X-Ray Excited Optical Luminescence and Portable Electron Probe Microanalyzer-Cathodoluminescence (EPMA-CL) Analyzers for On-Line and On-Site Analysis of Nonmetallic Inclusions in Steel.

The potential of the application of an X-ray excited optical luminescence (XEOL) analyzer and portable analyzers, composed of a cathodoluminescence (CL) spectrometer and electron probe microanalyzer (EPMA), to the on-line and on-site analysis of nonmetallic inclusions in steel is investigated as the first step leading to their practical use. MgAl2O4 spinel and Al2O3 particles were identified by capturing the luminescence as a result of irradiating X-rays in air on a model sample containing MgAl2O4 spinel and Al2O3 particles in the size range from 20 to 50 µm. We were able to identify the MgAl2O4 spinel and Al2O3 particles in the same sample using the portable CL spectrometer. In both cases, not all of the particles in the sample were identified because the luminescence intensities of the smaller Al2O3 in particular were too low to detect. These problems could be solved by using an X-ray tube with a higher power and increasing the beam current of the portable CL spectrometer. The portable EPMA distinguished between the MgAl2O4 spinel and Al2O3 particles whose luminescent colors were detected using the portable CL spectrometer. Therefore, XEOL analysis has potential for the on-line analysis of nonmetallic inclusions in steel if we have information on the luminescence colors of the nonmetallic inclusions. In addition, a portable EPMA-CL analyzer would be able to perform on-site analysis of nonmetallic inclusions in steel.

The ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation.

The severe defects in growth plate development caused by chondrocyte extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) gain or loss-of-function suggest that tight spatial and temporal regulation of mitogen-activated protein kinase signaling is necessary to achieve harmonious growth plate elongation and structure. We provide here evidence that neurofibromin, via its Ras guanosine triphosphatase -activating activity, controls ERK1/2-dependent fibroblast growth factor receptor (FGFR) signaling in chondrocytes. We show first that neurofibromin is expressed in FGFR-positive prehypertrophic and hypertrophic chondrocytes during growth plate endochondral ossification. Using mice lacking neurofibromin 1 (Nf1) in type II collagen-expressing cells, (Nf1col2(-/-) mutant mice), we then show that lack of neurofibromin in post-mitotic chondrocytes triggers a number of phenotypes reminiscent of the ones observed in mice characterized by FGFR gain-of-function mutations. Those include dwarfism, constitutive ERK1/2 activation, strongly reduced Ihh expression and decreased chondrocyte proliferation and maturation, increased chondrocytic expression of Rankl, matrix metalloproteinase 9 (Mmp9) and Mmp13 and enhanced growth plate osteoclastogenesis, as well as increased sensitivity to caspase-9 mediated apoptosis. Using wildtype (WT) and Nf1(-/-) chondrocyte cultures in vitro, we show that FGF2 pulse-stimulation triggers rapid ERK1/2 phosphorylation in both genotypes, but that return to the basal level is delayed in Nf1(-/-) chondrocytes. Importantly, in vivo ERK1/2 inhibition by daily injection of a recombinant form of C-type natriuretic peptide to post-natal pups for 18 days was able to correct the short stature of Nf1col2(-/-) mice. Together, these results underscore the requirement of neurofibromin and ERK1/2 for normal endochondral bone formation and support the notion that neurofibromin, by restraining RAS-ERK1/2 signaling, is a negative regulator of FGFR signaling in differentiating chondrocytes.

Mice lacking Nf1 in osteochondroprogenitor cells display skeletal dysplasia similar to patients with neurofibromatosis type I.

