Vol. 131, No 6 Printed in U.S A
Human Parathyroid Hormone-Related Peptide(107-l 11) Does not Inhibit Bone Resorption in Neonatal Mouse Calvariae TERUKI SONE, HIROAKI RYUICHI KASAI, YUKO AND CHOHEI SHIGENO
KOHNO, KIKUCHI,
HARUKI KIKUCHI, RYO TAKEUCHI,
TOSHIHIKO IKEDA, JUNJI KONISHI,
CalciumLaboratory, Departments of Radiologyand Nuclear Medicine (T.S., Ha.K., Y.K., R.T., J.K., C.S.) and Orthopedic Surgery (Hi.K., T.I., R.K.), Kyoto University Hospital, Sakyo, Kyoto 606-01, Japan ABSTRACT Recent analysis of the structure-function relationship of human PTH-related peptide (hPTHrP) has led to the discovery that its direct inhibitory activity on osteoclastic bone resorption resides fully in the 107-111 sequence of the peptide, as assessed by a bone resorption assay using isolated rat osteoclasts. Here we report that hPTHrP-(107-111) is inactive in neonatal mouse calvariae in culture. hPTHrP-(107-111). at doses of lo-“-lo-” M and incubation periods up to 96 h, did not affect either basal or agonist-stimulated ““Ca release from prelabeled neonatal mouse calvariae, while salmon calcitonin was a potent and powerful inhibitor of both basal and stimulated ‘“Ca release from bone. Moreover, salmon calcitonin, but not hPTHrP-(107-ill), inhibited the increase in osteoclast number in hPTHrP-(1-34)-treated bones. Fur-
thermore, hPTHrP-(107-139) also failed to inhibit 45Ca release and the hPTHrP-(1-34)-induced increase in osteoclast number in this organ culture model when tested under conditions identical to those for hPTHrP-(107-111). The addition of indomethacin to hPTHrP-(107ill)or hPTHrP-(107-139).treated bones was without effect, excluding the possibility that the direct inhibitory activity of these peptides on osteoclasts is ablated by a prostaglandin-mediated mechanism. Although the mechanism underlying the apparent inability of the carboxyl-terminal PTHrP fragments to inhibit osteoclastic bone resorption in neonatal mouse calvariae is unknown, it may involve the complex microenvironment of osteoclasts in intact bone, which contains a large variety of cell types other than osteoclasts. (Endocrinology 131: 2742-2746, 1992)
I?
TH-RELATED peptide (PTHrP) was initially identified by virtue of its expression by malignant tumors associated with syndromes of humoral hypercalcemia of malignancy (l-4). Homology with PTH is restricted to the aminoterminal 1-13 region of PTHrP and the amino acid sequence down-stream of this region diverges completely from that of PTH. Structure-function studies using a variety of synthetic PTHrP fragments have revealed at least three distinct regions with different biological activities: residues 1-34 responsible for PTH-like activities via interaction with PTH receptors in target cells including osteoblasts (2, 5-8), residues 35-108 for calcium and phosphate transport in the placenta (9, lo), and residues 107-139 for direct inhibitory activity on osteoclasts, the major effector of bone resorption (11). Detailed analysis of the structure-function relationship of the carboxyl-terminal 107-139 region of PTHrP molecule has revealed that its potent inhibitory activity on osteoclastic bone resorption is localized entirely in the highly conserved 107-l 11 sequence of the peptide (12). The inhibitory activity of hPTHrP-(107-111) on osteoclastic bone resorption appears to be direct, because bone resorption is inhibited in a purified osteoclast assay that is deficient in other bone cell types that are normally present in intact bone and mediate the resorptive message to osteoclasts (12, 13). To clarify the
effect of hPTHrP-(107-111) in intact bone where complex multicellular processes are involved in osteoclastic bone resorption, we employed a neonatal mouse calvarial organ culture system and examined whether hPTHrP-(107-111) is capable of inhibiting osteoclastic bone resorption induced by a variety of bone-resorbing hormones and local factors.
Received June 6, 1992. Address all correspondence and requests for reprints to: Chohei Shigeno, M.D., Ph.D., Calcium Laboratory, Department of Radiology and Nuclear Medicine, Kyoto University Hospital, Sakyo, Kyoto 60601, Japan.
Synthetic salmon calcitonin (sCT; 3969 IU/mg) was kindly provided by Yamanouchi Pharmaceuticals (Tokyo, Japan). Recombinant human interleukin-lol (rhIL-la; 2 x lo7 U/mg) was a gift from Otsuka Pharmaceuticals (Tokushima, Japan) (14). Synthetic human PTHrP-(l-34) was a gift from Dr. T. Noda, Asahi Kasei (Osaka, Japan) (8). lcu,25-
Materials
and Methods
Peptides Human PTHrP-(107-111) and PTHrP-(107-139) were synthesized using an automated solid phase peptide synthesizer (model 430A, Applied Biosystems, Foster City, CA). Deprotection and release of the peptides from the PAM resins was accomplished using anhydrous hydrogen fluoride with 10% anisol at 0 C for 2 h. After ethyl acetate extraction of the residual peptide-resin mixture, the peptides were extracted with 0.1 M aqueous acetic acid, separated from the resin by filtration through a scintered glass filter, and dried by lyophilization. The composition of the synthetic peptides was verified by quantitative amino acid analysis, using a Beckman System 6300 high performance amino acid analyzer. The purity of the synthetic peptides was determined by analytical reverse phase HPLC and amino acid sequence analysis, using an Applied Biosystems model 470A protein/peptide sequencer.
Other materials
2142
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PTHrP-(107-111)
AND
BONE
2743
RESORPTION
Dihydroxyvitamin D, [1,25-(OH)zDg] was a gift from Chugai Pharmaceuticals (Tokyo, Japan). Synthetic [Nle*,Nle’*,Tyr34]bovine PTH-(l34)NHz (NlePTH) was obtained from Bachem (Bubendorf, Switzerland). Prostaglandin E2 (PGE2) and indomethacin were obtained from Sigma (St. Louis, MO). All other reagents were of the highest quality available.
