Glutathione, N-acetylcysteine and Lipoic Acid Down-Regulate Starvation-Induced Apoptosis, RANKL/OPG Ratio and Sclerostin in Osteocytes: Involvement of JNK and ERK1/2 Signalling

Abstract

Osteocyte apoptosis due to microdamage and/or oxidative stress is related to increased local bone turnover and resorption observed in various bone diseases. Previous data on osteoblasts and osteoclasts have linked reactive oxygen species and antioxidants to bone remodelling. This study performs a comprehensive analysis on the effect of antioxidants such as glutathione (GSH), N-acetylcysteine and lipoic acid (LA) on starvation-induced osteocyte apoptosis and on cytokines involved in bone remodelling such as the receptor activator kB ligand (RANKL), osteoprotegerin (OPG) and sclerostin. For this study, apoptosis was induced by serum starvation in a murine osteocyte-like cell line MLO-Y4; this condition mimics in part osteocyte apoptosis due to microdamage. The results show that starvation-induced apoptosis and expression of RANKL, OPG and sclerostin are redox regulated processes. All antioxidants are able to inhibit the apoptosis due to starvation. They down-regulate the expression and the release of RANKL, the expression of sclerostin and RANKL/OPG ratio, whereas they only in part up-regulate OPG expression. Antioxidants mediate their effect on starvation-induced apoptosis by JNK signalling and on cytokine expression by both JNK and ERK1/2 activities. This study shows the possible involvement of biological antioxidants such as GSH and LA on redox regulated mechanisms related to apoptosis and expression of cytokines involved in bone remodelling. Moreover, it suggests that both JNK and ERK1/2 may be useful biological targets for drugs affecting bone diseases associated with increased oxidative stress.

Oxidative stress in bone remodeling: role of antioxidants

“…In fact, ROS elicit a spectrum of responses ranging from proliferation, growth, differentiation arrest to cell death, by activating numerous signalling pathways. Indeed, mitogen-activated protein kinase (MAPKs) such as extracellular signal-regulated kinases (ERK1/2), c-Jun-N terminal kinase (JNK) and p38 MAPK are involved in osteoblast or osteocytes apoptosis (15,43,44). High levels of ROS block and reduce the osteoblast activity and differentiation, therefore the mineralization and osteogenesis (8,45,46) ( Figure 1).…”

“…These events increase bone remodeling turnover with consequent alteration and decrease in bone mass. Antioxidants have opposing effects, they contribute to the differentiation of osteoblasts and bone formation (8,15,34,47), maintaining vital osteocytes which contribute to osteoblast activity and osteogenesis, while reduce the osteoclast differentiation and their activity ( Figure 1). There are several factors mainly produced by osteoblasts and osteocytes that regulate osteoclast and osteoblast activity and then bone remodeling, among these the most important are: the ligand of receptor activator of NF-kB (RANKL) and osteoprotegerin (OPG).…”

“…Their expression is sensitive to increased oxidative status, that induces RANKL up-regulation and OPG down-regulation through the activation of protein kinases (ERK1/2, JNK etc.) and/or other factors which affect specific transcription factors (8,15,27). RANKL activates the differentiation and activity of osteoclasts by interacting with specific receptors in preostoeclasts and mediates osteoclastogenesis and bone resorption; while OPG, produced by the activation of the signaling pathway Wnt/βcatenin, is a soluble receptor capable of binding and blocking RANKL, resulting in inhibition of osteoclast activity (30-32, 42, 48) The oxidative stress blocks the activation of osteoblasts and thus the production of OPG; under this condition, the action of RANKL prevails, and the differentiation and activity of osteoclasts are induced.…”

“…The expression of RANKL and OPG is regulated by various hormones and cytokines, but a fundamental event in the initiation and regulation of the bone formation and remodeling is the apoptosis of osteoblasts and osteocytes (27,32). Experimental data show that excessive apoptosis of osteocytes is correlated to an increased oxidative status causing an imbalance in favor of osteoclastogenesis (15,(50)(51)(52). There are few data on the molecular mechanisms that regulate these processes, but many studies are currently focused on the regulatory activities of the osteocytes.…”

“…Cys is the most abundant low-molecular thiols in extracellular fluids with concentrations ranging from 40 μM to 8-10 μM (12), and DHLA contains two thiol groups and is produced in almost every cell in small amounts. In vivo Cys and DHLA act directly on ROS and RNS (Reactive Nitrogen Species) as scavengers and by regeneration of other antioxidants such as vitamin C, E and GSH (13)(14)(15). The physiological redox state is maintained in equilibrium by various factors and mechanisms that regulate the activity of ROS-producing enzymes and antioxidants.…”