Ormeloxifene inhibits osteoclast differentiation in parallel to downregulating RANKL-induced ROS generation and suppressing the activation of ERK and JNK in murine RAW264.7 cells

Abstract

Ormeloxifene (Orm), a triphenylethylene compound, has been established as a selective estrogen receptor modulator (SERM) that suppresses the ovariectomy-induced bone resorption in rats. However, the precise mechanism underlying the bone-preserving action of Orm remains unclear. In this study, we evaluated the effect of Orm on osteoclast formation induced by receptor activator of nuclear factor kB ligand (RANKL) in the murine macrophage cell line RAW264.7. We also explored the mechanism of action of Orm by studying the RANKL-induced signaling pathways required for osteoclast differentiation. We found that Orm inhibited osteoclast formation from murine macrophage RAW264.7 cells induced by RANKL in a dose-dependent manner. Orm was able to abolish RANKL-induced reactive oxygen species (ROS) elevation and inhibited the transcriptional activation of two key RANKL-induced transcription factors namely activator protein-1 (AP-1) and NF-kB through mechanisms involving MAPKs. Activation of two MAPKs, i.e. ERK (MAPK1) and JNK (MAPK8), was alleviated by Orm effectively, which subsequently affected the activation of c-Jun and c-Fos, which are the essential components of the AP-1 transcription complex. Taken together, our results demonstrate that Orm potentially inhibits osteoclastogenesis by inhibiting ROS generation and thereby suppressing the activation of ERK1/2 (MAPK3/MAPK1) and JNK (MAPK8) and transcription factors (NF-kB and AP-1), which subsequently affect the regulation of osteoclastogenesis. These results provide a possible mechanism of action of Orm in regulating osteoclastogenesis, thereby supporting the beneficial bone-protective effects of this compound.

Activation of adenosine A2A receptor reduces osteoclast formation via PKA‐ and ERK1/2‐mediated suppression of NFκB nuclear translocation

“…The role of JNK in osteoclastogenesis has also previously been described (Cheng et al ., ; Kharkwal et al ., ). However, as with p38MAPK, intracellular signalling via this protein is secondary in our model; pJNK is activated at late time points (2–4 h) (see Supporting Information Figure S1B) and neither A 2A R activation/inhibition nor PKA silencing affected pJNK activation.…”

“…Of the three MAPKs studied here, only ERK1/2 is a target for adenosine A 2A receptor regulation. Although ERK1/2 plays a role in osteoclast survival (Miyazaki et al ., ) and differentiation (Saulnier et al ., ) our results remain controversial because prior reports document that different treatments such as bisphosphonates and statins (Tsubaki et al ., ) or ormeloxifene (Kharkwal et al ., ) exert their inhibitory action through inhibition of this MAPK. In contrast, we found that the A 2A R ligation both stimulated ERK 1/2 activation and inhibited osteoclast differentiation in a PKA‐dependent fashion.…”

Sitagliptin, An Anti-diabetic Drug, Suppresses Estrogen Deficiency-Induced OsteoporosisIn Vivo and Inhibits RANKL-Induced Osteoclast Formation and Bone Resorption In Vitro

“…Estrogen is essential to bone health for its antioxidant activity in osteoclasts and stimulating effects on osteoblasts (Kharkwal et al, 2012; Dou et al, 2016). Postmenopausal osteoporosis is a disease characterized by excessive bone destruction, which is a result of estrogen deficiency.…”

NLRP3 mediates osteolysis through inflammation‐dependent and ‐independent mechanisms

“…We find that NLRP3 assembles a functional inflammasome in WT and mutant pre‐OCs and OCs in the absence of proinflammatory LPS‐priming signals and exogenously added ATP. Although inflammasome‐activating signals may be dispensable for mutant cells, RANKL‐induced calcium fluxes, reactive oxygen species production, or mitochondrial alterations during OC differentiation are potential NLRP3 inflammasome activators in WT cells (26, 27). In addition, ATP and cellular debris from dying cells, including OCs, which have a short lifespan in cultures, may also activate this inflammasome.…”