Abstract
CSF-1 is a key factor in regulating bone remodeling; osteocytes express CSF-1 and its receptor. Viable osteocytes are essential for bone remodeling through cell-cell contact and secretion of factors that regulate osteoblasts and osteoclasts. Increased oxidative stress contributes to osteocyte death and correlates with bone loss during aging. The NADPH oxidase Nox4 is a major source of ROS in bone. CSF-1 decreases Nox4, suggesting that CSF-1 protects against oxidative stress. Here, we show that osteocyte apoptosis previously reported in our global CSF-1KO mice is associated with increased Nox4, as well as 4-HNE expression in osteocytes. Osteocytes isolated from CSF-1KO mice were less viable and showed increased intracellular ROS, elevated NADPH oxidase activity/Nox4 protein, activation of mTOR/S6K, and downstream apoptosis signals compared with WT osteocytes. Nox4 expression was also increased in CSF-1KO osteocytes and colocalized with MitoTracker Red in mitochondria. Notably, CSF-1 inhibited Nox4 expression and apoptosis cascade signals. In additional studies, shNox4 decreased these signals in CSF-1KO osteocytes, whereas overexpression of Nox4 in WT osteocytes activated the apoptosis pathway. To determine the role of CSF-1 in osteocytes, DMP1Cre-CSF-1cKO (CSF-1cKO) mice that lack CSF-1 in osteocytes/late osteoblasts were developed. Osteocyte defects in CSF-1cKO mice overlapped with those in CSF-1KO mice, including increased apoptosis, Nox4, and 4-HNE-expressing osteocytes. CSF-1cKO mice showed unbalanced cancellous bone remodeling with decreased bone formation and resorption. Continued exposure to high Nox4/ROS levels may further compromise bone formation and predispose to bone loss and skeletal fragility. Taken together, our findings suggest a novel link between CSF-1, Nox4-derived ROS, and osteocyte survival/function that is crucial for osteocyte-mediated bone remodeling. Results reveal new mechanisms by which CSF-1/oxidative stress regulate osteocyte homeostasis, which may lead to therapeutic strategies to improve skeletal health in aging. © 2018 American Society for Bone and Mineral Research.
Highlights
CSF-1 is a key factor that regulates mononuclear phagocytes in tissues undergoing active morphogenesis and remodeling
It is ubiquitously expressed in many cell types including osteocytes and osteoblasts, and is a constitutively active oxidase.[15,16,17,18,19] We showed that CSF-1 decreases Nox4 in cultured normal osteoblasts, suggesting that CSF-1 protects against oxidative stress.[20] little is known regarding the mechanisms by which CSF-1/oxidative stress regulates osteocyte homeostasis in skeletal tissues
CSF-1KO cells showed a similar osteocyte phenotype, except dentin matrix protein 1 (DMP1) was decreased as previously described in vivo.[8]. The CSF-1 transcript was absent in CSF-1KO osteocytes indicating an effective deletion of the CSF-1 gene
Summary
CSF-1 is a key factor that regulates mononuclear phagocytes in tissues undergoing active morphogenesis and remodeling. Previous studies have shown that CSF-1 protects against ischemic- or oxidant-induced cell injury and decreases oxidative stress.[12,13,14] Exposure of CSF-1-deficient mice to cerebral ischemia increased neuronal cell death, whereas CSF-1 administration improved cell survival.[12] Selective deletion of CSF-1 in the renal proximal tubule of transgenic mice, subjected to acute kidney injury, increased oxidative stress and tubulointerstitial fibrosis, whereas kidneys of WT controls were protected from these effects.[14] In bone, the NADPH oxidase Nox is a major source of ROS, including H2O2 and superoxide (O-2) It is ubiquitously expressed in many cell types including osteocytes and osteoblasts, and is a constitutively active oxidase.[15,16,17,18,19] We showed that CSF-1 decreases Nox in cultured normal osteoblasts, suggesting that CSF-1 protects against oxidative stress.[20] little is known regarding the mechanisms by which CSF-1/oxidative stress regulates osteocyte homeostasis in skeletal tissues. We hypothesized that CSF-1 deficiency in osteocytes increases Nox expression and oxidative stress, which impairs osteocyte survival/function and leads to altered bone remodeling
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