Tomohiro Hikata

A Systems Approach Reveals that the Myogenesis Genome Network Is Regulated by the Transcriptional Repressor RP58

We created a whole-mount in situ hybridization (WISH) database, termed EMBRYS, containing expression data of 1520 transcription factors and cofactors expressed in E9.5, E10.5, and E11.5 mouse embryos--a highly dynamic stage of skeletal myogenesis. This approach implicated 43 genes in regulation of embryonic myogenesis, including a transcriptional repressor, the zinc-finger protein RP58 (also known as Zfp238). Knockout and knockdown approaches confirmed an essential role for RP58 in skeletal myogenesis. Cell-based high-throughput transfection screening revealed that RP58 is a direct MyoD target. Microarray analysis identified two inhibitors of skeletal myogenesis, Id2 and Id3, as targets for RP58-mediated repression. Consistently, MyoD-dependent activation of the myogenic program is impaired in RP58 null fibroblasts and downregulation of Id2 and Id3 rescues MyoDs ability to promote myogenesis in these cells. Our combined, multi-system approach reveals a MyoD-activated regulatory loop relying on RP58-mediated repression of muscle regulatory factor (MRF) inhibitors.

Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development

Here we examined the involvement of Notch signaling in the endochondral ossification process, which is crucial for osteoarthritis (OA) development. Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpjfl/fl mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-CreERT;Rbpjfl/fl mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1 in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification and OA development, representing an extracellular therapeutic target of OA.

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.

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.

Risk factors for adjacent segment disease after posterior lumbar interbody fusion and efficacy of simultaneous decompression surgery for symptomatic adjacent segment disease.

Study Design: A retrospective study. Summary of Background Data: Posterior lumbar interbody fusion (PLIF) increases mechanical stress and can cause degenerative changes at the adjacent segment. However, the precise causes of adjacent segment disease (ASD) after PLIF are not known, and it is unclear whether simultaneous decompression surgery for symptomatic ASD is effective. Objective: To study, radiographically and symptomatically, the risk factors for adjacent segment disease (ASD) in the lumbar spine after L4/5 PLIF and to examine whether decompression surgery for the adjacent segment (L3/4) reduces the occurrence of symptomatic ASD. Methods: Fifty-four patients who underwent L4/5 PLIF for L4 degenerative spondylolisthesis and could be followed up for at least 2 years were included. Of these, 37 were treated simultaneously with decompression surgery at L3/4. We measured radiographic changes and assessed symptoms from the cranial adjacent segment. Results: Thirty-one patients (57.4%) met radiologic criteria for ASD. The length of follow-up (P=0.004) and simultaneous decompression surgery at L3/4 (P=0.009) were statistically significant factors for radiologic diagnosis of ASD. Seven patients (13.0%) had symptomatic ASD: 6 in the decompression group (16.2%) and 1 in the PLIF-only group (5.9%). Simultaneous decompression surgery did not reduce the incidence of symptomatic ASD (P=0.256). Local lordosis at the fused segment (P=0.005) and the sagittal angle of the facet joint at L3/4 (P=0.024) were statistically significant predictors of symptomatic ASD, which was accompanied by postoperative anterior listhesis above the fused segment (S group, 8.4%±8.0%; nonsymptomatic group: −0.7%±5.0%, P=0.024). Conclusions: Patients whose facet joint at the adjacent segment had a more sagittal orientation had postoperative anterior listhesis, which caused symptomatic ASD. Simultaneous decompression surgery without fusion at the adjacent level was not effective for these patients, but rather, there was a possibility that it induced symptomatic ASD.

