Hironari Masuda

Regulation of RANKL-induced osteoclastogenesis by TGF-β through molecular interaction between Smad3 and Traf6.

Previous studies have shown that transforming growth factor β (TGF‐β) promotes receptor activator of nuclear factor‐κB ligand (RANKL)–induced osteoclastogenesis. However, the underlying molecular mechanisms have not been elucidated. When TGF‐β signals were blocked either by a specific inhibitor of TGF‐β type 1 receptor kinase activity, SB431542, or by introducing a dominant‐negative mutant of TGF‐β type 2 receptor, RANKL‐induced osteoclastogenesis was almost completely suppressed. Blockade of Smad signaling by overexpression of Smad7 or c‐Ski markedly suppressed RANKL‐induced osteoclastogenesis, and retroviral induction of an activated mutant of Smad2 or Smad3 reversed the inhibitory effect of SB431542. Immunoprecipitation analysis revealed that Smad2/3 directly associates with the TRAF6‐TAB1‐TAK1 molecular complex, which is generated in response to RANKL stimulation and plays an essential role in osteoclast differentiation. TRAF6‐TAB1‐TAK1 complex formation was not observed when TGF‐β signaling was blocked. Analysis using deletion mutants revealed that the MH2 domain of Smad3 is necessary for TRAF6‐TAB1‐TAK1 complex formation, downstream signal transduction, and osteoclast formation. In addition, gene silencing of Smad3 in osteoclast precursors markedly suppressed RANKL‐induced osteoclast differentiation. In summary, TGF‐β is indispensable in RANKL‐induced osteoclastogenesis, and the binding of Smad3 to the TRAF6‐TAB1‐TAK1 complex is crucial for RANKL‐induced osteoclastogenic signaling.

Class IA phosphatidylinositol 3-kinase regulates osteoclastic bone resorption through protein kinase B-mediated vesicle transport.

Class IA phosphatidylinositol 3‐kinases (PI3Ks) are activated by growth factor receptors and regulate a wide range of cellular processes. In osteoclasts, they are activated downstream of αvβ3 integrin and colony‐stimulating factor‐1 receptor (c‐Fms), which are involved in the regulation of bone‐resorbing activity. The physiological relevance of the in vitro studies using PI3K inhibitors has been of limited value, because they inhibit all classes of PI3K. Here, we show that the osteoclast‐specific deletion of the p85 genes encoding the regulatory subunit of the class IA PI3K results in an osteopetrotic phenotype caused by a defect in the bone‐resorbing activity of osteoclasts. Class IA PI3K is required for the ruffled border formation and vesicular transport, but not for the formation of the sealing zone. p85α/β doubly deficient osteoclasts had a defect in macrophage colony‐stimulating factor (M‐CSF)–induced protein kinase B (Akt) activation and the introduction of constitutively active Akt recovered the bone‐resorbing activity. Thus, the class IA PI3K‐Akt pathway regulates the cellular machinery crucial for osteoclastic bone resorption, and may provide a molecular basis for therapeutic strategies against bone diseases.

Genomewide Comprehensive Analysis Reveals Critical Cooperation Between Smad and c‐Fos in RANKL‐Induced Osteoclastogenesis

We have previously reported that transforming growth factor β (TGF‐β) plays an essential role in receptor activator of nuclear factor‐κB ligand (RANKL)‐induced osteoclastogenesis. However, the detailed underlying molecular mechanisms still remain unclear. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) and chromatin immunoprecipitation (ChIP) followed by sequencing (FAIRE‐seq and ChIP‐seq) analyses indicated the cooperation of Smad2/3 with c‐Fos during osteoclastogenesis. Biochemical analysis and immunocytochemical analysis revealed that physical interaction between Smad2/3 and c‐Fos is required for their nuclear translocation. The gene expression of nuclear factor of activated T‐cells, cytoplasmic 1 (Nfatc1), a key regulator of osteoclastogenesis, was regulated by RANKL and TGF‐β, and c‐Fos binding to open chromatin sites was suppressed by inhibition of TGF‐β signaling by SB431542. Conversely, Smad2/3 binding to Nfatc1 was impaired by c‐Fos deficiency. These results suggest that TGF‐β regulates RANKL‐induced osteoclastogenesis through reciprocal cooperation between Smad2/3 and c‐Fos.

