Involvement of strawberry notch homologue 1 in neurite outgrowth of cortical neurons.

When the regulation of axonal and dendritic growth is altered, the neuronal network becomes disordered, which may contribute to the development of psychiatric disorders. Some genome analyses have suggested relationships between mutations in strawberry notch homologue 1 (SBNO1) and neurodevelopmental disorders. However, the function of SBNO1 has not yet been reported. Here, SBNO1 expression pattern during the development of the cerebral cortex in mice was examined. SBNO1 was strongly expressed in the cortical plate and its expression was maintained at a low level during the postnatal stage. CRISPR/Cas9-based knockout of Sbno1 in Neuro2A cultured cells showed delayed growth of neurites. A cortical neuron-specific conditional knockout mouse was constructed, which resulted in hypotrophy of axon bundles and dendrites in cortical neurons. Thus, when mutated, SBNO1 is a candidate gene for psychiatric diseases, such as schizophrenia, as suggested by human genome studies.

Peucedanum japonicum Thunb. ethanol extract suppresses RANKL-mediated osteoclastogenesis.

The constituents of Peucedanum japonicum Thunb. (PJ) exhibit biological and pharmacological activities, including anti-obesity, anti-oxidant and anti-allergic activities. The aim of the present study was to examine in vitro effects of PJ in RANKL-induced signaling pathways, which determine osteoclast differentiation. PJ ethanol extract (PEE) exhibited anti-osteoporotic activity by disrupting the phospholipase C (PLC)-Ca2+-c-Fos/cAMP response element-binding protein (CREB)-nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway during osteoclastogenesis. Murine bone marrow-derived macrophages (BMMs) were cultured and used to determine the effects of PJ in the receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclastogenesis. The effects of PEE in the RANKL-mediated signaling cascade were evaluated using a standard in vitro osteoclastogenesis system. PEE treatment of BMMs significantly reduced the number of RANKL-mediated tartrate resistant acid phosphatase (TRAP)-positive multinucleated cells (P<0.05 for 5 and 10 µg/ml PEE, P<0.01 for 25 and 50 µg/ml PEE), without cytotoxic effects. Furthermore, the expression of differentiation-related marker genes, including TRAP, Oscar, Cathepsin K, dendrocyte expressed seven transmembrane protein, ATPase H+ Transporting V0 Subunit D2 and NFATc1, were markedly suppressed. PEE induced a transient increase in free cytoplasmic Ca2+ ([Ca2+]i) mobilization via voltage-gated Ca2+ channels and PLC-sensitive pathways. Transient [Ca2+]i increase consequently resulted in the suppression of c-Fos, CREB and NFATc1 activities. These findings highlight the potential use of PJ in treating bone disorders caused by osteoclast overgrowth.

Lysosomal Ca2+ Signaling is Essential for Osteoclastogenesis and Bone Remodeling.

Lysosomal Ca2+ emerges as a critical component of receptor-evoked Ca2+ signaling and plays a crucial role in many lysosomal and physiological functions. Lysosomal Ca2+ release is mediated by the transient receptor potential (TRP) family member TRPML1, mutations that cause the lysosomal storage disease mucolipidosis type 4. Lysosomes play a key role in osteoclast function. However, nothing is known about the role of lysosomal Ca2+ signaling in osteoclastogenesis and bone metabolism. In this study, we addressed this knowledge gap by studying the role of lysosomal Ca2+ signaling in osteoclastogenesis, osteoclast and osteoblast functions, and bone homeostasis in vivo. We manipulated lysosomal Ca2+ signaling by acute knockdown of TRPML1, deletion of TRPML1 in mice, pharmacological inhibition of lysosomal Ca2+ influx, and depletion of lysosomal Ca2+ storage using the TRPML agonist ML-SA1. We found that knockdown and deletion of TRPML1, although it did not have an apparent effect on osteoblast differentiation and bone formation, markedly attenuated osteoclast function, RANKL-induced cytosolic Ca2+ oscillations, inhibited activation of NFATc1 and osteoclastogenesis-controlling genes, suppressed the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and markedly reduced the differentiation of bone marrow-derived macrophages into osteoclasts. Moreover, deletion of TRPML1 resulted in enlarged lysosomes, inhibition of lysosomal secretion, and attenuated the resorptive activity of mature osteoclasts. Notably, depletion of lysosomal Ca2+ with ML-SA1 similarly abrogated RANKL-induced Ca2+ oscillations and MNC formation. Deletion of TRPML1 in mice reduced the TRAP-positive bone surfaces and impaired bone remodeling, resulting in prominent osteopetrosis. These findings demonstrate the essential role of lysosomal Ca2+ signaling in osteoclast differentiation and mature osteoclast function, which play key roles in bone homeostasis. © 2016 American Society for Bone and Mineral Research.

