Fujio Okamoto

Selective inhibition of NF-kappa B blocks osteoclastogenesis and prevents inflammatory bone destruction in vivo.

Bone destruction is a pathological hallmark of several chronic inflammatory diseases, including rheumatoid arthritis and periodontitis. Inflammation-induced bone loss of this sort results from elevated numbers of bone-resorbing osteoclasts. Gene targeting studies have shown that the transcription factor nuclear factor-κB (NF-κB) has a crucial role in osteoclast differentiation, and blocking NF-κB is a potential strategy for preventing inflammatory bone resorption. We tested this approach using a cell-permeable peptide inhibitor of the IκB-kinase complex, a crucial component of signal transduction pathways to NF-κB. The peptide inhibited RANKL-stimulated NF-κB activation and osteoclastogenesis both in vitro and in vivo. In addition, this peptide significantly reduced the severity of collagen-induced arthritis in mice by reducing levels of tumor necrosis factor-α and interleukin-1β, abrogating joint swelling and reducing destruction of bone and cartilage. Therefore, selective inhibition of NF-κB activation offers an effective therapeutic approach for inhibiting chronic inflammatory diseases involving bone resorption.

The Association of Notch2 and NF-κB Accelerates RANKL-Induced Osteoclastogenesis

ABSTRACT Notch signaling plays a key role in various cell differentiation processes including bone homeostasis. However, the specific involvement of Notch in regulating osteoclastogenesis is still controversial. In the present study, we show that RANKL induces expression of Jagged1 and Notch2 in bone marrow macrophages during osteoclast differentiation. Suppression of Notch signaling by a selective γ-secretase inhibitor or Notch2 short hairpin RNA suppresses RANKL-induced osteoclastogenesis. In contrast, induction of Notch signaling by Jagged1 or by ectopic expression of intracellular Notch2 enhances NFATc1 promoter activity and expression and promotes osteoclastogenesis. Finally, we found that Notch2 and p65 interact in the nuclei of RANKL-stimulated cells and that both proteins are recruited to the NFATc1 promoter, driving its expression. Taken together, our results show a new molecular cross talk between Notch and NF-κB pathways that is relevant in osteoclastogenesis.

Protein tyrosine kinase inhibitors increase cytosolic calcium and inhibit actin organization as resorbing activity in rat osteoclasts

Although there is evidence that protein tyrosine kinase inhibitors (PTKIs) suppress bone resorption activity, the mechanism of action of these compounds on osteoclastic bone resorption remains obscure. In the present study, we investigated the effect of PTKIs on cytosolic Ca2+ concentration ([Ca2+]i) and on the cytoskeleton in rat osteoclasts. The PTKIs, genistein and herbimycin A, reversibly elevated [Ca2+]i measured by fura‐2 microfluorimetry. The PTKI‐induced increase was abolished by omission of extracellular Ca2+, but was not attenuated by depletion of Ca2+ stores. The PTKI‐induced increase was inhibited by addition of La3+ and Ni2+, but not abolished by dihydropyridine (DHP) Ca2+ channel blockers. Genistin, an inactive analogue of genistein, had no effect on [Ca2+]i. In the cytoskeleton assay, genistein rapidly disrupted the actin ring formation that serves as a marker for the resorbing state of osteoclasts. Disruption of the actin ring formation was also diminished in Ca2+‐free extracellular solution. These results suggest that PTKIs in rat osteoclasts elevate [Ca2+]i via activation of a DHP‐insensitive, nonspecific Ca2+ entry pathway and disrupt the formation of actin rings, resulting in suppression of bone resorption activity. The regulation of this Ca2+‐influx by PTKIs is likely to contribute to inhibition of bone resorption by these compounds. J. Cell. Physiol. 183:83–90, 2000.

