Proc. Natl. Acad. Sci.
USA
Vol. 85, pp. 5673-5677, August 1988
Medical Sciences
Direct action of the parathyroid hormone-like human hypercalcemic
factor on bone
(humoral hypercalcemia of malignancy/bone histomorphometry)
DAVID D. THOMPSON*, J. GREGORY SEEDOR, JOHN E. FISHER, MICHAEL ROSENBLATT,
AND GIDEON A. RODAN
Department of Biological Research and Molecular Biology, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486
Communicated by Alexander G. Bearn, March 28, 1988 (received for review January 28, 1988)
ABSTRACT Peptides containing residues 1-34 of the hu-
man parathyroid hormone (PTH)-like hypercalcemic factor
(hHCF), termed hHCF-(1-34)-NH2, produce effects similar to
those of PTH in several biological systems in vitro and in vivo.
However, there is conflicting evidence regarding the potency of
hHCF on bone and, by implication, its role in calcium mobili-
zation and the skeletal contribution to tumor-associated hyper-
calcemia. To resolve this conflict, the effects of infusing either
hHCF-(1-34)-NH2 or a peptide containing residues 1-34 of
bovine PTH [bPTH-(1-34)] into unrestrained thyroparathyroi-
dectomized rats on a low calcium diet were compared. Direct
effects on bone histology and serum calcium levels, which are
totally dependent on calcium mobilization from bone in these
animals, were examined. bPTH-(1-34) and hHCF-(1-34)-NH2
were equipotent in producing dose-dependent calcium mobili-
zation from bone. At an infusion rate of 0.1 nmol/hr, both
peptides produced hypercalcemia and extensive nephrocalcino-
sis. Histomorphometric analysis of tibiae from these animals
after 48 hr of peptide infusion showed a dose-related increase in
osteoclast number from 3-5 cells per mm2 at 0.01 nmol/hr to
L32 cells per mm2 at 0.1 nmol/hr of hHCF or bovine PTH.
These findings indicate that hHCF has a direct PTH-like effect
on bone and, in this model system, the hHCF-(1-34)-NH2 is
equipotent to bPTH-(1-34).
Albright (1) proposed that the syndrome of hypercalcemia of
malignancy could be produced in certain patients by ectopic
production of parathyroid hormone (PTH). Although radio-
immunoassays failed to detect elevated PTH levels in the
circulation of patients with humoral hypercalcemia of malig-
nancy (2, 3) and PTH mRNA could not be found in tumors
from these patients (4), extracts from such tumors exhibited
PTH-like activity in several bioassays (5-8). Proteins that
were similar to PTH in their N-terminal region (9-11) were
purified from at least four human tumors, and the comple-
mentary DNA sequences for two of these proteins were
determined (12, 13). These proteins are identical to each
other in 139 out of the 141 N-terminal amino acids and appear
to be encoded by a single gene, which generates several
mRNAs through alternative splicing (13). Eight of the 13
N-terminal amino acids are identical to PTH, whereas the
rest of the molecule is largely different.
A peptide containing residues 1-34 of human PTH-like
hypercalcemic factor [hHCF, termed hHCF-(1-34)-NH2]
was synthesized and its biological activity was compared to
that of residues 1-34 of bovine PTH [bPTH-(1-34)] (14, 15).
In all assays (including stimulation of bone and kidney
adenylate cyclase, urinary excretion of phosphorus and
cAMP, production of 1,25-dihydroxyvitamin D3 from 25-
hydroxyvitamin D3, the competition for binding to receptors,
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
5673
and the calcemic response in nephrectomized thyroparathy-
roidectomized rats), hHCF-(1-34)-NH2 was at least as potent
as bPTH-(1-34). In several instances in vivo, it was 6-10
times more potent than PTH (14). A notable exception was
the effect of hHCF-(1-34)-NH2 on bone resorption in rat
embryo long bone explants in vitro where its potency was
-6% that of bPTH-(1-34), suggesting that the action of hHCF
on bone may not contribute substantially to hypercalcemia
(15). To address this question, we developed an assay to
evaluate the ability of hHCF to mobilize calcium from bone
in vivo and compared its potency to that of bPTH-(1-34).