Bone resorption
assay
Bone resorption was assessed by the release of previously incorporated ?a from neonatal mouse calvariae, as previously described (15). Two-day-old mice (Sic-ddy) were injected SC with 7.4 X lo4 Bq [?a] CaClz (New England Nuclear, Boston, MA). Two days later, the parietal bones were excised, and each bone was cultured for 24-h in Ham’s F12 medium supplemented with 5% (vol/vol) heat-inactivated horse serum, 1.0 rnM CaCIZ, and 75 U/ml penicillin G at 37 C in a humidified 5% C02-95% air atmosphere. The medium was then changed with fresh medium, and culture was continued in the presence or absence of test substances for varying periods of time, as indicated. The radioactivity of “Ca in the medium and bone was separately determined by a liquid scintillation counter. Bone resorption was expressed as the percentage of ?a released into the medium (?a release) or the ratio of ?a release from treated vs. control bones.
Histological
analysis and measurement
of
analysis
Statistical differences Student’s t test.
were
assessed
by
47 NONE
-12
-10
log(hPTHrP-(107-l
-8
-6
I I)) (M)
B
osteoclast number
Mouse calvariae were cultured for 48 h, as described above, in control medium or medium containing test substances. They were then embedded in O.C.T. compound (Miles Laboratories, Elkhart, IN), frozen in liquid nitrogen, sectioned at 5-pm thickness with a cryostat microtome, and reacted cytochemically for tartrate-resistant acid phosphatase (TRAP). Briefly, calvarial sections were incubated for 1 h at room temperature in 0.1 M sodium acetate buffer (pH 5.0) containing 0.05 M sodium tartrate, naphthol AS-B1 phosphate (Sigma), and hexazonium pararosanilin. Osteoclasts were identified as TRAP-positive cells adhered to bone surface and counted in two sections, at 300-pm intervals, of three bones in each group. The average osteoclast number was expressed as the total number of TRAP-positive osteoclasts per mm bone surface.
Statistical
lo’
analysis
of variance
and
Results Bones were incubated for 72 h with hPTHrP-(107-111) at concentrations ranging from 10-‘2-10-6 M, either alone or in the presence of graded doses of hPTHrP-(l-34), a known stimulator of osteoclasticbone resorption (5). hPTHrP-(107111) had no effect on bone resorption in control or hPTHrP(1-34)-stimulated cultures (Fig. 1A). To examine the possible time dependenceof the effect of hPTHrP-( 107-l 11) on bone resorption, we incubated bones for varying periods of time with hPTHrP-(107-111) (lo-* M) in the presence or absence of hPTHrP-(l-34) (2 X 10e9M) and assessedbone resorption at 24-h intervals. sCT served as a direct inhibitor of osteoelastic bone resorption (seeFig. 2). The time-response curves for bones treated with and without hPTHrP-(107-111) were almost superimposable regardlessof hPTHrP-(l-34) stimulation. These results were in sharp contrast to those for sCT treatment, which resulted in nearly complete inhibition of bone resorption in hPTHrP-(1-34)-treated bones (Fig. 2A) and significant inhibition of resorption in control bones (Fig. 2B). It was of interest to determine whether hPTHrP-(107-111)
NONE
-12
-10
log(hPTHrP-(107-139))
-8
-6 (M)
FIG. 1. Dose-response study of the effects of hPTHrP-(107-111) and hPTHrP-(107-139) on 4”Ca release from prelabeled neonatal mouse calvariae. After 24-h preincubation, bones were cultured for 72 h in medium containing the indicated concentrations of hPTHrP-( 107-111) (A) or hPTHrP-(107-139) (B) in the presence (0, lOma M; 0,5 X 10-l” M) or absence (A) of hPTHrP-(l-34). Bone-resorbing activity is expressed as a percentage of the total Wa released into the medium. Points and bars represent the mean + SD of three similar experiments, each performed in quintuplicate. No significant difference was observed in each curve by analysis of variance.
modulates bone resorption stimulated by hormones and local factors other than hPTHrP, e.g. NlePTH, 1,25-(OH)2D3,rhILla, and PGEz, which stimulate bone resorption by osteoclasts in intact bone. In each case, sCT (10m8M) significantly inhibited bone resorption and in no case did hPTHrP-(107-111) (10e8M) significantly modulate bone resorption (Table 1). We further investigated the effect of hPTHrP-(107-139) on bone resorption in organ culture. Like hPTHrP-(107ill), hPTHrP-(107-139) failed to inhibit 45Carelease from prelabeled neonatal mouse calvariae at concentrations ranging from 10-‘2-10-6 M (Fig. 1B) and incubation periods up to 96 h (Fig. 2C). Histological evaluation of the effects of hPTHrP-(107111) and hPTHrP-(107-139) on mouse calvarial bones in culture revealed that neither of the two peptides affected osteoclast number in hPTHrP-(I-34)-treated bones. These results compare with those for sCT treatment, which almost completely abolished the hPTHrP-(1-34)-induced increase in osteoclastnumber (Table 2). Many osteotropic hormones and local factors act on bone cells other than osteoclaststo induce endogeneousproduction of PGE2, which causesosteoclastic bone resorption in intact bone (16-19). It was, thus, possibleto hypothesize that
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2744
PTHrP-(107-111) 25 -
oJ
I
I
I
24
48
72
Incubation
Time
AND
BONE
RESORPTION
I24
1
1992
401c
B
96
Endo.
48
(h)
Incubation
I 96
72 Time
O;4
(h)
48 Incubation
;2 Time
9b (h)
FIG. 2. Time dependence of the effect of hPTHrP-(107-111) and hPTHrP-(107-139) on %a release from prelabeled neonatal mouse calvariae. After 24-h preincubation, bones were further incubated for the indicated time periods up to 96 h under the following conditions. A and B, Bones were incubated with control medium only (O), hPTHrP-(107-111) (0, 10mR M), or sCT (A; lo-@ M) in the presence (A) or absence (B) of 2 x lo-’ M hPTHrP-(1-34). C, Bones were incubated with control medium only (0) or hPTHrP-(107-139) (0, lo-’ M) in the presence (- - -) or absence ) of 2 x 1O-9 M hPTHrP-(l-34). The amount of ‘%a released into the medium was determined every 24 h. Data are the mean + SEM from (two similar exneriments. each determined in auintunle. * and **. Statistically significant differences from the corresponding control groups at P . _ < 0.05 and P 2 0.01, respectively.
