Publications by authors named "Yuuki Imai"

108 Publications

Estrogen receptor α in mature osteoblasts regulates the late stage of bone regeneration.

Biochem Biophys Res Commun 2021 Jun 5;559:238-244. Epub 2021 May 5.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Ehime, Japan; Department of Pathophysiology, Ehime University Graduate School of Medicine, Ehime, Japan. Electronic address:

Estrogen deficiency impairs fracture healing and homeostasis of bone tissue. OVX-induced estrogen deficiency in mice attenuates fracture healing and changes the expression ratio of estrogen receptor (ER) α and ERβ in callus during the process of fracture healing. Therefore, ERs may be involved in the regulation of fracture healing. However, the roles of ERs in fracture healing are largely unknown. The purpose of this study was to clarify the significance of ERs during fracture healing using osteoblast-specific ER knockout mice in a mono-cortical drill hole bone regeneration model. The mature osteoblast-specific ER knockout mice were generated using osteocalcin (OCN)-Cre mice, and ERα and ERβ flox mice (OCN-Cre; ERα, ERα and OCN-Cre; ERβ, ERβ). Drill hole surgery was conducted on the tibiae of 8-week-old female mice. The mice were sacrificed 10 or 14 days after surgery and the bones were analyzed by DXA, μCT and bone histomorphometry. DXA analysis revealed that intact femoral BMD was significantly decreased in ERα mice compared with ERα mice, but there was no difference in bone mass between ERβ and ERβ mice. Micro CT analyses showed that the callus volume at the restricted drill hole site in tibiae was significantly less in ERα compared to ERα mice only at day 14 but not at day 10. In addition to femoral BMD, there was no significant difference in callus volume between ERβ and ERβ mice. Bone histomorphometric analyses showed that Ob.S/BS and N.Ob/B.Pm were significantly less in ERα mice compared with ERα mice only at day 10. In addition, Oc.S/BS and N.Oc/B.Pm were significantly less in ERα mice compared with ERα mice only at day 14. These results suggest that ERα but not ERβ in osteocalcin-positive osteoblasts may contribute to the late stage of bone regeneration.
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http://dx.doi.org/10.1016/j.bbrc.2021.04.112DOI Listing
June 2021

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

iScience 2021 Apr 13;24(4):102303. Epub 2021 Mar 13.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of as a target of AR in skeletal muscles. knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.
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http://dx.doi.org/10.1016/j.isci.2021.102303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041868PMC
April 2021

Topical co-administration of zoledronate with recombinant human bone morphogenetic protein-2 can induce and maintain bone formation in the bone marrow environment.

BMC Musculoskelet Disord 2021 Jan 20;22(1):94. Epub 2021 Jan 20.

Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahimachi, Abeno-ku, 545-8585, Osaka, Japan.

Background: Bone morphogenetic proteins (BMPs) induce osteogenesis in various environments. However, when BMPs are used alone in the bone marrow environment, the maintenance of new bone formation is difficult owing to vigorous bone resorption. This is because BMPs stimulate the differentiation of not only osteoblast precursor cells but also osteoclast precursor cells. The present study aimed to induce and maintain new bone formation using the topical co-administration of recombinant human BMP-2 (rh-BMP-2) and zoledronate (ZOL) on beta-tricalcium phosphate (β-TCP) composite.

Methods: β-TCP columns were impregnated with both rh-BMP-2 (30 µg) and ZOL (5 µg), rh-BMP-2 alone, or ZOL alone, and implanted into the left femur canal of New Zealand white rabbits (n = 56). The implanted β-TCP columns were harvested and evaluated at 3 and 6 weeks after implantation. These harvested β-TCP columns were evaluated radiologically using plane radiograph, and histologically using haematoxylin/eosin (H&E) and Masson's trichrome (MT) staining. In addition, micro-computed tomography (CT) was performed for qualitative analysis of bone formation in each group (n = 7).

Results: Tissue sections stained with H&E and MT dyes revealed that new bone formation inside the β-TCP composite was significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05). Micro-CT data also demonstrated that the bone volume and the bone mineral density inside the β-TCP columns were significantly greater in those impregnated with both rh-BMP-2 and ZOL than in those from the other experimental groups at 3 and 6 weeks after implantations (p < 0.05).

