Publications by authors named "Masaharu Takigawa"

201 Publications

RFX1-mediated CCN3 induction that may support chondrocyte survival under starved conditions.

J Cell Physiol 2021 Mar 3. Epub 2021 Mar 3.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

Cellular communication network factor (CCN) family members are multifunctional matricellular proteins that manipulate and integrate extracellular signals. In our previous studies investigating the role of CCN family members in cellular metabolism, we found three members that might be under the regulation of energy metabolism. In this study, we confirmed that CCN2 and CCN3 are the only members that are tightly regulated by glycolysis in human chondrocytic cells. Interestingly, CCN3 was induced under a variety of impaired glycolytic conditions. This CCN3 induction was also observed in two breast cancer cell lines with a distinct phenotype, suggesting a basic role of CCN3 in cellular metabolism. Reporter gene assays indicated a transcriptional regulation mediated by an enhancer in the proximal promoter region. As a result of analyses in silico, we specified regulatory factor binding to the X-box 1 (RFX1) as a candidate that mediated the transcriptional activation by impaired glycolysis. Indeed, the inhibition of glycolysis induced the expression of RFX1, and RFX1 silencing nullified the CCN3 induction by impaired glycolysis. Subsequent experiments with an anti-CCN3 antibody indicated that CCN3 supported the survival of chondrocytes under impaired glycolysis. Consistent with these findings in vitro, abundant CCN3 production by chondrocytes in the deep zones of developing epiphysial cartilage, which are located far away from the synovial fluid, was confirmed in vivo. Our present study uncovered that RFX1 is the mediator that enables CCN3 induction upon cellular starvation, which may eventually assist chondrocytes in retaining their viability, even when there is an energy supply shortage.
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http://dx.doi.org/10.1002/jcp.30348DOI Listing
March 2021

Bipartite regulation of cellular communication network factor 2 and fibroblast growth factor 1 genes by fibroblast growth factor 1 through histone deacetylase 1 and fork head box protein A1.

J Cell Commun Signal 2021 Mar 4;15(1):81-91. Epub 2021 Jan 4.

Department of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

Fibroblast growth factor 1 (FGF-1) is the first FGF family member, and it induces proliferation of fibroblasts and other types of the cells. However, recent studies are uncovering unexpected functions of this molecule. Our previous study redefined this growth factor as a catabolic molecule produced in cartilage upon metabolic insult. Indeed, FGF-1 was found to repress the gene expression of cellular communication network factor 2 (CCN2), which protects and regenerates cartilage, amplifying its own production through positive feedback regulation. In the present study, we investigated the molecular mechanism of this bipartite CCN2 repression and FGF1 activation by FGF-1 in chondrocytes. Repression of CCN2 and induction of FGF1 in human chondrocytic cells were both partly abolished by valproic acid, an inhibitor of histone deacetylase 1 (HDAC1), indicating the involvement of chromatin remodeling by histone acetylation in this system. In contrast, RNA degradation analysis suggested no contribution of post-transcriptional regulation of the mRNA stability to the effects conferred by FGF-1. Suspecting a regulation by a specific transcription factor, we next sought a candidate in silico from a large dataset. As a result, we found fork head box protein A1 (FOXA1) as the transcription factor that bound to both CCN2 and FGF1 loci. Functional analysis demonstrated that FOXA1 silencing significantly attenuated the CCN2 repression and FGF1 induction caused by FGF1. These findings collectively indicate that the bipartite regulation by FGF-1 is enabled by the combination of chromatin remodeling by HDACs and transcriptional modulation by FOXA1 with unknown transcriptional coactivators of opposite functionalities.
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http://dx.doi.org/10.1007/s12079-020-00600-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7905003PMC
March 2021

Triple knockdown of CDC37, HSP90-alpha and HSP90-beta diminishes extracellular vesicles-driven malignancy events and macrophage M2 polarization in oral cancer.

J Extracell Vesicles 2020 May 31;9(1):1769373. Epub 2020 May 31.

Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.

Evidence has been accumulating to indicate that extracellular vesicles (EVs), including exosomes, released by cancer cells can foster tumour progression. The molecular chaperones - CDC37, HSP90α and HSP90β play key roles in cancer progression including epithelial-mesenchymal transition (EMT), although their contribution to EVs-mediated cell-cell communication in tumour microenvironment has not been thoroughly examined. Here we show that triple depletion of the chaperone trio attenuates numerous cancer malignancy events exerted through EV release. Metastatic oral cancer-derived EVs (MEV) were enriched with HSP90α HSP90β and cancer-initiating cell marker CD326/EpCAM. Depletion of these chaperones individually induced compensatory increases in the other chaperones, whereas triple siRNA targeting of these molecules markedly diminished the levels of the chaperone trio and attenuated EMT. MEV were potent agents in initiating EMT in normal epithelial cells, a process that was attenuated by the triple chaperone depletion. The migration, invasion, and in vitro tumour initiation of oral cancer cells were significantly promoted by MEV, while triple depletion of CDC37/HSP90α/β reversed these MEV-driven malignancy events. In metastatic oral cancer patient-derived tumours, HSP90β was significantly accumulated in infiltrating tumour-associated macrophages (TAM) as compared to lower grade oral cancer cases. HSP90-enriched MEV-induced TAM polarization to an M2 phenotype, a transition known to support cancer progression, whereas the triple chaperone depletion attenuated this effect. Mechanistically, the triple chaperone depletion in metastatic oral cancer cells effectively reduced MEV transmission into macrophages. Hence, siRNA-mediated knockdown of the chaperone trio (CDC37/HSP90α/HSP90β) could potentially be a novel therapeutic strategy to attenuate several EV-driven malignancy events in the tumour microenvironment.

