Kazuhiro Chiba

Comparative study of spinopelvic sagittal alignment between patients with and without degenerative spondylolisthesis.

IntroductionTo date, few studies have focused on spinopelvic sagittal alignment as a predisposing factor for the development of degenerative spondylolisthesis (DS). The objectives of this study were to compare differences in spinopelvic sagittal alignment between patients with or without DS and to elucidate factors related to spinopelvic sagittal alignment.Materials and methodsA total of 100 patients with or without DS who underwent surgery for lumbar spinal canal stenosis were assessed in this study. Fifty patients with DS (DS group) and 50 age- and gender-matched patients without DS (non-DS group) were enrolled. Spinopelvic parameters including pelvic incidence (PI), sacral slope (SS), pelvic tilt (PT), L4 slope, L5 slope, thoracic kyphosis (TK), lumbar lordosis (LL) and sagittal balance were compared between the two groups. In the DS group, the percentage of vertebral slip (% slip) was also measured.ResultsSeveral spinopelvic parameters, PI, SS, L4 slope, L5 slope, TK and LL, in the DS group were significantly greater than those in the non-DS group, and PI had positive correlation with % slip (rxa0=xa00.35, pxa0<xa00.05). Degrees of correlations among spinopelvic parameters differed between the two groups. In the DS group, PI was more strongly correlated with SS (rxa0=xa00.82, pxa0<xa00.001) than with PT (rxa0=xa00.41, pxa0<xa00.01). In the non-DS group, PI was more strongly correlated with PT (rxa0=xa00.73, pxa0<xa00.001) than with SS (rxa0=xa00.38, pxa0<xa00.01).ConclusionsGreater PI may lead to the development and the progression of vertebral slip. Different compensatory mechanisms may contribute to the maintenance of spinopelvic sagittal alignment in DS and non-DS patients.

Prolyl Hydroxylase 3 (PHD3) Modulates Catabolic Effects of Tumor Necrosis Factor-α (TNF-α) on Cells of the Nucleus Pulposus through Co-activation of Nuclear Factor κB (NF-κB)/p65 Signaling

Background: The regulation of PHD expression and function under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of PHD3 is regulated by TNF-α and IL-1β. PHD3 controls TNF-α activity by modulating NF-κB signaling. Conclusion: PHD3 promotes the catabolic effects of TNF-α on nucleus pulposus cells. Significance: PHD3 may play an important role in pathogenesis of disc disease. Recent studies suggest a differential role of prolyl hydroxylase (PHD) isoforms in controlling hypoxia-inducible factor (HIF)-α degradation and activity in nucleus pulposus (NP) cells. However, the regulation and function of PHDs under inflammatory conditions that characterize disc disease are not yet known. Here, we show that in NP cells, TNF-α and IL-1β induce PHD3 expression through NF-κB. Lentiviral delivery of Sh-p65 and Sh-IKKβ confirms that cytokine-mediated PHD3 expression is NF-κB-dependent. It is noteworthy that although both cytokines induce HIF activity, mechanistic studies using Sh-HIF-1α and PHD3 promoter/enhancer constructs harboring well characterized hypoxia response element (HRE) show lack of HIF involvement in cytokine-mediated PHD3 expression. Loss-of-function studies clearly indicate that PHD3 serves as a co-activator of NF-κB signaling activity in NP cells; PHD3 interacts with, and co-localizes with, p65. We observed that when PHD3 is silenced, there is a significant decrease in TNF-α-induced expression of catabolic markers that include ADAMTS5, syndecan4, MMP13, and COX2, and at the same time, there is restoration of aggrecan and collagen type II expression. It is noteworthy that hydroxylase function of PHDs is not required for mediating cytokine-dependent gene expression. These findings show that by enhancing the activity of inflammatory cytokines, PHD3 may serve a critical role in degenerative disc disease.

A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.

Identification of a susceptibility locus for severe adolescent idiopathic scoliosis on chromosome 17q24.3.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity, affecting around 2% of adolescents worldwide. Genetic factors play an important role in its etiology. Using a genome-wide association study (GWAS), we recently identified novel AIS susceptibility loci on chromosomes 10q24.31 and 6q24.1. To identify more AIS susceptibility loci relating to its severity and progression, we performed GWAS by limiting the case subjects to those with severe AIS. Through a two-stage association study using a total of ∼12,000 Japanese subjects, we identified a common variant, rs12946942 that showed a significant association with severe AIS in the recessive model (Pu200a=u200a4.00×10−8, odds ratio [OR]u200a=u200a2.05). Its association was replicated in a Chinese population (combined Pu200a=u200a6.43×10−12, ORu200a=u200a2.21). rs12946942 is on chromosome 17q24.3 near the genes SOX9 and KCNJ2, which when mutated cause scoliosis phenotypes. Our findings will offer new insight into the etiology and progression of AIS.

