Elsevier

Bone

Volume 30, Issue 5, May 2002, Pages 726-732
Bone

Original article
Platelet-released supernatants stimulate formation of osteoclast-like cells through a prostaglandin/RANKL-dependent mechanism

https://doi.org/10.1016/S8756-3282(02)00697-XGet rights and content

Abstract

Platelets are activated at fracture sites or upon the insertion of implants as a consequence of vascular disruption and secrete the contents of their granules into the developing hematoma. The regeneration of injured tissue requires bone remodeling and the resorbing activity of osteoclasts. To test our hypothesis that platelets can stimulate osteoclastogenesis, we examined the effects of supernatants released from thrombin-activated platelets on osteoclast-like cell formation in murine bone marrow cultures. Histochemical analysis indicated the presence of bone-resorbing, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Transcripts that are characteristically expressed in native osteoclasts were increased in these cultures, as determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. The inhibition of both cyclooxygenases with indomethacin, as well as the addition of the cyclooxygenase-2 (COX-2)-selective antagonist, NS398, completely blocked osteoclast-like cell formation and decreased endogenous prostaglandin E2 production. Platelet-released supernatants stimulated the expression of receptor activator of NF-κB ligand (RANKL), whereas mRNA levels of osteoprotegerin (OPG) were decreased. The formation of osteoclast-like cells was prevented by recombinant OPG. Our results suggest that COX-2 activity is necessary for osteoclast-like cell formation in response to platelet-released supernatants, and that endogenously produced prostaglandin E2 can, in turn, increase the RANKL:OPG ratio, indicating that platelets can contribute to bone remodeling by stimulation of osteoclastogenesis.

Introduction

The differentiation of hematopoietic progenitor cells, presumably cells of the monocyte-macrophage lineage, into bone-resorbing osteoclasts is a multistep cascade that requires the presence of stromal cells and/or osteoblasts.30, 38 This cascade is initiated by local factors; for example, interleukin-1 (IL-1), IL-11, and prostaglandin E2 (PGE2), and systemic hormones such as 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and parathyroid hormone (PTH), which bind to their corresponding receptors on stromal cells/osteoblasts, thereby activating signaling pathways that can cause osteoclast (OCL) formation, including: (a) vitamin D receptor with its ligand 1,25(OH)2D3; (b) gp130, activated by cytokines of the IL-6 family; and (c) protein kinase A, activated by IL-1, PTH, and PGE2.40 Osteoclast formation has often been found to depend on the endogenous production of PGE2 and therefore on the conversion of arachidonic acid through cyclooxygenases (COX), particularly on the inducible version, COX-2.12, 23, 25, 32, 42 IL-1-mediated osteoclastogenesis depends on the enzymatic activity of COX-2 and can increase PGE2 production in stromal cells/osteoblasts.2, 35 PGE2 can in turn activate the cascade of osteoclastogenesis by binding to EP2/EP4 subtypes of PGE receptors.28, 34, 41

Stimulation via vitamin D receptor, gp130, and protein kinase A pathways leads to the expression of RANKL, the central mediator of osteoclastogenesis in stromal cells/osteoblasts.7, 14, 21, 39 RANKL has been proposed to be the common nomenclature for tumor necrosis factor (TNF)-related activation-induced cytokine (TRANCE), osteoclast differentiating factor (ODF), or osteoprotegerin ligand (OPGL).3 The interaction of RANKL with RANK, the latter being expressed in osteoclast precursor cells, provides the signal for osteoclastogenesis from hematopoietic progenitor cells.24, 46 RANKL is also important for the activation and survival of mature osteoclasts.16 OPG, being identical to osteoclast-inhibitory factor (OCIF) and TNF-receptor-like molecule 1, is a soluble protein that is expressed in multiple tissues and acts as a decoy receptor for RANKL, thus inhibiting its interaction with RANK.3, 37, 46 Knockout models further support these assumptions, wherein deletion of either RANKL or RANK results in the development of an osteopetrotic phenotype, whereas OPG−/− animals have severe osteoporosis.6, 10, 22 Recent studies have suggested that RANKL and OPG are involved in the regulation of skeletal repair, showing that the expression of RANKL was nearly undetectable in unfractured bones, but strongly induced throughout the period of fracture healing.20 Fractures, defects due to surgical intervention and the insertion of implants, are paralleled by vascular disruption, which causes platelets to secrete the contents of their granules into the developing hematoma.4, 5, 11, 18, 36 These granules contain a large number of factors, some of which are known to stimulate osteoclastogenesis, such as IL-1 and PGE2, whereas others (e.g., transforming growth factor [TGF]-β can inhibit this process.2, 3, 13, 29 Histologic observations have suggested that, after an injury, the formation of a blood clot occurs before resorption lacunae can be detected.9 It remains unknown, however, whether platelets that accumulate within the hematoma are involved in osteoclastogenesis.

In the present study, we examine the ability of supernatants from activated platelets to stimulate the development of tartrate-resistant acid phosphatase-positive (TRAP+)/multinucleated cells (MNC) in murine bone marrow cultures. To determine which signaling pathways play a role in this in vitro setting, cells were cultured in the presence of inhibitors of COX-1/COX-2, and OPG. We found that platelet-released supernatants stimulate osteoclastogenesis; the addition of the inhibitors of COX-2 and OPG completely diminished the formation of TRAP+/MNC. These findings suggest the following mechanism of osteoclast formation: Platelet-released supernatants stimulate the production of PGE2 in bone marrow cells, which in turn activates the RANKL-dependent pathway of osteoclastogenesis.

Section snippets

Isolation of platelet concentrates

Platelet concentrates from volunteer donors were prepared by standard apheresis procedures using an MCS+device (Hemonetics, Braintree, MA) and ACD-A as anticoagulant at a ratio of 1:10. The platelet concentrates were automatically leukodepleted by a negatively charged pall filter (LRF, XL) with <3 × 103 white blood cells per milliliter remaining in the preparation. The platelet concentrates were gently agitated at 21°C on a platelet agitator (Helmer Labs, Nobesville, IN) and aliquots were

PRS induces OCL formation

Murine bone marrow cells grown in the presence of PRS for 7 days showed the development of TRAP+/MNC with the ability to resorb bone, whereas, in control cultures, only negligible numbers of OCL (≤10) were detected. In five independent experiments, stimulated with PRS at dilution I, the number of TRAP+/MNC varied between 40 ± 26 and 320 ± 47. Figure 1A shows a characteristic dose response of OCL formation with a lowest significant concentration of PRS corresponding to 4 × 107 platelets per

Discussion

In the present study we have examined the ability of supernatants, released upon activation of platelets, to stimulate the formation of OCL in bone marrow cultures, and determined the signaling pathways involved in this process. Bone-resorbing factors, such as 1,25(OH)2D3, IL-11, IL-1, and PGE2, stimulated the formation of TRAP+/MNC, which are cells that satisfy the major criteria for definition of mature osteoclasts.43 Herein, we have demonstrated that PRS induces the development of TRAP+/MNC

Acknowledgements

R. Gruber is grateful to M.-T. Krauth for reviewing the manuscript. The authors thank R. Binder for helping establish the in vitro model, Prof. P. Pietschmann (Department of Pathophysiology, University of Vienna) for providing us with the OSF-1 mice, L. Amoyo for technical assistance, and Prof. W. Graninger (Department of Rheumatology, University of Vienna) for permission to use the PCR equipment. The work was supported by the Austrian Nationalbank, Grant No. 9269.

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