Publications by authors named "Tetsuya Matsuguchi"

69 Publications

Ultraviolet B irradiation decreases CXCL10 expression in keratinocytes through endoplasmic reticulum stress.

J Cell Biochem 2021 Apr 28. Epub 2021 Apr 28.

Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Ultraviolet radiation is one of the standard treatment selections for psoriasis. interferon (IFN)-γ and IFN-γ-induced CXCL10, which are highly expressed by keratinocytes in psoriasis lesion, are therapeutic targets for psoriasis. In this study, we found that ultraviolet B (UVB) irradiation inhibited IFN-γ signaling events, including STAT1 phosphorylation and induction of CXCL10 messenger RNA (mRNA) expression in keratinocytes. IFN-γ-induced expression of CXCL10 mRNA in HaCaT cells, a human keratinocyte cell line, and human epithelial keratinocytes were also inhibited by H O or endoplasmic reticulum (ER) stress inducers. Conversely, a mixture of antioxidants, Trolox and ascorbic acid, and the ER stress inhibitor salubrinal partially counteracted the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA expression in HaCaT cells. We also found that UVB and ER stress reduced IFN-γ receptor 1 protein levels in the plasma membrane fraction of keratinocytes. These observations suggested that ER stress and the generation of reactive oxygen species are essential for the inhibitory effect of UVB on IFN-γ-induced CXCL10 mRNA in keratinocytes.
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http://dx.doi.org/10.1002/jcb.29936DOI Listing
April 2021

Bone morphogenetic protein 9 (BMP9) directly induces Notch effector molecule Hes1 through the SMAD signaling pathway in osteoblasts.

FEBS Lett 2021 02 25;595(3):389-403. Epub 2020 Dec 25.

Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Japan.

Bone morphogenetic protein (BMP) 9 is one of the most osteogenic BMPs, but its mechanism of action has not been fully elucidated. Hes1, a transcriptional regulator with a basic helix-loop-helix domain, is a well-known effector of Notch signaling. Here, we find that BMP9 induces periodic increases of Hes1 mRNA and protein expression in osteoblasts, presumably through an autocrine negative feedback mechanism. BMP9-mediated Hes1 induction is significantly inhibited by an ALK inhibitor and overexpression of Smad7, an inhibitory Smad. Luciferase and ChIP assays revealed that two Smad-binding sites in the 5' upstream region of the mouse Hes1 gene are essential for transcriptional activation by BMP9. Thus, our data indicate that BMP9 induces Hes1 expression in osteoblasts via the Smad signaling pathway.
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http://dx.doi.org/10.1002/1873-3468.14016DOI Listing
February 2021

Glut1 expression is increased by p53 reduction to switch metabolism to glycolysis during osteoblast differentiation.

Biochem J 2020 05;477(10):1795-1811

Field of Oral Biochemistry, Department of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

The glycolytic system is selected for ATP synthesis not only in tumor cells but also in differentiated cells. Differentiated osteoblasts also switch the dominant metabolic pathway to aerobic glycolysis. We found that primary osteoblasts increased expressions of glycolysis-related enzymes such as Glut1, hexokinase 1 and 2, lactate dehydrogenase A and pyruvate kinase M2 during their differentiation. Osteoblast differentiation decreased expression of tumor suppressor p53, which negatively regulates Glut1 expression, and enhanced phosphorylation of AKT, which is regulated by phosphoinositol-3 kinase (PI3K). An inhibitor of PI3K enhanced p53 expression and repressed Glut1 expression. Luciferase reporter assay showed that p53 negatively regulated transcriptional activity of solute carrier family 2 member 1 gene promoter region. Inhibition of glycolysis in osteoblasts reduced ATP contents more significantly than inhibition of oxidative phosphorylation by carbonyl cyanide m-chlorophenyl hydrazine. These results have indicated that osteoblasts increase Glut1 expression through the down-regulation of p53 to switch their metabolic pathway to glycolysis during differentiation.
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http://dx.doi.org/10.1042/BCJ20190888DOI Listing
May 2020

CXCL13 is a differentiation- and hypoxia-induced adipocytokine that exacerbates the inflammatory phenotype of adipocytes through PHLPP1 induction.

