Akihiro Hakozaki

IL-27 Abrogates Receptor Activator of NF-κB Ligand-Mediated Osteoclastogenesis of Human Granulocyte-Macrophage Colony-Forming Unit Cells through STAT1-Dependent Inhibition of c-Fos

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Rα (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-κB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-κB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

Rheumatoid arthritis: a risk factor for deep venous thrombosis after total knee arthroplasty? Comparative study with osteoarthritis

BackgroundRecent advances in the understanding of blood coagulation processes favor an inflammatory basis for thrombotic events. In this study, thrombotic risk after total knee arthroplasty (TKA) was assessed and compared between patients with rheumatoid arthritis (RA) and those with osteoarthritis (OA).MethodsSubjects comprised 199 patients (238 knees) with RA and 156 patients (169 knees) with OA. Serum D-dimer levels were measured before and after the operation. Lowdose unfractionated heparin was given for 7 days when patients had a history of previous venous thromboembolism or had a D-dimer level or ≥10 μg/ml of D-dimer on postoperative day 1. Doppler ultrasonography (DUS) was routinely performed preoperatively and on postoperative day (POD) 7 for diagnosing a deep venous thrombosis (DVT).ResultsD-dimer levels on PODs 0, 1, and 7 were, respectively, 4.6, 37.2, and 11.2 μg/ml for RA and 1.8, 42.3, and 13.6 μg/ml for OA. The incidence of DUS-confirmed DVT was 20.6% in the RA group and 43.2% in the OA group, indicating a much higher incidence of postoperative DVT in OA patients (P < 0.001). Interestingly, when patients taking nonsteroidal antiinflammatory drugs (NSAIDs) or those >65 years of age were excluded, the incidence of DVT was comparable in the RA and OA groups. Symptomatic pulmonary embolism and DVT occurred in two and one OA patients and in one and two RA patients, respectively, with one postdischarge DVT included in each group.ConclusionsThe present study revealed that the incidence of DVT following TKA was significantly lower in RA patients than in those with OA. However, when the patients were matched for age and NSAID use, the incidence of DVT was equivalent in the two groups. These findings may allow us to reconsider a prophylactic regimen for venous thromboembolism in patients with RA.

Cell Surface Colony-Stimulating Factor 1 Can Be Cleaved by TNF-α Converting Enzyme or Endocytosed in a Clathrin-Dependent Manner

CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-α converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.

PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts

Cytokine signaling via various transcription factors regulates receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-mediated osteoclast differentiation from monocyte/macrophage lineage cells involved in propagation and resolution of inflammatory bone destruction. Protein inhibitor of activated STAT3 (PIAS3) was initially identified as a molecule that inhibits DNA binding of STAT3 and regulates many transcription factors through distinct mechanisms. To analyze PIAS3 function in osteoclasts in vivo, we have generated transgenic mice in which PIAS3 is specifically expressed in the osteoclast lineage using the tartrate-resistant acid phosphatase (TRAP) gene promoter. PIAS3 transgenic mice showed an osteopetrotic phenotype due to impairment of osteoclast differentiation. Overexpression of PIAS3 in RAW264.7 cells suppressed RANKL-induced osteoclastogenesis by inhibiting the expression of c-Fos and NFATc1. Interestingly, PIAS3 inhibits the transcriptional activity of microphthalmia-associated transcription factor (MITF) independent of sumoylation. Down-regulation of PIAS3 markedly enhances RANKL-mediated osteoclastogenesis in RAW264.7 cells. Furthermore, overexpression of PIAS3 in mouse primary osteoblast (POB), down-regulates RANKL expression induced by interleukin-6 (IL-6) cytokine family, and inhibits osteoclast formation from bone marrow macrophages (BMMs) in vitro coculture system. Down-regulation of PIAS3 leads to the accelerated expression of RANKL in POB stimulated with IL-6 and soluble IL-6 receptor (sIL-6R). Taken together, our results clearly indicate that PIAS3 negatively regulates RANKL-mediated osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblasts.

Receptor Activator of NF-κB (RANK) Ligand Induces Ectodomain Shedding of RANK in Murine RAW264.7 Macrophages

Osteoclastogenesis is a highly sophisticated process that involves a variety of membrane-bound proteins expressed in osteoblasts and osteoclast precursors. Over the past several years, proteolytic cleavage and release of the ectodomain of membrane-bound proteins, also referred to as ectodomain shedding, has emerged as an important posttranslational regulatory mechanism for modifying the function of cell surface proteins. In line with this notion, several membrane-bound molecules involved in osteoclastogenesis, including CSF-1R and receptor activator of NF-κB ligand (RANKL), are proteolytically cleaved and released from the cell surface. In this study, we investigated whether receptor activator of NF-κB (RANK), one of the most essential molecules in osteoclastogenesis, undergoes ectodomain shedding. The results showed that RANK is released in the form of a soluble monomeric protein and that TNF-α–converting enzyme is involved in this activity. We also identified potential cleavage sites in the juxtamembrane domain of RANK and found that rRANKL induces RANK shedding in a macrophage-like cell line RAW264.7 via TNFR-associated factor 6 and MAPK pathways. Furthermore, we found that RANKL-induced osteoclastogenesis is accelerated in TNF-α–converting enzyme-deficient osteoclast precursors. These observations suggest the potential involvement of ectodomain shedding in the regulation of RANK functions and may provide novel insights into the mechanisms of osteoclastogenesis.