Mutations in NF1 cause neurofibromatosis type I (NF1), a disorder characterized, among other clinical manifestations, by generalized and focal bony lesions. Dystrophic scoliosis and tibial pseudoarthrosis are the most severe skeletal manifestations for which treatment is not satisfactory, emphasizing the dearth of knowledge related to the biology of NF1 in bone cells. Using reporter mice, we report here that the mouse Col2α1-Cre promoter (collagen, type II, alpha 1) is active not only in chondrocytes but also in adult bone marrow osteoprogenitors giving rise to osteoblasts. Based on this finding, we crossed the Col2α1-Cre transgenic and Nf1(flox/flox) mice to determine whether loss of Nf1 in axial and appendicular osteochondroprogenitors recapitulates the skeletal abnormalities of NF1 patients. By microtomographic and X-rays studies, we show that Nf1(Col2)(-/-) mice display progressive scoliosis and kyphosis, tibial bowing and abnormalities in skull and anterior chest wall formation. These defects were accompanied by a low bone mass phenotype, high bone cortical porosity, osteoidosis, increased osteoclastogenesis and decreased osteoblast number, as quantified by histomorphometry and 3D-microtomography. Loss of Nf1 in osteochondroprogenitors also caused severe short stature and intervertebral disc defects. Blockade of the RAS/ERK activation characteristic of Nf1(-/-) osteoprogenitors by lovastatin during embryonic development could attenuate the increased cortical porosity observed in mutant pups. These data and the skeletal similarities between this mouse model and NF1 patients thus suggest that activation of the RAS/ERK pathway by Nf1 loss-of-function in osteochondroprogenitors is responsible for the vertebral and tibia lesions in NF1 patients, and that this molecular signature may represent a good therapeutic target.

Percutaneous Endoscopic Transforaminal Lumbar Interbody Fusion (PETLIF): Current Techniques, Clinical Outcomes, and Narrative Review.

Full endoscopic techniques are becoming more popular for degenerative lumbar pathologies. Percutaneous endoscopic lumbar interbody fusion (PETLIF) is a minimally invasive surgical technique for spondylolisthesis and lumbar spinal canal stenosis with instability. Nagahama first introduced PETLIF in 2019. This study investigated the clinical outcomes and complications of 24 patients who underwent PETLIF in our facility and compared them with previous studies. Literature searches were conducted on PubMed and Web of Science. The PETLIF surgical technique involves three steps to acquire disc height under general anesthesia. The procedure includes bone harvesting, spondylolisthesis reduction, endoscopic foraminoplasty, disc height expansion using an oval dilator, and intervertebral disc curettage. A cage filled with autologous bone is inserted into the disc space and secured with posterior fixation. Patients underwent PETLIF with an average operation time of 130.8 min and a blood loss of 24.0 mL. Postoperative hospital stays were 9.5 days. Improvement in VAS, disc height, spinal canal area, and % slip was observed, while lumbar lordosis remained unchanged. Complications included end plate injury, subsidence, and exiting nerve root injury. The differences between PETLIF and the extracted literature were found in patients' age, direct decompression, epidural or local anesthesia, approach, order of PPS, and cage insertion. In conclusion, PETLIF surgery is a practical, minimally invasive surgical technique for patients with lumbar degenerative diseases suffering from back and leg pain, demonstrating significant improvements in pain scores. However, it is essential to carefully consider the potential complications and continue to refine the surgical technique further to enhance the safety and efficacy of this procedure.

The transcription factor Znf219 regulates chondrocyte differentiation by assembling a transcription factory with Sox9.

Sox9 is an essential transcription factor for chondrogenesis by regulating the expression of chondrogenic genes. However, its regulatory mechanism is not fully understood. To address this, we attempted to identify the transcriptional partners of Sox9 by screening the cDNA library of the chondrogenic cell line ATDC5 using the collagen 2α1 (Col2α1) gene promoter fused to a luciferase reporter gene. One of the positive clones encoded the Znf219 gene. Whole mount in situ hybridization experiments indicated that Znf219 mRNA was specifically expressed in the developing limb buds where Col2α1 and Sox9 were strongly expressed. Znf219 markedly enhanced the transcriptional activity of Sox9 on the Col2a1 gene promoter. In addition, Znf219 is physically associated with Sox9 and is colocalized with Sox9 in the nucleus. We also found that overexpression of Znf219 profoundly increased Sox9-induced mRNA expression of Col2a1, aggrecan and Col11a2. Consistently, knockdown of Znf219 decreased the Sox9-induced mRNA expression of these genes. Furthermore, a dominant-negative mutant Znf219 inhibited Bmp2-induced chondrocyte differentiation. Our results suggest that Znf219 plays an important role in the regulation of chondrocyte differentiation as a transcriptional partner of Sox9.