Distinguishing the proapoptotic and antiresorptive functions of risedronate in murine osteoclasts: Role of the Akt pathway and the ERK/Bim axis

OBJECTIVE Nitrogen-containing bisphosphonates are one of the most successful therapeutics for osteoporosis. The aim of this study was to elucidate the functional mechanism of one of the typical nitrogen-containing bisphosphonates, risedronate. METHODS Osteoclasts generated from murine bone marrow macrophages were treated with risedronate in vitro, and its effects on apoptosis and bone-resorbing activity were examined. The mechanism of action of risedronate was examined by gene induction of constitutively active Akt-1 and constitutively active MEK-1, and by gene deletion of Bim. Bim(-/-) mice, in which osteoclasts were resistant to apoptosis, were treated with risedronate and analyzed radiographically, biochemically, and histologically. RESULTS Risedronate induced osteoclast apoptosis through the mitochondria-dependent pathway with an increased expression of Bim, and the proapoptotic effect of risedronate was suppressed by Bim deletion and constitutively active MEK-1 introduction. In contrast, the risedronate-induced suppression of bone resorption was completely reversed by inducing constitutively active Akt-1, but not by Bim deletion or constitutively active MEK-1 introduction. These results suggested that apoptosis and bone-resorbing activity of osteoclasts were regulated through the ERK/Bim axis and the Akt pathway, respectively, both of which were suppressed by risedronate. Although osteoclast apoptosis in response to risedronate administration was suppressed in the Bim(-/-) mice, risedronate treatment increased bone mineral density in Bim(-/-) mice at a level equivalent to that in wild-type mice. CONCLUSION Our findings indicate that the antiresorptive effect of risedronate in vivo is mainly mediated by the suppression of the bone-resorbing activity of osteoclasts and not by the induction of osteoclast apoptosis.

Bone resorption is regulated by cell-autonomous negative feedback loop of Stat5-Dusp axis in the osteoclast.

The transcription factor Stat5 inhibits the bone-resorbing function of osteoclasts, in part by inducing the expression of the phosphatases Dusp1 and Dusp2.

Remnant-Preserving Anterior Cruciate Ligament Reconstruction Using a Three-Dimensional Fluoroscopic Navigation System

Introduction Recently, remnant-preserving anterior cruciate ligament (ACL) reconstruction has been increasingly performed to achieve revascularization, cell proliferation, and recovery of high-quality proprioception. However, poor arthroscopic visualization makes accurate socket placement during remnant-preserving ACL reconstruction difficult. This study describes a surgical technique used to create an anatomical femoral socket with a three-dimensional (3D) fluoroscopy based navigation system during technically demanding remnant-preserving ACL reconstruction. Surgical Technique After a reference frame was attached to the femur, an intraoperative image of the distal femur was obtained, transferred to the navigation system and reconstructed into a 3D image. A navigation computer helped the surgeon visualize the entire lateral wall of the femoral notch and lateral intercondylar ridge, even when the remnant of the ruptured ACL impeded arthroscopic visualization of the bone surface. When a guide was placed, the virtual femoral tunnel overlapped the reconstructed 3D image in real time; therefore, only minimal soft tissue debridement was required. Materials and Methods We treated 47 patients with remnant-preserving ACL reconstruction using this system. The center of the femoral socket aperture was calculated according to the quadrant technique using 3D computed tomography imaging. Results The femoral socket locations were considered to be an anatomical footprint in accordance with previous cadaveric studies. Conclusions The 3D fluoroscopy-based navigation can assist surgeons in creating anatomical femoral sockets during remnant-preserving ACL reconstruction.

Iliotibial band irritation caused by the EndoButton after anatomic double-bundle anterior cruciate ligament reconstruction: report of two cases.