Conclusions: The topical co-administration of both rh-BMP-2 and ZOL on β-TCP composite promoted and maintained newly formed bone structure in the bone marrow environment.
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http://dx.doi.org/10.1186/s12891-021-03971-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7819170PMC
January 2021

DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration.

Biochem Biophys Res Commun 2021 01 10;534:79-85. Epub 2020 Dec 10.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Shitsukawa, Toon Ehime, 791-0295, Japan; Department of Pathophysiology, Ehime University Graduate School of Medicine, Shitsukawa, Toon Ehime, 791-0295, Japan; Division of Laboratory Animal Research, Advanced Research Support Center, Ehime University, Shitsukawa, Toon Ehime, 791-0295, Japan. Electronic address:

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.
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http://dx.doi.org/10.1016/j.bbrc.2020.11.116DOI Listing
January 2021

Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions.

Cell Commun Signal 2020 11 30;18(1):188. Epub 2020 Nov 30.

Division of Laboratory Animal Research, Advanced Research Support Center, Ehime University, Shitsukawa, Toon, Ehime, 791-0295, Japan.

Background: Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.

Methods: Immunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM.

Results: SM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM.

Conclusions: These findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.
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http://dx.doi.org/10.1186/s12964-020-00678-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708128PMC
November 2020

Estrogen Receptor β Controls Muscle Growth and Regeneration in Young Female Mice.

Stem Cell Reports 2020 09 20;15(3):577-586. Epub 2020 Aug 20.

Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics, Kumamoto University, 2-2-1 Honjo, Kumamoto 860-0811, Japan; Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; Center for Metabolic Regulation of Healthy Aging, Kumamoto University Faculty of Life Sciences, Nagasaki, Japan. Electronic address:

Estrogens are female sex hormones that are important for comprehensively maintaining muscle function, and an insufficiency affects muscle strength and regeneration in females. However, it is still unclear whether estrogen signaling is mediated through receptors. To investigate the specific role of estrogen receptor β (ERβ) in skeletal muscle and satellite cells (muscle stem cells), we generated muscle-specific ERβ-knockout (mKO) and satellite cell-specific ERβ-knockout (scKO) mice, respectively. Young female mKO mice displayed a decrease in fast-type dominant muscle mass. Female, but not male, scKO mice exhibited impaired muscle regeneration following acute muscle injury, probably due to reduced proliferation and increased apoptosis of satellite cells. RNA-sequencing analysis revealed that loss of ERβ in satellite cells altered gene expression of extracellular matrix components, including laminin and collagen. The results indicate that the estrogen-ERβ pathway is a sex-specific regulatory mechanism that controls muscle growth and regeneration in female mice.
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http://dx.doi.org/10.1016/j.stemcr.2020.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486216PMC
September 2020

The Musculoskeletal Knowledge Portal: Making Omics Data Useful to the Broader Scientific Community.

J Bone Miner Res 2020 09;35(9):1626-1633

Broad Institute of MIT & Harvard, Boston and Cambridge, MA, USA.

The development of high-throughput genotyping technologies and large biobank collections, complemented with rapid methodological advances in statistical genetics, has enabled hypothesis-free genome-wide association studies (GWAS), which have identified hundreds of genetic variants across many loci associated with musculoskeletal conditions. Similarly, basic scientists have valuable molecular cellular and animal data based on musculoskeletal disease that would be enhanced by being able to determine the human translation of their findings. By integrating these large-scale human genomic musculoskeletal datasets with complementary evidence from model organisms, new and existing genetic loci can be statistically fine-mapped to plausibly causal variants, candidate genes, and biological pathways. Genes and pathways identified using this approach can be further prioritized as drug targets, including side-effect profiling and the potential for new indications. To bring together these big data, and to realize the vision of creating a knowledge portal, the International Federation of Musculoskeletal Research Societies (IFMRS) established a working group to collaborate with scientists from the Broad Institute to create the Musculoskeletal Knowledge Portal (MSK-KP)(http://mskkp.org/). The MSK consolidates omics datasets from humans, cellular experiments, and model organisms into a central repository that can be accessed by researchers. The vision of the MSK-KP is to enable better understanding of the biological mechanisms underlying musculoskeletal disease and apply this knowledge to identify and develop new disease interventions. © 2020 American Society for Bone and Mineral Research (ASBMR).
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http://dx.doi.org/10.1002/jbmr.4147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8114232PMC
September 2020

Hedgehog Inhibitors Suppress Osteoclastogenesis in In Vitro Cultures, and Deletion of in Macrophage/Osteoclast Lineage Prevents Age-Related Bone Loss.