Clinical and radiographic outcomes of minimally invasive total knee arthroplasty through a lateral approach

PurposeWith increasing confidence and surgical experience, minimally invasive surgery (MIS) in total knee arthroplasty (TKA) is now being applied to more complicated cases. The present study assessed the feasibility of MIS-TKA using a lateral approach for valgus knees.MethodsSubjects comprised 26 patients with valgus knees who underwent MIS-TKA using a lateral subvastus approach. Five cases required a 1-cm snip of vastus lateralis obliquus, to shift the patella medially without eversion. Clinical scores and radiographic parameters of lateral MIS-TKA were examined and compared with those of 26 medial MIS-TKAs matched for preoperative patient characteristics.ResultsThe lateral MIS-TKA group showed slightly longer operative time and larger skin incision than the medial MIS-TKA group. Nevertheless, myoglobin index and pain on a visual analog scale on postoperative day 7 were significantly lower in the lateral MIS-TKA group than in the medial MIS-TKA group. Postoperative improvement of clinical scores was quite comparable between lateral and medial MIS-TKA groups. Radiographic assessment revealed that tibiofemoral mechanical axis aligned within ±3° from ideal in 24 of 26 patients after lateral MIS-TKA. MIS technique-related complications occurred in only 1 patient presenting with subsidence of the tibial component, due to malpositioning of the tibial component.ConclusionFrom the perspectives of postoperative pain, clinical scores, radiographic accuracy, and postoperative complication rate, lateral MIS-TKA achieved comparable or superior results to medial MIS-TKA. This technique may offer a promising technical option that can be utilized for most patients with valgus knee deformity.

Clinical significance of T2*-weighted gradient-echo MRI to monitor graft maturation over one year after anatomic double-bundle anterior cruciate ligament reconstruction: a comparative study with proton density-weighted MRI.

PURPOSE This study examined the feasibility of T2*-weighted imaging (T2*WI) gradient-echo MRI to reflect actual function of the graft after anatomic double-bundle ACL reconstruction. T2*WI and proton density-weighted imaging (PDWI) were compared in the assessment of ACL grafts. METHOD Sixty-one patients underwent T2*WI and PDWI at 3, 6, and 12 months postoperatively. Signal intensity of the anteromedial bundle (AMB) or posterolateral bundle (PLB) graft standardized to the intensity of the PCL was defined as signal intensity ratio (SIR). Correlations between degree of knee instability and SIR were assessed for each bundle, each time point, and each sequence. The diagnostic efficacy of T2*WI sequence to detect poorly functioning knee with anteroposterior translation ≥ 4 mm was assessed. RESULTS Significant correlations were observed between SIR and KT values for both AMB and PLB at 12 months on T2*WI (r=0.39 and 0.53, respectively), but not on PDWI. Notably, 9 of 10 patients with poorly functioning graft showing anteroposterior translation ≥ 4 mm at 12 months formed an isolated group with high T2*WI-SIR. Six of the 10 patients displayed an increase in SIR from 6 to 12 months. Defining anteroposterior translation ≥ 4 mm at 12 months as the diagnostic standard for poorly functioning graft, increasing T2*WI-SIR offered 60% sensitivity and > 90% specificity. CONCLUSIONS ACL graft intensity on T2*WI is more strongly associated with actual function of the graft than that on PDWI. An increasing trend in T2*WI-SIR from 6 to 12 months postoperatively represents a possible surrogate indicator for poorly functioning grafts.

Identification and analysis of function of a novel splicing variant of mouse receptor activator of NF-κB

Receptor activator of NF-κB (RANK) is a member of the tumor necrosis factor receptor (TNFR) family expressed in osteoclast precursors, and RANK–RANK ligand (RANKL) signaling is a key system for differentiation, activation and survival of osteoclasts. Here, we report the identification of a novel alternative splicing variant of mouse RANK gene (vRANK) that contains a new intervening exon between exon 1 and exon 2 of mouse full-length RANK (fRANK) mRNA. Since this novel exon contains the stop codon, vRANK encodes truncated amino acids that have a portion of the signal peptide of fRANK and an additional 19 amino acids that show no homology to previously reported domains. By transient transfection studies with vRANK–GFP and –Flag expressing constructs, vRANK was found localized mostly in the cytoplasm and partly in the cell membrane, but was not secreted into the culture supernatant. Under the stimulation of various factors, the expression of vRANK mRNA was almost parallel to that of fRANK in RAW264.7 cells not treated with M-CSF. Overexpression of vRANK, on the other hand, decreased TRACP (a marker of osteoclasts) mRNA expression as well as the number of TRACP-positive multinucleated giant cells. While the mRNA expression levels of NFATc1 (a master transcriptional factor of the osteoclast differentiation program) were not affected, apoptotic cells increased significantly in vRAN K-transfected cells treated with sRANKL. Taken together, these results suggest that vRANK is a novel osteoclast suppressor that reduces the number of RANKL-induced mature osteoclasts mainly by negating the anti-apoptotic effect of RANKL.