Abbreviations: CDC37: cell division control 37; EMT: epithelial-mesenchymal transmission; EV: extracellular vesicles; HNSCC: head and neck squamous cell carcinoma; HSP90: heat shock protein 90; TAM: tumour-associated macrophage.
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http://dx.doi.org/10.1080/20013078.2020.1769373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7580842PMC
May 2020

CCN3 (NOV) Drives Degradative Changes in Aging Articular Cartilage.

Int J Mol Sci 2020 Oct 13;21(20). Epub 2020 Oct 13.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

Aging is a major risk factor of osteoarthritis, which is characterized by the degeneration of articular cartilage. CCN3, a member of the CCN family, is expressed in cartilage and has various physiological functions during chondrocyte development, differentiation, and regeneration. Here, we examine the role of CCN3 in cartilage maintenance. During aging, the expression of mRNA in mouse primary chondrocytes from knee cartilage increased and showed a positive correlation with and mRNA. Increased accumulation of CCN3 protein was confirmed. To analyze the effects of CCN3 , either primary cultured human articular chondrocytes or rat chondrosarcoma cell line (RCS) were used. Artificial senescence induced by HO caused a dose-dependent increase in gene and CCN3 protein expression, along with enhanced expression of and mRNA and proteins, as well as SA-β gal activity. Overexpression of CCN3 also enhanced promoter activity via . Accordingly, the addition of recombinant CCN3 protein to the culture increased the expression of and mRNAs. We have produced cartilage-specific CCN3-overexpressing transgenic mice, and found degradative changes in knee joints within two months. Inflammatory gene expression was found even in the rib chondrocytes of three-month-old transgenic mice. Similar results were observed in human knee articular chondrocytes from patients at both mRNA and protein levels. These results indicate that CCN3 is a new senescence marker of chondrocytes, and the overexpression of CCN3 in cartilage may in part promote chondrocyte senescence, leading to the degeneration of articular cartilage through the induction of p53 and p21.
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http://dx.doi.org/10.3390/ijms21207556DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593953PMC
October 2020

Regulation of cellular communication network factor 2 (CCN2) in breast cancer cells via the cell-type dependent interplay between CCN2 and glycolysis.

J Oral Biosci 2020 09 11;62(3):280-288. Epub 2020 Aug 11.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. Electronic address:

Objectives: Anti-osteoclastic treatments for breast cancer occasionally cause medication-related osteonecrosis of the jaw. Moreover, elevated glycolytic activity, which is known as the Warburg effect, is usually observed in these breast cancer cells. Previously, we found that cellular communication network factor 2 (CCN2) production and glycolysis enhanced each other in chondrocytes. Here, we evaluated the interplay between CCN2 and glycolysis in breast cancer cells, as we suspected a possible involvement of CCN2 in the Warburg effect in highly invasive breast cancer cells.

Methods: Two human breast cancer cell lines with a distinct phenotype were used. Glycolysis was inhibited by using 2 distinct compounds, and gene silencing was performed using siRNA. Glycolysis and the expression of relevant genes were monitored via colorimetric assays and quantitative RT-PCR, respectively.

Results: Although CCN2 expression was almost completely silenced when treating invasive breast cancer cells with a siRNA cocktail against CCN2, glycolytic activity was not affected. Notably, the expression of glycolytic enzyme genes, which was repressed by CCN2 deficiency in chondrocytes, tended to increase upon CCN2 silencing in breast cancer cells. Inhibition of glycolysis, which resulted in the repression of CCN2 expression in chondrocytic cells, did not alter or strongly enhanced CCN2 expression in the invasive and non-invasive breast cancer cells, respectively.

Conclusions: High CCN2 expression levels play a critical role in the invasion and metastasis of breast cancer. Thus, a collapse in the intrinsic repressive machinery of CCN2 due to glycolysis may induce the acquisition of an invasive phenotype in breast cancer cells.
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http://dx.doi.org/10.1016/j.job.2020.07.001DOI Listing
September 2020

CTGF/CCN2 facilitates LRP4-mediated formation of the embryonic neuromuscular junction.

EMBO Rep 2020 08 17;21(8):e48462. Epub 2020 Jun 17.

Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan.

