Akiro Higashikawa

Phosphorylation of GSK-3β by cGMP-dependent protein kinase II promotes hypertrophic differentiation of murine chondrocytes

cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase-3beta (GSK-3beta; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3beta was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3beta. Analyses of mice with compound deficiencies in both protein kinases (Prkg2(-/-)Gsk3b(+/-)) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that beta-catenin levels decreased in Prkg2(-/-) mice, while overexpression of cGKII increased the accumulation and transactivation function of beta-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3beta. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3beta by cGKII.

Identification of the core element responsive to runt-related transcription factor 2 in the promoter of human type x collagen gene

OBJECTIVE Type X collagen and runt-related transcription factor 2 (RUNX-2) are known to be important for chondrocyte hypertrophy during skeletal growth and repair and development of osteoarthritis (OA) in mice. Aiming at clinical application, this study was undertaken to investigate transcriptional regulation of human type X collagen by RUNX-2 in human cells. METHODS Localization of type X collagen and RUNX-2 was determined by immunohistochemistry, and their functional interaction was examined in cultured mouse chondrogenic ATDC-5 cells. Promoter activity of the human type X collagen gene (COL10A1) was examined in human HeLa, HuH7, and OUMS27 cells transfected with a luciferase gene containing a 4.5-kb promoter and fragments. Binding to RUNX-2 was examined by electrophoretic mobility shift assay and chromatin immunoprecipitation. RESULTS RUNX-2 and type X collagen were co-localized in mouse limb cartilage and bone fracture callus. Gain and loss of function of RUNX-2 revealed that RUNX-2 is essential for type X collagen expression and terminal differentiation of chondrocytes. Human COL10A1 promoter activity was enhanced by RUNX-2 alone and more potently by RUNX-2 in combination with the coactivator core-binding factor beta in all 3 human cell lines examined. Deletion, mutagenesis, and tandem repeat analyses identified the core responsive element as the region between -89 and -60 bp (termed the hypertrophy box [HY box]), which showed specific binding to RUNX-2. Other putative RUNX-2 binding motifs in the human COL10A1 promoter did not respond to RUNX-2 in human cells. CONCLUSION Our findings indicate that the HY box is the core element responsive to RUNX-2 in human COL10A1 promoter. Studies on molecular networks related to RUNX-2 and the HY box will lead to treatments of skeletal growth retardation, bone fracture, and OA.

Kruppel-like factor 5 causes cartilage degradation through transactivation of matrix metalloproteinase 9.

Although degradation of cartilage matrix has been suggested to be a rate-limiting step for endochondral ossification during skeletal development, little is known about the transcriptional regulation. This study investigated the involvement of KLF5 (Krüppel-like factor 5), an Sp/KLF family member, in the skeletal development. KLF5 was expressed in chondrocytes and osteoblasts but not in osteoclasts. The heterozygous deficient (KLF5+/-) mice exhibited skeletal growth retardation in the perinatal period. Although chondrocyte proliferation and differentiation were normal, cartilage matrix degradation was impaired in KLF5+/- mice, causing delay in replacement of cartilage with bone at the primary ossification center in the embryonic limbs and elongation of hypertrophic chondrocyte layer in the neonatal growth plates. Microarray analyses identified MMP9 (matrix metalloproteinase 9) as a transcriptional target, since it was strongly up-regulated by adenoviral transfection of KLF5 in chondrogenic cell line OUMS27. The KLF5 overexpression caused gelatin degradation by stimulating promoter activity of MMP9 without affecting chondrocyte differentiation or vascular endothelial growth factor expression in the culture of chondrogenic cells; however, in osteoclast precursors, it affected neither MMP9 expression nor osteoclastic differentiation. KLF5 dysfunction by genetic heterodeficiency or RNA interference was confirmed to cause reduction of MMP9 expression in cultured chondrogenic cells. MMP9 expression was decreased in the limbs of KLF5+/- embryos, which was correlated with suppression of matrix degradation, calcification, and vascularization. We conclude that KLF5 causes cartilage matrix degradation through transcriptional induction of MMP9, providing the first evidence that transcriptional regulation of a proteinase contributes to endochondral ossification and skeletal development.

