Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels

Bone Research ARTICLE www.nature.com/boneres OPEN Mechanotransduction via the coordinated actions of integrins, PI3K signaling and Connexin hemichannels 1234567890();,: Manuel A. Riquelme1, Sumin Gu1, Rui Hua1 and Jean X. Jiang 1 Mechanical loading opens connexin 43 (Cx43) hemichannels (HCs), leading to the release of bone anabolic molecules, such as prostaglandins, from mechanosensitive osteocytes, which is essential for bone formation and remodeling. However, the mechanotransduction mechanism that activates HCs remains elusive. Here, we report a unique pathway by which mechanical signals are effectively transferred between integrin molecules located in different regions of the cell, resulting in HC activation. Both integrin α5 and αV were activated upon mechanical stimulation via either fluid dropping or flow shear stress (FSS). Inhibition of integrin αV activation or ablation of integrin α5 prevented HC opening on the cell body when dendrites were mechanically stimulated, suggesting mechanical transmission from the dendritic integrin αV to α5 in the cell body during HC activation. In addition, HC function was compromised in vivo, as determined by utilizing an antibody blocking αV activation and α5-deficient osteocyte-specific knockout mice. Furthermore, inhibition of integrin αV activation, but not that of α5, attenuated activation of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway upon mechanical loading, and the inhibition of PI3K/AKT activation blocked integrin α5 activation and HC opening. Moreover, HC opening was blocked only by an anti-integrin αV antibody at low but not high FSS levels, suggesting that dendritic αV is a more sensitive mechanosensor than α5 for activating HCs. Together, these results reveal a new molecular mechanism of mechanotransduction involving the coordinated actions of integrins and PI3K/ AKT in osteocytic dendritic processes and cell bodies that leads to HC opening and the release of key bone anabolic factors. Bone Research (2021)9:8 ; https://doi.org/10.1038/s41413-020-00126-w INTRODUCTION Bone continuously undergoes remodeling, which helps to maintain the proper structure and organization of the tissue. Mechanical loading, induced by healthy physical activity, promotes bone formation, and remodeling in association with enhancement of bone mass and strength.1 Osteocytes, the most abundant bone cell type, are thought to be the most likely mechanosensory cells in bone. Osteocytes were recently suggested to be the main factor regulating bone remodeling by orchestrating the functions of other bone cells as well as the remodeling of the bone matrix and are a potential therapeutic target for the treatment of osteoporosis.2–4 Osteocytes are embedded inside the bone mineral matrix, and the long dendritic processes of osteocytes form a network among neighboring osteocytes and cells on the bone surface.5 Osteocyte cell bodies and processes are surrounded by a fluid-filled space, creating an extensive lacunae-canalicular network.6,7 Interstitial fluid flow driven by extravascular pressure is thought to be a major form of mechanical stimulation for osteocytes.8–11 The small molecules generated by mechanical loading are likely transmitted between cells through gap junctions and between the cell and the extracellular matrix through hemichannels (HCs), which constitute half of all gap junction channels.12 HCs, formed by hexameric connexin molecules,13 have been demonstrated to be active in osteocytes in response to mechanical stress and are associated with the release of physiologically relevant anabolic molecules, such as prostaglandin E2 (PGE2), to the external environment.14,15 Prostaglandins released by bone cells are suggested to be skeletal anabolic agents, as they can increase bone mass in animals.16–18 As shown in our earlier study, HC activity is adaptively regulated by the magnitude and duration of flow shear stress (FSS).19 Our recent study also suggested that impairment of osteocytic HCs has a negative impact on cortical bone structure, strength, and osteocyte viability.20 Using a transwell filter system to separate cell bodies from dendritic processes, we demonstrated that dendritic processes sense mechanical stimulation and transmit signals to the cell body to open HCs.21 We further found that integrin α5β1 interacts directly with Cx43 and that this interaction is important for the opening of HCs on the cell body in response to mechanical loading.22 Mechanical stimulation facilitates the opening of Cx43 HCs, likely through PI3K activation.23 In addition, morphological and functional studies suggest that dendritic processes serve as osteocytic mechanosensory sites;21,24,25 integrins associated with other extracellular components serve as “tethering elements,” which connect the processes with the canalicular wall and amplify mechanical signals.8,26 One study showed that integrin αVβ3, located at the dendritic process, is responsible for the mechanosensory responses of osteocytes.27 Nonetheless, it remains unclear how mechanical signals are transmitted from the extended, long dendritic process to the cell body to open Cx43 HCs. In this study, we unveil a new intracellular mechanotransduction pathway in mechanically sensitive osteocytes via which 1 Department of Biochemistry and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA Correspondence: Jean X. Jiang () Received: 8 February 2020 Revised: 25 September 2020 Accepted: 14 October 2020 © The Author(s) 2021 Integrins and PI3K/AKT in mechanotransduction MA Riquelme et al. integrin αVβ3 at dendritic processes senses shear stress, transmits the signal to the cell body by activating intracellular PI3K-AKT signaling, and activates α5β1. This mechanotransduction leads to the opening of Cx43 HCs, which play an essential role in mediating the anabolic function of mechanical loading on bone. RESULTS Activation of integrin αVβ3 at dendritic processes opens HCs on the osteocyte cell body through PI3K-AKT signaling Integrin αVβ3 has been implicated as part of a “tethering element” connecting the dendrites of osteocytes to the canaliculi wall.8,25,26,28 Immunofluorescence labeling with antibodies specific for integrin αV or β3 showed that these integrin subunits are located at osteocyte dendritic processes (Fig. 1a). These two integrin subunits are also colocalized, suggesting that they form a αVβ3 heterodimers. We previously reported that FSS activates integrin α5β1 on the osteocyte cell body and that this activation and direct interaction between α5β1 and Cx43 opens HCs.22 MLOY4 osteocytic cells were subjected to FSS, and untreated cells were used as controls. Activation of integrin α5β1 was assessed based on increased binding to a GST-FNIII9-11 fragment;22,29 activation of αVβ3 was evaluated by either increased binding to WOW1 Fab30 or decreased binding with an inhibitory antibody targeting inactive integrin αV31 (Fig (...truncated)