Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling.

The mevalonate pathway plays a crucial role in bone metabolism. Here we examined roles of simvastatin in osteoclast function and differentiation induced by RANKL and BMP-2 using mouse macrophage-like MLC-6 cells and human osteoclast precursor cells. MLC-6 cells expressed BMP type-I and -II receptors and Smads as well as osteoclast markers including TRAP, RANK, cathepsin-K, M-CSF receptor, MMP-9 and calcitonin receptor. Treatment with RANKL and BMP-2 acted synergistically to stimulate RANK, TRAP and cathepsin-K expression in MLC-6 cells. Simvastatin suppressed osteoclastic activity shown by increases in RANK, TRAP and cathepsin-K expression induced by RANKL and BMP-2. In contrast simvastatin alone had no effects on the osteoclastic markers in MLC-6 cells. Simvastatin activated ERK, SAPK/JNK and AKT pathways and inactivated Ras in MLC-6 cells. Simvastatin had no effect on BMP-induced Smad1/5/8 phosphorylation regardless of RANKL stimulation. Since chemical inhibition of ERK, SAPK/JNK and AKT increased TRAP and cathepsin-K expression induced by BMP-2 and RANKL, these pathways are functionally involved in inhibition of osteoclastic activity. In addition, Src phosphorylation induced by RANKL, which is involved in osteoclast differentiation, was suppressed by simvastatin. We further confirmed an inhibitory mechanism of simvastatin on osteoclast differentiation using human osteoclast precursor cells which express BMP receptor and Smad signaling machinery. Simvastatin also activated ERK pathways and inactivated Src phosphorylation in human osteoclasts differentiated by M-CSF and RANKL treatments. The inhibition of TRAP and RANK expression by simvastatin was reversed by ERK inhibition, whereas Src inhibitor enhanced simvastatin-induced suppression of osteoclast markers. Collectively, our data show that simvastatin inhibits osteoclastic differentiation through inhibiting Src as well as enhancing MAPK/AKT pathways.

Yamashita M ，Otsuka F ，Mukai T ，Yamanaka R ，Otani H ，Matsumoto Y ，Nakamura E ，Takano M ，Sada KE ，Makino H ... -  《-》

Simvastatin antagonizes tumor necrosis factor-alpha inhibition of bone morphogenetic proteins-2-induced osteoblast differentiation by regulating Smad signaling and Ras/Rho-mitogen-activated protein kinase pathway.

Yamashita M ，Otsuka F ，Mukai T ，Otani H ，Inagaki K ，Miyoshi T ，Goto J ，Yamamura M ，Makino H ... -  《-》

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling.

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up-regulated by receptor activator of nuclear factor-kappaB ligand (RANKL) during osteoclast differentiation. EGFR-specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL-dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL-mediated activation of osteoclastogenic signaling pathways, including c-Jun N-terminal kinase (JNK), NF-kappaB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide-3 kinase (PI3K)-Akt/PKB pathway and subsequent activation of BAD and caspases-9 and -3 may be responsible for the EGFR inhibition-induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor-kappaB (RANK) and Grb2-associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL-activated signaling pathways by cross-talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co-receptor in osteoclast differentiation and survival.

Yi T ，Lee HL ，Cha JH ，Ko SI ，Kim HJ ，Shin HI ，Woo KM ，Ryoo HM ，Kim GS ，Baek JH ... -  《-》

BAPTA-AM, an intracellular calcium chelator, inhibits RANKL-induced bone marrow macrophages differentiation through MEK/ERK, p38 MAPK and Akt, but not JNK pathways.

To examine the roles of intracellular calcium in RANKL-induced bone marrow macrophages (BMMs) differentiation, the effects of intracellular calcium chelator BAPTA-AM on RANKL-induced BMMs differentiation, and the activation of its relating signal proteins (MAPKs, and the PI3K/Akt) were studied. BMMs were cultured with various concentrations of BAPTA-AM in the presence of M-CSF (25 ng/ml) and RANKL (25 ng/ml) for 7 days, osteoclastogenic ability, cytosolic free Ca(2+) concentration, osteoclast survival and the expression of phosphorylated ERK1/2, SAPK/JNK, Akt and p38 MAPK were measured by TRAP staining, spectrofluorometer and Western blotting. BAPTA-AM inhibited osteoclastogenesis and osteoclast survival of BMMs by RANKL induction. In osteoclasts without the pretreatment of BAPTA-AM, the increased response of [Ca(2+)](i) was observed within 15 min and the maximum was about 1.2 times that of control. This response was sustained for 30 min and returned to the control level at 1h after RANKL-inducing, and the increased response of [Ca(2+)](i) was completely abolished and sustained to at least 8h by BAPTA-AM. Although immunoblotting data revealed that RANKL could activate the phosphorylation of ERK1/2, SAPK/JNK, Akt and p38 MAPK, the expression of ERK1/2, Akt and p38 MAPK phosphorylation was inhibited by BAPTA-AM dose-dependently. These results revealed that BAPTA-AM inhibit osteoclastogenic ability of BMMs via suppressing the increase of [Ca(2+)](i) which lead to inhibit RANKL-induced the phosphorylation of ERK, Akt and p38 MAPK, but not JNK. This finding may be useful in the development of an osteoclastic inhibitor that targets intracellular signaling factors.

Zhou S ，Yuan X ，Liu Q ，Zhang X ，Pan X ，Zang L ，Xu L ... -  《-》

Receptor activator of NF-kappaB ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells.

Fujisaki K ，Tanabe N ，Suzuki N ，Kawato T ，Takeichi O ，Tsuzukibashi O ，Makimura M ，Ito K ，Maeno M ... -  《LIFE SCIENCES》