Int J Mol Sci 2020 Apr 15;21(8). Epub 2020 Apr 15.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime 791-0295, Japan.

The functional role of the Hedgehog (Hh)-signaling pathway has been widely investigated in bone physiology/development. Previous studies have, however, focused primarily on functions in bone formation, while its roles in bone resorption have not been fully elucidated. Here, we found that cyclopamine (smoothened (Smo) inhibitor), GANT-58 (GLI1 inhibitor), or GANT-61 (GLI1/2 inhibitor) significantly inhibited RANKL-induced osteoclast differentiation of bone marrow-derived macrophages. Although the inhibitory effects were exerted by cyclopamine or GANT-61 treatment during 0-48 h (early stage of osteoclast differentiation) or 48-96 h (late stage of osteoclast differentiation) after RANKL stimulation, GANT-58 suppressed osteoclast formation only during the early stage. These results suggest that the Smo-GLI1/2 axis mediates the whole process of osteoclastogenesis and that GLI1 activation is requisite only during early cellular events of osteoclastogenesis. Additionally, macrophage/osteoclast-specific deletion of Smo in mice was found to attenuate the aging phenotype characterized by trabecular low bone mass, suggesting that blockage of the Hh-signaling pathway in the osteoclast lineage plays a protective role against age-related bone loss. Our findings reveal a specific role of the Hh-signaling pathway in bone resorption and highlight that its inhibitors show potential as therapeutic agents that block osteoclast formation in the treatment of senile osteoporosis.
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http://dx.doi.org/10.3390/ijms21082745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216259PMC
April 2020

Evaluation of the structure of the otoconial layer using micro-computed tomography.

Auris Nasus Larynx 2020 Oct 13;47(5):734-739. Epub 2020 Mar 13.

Department of Otolaryngology, Head and Neck Surgery, Ehime University Graduate School of Medicine, Ehime, Japan.

Objective: Estrogen deficiency caused by bilateral ovariectomy (OVX) has been reported to lead to morphological changes in otoconia. Thus, we examined the morphological changes in the otoconial layer after OVX. We also investigated whether micro-computed tomography (µCT) is useful for the detection of morphological changes in the otoconial layer.

Methods: The otic capsules of C57BL/6 J mice were removed and evaluated using histological techniques and µCT at 2, 4, and 8 weeks after OVX or sham surgery. The volume of the utricle otoconial layer was measured and compared between the OVX and sham groups. The µCT scan and histological study results were also compared.

Results: The volume of the utricle otoconial layer was significantly increased 4 weeks after OVX compared to the sham group in both histological and µCT studies (p < 0.05). The volume of the otoconial layer measured using µCT was significantly correlated with the histological study results (p < 0.05).

Conclusion: The volume of the utricle otoconial layer increased after OVX. These morphological changes could be detected by µCT.
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http://dx.doi.org/10.1016/j.anl.2020.02.013DOI Listing
October 2020

Recent Insights into Long Bone Development: Central Role of Hedgehog Signaling Pathway in Regulating Growth Plate.

Int J Mol Sci 2019 Nov 20;20(23). Epub 2019 Nov 20.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City 791-0295, Japan.