At the neuromuscular junction (NMJ), lipoprotein-related receptor 4 (LRP4) mediates agrin-induced MuSK phosphorylation that leads to clustering of acetylcholine receptors (AChRs) in the postsynaptic region of the skeletal muscle. Additionally, the ectodomain of LRP4 is necessary for differentiation of the presynaptic nerve terminal. However, the molecules regulating LRP4 have not been fully elucidated yet. Here, we show that the CT domain of connective tissue growth factor (CTGF/CCN2) directly binds to the third beta-propeller domain of LRP4. CTGF/CCN2 enhances the binding of LRP4 to MuSK and facilitates the localization of LRP4 on the plasma membrane. CTGF/CCN2 enhances agrin-induced MuSK phosphorylation and AChR clustering in cultured myotubes. Ctgf-deficient mouse embryos (Ctgf ) have small AChR clusters and abnormal dispersion of synaptic vesicles along the motor axon. Ultrastructurally, the presynaptic nerve terminals have reduced numbers of active zones and mitochondria. Functionally, Ctgf embryos exhibit impaired NMJ signal transmission. These results indicate that CTGF/CCN2 interacts with LRP4 to facilitate clustering of AChRs at the motor endplate and the maturation of the nerve terminal.
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http://dx.doi.org/10.15252/embr.201948462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403661PMC
August 2020

Roles of Interaction between CCN2 and Rab14 in Aggrecan Production by Chondrocytes.

Int J Mol Sci 2020 Apr 16;21(8). Epub 2020 Apr 16.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8525, Japan.

To identify proteins that cooperate with cellular communication network factor 2 (CCN2), we carried out GAL4-based yeast two-hybrid screening using a cDNA library derived from the chondrocytic cell line HCS-2/8. Rab14 GTPase (Rab14) polypeptide was selected as a CCN2-interactive protein. The interaction between CCN2 and Rab14 in HCS-2/8 cells was confirmed using the in situ proximity ligation assay. We also found that CCN2 interacted with Rab14 through its IGFBP-like domain among the four domains in CCN2 protein. To detect the colocalization between CCN2 and Rab14 in the cells in detail, CCN2, wild-type Rab14 (Rab14WT), a constitutive active form (Rab14CA), and a dominant negative form (Rab14DN) of Rab14 were overexpressed in monkey kidney-tissue derived COS7 cells. Ectopically overexpressed Rab14 showed a diffuse cytosolic distribution in COS7 cells; however, when Rab14WT was overexpressed with CCN2, the Rab14WT distribution changed to dots that were evenly distributed within the cytosol, and both Rab14 and CCN2 showed clear colocalization. When Rab14CA was overexpressed with CCN2, Rab14CA and CCN2 also showed good localization as dots, but their distribution was more widespread within cytosol. The coexpression of Rab14DN and CCN2 also showed a dotted codistribution but was more concentrated in the perinuclear area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the reduction in or mRNA by their respective siRNA significantly enhanced the expression of ER stress markers, and mRNA in HCS-2/8 chondrocytic cells, suggesting that ER and Golgi stress were induced by the inhibition of membrane vesicle transfer via the suppression of CCN2 or Rab14. Moreover, to study the effect of the interaction between CCN2 and its interactive protein Rab14 on proteoglycan synthesis, we overexpressed Rab14WT or Rab14CA or Rab14DN in HCS-2/8 cells and found that the overexpression of Rab14DN decreased the extracellular proteoglycan accumulation more than the overexpression of Rab14WT/CA did in the chondrocytic cells. These results suggest that intracellular CCN2 is associated with Rab14 on proteoglycan-containing vesicles during their transport from the Golgi apparatus to endosomes in chondrocytes and that this association may play a role in proteoglycan secretion by chondrocytes.
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http://dx.doi.org/10.3390/ijms21082769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215643PMC
April 2020

Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (): CRISPR against Cancer.

Cancers (Basel) 2020 Apr 4;12(4). Epub 2020 Apr 4.

Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.
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http://dx.doi.org/10.3390/cancers12040881DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226423PMC
April 2020

Antiparkinson Drug Benztropine Suppresses Tumor Growth, Circulating Tumor Cells, and Metastasis by Acting on SLC6A3/DAT and Reducing STAT3.

Cancers (Basel) 2020 Feb 24;12(2). Epub 2020 Feb 24.

Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama 700-8525, Japan.

Tumor growth, progression, and therapy resistance are crucial factors in the prognosis of cancer. The properties of three-dimensional (3D) tumor-like organoids (tumoroids) more closely resemble in vivo tumors compared to two-dimensionally cultured cells and are therefore effectively used for assays and drug screening. We here established a repurposed drug for novel anticancer research and therapeutics using a 3D tumoroid-based screening system. We screened six pharmacologically active compounds by using an original tumoroid-based multiplex phenotypic screening system with a matrix metalloproteinase 9 (MMP9) promoter-driven fluorescence reporter for the evaluation of both tumoroid formation and progression. The antiparkinson drug benztropine was the most effective compound uncovered by the screen. Benztropine significantly inhibited in vitro tumoroid formation, cancer cell survival, and MMP9 promoter activity. Benztropine also reduced the activity of oncogenic signaling transducers and trans-activators for MMP9, including STAT3, NF-κB, and β-catenin, and the properties of cancer stem cells/cancer-initiating cells. Benztropine and GBR-12935 directly targeted the dopamine transporter DAT/SLC6A3, whose genetic alterations such as amplification were correlated with poor prognosis for cancer patients. Benztropine also inhibited the tumor growth, circulating tumor cell (CTC) number, and rate of metastasis in a tumor allograft model in mice. In conclusion, we propose the repurposing of benztropine for anticancer research and therapeutics that can suppress tumor progression, CTC, and metastasis of aggressive cancers by reducing key pro-tumorigenic factors.
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http://dx.doi.org/10.3390/cancers12020523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072357PMC
February 2020

Suppression of adipocyte differentiation by low-intensity pulsed ultrasound via inhibition of insulin signaling and promotion of CCN family protein 2.

