PTH1R translocation to primary cilia in mechanically-stimulated ostecytes prevents osteoclast formation via regulation of CXCL5 and IL-6 secretion.

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6.

Primary cilia and PTH1R interplay in the regulation of osteogenic actions.

Primary cilia are subcellular structures specialized in sensing different stimuli in a diversity of cell types. In bone, the primary cilium is involved in mechanical sensing and transduction of signals that regulate the behavior of mesenchymal osteoprogenitors, osteoblasts and osteocytes. To perform its functions, the primary cilium modulates a plethora of molecules including those stimulated by the parathyroid hormone (PTH) receptor type I (PTH1R), a master regulator of osteogenesis. Binding of the agonists PTH or PTH-related protein (PTHrP) to the PTH1R or direct agonist-independent stimulation of the receptor activate PTH1R signaling pathways. In turn, activation of PTH1R leads to regulation of bone formation and remodeling. Herein, we describe the structure, function and molecular partners of primary cilia in the context of bone, playing special attention to those signaling pathways that are mediated directly or indirectly by PTH1R in association with primary cilia during the process of osteogenesis.