ELSEVIER ORIGINAL ARTICLES Bone Vol, 22, No. 3 March 1998:189-194 Localization of Parathyroid Hormone-Related Protein in Osteoclasts by In Situ Hybridization and Immunohistochemistry V. KARTSOGIANNIS, N. UDAGAWA, K. W. NG, T. J. MARTIN, J. M. MOSELEY, and H. ZHOU Department of Medicine, The University of Melbourne and St. Vincent's Institute of Medical Research, St. Vincent's Hospital, Fitzroy, Victoria, Australia Using immunohistology with two specific antisera raised against N-terminal parathyroid hormone-related protein (PTHrP) and in situ hybridization (riboprobe to common coding exon), evidence is provided for the expression of PTHrP by mouse, rabbit, and human osteoclasts derived from several in vitro and in vivo sources. In cocultures of mouse bone marrow and calvarial cells treated with 1,25- dihydroxyvitamin D3, the generated osteoclasts expressed both PTHrP messenger RNA (mRNA) and protein. In addi- tion, PTHrP was detected in the majority of actively resorb- ing osteoclasts in sections of newborn and adult mouse long bones. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in osteoclasts at active bone resorption sites as well as in actively synthesizing osteoblasts and bone lining cells. Localization of PTHrP was also demonstrated in osteoclast-like cells of human giant cell tumors from bone. In some of these tumors, a small proportion of the multinucle- ated cells expressed tartrate resistant acid phosphatase (TRAP), but not PTHrP mRNA or protein. Finally, both mRNA and protein for PTHrP were expressed in osteoclasts in sections of bone or joints from patients with Paget's disease, rheumatoid arthritis, and osteoarthritis. These ob- servations raise the possibility that PTHrP might participate in osteoclast function. (Bone 22:189-194; 1998) © 1998 by Elsevier Science Inc. All rights reserved. Key Words: Parathyroid hormone-related protein; In situ hy- bridization; Immunohistochemistry; Osteoclasts. Introduction Parathyroid hormone-related protein (PTHrP) was discovered as a bone resorbing hormone produced in excess by certain cancers, and responsible for the syndrome of humoral hypercalcemia of malignancy (reviewed in Grill et al. j ~). However, it is now well established that PTHrP acts as a local regulator in many normal tissues (reviewed in Moseley and Gillespie 26 and Philbrick et al.3°). The discovery of the local production of PTHrP at sites of parathyroid hormone (PTH) action in several tissues has led to Address for correspondence and reprints: Dr. Hong Zhou, Department of Medicine, The University of Melbourne, St. Vincent's Hospital, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia. E-mail: h.zhou@ medicine.unimelb.edu.au the view that those actions previously attributed to PTH are more likely performed by PTHrP as a paracrine effector in those tissues. Experimental evidence suggests that PTHrP appears to act in a paracrine fashion in a number of normal developmental and physiological processes, some of which include: embryogenesis and neonatal development, cellular growth and differentiation, smooth muscle relaxation at many sites, reproduction and lacta- tion, and placental calcium transfer. Its actions in calcium trans- port and as a smooth muscle relaxant at many locations are indisputable (reviewed in de Papp and Stewart7). In addition to evidence that PTHrP is produced by osteoblasts and chondrocytes, 2°'23'25 studies of mice with targeted disruption of the PTHrP gene revealed that PTHrP is critically important in endochondral bone formation during fetal development and is absolutely necessary for normal bone integrity in the postnatal skeleton.Z,3, Js,19,24 We have shown previously 21'35 that PTHrP is synthesized by osteoblasts during intramembranous bone forma- tion and may therefore be important in that process as well. In the course of studying osteoclast formation in vitro, we noted that these cells also expressed PTHrP. This report describes PTHrP expression in osteoclasts from several in vitro and in vivo sources, and species by immunohistochemistry, and in situ hybridization. Materials and Methods Mouse bone marrow cell cultures were prepared as previously described. 34 Bone marrow cells from long bones of 6-9 week old male C57B1/6J mice were cocultured with calvarial cells ob- tained from newborn mice in collagen gels in the presence of 10-nmol/L 1,25-dixydroxyvitamin D 3. After 7 days, cells were treated with collagenase (0.2%), and the dispersed cells subcul- tured on 13 mm diam Thermanox coverslips (Nunc) in 24-well plates before processing for in situ hybridization or immunohis- tochemistry. The expression of calcitonin receptors (CTRs) was also assessed by autoradiography using [125I]-salmon calcitonin as previously described) 6 Long bones were obtained from the femora and tibiae of newborn and adult healthy C57B l/6J male mice. Rabbit bone tissue harvests were obtained using an in vivo intramembranous bone formation rabbit model as previously described. 4° This model uses a biocompatible intraosseous tita- nium device implanted into the rabbit tibia, which allows bone tissue to be harvested repeatedly under controlled conditions during a defined period of time. © 1998 by Elsevier Science Inc. 189 8756-3282/98/$19.00 All rights reserved. Pit $8756-3282(97)00278-0