MATERIALS AND METHODS
Thyroparathyroidectomy was performed on 200-g male
Sprague-Dawley rats. At the time of thyroparathyroidec-
tomy, a femoral vein catheter (o.d., 0.28 mm) was installed
and guided subcutaneously to the scapular region from which
it exited through a small incision. The catheter was stabilized
by using a rodent jacket (Harvard Bioscience, South Natick,
MA) and attached to a 22-gauge miniature single-channel
swivel (Harvard Bioscience). The swivel was then connected
to a microinjection pump (Harvard Bioscience) calibrated to
an infusion rate of 41.7 ,ul/hr (1 ml/24 hr). This arrangement
permitted free movement of the animal and allowed us to
change syringes daily. Immediately after surgery, saline
infusion was started to maintain catheter patency. Twelve
hours after surgery, animals were placed on a calcium-
deficient diet (<0.009% Ca) (Toverud, ICN Nutritional,
Cleveland). Infusion of hHCF-(1-34)-NH2 or bPTH-(1-34) at
rates of 0.01, 0.03, 0.06, and 0.10 nmol/hr in saline was
started 24 hr after surgery (four animals per group). Animals
could not be maintained on saline infusion alone over the
course of this study due to extreme hypocalcemia resulting in
death.
Peptides. The hHCF-(1-34)-NH2 was synthesized as de-
scribed (14). bPTH-(1-34) was purchased from Bachem
(Torrance, CA).
Serum Chemistry. Blood samples were obtained retroor-
bitally from the eye at the time of thyroparathyroidectomy at
the onset of infusion (time 0), and at 24 and 48 hr after the
onset of infusion. Serum calcium (mg/dl) was determined by
atomic absorption spectrophotometry. When the sample size
was sufficient, serum phosphorus (mg/dl) was also deter-
mined (16).
Histology. After 48 hr of continuous infusion, animals were
sacrificed, and the tibiae were removed and fixed in 70%
(vol/vol) ethanol. The bones were dehydrated in an ethanol
series and embedded in methyl methacrylate. Coronal sec-
tions (6 ,um thick) were removed from the proximal tibiae and
stained with Masson's trichrome stain. Histomorphometric
Abbreviations: PTH, parathyroid hormone; hHCF, human PTH-like
hypercalcemic factor; bPTH, bovine PTH.
*To whom reprint requests should be addressed.
5674 Medical Sciences: Thompson et al.
quantification of osteoclast number per mm2 of marrow and
per mm of trabecular surface was carried out with a semi-
automatic real-color Magiscan image analyzer (Joyce-Loebl)
and a Nikon Microphot-Fx microscope ( x 488). Osteoclasts
were stained red with Masson's trichrome stain and were
readily detected with the Sony model DXC-M3AP color
video camera attached to the microscope and the Magiscan
image analyzer. Trabecular bone surfaces (stained blue) were
quantified with the "Bone" software package developed for
bone histomorphometry (Joyce-Loebl). The tibial metaphy-
seal region analyzed was located in the secondary spongiosa
underlying the epiphyseal growth plate. At the time of
sacrifice, kidneys were also removed, fixed in 70% and 95%
(vol/vol) ethanol, and embedded in glycol methacrylate.
Coronal sections 4.5 Am thick were removed from the
kidneys and stained with von Kossa stain (a toluidine blue
counterstain) and hematoxylin and eosin.
RESULTS
Twenty-four hours after thyroparathyroidectomy, serum cal-
cium levels declined to =5 mg/dl (Fig. 1). Nearly identical
calcemic responses were noted for the two peptides at any
given dose. Infusion of hHCF-(1-34)-NH2 or bPTH-(1-34) at
0.01 nmol/hr sustained the calcium level after thyroparathy-
roidectomy for 48 hr (Fig. 1). Infusion of hHCF-(1-34)-NH2
or bPTH-(1-34) at 0.03 nmol/hr did not change the level of
serum calcium (5 mg/dl) over the first 24 hr but increased
l0
ci
u animals infused with bPTH. No mineralization was noted in
8 kidneys from rats infused with either peptide at 0.01 nmol/hr.
B
13
la 11
I-,
to,
9-
7.~
Time, hr
FIG. 1. Serum calcium values (mean ± SEM) from thyropa-
rathyroidectomized rats (four rats per group) were infused for 48 hr
at 0.1 nmol/hr (A), 0.06 nmol/hr (&), 0.03 nmol/hr (m), or 0.01
nmol/hr (e) with hHCF-(1-34)-NH2 (A) or bPTH-(1-34)-NH2 (B).
Proc. Natl. Acad. Sci. USA 85 (1988)
8Co
04
2
G
0 10 20 30 40 50
Time, hr
FIG. 2. Serum phosphorus values (mean + SEM) from rats
infused for 48 hr with hHCF-(1-34)-NH2 at 0.06 nmol/hr (A), 0.03
nmol/hr (n), or 0.01 nmol/hr (e) or with bPTH-(1-34) at 0.03 nmol/hr
(o) or 0.1 nmol/hr (A).
calcium levels to 410 mg/dl by 48 hr (Fig. 1). In animals
infused with hHCF-(1-34)-NH2 or bPTH-(1-34) at 0.06 or
0.10 nmol/hr, calcium levels reached 10 mg/dl by 24 hr. With
hHCF-(1-34)-NH2 or bPTH-(1-34) at 0.1 nmol/hr, calcium
continued to increase and reached hypercalcemic levels (12.5
mg/dl) at 48 hr. Serum phosphorus levels declined in a
dose-dependent manner in animals receiving either peptide.