The longitudinal growth of long bone, regulated by an epiphyseal cartilaginous component known as the "growth plate", is generated by epiphyseal chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossification, a sequential bone replacement of cartilaginous tissue, and any failure in this process causes a wide range of skeletal disorders. Therefore, the cellular and molecular characteristics of the growth plate are of interest to many researchers. Hedgehog (Hh), well known as a mitogen and morphogen during development, is one of the best known regulatory signals in the developmental regulation of the growth plate. Numerous animal studies have revealed that signaling through the Hh pathway plays multiple roles in regulating the proliferation, differentiation, and maintenance of growth plate chondrocytes throughout the skeletal growth period. Furthermore, over the past few years, a growing body of evidence has emerged demonstrating that a limited number of growth plate chondrocytes transdifferentiate directly into the full osteogenic and multiple mesenchymal lineages during postnatal bone development and reside in the bone marrow until late adulthood. Current studies with the genetic fate mapping approach have shown that the commitment of growth plate chondrocytes into the skeletal lineage occurs under the influence of epiphyseal chondrocyte-derived Hh signals during endochondral bone formation. Here, we discuss the valuable observations on the role of the Hh signaling pathway in the growth plate based on mouse genetic studies, with some emphasis on recent advances.
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http://dx.doi.org/10.3390/ijms20235840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6928971PMC
November 2019

Micro-CT imaging analysis for the effects of ibandronate and eldecalcitol on secondary osteoporosis and arthritis in adjuvant-induced arthritis rats.

Biomed Res 2019 ;40(5):197-205

Department of Orthopedic Surgery, Akita University Graduate School of Medicine.

We investigated the effects of ibandronate, a bisphosphonate; eldecalcitol, an active vitamin D3 analogue; and combination treatment with both agents on secondary osteoporosis and arthritis using rats with adjuvant-induced arthritis. Arthritis was induced in 8-week-old male Lewis rats. Rats were randomized into four treatment groups and an untreated normal control group: ibandronate, eldecalcitol, ibandronate + eldecalcitol, vehicle, and control. Paw thickness was measured to evaluate arthritis. Joint destruction was evaluated histomorphometrically by the ankle joint stained with Fast Green and safranin O. The femur and lumbar spine were scanned using dual-energy X-ray absorptiometry, and the distal femur was scanned using micro-computed tomography for bone mineral density (BMD) and trabecular microstructural evaluations. Ibandronate and/or eldecalcitol increased BMD in both the lumbar vertebrae and femur and improved several microstructural parameters (bone volume/total volume, structure model index, trabecular number, and trabecular separation of the distal femur). In addition, there was an additive effect of combination treatment compared with single treatments for most trabecular parameters, including BMD and bone volume. However, ibandronate and/or eldecalcitol did not inhibit arthritis and joint destruction. Combination treatment with ibandronate and eldecalcitol may be effective for secondary osteoporosis associated with arthritis.
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http://dx.doi.org/10.2220/biomedres.40.197DOI Listing
February 2020

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

J Biol Chem 2019 09 31;294(38):14135-14148. Epub 2019 Jul 31.

Division of Cell-Free Sciences, Proteo-Science Center (PROS), 3 Bunkyo-cho, Matsuyama, Ehime 790-8577, Japan

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in -deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). -knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLD variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.
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http://dx.doi.org/10.1074/jbc.RA119.010119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755803PMC
September 2019

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

Int J Cancer 2020 03 22;146(5):1369-1382. Epub 2019 Jul 22.

Department of Pathophysiology, Ehime University Graduate School of Medicine, Toon, Japan.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.
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http://dx.doi.org/10.1002/ijc.32554DOI Listing
March 2020

A simple mouse model of pericardial adhesions.

J Cardiothorac Surg 2019 Jun 28;14(1):124. Epub 2019 Jun 28.

Department of Cardiovascular and Thoracic Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime, 791-0295, Japan.

Background: Postoperative pericardial adhesions are considered a risk factor for redo cardiac surgery. Several large- and medium-size animal models of pericardial adhesions have been reported, but small animal models for investigating the development of anti-adhesion materials and molecular mechanisms of this condition are lacking. In this study, we aimed to establish a simple mouse model of pericardial adhesions to address this gap.

Methods: We administered blood, minocycline, picibanil, and talc into the murine pericardial cavity via one-shot injection. Micro-computed tomography analyses of contrast agent-injected mice were carried out for methodological evaluation. We investigated various dosages and treatment durations for molecules identified to be inducers of pericardial adhesion. The adhesive grade was quantified by scoring the strength and volume of adhesion tissues at sacrificed time points. Histological staining with hematoxylin and eosin and Masson's trichrome, and immunostaining for F4/80 or αSMA was performed to investigate the structural features of pericardial adhesions, and pathological features of the pericardial adhesion tissue were compared with human clinical specimens.