J Cell Biochem 2020 Feb 17. Epub 2020 Feb 17.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.

Adipocyte differentiation is regulated by several transcription factors such as the CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor-γ (PPARγ). Here, we demonstrate that low-intensity pulsed ultrasound (LIPUS) suppressed differentiation into mature adipocytes via multiple signaling pathways. When C3H10T1/2, a mesenchymal stem cell line, was treated with LIPUS (3.0 MHz, 60 mW/cm ) for 20 minutes once a day for 4 days during adipogenesis, and both the number of lipid droplets and the gene expression of PPARγ and C/EBPα were significantly decreased. Furthermore, LIPUS treatment decreased the phosphorylation of the insulin receptor and also that of Akt and ERK1/2, which are located downstream of this receptor. Next, we showed that LIPUS suppressed the gene expression of angiotensinogen (AGT), which is an adipokine produced by mature adipocytes, as well as that of angiotensin-converting enzyme 1 (ACE1) and angiotensin receptor type 1 (AT R) during adipogenesis of pre-adipogenic 3T3-L1 cells. Next, the translocation of Yes-associated protein (YAP) into the nucleus of 3T3-L1 cells was promoted by LIPUS, leading to upregulation of CCN family protein 2 (CCN2), a cellular communication network factor. Moreover, forced expression of CCN2 in 3T3-L1 cells decreased PPARγ gene expression, but it did not increase alkaline phosphatase and osterix gene expression. Finally, gene silencing of CCN2 in C3H10T1/2 cells diminished the effect of LIPUS on the gene expression of PPARγ and C/EBPα. These findings suggest that LIPUS suppressed adipogenesis through inhibition of insulin signaling and decreased PPARγ expression via increased CCN2 production, resulting in a possible decrease of mature adipocytes.
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http://dx.doi.org/10.1002/jcb.29680DOI Listing
February 2020

Selective Agonists of Nuclear Retinoic Acid Receptor Gamma Inhibit Growth of HCS-2/8 Chondrosarcoma Cells.

J Orthop Res 2020 05 19;38(5):1045-1051. Epub 2019 Dec 19.

Department of Orthopaedics, University of Maryland School of Medicine, Baltimore, Maryland.

Chondrosarcoma is the second most common primary bone sarcoma. Treatment of chondrosarcoma is limited to surgery due to radiation and chemotherapy resistance of this cancer. An ideal treatment for chondrosarcoma would be a well-tolerated, minimally invasive local or systemic treatment modality to halt or slow tumor growth prior to resection of local, unresectable local, or metastatic disease. Palovarotene, an agonist of nuclear retinoic acid receptor γ (RARγ) has shown therapeutic action for treatment of heterotopic ossification and osteochondroma without serious adverse effects in animal models. We hypothesized that selective agonists of RARγ would have an inhibitory effect on chondrosarcoma. All human chondrosarcoma specimens expressed RARγ as determined by immunohistochemical staining. The ΗCS-2/8 chondrosarcoma cell line, established from low-grade human chondrosarcoma, was used to examine the actions of RARγ agonists. In ΗCS2/8 pellet cultures, RARγ agonist treatment reduced the mass size and significantly decreased total glycosaminoglycan, protein amounts, and gene expression levels of cartilage matrix molecules when compared with control groups. Systemic treatment with RARγ agonists significantly inhibited the growth of ΗCS-2/8 cell transplants in vivo. Furthermore, local injection of RARγ agonist-loaded poly-lactic acid nanoparticles induced regression of the mass size of the transplants. Histologic analysis demonstrated that RARγ agonist treatment inhibited cell proliferation activity and stimulated encapsulation of the tumor. These findings indicate that RARγ agonists, including palovarotene, may have an anti-tumor effect on low-grade chondrosarcomas. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1045-1051, 2020.
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http://dx.doi.org/10.1002/jor.24555DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162703PMC
May 2020

Jiadifenolide induces the expression of cellular communication network factor (CCN) genes, and CCN2 exhibits neurotrophic activity in neuronal precursor cells derived from human induced pluripotent stem cells.

Biochem Biophys Res Commun 2019 11 7;519(2):309-315. Epub 2019 Sep 7.

Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan. Electronic address:

Jiadifenolide has been reported to have neurotrophin-like activity in primary rat cortical neurons, and also possesses neurotrophic effects in neuronal precursor cells derived from human induced pluripotent stem cells (hiPSCs), as we have previously reported. However, the molecular mechanisms by which jiadifenolide exerts its neurotrophic effects in rat and human neurons are unknown. Thus, we aimed to investigate the molecular mechanisms and pathways by which jiadifenolide promotes neurotrophic effects. Here, we found that jiadifenolide activated cellular communication network factor (CCN) signaling pathways by up-regulating mRNA level expression of CCN genes in human neuronal cells. We also found that CCN2 (also known as connective tissue growth factor, CTGF) protein promotes neurotrophic effects through activation of the p44/42 mitogen-activated protein kinase signaling pathway. This is the first discovery which links neurotrophic activity with CCN signaling.
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http://dx.doi.org/10.1016/j.bbrc.2019.09.003DOI Listing
November 2019

Roles of matricellular CCN2 deposited by osteocytes in osteoclastogenesis and osteoblast differentiation.