A Narrative Review of Full-Endoscopic Lumbar Discectomy Using Interlaminar Approach.

Lumbar disc herniation (LDH) is a frequently encountered pathologic condition in orthopedic daily practice. Discectomy is considered when patients with LDH experience persistent limb or lumbar pain or neurologic deficits. Various minimally invasive techniques are available for discectomy. Among these techniques, full-endoscopic lumbar discectomy (FED) is one of the least invasive options. During FED, removal of LDH is accomplished using 2 major approaches: transforaminal (TF) or interlaminar (IL). The prototype FED was percutaneous nucelotomy. An endoscope was integrated to percutaneous nucelotomy and TF endoscopic lumbar discectomy (TELD) was first derived. IL endoscopic lumbar discectomy (IELD) was introduced years after TELD. TELD and IELD can compensate for the shortcomings of each other and endoscopic spine surgeons need to acquire both techniques to deal with all type of LDHs. Because of its long history, the TF approach seems to represent the major approach for FED, but the IL approach has numerous benefits in particular types of LDH. The present article focuses on IELD and reviews the history, surgical techniques, indications and contraindications, clinical outcomes, and complications. This review will contribute to improved understanding of IELD as an important technique in full-endoscopic spine surgery.

Spinal Metastases without Pedicle Signs on Radiograph and their Associated Clinical and Radiological Features.

BACKGROUND: The pedicle sign is a radiographic indicator of spinal metastases. However, it is not only the pedicle sign that is important in radiographic diagnosis of bone metastases. In the present study, the radiological features of symptomatic spinal metastases in patients without the pedicle sign were retrospectively examined. MATERIALS AND METHODS: Among 186 patients with symptomatic spinal metastases who visited our department between January 1, 2011, and December 31, 2017, 64 without the pedicle sign and with available computed tomography (CT) and magnetic resonance imaging (MRI) data in the first visit were enrolled and their data were analyzed. One author evaluated radiographs for findings suggestive of spinal metastases, CT to assess bone destruction, and MRI to evaluate the extent of lesions. Clinical variables were also examined and compared between patients with and without bone changes on radiography. RESULTS: Bone changes strongly suggesting bone metastasis, other than the pedicle sign, were observed in 31 out of 64 patients: bone cortical disappearance in 20, increased radiolucency of the central area in the vertebral body in 8, an irregular osteoblastic change in 5, and asymmetrical vertebral collapse in 10. An analysis of CT data revealed that intertrabecular, mildly osteolytic, and mildly osteoblastic types were more frequent in patients without any changes suggestive of bone metastases on radiographs. CONCLUSION: Radiographic findings other than the pedicle sign are useful for diagnosing bone metastases. The key to a radiographic diagnosis of spinal metastases is to pay attention to changes in the bone cortex of all vertebral components on radiographs in addition to the pedicle.

Accuracy and safety of percutaneous pedicle screw placement using the K-wireless minimally invasive spine percutaneous pedicle screw system in Japan: A randomized active controlled study.

