Kosei Ijiri

The control of chondrogenesis

Chondrogenesis is the earliest phase of skeletal development, involving mesenchymal cell recruitment and migration, condensation of progenitors, and chondrocyte differentiation, and maturation and resulting in the formation of cartilage and bone during endochondral ossification. This process is controlled exquisitely by cellular interactions with the surrounding matrix, growth and differentiation factors, and other environmental factors that initiate or suppress cellular signaling pathways and transcription of specific genes in a temporal‐spatial manner. Vertebrate limb development is controlled by interacting patterning systems involving prominently the fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and hedgehog pathways. Both positive and negative signaling kinases and transcription factors, such as Sox9 and Runx2, and interactions among them determine whether the differentiated chondrocytes remain within cartilage elements in articular joints or undergo hypertrophic maturation prior to ossification. The latter process requires extracellular matrix remodeling and vascularization controlled by mechanisms that are not understood completely. Recent work has revealed novel roles for mediators such as GADD45β, transcription factors of the Dlx, bHLH, leucine zipper, and AP‐1 families, and the Wnt/β‐catenin pathway that interact at different stages during chondrogenesis. J. Cell. Biochem.

Defining the roles of inflammatory and anabolic cytokines in cartilage metabolism

In osteoarthritis (OA), adult articular chondrocytes undergo phenotypic modulation in response to alterations in the environment owing to mechanical injury and inflammation. These processes not only stimulate the production of enzymes that degrade the cartilage matrix but also inhibit repair. With the use of in vitro and in vivo models, new genes, not known previously to act in cartilage, have been identified and their roles in chondrocyte differentiation during development and in dysregulated chondrocyte function in OA have been examined. These new genes include growth arrest and DNA damage (GADD)45β and the epithelial-specific ETS (ESE)-1 transcription factor, induced by bone morphogenetic protein (BMP)-2 and inflammatory cytokines, respectively. Both genes are induced by NF-κB, suppress COL2A1 and upregulate matrix meatalloproteinase-13 (MMP-13) expression. These genes have also been examined in mouse models of OA, in which discoidin domain receptor 2 is associated with MMP-13-mediated remodelling, in order to understand their roles in physiological cartilage homoeostasis and joint disease.

A novel role for GADD45beta as a mediator of MMP-13 gene expression during chondrocyte terminal differentiation

The growth arrest and DNA damage-inducible 45β (GADD45β) gene product has been implicated in the stress response, cell cycle arrest, and apoptosis. Here we demonstrated the unexpected expression of GADD45β in the embryonic growth plate and uncovered its novel role as an essential mediator of matrix metalloproteinase-13 (MMP-13) expression during terminal chondrocyte differentiation. We identified GADD45β as a prominent early response gene induced by bone morphogenetic protein-2 (BMP-2) through a Smad1/Runx2-dependent pathway. Because this pathway is involved in skeletal development, we examined mouse embryonic growth plates, and we observed expression of Gadd45β mRNA coincident with Runx2 protein in pre-hypertrophic chondrocytes, whereas GADD45β protein was localized prominently in the nucleus in late stage hypertrophic chondrocytes where Mmp-13 mRNA was expressed. In Gadd45β-/- mouse embryos, defective mineralization and decreased bone growth accompanied deficient Mmp-13 and Col10a1 gene expression in the hypertrophic zone. Transduction of small interfering RNA-GADD45β in epiphyseal chondrocytes in vitro blocked terminal differentiation and the associated expression of Mmp-13 and Col10a1 mRNA in vitro. Finally, GADD45β stimulated MMP-13 promoter activity in chondrocytes through the JNK-mediated phosphorylation of JunD, partnered with Fra2, in synergy with Runx2. These observations indicated that GADD45β plays an essential role during chondrocyte terminal differentiation.

Differential expression of GADD45β in normal and osteoarthritic cartilage: Potential role in homeostasis of articular chondrocytes

OBJECTIVE Our previous study suggested that growth arrest and DNA damage-inducible protein 45beta (GADD45beta) prolonged the survival of hypertrophic chondrocytes in the developing mouse embryo. This study was undertaken, therefore, to investigate whether GADD45beta plays a role in adult articular cartilage. METHODS Gene expression profiles of cartilage from patients with late-stage osteoarthritis (OA) were compared with those from patients with early OA and normal controls in 2 separate microarray analyses. Histologic features of cartilage were graded using the Mankin scale, and GADD45beta was localized by immunohistochemistry. Human chondrocytes were transduced with small interfering RNA (siRNA)-GADD45beta or GADD45beta-FLAG. GADD45beta and COL2A1 messenger RNA (mRNA) levels were analyzed by real-time reverse transcriptase-polymerase chain reaction, and promoter activities were analyzed by transient transfection. Cell death was detected by Hoechst 33342 staining of condensed chromatin. RESULTS GADD45beta was expressed at higher levels in cartilage from normal donors and patients with early OA than in cartilage from patients with late-stage OA. All chondrocyte nuclei in normal cartilage immunostained for GADD45beta. In early OA cartilage, GADD45beta was distributed variably in chondrocyte clusters, in middle and deep zone cells, and in osteophytes. In contrast, COL2A1, other collagen genes, and factors associated with skeletal development were up-regulated in late OA, compared with early OA or normal cartilage. In overexpression and knockdown experiments, GADD45beta down-regulated COL2A1 mRNA and promoter activity. NF-kappaB overexpression increased GADD45beta promoter activity, and siRNA-GADD45beta decreased cell survival per se and enhanced tumor necrosis factor alpha-induced cell death in human articular chondrocytes. CONCLUSION These observations suggest that GADD45beta might play an important role in regulating chondrocyte homeostasis by modulating collagen gene expression and promoting cell survival in normal adult cartilage and in early OA.