Impact of sagittal spinopelvic alignment on clinical outcomes after decompression surgery for lumbar spinal canal stenosis without coronal imbalance

OBJECT The object of this study was to investigate correlations between sagittal spinopelvic alignment and improvements in clinical and quality-of-life (QOL) outcomes after lumbar decompression surgery for lumbar spinal canal stenosis (LCS) without coronal imbalance. METHODS The authors retrospectively reviewed data from consecutive patients treated for LCS with decompression surgery in the period from 2009 through 2011. They examined correlations between preoperative or postoperative sagittal vertical axis (SVA) and radiological parameters, clinical outcomes, and health-related (HR)QOL scores in patients divided according to SVA. Clinical outcomes were assessed according to Japanese Orthopaedic Association (JOA) and visual analog scale (VAS) scores. Health-related QOL was evaluated using the Roland-Morris Disability Questionnaire (RMDQ) and the JOA Back Pain Evaluation Questionnaire (JOABPEQ). RESULTS One hundred nine patients were eligible for inclusion in the study. Compared to patients with normal sagittal alignment prior to surgery (Group A: SVA < 50 mm), those with preoperative sagittal imbalance (Group B: SVA ≥ 50 mm) had significantly smaller lumbar lordosis and thoracic kyphosis angles and larger pelvic tilt. In Group B, there was a significant decrease in postoperative SVA compared with the preoperative SVA (76.3 ± 29.7 mm vs. 54.3 ± 39.8 mm, p = 0.004). The patients in Group B with severe preoperative sagittal imbalance (SVA > 80 mm) had residual sagittal imbalance after surgery (82.8 ± 41.6 mm). There were no significant differences in clinical and HRQOL outcomes between Groups A and B. Compared to patients with normal postoperative SVA (Group C: SVA < 50 mm), patients with a postoperative SVA ≥ 50 mm (Group D) had significantly lower JOABPEQ scores, both preoperative and postoperative, for walking ability (preop: 36.6 ± 26.3 vs. 22.7 ± 26.0, p = 0.038, respectively; postop: 71.1 ± 30.4 vs. 42.5 ± 29.6, p < 0.001) and social functioning (preop: 38.7 ± 18.5 vs. 30.2 ± 16.7, p = 0.045; postop: 67.0 ± 25.8 vs. 49.6 ± 20.0, p = 0.001), as well as significantly higher postoperative RMDQ (4.9 ± 5.2 vs. 7.9 ± 5.7, p = 0.015) and VAS scores for low-back pain (2.68 ± 2.69 vs. 3.94 ± 2.59, p = 0.039). CONCLUSIONS Preoperative sagittal balance was not significantly correlated with clinical or HRQOL outcomes after decompression surgery in LCS patients without coronal imbalance. Decompression surgery improved the SVA value in patients with preoperative sagittal imbalance; however, the patients with severe preoperative sagittal imbalance (SVA > 80 mm) had residual imbalance after decompression surgery. Both clinical and HRQOL outcomes were negatively affected by postoperative residual sagittal imbalance.

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.

Complications Associated With Spine Surgery in Patients Aged 80 Years or Older: Japan Association of Spine Surgeons with Ambition (JASA) Multicenter Study:

Study Design: Retrospective study of registry data. Objectives: Aging of society and recent advances in surgical techniques and general anesthesia have increased the demand for spinal surgery in elderly patients. Many complications have been described in elderly patients, but a multicenter study of perioperative complications in spinal surgery in patients aged 80 years or older has not been reported. Therefore, the goal of the study was to analyze complications associated with spine surgery in patients aged 80 years or older with cervical, thoracic, or lumbar lesions. Methods: A multicenter study was performed in patients aged 80 years or older who underwent 262 spinal surgeries at 35 facilities. The frequency and severity of complications were examined for perioperative complications, including intraoperative and postoperative complications, and for major postoperative complications that were potentially life threatening, required reoperation in the perioperative period, or left a permanent injury. Results: Perioperative complications occurred in 75 of the 262 surgeries (29%) and 33 were major complications (13%). In multivariate logistic regression, age over 85 years (hazard ratio [HR] = 1.007, P = 0.025) and estimated blood loss ≥500 g (HR = 3.076, P = .004) were significantly associated with perioperative complications, and an operative time ≥180 min (HR = 2.78, P = .007) was significantly associated with major complications. Conclusions: Elderly patients aged 80 years or older with comorbidities are at higher risk for complications. Increased surgical invasion, and particularly a long operative time, can cause serious complications that may be life threatening. Therefore, careful decisions are required with regard to the surgical indication and procedure in elderly patients.