Background: Minimally invasive lumbar fusion has recently become a widely used technique worldwide. This randomized active controlled study was conducted to demonstrate the non-inferiority of the K-wireless Minimally Invasive Spine (MIS) Percutaneous Pedicle Screw (PPS) system compared with use of the six pedicle screw systems currently used in our practices with respect to the accuracy of pedicle screw placement.Also to compare the screw-insertion time and number of fluoroscopic observations during screw insertion between the groups. Methods: A total of 80 patients with degenerative spinal diseases or vertebral fractures were assigned, including 41 patients in the K-wireless MIS PPS system group (K-wireless group) and 39 in the control group (K-wire group).The accuracy of the screw insertion, screw-insertion time, number of fluoroscopic observations during screw insertion, and the incidence of adverse events were compared between the K-wireless group and the K-wire group. The accuracy rate was calculated as the number of screws with no breach divided by the total number of screws. Results: The accuracy rates of screw insertion were 85.7% and 75.0% in the K-wireless and K-wire groups, respectively, with an intergroup difference of 10.7% (95% confidence interval: 2.3-19.1%). The K-wireless group demonstrated non-inferiority compared with the K-wire group. The mean screw-insertion time was significantly shorter in the K-wireless group (2.62 and 2.97 min in the K-wireless and K-wire groups, respectively; P=0.005). There were also significantly fewer fluoroscopies in the K-wireless group (10.7 and 17.4 in the K-wireless and K-wire groups, respectively; P<0.001). There were no device-related or study treatment-related adverse events in either group. Conclusions: The accuracy of pedicle screw insertion using the K-wireless MIS PPS system was not inferior to that of existing products. In terms of safety, no product-related or treatment-related adverse events were identified in this study and no new safety concerns were noted.

Risk Factors and Surgical Management of Recurrent Herniation after Full-Endoscopic Lumbar Discectomy Using Interlaminar Approach.

Full-endoscopic lumbar discectomy (FED) is one of the least invasive procedures for lumbar disc herniation. Patients who receive FED for lumbar disc herniation may develop recurrent herniation at a frequency similar to conventional procedures. Reoperation and risk factors of recurrent lumbar disc herniation were investigated among 909 patients who received FED using an interlaminar approach (FED-IL). Sixty-five of the 909 patients received reoperation for recurrent herniation. Disc height, smoking, diabetes mellitus (DM), subligamentous extrusion (SE) type, and Modic change were identified as the risk factors for recurrence. Other indicators such as LL, Cobb angle, disc migration, age, sex, and body mass index (BMI) did not reach significance. Among 65 patients, reoperation was performed within 14 days following FED-IL (very early) in 7 patients, from 15 days to 3 months (early) in 14 patients, from 3 months to 1 year (midterm) in 17 patients, and after more than 1 year (late) in 27 patients. The very early group included a greater number of males, and the mean age was significantly lower in comparison to other groups. All patients in the very early group received FED-IL for reoperation. Reoperation within 2 weeks allows FED-IL to be performed without adhesion. Fusion surgery was performed on three cases in the early and midterm groups and on 10 cases in the late group, which increased over time as degenerative change and adhesion progressed. The procedure selected to treat recurrent herniation mostly depends on the surgeon's preference. Revision FED-IL is the first choice for recurrent herniation in terms of minimizing surgical burden, whereas fusion surgery offers the advantage that discectomy can be performed through unscarred tissues. FED-IL is recommended for recurrent herniation within 2 weeks before adhesion progresses.

Cine MRI Is Useful for the Diagnosis of Intradural Arachnoid Cyst with Spinal Arachnoid Web: A Case Report.

CASE: A 28-year-old woman developed gait disturbance due to lower limb weakness 3 years before presentation. Conventional magnetic resonance imaging (MRI) findings were inconclusive; therefore, we performed cine MRI, which confirmed the presence of a pulsatile cyst on the posterior thoracic spinal cord. The cyst compressed the spinal cord, and its pulsations synchronized with the patient's heartbeats. We resected the intradural arachnoid cyst and thickened arachnoid membrane. The gait disturbance improved after surgery. CONCLUSIONS: Cine MRI can be used to identify a pulsating arachnoid cyst that cannot be visualized with a conventional MRI. Cine MRI is useful in patients with unexplained spinal symptoms.

Dmrt2 promotes transition of endochondral bone formation by linking Sox9 and Runx2.