Biochem J 2019 11;476(22):3533-3548

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.

Hypoxia in adipose tissue is regarded as a trigger that induces dysregulation of the secretory profile in adipocytes. Similarly, local dysregulation of adipocytokine secretion is an initial event in the deleterious effects of obesity on metabolism. We previously reported that CXCL13 is highly produced during adipogenesis, however little is known about the roles of CXCL13 in adipocytes. Here, we found that hypoxia, as modeled by 1% O2 or exposure to the hypoxia-mimetic reagent desferrioxamine (DFO) has strong inductive effects on the expression of CXCL13 and CXCR5, a CXCL13 receptor, in both undifferentiated and differentiated adipocytes and in organ-cultured white adipose tissue (WAT). CXCL13 was also highly expressed in WAT from high fat diet-fed mice. Hypoxic profile, typified by increased expression of interleukin-6 (IL-6) and plasminogen activator inhibitor-1 (PAI-1) and decreased expression of adiponectin, was significantly induced by CXCL13 treatment during adipogenic differentiation. Conversely, the treatment of adipocytes with a neutralizing-antibody against CXCL13 as well as CXCR5 knockdown by specific siRNA effectively inhibited DFO-induced inflammation. The phosphorylation of Akt2, a protective factor of adipose inflammation, was significantly inhibited by CXCL13 treatment during adipogenic differentiation. Mechanistically, CXCL13 induces the expression of PHLPP1, an Akt2 phosphatase, through focal adhesion kinase (FAK) signaling; and correspondingly we show that CXCL13 and DFO-induced IL-6 and PAI-1 expression was blocked by Phlpp1 knockdown. Furthermore, we revealed the functional binding sites of PPARγ2 and HIF1-α within the Cxcl13 promoter. Taken together, these results indicate that CXCL13 is an adipocytokine that facilitates hypoxia-induced inflammation in adipocytes through FAK-mediated induction of PHLPP1 in autocrine and/or paracrine manner.
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http://dx.doi.org/10.1042/BCJ20190709DOI Listing
November 2019

BMP9 prevents induction of osteopontin in JNK-inactivated osteoblasts via Hey1-Id4 interaction.

Int J Biochem Cell Biol 2019 11 21;116:105614. Epub 2019 Sep 21.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima, 890-8544, Japan.

Osteopontin (OPN) is an osteoblast-derived secretory protein that plays a role in bone remodeling, osteoblast responsiveness, and inflammation. We recently found that osteoblast differentiation is type-specific, with conditions of JNK inactivation inducing osteoblasts that preferentially express OPN (OPN-type). Since OPN-type osteoblasts highly express osteogenesis-inhibiting proteins and Rankl, an important inducer of osteoclastogenesis, an increased appearance of OPN-type osteoblasts may be associated with inefficient and poor-quality bone regeneration. However, whether specific osteogenic inducers can modulate OPN-type osteoblast differentiation is completely unknown. Here, we demonstrate that bone morphogenic protein 9 (BMP9) prevents induction of OPN-type osteoblast differentiation under conditions of JNK inhibition. Although JNK inactivation suppressed both BMP2- and BMP9-induced matrix mineralization and osteocalcin expression, the expression of Rankl and specific cytokines such as Gpha2, Esm1, and Sfrp1 under similar conditions was increased in all cells except those treated with BMP9. Increased expression of Id4, a critical transcriptional regulator of OPN-type osteoblast differentiation, was similarly prevented only in BMP9-treated cells. We also found that BMP9 specifically induces the expression of Hey1, a bHLH transcriptional repressor, and that Id4 inhibits the suppressive effects of Hey1 on Opn promoter activity by forming Id4-Hey1 complexes in osteoblasts. Using site-direct mutagenesis, ChIP, and immunoprecipitation, we elucidated that BMP9-induced overexpression of Hey1 can overcome the effects of Id4 and suppress OPN expression. We further found that p38 activation and JNK inactivation are involved in BMP9-induced Hey1 expression. Collectively, these data suggest that BMP9 is a unique osteogenic inducer that regulates OPN-type osteoblast differentiation.
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http://dx.doi.org/10.1016/j.biocel.2019.105614DOI Listing
November 2019

BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.