TABLE
1. Effects
stimulated
by various
of hPTHrP-(107-111) and sCT on 45Ca release bone resorbing agents Bone-resorbing
Stimulator
Untreated
None 1,25-(OH)*D3 (1 nM) NlePTH (2 nM) hPTHrP-(l-34) (2 nM) rhIL-lot (100 pg/ml) PGE, (1 PM)
1.00 2.49 2.51 2.60 2.38 2.41
f f k k + +
activity
(T/C ratio)
bPTHrP-(107-111)
0.03 0.35 0.21 0.30 0.32 0.08
1.03 2.40 2.45 2.57 2.29 2.48
f + + + + +
sCT
0.85 0.95 1.02 1.10 0.93 1.09
0.06 0.17 0.20 0.11 0.20 0.13
+ + + + + +
0.07” 0.05* 0.09* 0.08* 0.06b O.lob
After 24-h preincubation, bones were cultured for 72 h in various conditions, as indicated above. sCT and hPTHrP-(107-111) were tested at lo-’ M each. Bone-resorbing activity is expressed as a treated to control (T/C) ratio to facilitate comparisons between multiple experiments. Data are the mean + SEM (n = 5). ’ P < 0.05 us. control. b P < 0.01 us. control.
TABLE
2. Effect of hPTHrP-(107-ill), sCT on osteoclast number in neonatal with or without hPTHrP-(l-34)
hPTHrP-(107-139), and mouse calvariae incubated
No. of osteoclast/mm Treatment None hPTHrP-(107-111) hPTHrP-(107-139) sCT (10-R M)
Without hPTHrP-(l-34)
(lOmR M) (lo-’ M)
3.9 4.1 4.0 3.8
+ * f +
With
1.8 2.0 1.9 1.5
bone surface hPTHrP-(l-34) (2 X 1o-9 M)
15.8 17.9 14.9 4.5
+ k f +
2.8 3.2 3.0 1.8”
After 24-h preincubation, bones were cultured for 48 h in various conditions, as indicated above. Data are the mean k SEM of three determinations. ’ Statistically significant difference from the hPTHrP-(l-34) alone group (P < 0.05).
hPTHrP-(107-111) and hPTHrP-(107-139) act on some cell types present in neonatal mouse calvariae to stimulate PGEl synthesis, which, in turn, obliterates the direct inhibitory effects of these peptides on osteoclasts.To test this hypothesis, we incubated bones with each of the two carboxyl-
terminal PTHrP peptides in the presence or absence of indomethacin. Indomethacin (10m6M) did not influence the effect of either of the two carboxyl-terminal PTHrP peptides (lo-@ M each) on basal bone resorption or bone resorption induced by PTHrP-(1-34) (2 X 10m9M; Table 3).
Discussion
We have found that the 107-l 11 sequenceof the hPTHrP molecule doesnot have the ability to inhibit osteoclasticbone resorption in bone organ culture. The apparent inability of hPTHrP-(107-111) to inhibit bone resorption in intact bone contrasts with its potent inhibitory activity on bone resorption in an isolated osteoclast assay previously shown by TABLE
3. Effect of indomethacin on basal and hPTHrP-(l-34)stimulated %a release from prelabeled neonatal mouse calvariae treated with hPTHrP-(107-111) or hPTHrP-(107-139) Bone-resorbing activity (T/C ratio)
Treatment
Without
In the absence of hPTHrP-(1-34) nM) None hPTHrP-(107-111) (10 nM) hPTHrP-(107-139) (10 nM) In the presence of hPTHrP-(l-34)
indomethacin
With indomethacin
1.00 + 0.10 1.03 + 0.10
1.04 + 0.08 0.95 + 0.06
1.01
0.97
(2
+ 0.05
+ 0.12
(2 nM) None hPTHrP-(107-111) hPTHrP-(107-139)
(10 nM) (10 nM)
2.23 + 0.16 1.95 -t 0.10 2.19
+ 0.09
2.22 + 0.29 2.14 f 0.20 2.27 + 0.16
After 24-h preincubation, bones were cultured for 72 h in various conditions, as indicated above. Indomethacin was added at 1 pM. Boneresorbing activity is expressed as a treated to control (T/C) ratio to facilitate comparisons between multiple experiments. Data are the mean f SEM (n = 5). There is no statistically significant difference between the without indomethacin and with indomethacin groups in each treatment.
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PTHrP-(107-111)
AND
Fenton et al. (12). We have no clear explanation for this difference. However, this difference may be due to the difference in microenvironment of osteoclasts between the two assay systems. hPTHrP-(107-111) was shown to have inhibitory activity on osteoclastic bone resorption in a bioassay that effectively lacks many bone cell types other than osteoclasts. In particular, the isolated osteoclast assay is devoid of osteoblasts, which are believed to be target cells for most, if not all, bone-resorbing hormones and local factors and transduce the resorptive message to osteoclasts in intact bone (20-22). The observed difference closely resembles the difference in the mode of action of PGEz on bone, which acts as a direct inhibitor of osteoclast function, as assessed by an isolated osteoclast bone resorption assay (23), yet induces osteoclastic bone resorption indirectly in intact bone via stimulatory effects on osteoblasts (23, 24). We, therefore, hypothesized that PTHrP-(107-111) can have an additional effect on some cell types other than osteoclasts in intact bone, which are induced to produce some bone-resorbing factor(s), thereby masking the direct inhibitory effect of the peptide on osteoclasts. One candidate for this mediator is PGE, (19). The addition of indomethacin had no effect on bone resorption in neonatal mouse calvariae treated with either hPTHrP(107-111) or hPTHrP-(107-139), which makes PG-mediated effects unlikely. Nonetheless, we cannot exclude the possibility that some signaling pathway other than a PG-mediated mechanism may underlie the apparent failure of PTHrP(107-111) in intact bone. This hypothesis deserves further study. Alternatively, an additional peptide sequence beyond the Trp”’ residue may be required to generate osteoclastic inhibitory activity in our organ culture model. To test this possibility, we synthesized and tested hPTHrP-(107-139) for effects in mouse calvariae in culture. Like hPTHrP-(107-11 l), hPTHrP-(107-139) did not modulate either basal bone resorption or bone resorption stimulated by hPTHrP-(l-34). Our results differ from those of Fenton et al. (12), who found that hPTHrP-(107-139) is a potent inhibitor of bone resorption in cultured fetal rat long bones, osteoblast/osteoclast coculture, as well as purified osteoclast assay. This difference may be due to either species difference or differences in the relative maturity of bone cell types present in the two assay systems (25, 26) as well as in the mode of bone formation (intramembranous us. endochondral bone formation). For example, transforming growth factor-0 inhibits bone resorption in fetal rat long bone cultures (27), while in neonatal mouse calvarial cultures, it stimulates bone resorption (19). Indeed, the apparent failure of PTHrP-(107-139) to inhibit bone resorption in cultured mouse calvariae was unexpected, because these bones contain mature osteoclasts as the major population of cells in the osteoclast lineage, which should be the target cells for the direct inhibitory action of PTHrP(107-139) (11). There is evidence that PTHrP is expressed at sites of both endochondral and intramembranous bone formation in human fetus (28). PTHrP is released by resorbing rat long bone in culture (29). These data support the intriguing hypothesis that PTHrP plays a distinct role in normal skeletal develop-