Endochondral bone formation is fundamental for skeletal development. During this process, chondrocytes undergo multiple steps of differentiation and coordinated transition from a proliferating to a hypertrophic stage, which is critical to advance skeletal development. Here, we identified the transcription factor Dmrt2 (double-sex and mab-3 related transcription factor 2) as a Sox9-inducible gene that promotes chondrocyte hypertrophy in pre-hypertrophic chondrocytes. Epigenetic analysis further demonstrated that Sox9 regulates Dmrt2 expression through an active enhancer located 18 kb upstream of the Dmrt2 gene and that this enhancer's chromatin status is progressively activated through chondrocyte differentiation. Dmrt2-knockout mice exhibited a dwarf phenotype with delayed initiation of chondrocyte hypertrophy. Dmrt2 augmented hypertrophic chondrocyte gene expression including Ihh through physical and functional interaction with Runx2. Furthermore, Dmrt2 deficiency reduced Runx2-dependent Ihh expression. Our findings suggest that Dmrt2 is critical for sequential chondrocyte differentiation during endochondral bone formation and coordinates the transcriptional network between Sox9 and Runx2.

C-arm fluoroscopy for tracheal intubation in a patient with severe cervical spine pathology.

Tracheal intubation is challenging in patients with severe cervical spine pathology. In such cases, awake fiberoptic intubation is the gold standard and safest option for tracheal intubation. However, this technique requires the patient's understanding and cooperation, and therefore, may be contraindicated in patients with refusal or poor tolerance. Herein, we report successful orotracheal intubation in a patient with limited mouth opening and severe cervical spine rigidity under general anesthesia using an extraglottic airway device and a gum-elastic bougie under C-arm fluoroscopic guidance.

Clinical and Radiological Outcomes of Corrective Surgery on Adult Spinal Deformity Patients: Comparison of Short and Long Fusion.

Despite the accumulated knowledge of spinal alignment and clinical outcomes the full corrective surgery cannot be applied to all the deformity patients as it requires considerable surgical burden to the patients. The aim of this study was to investigate the clinical and radiological outcomes of the patients who have received short and long fusion for ASD. A total of 21 patients who received surgical reconstructive spinal fusion procedures and were followed up for at least one year were retrospectively reviewed. Sixteen cases have received spinal corrective surgery that upper instrumented vertebrate (UIV) was thoracic level (group T), or 5 cases were with UIV in lumbar level (group L). Group L had shorter operation time, smaller intraoperative estimated blood loss, and shorter postoperative hospitalization days. Group T tends to improve more in the magnitude of VAS of lumbar pain compared to group L. Improvement of spinal alignment revealed the advantage of long fusion compared to short fusion, in Cobb angle, sagittal vertical axis (SVA), lumbar lordosis (LL), PI-LL C7 plum line (C7PL), and center sacral vertebral line (CSVL). Pelvic tilt (PT) did not differ between the groups. Disc lordosis was the most acquired in XLIF compared to TLIF and PLF and maintained one year. There were 9 adverse events, 3 cases of pulmonary embolism (PE), one case of delirium, and 6 cases of proximal junctional kyphosis. Current study elucidated that long fusion, UIV, is thoracic and can achieve better spinal alignment compared to short fusion, UIV, in lumbar. XLIF demonstrated strong ability to reconstruct the deformity on intervertebral space that is better to apply as much intervertebral space as possible. For the ASD patients with complications, short fusion can be one of the options.

Expression of phospholipase D2 in human colorectal carcinoma.