FASEB J 2019 11 31;33(11):12124-12134. Epub 2019 Jul 31.

Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Bone morphogenetic protein (BMP)9 has been reported to be the most potent BMP to induce bone formation. However, the details of BMP9-transduced intracellular signaling remain ambiguous. Here, we have investigated signal transduction mechanisms of BMP9 in comparison to BMP2, another potent inducer of bone formation, in osteoblasts. In a mouse osteoblast cell line, BMP9 induced higher mRNA levels of alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) than BMP2 within 2 h. Unlike BMP2, BMP9 induced rapid phosphorylation of glycogen synthase kinase 3-β (GSK3-β) and protein kinase B (Akt) and increased the cellular protein content of β-catenin. BMP9 moderately increased mRNA levels of several canonical Wingless-related integration site to lower degrees than BMP2. Furthermore, BMP9-induced GSK3-β phosphorylation was not inhibited by pretreatment with actinomycin D, cycloheximide, or Brefeldin A, indicating it is independent of Wnt protein secretion. BMP9-induced GSK3-β phosphorylation was abrogated by Akt or class I PI3K-specific inhibitors. Moreover, inactivation of GSK3-β by LiCl did not further promote ALP and Runx2 mRNA induction by BMP9 as significantly as that by BMP2. Notably, BMP9-induced GSK3-β phosphorylation was inhibited by small interfering RNA against endoglin and GIPC PDZ domain-containing family, member 1. Taken together, our present findings have indicated that BMP9 directly activates GSK3β-β-catenin signaling pathway through class I PI3K-Akt Axis in osteoblasts, which may be essential for the potent osteoinductive activity of BMP9.-Eiraku, N., Chiba, N., Nakamura, T., Amir, M. S., Seong, C.-H., Ohnishi, T., Kusuyama, J., Noguchi, K., Matsuguchi, T. BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts.
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http://dx.doi.org/10.1096/fj.201900733RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6902712PMC
November 2019

Low intensity pulsed ultrasound (LIPUS) maintains osteogenic potency by the increased expression and stability of Nanog through spleen tyrosine kinase (Syk) activation.

Cell Signal 2019 10 19;62:109345. Epub 2019 Jun 19.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.

Mesenchymal stem cells (MSCs) are a powerful tool for cell-based, clinical therapies like bone regeneration. Therapeutic use of cell transplantation requires many cells, however, the expansion process needed to produce large quantities of cells reduces the differentiation potential of MSCs. Here, we examined the protective effects of low intensity pulsed ultrasound (LIPUS) on the maintenance of osteogenic potency. Primary osteoblastic cells were serially passaged between 2 and 12 times with daily LIPUS treatment. We found that LIPUS stimulation maintains osteogenic differentiation capacity in serially passaged cells, as characterized by improved matrix mineralization and Osteocalcin mRNA expression. Decreased expression of Nanog, Sox2, and Msx2, and increased expression of Pparg2 from serial passaging was recovered in LIPUS-stimulated cells. We found that LIPUS stimulation not only increased but also sustained expression of Nanog in primary osteoblasts and ST2 cells, a mouse mesenchymal stromal cell line. Nanog overexpression in serially passaged cells mimicked the recuperative effects of LIPUS on osteogenic potency, highlighting the important role of Nanog in LIPUS stimulation. Additionally, we found that spleen tyrosine kinase (Syk) is an important signaling molecule to induce Nanog expression in LIPUS-stimulated cells. Syk activation was regulated by both Rho-associated kinase 1 (ROCK1) and extracellular ATP in a paracrine manner. Interestingly, the LIPUS-induced increase in Nanog mRNA expression was regulated by ATP-P2X4-Syk Y323 activation, while the improvement of Nanog protein stability was controlled by the ROCK1-Syk Y525/526 pathway. Taken together, these results indicate that LIPUS stimulation recovers and maintains the osteogenic potency of serially passaged cells through a Syk-Nanog axis.
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http://dx.doi.org/10.1016/j.cellsig.2019.109345DOI Listing
October 2019

Low-intensity pulsed ultrasound promotes bone morphogenic protein 9-induced osteogenesis and suppresses inhibitory effects of inflammatory cytokines on cellular responses via Rho-associated kinase 1 in human periodontal ligament fibroblasts.