Results: Administration of talc resulted in the most extensive pericardial adhesions. Micro-computed tomography imaging data confirmed that accurate injection into the pericardial cavity was achieved. We found the optimal condition for the formation of strong pericardial adhesions to be injection of 2.5 mg/g talc for 2 weeks. Furthermore, histological analysis showed that talc administration led to an invasion of myofibroblasts and macrophages in the pericardial cavity and epicardium, consistent with pathological findings in patients with left ventricular assistive devices.

Conclusions: We successfully established a simple mouse model of talc-induced pericardial adhesions, which mimics human pathology and could contribute to solving the clinical issues related to pericardial adhesions.
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http://dx.doi.org/10.1186/s13019-019-0940-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599257PMC
June 2019

Fibroblast Growth Factor 9 Is Upregulated Upon Intervertebral Mechanical Stress-Induced Ligamentum Flavum Hypertrophy in a Rabbit Model.

Spine (Phila Pa 1976) 2019 Oct;44(20):E1172-E1180

Department of Orthopedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan.

Study Design: Case-control study of an animal model.

Objective: To investigate the factors that are upregulated and potentially related to degenerative changes in the ligamentum flavum (LF) upon mechanical stress concentration.

Summary Of Background Data: LF hypertrophy is reported to be associated with mechanical stress. However, few studies, using exhaustive analysis with control subjects, on the molecular mechanisms of LF hypertrophy have been published.

Methods: Fourteen rabbits were used for this study. The first group underwent L2-3 and L4-5 posterolateral fusion with instrumentation and resection of the L3-4 supraspinal muscle to concentrate the mechanical stress on L3-4, whereas the other group underwent a sham operation. The deep layer of the LF from L2-3 to L4-5 in both groups was harvested after 16 weeks. Gene expression was evaluated exhaustively using DNA microarray and real-time polymerase chain reaction (RT-PCR). Fibroblast growth factor 9 (FGF9) protein expression was subsequently examined by immunohistological staining.

Results: A total of 680 genes were found to be upregulated upon mechanical stress concentration and downregulated upon mechanical shielding compared with those in the sham group. Functional annotation analysis revealed that these genes not only included those related to the extracellular matrix but also those related to certain FGF families. On RT-PCR validation and immunohistological analysis, we identified that the FGF9 protein increases in the LF upon mechanical stress, especially in the area wherein degenerative changes were frequently identified in the previous literature.

Conclusion: FGF9 and its pathway are suggested to contribute to the degenerative changes in the LF following mechanical stress. This finding will be helpful in further understanding the molecular mechanism of human LF degeneration.

Level Of Evidence: N/A.
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http://dx.doi.org/10.1097/BRS.0000000000003089DOI Listing
October 2019

The effects of resistance training on bone mineral density and bone quality in type 2 diabetic rats.

Physiol Rep 2019 03;7(6):e14046

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Ehime, Japan.

Resistance training (RT) has been known to be effective in maintaining and improving bone strength, which is based on bone mineral density (BMD) and bone quality. However, it is not clear whether RT is effective in improving bone strength in patients with type-2 diabetes mellitus (T2DM), who have a high risk of fracture. Therefore, we tested the effects of a 6-week RT regimen using percutaneous electrical stimulation in T2DM model rats, male Otsuka Long-Evans Tokushima Fatty (OLETF), and its control, Long-Evans Tokushima Otsuka (LETO). After 6 weeks of RT, tibial BMD in RT legs was significantly higher than that in control (CON) legs in both groups. In diaphyseal cortical bone, bone area/tissue area, and cortical thickness was significantly increased in RT legs compared with CON legs in both groups. Cortical porosity was highly observed in OLETF compared with LETO, but RT improved cortical porosity in both groups. Interestingly, trabecular number, trabecular thickness and trabecular space as well as BMD and bone volume/tissue volume in proximal tibial metaphyseal trabecular bone were significantly improved in RT legs compared with CON legs in both groups. In contrast, connectivity density and structural model index were not affected by RT. These results indicate that the 6-week RT regimen effectively increased BMD and improved bone quality in T2DM model rats as well as control rats. Therefore, RT may have the potential to improve bone strength and reduce fracture risk, even in patients with T2DM.
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http://dx.doi.org/10.14814/phy2.14046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436184PMC
March 2019

Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin.