RANKL-induced TRPV2 expression regulates osteoclastogenesis via calcium oscillations

The receptor activator of NFκB ligand (RANKL) induces Ca(2+) oscillations and activates the Nuclear Factor of Activated T cells 1 (NFATc1) during osteoclast differentiation (osteoclastogenesis). Ca(2+) oscillations are an important trigger signal for osteoclastogenesis, however the molecular basis of Ca(2+) permeable influx pathways serving Ca(2+) oscillations has not yet been identified. Using a DNA microarray, we found that Transient Receptor Potential Vanilloid channels 2 (TRPV2) are expressed significantly in RANKL-treated RAW264.7 cells (preosteoclasts) compared to untreated cells. Therefore, we further investigated the expression and functional role of TRPV2 on Ca(2+) oscillations and osteoclastogenesis. We found that RANKL dominantly up-regulates TRPV2 expression in preosteoclasts, and evokes spontaneous Ca(2+) oscillations and a transient inward cation current in a time-dependent manner. TRPV inhibitor ruthenium red and tetracycline-induced TRPV2 silencing significantly decreased both the frequency of Ca(2+) oscillations and the transient inward currents in RANKL-treated preosteoclasts. Silencing of store-operated Ca(2+) entry (SOCE) proteins similarly suppressed both RANKL-induced oscillations and currents in preosteoclasts. Furthermore, suppression of TRPV2 also reduced RANKL-induced NAFTc1 expression, its nuclear translocation, and osteoclastogenesis. In summary, Ca(2+) oscillations in preosteoclasts are triggered by RANKL-dependent TRPV2 and SOCE activation and intracellular Ca(2+) release. Subsequent activation of NFATc1 promotes osteoclastogenesis.

Intracellular ClC-3 chloride channels promote bone resorption in vitro through organelle acidification in mouse osteoclasts.

ClC-7 Cl(-) channels expressed in osteoclasts are important for bone resorption since it has been shown that disruption of the ClCN7 gene in mice leads to severe osteopetrosis. We have previously reported that Cl(-) currents recorded from mouse osteoclasts resemble those of ClC-3 Cl(-) channels. The aim of the present study was to determine the expression of ClC-3 channels in mouse osteoclasts and their functional role during bone resorption. We detected transcripts for both ClC-7 and ClC-3 channels in mouse osteoclasts by RT-PCR. The expression of ClC-3 was confirmed by immunocytochemical staining. Mouse osteoclasts lacking ClC-3 Cl(-) channels (ClC-3(-/-) osteoclasts) derived from ClCN3 gene-deficient mice (ClC-3(-/-)) showed lower bone resorption activity compared with ClC-3+/+ osteoclasts derived from wild-type mice (ClC-3+/+). Treatment of ClC-3+/+ osteoclasts with small interfering RNA (siRNA) against ClC-3 also significantly reduced bone resorption activity. Electrophysiological properties of basal and hypotonicity-induced Cl(-) currents in ClC-3(-/-) osteoclasts did not differ significantly from those in ClC-3+/+ osteoclasts. Using immunocytochemistry, ClC-3 was colocalized with lysosome-associated membrane protein 2. Using pH-sensitive dyes, organelle acidification activity in ClC-3(-/-) osteoclasts was weaker than in ClC-3+/+ osteoclasts. Treatment of ClC-3+/+ osteoclasts with siRNA against ClC-3 also reduced the organelle acidification activity. In conclusion, ClC-3 Cl(-) channels are expressed in intracellular organelles of mouse osteoclasts and contribute to osteoclastic bone resorption in vitro through organelle acidification.

Calcitonin inhibits proton extrusion in resorbing rat osteoclasts via protein kinase A

Although calcitonin is well known to be a potent inhibitor of bone resorption, it remains unknown how it regulates osteoclastic H+ transport. In this study, we examined the effects of calcitonin on H+ extrusion in cultured rat resorbing osteoclasts using an intracellular pH (pHi) indicator, BCECF [2′7′-bis-(2-carboxyethyl)- 5-carboxyfluorescein]. Resorbing osteoclasts were identified by their formation of resorbing pits on calcium phosphate-coated quartz coverslips. Both basal pHi and H+ extrusion activity were significantly higher compared to non-resorbing osteoclasts. Two types of H+-extruding systems were identified by pharmacological and immunocytochemical means: a bafilomycin-A1-sensitive and an amiloride-sensitive system [H+ extrusion mediated by a vacuolar type proton pump (V-ATPase) and by a Na+/H+ exchanger (NHE), respectively]. Calcitonin inhibited both H+ extrusion activities in a dose-dependent manner and this action was mimicked by protein kinase A (PKA) activators, but not by protein kinase C (PKC) activators. Pretreatment with PKA inhibitors completely suppressed calcitonin-induced inhibition, whereas neither PKC inhibitors nor calcium chelators suppressed it. These results indicate that calcitonin inhibits H+ extrusion generated by V-ATPase and NHE via PKA activation. These inhibitory mechanisms of H+ transport by calcitonin are important for the regulation of bone resorption.

