0013-7227/9l/1296-3424$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 6 Printed in U.S.A.
A POTENT INHIBITOR OF OSTEOCLASTIC BONE RESORPTION WITHIN A HIGHLY CONSERVED PENTAPEPTIDE REGION OF PARATHYROID HORMONE-RELATED PROTEIN; PTHrP[107-lll] A. J. Fenton, B. E. Kemp, R. G. Hammonds Jr, K. Mitchelhill, J. M. Moseley, T. J. Martin and G. C. Nicholson Dept of Medicine, The University of Melbourne, The Geelong Hospital, Geelong, Victoria, Australia 3220 (AJF, GCN), St Vincent's Institute of Medical Research and Dept of Medicine, The University of Melbourne, Fitzroy, Victoria, Australia 3065 (BEK, KM, JMM, TJM), Genentech Inc, Sth San Francisco, California 94080 (RGH) ABSTRACT: We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-l 11]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.
Parathyroid hormone-related protein (PTHrP) is produced by a variety of cancers and is thought to be responsible for the clinical syndrome of humoral hypercalcemia of malignancy (1-3). The amino-terminal region contains a PTH-like sequence responsible for indirectly stimulating osteoclastic bone resorption by acting on osteoblasts and restricting calcium excretion by the kidney (4-6). From studies of the action of PTHrP[l-141] on populations of osteoclasts disaggregated from neonatal rat bones we found that recombinant hPTHrP[l-141] had a direct effect on osteoclasts to inhibit bone resorption whereas no such inhibition was obtained with the synthetic amino-terminal peptides hPTHrP[l-34] and hPTHrP[l-108]. This osteoclast inhibitory activity was localised to a carboxyl-terminal fragment, hPTHrP[107-139] (7). These findings indicate that processing of PTHrP[l-141] may result in peptides containing an amino-terminal portion which activates bone resorption by acting indirectly via osteoblasts, or a carboxylterminal portion which inhibits bone resorption by acting directly on osteoclasts. In the present manuscript we report that the highly conserved pentapeptide, TRSAW, corresponding to residues [107-111] in PTHrP[107-139] is responsible for the inhibition of bone resoiption by osteoclasts. Bone resoiption was assessed by quantification of resorption pits formed by osteoclasts disaggregated from neonatal rat bones and settled onto thin slices of devitalised bovine cortical bone (7,8). MATERIALS AND METHODS
Isolated osteoclast bone resorption assay: Osteoclasts were isolated from neonatal Sprague-Dawley rats, as previously described (7), by curetting the long bones into culture medium (HEPES-buffered Medium 199 [Flow, Melbourne, Australia]). The resulting suspension containing a mixed cell population was allowed to settle onto 4mm x 4mm x 100 |im slices of devitalised bovine cortical bone for 20 minutes before rinsing. After this brief settling period, the majority of contaminating cells are removed in the rinsing step, resulting in a functionally pure population of osteoclasts (approximately 35 osteoclasts per bone slice). The bone slices were then incubated in tissue culture medium (Eagle's Minimum Essential Medium [Flow] supplemented with 500,000 Received in Iowa City, Iowa, Sept. 13, 1991.
iU/ml benzylpenicillin and 10% heat-inactivated fetal bovine serum) at 37 C in 95% air, 5% CO2, pH 7.2 in the presence or absence of test agents. The assay was routinely performed in the absence of any agent known to stimulate bone resoiption, such as PTH or PTHrP[l-34]. After 24 hours, the cells were fixed, reacted cytochemically for tartrate-resistant acid phosphatase (TRACP) (9) and the number of multinucleated cells with strong reactions for TRACP (i.e. osteoclasts) counted. The cells were then stripped by ultrasonication in 0.25M NH4OH prior to processing for scanning electron microscopy. The entire surface of each bone slice was scanned and the number of resoiption pits counted. Resoiption was routinely assessed by counting the number of resoiption pits per multinucleated osteoclast on each bone slice, although in selected experiments, the plan areas of all resorption pits were also measured using digitising morphometry. While treatment with hPTHrP[107-139] and hPTHrP[107-lll] reduced the total plan area of bone resorbed per bone slice (Control: 12,302±345nm2 [n=14], PTHrP[107-139]: 32931680 Hm2 [n=14] (p
Vol. 129, No. 6 Printed in U.S.A.