Rats infused with hHCF-(1-34)-NH2 at 0.01 nmol/hr showed
no significant changes in phosphorus levels over the exper-
imental period (Fig. 2). With infusion ofeither peptide at 0.03,
0.06, or 0.10 nmol/hr, dose-related nephrocalcinosis was
observed. Animals infused with hHCF appeared to have
more extensive nephrocalcinosis by gross examination than
Calcification was restricted to the collecting ducts and
proximal and distal tubules as determined by von Kossa
positive staining.
Examination of the tibiae by histological methods provided
direct evidence for the action of hHCF-(1-34)-NH2 and
bPTH-(1-34) on bone and for the equipotency of the two
peptides in vivo (Fig. 3). Many osteoclasts were seen at 48 hr
on cortical and trabecular bone surfaces at the three highest
30
0,
20.
10
Infusion rate, nmol/hr
FIG. 3. Histomorphometric quantification of the number of
osteoclasts per mm2 (mean ± SEM) of proximal tibial metaphysis
after 48 hr of infusion with the indicated concentrations of hHCF-(1-
34)-NH2 (open bars) or bPTH-(1-34) (cross-hatched bars).
 Medical Sciences: Thompson et al.
infusion doses. Osteoclast number was related to the peptide
dose and nearly identical effects were produced by equal
infusion rates of hHCF-(1-34)-NH2 and bPTH-(1-34) (Fig. 4).
Infusion of either peptide at 0.01 nmol/hr was associated with
S5 osteoclasts per mm2; infusion at 0.10 nmol/hr was
associated with w32 osteoclasts per mm2. Many multinucle-
ated osteoclasts were apposed directly to trabecular surfaces,
and, in animals receiving either peptide at 0.06 and 0.1
nmol/hr, virtually all subepiphyseal trabecular surfaces con-
tained osteoclasts (Fig. 4). The cortical bone of animals
treated with the higher two doses of the peptides also
contained osteoclasts at both the periosteal and endosteal
surfaces. The number of osteoclasts associated with trabec-
ular surface (Table 1) correlated well with serum calcium
levels observed at 48 hr.
DISCUSSION
An assay system was designed to directly evaluate the ability
of a tumor-secreted peptide, hHCF, to act on bone cells and
Proc. Natl. Acad. Sci. USA 85 (1988) 5675
to mobilize calcium from bone in vivo. This system employs
unrestrained thyroparathyroidectomized animals that are on
a calcium-deficient diet and that receive the test substance by
intravenous infusion. This assay system also evaluates the
potential contribution of bone, the only major source of
calcium available, to the maintenance or elevation of serum
calcium levels in animals with the hypercalcemia syndrome
produced by hHCF-(1-34)-NH2. The continuous intravenous
administration of the factor may mimic the uncontrolled and
constant secretion of such a peptide by a tumor. Our studies
also provide, at least for the rat model, an estimate of the
relationship between the infusion rate of the peptide and the
calcemic response attained, which results from the combined
effects of bone resorption and renal disposition of calcium
and phosphate.
The homeostatic replacement dose of bPTH-(1-34), ad-
ministered by continuous infusion, was found to be 0.1-0.15
nmol-kg-lhr-1; this infusion rate corresponds to the calci-

FIG. 4. Photomicrographs of Masson's trichrome-stained 6-jsm sections from the proximal tibial metaphyses of rats infused with either
hHCF-(1-34)-NH2 or bPTH-(1-34) for 48 hr. Infusion of peptide at an infusion rate of0.10 nmol/hr results in abundant multinucleated osteoclasts
(arrows) on trabecular surfaces (blue) (A and B), while infusion at a rate of 0.01 nmol/hr fails to elicit this response (C and D). (A) hHCF-(1-
34)-NH2 at 0.10 nmol/hr. (B) bPTH-(1-34) at 0.10 nmol/hr. (C) hHCF-(1-34)-NH2 at 0.01 nmol/hr. (D) bPTH-(1-34) at 0.01 nmol/hr. (x 700.)
5676 Medical Sciences: Thompson et al.
Table 1. Effect of hHCF-(1-34)-NH2 and bPTH-(1-34) infusion
on the number of osteoclasts apposed to trabecular bone
Treatment Osteoclasts per mm of Serum calcium,
Peptide Dose, nmol/hr trabecular surface mg/dl
hHCF 0.10 11.1 (5.1) 13.3 (1.1)
bPTH 0.10 8.3 (1.1) 12.9 (3.4)
hHCF 0.06 5.8 (1.6) 11.0 (0.8)
bPTH 0.06 8.5 (2.5) 13.1 (0.8)
hHCF 0.03 4.9 (2.5) 10.5 (1.4)
bPTH 0.03 3.9 (0.4) 11.3 (0.9)
hHCF 0.01 1.3 (0.1) 4.2 (0.2)
bPTH 0.01 0.9 (0.1) 4.3 (0.3)
The number of osteoclasts and the trabecular bone surface were
determined for each condition in the proximal tibial metaphysis.