J Cell Biochem 2019 09 21;120(9):14657-14669. Epub 2019 Apr 21.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Periodontal ligament fibroblasts (PDLFs) have osteogenic capacity, producing bone matrix proteins. Application of bone morphogenic proteins (BMPs) to PDLFs is a promising approach for periodontal regeneration. However, in chronic bone metabolic disorders, such as periodontitis, proper control of accompanying inflammation is essential for optimizing the effects of BMPs on PDLFs. We have previously shown that low-intensity pulsed ultrasound (LIPUS), a medical technology that induces mechanical stress using sound waves, significantly promotes osteogenesis in mesenchymal stem cells. Here, we demonstrate that LIPUS promotes the BMP9-induced osteogenic differentiation of PDLFs. In contrast, BMP2-induced osteogenic differentiation was not altered by LIPUS, probably due to the LIPUS-induced secretion of Noggin, a BMP2 antagonist, from PDLFs. To examine if LIPUS affects inflammatory responses of PDLFs to lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (LPS-PG), we also simultaneously treated PDLFs with LIPUS and LPS-PG. Treatment with LIPUS significantly inhibited the phosphorylation of ERKs, TANK-binding kinase 1, and interferon regulatory factor 3 in LPS-PG-stimulated PDLFs, in addition to inhibiting the degradation of IκB. Furthermore, LIPUS treatment reduced messenger RNA (mRNA) expression of interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-8, C-C motif chemokine ligand 2, C-X-C motif chemokine ligand 1 (CXCL1), CXCL10 and receptor activator of nuclear factor kappa-B ligand, and also diminished IL-1ß and tumor necrosis factor a (TNFa)-induced inflammatory reactions. Phosphorylation of Rho-associated kinase 1 (ROCK1) was induced by LIPUS, while ROCK1-specific inhibitor prevented the promotive effects of LIPUS on p38 phosphorylation, mRNA expression of CXCL1 and Noggin, and osteogenesis. The suppressive effects of LIPUS on LPS-PG-stimulated inflammatory reactions were also prevented by ROCK1 inhibition. Moreover, LIPUS treatment blocked inhibitory effects of LPS-PG and IL-1ß on osteogenesis. These results indicate that LIPUS suppresses inflammatory effects of LPS-PG, IL-1ß, and TNFa and also promotes BMP9-induced osteogenesis through ROCK1 in PDLFs.
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http://dx.doi.org/10.1002/jcb.28727DOI Listing
September 2019

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

FASEB J 2019 06 18;33(6):7331-7347. Epub 2019 Mar 18.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).
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http://dx.doi.org/10.1096/fj.201802465RDOI Listing
June 2019

Constitutive activation of p46JNK2 is indispensable for C/EBPδ induction in the initial stage of adipogenic differentiation.

Biochem J 2017 10 5;474(20):3421-3437. Epub 2017 Oct 5.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan

Adipogenic differentiation plays a vital role in energy homeostasis and endocrine system. Several transcription factors, including peroxisome proliferator-activated receptor gamma 2 and CCAAT-enhancer-binding protein (C/EBP) α, β, and δ, are important for the process, whereas the stage-specific intracellular signal transduction regulating the onset of adipogenesis remains enigmatic. Here, we explored the functional role of c- N-terminal kinases (JNKs) in adipogenic differentiation using differentiation models of 3T3-L1 cells and primary adipo-progenitor cells. JNK inactivation with either a pharmacological inhibitor or JNK2-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the down-regulation of , (), , and expressions. Conversely, increased adipogenesis was observed by the inducible overexpression of p46JNK2 (JNK2-1), whereas it was not observed by that of p54JNK2 (JNK2-2), indicating a distinct role of p46JNK2. The essential role of JNK appears restricted to the early stage of adipogenic differentiation, as JNK inhibition in the later stages did not influence adipogenesis. Indeed, JNK phosphorylation was significantly induced at the onset of adipogenic differentiation. As for the transcription factors involved in early adipogenesis, JNK inactivation significantly inhibited the induction of , but not , during the initial stage of adipogenic differentiation. JNK activation increased mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes.
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http://dx.doi.org/10.1042/BCJ20170332DOI Listing
October 2017

Spleen tyrosine kinase influences the early stages of multilineage differentiation of bone marrow stromal cell lines by regulating phospholipase C gamma activities.