Neurologic Level Diagnosis of Cervical Stenotic Myelopathy

Study Design. A cross-sectional analysis. Objective. To elucidate the accuracy of neurologic level diagnosis of cervical stenotic myelopathy. Summary of Background Data. Neurologic level diagnosis in cervical myelopathy has not been well established. Methods. A total of 106 patients with cervical stenotic myelopathy, with a single-level intramedullary high-intensity area confirmed on both preoperative and postoperative T2-weighted magnetic resonance imaging (MRI), were included in this study. We performed a level diagnosis on the basis of neurologic signs (the uppermost muscle with weakness, diminished or exaggerated deep tendon reflex, the uppermost level of sensory disturbance of the upper extremities) and compared it with a level diagnosis made by T2-weighted MRI. The sensitivity, specificity, and accuracy of neurologic signs on our index corresponding to each intervertebral level were calculated. Results. The averages of sensitivity, specificity, and accuracy were 42%, 80%, and 70%, respectively, in the uppermost muscle with weakness, 66%, 89%, and 83% in deep tendon reflex, and 74%, 91%, and 87% in the sensory disturbance area. The positive and negative predictive values were 40% and 91%, respectively, in the uppermost muscle with weakness, 66% and 89% in deep tendon reflex, and 74% and 91% in the sensory disturbance area. Accuracy of a diagnosis based on muscle weakness was less high, the reason being that in many patients, the uppermost muscle with weakness was extensor digiti communis or the intrinsic muscles of the hands, and this led to a lower sensitivity. Conclusions. The average accuracy of neurologic level diagnosis based on the index we proposed was ≥70%. The level diagnosis by a sensory disturbance area showed the highest accuracy (87%).

Akt1 in murine chondrocytes controls cartilage calcification during endochondral ossification under physiologic and pathologic conditions.

OBJECTIVE To examine the role of the phosphoinositide-dependent serine/threonine protein kinase Akt1 in chondrocytes during endochondral ossification. METHODS Skeletal phenotypes of homozygous Akt1-deficient (Akt1(-/-)) mice and their wild-type littermates were compared in radiologic and histologic analyses. An experimental osteoarthritis (OA) model was created by surgically inducing instability in the knee joints of mice. For functional analyses, we used primary costal and articular chondrocytes from neonatal mice and mouse chondrogenic ATDC5 cells with retroviral overexpression of constitutively active Akt1 or small interfering RNA (siRNA) for Akt1. RESULTS Among the Akt isoforms (Akt1, Akt2, and Akt3), Akt1 was the most highly expressed in chondrocytes, and the total level of Akt protein was decreased in Akt1(-/-) chondrocytes, indicating a dominant role of Akt1. Akt1(-/-) mice exhibited dwarfism with normal proliferative and hypertrophic zones but suppressed cartilage calcification in the growth plate compared with their wild-type littermates. In mice with surgically induced OA, calcified osteophyte formation, but not cartilage degradation, was prevented in the Akt1(-/-) joints. Calcification was significantly suppressed in cultures of Akt1(-/-) chondrocytes or ATDC5 cells overexpressing siRNA for Akt1 and was enhanced in ATDC5 cells overexpressing constitutively active Akt1. Neither proliferation nor hypertrophic differentiation was affected by the gain or loss of function of Akt1. The expression of ANK and nucleotide pyrophosphatase/phosphodiesterase 1, which accumulate pyrophosphate, a crucial calcification inhibitor, was enhanced by Akt1 deficiency or siRNA for Akt1 and was suppressed by constitutively active Akt1. CONCLUSION Our findings indicate that Akt1 in chondrocytes controls cartilage calcification by inhibiting pyrophosphate during endochondral ossification in skeletal growth and during osteophyte formation in OA.

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1−/−) or when osteoblasts were from PTH1R−/− mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010.

Aorta movement in patients with scoliosis after posterior surgery.

Study Design. Retrospective analysis. Objective. To evaluate movement of the aorta in patients with scoliosis who have undergone the posterior correction and fusion. Summary of Background Data. Surgeons check preoperative imaging for pedicle screw placement, but past analyses indicated that the aorta shifts after scoliosis surgery. Few studies, however, evaluated the aorta movement in detail. Methods. A total of 22 patients with a right thoracic curve underwent posterior instrumentation and fusion. The average age at surgery was 17.2 years. The average of the preoperative Cobb angle was 65.2° which decreased to 20.0°. Computed-tomographic data were analyzed by multiplanar reconstruction. In our coordinate system, the middle of the base of the left superior facet was set as the origin and a line connecting the middle points of both bases of the superior facets was defined as the X-axis. We defined the angle and the distance to describe the aorta position and analyzed the movement of the aorta relative to the spine. Deformity parameters were examined to determine their correlation with the aorta parameters. We simulated variable pedicle screw placement and defined a warning pedicle when the aorta enters the expected area of the screw and examined them in 24 scenarios. Results. The aorta moved 4.7 ± 3.0 mm on an average. The aorta had a tendency to migrate in the anteromedial direction and this movement correlated with preoperative apical vertebral translation, preoperative sagittal alignment, and change of sagittal alignment. The ratio of warning pedicles at the middle thoracic level (T7–T9) increased after deformity correction. Conclusion. The aorta moved anteromedially relative to the spine after the posterior correction and the risk of the aorta by a pedicle screw increased by correction of the deformity at the middle thoracic spine. Surgeons are recommended to anticipate the aorta movement in the surgical planning.

