Yasuhiro Ishidou

Distinct and overlapping patterns of localization of bone morphogenetic protein (BMP) family members and a BMP type II receptor during fracture healing in rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.

Expression and localization of bone morphogenetic proteins (BMPs) and BMP receptors in ossification of the ligamentum flavum

To clarify the pathogenesis of ossification of the ligamentum flavum (OLF), we examined the expression and localization of bone morphogenetic proteins (BMPs) and their receptors (BMPRs) in the ligamentum flavum of the patients with OLF by immunohistochemical staining and compared them with staining patterns in control patients. The BMPRs appeared extensively in mature and immature chondrocytes around the calcified zone and in spindle-shaped cells and round cells in the remote part from ossified foci in examined tissue of OLF. The ligands for BMPRs, BMP-2/-4 and osteogenic protein-1 (OP-1)/BMP-7, colocalized in OLF patients. In the control cases, expression of BMPs and BMPRs was observed around the calcified zone at the insertion of the ligamentum flavum to the bone, and limited expression was found in the smaller range. Thus, the expression profile of BMPs and BMPRs in OLF patients was entirely different from the control patients, suggesting that BMPs may be involved in promoting endochondral ossification at ectopic ossification sites in OLF, and that ossification activity is continuous in these patients.

Early Effects of Electrical Stimulation on Osteogenesis

The mechanism by which electrical stimulation causes osteogenesis is unknown. Bone marrow of the rabbit was stimulated by direct electrical current or electromagnetic fields to clarify the mechanism of osteogenesis by electrical stimulation. A total of 105 rabbits were separated into five groups: a direct current stimulation by Kirshner wire insertion group; a pulsed electromagnetic fields stimulation (PEMF) group; a PEMF with Kirshner wire insertion group; a Kirshner wire insertion group; and an intramedullary drilling control group. Measurement of intramedullary new bone formation and determination of alkaline phosphatase activity within the bone marrow were performed. Argylophilic nuclear organizer region (AgNOR) staining was done to evaluate the change in proliferative activity of the osteoblasts during electrical stimulation. In the direct current stimulation group and the PEMF accompanied by the insertion of the Kirshner wire group, alkaline phosphatase activity in the bone marrow and AgNOR staining increased at 7 days after surgery. At 14 days after surgery, alkaline phosphatase activity and proliferative activity of osteoblast were significantly higher in these two groups than in the other groups (PEMF group, Kirshner wire insertion alone group, intramedullary drilling group). Intramedullary new bone formation was most active in the direct current stimulation group. Electromagnetic stimulation of the inserted Kirshner wire also promoted bone formation significantly. The Kirshner wire insertion alone group and the intramedullary drilling group showed bone formation, but it was significantly less. Electromagnetic stimulation without the insertion of the Kirshner wire showed little bone formation. These findings revealed that the degree of osteogenesis induced by electrical stimulation is influenced by the tissue environment, and that osteogenesis is promoted markedly when electrical stimulation is provided in the environment of inflammation and reactive cells.

Functional impact of human collagen α2 (XI) gene polymorphism in pathogenesis of ossification of the posterior longitudinal ligament of the spine

Ossification of the posterior longitudinal ligament (OPLL) of the spine is the leading cause of myelopathy in Japan. In earlier studies, we provided genetic linkage and allelic association evidence of distinct differences in the human collagen α2(XI) gene (COL11A2) that might constitute inherited predisposition to OPLL.(1) In the present study, a strong allelic association with non‐OPLL (p = 0.0003) was observed with an intron 6 polymorphism [intron 6 (−4A)], in which the intron 6 (−4A) allele is more frequently observed in non‐OPLL subjects than in OPLL patients. In addition, a newly identified polymorphism in exon 6 [exon 6 (+28A)] was in linkage disequilibrium with the intron 6 (−4A). The functional impact of the polymorphisms was analyzed by comparing the differences in messenger RNA (mRNA) splicing by reverse‐transcription polymerase chain reaction (RT‐PCR) analysis in cultured cells from the interspinous ligament and an in vitro exon trapping study. The intron 6 (−4A) allele resulted in skipping exon 6 and retaining exon 7, while the exon 6 (+28A) allele was not associated with alteration in mRNA splicing. Similar mRNA species were observed in undifferentiated osteoblast (Ob) cells and in cells from posterior longitudinal ligament of non‐OPLL subjects. The region containing exons 6‐8 is an acidic subdomain presumably exposed to the surface that could interact with molecules of the extracellular matrix. Accordingly, retaining exon 7 together with removal of exon 6 observed in intron 6 (−4A) could play a protective role in the ectopic ossification process because the same pattern was observed in undifferentiated Ob cells and nonossified posterior longitudinal ligament cells.

