Katsuyuki Imamura

SnoN Suppresses Maturation of Chondrocytes by Mediating Signal Cross-talk between Transforming Growth Factor-β and Bone Morphogenetic Protein Pathways

Background: BMP signaling promotes chondrocyte maturation and, subsequently, endochondral ossification, whereas TGF-β signaling is inhibitory. Results: TGF-β induced SnoN to suppress BMP signaling and chondrocyte hypertrophy. Conclusion: SnoN mediates a signal cross-talk between TGF-β and BMP to regulate chondrocyte maturation. Significance: Our data revealed an effector of TGF-β signaling as a putative therapeutic molecular target for cartilage/bone regeneration or osteoarthritis. Hypertrophic maturation of chondrocytes is a crucial step in endochondral ossification, whereas abnormally accelerated differentiation of hypertrophic chondrocytes in articular cartilage is linked to pathogenesis of osteoarthritis. This cellular process is promoted or inhibited by bone morphogenetic protein (BMP) or transforming growth factor-β (TGF-β) signaling, respectively, suggesting that these signaling pathways cross-talk during chondrocyte maturation. Here, we demonstrated that expression of Tgfb1 was increased, followed by phosphorylation of Smad2, during BMP-2-induced hypertrophic maturation of ATDC5 chondrocytes. Application of a TGF-β type I receptor inhibitor compound, SB431542, increased the expression of Id1, without affecting the phosphorylation status of Smad1/5/8, indicating that the activated endogenous TGF-β pathway inhibited BMP signaling downstream of the Smad activation step. We searched for TGF-β-inducible effectors that are able to inhibit BMP signaling in ATDC5 cells and identified SnoN. Overexpression of SnoN suppressed the activity of a BMP-responsive luciferase reporter in COS-7 cells as well as expression of Id1 in ATDC5 cells and, subsequently, the expression of Col10a1, a hallmark of hypertrophic chondrocyte maturation. siRNA-mediated loss of SnoN showed opposite effects in BMP-treated ATDC5 cells. In adult mice, we found the highest level of SnoN expression in articular cartilage. Importantly, SnoN was expressed, in combination with phosphorylated Smad2/3, in prehypertrophic chondrocytes in the growth plate of mouse embryo bones and in chondrocytes around the ectopically existing hypertrophic chondrocytes of human osteoarthritis cartilage. Our results indicate that SnoN mediates a negative feedback mechanism evoked by TGF-β to inhibit BMP signaling and, subsequently, hypertrophic maturation of chondrocytes.

Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

Background: It is not clear how BMP-induced chondrocyte maturation is cell-autonomously terminated. Results: BMP-2 induced the ceramide-generating enzyme neutral sphingomyelinase 2 (nSMase2) in chondrocytes, whereas silencing of nSMase2 enhanced maturation in an Akt signaling-dependent manner. Conclusion: nSMase2 signaling regulates BMP-induced chondrocyte maturation as a negative feedback mechanism. Significance: This study elucidated the novel link between BMP and lipid signaling in chondrogenesis. Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C2-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C2-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.

Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

Background: The mechanisms by which Hivep3 regulates the osteochondrogenesis remain elusive. Results: Knockdown of Hivep3 down-regulated Alg2 expression. Alg2 suppressed osteoblast differentiation by inhibiting the activity of Runx2. Alg2 silencing suppressed the expression of Creb3l2 and chondrogenesis. Conclusion: Alg2 may be a modulator of osteochondrogenesis. Significance: This is the first report to describe the association of an Alg gene with osteochondrogenesis. Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

TGF-β signalling and PEG10 are mutually exclusive and inhibitory in chondrosarcoma cells

Histological distinction between enchondroma and chondrosarcoma is difficult because of a lack of definitive biomarkers. Here, we found highly active transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signalling in human chondrosarcomas compared with enchondromas by immunohistochemistry of phosphorylated SMAD3 and SMAD1/5. In contrast, the chondrogenic master regulator SOX9 was dramatically down-regulated in grade 1 chondrosarcoma. Paternally expressed gene 10 (PEG10) was identified by microarray analysis as a gene overexpressed in chondrosarcoma SW1353 and Hs 819.T cells compared with C28/I2 normal chondrocytes, while TGF-β1 treatment, mimicking higher grade tumour conditions, suppressed PEG10 expression. Enchondroma samples exhibited stronger expression of PEG10 compared with chondrosarcomas, suggesting a negative association of PEG10 with malignant cartilage tumours. In chondrosarcoma cell lines, application of the TGF-β signalling inhibitor, SB431542, increased the protein level of PEG10. Reporter assays revealed that PEG10 repressed TGF-β and BMP signalling, which are both SMAD pathways, whereas PEG10 knockdown increased the level of phosphorylated SMAD3 and SMAD1/5/9. Our results indicate that mutually exclusive expression of PEG10 and phosphorylated SMADs in combination with differentially expressed SOX9 is an index to distinguish between enchondroma and chondrosarcoma, while PEG10 and TGF-β signalling are mutually inhibitory in chondrosarcoma cells.

Lateral Approach to C2 Root Schwannoma Located Ventral to the Spinal Cord: A Case Report

脊髄腹側にあるC2前根糸由来の神経症腫に対し側方進入にて手術施行したので報告する．（症例）31歳男性．2003年2月上位頚椎のdumb-bell腫瘍に対し，後方進入にて環椎後弓切除・腫瘍摘出術施行．病理はschwannomaであった．myelopathyは軽快するも，MRI上脊柱管内に腫瘍は遺残．2004年9月左上肢の筋力低下出現．手術目的にて2005年6月入院．四肢DTRの亢進，左上下肢の筋力低下を認め，JOA scoreは11/17．脊髄は環椎レベルで腹側から著明に圧迫されており，側方進入摘出術を施行．胸鎖乳突筋後方より進入，腫瘍を内減圧し骨削除は行わずに全摘し得た．腫瘍はC2神経根前根と連続していた．術後大後頭神経領域の知覚低下出現するもmyelopathyは軽快した．（考察）上位頚椎部の脊髄腫瘍は呼吸麻痺や重篤な麻痺を来しうるため，特に良性の場合全摘が望ましい．腫瘍が脊髄腹側に存在する場合，側方進入摘出術は有用な方法と考える．

Treatment of Femoral Fracture with MRSA Myelitis

We treated an 81-year-old female who had femoral fracture with osteomyelitis by MRSA. She underwent osteosynthesis of the femoral neck fracture in 2000. Two years after the operation, her thigh became infected, for which she underwent operations such as removal of prosthesis, continuous irrigation, and plugging of PMMA cement beads with VCM. Unfortunately, she suffered a femoral fracture again. We performed osteosynthesis using the gamma-nail and grafted calcium phosphate cement (Biopex) with VCM to the bone defect, and obtained good results Biopex is useful for grafting to the bone defect when osteosynthesis is required for the treatment of fractures with osteomyelitis by MRSA.