The relationship between findings on magnetic resonance imaging and previous history of low back pain

The objective of this study was to evaluate the relationship between magnetic resonance imaging (MRI) findings and previous low back pain (LBP) in participants without current LBP. Current LBP was defined as LBP during the past month. Previous LBP was defined as a history of medical consultation for LBP. Ninety-one participants without current LBP were included. Sagittal T2-weighted MRI was used to assess the intervertebral space from T12/L1 to L5/S1. These images were classified into five grades based on the Pfirrmann grading system. Furthermore, we evaluated the presence of disk bulging, high-intensity zone, and spondylolisthesis. We compared the MRI findings between groups with (27 participants) and without (64 participants) previous LBP without current LBP. Intraobserver and interobserver kappa values were evaluated. Participants had an average age of 34.9 years; 47 were female and 44 were male; and their average body mass index was 21.8 kg/m2. Compared to the group of participants without previous LBP, the group of participants with previous LBP had a significantly higher incidence of disk degeneration such as a Pfirrmann grade ≥3, disk bulging, and high-intensity zone in the analyses adjusted by age and sex. There were no significant differences in spondylolisthesis between the groups. An odds ratio of >10 was only found for Pfirrmann grade ≥3, ie, a Pfirrmann grade ≥3 was strongly associated with a history of previous LBP in participants without current LBP.

Validation study of a diagnostic scoring system for sacroiliac joint-related pain

Background There are no specific radiological findings for the diagnosis of sacroiliac joint-related pain. A diagnostic scoring system had been developed in 2017. The score comprised the sum of scores of six items. The score ranged from 0 to 9 points, and the cutoff was calculated as 4. Objective To evaluate the validity of the diagnostic scoring system for sacroiliac joint-related pain. Patients and methods The sacroiliac joint-related pain group (n=31) comprised patients diagnosed with sacroiliac joint-related pain based on patient history, physical findings, and responses to analgesic periarticular injection. In addition, it was confirmed that they had no other lumbar or hip joint diseases. The non-sacroiliac joint-related pain group (n=123) comprised patients with low back pain due to a reason other than sacroiliac joint-related pain. We evaluated scores for all subjects. We analyzed the differences in each item between both groups and performed receiver-operating characteristic curve analysis to evaluate the score validity. Results There were no significant differences in patient characteristics between groups. There were significant differences for the following four of six items: one-finger test results (P<0.0001), pain while sitting on a chair (P=0.0141), sacroiliac joint shear test results (P<0.0001), and tenderness of the posterosuperior iliac spine (P<0.0001). The cut-off value was 5 points, the area under the curve was 0.80239, sensitivity was 77.4%, and specificity was 76.4%. Conclusion The score demonstrated moderate validity for diagnosing sacroiliac joint-related pain.

The associations between magnetic resonance imaging findings and low back pain: A 10-year longitudinal analysis.

Purpose To conduct a 10-year longitudinal analysis of the relationship between magnetic resonance imaging (MRI) findings and low back pain (LBP). Materials and methods Ninety-one volunteers with a history of LBP, but without current LBP were recruited between 2005 and 2006. Participants’ baseline demographics and MRI findings were recorded. All volunteers were invited for a follow-up MRI in 2016; of these, 49 volunteers (53.8%) participated in the follow-up. We enquired whether they had LBP history during the 10 years between the baseline and follow-up examinations. Sagittal T1 and T2-weighted MRI were used to assess the intervertebral space from T12/L1 to L5/S1. We evaluated the presence of disc degeneration by Pfirrmann’s grading system, disc bulging, high intensity zone (HIZ), spondylolisthesis, and any type of Modic changes in the follow-up MRIs. We compared the follow-up MRI findings with the baseline findings; the progress of each finding over the 10 years were also compared between the groups with (n = 36) and without (n = 13) LBP. Results Average age of the study participants at follow-up was 44.8 years; 25 were female and 24 were male. Average age, sex, body mass index, and smoking habits of those who did and did not participate in the follow-up study, as well as the demographic characteristics of those who did and did not have LBP history during the 10 years, were not significantly different. Compared with the group without LBP history, the group that had LBP history during the 10 years did not have a significantly increased prevalence of disc degeneration, disc bulging, and HIZ in the follow-up and baseline MRIs. Spondylolisthesis and any type of Modic changes were also not associated with LBP history during the 10 years. Conclusions Follow-up MRI findings consistent with Pfirrmann grading ≥4, disc bulging, HIZ, spondylolisthesis, and any type of Modic changes were not associated with LBP history during the 10 years between the baseline and follow-up study. The progresses of these findings were also not associated with the LBP history. In addition, baseline MRI findings were not associated with LBP history during the 10 years; therefore, our data suggest that baseline MRI findings cannot predict future LBP.