Bone 2019 05 13;122:93-100. Epub 2019 Feb 13.

Department of Pathophysiology, Graduate School of Medicine, Ehime University, Toon, Ehime, Japan; Division of Laboratory Animal Research, Advanced Research Support Center, Ehime University, Toon, Ehime, Japan; Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Toon, Ehime, Japan. Electronic address:

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.
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http://dx.doi.org/10.1016/j.bone.2019.02.011DOI Listing
May 2019

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8 T Cells.

J Immunol 2019 02 9;202(4):1088-1098. Epub 2019 Jan 9.

Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan;

Although the methylation status of histone H3K27 plays a critical role in CD4 T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8 T cell-dependent immune response remains unclear. We therefore generated T cell-specific Cd4-Cre Tg ( KO) mice to determine the role of Utx in CD8 T cells. Wild-type (WT) and KO mice were infected with expressing OVA to analyze the immune response of Ag-specific CD8 T cells. There was no significant difference in the number of Ag-specific CD8 T cells upon primary infection between WT and KO mice. However, deficiency resulted in more Ag-specific CD8 T cells upon secondary infection. Adoptive transfer of KO CD8 T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including encoding Blimp1, in KO CD8 T cells. We confirmed that the trimethylation level of histone H3K27 in the gene loci in the KO cells was higher than in the WT cells. The treatment of CD8 T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8 T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8 T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8 T cells.
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http://dx.doi.org/10.4049/jimmunol.1801083DOI Listing
February 2019

[ASBMR 2018 report on basic research.]

Authors:
Yuuki Imai

Clin Calcium 2019;29(1):99-103

Division of Integrative Pathophysiology, Proteo-Science Center Department of Pathophysiology, Graduate School of Medicine Division of Laboratory Animal Research, Advanced Research Support Center Ehime University, Toon, Ehime, Japan.

This is a brief report on ASBMR 2018 held at Montreal, Quebec, Canada, focusing on basic research. Topics of ASBMR 2018 were varied among wide research fields, however, this report focuses on several topics because of spatio-temporal restriction of attendees. Also, the selected topics were very limited according to the author's interests.
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http://dx.doi.org/CliCa190199103DOI Listing
August 2019

Raman Spectroscopic Analysis to Detect Reduced Bone Quality after Sciatic Neurectomy in Mice.

Molecules 2018 Nov 25;23(12). Epub 2018 Nov 25.

Department of Bone and Joint Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon 791-0295, Ehime, Japan.

Bone mineral density (BMD) is a commonly used diagnostic indicator for bone fracture risk in osteoporosis. Along with low BMD, bone fragility accounts for reduced bone quality in addition to low BMD, but there is no diagnostic method to directly assess the bone quality. In this study, we investigated changes in bone quality using the Raman spectroscopic technique. Sciatic neurectomy (NX) was performed in male C57/BL6J mice (NX group) as a model of disuse osteoporosis, and sham surgery was used as an experimental control (Sham group). Eight months after surgery, we acquired Raman spectral data from the anterior cortical surface of the proximal tibia. We also performed a BMD measurement and micro-CT measurement to investigate the pathogenesis of osteoporosis. Quantitative analysis based on the Raman peak intensities showed that the carbonate/phosphate ratio and the mineral/matrix ratio were significantly higher in the NX group than in the Sham group. There was direct evidence of alterations in the mineral content associated with mechanical properties of bone. To fully understand the spectral changes, we performed principal component analysis of the spectral dataset, focusing on the matrix content. In conclusion, Raman spectroscopy provides reliable information on chemical changes in both mineral and matrix contents, and it also identifies possible mechanisms of disuse osteoporosis.
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http://dx.doi.org/10.3390/molecules23123081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321365PMC
November 2018

The tumor suppressor menin prevents effector CD8 T-cell dysfunction by targeting mTORC1-dependent metabolic activation.

Nat Commun 2018 08 17;9(1):3296. Epub 2018 Aug 17.

