrate that adrenomedullin mRNA and peptide were both highly
expressed in primary osteoblastic cells.
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shows an autoradiograph of a Northern
blot of total RNA extracted from treated and control cells, hybridized
to an adrenomedullin cDNA probe, and then rehybridized to a GAPDH cDNA
probe. In this autoradiograph, the adrenomedullin band seems to migrate
slightly slower in lanes 1–3, in comparison with lanes 4 and 5. This
probably results from a small distortion in the gel and the higher
amounts of RNA loaded in lanes 1–3. This experiment was repeated
twice, with similar results, and a quantitative analysis of these
results is presented in Fig. 4B. TGF-ß inhibited adrenomedullin RNA
expression, IGF-I had a moderate inhibitory effect, and PTH did not
affect adrenomedullin RNA levels. Hydrocortisone stimulated the
expression of adrenomedullin RNA, similar to its effect in vascular
smooth muscle cells.
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, we detected
specific high-affinity binding of adrenomedullin to both osteoblast
membranes and to intact cells.
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) could be fitted to a two-binding-site
model significantly better than to a one-site model (F = 13.24,
P = 0.0001). Analysis of three binding experiments
showed that 125I-adrenomedullin binds to a
high-affinity site with inhibitory concentration 50%
(IC50) = 0.41 ± 0.16
nM, and to a low-affinity one with
IC50 = 612 ± 250
nM. The maximal binding of
125I-adrenomedullin to the cell membrane
preparation was 15.1 ± 0.69 fmol/mg protein. The maximal binding
of 125I-adrenomedullin to the intact cells
was 4.84 ± 0.12 fmol/106 cell, that
corresponds to 2910 binding sites/cell. Analyzing the data by nonlinear
regression showed again that the results could be better fitted to a
two-binding-site model than to a one-site model (F = 33.2,
P < 0.0001, Fig. 5B). The IC50
values were 1.9 ± 0.65 nM for the
high-affinity site and 69.7 ± 5.7 nM for
the low-affinity site. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies in our laboratory established that adrenomedullin stimulates proliferation of osteoblasts to a degree comparable with that produced by other osteoblast growth factors, such as TGF-ß and IGF-I (18). We have also shown that adrenomedullin increases protein synthesis in osteoblastic cells in vitro and increases the area of mineralized and unmineralized bone in vivo. These results suggest that adrenomedullin may have a role in the regulation of bone metabolism. Our present study focuses on the expression of genes that might mediate the effects of adrenomedullin in osteoblasts, as well as the expression of the adrenomedullin gene itself. The coexpression of the adrenomedullin ligand and the adrenomedullin receptors in primary osteoblasts and the observation that adrenomedullin binds with high affinity to these cells suggest that the stimulation of osteoblast proliferation and activity by adrenomedullin might involve an autocrine/paracrine mechanism.
Previous studies have suggested an autocrine/paracrine mode of action of adrenomedullin in other tissues and cell types. Tsuruda et al. (25) recently showed that cultured neonatal cardiac fibroblasts produce and secrete adrenomedullin, and that the secreted adrenomedullin may inhibit proliferation and protein synthesis of these cells. They, therefore, suggested an autocrine/paracrine role of adrenomedullin in modulating cardiac fibroblast growth. Miller et al. (26) demonstrated the coexpression of adrenomedullin and adrenomedullin receptor in a variety of human cancer cell lines of diverse origin. They suggested the existence of a potential autocrine growth mechanism that could drive neoplastic proliferation. Montuenga et al. (3) followed the expression of adrenomedullin and its receptor during development of mouse and rat embryos. They found that the localization of the receptor paralleled that of adrenomedullin itself, suggesting a paracrine/autocrine mode of action. Among other organs and tissues, they identified adrenomedullin, by immunohistochemistry, in maturing cartilage, in hypertrophic cartilage, and in osteoblasts of the developing bone. In situ hybridization showed adrenomedullin and adrenomedullin receptor RNA expression in cartilage. It is important to note that the two latter studies that showed expression of adrenomedullin receptor gene were published before the cloning of RAMPs, and they assessed only the expression of the L1 adrenomedullin receptor.
A number of different adrenomedullin receptors have been described in the literature. The first protein that was shown to bind adrenomedullin specifically is L1, which was isolated from a rat lung cDNA library (13). A putative human adrenomedullin receptor, which displays 73% homology to L1, was isolated subsequently (27). There is some controversy regarding L1 and its human homologue. The results described by Kapas et al., which demonstrated high-affinity binding and increased levels of cAMP in COS cells that were transfected with L1 and treated with adrenomedullin, could not be reproduced by other laboratories (16, 28, 29). Another receptor that is sometimes described as adrenomedullin receptor is RDC-1 (30, 31), a receptor originally isolated from a dog thyroid library. However, this receptor has much higher affinity for CGRP than for adrenomedullin and, therefore, was not included in our study. The cloning of the RAMP accessory factors, which, when coexpressed with CRLR or CTR, determine their ligand specificity, was a major breakthrough in the study of receptors for the CGRP peptide family (16). Since the cloning of RAMPs, five novel receptors have been described: a CGRP receptor (CRLR and RAMP1), two adrenomedullin receptors (CRLR and RAMP2 or 3), and two amylin receptors (CTR and RAMP1 or 3) (32, 33). In the present study, we demonstrated the expression of RNA for L1, CRLR, RAMP2, and RAMP3, which form the three receptors that are considered the most likely to be specific adrenomedullin receptors.
