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.
