Molecular mechanosensors in osteocytes

Bone Research REVIEW ARTICLE www.nature.com/boneres OPEN Molecular mechanosensors in osteocytes Lei Qin1, Wen Liu1, Huiling Cao1 and Guozhi Xiao1 1234567890();,: Osteocytes, the most abundant and long-lived cells in bone, are the master regulators of bone remodeling. In addition to their functions in endocrine regulation and calcium and phosphate metabolism, osteocytes are the major responsive cells in force adaptation due to mechanical stimulation. Mechanically induced bone formation and adaptation, disuse-induced bone loss and skeletal fragility are mediated by osteocytes, which sense local mechanical cues and respond to these cues in both direct and indirect ways. The mechanotransduction process in osteocytes is a complex but exquisite regulatory process between cells and their environment, between neighboring cells, and between different functional mechanosensors in individual cells. Over the past two decades, great efforts have focused on finding various mechanosensors in osteocytes that transmit extracellular mechanical signals into osteocytes and regulate responsive gene expression. The osteocyte cytoskeleton, dendritic processes, Integrin-based focal adhesions, connexin-based intercellular junctions, primary cilium, ion channels, and extracellular matrix are the major mechanosensors in osteocytes reported so far with evidence from both in vitro and in vitro studies. This review aims to give a systematic introduction to osteocyte mechanobiology, provide details of osteocyte mechanosensors, and discuss the roles of osteocyte mechanosensitive signaling pathways in the regulation of bone homeostasis. Bone Research (2020)8:23 ; https://doi.org/10.1038/s41413-020-0099-y INTRODUCTION Osteocytes are the most abundant and long-lived cell type in bone, accounting for 90%–95% of total bone cells in the adult skeleton.1 Although osteocytes are terminally differentiated cells derived from osteoblasts, bone contains ten times more osteocytes than osteoblasts.2 Over the last two to three decades, osteocytes, previously seen as a “passive placeholder” in mineralized bone, have emerged as a new multifunctional “superstar” in bone research.1 First, osteocytes are the master regulator of bone homeostasis through their direct regulation of local calcium abundance in mineralization and indirect control of osteoblast (bone-forming cell) and osteoclast (bone-resorbing cell) activities by the secretion of important regulatory factors.3–5 Second, osteocytes are endocrine cells that regulate phosphate metabolism in multiple organs, such as the kidney and parathyroid.1,6–8 Last, but the most importantly, osteocytes function as the principal regulators of bone mechanosensation and mechanotransduction.1,9–11 Mechanical stimuli induce and regulate various cellular functions, such as gene expression, protein synthesis, cell proliferation, and differentiation.12,13 Galileo was a pioneer who observed and described that in bone tissue “loading is required to preserve bone mass.”10 In 1892, the German surgeon Julius Wolff introduced his famous “Wolff’s Law,” stating that bone growth and remodeling occur in response to forces placed upon bone in a healthy person.10,14 In the 1980s, Harold Frost was the first to use the word “mechanostat” to describe the mechanism underlying this load-induced bone adaptation process and identify osteocytes as the “mechanostat” of bone.10,15 During mechanical stimulation from daily activities, whole-body mechanics are transduced to the organ level, tissue level, and finally, cellular level.16 In bone tissue, osteocytes have been suggested to be the main cell type responsive to mechanical stimulation.1,10,16 Direct evidence for the mechanosensitive function of osteocytes was revealed in a study showing that transgenic mice with specific osteocyte ablation failed to respond to unloading-induced bone loss.17 The mechanical environment in the mineralized extracellular matrix (ECM), in which osteocytes are embedded, presents a dynamic combination of various biophysical stimuli, including strain, stress, shear, osmotic pressure, fluid flow, streaming potentials, and acceleration.18 Among these stimuli, the shear stress of fluid flow from loading is the main force stimulation applied to osteocytes.9,16 The essential role of shear stress in osteocytes is determined by the natural physical environment of these cells, with osteocytes embedded in a lacuno-canalicular system (LCS) (Fig. 1). Transmission electron microscopy (TEM) analysis of fine murine bone sections revealed an average distance of 0.7 μm (0.1–2.0 μm) in the osteocyte lacuna, the space between the osteocyte cell body and mineralized ECM.19 A layer of collagen fibrils called the pericellular matrix (PCM), which is distinct from mineralized ECM, surrounds the osteocyte cell body in the lacuna. The PCM has a thickness of 0.5–1.0 μm and does not directly interact with the osteocyte cell surface, leaving a 50–80-nm space between cells and the PCM.20 In the osteocyte canaliculi, the canalicular diameter ranges from 210–260 nm.21,22 Moreover, collagen matrix projections from mineralized substrate form “hill-like” structures in osteocyte canaliculi that directly link the matrix and osteocyte dendrites. These structures are called “collagen hillocks”20 or “canalicular projections,”22 and an average internal space of 130 ± 40 nm exists between two projections.20 At the interface between collagen hillocks and osteocyte dendrites, Integrin-mediated focal 1 Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Shenzhen Key Laboratory of Cell Microenvironment, and School of Medicine, Southern University of Science and Technology, Shenzhen 518055, China Correspondence: Huiling Cao () or Guozhi Xiao () Received: 20 January 2020 Revised: 7 April 2020 Accepted: 17 April 2020 © The Author(s) 2020 Molecular mechanosensors in osteocytes L Qin et al. 2 a b c Gap junctions Focal adhesions Primary cilium Cx43 Integrins 130±40 nm MTs 480-550 nm IFs Collagen hillocks F-actin Cytoskeleton Piezo VSC III II IV Ion channels 50-80 nm 0.5-1.0 µm Peri-cellular matrix Fig. 1 Osteocytes in the LCS of the bone environment. a SEM image of acid-etched resin-embedded cortical bone sections reveals an ellipsoid cell shape and extensive canaliculi connections among osteocytes.8 b Magnified SEM image of a single osteocyte highlighted in the yellow square in a. c Illustration of osteocytes in the LCS of the bone environment. Magnified cartoon image of two adjacent osteocytes highlighted in the yellow square in a. The important aspects of osteocytes are highlighted in magnified cartoon images: focal adhesions, gap junctions, the primary cilium, cell cytoskeleton, ion channels, pericellular matrix at the lacunar region, and collagen hillocks at the canalicular region. [Panels a and b from Bonewald et al.,8 reprinted with permission] adhesions (FAs) link the cell membrane and matrix23 and further transmit (...truncated)