Implicación de las conexinas, integrinas y cilio primario en la actividad de las células óseas / Involvement of connexins, integrins and primary cilium in the activity of bone cells

Introducción: los osteocitos son capaces de detectar diferentes señales, transducirlas en respuestas biológicas y trasmitirlasa los osteoblastos y osteoclastos, permitiendo el mantenimiento de la homeostasis ósea. La mecanotransducción ósea esposible gracias a que los osteocitos presentan diferentes estructuras mecanosensoras como las conexinas (Cx), las integrinas,el cilio primario e incluso receptores acoplados a proteínas G como el receptor de la parathormona tipo 1 (PTH1R).Objetivo: analizar la posible interacción de los diferentes elementos mecanosensores de los osteocitos y ver su influen-cia en la respuesta biológica.Material y métodos: se trabajó con las líneas celulares osteocíticas MLO-Y4 Cx43+/+ (scrambled (SCR) y ARNi α2) yCx43-/-.Resultados y conclusión: los resultados obtenidos muestran que la Cx43 y la integrina α2 se encuentran involucradas enel aumento de la longitud del cilio primario, afectando potencialmente a su funcionalidad como mecanosensor (SCR vs.ARNi α2, p < 0,0001 SCR vs. Cx43-/- y p < 0,0001 ARNi α2 vs. Cx43-/-). La integrina α2 también influyó en la localizacióncelular de Cx43 promoviendo que esta se encuentre en la membrana plasmática. También se observó que la activación dePTH1R por agonistas como parathormona (PTH) y proteína relacionada con la parathormona (PTHrP) inducen la fosforilaciónde la quinasa ERK 1/2, y estos efectos podrían verse afectados por la deficiencia en Cx43, pero no parecen ser mediadospor el silenciamiento de integrina α2. Finalmente, se observó que la presencia de la Cx43 y de integrina α2 en los osteoci-tos aumenta su capacidad de adhesión (Cx43+/+ SCR y ARNi α2 vs. CX43-/- p < 0,001 y p = 0,0039) y que la deficienciaen Cx43 provoca un incremento de la mortalidad de estas células (Cx43-/- vs. Cx43+/+ p = 0,0074).(AU)

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.

Primary cilia mediate parathyroid hormone receptor type 1 osteogenic actions in osteocytes and osteoblasts via Gli activation.

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.