BONE
RESORPTION
2745
ment and physiology, possibly through local mechanisms unrelated to PTH-like activity. Determination of the microenvironmental requirement for the biological activity of the carboxyl-terminal fragments of hPTHrP and its physiological relevance remains to be elucidated. References 1 Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, Rodda Cl’, Ebeling PR, Hudson PJ, Zajac JD, Martin TJ 1987 Parathyroid hormone-related protein purified from a human
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Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI 1987 A parathyroid hormone-related protein implicated in malignant cloning and expression. Science 237:893-896
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Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF 1987 Identification of a novel 17,000dalton parathyroid hormone-like adenylate tein from a tumor associated with humoral nancy. J Biol Chem 262:7151-7156
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Strewler GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC, Rosenblatt M, Nissenson RA 1987 Parathyroid hormonelike protein from homology
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Kemp 8, Moseley J, Rodda C, Ebeling P, Wettenhall R, Stapleton D, Diefenbach-Jagger H, Ure F, Michelangeli V, Simmons H, Raisz L, Martin TJ 1987 Parathyroid hormone-related protein of 1malignancy: active synthetic fragments. Science 238:1568-1570 Jlippner H, Abou-Samura A-B, Uneno S, Gu W-X, Potts J’I’J, Segre GV 1988 The parathyroid hormone-like peptide associated with
humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem 263:8557-8560 7. Nissenson RA, Diep D, Strewler GJ 1988 Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormonelike protein from human malignancies: binding to parathyroid hormone receptors and activiation of adenylate cyclase in bone cells and kidney. J Biol Chem 263:12866-12871 8
Shigeno C, Yamamoto I, Kitamura N, Noda T, Lee K, Sone T, Shiomi K, Ohtaka A, Fujii N, Yajima H , Konishi J 1988 Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells. J Biol Chem 263:18369-18377
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Rodda CP, Kubota M, Heath JA, Ebeling PR, Mosely JM, Care AD, Caple IW , Martin TJ 1988 Evidence for a novel parathyroid hormone-related protein in fetal Iamb parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcemia of malignancy. J Endocrinol 117:261-271
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Care AD, Abbas SK, Pickard DW, Barri M, Drinkhill M, Findlay JBC, White IR, Caple IW 1990 Stimulation of bovine placenta transport human
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75:605-608
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Fenton AJ, Kemp BE, Kent GN, Moseley JM, Zheng MH, Rowe DJ, Britto JM, Martin TJ, Nicholson GC 1991 A carboxyl-terminal peptide from the parathyroid hormone-related protein resorption by osteoclasts. Endocrinology 129:1762-1768
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Fenton AJ, Kemp BE, Hammonds Jr RG, Mitchelhill JM, Martin TJ, Nicholson GC 1991 A potent inhibitor
inhibits
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K, Moseley
of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP-(107-111). Endocrinology 129:3424-3426 13. Chambers T, Revel1 P, Fuller K, Athanasou N 1984 Resorption of bone by isolated rabbit osteoclasts. J Cell Sci 66:383-399 14. Yamamoto I, Kawano M, Sone T, Iwato K, Tanaka H, Ishikawa
H, Kitamura N, Lee K, Shigeno C, Konishi J, Tanabe 0, Nobuyoshi M, Ohmoto Y, Hirai Y, Higuchi M, Ohsawa T, Kuramoto
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Production of interleukin I beta, a potent bone resorbing by cultured human myeloma cells. Cancer Res 49:4242-
Shigeno C, Yamamoto I, Dokoh S, Hino M, Aoki J, Yamada K, Morita R, Kameyama M, Torizuka K 1985 Identification of 1,24(R)dihydroxy-vitamin cancer-associated 768
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PMJ, Chambers TJ 1987 1,25-Dihydroxyvitamin DB rat osteoblastic cells to release a soluble factor that osteoclastic bone resorption. J Clin Invest 80:425-429 22. McSheehy PMJ, Chambers TJ 1986 Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption. Endocrinology 119:1654-1659 23. Chambers TJ, McSheehy PMJ, Thompson BM, Fuller K 1985 The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology 60:234-239 24. Klein DC, Raisz LG 1970 Prostaalandins: stimulation of bone resorption in tissue culture. Endocrinology 86:1436 25. Al-Humidan A, Ralston S. Hughes D. Chaoman K. Aarden L. Russell R, Go&en M 1991. Intezeukin-6 does not stimulate bone resorption in neonatal mouse calvariae. J Bone Mineral Res 6:3-8 26. Roodman G 1992 Perspectives: interleukin-6: an osteotropic factor? J Bone Mineral Res 7:475-478 27. Pfeilschifter J, Seyedin S, Mundy G 1988 Transforming growth factor beta inhibits bone resorption in fetal rat long bone cultures. J Clin Invest 82:680-685 28. Moseley JM, Hayman JA, Danks JA, Alcorn D, Grill V, Southby J, Horton MA 1991 Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 73:478-484 29. Bergman P, Nijs-DeWolf N, Pepersack T, Corvilain J 1990 Release of parathyroid hormonelike peptides by fetal rat long bones in culture. J Bone Mineral Res 5:741-753 21.