Surgical results and related factors for ossification of posterior longitudinal ligament of the thoracic spine: A multi-institutional retrospective study

Study Design. Retrospective multi-institutional study Objective. To describe the surgical outcomes in patients with ossification of the posterior longitudinal ligament in the thoracic spine (T-OPLL) and to clarify factors related to the surgical outcomes. Summary of Background Data. Detailed analyses of surgical outcomes of T-OPLL have been difficult because of the rarity of this disease. Methods. The subjects were 154 patients with T-OPLL who were surgically treated at 34 institutions between 1998 and 2002. The surgical procedures were laminectomy in 36, laminoplasty in 51, anterior decompression via anterior approach in 25 and via posterior approach in 29, combined anterior and posterior fusion in 8, and sternum splitting approach in 5 patients. Instrumentation was conducted in 52 patients. Assessments were made on (1) The Japanese Orthopedic Association (JOA) scores (full score, 11 points), its recovery rates, (2) factors related to surgical results, and (3) complications and their consequences. Results. (1) The mean JOA score before surgery was 4.6 ± 2.0 and, 7.1 ± 2.5 after surgery. The mean recovery rate was 36.8% ± 47.4%. (2) The recovery rate was 50% or higher in 72 patients (46.8%). Factors significantly related to this were location of the maximum ossification (T1–T4) (odds ratio, 2.43–4.17) and the use of instrumentation (odds ratio, 3.37). (3) The frequent complications were deterioration of myelopathy immediately after surgery in 18 (11.7%) and dural injury in 34 (22.1%) patients. Conclusion. The factors significantly associated with favorable surgical results were maximum ossification located at the upper thoracic spine and use of instrumentation. T-OPLL at the nonkyphotic upper thoracic spine can be treated by laminoplasty that is relatively a safe surgical procedure for neural elements. The use of instrumentation allows correction of kyphosis or prevention of progression of kyphosis, thereby, enhancing and maintaining decompression effect, and its use should be considered with posterior decompression.

Functional folate receptor beta-expressing macrophages in osteoarthritis synovium and their M1/M2 expression profiles

Objective: The distribution of folate receptor (FR)-β+ macrophages and their M1/M2 expression profiles were examined in osteoarthritis (OA) synovial tissues, and compared to those in rheumatoid arthritis (RA) synovial tissues and CD163+ macrophages in both OA and RA synovial tissues. Method: The phenotypes and fluorescein isothiocyanate (FITC)–folate uptake of FR-β+ synovial macrophages were analysed by flow cytometry. The distribution of FR-β+ macrophages in OA and RA synovial tissues was examined by immunofluorescent microscopy. Tumour necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), interleukin (IL)-10, and transforming growth factor (TGF)-β expression in FR-β+ macrophages was detected by double-immunostaining in both OA and RA synovial tissues. Results: FR-β+ macrophages were predominantly present in the synovial lining layer in OA patients. The proportion of CD163–FR-β+ cells in synovial mononuclear cells (MNCs) was increased in OA compared to RA synovial tissues. FR-βhigh macrophages from OA synovial tissues represented the majority of folic acid-binding cells. Although FR-β+ or CD163+ macrophages in the synovial tissues of OA and RA patients expressed a mixed pattern of M1 and M2 macrophage markers, there were more M2 markers expressing synovial macrophages in OA than in RA patients. Conclusions: The distribution and M1/M2 expression profiles of FR-β+ synovial macrophages were different between OA and RA synovial tissues. Thus, the findings underscore that the M1/M2 paradigm using surface markers FR-β and CD163 is an oversimplification of macrophage subsets. Functional FR-β present on OA synovial macrophages provides a potential tool for the diagnosis and treatment of OA.

ESE-1 is a potent repressor of type II collagen gene (COL2A1) transcription in human chondrocytes.

