Introduction Osteoblasts are derived from mesenchymal progenitor cells and are the main functional cells for bone formation [[1], [2], [3]]. They are responsible for the synthesis, secretion and mineralization of bone matrix and can secrete various bioactive substances for bone formation and reconstruction [[1], [2], [3]]. In the pathogenesis of osteoporosis, uncontrolled reactive oxygen species (ROS) production and excessive oxidative injury will lead to significant damage and death of osteo

Abstract

Excessive oxidative stress will cause significant injury to osteoblasts, serving as one major pathological mechanism of osteoporosis. Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein and is expressed in the bone. We here explored its potential activity against hydrogen peroxide (H2O2)-induced oxidative injury in cultured osteoblasts. In primary murine and human osteoblasts, NLGN3 stimulation dose-dependently induced Akt, Erk1/2 and S6K activation. NLGN3 pretreatment ameliorated H2O2-induced cytotoxicity and death in osteoblasts. Moreover, H2O2-induced reactive oxygen species (ROS) production and oxidative injury were alleviated with NLGN3 pretreatment in cultured osteoblasts. Further studies showed that NLGN3 activated Nrf2 signaling cascade and induced Nrf2 protein Serine-40 phosphorylation, Keap1-Nrf2 dissociation, Nrf2 protein stabilization and nuclear translocation in osteoblasts. NLGN3 also increased antioxidant response element (ARE) activity and induced expression of Nrf2-ARE-dependent genes (HO1, GCLC and NQO1) in osteoblasts. Moreover NLGN3 mitigated osteoblast oxidative injury by dexamethasone or sodium fluoride (NaF). Nrf2 cascade activation is essential for NLGN3-induced cytoprotective activity in osteoblasts. Nrf2 shRNA or knockout (KO) abolished NLGN3-induced osteoblast cytoprotection against H2O2. Contrarily forced Nrf2 cascade activation by Keap1 KO mimicked NLGN3-induced anti-oxidative activity in murine osteoblasts. Importantly, NLGN3-induced Serine-40 phosphorylation and Nrf2 cascade activation were blocked by an Akt inhibitor MK-2206 or by Akt1 shRNA. Importantly, Akt inhibition, Akt1 silencing or Nrf2 S40T mutation largely inhibited NLGN3-induced osteoblast cytoprotection against H2O2. At last, we showed that NLGN3 mRNA and protein expression was significantly downregulated in necrotic bone tissues of dexamethasone-taken patients. Taken together, NLGN3 activated Akt-dependent Nrf2 cascade to protect osteoblasts from oxidative stress.

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Introduction

Osteoblasts are derived from mesenchymal progenitor cells and are the main functional cells for bone formation [[1], [2], [3]]. They are responsible for the synthesis, secretion and mineralization of bone matrix and can secrete various bioactive substances for bone formation and reconstruction [[1], [2], [3]]. In the pathogenesis of osteoporosis, uncontrolled reactive oxygen species (ROS) production and excessive oxidative injury will lead to significant damage and death of osteoblasts [[4], [5], [6]], causing bone mass reduction and microstructure disruption [[4], [5], [6]]. In in vitro experiment system, hydrogen peroxide (H2O2) or other oxidative stimuli were added to cultured osteoblasts/osteoblastic cells [[7], [8], [9], [10], [11], [12], [13]]. H2O2 treatment in osteoblasts/osteoblastic cells was shown to cause oxidative stress, protein denaturation, lipid peroxidation as well as DNA damage, eventually cell death and apoptosis [[8], [9], [10], [11],13].

Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein, essential for synapse development and function [14,15]. Its mutation is associated with autism [14]. Interestingly, early studies have shown that neuronal activity could enable neurons to secrete NLGN3, thereby promoting surrounding glioma cell growth and proliferation [[16], [17], [18], [19]]. NLGN3 was shown to phosphorylate multiple receptor tyrosine kinases (RTKs) and other receptors in glioma cells, activating downstream signaling cascades, including PI3K-Akt-mTOR and Erk-MAPK [[16], [17], [18], [19]]. Zelano et al., have shown that NLGN3 is expressed in the bone [20] and its expression was downregulated in spinal motoneurons after axotomy [20]. We here explored its potential activity against H2O2-induced oxidative injury in cultured osteoblasts.

One of the best characterized anti-oxidant cascades is nuclear-factor-E2-related factor 2 (Nrf2) [[21], [22], [23]]. Under the unstimulated basal condition, Nrf2 protein in the cytosol associates with its suppressor protein Kelch-1ike ECH- associated protein l (Keap1). The latter, together with Cullin 3 (Cul3), initiates Nrf2 proteasomal degradation through the ubiquitin system [[21], [22], [23]]. Activated Nrf2 protein, i.e. through phosphorylation, separates from Keap1. This will strop Nrf2 ubiquitin process and cause its stabilization [[21], [22], [23]]. The stabilized Nrf2 protein then enters nucleus and binds to anti-oxidant response element (ARE) along with small Maf proteins, promoting transcription and expression of over 250 Nrf2 cascade genes [[21], [22], [23]]. Proteins encoded by Nrf2-cascade genes are mostly antioxidant and cytoprotective proteins or detoxification enzymes, including ?-glutamyl cysteine ligase catalytic subunit (GCLC), the modified subunit (GCLM), NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase 1 (HO1) [[21], [22], [23]]. Activation of Nrf2 cascade, through genetic means or various pharmacological agents, can protect osteoblasts/osteoblastic cells from H2O2 [7,10,13,[24], [25], [26], [27]] and other oxidative stimuli [[28], [29], [30]]. The results of this study will show that NLGN3 activates Nrf2 cascade to protect osteoblasts from H2O2.