Japanese orthopaedic association back pain evaluation questionnaire (JOABPEQ) as an outcome measure for patients with low back pain: reference values in healthy volunteers

BackgroundIn 2007, the Japanese orthopaedic association back pain evaluation questionnaire (JOABPEQ) was established to overcome the limitations of the original JOA scoring system developed in 1986. Although this new self-administered questionnaire is a more accurate outcome measure for evaluating patients with low back pain, physicians were unable to as certain the exact status of a patient at a single time point because of a lack of reference values. This study aimed to establish the reference values of JOABPEQ in different age and gender groups using data obtained from healthy volunteers.MethodsThis study was conducted in 21 university hospitals and affiliated hospitals from October 2012 to July 2013. The JOABPEQ includes 25 questions that yield five domains to evaluate individuals with low back pain from five different perspectives. A total of 1,456 healthy volunteers (719 men, 737 women; age range, 20–89xa0years) answered the questionnaire. The differences in scores according to age and gender were examined by non-parametric tests.ResultsThe JOABPEQ scores significantly decreased with age in the domains of lumbar spine dysfunction, gait disturbance, and social life dysfunction. In these three domains, the median scores approached the 100 possible points in individuals aged 20–70 for both genders. However, the median scores for lumbar spine dysfunction and social life dysfunction decreased to 83.0 and 65.0–78.0 points, respectively, in individuals in their 80xa0s and 70–80xa0s, respectively; and the scores for gait disturbance decreased to 93.0 and 71.0 points for males and females in their 80xa0s. Overall, the median scores for pain-related and psychological disorders were 100 and 60.0–72.0 points, respectively.ConclusionThe reference values for JOABPEQ according to age and gender were established herein. Patients with low back pain should be evaluated with this new self-administered questionnaire taking these reference values into account.

HIF-1-PHD2 axis controls expression of syndecan 4 in nucleus pulposus cells

Intervertebral disc degeneration is the leading cause of chronic back pain. Recent studies show that raised level of SDC4, a cell‐surface heparan sulfate (HS) proteoglycan, plays a role in pathogenesis of disc degeneration. However, in nucleus pulposus (NP) cells of the healthy intervertebral disc, the mechanisms that control expression of SDC4 and its physiological function are unknown. Hypoxia induced SDC4 mRNA and protein expression by ~2.4‐ and 4.4‐fold (P<0.05), respectively, in NP cells. While the activity of the SDC4 promoter containing hypoxia response element (HRE) was induced 2‐fold (P<0.05), the HRE mutation decreased the activity by 40% in hypoxia. Transfections with plasmids coding prolyl‐4‐hydroxylase domain protein 2 (PHD2) and ShPHD2 show that hypoxic expression of SDC4 mRNA and protein is regulated by PHD2 through controlling hypoxia‐inducible factor 1α (HIF‐1α) levels. Although overexpression of HIF‐1 α significantly increased SDC4 protein levels, stable suppression of HIF‐1α and HIF‐1β decreased SDC4 expression by 50% in human NP cells. Finally, suppression of SDC4 expression, as well as HS function, resulted in an ~2‐fold increase in sex‐determining region Y (SRY)‐box 9 (Sox9) mRNA, and protein (P<0.05) and simultaneous increase in Sox9 transcriptional activity and target gene expression. Taken together, our findings suggest that in healthy discs, SDC4, through its HS side chains, contributes to maintenance of the hypoxic tissue niche by controlling baseline expression of Sox9.—Fujita, N., Hirose, Y., Tran, C. M., Chiba, K., Miyamoto, T., Toyama, Y., Shapiro, I. M., Risbud, M. V. HIF‐1‐PHD2 axis controls expression of syndecan 4 in nucleus pulposus cells. FASEB J. 28, 2455–2465 (2014). www.fasebj.org

FIH-1-Mint3 Axis Does Not Control HIF-1α Transcriptional Activity in Nucleus Pulposus Cells

Background: Cells of the hypoxic nucleus pulposus (NP) require tightly regulated HIF-1 signaling for proper function. Results: Although overexpressed FIH-1 can regulate HIF-1 activity in NP cells, silencing endogenous FIH-1 results in no change in HIF-1 target gene expression. Conclusion: FIH-1 does not represent a major mechanism of controlling HIF-1-dependent transcription in NP cells. Significance: This study describes a physiological adaptation of NP cells. The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxia-sensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1α-C-TAD and HIF-2α-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.