Sci Rep 2019 07 29;9(1):10913. Epub 2019 Jul 29.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

In this study, we investigated the effect of CCN2 (cellular communication network factor 2), previously termed connective tissue growth factor, deposited in bone matrix on osteoclastogenesis and osteoblast differentiation. To mimic the bone matrix environment, osteocytic MLO-Y4 cells had been embedded in collagen-gel with recombinant CCN2 (rCCN2), and mouse macrophage-like RAW264.7 cells were inoculated on the gel and treated with receptor activator of NF-κB ligand (RANKL). NFATc1 and cathepsin K (CTSK) productions were more increased in the combination of RAW264.7 and MLO-Y4 cells treated with rCCN2 than the combination without rCCN2. Next, we isolated an osteocyte-enriched population of cells and osteoclast progenitor cells from wild type and tamoxifen-inducible Ccn2-deficient (KO) mice and performed similar analysis. NFATc1 and CTSK productions were decreased in the KO osteocyte-enriched population at 6 months after the tamoxifen injection, regardless of the origin of the osteoclast progenitor cells. Interestingly, CTSK production was rather increased in KO osteocytes at 1 year after the injection. Finally, the combination of osteoblastic MC3T3-E1 and MLO-Y4 cells in rCCN2-containing bone matrix revealed the up-regulation of osteoblastic marker genes. These findings suggest that CCN2 supplied by osteocytes regulates both osteoclastogenesis and osteoblast differentiation.
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http://dx.doi.org/10.1038/s41598-019-47285-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662664PMC
July 2019

A Reporter System Evaluates Tumorigenesis, Metastasis, β-catenin/MMP Regulation, and Druggability.

Tissue Eng Part A 2019 10 14;25(19-20):1413-1425. Epub 2019 Jun 14.

Department of Dental Pharmacology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.

Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of were found in colon cancer cases. We found that proximal promoter sequences of in murine and human contained conserved binding sites for transcription factors β-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by β-catenin signaling stimulator lithium chloride. The promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the promoter , potentially through inhibition of β-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as β-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and β-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.
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http://dx.doi.org/10.1089/ten.TEA.2018.0348DOI Listing
October 2019

Possible reparative effect of low-intensity pulsed ultrasound (LIPUS) on injured meniscus.

J Cell Commun Signal 2019 Jun 20;13(2):193-207. Epub 2018 Nov 20.

Advanced Research Center for Oral and Craniofacial Sciences (ARCOCS), Okayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

Menisci are a pair of crescent-shaped fibrocartilages, particularly of which their inner region of meniscus is an avascular tissue. It has characteristics similar to those of articular cartilage, and hence is inferior in healing. We previously reported that low-intensity pulsed ultrasound (LIPUS) treatment stimulates the production of CCN2/CTGF, a protein involved in repairing articular cartilage, and the gene expression of major cartilage matrices such as type II collagen and aggrecan in cultured chondrocytes. Therefore, in this present study, we investigated whether LIPUS has also favorable effect on meniscus cells and tissues. LIPUS applied with a 60 mW/cm intensity for 20 min stimulated the gene expression and protein production of CCN2 via ERK and p38 signaling pathways, as well as gene expression of SOX9, aggrecan, and collagen type II in human inner meniscus cells in culture, and slightly stimulated the gene expression of CCN2 and promoted the migration in human outer meniscus cells in culture. LIPUS also induced the expression of Ccn2, Sox9, Col2a1, and Vegf in rat intact meniscus. Furthermore, histological evaluations showed that LIPUS treatment for 1 to 4 weeks promoted healing of rat injured lateral meniscus, as evidenced by better and earlier angiogenesis and extracellular matrix synthesis. The data presented indicate that LIPUS treatment might prevent meniscus from degenerative change and exert a reparative effect on injured meniscus via up-regulation of repairing factors such as CCN2 and that it might thus be useful for treatment of an injured meniscus as a non-invasive therapy.
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http://dx.doi.org/10.1007/s12079-018-0496-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498275PMC
June 2019

Depletion of Lipid Efflux Pump ABCG1 Triggers the Intracellular Accumulation of Extracellular Vesicles and Reduces Aggregation and Tumorigenesis of Metastatic Cancer Cells.

Front Oncol 2018 10;8:376. Epub 2018 Oct 10.

Department of Dental Pharmacology, Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama University, Okayama, Japan.

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of is found in 10-35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors . Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs and their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.
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http://dx.doi.org/10.3389/fonc.2018.00376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191470PMC
October 2018

CCN2/CTGF binds the small leucine rich proteoglycan protein Tsukushi.

J Cell Commun Signal 2019 Mar 20;13(1):113-118. Epub 2018 Sep 20.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama, 700-8525, Japan.