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline. PA acts as a second messenger in cell proliferation; therefore PLD is believed to play an important role in carcinogenesis. PLD activity has been reported to be elevated in human breast, gastric, renal cell and colorectal carcinomas, compared with adjacent non-neoplastic tissues. The activity of PLD was also correlated with nuclear grade in breast cancer, tumor size in gastric carcinoma, and nodal involvement and deeper invasion in colorectal carcinoma. However, the number of cases in each study was small. The aim of this study was to investigate the expression level of PLD2 and its association with clinicopathological features in human colorectal carcinoma. Ninety-seven colorectal carcinomas were obtained from surgery. Expression level of PLD2 was assessed by real-time PCR. The prognostic relevance of PLD2 expression level in patients with colorectal carcinoma was also analyzed by the survival analysis of mortality follow-up data covering the period 2000-2004. PLD expression level was varied from tumor to tumor. Expression level of PLD was significantly correlated with tumor size (P<0.05); it was independent of lymph node metastasis, extent of invasion, pathological classification, distant metastasis and Dukes' stage. PLD expression level was also significantly correlated with survival of patients with colorectal carcinoma (P<0.05). These findings suggested that PLD2 plays an important role in progression of colorectal carcinoma and that PLD2 could be a target for therapy in colorectal carcinoma.

The transcription factor Foxc1 is necessary for Ihh-Gli2-regulated endochondral ossification.

Indian hedgehog (Ihh) regulates endochondral ossification in both a parathyroid hormone-related protein (PTHrP)-dependent and -independent manner by activating transcriptional mediator Gli2. However, the molecular mechanisms underlying these processes remain elusive. Here by using in vivo microarray analysis, we identify forkhead box C1 (Foxc1) as a transcriptional partner of Gli2. Foxc1 stimulates expression of Ihh target genes, including PTHrP and Col10a1, through its physical and functional interaction with Gli2. Conversely, a dominant negative Foxc1 inhibits the Ihh target gene expression. In a spontaneous loss of Foxc1 function mouse (Foxc1(ch/ch)), endochondral ossification is delayed and the expression of Ihh target genes inhibited. Moreover, the pathological Foxc1 missense mutation observed in the Axenfeld-Rieger syndrome impairs Gli2-Foxc1 association as well as Ihh function. Our findings suggest that Foxc1 is an important transcriptional partner of Ihh-Gli2 signalling during endochondral ossification, and that disruption of the Foxc1-Gli2 interaction causes skeletal abnormalities observed in the Axenfeld-Rieger syndrome.

Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1.

Individuals with neurofibromatosis type-1 (NF1) can manifest focal skeletal dysplasias that remain extremely difficult to treat. NF1 is caused by mutations in the NF1 gene, which encodes the RAS GTPase-activating protein neurofibromin. We report here that ablation of Nf1 in bone-forming cells leads to supraphysiologic accumulation of pyrophosphate (PPi), a strong inhibitor of hydroxyapatite formation, and that a chronic extracellular signal-regulated kinase (ERK)-dependent increase in expression of genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank, causes this phenotype. Nf1 ablation also prevents bone morphogenic protein-2-induced osteoprogenitor differentiation and, consequently, expression of alkaline phosphatase and PPi breakdown, further contributing to PPi accumulation. The short stature and impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts can be corrected by asfotase-α enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization. They also suggest that altered PPi homeostasis contributes to the skeletal dysplasias associated with NF1 and that some of the NF1 skeletal conditions could be prevented pharmacologically.

Silent information regulator (Sir)T1 inhibits NF-κB signaling to maintain normal skeletal remodeling.

Silent information regulator T1 (SirT1) is linked to longevity and negatively controls NF-κB signaling, a crucial mediator of survival and regulator of both osteoclasts and osteoblasts. Here we show that NF-κB repression by SirT1 in both osteoclasts and osteoblasts is necessary for proper bone remodeling and may contribute to the mechanisms linking aging and bone loss. Osteoclast- or osteoblast-specific SirT1 deletion using the Sirt(flox/flox) mice crossed to lysozyme M-cre and the 2.3 kb col1a1-cre transgenic mice, respectively, resulted in decreased bone mass caused by increased resorption and reduced bone formation. In osteoclasts, lack of SirT1 promoted osteoclastogenesis in vitro and activated NF-κB by increasing acetylation of Lysine 310. Importantly, this increase in osteoclastogenesis was blocked by pharmacological inhibition of NF-κB. In osteoblasts, decreased SirT1 reduced osteoblast differentiation, which could also be rescued by inhibition of NF-κB. In further support of the critical role of NF-κB signaling in bone remodeling, elevated NF-κB activity in IκBα(+/-) mice uncoupled bone resorption and formation, leading to reduced bone mass. These findings support the notion that SirT1 is a genetic determinant of bone mass, acting in a cell-autonomous manner in both osteoblasts and osteoclasts, through control of NF-κB and bone cell differentiation.