WHI-131 Promotes Osteoblast Differentiation and Prevents Osteoclast Formation and Resorption in Mice.

The small molecule WHI-131 is a potent therapeutic agent with anti-inflammatory, antiallergic, and antileukemic potential. However, the regulatory effects of WHI-131 on osteoblast and osteoclast activity are unclear. We examined the effects of WHI-131 on osteoblast and osteoclast differentiation with respect to bone remodeling. The production of receptor activator of nuclear factor kappa-B ligand (RANKL) by osteoblasts in response to interleukin (IL)-1 or IL-6 stimulation decreased by 56.8% or 50.58%, respectively, in the presence of WHI-131. WHI-131 also abrogated the formation of mature osteoclasts induced by IL-1 or IL-6 stimulation. Moreover, WHI-131 treatment decreased RANKL-induced osteoclast differentiation of bone marrow-derived macrophages, and reduced the resorbing activity of mature osteoclasts. WHI-131 further decreased the mRNA and protein expression levels of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) by almost twofold, and significantly downregulated the mRNA expression of the following genes: tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), DC-STAMP, OC-STAMP, ATP6v0d2, and cathepsin K (CtsK) compared with the control group. WHI-131 further suppressed the phosphorylation of protein kinase B (Akt) and degradation of inhibitor of kappa B (IκB); Ca(2+) oscillation was also affected, and phosphorylation of the C-terminal Src kinase (c-Src)-Bruton agammaglobulinemia tyrosine kinase (Btk)-phospholipase C gamma 2 (PLCγ2) (c-Src-Btk-PLCg2 calcium signaling pathway) was inhibited following WHI-131 treatment. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway was activated by WHI-131, accompanied by phosphorylation of STAT3 Ser727 and dephosphorylation of STAT6. In osteoblasts, WHI-131 caused an approximately fourfold increase in alkaline phosphatase activity and Alizarin Red staining intensity. Treatment with WHI-131 increased the mRNA expression levels of genes related to osteoblast differentiation, and induced the phosphorylation of Akt, p38, and Smad1/5/8. Furthermore, 5-week-old ICR mice treated with WHI-131 exhibited antiresorbing effects in a lipopolysaccharide-induced calvaria bone loss model in vivo and increased bone-forming activity in a calvarial bone formation model. Therefore, the results of this study show that WHI-131 plays a dual role by inhibiting osteoclast differentiation and promoting osteoblast differentiation. Thus, WHI-131 could be a useful pharmacological agent to treat osteoporosis by promoting bone growth and inhibiting resorption.

Harpagoside Inhibits RANKL-Induced Osteoclastogenesis via Syk-Btk-PLCγ2-Ca(2+) Signaling Pathway and Prevents Inflammation-Mediated Bone Loss.

Harpagoside (HAR) is a natural compound isolated from Harpagophytum procumbens (devil's claw) that is reported to have anti-inflammatory effects; however, these effects have not been investigated in the context of bone development. The current study describes for the first time that HAR inhibits receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in vitro and suppresses inflammation-induced bone loss in a mouse model. HAR also inhibited the formation of osteoclasts from mouse bone marrow macrophages (BMMs) in a dose-dependent manner as well as the activity of mature osteoclasts, including filamentous actin (F-actin) ring formation and bone matrix breakdown. This involved a HAR-induced decrease in extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) phosphorylation, leading to the inhibition of Syk-Btk-PLCγ2-Ca(2+) in RANKL-dependent early signaling, as well as the activation of c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1), which resulted in the down-regulation of various target genes. Consistent with these in vitro results, HAR blocked lipopolysaccharide (LPS)-induced bone loss in an inflammatory osteoporosis model. However, HAR did not prevent ovariectomy-mediated bone erosion in a postmenopausal osteoporosis model. These results suggest that HAR is a valuable agent against inflammation-related bone disorders but not osteoporosis induced by hormonal abnormalities.