McSheehy
Endo. Voll31.
stimulates increases
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Human Parathyroid Hormone-Related Peptide(107-l 11) Does not Inhibit Bone Resorption in Neonatal Mouse Calvariae TERUKI SONE, HIROAKI RYUICHI KASAI, YUKO AND CHOHEI SHIGENO
KOHNO, KIKUCHI,
HARUKI KIKUCHI, RYO TAKEUCHI,
TOSHIHIKO IKEDA, JUNJI KONISHI,
CalciumLaboratory, Departments of Radiologyand Nuclear Medicine (T.S., Ha.K., Y.K., R.T., J.K., C.S.) and Orthopedic Surgery (Hi.K., T.I., R.K.), Kyoto University Hospital, Sakyo, Kyoto 606-01, Japan ABSTRACT Recent analysis of the structure-function relationship of human PTH-related peptide (hPTHrP) has led to the discovery that its direct inhibitory activity on osteoclastic bone resorption resides fully in the 107-111 sequence of the peptide, as assessed by a bone resorption assay using isolated rat osteoclasts. Here we report that hPTHrP-(107-111) is inactive in neonatal mouse calvariae in culture. hPTHrP-(107-111). at doses of lo-“-lo-” M and incubation periods up to 96 h, did not affect either basal or agonist-stimulated ““Ca release from prelabeled neonatal mouse calvariae, while salmon calcitonin was a potent and powerful inhibitor of both basal and stimulated ‘“Ca release from bone. Moreover, salmon calcitonin, but not hPTHrP-(107-ill), inhibited the increase in osteoclast number in hPTHrP-(1-34)-treated bones. Fur-
thermore, hPTHrP-(107-139) also failed to inhibit 45Ca release and the hPTHrP-(1-34)-induced increase in osteoclast number in this organ culture model when tested under conditions identical to those for hPTHrP-(107-111). The addition of indomethacin to hPTHrP-(107ill)or hPTHrP-(107-139).treated bones was without effect, excluding the possibility that the direct inhibitory activity of these peptides on osteoclasts is ablated by a prostaglandin-mediated mechanism. Although the mechanism underlying the apparent inability of the carboxyl-terminal PTHrP fragments to inhibit osteoclastic bone resorption in neonatal mouse calvariae is unknown, it may involve the complex microenvironment of osteoclasts in intact bone, which contains a large variety of cell types other than osteoclasts. (Endocrinology 131: 2742-2746, 1992)
I?
TH-RELATED peptide (PTHrP) was initially identified by virtue of its expression by malignant tumors associated with syndromes of humoral hypercalcemia of malignancy (l-4). Homology with PTH is restricted to the aminoterminal 1-13 region of PTHrP and the amino acid sequence down-stream of this region diverges completely from that of PTH. Structure-function studies using a variety of synthetic PTHrP fragments have revealed at least three distinct regions with different biological activities: residues 1-34 responsible for PTH-like activities via interaction with PTH receptors in target cells including osteoblasts (2, 5-8), residues 35-108 for calcium and phosphate transport in the placenta (9, lo), and residues 107-139 for direct inhibitory activity on osteoclasts, the major effector of bone resorption (11). Detailed analysis of the structure-function relationship of the carboxyl-terminal 107-139 region of PTHrP molecule has revealed that its potent inhibitory activity on osteoclastic bone resorption is localized entirely in the highly conserved 107-l 11 sequence of the peptide (12). The inhibitory activity of hPTHrP-(107-111) on osteoclastic bone resorption appears to be direct, because bone resorption is inhibited in a purified osteoclast assay that is deficient in other bone cell types that are normally present in intact bone and mediate the resorptive message to osteoclasts (12, 13). To clarify the
effect of hPTHrP-(107-111) in intact bone where complex multicellular processes are involved in osteoclastic bone resorption, we employed a neonatal mouse calvarial organ culture system and examined whether hPTHrP-(107-111) is capable of inhibiting osteoclastic bone resorption induced by a variety of bone-resorbing hormones and local factors.
Received June 6, 1992. Address all correspondence and requests for reprints to: Chohei Shigeno, M.D., Ph.D., Calcium Laboratory, Department of Radiology and Nuclear Medicine, Kyoto University Hospital, Sakyo, Kyoto 60601, Japan.
Synthetic salmon calcitonin (sCT; 3969 IU/mg) was kindly provided by Yamanouchi Pharmaceuticals (Tokyo, Japan). Recombinant human interleukin-lol (rhIL-la; 2 x lo7 U/mg) was a gift from Otsuka Pharmaceuticals (Tokushima, Japan) (14). Synthetic human PTHrP-(l-34) was a gift from Dr. T. Noda, Asahi Kasei (Osaka, Japan) (8). lcu,25-
Materials
and Methods
Peptides Human PTHrP-(107-111) and PTHrP-(107-139) were synthesized using an automated solid phase peptide synthesizer (model 430A, Applied Biosystems, Foster City, CA). Deprotection and release of the peptides from the PAM resins was accomplished using anhydrous hydrogen fluoride with 10% anisol at 0 C for 2 h. After ethyl acetate extraction of the residual peptide-resin mixture, the peptides were extracted with 0.1 M aqueous acetic acid, separated from the resin by filtration through a scintered glass filter, and dried by lyophilization. The composition of the synthetic peptides was verified by quantitative amino acid analysis, using a Beckman System 6300 high performance amino acid analyzer. The purity of the synthetic peptides was determined by analytical reverse phase HPLC and amino acid sequence analysis, using an Applied Biosystems model 470A protein/peptide sequencer.
Other materials
2142
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PTHrP-(107-111)
AND
BONE
2743
RESORPTION
Dihydroxyvitamin D, [1,25-(OH)zDg] was a gift from Chugai Pharmaceuticals (Tokyo, Japan). Synthetic [Nle*,Nle’*,Tyr34]bovine PTH-(l34)NHz (NlePTH) was obtained from Bachem (Bubendorf, Switzerland). Prostaglandin E2 (PGE2) and indomethacin were obtained from Sigma (St. Louis, MO). All other reagents were of the highest quality available.
Bone resorption
assay
Bone resorption was assessed by the release of previously incorporated ?a from neonatal mouse calvariae, as previously described (15). Two-day-old mice (Sic-ddy) were injected SC with 7.4 X lo4 Bq [?a] CaClz (New England Nuclear, Boston, MA). Two days later, the parietal bones were excised, and each bone was cultured for 24-h in Ham’s F12 medium supplemented with 5% (vol/vol) heat-inactivated horse serum, 1.0 rnM CaCIZ, and 75 U/ml penicillin G at 37 C in a humidified 5% C02-95% air atmosphere. The medium was then changed with fresh medium, and culture was continued in the presence or absence of test substances for varying periods of time, as indicated. The radioactivity of “Ca in the medium and bone was separately determined by a liquid scintillation counter. Bone resorption was expressed as the percentage of ?a released into the medium (?a release) or the ratio of ?a release from treated vs. control bones.