Arid5b facilitates chondrogenesis by recruiting the histone demethylase Phf2 to Sox9-regulated genes.

Histone modification, a critical step for epigenetic regulation, is an important modulator of biological events. Sox9 is a transcription factor critical for endochondral ossification; however, proof of its epigenetic regulation remains elusive. Here we identify AT-rich interactive domain 5b (Arid5b) as a transcriptional co-regulator of Sox9. Arid5b physically associates with Sox9 and synergistically induces chondrogenesis. Growth of Arid5b(-/-) mice is retarded with delayed endochondral ossification. Sox9-dependent chondrogenesis is attenuated in Arid5b-deficient cells. Arid5b recruits Phf2, a histone lysine demethylase, to the promoter region of Sox9 target genes and stimulates H3K9me2 demethylation of these genes. In the promoters of chondrogenic marker genes, H3K9me2 levels are increased in Arid5b(-/-) chondrocytes. Finally, we show that Phf2 knockdown inhibits Sox9-induced chondrocyte differentiation. Our findings establish an epigenomic mechanism of skeletal development, whereby Arid5b promotes chondrogenesis by facilitating Phf2-mediated histone demethylation of Sox9-regulated chondrogenic gene promoters.

Regulation of endochondral ossification by transcription factors.

Endochondral ossification is very unique and complex biological event which is associated with skeletal development and tissue partnering. Genetic studies and gene-targeting approaches identified several transcription factors that play important roles in endochondral ossification. These transcription factors sequentially and harmoniously regulate each step of endochondral ossification, and consequently maintain the spatio-temporal control of the program. Importantly, these transcription factors form large protein complex to control chromatin remodeling, histone modification, transcription and splicing steps during endochondral ossification. It is also important to understand how these transcription factors regulate expression of their target genes. Biochemical and molecular cloning techniques largely contributed to identification of the components of the transcriptional complex and the target genes. Most recently, importance of endoplasmic reticulum (ER) stress in endochondral ossification has been reported. A transcription factor, BBF2H7, functions as an ER stress sensor in chondrocytes through regulation of appropriate secretion of chondrogenic matrices. We would like to discuss how the transcription factors regulate endochondral ossification.

Arid5a cooperates with Sox9 to stimulate chondrocyte-specific transcription.

SRY-box-containing gene 9 (Sox9) is an essential transcription factor in chondrocyte lineage determination and differentiation. Recent studies demonstrated that Sox9 controls the transcription of chondrocyte-specific genes in association with several other transcriptional regulators. To further understand the molecular mechanisms by which Sox9 influences transcriptional events during chondrocyte differentiation, we attempted to identify transcriptional partners of Sox9 and to examine their roles in chondrocyte differentiation. We isolated AT-rich interactive domain-containing protein 5a (Arid5a; also known as Mrf1) as an activator of the Col2a1 gene promoter from an ATDC5 cDNA library. Arid5a was highly expressed in cartilage and induced during chondrocyte differentiation. Furthermore, Arid5a physically interacted with Sox9 in nuclei and up-regulated the chondrocyte-specific action of Sox9. Overexpression of Arid5a stimulated chondrocyte differentiation in vitro and in an organ culture system. In contrast, Arid5a knockdown inhibited Col2a1 expression in chondrocytes. In addition, Arid5a binds directly to the promoter region of the Col2a1 gene and stimulates acetylation of histone 3 in the region. Our results suggest that Arid5a may directly interact with Sox9 and thereby enhance its chondrocyte-specific action.