Expression and functional proteomic analyses of osteocytes from Xenopus laevis tested under mechanical stress conditions: preliminary observations on an appropriate new animal model

Abstract

Hitherto, the role of the osteocyte as transducer of mechanical stimuli into biological signals is far from settled. In this study, we used an appropriate model represented by the cortex of Xenopus laevis long bone diaphysis lacking (unlike the mammalian one) of vascular structures and containing only osteocytes inside the bone matrix. These structural features allow any change of protein profile that might be observed upon different experimental conditions, such as bone adaptation to stress/mechanical loading, to be ascribed specifically to osteocytes. The study was conducted by combining ultrastructural observations and two-dimensional electrophoresis for proteomic analysis. The osteocyte population was extracted from long bones of lower limbs of amphibian skeletons after different protocols (free and forced swimming). The experiments were performed on 210 frogs subdivided into five trials, each including free swimming frogs (controls) and frogs submitted to forced swimming (stressed). The stressed groups were obliged to swim (on movable spheres covering the bottom of a pool on a vibrating plate) continuously for 8 h, and killed 24 h later along with the control groups. Long bones free of soft tissues (periosteum, endosteum and bone marrow), as well as muscles of posterior limbs, were processed and analyzed for proteins differentially expressed or phosphorylated between the two sample groups. The comparative analysis showed that protein phosphorylation profiles differ between control and stressed groups. In particular, we found in long bones of stressed samples that both Erk1/2 and Akt are hyperphosphorylated; moreover, the different phosphorylation of putative Akt substrates (recognized by specific Akt phosphosubstrates-antibody) in stressed vs. control samples clearly demonstrated that Akt signaling is boosted by forced swimming (leading to an increase of mechanical stress) of amphibian long bones. In parallel, we found in posterior limb muscles that the expression of heat shock protein HSP27 and HSP70 stress markers increased upon the forced swimming condition. Because the cortexes of frog long bones are characterized by the presence of only osteocytes, all our results establish the suitability of the X. laevis animal model to study the bone response to stress conditions mediated by this cell type and pave the way for further analysis of the signaling pathways involved in these signal transduction mechanisms.

WISP-2 expression induced by Teriparatide treatment affects in vitro osteoblast differentiation and improves in vivo osteogenesis

“…As proposed by many authors (R. A. Dodds et al, 1993;Marotti and Palumbo, 2007;Skerry et al, 1989a,b) and largely demonstrated by Bonewald (2011) and Nakashima (Nakashima et al, 2011) osteocytes are considered the mechanosensors of bone (Bertacchini et al, 2017;Maycas et al, 2015;Nakashima et al, 2012) and are actively involved in orchestrating the function of both bone-forming osteoblasts and bone-resorbing osteoclasts. However, the role of osteocytes in osteoblast regulation, under both physiologic and pathologic conditions, is not fully characterized.…”

Osteocyte pericellular and perilacunar matrices as markers of bone–implant mechanical integrity

“…This porous network allows for the transport of interstitial fluid from the vascular canals to the cells. Within this porous network, the cells are surrounded by a glycoproteic pericellular matrix less than 100 nm in thickness (PCM) (You et al, 2004), mainly made of perlecan (Thompson et al, 2011), and are attached to the wall of the lacuna through tethering perlecan fibers (Bertacchini et al, 2017;McNamara et al, 2009) with an average spacing of 40 nm (You et al, 2004). This leaves a space, the pericellular space, between the PCM and the lacunar wall, where interstitial fluids can flow from the vascular canals to the osteocytes.…”

The Osteocyte: From “Prisoner” to “Orchestrator”

“…As regards the transmission of mechanical signals, both recent and less recent literature indicates the osteocyte as the main strain-sensitive bone cell [ 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ]. We have shown [ 42 , 43 ] that shear stress-activated osteocytes are capable of steadily increasing and maintaining the basal current produced by the ionic fluxes (streaming potentials), which occur inside the lacuno-canalicular microcavities in response to pulsing mechanical loading ( Figure 8 ).…”

“…Recently [ 58 ], as far as the bone adaptation-related cell-signaling is concerned, both Erk1/2 and Akt were showed to be hyperphosphorylated in frog long bones of stressed samples (forced swimming) suggesting that among the putative osteocyte signal transduction mechanisms, Akt signaling is boosted by increased mechanical stresses. Moreover, the authors confirmed again that the increase of osteocyte gap junction number is dependent on mechanical loading ( Figure 10 ).…”