acid-phosphatase and salubrinal

Inactivating bone-resorbing osteoclasts is a prime therapeutic strategy for the prevention of bone loss in patients with osteopenia and osteoporosis. Synthetic agents such as salubrinal and guanabenz, which attenuate stress to the endoplasmic reticulum, are reported to inhibit development of osteoclasts. However, the mechanism of their inhibitory action on osteoclasts is largely unknown. Using genome-wide expression profiles, we predicted key transcription factors that downregulated nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), a master transcription factor for osteoclastogenesis. Principal component analysis (PCA) predicted a list of transcription factors that were potentially responsible for reversing receptor activator of nuclear factor kappa-B ligand (RANKL)-driven stimulation of osteoclastogenesis. A partial silencing of NFATc1 allowed a selection of transcription factors that were likely to be located upstream of NFATc1. We validated the predicted transcription factors by focusing on two AP-1 transcription factors (c-Fos and JunB) using RAW264.7 pre-osteoclasts as well as primary bone marrow cells. As predicted, their mRNA and protein levels were elevated by RANKL, and the elevation was suppressed by salubrinal and guanabenz. A partial silencing of c-Fos or JunB by RNA interference decreased NFATc1 as well as tartrate-resistant acid phosphatase (TRAP) mRNA. Collectively, a systems-biology approach allows the prediction of a RANKL-salubrinal/guanabenz-NFATc1 regulatory axis, and in vitro assays validate an involvement of AP-1 transcription factors in suppression of osteoclastogenesis.

Topics: Acid Phosphatase; Animals; Bone Marrow Cells; Cell Differentiation; Cells, Cultured; Cinnamates; Down-Regulation; Guanabenz; Isoenzymes; Mice; NFATC Transcription Factors; Osteoblasts; Osteogenesis; Principal Component Analysis; Proto-Oncogene Proteins c-fos; RANK Ligand; RNA Interference; RNA, Messenger; RNA, Small Interfering; Tartrate-Resistant Acid Phosphatase; Thiourea; Transcription Factor AP-1; Transcription Factors

In response to various stresses including viral infection, nutrient deprivation, and stress to the endoplasmic reticulum, eukaryotic translation initiation factor 2 alpha (eIF2α) is phosphorylated to cope with stress induced apoptosis. Although bone cells are sensitive to environmental stresses that alter the phosphorylation level of eIF2α, little is known about the role of eIF2α mediated signaling during the development of bone-resorbing osteoclasts. Using two chemical agents (salubrinal and guanabenz) that selectively inhibit de-phosphorylation of eIF2α, we evaluated the effects of phosphorylation of eIF2α on osteoclastogenesis of RAW264.7 pre-osteoclasts as well as development of MC3T3 E1 osteoblast-like cells. The result showed that salubrinal and guanabenz stimulated matrix deposition of osteoblasts through upregulation of activating transcription factor 4 (ATF4). The result also revealed that these agents reduced expression of the nuclear factor of activated T cells c1 (NFATc1) and inhibited differentiation of RAW264.7 cells to multi-nucleated osteoclasts. Partial silencing of eIF2α with RNA interference reduced suppression of salubrinal/guanabenz-driven downregulation of NFATc1. Collectively, we demonstrated that the elevated phosphorylation level of eIF2α not only stimulates osteoblastogenesis but also inhibit osteoclastogenesis through regulation of ATF4 and NFATc1. The results suggest that eIF2α-mediated signaling might provide a novel therapeutic target for preventing bone loss in osteoporosis.

Topics: Acid Phosphatase; Activating Transcription Factor 4; Animals; Cells, Cultured; Cinnamates; Down-Regulation; Eukaryotic Initiation Factor-2; Genes, fos; Guanabenz; Isoenzymes; Mice; NFATC Transcription Factors; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteoporosis; Phosphorylation; Receptors, Cell Surface; RNA, Messenger; Tartrate-Resistant Acid Phosphatase; Thiourea