PAR4 activation promotes osteoclast formation and resorptive function

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Introduction Osteoclastogenesis involves several steps: proliferation of precursors, early differentiation into mononuclear cells, and fusion into multinucleated osteoclasts. RANKL (receptor activator of nuclear factor kB ligand), produced by osteoblasts, is the key regulator. Thrombin, the key enzyme of coagulation enhances the release of osteoclastogenic factors by osteoblasts but paradoxically impedes the initial stages of RANKL-induced osteoclast differentiation in precursor cells, independently of its proteolytic activity. In various other cell types, thrombin mediates intracellular signaling via protease-activated receptors 1 and 4 (PAR1/PAR4). A transient expression of PAR1 was observed in osteoclast precursor cells, while PAR4 expression has not yet been investigated. We aimed to assess the influence of PAR-activation on the differentiation of osteoclast precursors into mature osteoclasts. Method Peripheral blood mononuclear cells (PBMCs) from whole blood were isolated followed by positive magnetic sorting for CD14+monocytes. Monocytes were expanded to macrophages with 50 ng/ml recombinant macrophage colony-stimulating factor (rhM-CSF), and osteoclast differentiation was induced by adding 50 ng/ml RANKL. We tested the impact of PAR activation by adding peptides specifically activating PAR1 (PAR1-AP, 32 µM) or PAR4 (PAR4-AP, 50 µM) to the culture medium. Cells were fixed, stained for TRAP (tartrate-resistant acid phosphatase), and counterstained with hematoxylin. Tartrate-Resistant Acid Phosphatase in the conditioned medium (TRAcP) was quantified with a fluorometric assay. PAR4 expression was determined via Western blot analysis. Results In our in-vitro differentiation assay, we observed an expedited osteoclast differentiation process upon the addition of PAR4-AP, as opposed to PAR1-AP. Preliminary data suggested that this acceleration manifests within 1-2 days of introducing RANKL, corresponding to an increase in TRAcP activity in the medium. Western blot analysis unveiled a robust expression of PAR4 in precursor cells treated with RANKL. Conclusion Accelerated differentiation upon PAR4 activation of osteoclast precursor cells appears to contradict previous reports on the inhibiting activity of thrombin acting via PAR1 or independently of its proteolytic activity. PAR4 may serve as a regulator to fine-tune the action of thrombin. Additionally, Cathepsin K functions as a ligand for PAR4. As its secretion is a distinctive feature of osteoclast formation, this may potentially signify the presence of a positive feedback loop. Further functional studies employing physiological PAR4 ligands and antagonists are necessary to elucidate the precise role of PAR4 in osteoclastogenesis. Publication History Article published online: 26 February 2024 © 2024. Thieme. All rights reserved. Georg Thieme Verlag Rüdigerstraße 14, 70469 Stuttgart, Germany

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