Calcitonin in human odontoclasts regulates root resorption activity via protein kinase A

Calcitonin is a known inhibitor of osteoclastic bone resorption, but it remains uncertain whether calcitonin also regulates human odontoclastic activity, particularly during the physiological process of root resorption. In this study, we examined the expression of calcitonin receptors in human odontoclasts and the effect of calcitonin on root resorption, using immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Actin-ring formation was used to assess cytostructural changes during resorption activity. Our results show that calcitonin receptors are expressed in human odontoclasts freshly isolated from deciduous teeth of the periodontal region. Calcitonin inhibited actin-ring formation and resorption activity. This calcitonin-induced inhibition was mimicked by forskolin and dibutyryl-adenosine 3″,5″-cyclic monophosphate (db-cAMP), which are protein kinase A (PKA) activators, but not by phorbol 12-myristate 13-acetate, a protein kinase C activator. Pretreatment with adenosine 3″,5″-cyclic monophosphothioate Rp diastereomer (Rp-cAMPS), a PKA inhibitor, suppressed the calcitonin-induced inhibition of actin-ring formation. These results indicate that calcitonin receptor activation suppresses odontoclastic root resorption via PKA, a signaling pathway different from that in human osteoclasts.

Expression of mouse osteoclast K-Cl Co-transporter-1 and its role during bone resorption.

To assess the role of Cl− transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl− co‐transporters (KCCs). KCC1 and chloride channel‐7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)‐butylindazone (DIOA), KCC1 antisense oligo‐nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl− extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl− extrusion mechanism accompanying the H+ extrusion during bone resorption.

Characteristics of ClC7 Cl− channels and their inhibition in mutant (G215R) associated with autosomal dominant osteopetrosis type II in native osteoclasts and hClcn7 gene-expressing cells

ClC7 Cl− channels (Clcn7) are crucial for osteoclastic bone resorption and have heterozygous mutation in autosomal osteopetrosis type II (ADO II) patients. Although extracellular acidification is known to induce ClC7 Cl− currents in Clcn7-transfected oocytes, other characteristics of this acid-induced Cl− current, as well as the effects of mutant Clcn7 in ADO II, remain to be determined. The present study showed that extracellular acidification evoked outward Cl− currents in mouse osteoclasts. Expression of wild-type human Clcn7 in HEK293 cells also induced a significant increase in acid-activated Cl− currents. These acid-activated Cl− currents were independent of intracellular acidification and [Ca2+]i increase. HEK293 cells with the Clcn7 mutation associated with ADO II at G215R did not display these Cl− currents. These results suggest that osteoclastic ClC7 Cl− channels are activated under extracellar acidification and suppressed in Clcn7 mutant associated with ADO II during bone resorption.

Antibodies against ClC7 inhibit extracellular acidification-induced Cl− currents and bone resorption activity in mouse osteoclasts

The Cl− channel/transporter ClC7 is crucial for osteoclastic bone resorption and might become a therapeutic target for osteoporosis. In this study, we raised anti-ClC7 polyclonal antibodies against three different peptide sequences, including G215, P249, and R286, which are the mutation regions found in autosomal dominant osteopetrosis type II patients and examined the effects of these antibodies on the ClC7 Cl− current induced by extracellular acidification (acid-activated Cl− current) using the whole-cell patch clamp technique and bone resorption activity in mouse osteoclasts. Intracellular dialysis of osteoclasts with antibodies to intracellular G215 (Ab-G215) and extracellular application of antibodies to extracellular P249 (Ab-P249) or R286 (Ab-R286) inhibited the acid-activated Cl− current. These antibodies also suppressed the acid-activated Cl− current in ClC7 overexpressing Raw264.7 cells; however, Cl− currents evoked by hypotonic stimulation and the inherent inwardly rectifying K+ currents in mouse osteoclasts were unaffected by these antibodies. Furthermore, extracellularly applied Ab-P249 and Ab-R286 also reduced bone resorption activity. Our results demonstrate that these antibodies specifically block ClC7 in mouse osteoclasts. Thus, anti-ClC7 antibodies have potential promise for treatment of osteoporosis.