Role of GLI2 in the growth of human osteosarcoma

The Hedgehog pathway functions as an organizer in embryonic development. Aberrant activation of the Hedgehog pathway has been reported in various types of malignant tumours. The GLI2 transcription factor is a key mediator of Hedgehog pathway but its contribution to neoplasia is poorly understood. To establish the role of GLI2 in osteosarcoma, we examined its expression by real‐time PCR using biopsy tissues. To examine the function of GLI2, we evaluated the growth of osteosarcoma cells and their cell cycle after GLI2 knockdown. To study the effect of GLI2 activation, we examined mesenchymal stem cell growth and the cell cycle after forced expression of GLI2. We found that GLI2 was aberrantly over‐expressed in human osteosarcoma biopsy specimens. GLI2 knockdown by RNA interferences prevented osteosarcoma growth and anchorage‐independent growth. Knockdown of GLI2 promoted the arrest of osteosarcoma cells in G1 phase and was accompanied by reduced protein expression of the cell cycle accelerators cyclin D1, SKP2 and phosphorylated Rb. On the other hand, knockdown of GLI2 increased the expression of p21cip1. In addition, over‐expression of GLI2 promoted mesenchymal stem cell proliferation and accelerated their cell cycle progression. Finally, evaluation of mouse xenograft models showed that GLI2 knockdown inhibited the growth of osteosarcoma in nude mice. Our findings suggest that inhibition of GLI2 may represent an effective therapeutic approach for patients with osteosarcoma. Copyright

Role of GOLPH3 and GOLPH3L in the proliferation of human rhabdomyosarcoma.

GOLPH3 was originally identified by proteomic analyses of Golgi proteins localized in the trans-Golgi network. Recently, it was reported that GOLPH3 is up-regulated in various types of malignancies, including melanoma, colon cancer and lung cancer. However, the mechanism through which GOLPH3 is involved in the pathogenesis of rhabdomyosarcoma remains unidentified. In order to explore the function of GOLPH3 and its isoform, GOLPH3L, in the pathogenesis of rhabdomyosarcoma, we investigated the expression and knockdown effects of GOLPH3 and GOLPH3L in human rhabdomyosarcoma. Western blot analysis and real-time PCR revealed that human rhabdomyosarcoma cell lines and biopsy specimens exhibited an increased expression of GOLPH3 and GOLPH3L. GOLPH3 and GOLPH3L knockdown by siRNA prevented the proliferation of human rhabdomyosarcoma cell lines. In addition, double-knockdown of GOLPH3 and GOLPH3L also prevented the proliferation of rhabdomyosarcoma cell lines. Our findings improve the understanding of rhabdomyosarcoma pathogenesis and suggest that the knockdown of GOLPH3 or GOLPH3L may be an effective treatment for rhabdomyosarcoma.

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: Functional analysis of p62 and autophagy in hypoxic neuronal cells

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Arsenic Trioxide Prevents Osteosarcoma Growth by Inhibition of GLI Transcription via DNA Damage Accumulation

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma.

GLI2 is a novel therapeutic target for metastasis of osteosarcoma.

Aberrant activation of the Hedgehog (Hh) pathway has been reported in several malignancies. We previously demonstrated that knockdown of GLI2 inhibited proliferation of osteosarcoma cells through regulation of the cell cycle. In this study, we analyzed the function of GLI2 in the pathogenesis of osteosarcoma metastasis. Immunohistochemical studies showed that GLI2 was overexpressed in patient osteosarcoma specimens. Knockdown of GLI2 inhibited migration and invasion of osteosarcoma cells. In contrast, the forced expression of constitutively active GLI2 in mesenchymal stem cells promoted invasion. In addition, xenograft models showed that knockdown of GLI2 decreased lung metastasis of osteosarcomas. To examine clinical applications, we evaluated the efficacy of arsenic trioxide (ATO), which is a Food and Drug Administration‐approved antitumor drug, on osteosarcoma cells. ATO treatment suppressed the invasiveness of osteosarcoma cells by inhibiting the transcriptional activity of GLI2. In addition, the combination of Hh inhibitors including ATO, vismodegib and GANT61 prevented migration and metastasis of osteosarcoma cells. Consequently, our findings suggested that GLI2 regulated metastasis as well as the progression of osteosarcomas. Inhibition of the GLI2 transcription may be an effective therapeutic method for preventing osteosarcoma metastasis.

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.