The results of our binding studies show, for the first time, high-affinity binding of 125I-adrenomedullin to primary osteoblasts. Binding of 125I-adrenomedullin was measured in cell membrane preparations as well as in intact cells. The adrenomedullin binding results produced a curve that could be best fitted to a two-site competition model. In the membrane preparation, the presence of two binding sites could be attributable to interaction of the 125I-adrenomedullin with a G protein-coupled receptor. As the cellular GTP is washed away, a fraction of the receptors are associated with G proteins, whereas others are not. This forms a heterogeneous population of receptors with different affinities for the 125I-adrenomedullin ligand. However, the results of the intact cell study also indicate the existence of two binding activities with different affinities for the ligand. We conclude, therefore, that primary osteoblasts have at least two types of functional adrenomedullin receptors. There is some variability between cell membrane and whole-cell adrenomedullin binding results in the present study. For the high-affinity binding site, IC50 values are 0.41 ± 0.16 nM in the membrane experiment and 1.9 ± 0.65 nM in the intact cells, whereas for the low affinity site, IC50 is 612 ± 250 nM in the membrane experiment and 69.7 ± 5.7 nM in intact cells. There is also a difference in the extent of nonspecific binding, which is much higher in the cell membrane experiment. A number of factors could contribute to the differences in the results of the two experimental approaches. The disruption of the cells in the membrane binding study could have exposed some intracellular binding sites for adrenomedullin; whereas in the intact cells, only binding to the plasma membrane is measured. As discussed above, GTP concentrations could be different in the two experiments. Some technical details, such as working in siliconized tubes vs. tissue culture dishes and separating bound from free ligand using centrifugation vs. aspirating medium from adherent cells could have also contributed to these differences. Similar variability in IC50 values has been found for related peptides studied in these distinct experimental conditions (34).
Our gene expression studies, using the RT-PCR technique, were performed using RNA from primary osteoblasts as well as that of the osteoblast-like cell line, UMR106–06. Although the primary osteoblast cultures prepared in our laboratory consistently show very high percentages of alkaline phosphatase-positive cells, these cultures cannot be considered as absolutely homogenous. Therefore, the clonal cell line UMR106–06, which was originally derived from a rat osteogenic sarcoma with an osteoblastic phenotype, was studied in parallel with the primary osteoblasts. It has been reported previously that this particular subclone of the UMR106 cell line expresses a CTR (35). The results of the RT-PCR experiment showed that six of the seven genes tested were present in both primary osteoblasts and UMR106–06, whereas CTR was only expressed in UMR106–06. Therefore, it seems that these six genes are truly expressed in osteoblastic cells and were not amplified from a small population of nonosteoblastic cells in the primary cultures. RAMP expression was further analyzed by Northern blot. This experiment not only confirmed the expression of RAMPs in the osteoblasts but also enabled us to quantify the relative levels of expression of the three RAMPs. Very high expression of RAMP2 mRNA was detected in both primary osteoblasts and UMR106–06 cells. RAMP1 mRNA is expressed at a low level in primary osteoblasts and at a very low level in UMR106–06. RAMP3 mRNA is expressed in primary osteoblasts and could also be detected in UMR106–06 by RT-PCR; but in this cell-line, the RAMP3 RNA level is below the sensitivity threshold of the Northern blot.
The regulation of adrenomedullin gene transcription and peptide
synthesis has been studied in detail in smooth muscle and endothelial
cells, because of the interest in its action as a potent vasodilator.
Adrenomedullin expression was found to be regulated by a range of
cytokines, growth factors, and hormones, including tumor necrosis
factors and ß, interleukin-1 and ß, and dexamethasone. Other
hormones and growth factors, such as fibroblast growth factor,
epidermal growth factor, and platelet-derived growth factor, were found
to have little effect (17). Studies of the regulation of
adrenomedullin expression in other tissues and cell types revealed that
some factors have similar effects, whereas others act in a
tissue-specific manner. In the present study, we looked at the effect
of major regulators of bone cell growth on adrenomedullin transcription
in primary osteoblasts. We have found that TGF-ß inhibits
adrenomedullin RNA expression in primary osteoblasts, whereas IGF-I has
a moderate inhibitory effect, and PTH does not alter adrenomedullin RNA
levels. We conclude that changes in adrenomedullin expression do not
seem to be central to the growth effect of IGF-I and PTH in primary
osteoblasts. The physiological significance of inhibition of
adrenomedullin RNA levels by TGF-ß is yet to be defined.