The epithelium‐specific ETS (ESE)‐1 transcription factor is induced in chondrocytes by interleukin‐1β (IL‐1β). We reported previously that early activation of EGR‐1 by IL‐1β results in suppression of the proximal COL2A1 promoter activity by displacement of Sp1 from GC boxes. Here we report that ESE‐1 is a potent transcriptional suppressor of COL2A1 promoter activity in chondrocytes and accounts for the sustained, NF‐κB‐dependent inhibition by IL‐1β. Of the ETS factors tested, this response was specific to ESE‐1, since ESE‐3, which was also induced by IL‐1β, suppressed COL2A1 promoter activity only weakly. In contrast, overexpression of ETS‐1 increased COL2A1 promoter activity and blocked the inhibition by IL‐1β. These responses to ESE‐1 and ETS‐1 were confirmed using siRNA‐ESE1 and siRNA‐ETS1. In transient cotransfections, the inhibitory responses to ESE‐1 and IL‐1β colocalized in the −577/−132 bp promoter region, ESE‐1 bound specifically to tandem ETS sites at −403/−381 bp, and IL‐1‐induced binding of ESE‐1 to the COL2A1 promoter was confirmed in vivo by ChIP. Our results indicate that ESE‐1 serves a potent repressor function by interacting with at least two sites in the COL2A1 promoter. However, the endogenous response may depend upon the balance of other ETS factors such as ETS‐1, and other IL‐1‐induced factors, including EGR‐1 at any given time. Intracellular ESE‐1 staining in chondrocytes in cartilage from patients with osteoarthritis (OA), but not in normal cartilage, further suggests a fundamental role for ESE‐1 in cartilage degeneration and suppression of repair. J. Cell. Physiol. 215: 562–573, 2008.

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

GADD45β Enhances Col10a1 Transcription via the MTK1/MKK3/6/p38 Axis and Activation of C/EBPβ-TAD4 in Terminally Differentiating Chondrocytes

GADD45β (growth arrest- and DNA damage-inducible) interacts with upstream regulators of the JNK and p38 stress response kinases. Previously, we reported that the hypertrophic zone of the Gadd45β−/− mouse embryonic growth plate is compressed, and expression of type X collagen (Col10a1) and matrix metalloproteinase 13 (Mmp13) genes is decreased. Herein, we report that GADD45β enhances activity of the proximal Col10a1 promoter, which contains evolutionarily conserved AP-1, cAMP-response element, and C/EBP half-sites, in synergism with C/EBP family members, whereas the MMP13 promoter responds to GADD45β together with AP-1, ATF, or C/EBP family members. C/EBPβ expression also predominantly co-localizes with GADD45β in the embryonic growth plate. Moreover, GADD45β enhances C/EBPβ activation via MTK1, MKK3, and MKK6, and dominant-negative p38αapf, but not JNKapf, disrupts the combined trans-activating effect of GADD45β and C/EBPβ on the Col10a1 promoter. Importantly, GADD45β knockdown prevents p38 phosphorylation while decreasing Col10a1 mRNA levels but does not affect C/EBPβ binding to the Col10a1 promoter in vivo, indicating that GADD45β influences the transactivation function of DNA-bound C/EBPβ. In support of this conclusion, we show that the evolutionarily conserved TAD4 domain of C/EBPβ is the target of the GADD45β-dependent signaling. Collectively, we have uncovered a novel molecular mechanism linking GADD45β via the MTK1/MKK3/6/p38 axis to C/EBPβ-TAD4 activation of Col10a1 transcription in terminally differentiating chondrocytes.

Neurological complications of cervical laminoplasty for patients with ossification of the posterior longitudinal ligament - A multi-institutional retrospective study

Study Design. Retrospective multi-institutional study. Objective. To investigate the incidence of neurological deficits after cervical laminoplasty for ossification of the posterior longitudinal ligament (OPLL). Summary of Background Data. According to analysis of long-term results, laminoplasty for cervical OPLL has been reported as a safe and effective alternative procedure with few complications. However, perioperative neurological complication rates of laminoplasty for cervical OPLL have not been well described. Methods. Subjects comprised 581 patients (458 men and 123 women; mean age: 62 ± 10 years; range: 30–86 years) who had undergone laminoplasty for cervical OPLL at 27 institutions between 2005 and 2008. Continuous-type OPLL was seen in 114, segmental-type in 146, mixed-type in 265, local-type in 24, and not judged in 32 patients. Postoperative neurological complications within 2 weeks after laminoplasty were analyzed in detail. Cobb angle between C2 and C7 (C2/C7 angle), maximal thickness, and occupying rate of OPLL were investigated. Pre- and postoperative magnetic resonance imaging was performed on patients with postoperative neurological complications. Results. Open-door laminoplasty was conducted in 237, double-door laminoplasty in 311, and other types of laminoplasty in 33 patients. Deterioration of lower-extremity function occurred after laminoplasty in 18 patients (3.1%). Causes of deterioration were epidural hematoma in 3, spinal cord herniation through injured dura mater in 1, incomplete laminoplasty due to vertebral artery injury while making a trough in 1, and unidentified in 13 patients. Prevalence of unsatisfactory recovery not reaching preoperative level by 6-month follow-up was 7/581 (1.2%). Mean occupying rate of OPLL for patients with deteriorated lower-extremity function was 51.2 ± 13.6% (range, 21.0%–73.3%), significantly higher than the 42.3 ± 13.0% for patients without deterioration. OPLL thickness was also higher in patients with deterioration (mean, 6.6 ± 2.2 mm) than in those without deterioration (mean, 5.7 ± 2.0 mm). No significant difference in C2/C7 lordotic angle was seen between groups. Conclusion. Although most neurological deterioration can be expected to recover to some extent, the frequency of short-term neurological complications was higher than the authors expected.