Mechanical stimulation of primary cilia in osteocytes and osteoblasts has been proposed as a mechanism that participates in bone cell survival and skeletal remodeling. Among different signaling pathways stimulated by primary cilia, the hedgehog signaling pathway has been associated with the regulation of bone development. Parathyroid hormone (PTH)-related protein (PTHrP) signaling through PTH 1 receptor (PTH1R) also regulates bone cell survival and remodeling and has been associated with the hedgehog pathway during skeletal development. We hypothesize that primary cilia and PTH1R concomitantly regulate bone remodeling and cell survival and aim to describe the mechanisms that mediate these effects in osteocytes and osteoblasts. Colocalization of PTH1R with primary cilia was observed in control and PTHrP-stimulated MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells. Activation of PTH1R by PTHrP increased cell survival, osteoblast gene expression (osteocalcin, runt-related transcription factor 2, and bone alkaline phosphatase) and the expression of the hedgehog transcription factor Gli-1 in osteocytes and osteoblasts. These effects were abrogated by small interfering RNAs for the primary cilia protein IFT88 or by a primary cilia specific inhibitor (chloral hydrate). Preincubation of MLO-Y4 osteocytic and MC3T3-E1 osteoblastic cells with the Gli-1 antagonist GANT61 inhibited PTHrP prosurvival actions but did not affect PTHrP-induced overexpression of osteogenic genes. Mechanical stimulation by fluid flow increased the number of primary cilia-presenting cells in osteocytes and osteoblasts. We propose that PTH1R activation induces prosurvival actions via primary cilia-and Gli-1-dependent mechanism and modulates osteogenic responses via a primary cilia-dependent and Gli-1-independent pathway in osteocytes and osteoblasts.

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.

ResumenDiferentes estudios apoyan la idea de que la integridad del esqueleto depende de la percepción de estímulos mecánicos que promuevan una actividad celular encaminada al mantenimiento, reparación o adaptación de la morfología o estructura de este tejido. Esta revisión pretende abordar, desde un punto de vista general, cuáles son las células mecanosensibles en el entorno óseo, las estructuras biológicas que permiten a estas células percibir los estímulos físicos y cómo estos son transformados finalmente en señales biológicas que modulan la actividad celular, proceso conocido como mecanotransducción. Asumiendo la complejidad de las interacciones celulares que regulan el funcionamiento y la integridad del tejido óseo, es más que posible que el conocimiento de los mecanismos, tanto moleculares como celulares, que modulan la respuesta del hueso a las fuerzas mecánicas pueda resultar útil para entender la fisiología del esqueleto y la fisiopatología de las enfermedades esqueléticas, y contribuir al desarrollo de intervenciones que mejoren la resistencia ósea.

ResumenDiferentes estudios apoyan la idea de que la integridad del esqueleto depende de la percepción de estímulos mecánicos que promuevan una actividad celular encaminada al mantenimiento, reparación o adaptación de la morfología o estructura de este tejido. Esta revisión pretende abordar, desde un punto de vista general, cuáles son las células mecanosensibles en el entorno óseo, las estructuras biológicas que permiten a estas células percibir los estímulos físicos y cómo estos son transformados finalmente en señales biológicas que modulan la actividad celular, proceso conocido como mecanotransducción. Asumiendo la complejidad de las interacciones celulares que regulan el funcionamiento y la integridad del tejido óseo, es más que posible que el conocimiento de los mecanismos, tanto moleculares como celulares, que modulan la respuesta del hueso a las fuerzas mecánicas pueda resultar útil para entender la fisiología del esqueleto y la fisiopatología de las enfermedades esqueléticas, y contribuir al desarrollo de intervenciones que mejoren la resistencia ósea.

Objetives: Bone tissue can adapt to environmental stimuli by altering its morphology and metabolism. Different bone cells communicate with each other through communicating junctions (CJs). Connexin 43 (Cx43) is the most abundant CJ protein with key functions in signal transduction and in response to hormonal and mechanical stimuli. Another mechanosensor element of osteocytes is the primary cilium, formed by microtubules and which develops in the cell cycle's G0 phase. Our study aims to determine Cx43 and primary cilium involvement in osteocytic activity, to analyze the possible interaction between these two mechanosensors and to assess the role they play in the detection and response of osteocytes to mechanical stimuli and stimulation of the parathormone type 1 receptor (PTH1R) by its ligand, the parathormone-related protein (PTHrP) (1-36). Material and methods:The control MLO-Y4 (Cx43 +/+) osteocyte cell line was compared to Cx43-deficient MLO-Y4 (Cx43 -/-). The expression analysis of intraflagellar transport protein 88 (IFT88), Cx43 and phosphorylation of the extracellular signal regulatory kinase (P-ERK) was determined by Western blot. To characterize the possible colocalization between the primary cilium and Cx43, an immunofluorescence was carried out. To simulate mechanical stimulation in vitro, cells were subjected to mechanical stress of 10 dynes/cm2 by fluid flow for 10 minutes. Results:The results obtained show that the number of cells with primary cilium does not vary due to the expression of Cx43 (p = 0.089). In cells with Cx43 presence, mechanical stimulation by fluid flow and PTHrP increase the phosphorylation of extracellular signal-regulated kinases (ERK) compared to unstimulated cells (p = 0.049 and p = 0.011, respectively). Conclusions: The primary cilium and Cx43 act as mechanosensing elements for osteocytes. Deficiency in Cx43 does not influence ciliogenesis or activation by mechanical stimulation of pro-survival signaling pathways in osteocytes.