Histological
analysis and measurement
of
analysis
Statistical differences Student’s t test.
were
assessed
by
47 NONE
-12
-10
log(hPTHrP-(107-l
-8
-6
I I)) (M)
B
osteoclast number
Mouse calvariae were cultured for 48 h, as described above, in control medium or medium containing test substances. They were then embedded in O.C.T. compound (Miles Laboratories, Elkhart, IN), frozen in liquid nitrogen, sectioned at 5-pm thickness with a cryostat microtome, and reacted cytochemically for tartrate-resistant acid phosphatase (TRAP). Briefly, calvarial sections were incubated for 1 h at room temperature in 0.1 M sodium acetate buffer (pH 5.0) containing 0.05 M sodium tartrate, naphthol AS-B1 phosphate (Sigma), and hexazonium pararosanilin. Osteoclasts were identified as TRAP-positive cells adhered to bone surface and counted in two sections, at 300-pm intervals, of three bones in each group. The average osteoclast number was expressed as the total number of TRAP-positive osteoclasts per mm bone surface.
Statistical
lo’
analysis
of variance
and
Results Bones were incubated for 72 h with hPTHrP-(107-111) at concentrations ranging from 10-‘2-10-6 M, either alone or in the presence of graded doses of hPTHrP-(l-34), a known stimulator of osteoclasticbone resorption (5). hPTHrP-(107111) had no effect on bone resorption in control or hPTHrP(1-34)-stimulated cultures (Fig. 1A). To examine the possible time dependenceof the effect of hPTHrP-( 107-l 11) on bone resorption, we incubated bones for varying periods of time with hPTHrP-(107-111) (lo-* M) in the presence or absence of hPTHrP-(l-34) (2 X 10e9M) and assessedbone resorption at 24-h intervals. sCT served as a direct inhibitor of osteoelastic bone resorption (seeFig. 2). The time-response curves for bones treated with and without hPTHrP-(107-111) were almost superimposable regardlessof hPTHrP-(l-34) stimulation. These results were in sharp contrast to those for sCT treatment, which resulted in nearly complete inhibition of bone resorption in hPTHrP-(1-34)-treated bones (Fig. 2A) and significant inhibition of resorption in control bones (Fig. 2B). It was of interest to determine whether hPTHrP-(107-111)
NONE
-12
-10
log(hPTHrP-(107-139))
-8
-6 (M)
FIG. 1. Dose-response study of the effects of hPTHrP-(107-111) and hPTHrP-(107-139) on 4”Ca release from prelabeled neonatal mouse calvariae. After 24-h preincubation, bones were cultured for 72 h in medium containing the indicated concentrations of hPTHrP-( 107-111) (A) or hPTHrP-(107-139) (B) in the presence (0, lOma M; 0,5 X 10-l” M) or absence (A) of hPTHrP-(l-34). Bone-resorbing activity is expressed as a percentage of the total Wa released into the medium. Points and bars represent the mean + SD of three similar experiments, each performed in quintuplicate. No significant difference was observed in each curve by analysis of variance.
modulates bone resorption stimulated by hormones and local factors other than hPTHrP, e.g. NlePTH, 1,25-(OH)2D3,rhILla, and PGEz, which stimulate bone resorption by osteoclasts in intact bone. In each case, sCT (10m8M) significantly inhibited bone resorption and in no case did hPTHrP-(107-111) (10e8M) significantly modulate bone resorption (Table 1). We further investigated the effect of hPTHrP-(107-139) on bone resorption in organ culture. Like hPTHrP-(107ill), hPTHrP-(107-139) failed to inhibit 45Carelease from prelabeled neonatal mouse calvariae at concentrations ranging from 10-‘2-10-6 M (Fig. 1B) and incubation periods up to 96 h (Fig. 2C). Histological evaluation of the effects of hPTHrP-(107111) and hPTHrP-(107-139) on mouse calvarial bones in culture revealed that neither of the two peptides affected osteoclast number in hPTHrP-(I-34)-treated bones. These results compare with those for sCT treatment, which almost completely abolished the hPTHrP-(1-34)-induced increase in osteoclastnumber (Table 2). Many osteotropic hormones and local factors act on bone cells other than osteoclaststo induce endogeneousproduction of PGE2, which causesosteoclastic bone resorption in intact bone (16-19). It was, thus, possibleto hypothesize that
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2744
PTHrP-(107-111) 25 -
oJ
I
I
I
24
48
72
Incubation
Time
AND
BONE
RESORPTION
I24
1
1992
401c
B
96
Endo.
48
(h)
Incubation
I 96
72 Time
O;4
(h)
48 Incubation
;2 Time
9b (h)
FIG. 2. Time dependence of the effect of hPTHrP-(107-111) and hPTHrP-(107-139) on %a release from prelabeled neonatal mouse calvariae. After 24-h preincubation, bones were further incubated for the indicated time periods up to 96 h under the following conditions. A and B, Bones were incubated with control medium only (O), hPTHrP-(107-111) (0, 10mR M), or sCT (A; lo-@ M) in the presence (A) or absence (B) of 2 x lo-’ M hPTHrP-(1-34). C, Bones were incubated with control medium only (0) or hPTHrP-(107-139) (0, lo-’ M) in the presence (- - -) or absence ) of 2 x 1O-9 M hPTHrP-(l-34). The amount of ‘%a released into the medium was determined every 24 h. Data are the mean + SEM from (two similar exneriments. each determined in auintunle. * and **. Statistically significant differences from the corresponding control groups at P . _ < 0.05 and P 2 0.01, respectively.
TABLE
1. Effects
stimulated
by various
of hPTHrP-(107-111) and sCT on 45Ca release bone resorbing agents Bone-resorbing
Stimulator
Untreated
None 1,25-(OH)*D3 (1 nM) NlePTH (2 nM) hPTHrP-(l-34) (2 nM) rhIL-lot (100 pg/ml) PGE, (1 PM)
1.00 2.49 2.51 2.60 2.38 2.41
f f k k + +
activity
(T/C ratio)
bPTHrP-(107-111)
0.03 0.35 0.21 0.30 0.32 0.08
1.03 2.40 2.45 2.57 2.29 2.48
f + + + + +
sCT
0.85 0.95 1.02 1.10 0.93 1.09
0.06 0.17 0.20 0.11 0.20 0.13
+ + + + + +
0.07” 0.05* 0.09* 0.08* 0.06b O.lob
After 24-h preincubation, bones were cultured for 72 h in various conditions, as indicated above. sCT and hPTHrP-(107-111) were tested at lo-’ M each. Bone-resorbing activity is expressed as a treated to control (T/C) ratio to facilitate comparisons between multiple experiments. Data are the mean + SEM (n = 5). ’ P < 0.05 us. control. b P < 0.01 us. control.