Attenuated RANKL-induced cytotoxicity by Portulaca oleracea ethanol extract enhances RANKL-mediated osteoclastogenesis.

BACKGROUND: Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. METHODS: In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca(2+) mobilization, nuclear factor of activated T-cell c1 (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. RESULTS: Our results demonstrated that POEE suppressed RANKL-induced Ca(2+) oscillations by inhibition of Ca(2+) release from internal Ca(2+) stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. CONCLUSIONS: These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis.

Anti-osteoclastogenic activity of praeruptorin A via inhibition of p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca2+ oscillation.

BACKGROUND: A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet. MATERIALS AND METHODS: The effect of praeruptorin A on the differentiation of bone marrow-derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca(2+) levels were also measured. RESULTS: Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca(2+) oscillation without any changes in the phosphorylation of PLCγ. CONCLUSION: Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCγ-independent Ca(2+) oscillation.

Oleanolic acid acetate inhibits osteoclast differentiation by downregulating PLCγ2-Ca(2+)-NFATc1 signaling, and suppresses bone loss in mice.

Owing to their potential pharmacological activities in human disease, natural plant-derived compounds have recently become the focus of increased research interest. In this study, we first isolated oleanolic acid acetate (OAA), a triterpenoid compound, from Vigna angularis (azuki bean) to discover anti-bone resorptive agents. Many studies have identified and described the various medicinal effects of V. angularis extract. However, the pharmacological effect of OAA-derived V. angularis extract, particularly the effect on osteoclastogenesis, is not known. Therefore, we investigated the effect and mechanism of OAA in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. OAA inhibited RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. Interestingly, OAA significantly inhibited Btk phosphorylation, phospholipase Cγ2 (PLCγ2) phosphorylation, calcium ion (Ca(2+)) oscillation, and nuclear factor of activated T cell c1 (NFATc1) expression in RANKL-stimulated BMMs, but did not affect RANKL-induced mitogen-activated protein kinase. OAA also inhibited the bone-resorbing activity of mature osteoclasts. Furthermore, mice treated with OAA demonstrated marked attenuation of lipopolysaccharide-induced bone erosion based on micro-computed tomography and histologic analysis of femurs. Taken together, the results suggested that OAA inhibited RANKL-mediated osteoclastogenesis via PLCγ2-Ca(2+)-NFATc1 signaling in vitro and suppressed inflammatory bone loss in vivo.

Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation.

Chrysanthemum zawadskii Herbich var. latilobum Kitamura, known as "Gujulcho" in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects of C. zawadskii Herbich var. latilobum Kitamura ethanol extract (CZE) on osteoclast differentiation induced by treatment with the receptor activator of NF- κ B ligand (RANKL). CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca(2+)-oscillation via the inactivation of PLC γ 2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activation in vitro and in vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

Treatment of eggshell with casein phosphopeptide reduces the severity of ovariectomy-induced bone loss.

It has been generally accepted that calcium intake prevents bone loss, and frequent fracture resulted from osteoporosis. However, it is still elusive as to how effective sole calcium intake is in preventing or attenuating the severity of osteoporosis. Here, we demonstrate the effects of eggshell-casein phosphopeptide (ES-CPP), and compared these effects those of calcium supplement, for restoring ovariectomy-mediated bone loss. CPP, synthesized from the hydrolysis of casein (0.5%) using trypsin, was added to the grinded ES and was then administered to the ovariectomized (OVX) rat at 100 mg/kg for 4 weeks. Urine and feces from each group were collected each day, and were used to calculate the apparent calcium absorption rate in a day. After 4 weeks incubation, blood and femoral bones were isolated for the analysis of parameters representing osteoporosis. The apparent calcium absorption rate was significantly increased in the ES-CPP treated groups, in comparison to both the OVX and the commercial calcium supplement (CCS) treated group. Notably, treatment with ES-CPP markedly enhanced the calcium content in femoral bone and the relative weight of femoral bone to body weight, though calcium content in serum was barely changed by treatment with ES-CPP. Parameters of osteoporosis, such as osteocalcin in serum and bone mineral density, were rescued by treatment with ES-CPP, compared to treatment with commercial calcium supplement. This finding strongly suggests the possible use of ES-CPP in preventing or attenuating the severity of postmenopausal osteoporosis.