A POTENT INHIBITOR OF OSTEOCLASTIC BONE RESORPTION WITHIN A HIGHLY CONSERVED PENTAPEPTIDE REGION OF PARATHYROID HORMONE-RELATED PROTEIN; PTHrP[107-lll] A. J. Fenton, B. E. Kemp, R. G. Hammonds Jr, K. Mitchelhill, J. M. Moseley, T. J. Martin and G. C. Nicholson Dept of Medicine, The University of Melbourne, The Geelong Hospital, Geelong, Victoria, Australia 3220 (AJF, GCN), St Vincent's Institute of Medical Research and Dept of Medicine, The University of Melbourne, Fitzroy, Victoria, Australia 3065 (BEK, KM, JMM, TJM), Genentech Inc, Sth San Francisco, California 94080 (RGH) ABSTRACT: We have recently shown that a carboxyl-terminal sequence of parathyroid hormone-related protein, PTHrP[107-139], is a potent direct inhibitor of osteoclastic bone resorption. We now report that this bone resorption inhibitory activity, which we have called osteostatin, is entirely contained within the highly conserved pentapeptide PTHrP[107-l 11]. Our results indicate that processing at residue 106 and a free amino terminus is required for full activity of the peptide. The retroinverted peptide is considerably less potent than the parent peptide. The retention of full biological activity in such a short fragment was unexpected. This data provides the basis for the development of further analogs with potential therapeutic use in disorders associated with increased osteoclastic bone resorption.
Parathyroid hormone-related protein (PTHrP) is produced by a variety of cancers and is thought to be responsible for the clinical syndrome of humoral hypercalcemia of malignancy (1-3). The amino-terminal region contains a PTH-like sequence responsible for indirectly stimulating osteoclastic bone resorption by acting on osteoblasts and restricting calcium excretion by the kidney (4-6). From studies of the action of PTHrP[l-141] on populations of osteoclasts disaggregated from neonatal rat bones we found that recombinant hPTHrP[l-141] had a direct effect on osteoclasts to inhibit bone resorption whereas no such inhibition was obtained with the synthetic amino-terminal peptides hPTHrP[l-34] and hPTHrP[l-108]. This osteoclast inhibitory activity was localised to a carboxyl-terminal fragment, hPTHrP[107-139] (7). These findings indicate that processing of PTHrP[l-141] may result in peptides containing an amino-terminal portion which activates bone resorption by acting indirectly via osteoblasts, or a carboxylterminal portion which inhibits bone resorption by acting directly on osteoclasts. In the present manuscript we report that the highly conserved pentapeptide, TRSAW, corresponding to residues [107-111] in PTHrP[107-139] is responsible for the inhibition of bone resoiption by osteoclasts. Bone resoiption was assessed by quantification of resorption pits formed by osteoclasts disaggregated from neonatal rat bones and settled onto thin slices of devitalised bovine cortical bone (7,8). MATERIALS AND METHODS
Isolated osteoclast bone resorption assay: Osteoclasts were isolated from neonatal Sprague-Dawley rats, as previously described (7), by curetting the long bones into culture medium (HEPES-buffered Medium 199 [Flow, Melbourne, Australia]). The resulting suspension containing a mixed cell population was allowed to settle onto 4mm x 4mm x 100 |im slices of devitalised bovine cortical bone for 20 minutes before rinsing. After this brief settling period, the majority of contaminating cells are removed in the rinsing step, resulting in a functionally pure population of osteoclasts (approximately 35 osteoclasts per bone slice). The bone slices were then incubated in tissue culture medium (Eagle's Minimum Essential Medium [Flow] supplemented with 500,000 Received in Iowa City, Iowa, Sept. 13, 1991.
iU/ml benzylpenicillin and 10% heat-inactivated fetal bovine serum) at 37 C in 95% air, 5% CO2, pH 7.2 in the presence or absence of test agents. The assay was routinely performed in the absence of any agent known to stimulate bone resoiption, such as PTH or PTHrP[l-34]. After 24 hours, the cells were fixed, reacted cytochemically for tartrate-resistant acid phosphatase (TRACP) (9) and the number of multinucleated cells with strong reactions for TRACP (i.e. osteoclasts) counted. The cells were then stripped by ultrasonication in 0.25M NH4OH prior to processing for scanning electron microscopy. The entire surface of each bone slice was scanned and the number of resoiption pits counted. Resoiption was routinely assessed by counting the number of resoiption pits per multinucleated osteoclast on each bone slice, although in selected experiments, the plan areas of all resorption pits were also measured using digitising morphometry. While treatment with hPTHrP[107-139] and hPTHrP[107-lll] reduced the total plan area of bone resorbed per bone slice (Control: 12,302±345nm2 [n=14], PTHrP[107-139]: 32931680 Hm2 [n=14] (p