J Cell Physiol 2018 Mar 28;233(3):2549-2559. Epub 2017 Sep 28.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

Bone marrow stromal cells (BMSCs) are multipotent cells that can differentiate into adipocytes and osteoblasts. Inadequate BMSC differentiation is occasionally implicated in chronic bone metabolic disorders. However, specific signaling pathways directing BMSC differentiation have not been elucidated. Here, we explored the roles of spleen tyrosine kinase (Syk) in BMSC differentiation into adipocytes and osteoblasts. We found that Syk phosphorylation was increased in the early stage, whereas its protein expression was gradually decreased during the adipogenic and osteogenic differentiation of two mouse mesenchymal stromal cell lines, ST2 and 10T(1/2), and a human BMSC line, UE6E-7-16. Syk inactivation with either a pharmacological inhibitor or Syk-specific siRNA suppressed adipogenic differentiation, characterized by decreased lipid droplet appearance and the gene expression of fatty acid protein 4 (Fabp4), peroxisome proliferator-activated receptor γ2 (Pparg2), CCAAT/enhancer binding proteins α (C/EBPα), and C/EBPβ. In contrast, Syk inhibition promoted osteogenic differentiation, represented by increase in matrix mineralization and alkaline phosphatase (ALP) activity, as well as the expression levels of osteocalcin, runt-related transcription factor 2 (Runx2), and distal-less homeobox 5 (Dlx5) mRNAs. We also found that Syk-induced signals are mediated by phospholipase C γ1 (PLCγ1) in osteogenesis and PLCγ2 in adipogenesis. Notably, Syk-activated PLCγ2 signaling was partly modulated through B-cell linker protein (BLNK) in adipogenic differentiation. On the other hand, growth factor receptor-binding protein 2 (Grb2) was involved in Syk-PLCγ1 axis in osteogenic differentiation. Taken together, these results indicate that Syk-PLCγ signaling has a dual role in regulating the initial stage of adipogenic and osteogenic differentiation of BMSCs.
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http://dx.doi.org/10.1002/jcp.26130DOI Listing
March 2018

Low-Intensity Pulsed Ultrasound (LIPUS) Promotes BMP9-Induced Osteogenesis and Suppresses Inflammatory Responses in Human Periodontal Ligament-Derived Stem Cells.

J Orthop Trauma 2017 Jul;31(7):S4

Departments of *Oral Biochemistry; and †Periodontology, Graduate School of Medical and Dental Sciences, Kagoshima University.

Objective: We previously reported that low intensity pulsed ultrasound (LIPUS) promotes marrow stromal cell (MSC) osteogenesis and suppresses the LPS-induced inflammatory response in osteoblasts. Here, we examined the effects of LIPUS on human periodontal ligament-derived stem cells (hPDLSCs) in chronic inflammatory bone disease, such as periodontitis.

Materials And Methods: hPDLSCs were collected from 3 healthy third molars. hPDLSCs were induced to differentiate by either recombinant BMP2 or BMP9 with or without daily LIPUS treatment (20 min/d). hPDLSCs were also stimulated by Porphyromonas gingivalis-derived LPS (LPS-PG), IL-1beta, and TNF-alpha with or without LIPUS. Matrix mineralization was evaluated by alizarin red S staining. The expression of genes for osteogenic makers and for inflammatory cytokines were analyzed by real time RT-PCR.