Extracellular molecules coordinate the multiple signaling pathways spatiotemporally to exchange information between cells during development. Understanding the regulation of these signal molecule-dependent pathways elucidates the mechanism of intercellular crosstalks. CCN2/CTGF is one of the CCN family members that binds BMP2, fibronectin, aggrecan, FGFR2 - regulating cartilage and bone formation, angiogenesis, wound repair etc. Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family, binds nodal/Vg1/TGF-β1, BMP4/chordin, Delta, FGF8, Frizzled4, and is involved in the early body formation, bone growth, wound healing, retinal stem cell regulation etc. These two secreted molecules are expressed in similar tissues and involved in several biological events by functioning as extracellular signaling modulators. Here, we examine the molecular interaction between CCN2 and TSK biochemically. Co-precipitation assay and Surface Plasmon Resonance measurement showed their direct binding with the Kd value 15.3 nM. Further, the Solid-phase Binding Assay indicated that TSK binds to IGFBP and CT domains of CCN2. Our data suggest that CCN2 and TSK exert their function together in the body formation.
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http://dx.doi.org/10.1007/s12079-018-0487-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381378PMC
March 2019

The BMP-2 mutant L51P: a BMP receptor IA binding-deficient inhibitor of noggin.

J Bone Miner Metab 2019 Mar 17;37(2):199-205. Epub 2018 Apr 17.

Physiological Chemistry II, Theodor-Boveri-Institute for Biocenter of Würzburg University, Würzburg, Germany.

The antagonist-specific regulation in tissue engineering constitutes important attempts to achieve an improved and rapid bone regeneration by controlling the natural biological response of the natural body growth factors. L51P is molecularly engineered bone morphogentic protein-2 (BMP-2) variant with a substitution of the 51st leucine with a proline residue. L51P is deficient in BMP receptor binding, but maintains its structure and affinity for inhibitory proteins such as noggin, chordin, and gremlin. These modifications convert the BMP-2 variant L51P into a receptor-inactive inhibitor of BMP antagonists. This current approach may prevent the uncontrolled bone overgrowth using high concentration of BMPs and thus regulates the possible growth factor's high-dose side effects. Exploring of L51P biological functions is required to broad our understanding of BMP mutant biological functions and their potential clinical applications. The progress of L51P researches would hopefully lead to the development of multiple applications for using the L51P in bone and fracture healing disorders.
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http://dx.doi.org/10.1007/s00774-018-0925-0DOI Listing
March 2019

Organoids with cancer stem cell-like properties secrete exosomes and HSP90 in a 3D nanoenvironment.

PLoS One 2018 7;13(2):e0191109. Epub 2018 Feb 7.

Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States of America.

Ability to form cellular aggregations such as tumorspheres and spheroids have been used as a morphological marker of malignant cancer cells and in particular cancer stem cells (CSC). However, the common definition of the types of cellular aggregation formed by cancer cells has not been available. We examined morphologies of 67 cell lines cultured on three dimensional morphology enhancing NanoCulture Plates (NCP) and classified the types of cellular aggregates that form. Among the 67 cell lines, 49 cell lines formed spheres or spheroids, 8 cell lines formed grape-like aggregation (GLA), 8 cell lines formed other types of aggregation, and 3 cell lines formed monolayer sheets. Seven GLA-forming cell lines were derived from adenocarcinoma among the 8 lines. A neuroendocrine adenocarcinoma cell line PC-3 formed asymmetric GLA with ductal structures on the NCPs and rapidly growing asymmetric tumors that metastasized to lymph nodes in immunocompromised mice. In contrast, another adenocarcinoma cell line DU-145 formed spheroids in vitro and spheroid-like tumors in vivo that did not metastasize to lymph nodes until day 50 after transplantation. Culture in the 3D nanoenvironment and in a defined stem cell medium enabled the neuroendocrine adenocarcinoma cells to form slowly growing large organoids that expressed multiple stem cell markers, neuroendocrine markers, intercellular adhesion molecules, and oncogenes in vitro. In contrast, the more commonly used 2D serum-contained environment reduced intercellular adhesion and induced mesenchymal transition and promoted rapid growth of the cells. In addition, the 3D stemness nanoenvironment promoted secretion of HSP90 and EpCAM-exosomes, a marker of CSC phenotype, from the neuroendocrine organoids. These findings indicate that the NCP-based 3D environment enables cells to form stem cell tumoroids with multipotency and model more accurately the in vivo tumor status at the levels of morphology and gene expression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0191109PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802492PMC
March 2018

[The role of osteocytes in bone remodeling.]

Clin Calcium 2017;27(12):1697-1703

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

Bone remodeling has important roles in the functions of bone tissues, such as supporting the body and mineral storage. Osteocytes, which are the most abundant cells in bone tissues, detect the mechanical loading and regulate both bone formation by osteoblasts and bone resorption by osteoclasts. However, its mechanism is still unknown. In this review, we summarize how osteocytes detect mechanical stress and discuss the potential role of CCN2/CTGF as a novel bone remodeling factor produced by osteocytes.
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http://dx.doi.org/CliCa171216971703DOI Listing
April 2018

Physiological role of urothelial cancer-associated one long noncoding RNA in human skeletogenic cell differentiation.