Hydrocortisone, which has been found to increase RNA levels in vascular
smooth muscle cells, had a similar effect in the primary osteoblast
cultures. It would be of major interest to study the effects of a
number of other bone growth regulators on adrenomedullin expression. We
also tried to study the effects of the bone growth regulators on
adrenomedullin peptide synthesis, but this was prevented by the low
affinity of the available rat adrenomedullin antibodies. Further
elucidation of the regulation of adrenomedullin transcription and
translation is an important area for future work.
Adrenomedullin shows approximately 20% sequence identity with amylin and CGRP. In our previous work, we have shown that adrenomedullin and amylin stimulate proliferation of primary osteoblasts in vitro (18, 36). The effects of adrenomedullin and amylin on osteoblast proliferation are similar, in terms of maximal effects, kinetics, and the ability to stimulate both quiescent and actively growing cells. Adrenomedullin is mitogenic to osteoblasts in lower concentrations than is amylin. The effects of both hormones are blocked by the amylin antagonists, amylin (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) and reduced amylin. CGRP also stimulates osteoblast proliferation, but it is only effective at a 100-fold higher concentration than amylin, and there is no additivity between maximal doses of CGRP and amylin (37). These data suggested that CGRP may be acting in osteoblasts through an amylin or adrenomedullin receptor. It is interesting to examine these findings in light of our new results, which characterize the type of receptors expressed in the primary osteoblasts. Recent evidence suggests that RAMP function is influenced by coexpression of other RAMP proteins in the same cell. It has been shown that in oocytes, coexpression of RAMP2 or 3 can completely inhibit the activity of RAMP1 (29). In contrast, Buhlmann et al. showed that coexpression of RAMP1 and RAMP2 inhibits the adrenomedullin receptor and produces CGRP-specific receptor (38). Our study of CGRP effects in primary osteoblasts suggested that no specific CGRP receptor is expressed in these cells. This could be a result of the low level of expression of RAMP1, compared with RAMP2, or it is possible that in these cells there is inhibition of CGRP receptor and activation of adrenomedullin receptor, similar to the results from the oocyte study. The best characterized amylin receptors, at present, are the combinations of CTR with either RAMP1 or RAMP3 (32, 33). This raises an interesting question: although amylin clearly stimulates primary osteoblasts to proliferate, there is evidence from this study, as well as from others, that CTR itself is not expressed in these cells; therefore, the receptor that binds and mediates amylin’s effect in primary osteoblasts still needs to be identified.
Previous studies in our laboratory established adrenomedullin as a potent stimulator of osteoblast proliferation and protein synthesis that increases bone mass and is active at periphysiological concentrations. In the present study, we show specific binding of adrenomedullin to primary osteoblast cultures and characterize the adrenomedullin receptors expressed in these cells. We also show expression of adrenomedullin peptide in primary osteoblasts. Taken together, our results raise the possibility that adrenomedullin plays a role in the paracrine/autocrine regulation of bone metabolism. A major challenge in osteoporosis research, at present, is the development of a therapy that increases bone mass by stimulation of osteoblast activity. It is possible that new candidate drugs for the treatment of osteoporosis will emerge from this group of peptides with anabolic effects in osteoblasts.
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Acknowledgments |
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Footnotes |
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Received August 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thyroid-stimulating hormone thyrotroph
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 M Nakamura, S Morimoto, Q Yang, T Hisamatsu, N Hanai, Y Nakamura, I Mori, and K Kakudo Osteoclast-like cells express receptor activity modifying protein 2: application of laser capture microdissection J. Mol. Endocrinol., February 1, 2005; 34(1): 257 - 261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Grey, T. Banovic, Q. Zhu, M. Watson, K. Callon, K. Palmano, J. Ross, D. Naot, I. R. Reid, and J. Cornish The Low-Density Lipoprotein Receptor-Related Protein 1 Is a Mitogenic Receptor for Lactoferrin in Osteoblastic Cells Mol. Endocrinol., September 1, 2004; 18(9): 2268 - 2278. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Cornish, K. E. Callon, D. Naot, K. P. Palmano, T. Banovic, U. Bava, M. Watson, J.-M. Lin, P. C. Tong, Q. Chen, et al. Lactoferrin Is a Potent Regulator of Bone Cell Activity and Increases Bone Formation in Vivo Endocrinology, September 1, 2004; 145(9): 4366 - 4374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Olson, L. E. Deal, and M. Piesman Epinephrine-Secreting Pheochromocytoma Presenting with Cardiogenic Shock and Profound Hypocalcemia Ann Intern Med, May 18, 2004; 140(10): 849 - 851. [Full Text] [PDF]
|
|
|
|
|
|
J. Cornish, M. T. Gillespie, K. E. Callon, N. J. Horwood, J. M. Moseley, and I. R. Reid Interleukin-18 Is a Novel Mitogen of Osteogenic and Chondrogenic Cells Endocrinology, April 1, 2003; 144(4): 1194 - 1201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Villa, C. Dal Fiume, A. Maestroni, A. Rubinacci, F. Ravasi, and F. Guidobono Human osteoblast-like cell proliferation induced by calcitonin-related peptides involves PKC activity Am J Physiol Endocrinol Metab, March 1, 2003; 284(3): E627 - E633. [Abstract] [Full Text] [PDF]
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