Serum calcium was measured 48 hr after infusion. Results are
presented as the mean offour samples and values in parentheses are
SEM.
um-normalizing amounts of PTH delivered by Alzet pumps to
thyroparathyroidectomized rats fed a calcium-enriched diet
(17). This observation demonstrates the dominant role of the
(rat) skeleton in defense against hypocalcemia. Infusion of
bPTH-(1-34) at 0.01 nmol/hr (=0.05 nmol kg- 1'hr- 1) failed
to raise calcium levels, but sustained the hypocalcemic level
of 5 mg/dl after thyroparathyroidectomy. Infusion of saline
alone resulted in hypocalcemic death. An infusion rate of
0.03 nmol/hr (0.15 nmol-kg-1 hr-1) raised calcium levels
only slightly or not at all after 24 hr, but animals became
normocalcemic by 48 hr. One possible explanation for this
finding is that the infusion rate of 0.03 nmol/hr may recruit
new osteoclasts but may be insufficient to activate existing
osteoclasts (18). The higher rates of 0.06 nmol/hr and 0.1
nmol/hr increased calcium to normal levels at 24 hr and
further increased calcium levels thereafter. These concen-
trations also caused extensive nephrocalcinosis. The infusion
at 0.1 nmol/hr produced hypercalcemia in all animals by 48
hr. The faster calcemic response to the two higher infusion
rates could reflect activation of existing osteoclasts.
Histomorphometric analysis after 48 hr of peptide infusion
showed good correlation between the number of osteoclasts
per mm2 of marrow or per mm of trabecular bone surface and
the serum calcium and phosphate levels achieved. Animals
receiving the lowest concentration of peptide had 2-5 osteo-
clasts per mm2, which is comparable to the number of
osteoclasts found in controls (19). In animals in which
normocalcemic levels were reached, 20 osteoclasts per mm2
were observed; hypercalcemic animals had =30 osteoclasts
per mm2. These findings are consistent with the reported
increase in the number of osteoclasts in bone biopsies from
patients with humoral hypercalcemia of malignancy (20); a
similar finding is commonly associated with hyperparathy-
roidism (21). The trabecular bone volume, estimated at a
defined subepiphyseal site, was commensurately reduced
(data not shown). At the higher rates of peptide infusion (0.06
and 0.1 nmol/hr), there was pronounced osteoclastic resorp-
tion of cortical bone, a characteristic feature of hyperpara-
thyroidism. The histologic evidence thus fully supports the
conclusion that bone is the major source of calcium mobili-
zation in these animals and that the rat skeleton responds
identically to bPTH-(1-34) and hHCF-(1-34)-NH2. This point
is emphasized by the finding that hypercalcemia is produced
by both peptides at the same infusion rate (-0.5 nmol kg- 1
hr- 1) Similarly, the highest doses of hHCF and bPTH
produced the most marked hypophosphatemia and the great-
est degree of nephrocalcinosis.
Differences between results obtained by Kemp et al. (15)
with rat embryo long bone explants in vitro and those
obtained in vivo in this study are not readily apparent at this
time. Both studies used the same sequence for hHCF-(1-
Proc. Natl. Acad Sci. USA 85 (1988)
34)-NH2 in the same species. There may be differences in
peptide metabolism in the two systems. Although this study
indicates that hHCF-(1-34)-NH2 possesses equivalent activ-
ity to PTH-(1-34), which is very similar to that of PTH-(1-
84)-NH2, it is not yet known whether additional potency or
activity can be achieved by adding residues on the C-terminal
side of residue 34 in the hHCF sequence.
In conclusion, these studies demonstrate a striking simi-
larity in the action and potency of bPTH and hHCF on bone
and mineral ion fluxes in this in vivo system. These findings
also indicate that bone resorption and the associated calcium
release can serve as an important mechanism for the gener-
ation of the hypercalcemic paraneoplastic syndrome, obser-
vations that are consistent with the concept put forth by
Albright (1).
Note. While this paper was under review, Stewart et al. (22) reported
that the hHCF fragment containing residues 1-36 is equipotent with
bPTH-(1-34) in vitro and in vivo. These findings strengthen our
conclusions based on data for the fragment hHCF-(1-34)-NH2
presented in this study.
We thank Dr. Ruth Nutt and Mr. Jay Levy for the synthesis of
hHCF and Ms. Dianne McDonald for preparation of the manuscript.
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