Department of Immunology, Graduate School of Medicine, Ehime University, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism.
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http://dx.doi.org/10.1038/s41467-018-05854-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098065PMC
August 2018

Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone.

Histochem Cell Biol 2018 Apr 22;149(4):365-373. Epub 2018 Jan 22.

Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, 791-0295, Japan.

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.
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http://dx.doi.org/10.1007/s00418-018-1641-5DOI Listing
April 2018

DANFIN functions as an inhibitor of transcription factor NF-κB and potentiates the antitumor effect of bortezomib in multiple myeloma.

Biochem Biophys Res Commun 2018 01 25;495(3):2289-2295. Epub 2017 Dec 25.

Division of Cell-Free Sciences, Proteo-Science Center (PROS), Ehime University, Japan. Electronic address:

Nuclear factor-κB (NF-κB) proteins are transcription factors that play key roles in regulating most immune responses and cell death. Constitutively active NF-κB has been shown to exhibit chemoresistance by inducing anti-apoptosis in tumor cells. Multiple myeloma is known as a constitutive NF-κB activating disease, and the proteasome inhibitor bortezomib is used to treat multiple myeloma and mantle cell lymphoma. We demonstrate here that DANFIN (N,N'-bis-(2,4-dimethyl-phenyl)-ethane-1,2-diamine) functions as an inhibitor of the p65 family proteins and induces chemosensitization to bortezomib in multiple myeloma. DANFIN was found to be an inhibitor of interactions between p65 and IκBα without the inhibition of the DNA binding activity of the p65 protein. In addition, DANFIN affected the IκBα binding region in Rel Homology Domain (RHD) and suppressed the nuclear translocalization of the p65 protein in cells. Furthermore, in multiple myeloma cells, DANFIN suppressed the expression level of NF-κB target genes and induced apoptosis. The combination therapy of DANFIN with bortezomib dramatically enhanced the apoptosis of multiple myeloma cells and indicated a remarkable anti-tumor effect in a multiple-myeloma xenograft mouse model.
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http://dx.doi.org/10.1016/j.bbrc.2017.12.142DOI Listing
January 2018

The HIV co-receptor CCR5 regulates osteoclast function.

Nat Commun 2017 12 20;8(1):2226. Epub 2017 Dec 20.

Division of Bio-Imaging, Proteo-Science Center (PROS), Ehime University, Ehime, 791-0295, Japan.

C-C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5 ) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts.
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http://dx.doi.org/10.1038/s41467-017-02368-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738403PMC
December 2017

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University, Toon, Ehime 791-0295, Japan

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice ( ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including , which is reported to be an IL1-related gene and to affect chondrocyte differentiation. knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.
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http://dx.doi.org/10.1242/dev.157412DOI Listing
January 2018

Genome-wide Association Study of Idiopathic Osteonecrosis of the Femoral Head.

Sci Rep 2017 11 8;7(1):15035. Epub 2017 Nov 8.

Division of Integrative Pathophysiology, Proteo-Science Center, Ehime University Graduate School of Medicine, Shitsukawa, Toon-city, Ehime, 791-0295, Japan.

Idiopathic osteonecrosis of the femoral head (IONFH) is an ischemic disorder that causes bone necrosis of the femoral head, resulting in hip joint dysfunction. IONFH is a polygenic disease and steroid and alcohol have already known to increase its risk; however, the mechanism of IONFH remains to be elucidated. We performed a genome-wide association study using ~60,000 subjects and found two novel loci on chromosome 20q12 and 12q24. Big data analyses identified LINC01370 as a candidate susceptibility gene in the 20q12 locus. Stratified analysis by IONFH risk factors suggested that the 12q24 locus was associated with IONFH through drinking capacity. Our findings would shed new light on pathophysiology of IONFH.
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http://dx.doi.org/10.1038/s41598-017-14778-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5678103PMC
November 2017

[Calcium and bone metabolism across women's life stages. Molecular mechanisms underlying bone metabolism by estrogen.]

Authors:
Yuuki Imai

Clin Calcium 2017;27(5):635-641

Division of Integrative Pathophysiology, Proteo-Science Center, Department of Pathophysiology, Graduate School of Medicine, Division of Experimental Animal Research, Advanced Research Support Center, Ehime University, Japan.