TABLE
2. Effect of hPTHrP-(107-ill), sCT on osteoclast number in neonatal with or without hPTHrP-(l-34)
hPTHrP-(107-139), and mouse calvariae incubated
No. of osteoclast/mm Treatment None hPTHrP-(107-111) hPTHrP-(107-139) sCT (10-R M)
Without hPTHrP-(l-34)
(lOmR M) (lo-’ M)
3.9 4.1 4.0 3.8
+ * f +
With
1.8 2.0 1.9 1.5
bone surface hPTHrP-(l-34) (2 X 1o-9 M)
15.8 17.9 14.9 4.5
+ k f +
2.8 3.2 3.0 1.8”
After 24-h preincubation, bones were cultured for 48 h in various conditions, as indicated above. Data are the mean k SEM of three determinations. ’ Statistically significant difference from the hPTHrP-(l-34) alone group (P < 0.05).
hPTHrP-(107-111) and hPTHrP-(107-139) act on some cell types present in neonatal mouse calvariae to stimulate PGEl synthesis, which, in turn, obliterates the direct inhibitory effects of these peptides on osteoclasts.To test this hypothesis, we incubated bones with each of the two carboxyl-
terminal PTHrP peptides in the presence or absence of indomethacin. Indomethacin (10m6M) did not influence the effect of either of the two carboxyl-terminal PTHrP peptides (lo-@ M each) on basal bone resorption or bone resorption induced by PTHrP-(1-34) (2 X 10m9M; Table 3).
Discussion
We have found that the 107-l 11 sequenceof the hPTHrP molecule doesnot have the ability to inhibit osteoclasticbone resorption in bone organ culture. The apparent inability of hPTHrP-(107-111) to inhibit bone resorption in intact bone contrasts with its potent inhibitory activity on bone resorption in an isolated osteoclast assay previously shown by TABLE
3. Effect of indomethacin on basal and hPTHrP-(l-34)stimulated %a release from prelabeled neonatal mouse calvariae treated with hPTHrP-(107-111) or hPTHrP-(107-139) Bone-resorbing activity (T/C ratio)
Treatment
Without
In the absence of hPTHrP-(1-34) nM) None hPTHrP-(107-111) (10 nM) hPTHrP-(107-139) (10 nM) In the presence of hPTHrP-(l-34)
indomethacin
With indomethacin
1.00 + 0.10 1.03 + 0.10
1.04 + 0.08 0.95 + 0.06
1.01
0.97
(2
+ 0.05
+ 0.12
(2 nM) None hPTHrP-(107-111) hPTHrP-(107-139)
(10 nM) (10 nM)
2.23 + 0.16 1.95 -t 0.10 2.19
+ 0.09
2.22 + 0.29 2.14 f 0.20 2.27 + 0.16
After 24-h preincubation, bones were cultured for 72 h in various conditions, as indicated above. Indomethacin was added at 1 pM. Boneresorbing activity is expressed as a treated to control (T/C) ratio to facilitate comparisons between multiple experiments. Data are the mean f SEM (n = 5). There is no statistically significant difference between the without indomethacin and with indomethacin groups in each treatment.
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PTHrP-(107-111)
AND
Fenton et al. (12). We have no clear explanation for this difference. However, this difference may be due to the difference in microenvironment of osteoclasts between the two assay systems. hPTHrP-(107-111) was shown to have inhibitory activity on osteoclastic bone resorption in a bioassay that effectively lacks many bone cell types other than osteoclasts. In particular, the isolated osteoclast assay is devoid of osteoblasts, which are believed to be target cells for most, if not all, bone-resorbing hormones and local factors and transduce the resorptive message to osteoclasts in intact bone (20-22). The observed difference closely resembles the difference in the mode of action of PGEz on bone, which acts as a direct inhibitor of osteoclast function, as assessed by an isolated osteoclast bone resorption assay (23), yet induces osteoclastic bone resorption indirectly in intact bone via stimulatory effects on osteoblasts (23, 24). We, therefore, hypothesized that PTHrP-(107-111) can have an additional effect on some cell types other than osteoclasts in intact bone, which are induced to produce some bone-resorbing factor(s), thereby masking the direct inhibitory effect of the peptide on osteoclasts. One candidate for this mediator is PGE, (19). The addition of indomethacin had no effect on bone resorption in neonatal mouse calvariae treated with either hPTHrP(107-111) or hPTHrP-(107-139), which makes PG-mediated effects unlikely. Nonetheless, we cannot exclude the possibility that some signaling pathway other than a PG-mediated mechanism may underlie the apparent failure of PTHrP(107-111) in intact bone. This hypothesis deserves further study. Alternatively, an additional peptide sequence beyond the Trp”’ residue may be required to generate osteoclastic inhibitory activity in our organ culture model. To test this possibility, we synthesized and tested hPTHrP-(107-139) for effects in mouse calvariae in culture. Like hPTHrP-(107-11 l), hPTHrP-(107-139) did not modulate either basal bone resorption or bone resorption stimulated by hPTHrP-(l-34). Our results differ from those of Fenton et al. (12), who found that hPTHrP-(107-139) is a potent inhibitor of bone resorption in cultured fetal rat long bones, osteoblast/osteoclast coculture, as well as purified osteoclast assay. This difference may be due to either species difference or differences in the relative maturity of bone cell types present in the two assay systems (25, 26) as well as in the mode of bone formation (intramembranous us. endochondral bone formation). For example, transforming growth factor-0 inhibits bone resorption in fetal rat long bone cultures (27), while in neonatal mouse calvarial cultures, it stimulates bone resorption (19). Indeed, the apparent failure of PTHrP-(107-139) to inhibit bone resorption in cultured mouse calvariae was unexpected, because these bones contain mature osteoclasts as the major population of cells in the osteoclast lineage, which should be the target cells for the direct inhibitory action of PTHrP(107-139) (11). There is evidence that PTHrP is expressed at sites of both endochondral and intramembranous bone formation in human fetus (28). PTHrP is released by resorbing rat long bone in culture (29). These data support the intriguing hypothesis that PTHrP plays a distinct role in normal skeletal develop-
BONE
RESORPTION
2745
ment and physiology, possibly through local mechanisms unrelated to PTH-like activity. Determination of the microenvironmental requirement for the biological activity of the carboxyl-terminal fragments of hPTHrP and its physiological relevance remains to be elucidated. References 1 Moseley JM, Kubota M, Diefenbach-Jagger H, Wettenhall REH, Kemp BE, Suva LJ, Rodda Cl’, Ebeling PR, Hudson PJ, Zajac JD, Martin TJ 1987 Parathyroid hormone-related protein purified from a human
lung
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Proc Nat1 Acad
Sci USA
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Suva LJ, Winslow GA, Wettenhall REH, Hammonds RG, Moseley JM, Diefenbach-Jagger H, Rodda CP, Kemp BE, Rodriguez H, Chen EY, Hudson PJ, Martin TJ, Wood WI 1987 A parathyroid hormone-related protein implicated in malignant cloning and expression. Science 237:893-896
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Burtis WJ, Wu T, Bunch C, Wysolmerski JJ, Insogna KL, Weir EC, Broadus AE, Stewart AF 1987 Identification of a novel 17,000dalton parathyroid hormone-like adenylate tein from a tumor associated with humoral nancy. J Biol Chem 262:7151-7156
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6.