Matrix metalloproteinase 13 in the ligamentum flavum from lumbar spinal canal stenosis patients with and without diabetes mellitus

BackgroundLumbar spinal canal stenosis (LSCS) is one of the most common spinal disorders in the elderly, and ligamentum flavum (LF) hypertrophy is an important cause of LSCS. Matrix metalloproteinase 13 (MMP13) can degrade fibrillar collagens and elastic microfibrils, and is involved in inflammation and fibrosis. The purpose of this study was to compare the expression of MMP13 in the LF from LSCS patients with diabetes mellitus [DM (+)] with that in the LF from patients without DM [DM (−)] and to analyze the relationship among DM, MMP13 expression, and LF hypertrophy.MethodsLFs from 11 DM (+) and 24 DM (−) LSCS patients were analyzed in this study. Histology analysis using hematoxylin and eosin and Masson’s trichrome stain was performed for each LF. The expression of MMP13 was analyzed by quantitative real-time PCR. The thickness of LF was measured by CT.ResultsIn the LF from DM (+) LSCS patients, the elastic fibers were more disorganized and had lower volumes than in the LF from DM (−) LSCS patients, while more fibrotic tissue was observed in the LF from DM (+) than from DM (−) LSCS patients. MMP13 expression was significantly higher in the LF from DM (+) LSCS patients (0.46xa0±xa00.61 vs. 0.05xa0±xa00.09, Pxa0=xa00.002). The LF from the DM (+) LSCS patients was significantly thicker than that from the DM (−) LSCS patients (5.0xa0±xa00.9 vs. 3.1xa0±xa00.8xa0mm, Pxa0<xa00.01), and the thickness was correlated with the expression of MMP13 (correlation coefficientxa0=xa00.43, Pxa0=xa00.01, Pearsons correlation test).ConclusionDM-related MMP13 expression can be one of the factors contributing to fibrosis and hypertrophy of the LF. Further research on the mechanism of this process may lead to new therapies for LF hypertrophy.

GRIP1 enhances estrogen receptor α-dependent extracellular matrix gene expression in chondrogenic cells

OBJECTIVEnThe role of postmenopause on the pathogenesis of cartilage degeneration has been an open question. We assessed cartilage degeneration in estrogen receptor (ER)alpha null mice and examined the role of glucocorticoid receptor-interacting protein 1 (GRIP1) in the ERalpha-dependent transcription of a type II collagen gene (col2a1) with special reference to a crosstalk with the transforming growth factor (TGF)-beta signaling pathway.nnnMETHODSnThe vertebral cartilaginous endplate from female ERalpha null mice was subjected to histological analyses. Col2a1 expression of primary chondrocytes (PCs) obtained from ERalpha null mice after 17beta-estradiol (E(2)) and TGF-beta1 stimulation was examined by reverse transcription polymerase chain reaction (RT-PCR). Estrogen response element (ERE) or col2a1 promoter-enhancer luciferase reporter system was used to investigate the crosstalk among ERalpha, GRIP1, and MKK6. Col2a1 expression and glycosaminoglycan (GAG) content were measured in ATDC5 cells treated with GRIP1 small interfering RNA (siRNA).nnnRESULTSnERalpha deficiency clearly accelerated impairment of the vertebral cartilaginous endplate. E(2) and TGF-beta1 stimulation increased col2a1 expression in PC from wild-type mice, but not that from ERalpha null mice. The same stimulation increased the col2a1 promoter-enhancer reporter activity, and the elevated activity was decreased by dominant-negative ERalpha and p38 mitogen-activated protein kinase (MAPK) inhibitor. GRIP1 increased the E(2)-dependent ERE activation in the presence of ERalpha and constitutive-active MKK6. GRIP1 siRNA repressed col2a1 expression and GAG production in ATDC5 cells.nnnCONCLUSIONSnCrosstalks between ERalpha/GRIP1 and TGF-beta/MKK6/p38 MAPK pathway have protective roles on cartilage metabolism via regulating the extracellular matrices expression. The finding may lead to the development of a novel therapeutic approach for cartilage degeneration.