A Retrospective Cohort Study Comparing the Safety and Efficacy of Minimally Invasive Versus Open Surgical Techniques in the Treatment of Spinal Metastases

Study Design: A retrospective cohort study. Objective: This study was conducted to assess the invasiveness, efficacy, and safety of minimally invasive spine stabilization (MISt) for metastatic spinal tumor patients with short life expectancy. Summary of Background Data: Conventional open surgery for metastatic spinal tumors has the disadvantages of significant blood loss, potential infection, damage to back muscles, and extended hospital stays. The minimally invasive spine surgery has changed the treatment of metastatic spinal tumors radically and fundamentally. Materials and Methods: We retrospectively reviewed data from 50 consecutive patients registered with the Keio Spine Research Group (KSRG) who underwent posterior palliative surgery for metastatic spinal tumors from January 2009 to June 2015. Of these, 25 patients underwent MISt surgery (M group), and 25 underwent conventional open surgery (C group). The patients were assessed by demographic data, surgical invasiveness, complications, pain improvement, and neurological recovery. Results: The 2 groups did not differ significantly in baseline characteristics. The M group had significantly less blood loss (M, 340.1 mL; C, 714.3 mL; P=0.005), less postoperative drainage (M, 136.0 mL; C, 627.0 mL; P<0.001), lower rates of red blood cell transfusion (M, 3 cases; C, 10 cases; P=0.029), and a shorter postoperative period of bed rest (M, 2.0 d; C, 3.6 d; P<0.001), compared with the C group. The perioperative complication rates were significantly lower (P=0.012) in the M group (3 patients, 12%) than in the C group (11 patients, 44%). Neurological deficits and pain improved significantly and comparably in the 2 groups after surgery. Conclusions: MISt is a less invasive and effective alternative surgery to conventional open surgery for metastatic spinal tumors. MISt should be considered as a valid option for the treatment of metastatic spinal tumor patients with a short life expectancy. Level of Evidence: Level 3.

Risk Factors for Delirium After Spine Surgery in Extremely Elderly Patients Aged 80 Years or Older and Review of the Literature: Japan Association of Spine Surgeons with Ambition Multicenter Study

Study Design: Retrospective database analysis. Objective: Spine surgeries in elderly patients have increased in recent years due to aging of society and recent advances in surgical techniques, and postoperative complications have become more of a concern. Postoperative delirium is a common complication in elderly patients that impairs recovery and increases morbidity and mortality. The objective of the study was to analyze postoperative delirium associated with spine surgery in patients aged 80 years or older with cervical, thoracic, and lumbar lesions. Methods: A retrospective multicenter study was performed in 262 patients 80 years of age or older who underwent spine surgeries at 35 facilities. Postoperative complications, incidence of postoperative delirium, and hazard ratios of patient-specific and surgical risk factors were examined. Results: Postoperative complications occurred in 59 of the 262 spine surgeries (23%). Postoperative delirium was the most frequent complication, occurring in 15 of 262 patients (5.7%), and was significantly associated with hypertension, cerebrovascular disease, cervical lesion surgery, and greater estimated blood loss (P < .05). In multivariate logistic regression using perioperative factors, cervical lesion surgery (odds ratio = 4.27, P < .05) and estimated blood loss ≥300 mL (odds ratio = 4.52, P < .05) were significantly associated with postoperative delirium. Conclusions: Cervical lesion surgery and greater blood loss were perioperative risk factors for delirium in extremely elderly patients after spine surgery. Hypertension and cerebrovascular disease were significant risk factors for postoperative delirium, and careful management is required for patients with such risk factors.