Results: LIPUS promoted BMP9-induced osteogenesis of hPDLSCs based on increases in both cell calcification and osteogenic marker expression. In contrast, LIPUS did not affect BMP2-induced osteogenic differentiation. LIPUS-induced Noggin expression was potentially involved in the differential response of the cells. Either LPS-PG, IL-1beta, or TNF-alpha-induced ERK phosphorylation and IL-8, CCL2, and RANKL expression were decreased in LIPUS-treated hPDLSCs. Moreover, the inhibitory effects of LPS-PG and IL-1beta on osteogenesis of hPDLSCs were significantly blocked by LIPUS.

Discussion: LIPUS is an effective tool to promote osteogenic differentiation under inflammatory conditions.
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http://dx.doi.org/10.1097/01.bot.0000520897.92470.70DOI Listing
July 2017

Osteopontin inhibits osteoblast responsiveness through the down-regulation of focal adhesion kinase mediated by the induction of low-molecular weight protein tyrosine phosphatase.

Mol Biol Cell 2017 May 22;28(10):1326-1336. Epub 2017 Mar 22.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8544, Japan

Osteopontin (OPN) is an osteogenic marker protein. Osteoblast functions are affected by inflammatory cytokines and pathological conditions. OPN is highly expressed in bone lesions such as those in rheumatoid arthritis. However, local regulatory effects of OPN on osteoblasts remain ambiguous. Here we examined how OPN influences osteoblast responses to mechanical stress and growth factors. Expression of NO synthase 1 () and was increased by low-intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of expression was abrogated by both exogenous OPN overexpression and recombinant OPN treatment, whereas it was promoted by OPN-specific siRNA and OPN antibody. Moreover, LIPUS-induced phosphorylation of focal adhesion kinase (FAK), a crucial regulator of mechanoresponses, was down-regulated by OPN treatments. OPN also attenuated hepatocyte growth factor-induced vitamin D receptor () expression and platelet-derived growth factor-induced cell mobility through the repression of FAK activity. Of note, the expression of low-molecular weight protein tyrosine phosphatase (LMW-PTP), a FAK phosphatase, was increased in both OPN-treated and differentiated osteoblasts. CD44 was a specific OPN receptor for LWW-PTP induction. Consistently, the suppressive influence of OPN on osteoblast responsiveness was abrogated by LMW-PTP knockdown. Taken together, these results reveal novel functions of OPN in osteoblast physiology.
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http://dx.doi.org/10.1091/mbc.E16-10-0716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426847PMC
May 2017

Hepatocyte growth factor reduces CXCL10 expression in keratinocytes.

FEBS Lett 2016 Oct 18;590(20):3595-3605. Epub 2016 Oct 18.

Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Japan.

Keratinocytes secrete vascular endothelial growth factor (VEGF) and angioregulatory chemokines during cutaneous wound healing. Hepatocyte growth factor (HGF) promotes skin re-epithelialization by increasing VEGF expression in keratinocytes. Here, we investigated the regulatory roles of HGF in the expression of genes encoding angiogenic and angiostatic chemokines in keratinocytes and found that HGF specifically inhibits mRNA expression of the angiostatic chemokine CXCL10 in both mouse primary keratinocytes and in the human keratinocyte cell line HaCaT through the MEK/ERK cascade. Furthermore, HGF inhibited tumor necrosis factor-α-induced CXCL10 expression at both mRNA and protein levels in HaCaT cells. Thus, HGF may orchestrate angiogenesis in wounded skin by modulating both VEGF and CXCL10 expression in keratinocytes.
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http://dx.doi.org/10.1002/1873-3468.12452DOI Listing
October 2016

Investigating the associations between adiposity, life course overweight trajectories, and telomere length.

Aging (Albany NY) 2016 09;8(11):2689-2701

Medical Research Council Unit for Lifelong Health and Ageing at UCL, University College London, London WC1B 5JU, UK.