Osteoporosis, which patients are estimated as more than 13 million in Japan, is mainly caused by postmenopausal osteoporosis. Estrogen deficiency induced by menopause can disturb endocrine feedback and homeostasis, followed by bone loss by increased bone resorption with high bone turnover. Recent studies using systemic or conditional Estrogen Receptor α(ERα)gene knockout mice have unveiled molecular mechanisms underlying bone metabolism. In this review, it will be discussed that direct and indirect effects of estrogen and its metabolites for bone metabolism.
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http://dx.doi.org/CliCa1705635641DOI Listing
October 2017

The CUL3-SPOP-DAXX axis is a novel regulator of VEGFR2 expression in vascular endothelial cells.

Sci Rep 2017 02 20;7:42845. Epub 2017 Feb 20.

Division of Cell Growth and Tumor Regulation, Proteo-Science Center (PROS), Ehime University, Toon, Ehime, Japan.

Vascular endothelial cell growth factor receptor 2 (VEGFR2) is an essential receptor for the homeostasis of endothelial cells. In this study, we showed that NEDD8-conjugated Cullin3 (CUL3)-based ubiquitin E3 (UbE3) ligase plays a crucial role in VEGFR2 mRNA expression. Human umbilical vein endothelial cells treated with MLN4924, an inhibitor of NEDD8-activating enzyme, or with CUL3 siRNA drastically lost their response to VEGF due to the intense decrease in VEGFR2 expression. Moreover, speckle-type POZ protein (SPOP) and death-domain associated protein (DAXX) were involved in the CUL3 UbE3 ligase complex as a substrate adaptor and a substrate, respectively. Knockdown of SPOP and CUL3 led to the upregulation of DAXX protein and downregulation of VEGFR2 levels. These levels were inversely correlated with one another. In addition, simultaneous knockdown of SPOP and DAXX completely reversed the downregulation of VEGFR2 levels. Moreover, the CUL3-SPOP-DAXX axis had the same effects on NOTCH1, DLL4 and NRP1 expression. Taken together, these findings suggest that the CUL3-SPOP-DAXX axis plays a very important role in endothelial cell function by targeting key angiogenic regulators.
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http://dx.doi.org/10.1038/srep42845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5317005PMC
February 2017

Local co-application of zoledronate promotes long-term maintenance of newly formed bone induced by recombinant human bone morphogenetic protein 2.

Biochem Biophys Res Commun 2016 Nov 14;480(3):314-320. Epub 2016 Oct 14.

Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3, Asahimachi, Abeno-ku, Osaka, 545-8585, Japan.

Bone Morphogenetic Proteins (BMPs) strongly induce the recruitment and differentiation of mesenchymal progenitor cells into mature osteoblasts, but also directly and indirectly stimulate differentiation of osteoclast progenitor cells and acceleration of mature osteoclasts function leading excessive bone resorption. Bisphosphonates, such as zoledronate (ZOL), inhibit osteoclasts function and osteoclasts mediated bone resorption. The short or middle term effect of BMPs and bisphosphonates on bone formation were previously reported, but there was no study that argue about the long term effect of bisphosphonates on BMP-induced bone anabolism. The present study demonstrated that the local administration of ZOL with recombinant human BMP-2 (rh-BMP-2) using beta tricalcium phosphate (β-TCP) as a carrier had superior efficacy not only to augment the BMP-induced new ectopic bone formation but to maintain the trabecular bone structure inside the new bone for long period. Histological analysis showed that rh-BMP-2/β-TCP composite induced trabecular bone resorption especially inside the new bone nodules over time, whereas no trabecular bone resorption was seen in rh-BMP-2/ZOL/β-TCP composite reducing the number of TRAP-positive cells. Thus, inhibition of bone resorption by bisphosphonate, such as ZOL, would be one of the advantageous ways to augment the new bone formation induced by rh-BMP-2, and moreover local co-application of ZOL using β-TCP as a carrier can be a useful material for long term suppression of osteoclastic resorption and thereby maintain the structure of new bone formation induced by rh-BMP-2.
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http://dx.doi.org/10.1016/j.bbrc.2016.10.034DOI Listing
November 2016