J Cell Physiol 2018 06 15;233(6):4825-4840. Epub 2018 Jan 15.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

A vast number of long-noncoding RNAs (lncRNA) are found expressed in human cells, which RNAs have been developed along with human evolution. However, the physiological functions of these lncRNAs remain mostly unknown. In the present study, we for the first time uncovered the fact that one of such lncRNAs plays a significant role in the differentiation of chondrocytes and, possibly, of osteoblasts differentiated from mesenchymal stem cells, which cells eventually construct the human skeleton. The urothelial cancer-associated 1 (UCA1) lncRNA is known to be associated with several human malignancies. Firstly, we confirmed that UCA1 was expressed in normal human chondrocytes, as well as in a human chondrocytic cell line; whereas it was not detected in human bone marrow mesenchymal stem cells (hBMSCs). Of note, although UCA1 expression was undetectable in hBMSCs, it was markedly induced along with the differentiation toward chondrocytes, suggesting its critical role in chondrogenesis. Consistent with this finding, silencing of the UCA1 gene significantly repressed the expression of chondrogenic genes in human chondrocytic cells. UCA1 gene silencing and hyper-expression also had a significant impact on the osteoblastic phenotype in a human cell line. Finally, forced expression of UCA1 in a murine chondrocyte precursor, which did not possess a UCA1 gene, overdrove its differentiation into chondrocytes. These results indicate a physiological and important role of this lncRNA in the skeletal development of humans, who require more sustained endochondral ossification and osteogenesis than do smaller vertebrates.
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http://dx.doi.org/10.1002/jcp.26285DOI Listing
June 2018

Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes.

PLoS One 2017 16;12(11):e0188014. Epub 2017 Nov 16.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0188014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690650PMC
December 2017

Metabolic regulation of the CCN family genes by glycolysis in chondrocytes.

J Cell Commun Signal 2018 Mar 11;12(1):245-252. Epub 2017 Nov 11.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

The CCN family consists of 6 genes in the mammalian genome and produces multifunctional proteins involved in a variety of biological processes. Recent reports indicate the profound roles of CCN2 in energy metabolism in chondrocytes, and Ccn2 deficiency is known to alter the expression of 2 other family members including Ccn3. However, almost nothing is known concerning the regulation of the CCN family genes by energy metabolism. In order to gain insight into this critical issue, we initially and comprehensively evaluated the effect of inhibition of glycolysis on the expression of all of the CCN family genes in chondrocytic cells. Upon the inhibition of a glycolytic enzyme, repression of CCN2 expression was observed, whereas CCN3 expression was conversely induced. Similar repression of CCN2 was conferred by the inhibition of aerobic ATP production, which, however, did not induce CCN3 expression. In contrast, glucose starvation significantly enhanced the expression of CCN3 in those cells. The results of a reporter gene assay using a molecular construct containing a CCN3 proximal promoter revealed a dose-dependent induction of the CCN3 promoter activity by the glycolytic inhibitor in chondrocytic cells. These results unveiled a critical role of glycolytic activity in the regulation of CCN2 and CCN3, which activity mediated the mutual regulation of these 2 major CCN family members in chondrocytes.
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http://dx.doi.org/10.1007/s12079-017-0420-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842183PMC
March 2018

An early history of CCN2/CTGF research: the road to CCN2 via hcs24, ctgf, ecogenin, and regenerin.

J Cell Commun Signal 2018 Mar 26;12(1):253-264. Epub 2017 Oct 26.

Advanced Research Center for Oral and Craniofacial Sciences (ARCOCS), Okayama University Dental School/Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama, 700-8525, Japan.

The principal aim of this historical review is to present the processes by which the different aspects of CCN2/CTGF/Hcs24 were discovered by different groups and how much CCN2/CTGF, by being integrated into CCN family, has contributed to the establishment of the basic concepts regarding the role and functions of this new class of proteins. This review should be particularly useful to new investigators who have recently entered this exciting field of study and also provides a good opportunity to acknowledge the input of those individuals who participated in the development of this scientific field.
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http://dx.doi.org/10.1007/s12079-017-0414-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842178PMC
March 2018

Low-intensity pulsed ultrasound stimulation promotes osteoblast differentiation through hedgehog signaling.

J Cell Biochem 2018 06 28;119(6):4352-4360. Epub 2018 Feb 28.

Department of Oral and Maxillofacial Surgery, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.

Low-intensity pulsed ultrasound (LIPUS) has been used as an adjunct to fracture healing therapies, but the mechanisms underlying its action are not known. We reported that sonic hedgehog (SHH) signaling was activated in osteoblasts at the dynamic remodeling site of a bone fracture. Mechanical stimulation is a crucial factor in bone remodeling, and it is related to the primary cilia as a sensor of hedgehog signaling. Here we observed that LIPUS promoted callus formation in accord with Gli2-positive cells after 14 days at the mouse femur fractured site compared with a control group. An immunofluorescence analysis showed that the numbers of primary cilia and cilia/osterix double-positive osteoblasts were increased at the fracture site by LIPUS. LIPUS stimulated not only the number and the length of primary cilia, but also the levels of ciliated protein, Ift88 mRNA, and SHH, Gli1, and Gli2 in MC3T3-E1 cells. Further experiments revealed that LIPUS stimulated osteogenic differentiation in the presence of smoothened agonist (SAG) treatment. These results indicate that LIPUS stimulates osteogenic differentiation and the maturation of osteoblasts by a primary cilium-mediated activation of hedgehog signaling.
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http://dx.doi.org/10.1002/jcb.26418DOI Listing
June 2018

UPR transducer BBF2H7 allows export of type II collagen in a cargo- and developmental stage-specific manner.