cyclase-stimulating prohypercalcemia of malig-
Strewler GJ, Stern PH, Jacobs JW, Eveloff J, Klein RF, Leung SC, Rosenblatt M, Nissenson RA 1987 Parathyroid hormonelike protein from homology
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hypercalcemia:
human renal carcinoma cells: structural with parathyroid hormone. J CIin Invest
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Kemp 8, Moseley J, Rodda C, Ebeling P, Wettenhall R, Stapleton D, Diefenbach-Jagger H, Ure F, Michelangeli V, Simmons H, Raisz L, Martin TJ 1987 Parathyroid hormone-related protein of 1malignancy: active synthetic fragments. Science 238:1568-1570 Jlippner H, Abou-Samura A-B, Uneno S, Gu W-X, Potts J’I’J, Segre GV 1988 The parathyroid hormone-like peptide associated with
humoral hypercalcemia of malignancy and parathyroid hormone bind to the same receptor on the plasma membrane of ROS 17/2.8 cells. J Biol Chem 263:8557-8560 7. Nissenson RA, Diep D, Strewler GJ 1988 Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormonelike protein from human malignancies: binding to parathyroid hormone receptors and activiation of adenylate cyclase in bone cells and kidney. J Biol Chem 263:12866-12871 8
Shigeno C, Yamamoto I, Kitamura N, Noda T, Lee K, Sone T, Shiomi K, Ohtaka A, Fujii N, Yajima H , Konishi J 1988 Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells. J Biol Chem 263:18369-18377
9
Rodda CP, Kubota M, Heath JA, Ebeling PR, Mosely JM, Care AD, Caple IW , Martin TJ 1988 Evidence for a novel parathyroid hormone-related protein in fetal Iamb parathyroid glands and sheep placenta: comparisons with a similar protein implicated in humoral hypercalcemia of malignancy. J Endocrinol 117:261-271
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Care AD, Abbas SK, Pickard DW, Barri M, Drinkhill M, Findlay JBC, White IR, Caple IW 1990 Stimulation of bovine placenta transport human
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Fenton AJ, Kemp BE, Kent GN, Moseley JM, Zheng MH, Rowe DJ, Britto JM, Martin TJ, Nicholson GC 1991 A carboxyl-terminal peptide from the parathyroid hormone-related protein resorption by osteoclasts. Endocrinology 129:1762-1768
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Fenton AJ, Kemp BE, Hammonds Jr RG, Mitchelhill JM, Martin TJ, Nicholson GC 1991 A potent inhibitor
inhibits
bone
K, Moseley
of osteoclastic bone resorption within a highly conserved pentapeptide region of parathyroid hormone-related protein; PTHrP-(107-111). Endocrinology 129:3424-3426 13. Chambers T, Revel1 P, Fuller K, Athanasou N 1984 Resorption of bone by isolated rabbit osteoclasts. J Cell Sci 66:383-399 14. Yamamoto I, Kawano M, Sone T, Iwato K, Tanaka H, Ishikawa
H, Kitamura N, Lee K, Shigeno C, Konishi J, Tanabe 0, Nobuyoshi M, Ohmoto Y, Hirai Y, Higuchi M, Ohsawa T, Kuramoto
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Production of interleukin I beta, a potent bone resorbing by cultured human myeloma cells. Cancer Res 49:4242-
Shigeno C, Yamamoto I, Dokoh S, Hino M, Aoki J, Yamada K, Morita R, Kameyama M, Torizuka K 1985 Identification of 1,24(R)dihydroxy-vitamin cancer-associated 768
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1991 Mechanism of regulation of prostaglandin thyroid hormone, interleukin-1, and cortisol parietal bones. Endocrinology 128:2503-2510 19.
AND
production by parain cultured mouse
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factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc Nat1 Acad Sci USA 82:4535-4538 20. Rodan GA, Martin TJ 1981 Role of osteoblasts in hormonal control of bone resorption-a hypothesis. Calcif Tissue Int 33:349-351
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PMJ, Chambers TJ 1987 1,25-Dihydroxyvitamin DB rat osteoblastic cells to release a soluble factor that osteoclastic bone resorption. J Clin Invest 80:425-429 22. McSheehy PMJ, Chambers TJ 1986 Osteoblast-like cells in the presence of parathyroid hormone release soluble factor that stimulates osteoclastic bone resorption. Endocrinology 119:1654-1659 23. Chambers TJ, McSheehy PMJ, Thompson BM, Fuller K 1985 The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones. Endocrinology 60:234-239 24. Klein DC, Raisz LG 1970 Prostaalandins: stimulation of bone resorption in tissue culture. Endocrinology 86:1436 25. Al-Humidan A, Ralston S. Hughes D. Chaoman K. Aarden L. Russell R, Go&en M 1991. Intezeukin-6 does not stimulate bone resorption in neonatal mouse calvariae. J Bone Mineral Res 6:3-8 26. Roodman G 1992 Perspectives: interleukin-6: an osteotropic factor? J Bone Mineral Res 7:475-478 27. Pfeilschifter J, Seyedin S, Mundy G 1988 Transforming growth factor beta inhibits bone resorption in fetal rat long bone cultures. J Clin Invest 82:680-685 28. Moseley JM, Hayman JA, Danks JA, Alcorn D, Grill V, Southby J, Horton MA 1991 Immunohistochemical detection of parathyroid hormone-related protein in human fetal epithelia. J Clin Endocrinol Metab 73:478-484 29. Bergman P, Nijs-DeWolf N, Pepersack T, Corvilain J 1990 Release of parathyroid hormonelike peptides by fetal rat long bones in culture. J Bone Mineral Res 5:741-753 21.
McSheehy
Endo. Voll31.
stimulates increases
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