Two patients underwent arthroscopic anatomic double-bundle anterior cruciate ligament (ACL) reconstruction using the EndoButton for femoral fixation. The femoral tunnels were created by the inside-out technique through a far anteromedial portal. The patients postoperatively developed moderate lateral knee pain without instability. At the second-look arthroscopic evaluation, the two EndoButtons were removed. Both patients were completely asymptomatic several months after implant removal, implying that the EndoButtons caused the mechanical irritation in the iliotibial band. This is the first report describing removal of EndoButtons because of pain caused by friction with the iliotibial band. In anatomic ACL reconstruction, if the femoral tunnel exit is positioned near the lateral femoral epicondyle, care should be taken to prevent iliotibial band friction syndrome that could result because of the EndoButton.

Antiapoptotic molecule Bcl‐2 is essential for the anabolic activity of parathyroid hormone in bone

The antiapoptotic molecule Bcl‐2 inhibits apoptosis by preventing cytochrome c release from mitochondria. Although several studies have indicated the importance of Bcl‐2 in maintaining skeletal integrity, the detailed cellular and molecular mechanisms remain elusive. Since Bcl‐2−/− mice die before six weeks of age on account of renal failure and cannot be compared to adult wild‐type mice, we generated Bcl‐2−/−Bim+/− mice, in which a single Bim allele was inactivated, and compared them with their Bcl‐2+/−Bim+/− littermates. Bcl‐2−/−Bim+/− mice grew normally, but exhibited decreased bone mass compared to Bcl‐2+/−Bim+/− mice, mainly due to impaired osteoblast function. Interestingly, the anabolic effect of parathyroid hormone (PTH) was not observed in Bcl‐2−/−Bim+/− mice. This data demonstrates that Bcl‐2 is indispensable for the anabolic activity of PTH in bone.

Individual and combining effects of anti-RANKL monoclonal antibody and teriparatide in ovariectomized mice

We examined the individual and combined effects of teriparatide and anti-RANKL (receptor activator of nuclear factor κB ligand) monoclonal antibody in ovariectomized mice. Three-month-old female C57BL/6 mice were ovariectomized (OVX) or sham operated. Four weeks after OVX, they were assigned to 3 different groups to receive anti-RANKL monoclonal antibody (Ab) alone (5 mg/kg single injection at 4 weeks after OVX, Ab group), teriparatide alone (80 μg/kg daily injection for 4 weeks from 4 weeks after OVX, PTH group), or mAb plus teriparatide (Ab + PTH group). Mice were sacrificed 8 weeks after OVX. Bone mineral density (BMD) was measured at the femur and lumbar spine. Hind limbs were subjected to histological and histomorphometric analysis. Serum osteocalcin and CTX-I levels were measured to investigate the bone turnover. Compared with Ab group, Ab + PTH group showed a significant increase in BMD at distal femur and femoral shaft. Cortical bone volume was significantly increased in PTH and Ab + PTH groups compared with Ab group. Bone turnover in Ab + PTH group was suppressed to the same degree as in Ab group. The number of TRAP-positive multinucleated cells was markedly reduced in Ab and Ab + PTH groups. These results suggest that combined treatment of teriparatide with anti-RANKL antibody has additive effects on BMD in OVX mice compared with individual treatment.

Anti-apoptotic Bcl-2 family member Mcl-1 regulates cell viability and bone-resorbing activity of osteoclasts

Myeloid cell leukemia sequence 1 (Mcl-1) is an anti-apoptotic Bcl-2 family protein and an immediate early gene expressed during myeloid leukemia cell line differentiation. We analyzed the expression and function of Mcl-1 in osteoclasts. Mcl-1 protein exhibited a short half-life in osteoclasts caused by its degradation in the ubiquitin-proteasome system. Mcl-1 had no effect on osteoclast differentiation, but its overexpression prolonged osteoclast survival and suppressed the bone-resorbing activity of these cells, as determined by pit formation assay. Conversely, Mcl-1 depletion suppressed osteoclast survival and increased bone resorption. This negative role for Mcl-1 on the bone-resorptive activities of osteoclasts may be caused by the increase in adenosine triphosphate/adenosine diphosphate ratio. Finally, we showed that the local deletion of Mcl-1 by the injection of the Cre adenovirus into the calvaria of Mcl1(fl/fl) mice significantly affected GST-RANKL-induced bone resorption in vivo. These results demonstrated that Mcl-1 positively regulates cell viability and negatively regulates the bone-resorbing activity of osteoclasts both in vitro and in vivo.