J Cell Biol 2017 06 12;216(6):1761-1774. Epub 2017 May 12.

Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan

The unfolded protein response (UPR) handles unfolded/misfolded proteins accumulated in the endoplasmic reticulum (ER). However, it is unclear how vertebrates correctly use the total of ten UPR transducers. We have found that ER stress occurs physiologically during early embryonic development in medaka fish and that the smooth alignment of notochord cells requires ATF6 as a UPR transducer, which induces ER chaperones for folding of type VIII (short-chain) collagen. After secretion of hedgehog for tissue patterning, notochord cells differentiate into sheath cells, which synthesize type II collagen. In this study, we show that this vacuolization step requires both ATF6 and BBF2H7 as UPR transducers and that BBF2H7 regulates a complete set of genes (, , , and ) essential for the enlargement of COPII vesicles to accommodate long-chain collagen for export, leading to the formation of the perinotochordal basement membrane. Thus, the most appropriate UPR transducer is activated to cope with the differing physiological ER stresses of different content types depending on developmental stage.
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http://dx.doi.org/10.1083/jcb.201609100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461018PMC
June 2017

A Tumor Suppressor Gene Product, Platelet-Derived Growth Factor Receptor-Like Protein Controls Chondrocyte Proliferation and Differentiation.

J Cell Biochem 2017 11 23;118(11):4033-4044. Epub 2017 May 23.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama-City, Okayama, 700-8525, Japan.

The platelet-derived growth factor receptor-like (PDGFRL) gene is regarded as a tumor suppressor gene. However, nothing is known about the molecular function of PDGFRL. In this study, we initially clarified its function in chondrocytes. Among all cell lines examined, the PDGFRL mRNA level was the highest in chondrocytic HCS-2/8 cells. Interestingly, the proliferation of chondrocytic HCS-2/8 cells was promoted by PDGFRL overexpression, whereas that of the breast cancer-derived MDA-MB-231 cells was inhibited. Of note, in PDGFRL-overexpressing HCS-2/8 cells, the expression of chondrocyte differentiation marker genes, SOX9, ACAN, COL2A1, COL10A1, and ALP, was decreased. Moreover, we confirmed the expression of PDGFRL mRNA in normal cartilage tissue and chondrocytes. Eventually, the expression of PDGFRL mRNA in condrocytes except in the case of hypertrophic chondrocytes was demonstrated in vivo and in vitro. These findings suggest that PDGFRL plays the different roles, depending upon cell types. Particularly, in chondrocytes, PDGFRL may play a new and important role which is distinct from the function previously reported. J. Cell. Biochem. 118: 4033-4044, 2017. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jcb.26059DOI Listing
November 2017

Catabolic effects of FGF-1 on chondrocytes and its possible role in osteoarthritis.

J Cell Commun Signal 2017 Sep 25;11(3):255-263. Epub 2017 Mar 25.

Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.

Fibroblast growth factor 1 (FGF-1) is a classical member of the FGF family and is produced by chondrocytes cultured from osteoarthritic patients. Also, this growth factor was shown to bind to CCN family protein 2 (CCN2), which regenerates damaged articular cartilage and counteracts osteoarthritis (OA) in an animal model. However, the pathophysiological role of FGF-1 in cartilage has not been well investigated. In this study, we evaluated the effects of FGF-1 in vitro and its production in vivo by use of an OA model. Treatment of human chondrocytic cells with FGF-1 resulted in marked repression of genes for cartilaginous extracellular matrix components, whereas it strongly induced matrix metalloproteinase 13 (MMP-13), representing its catabolic effects on cartilage. Interestingly, expression of the CCN2 gene was dramatically repressed by FGF-1, which repression eventually caused the reduced production of CCN2 protein from the chondrocytic cells. The results of a reporter gene assay revealed that this repression could be ascribed, at least in part, to transcriptional regulation. In contrast, the gene expression of FGF-1 was enhanced by exogenous FGF-1, indicating a positive feedback system in these cells. Of note, induction of FGF-1 was observed in the articular cartilage of a rat OA model. These results collectively indicate a pathological role of FGF-1 in OA development, which includes an insufficient cartilage regeneration response caused by CCN2 down regulation.
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http://dx.doi.org/10.1007/s12079-017-0384-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559396PMC
September 2017

Novel role of CCN3 that maintains the differentiated phenotype of articular cartilage.

J Bone Miner Metab 2017 Nov 16;35(6):582-597. Epub 2016 Nov 16.

Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School, Okayama, Japan.

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.
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http://dx.doi.org/10.1007/s00774-016-0793-4DOI Listing
November 2017