Obesity may accelerate ageing through chronic inflammation. To further examine this association, we assessed current adiposity, adiposity at early adulthood and life course overweight trajectories in relation to leukocyte telomere length (LTL). We included a total of 7,008 nationally representative U.S. residents and collected information on objectively measured body mass index (BMI), waist circumference and percent body fat. BMI at age 25 and overweight trajectories were assessed using self-reported history. Leukocyte telomere length (LTL) relative to a standard DNA reference (T/S ratio) was quantified by polymerase chain reaction (PCR). Linear regression models were used to examine the difference in LTL across adiposity measures at examination, BMI at age 25, and overweight trajectories. A 0.2% decrease in telomere length (95% CI: -0.3 to -0.07%) was observed for every kg/m2 increase in BMI, whereas a unit increase in waist circumference (cm) and percent body fat contributed to a 0.09% and 0.01% decrease in LTL, respectively. Higher BMI and being obese at age 25 contributed to lower LTL at older ages. Associations between weight loss through life course and LTL were observed, which further marked the importance of life course adiposity dynamics as a determinant of ageing.
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http://dx.doi.org/10.18632/aging.101036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5191863PMC
September 2016

Circulating Prostate-Specific Antigen and Telomere Length in a Nationally Representative Sample of Men Without History of Prostate Cancer.

Prostate 2017 01 27;77(1):22-32. Epub 2016 Aug 27.

PILAR Research and Education, Cambridge, United Kingdom.

Background: We investigated the association of prostate-specific antigen (PSA) with leukocyte telomere length, which may be altered in preclinical prostate malignancies.

Methods: This study was based on the 2001-2002 U.S. National Health and Nutrition Examination Survey (NHANES). A subsample of 1,127 men aged 40-85 years without prior history of prostate cancer who provided informed consent and blood samples were selected. Leukocyte telomere length (LTL) relative to standard DNA reference (T/S ratio) was quantified by polymerase chain reaction (PCR). Survey-weighted multivariable linear regression was performed to examine T/S ratio across quintiles of total and free PSA and free-to-total PSA ratio (%fPSA). A sensitivity analysis was performed by excluding men dying from prostate cancer during follow-up through to December 31, 2006. Stratification analyses were carried out to assess any effect modification by age group, race, body mass index (BMI), and levels of C-reactive protein (CRP), a marker of inflammation.

Results: Higher total PSA levels were associated to longer LTL, with approximately 8% increase in log-transformed T/S ratio (95% confidence interval [CI]: 2-13%) among men in the highest quintile of total PSA compared to the lowest in the fully adjusted model (P  = 0.01). No significant association was found for free PSA or %fPSA, although nonlinearity between all PSA measures and T/S ratio was indicated. Similar results were found after excluding men who died from prostate cancer during follow-up. We also found the associations between total PSA and T/S ratio to be strongest among non-Hispanic blacks, non-obese men (BMI <30 kg/m ), and those with low CRP. However, a significant interaction was only found between total PSA and race/ethnicity (P  = 0.01).

Conclusion: Total PSA levels were strongly associated to LTL, particularly among non-Hispanic blacks. Our findings support a potential link between PSA and specific mechanisms contributing to prostate cancer development. Prostate 77:22-32, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/pros.23245DOI Listing
January 2017

CXCL3 positively regulates adipogenic differentiation.

J Lipid Res 2016 10 10;57(10):1806-1820. Epub 2016 Aug 10.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. Electronic address:

Chemokines are a family of cytokines inducing cell migration and inflammation. Recent reports have implicated the roles of chemokines in cell differentiation. However, little is known about the functional roles of chemokines in adipocytes. Here, we explored gene expression levels of chemokines and chemokine receptors during adipogenic differentiation. We have found that two chemokines, chemokine (C-X-C motif) ligand 3 (CXCL3) and CXCL13, as well as CXC chemokine receptor 2 (CXCR2), a CXCL3 receptor, are highly expressed in mature adipocytes. When 3T3-L1 cells and ST2 cells were induced to differentiate, both the number of lipid droplets and the expression levels of adipogenic markers were significantly promoted by the addition of CXCL3, but not CXCL13. Conversely, gene knockdown of either CXCL3 or CXCR2 by specific siRNA effectively inhibited the course of adipogenic differentiation. CXCL3 treatment of 3T3-L1 cells significantly induced the phosphorylation of ERK and c-jun N-terminal kinase (JNK). Furthermore, CXCL3-induced CCAAT-enhancer binding protein (C/EBP)β and δ expression was suppressed by both ERK and JNK-specific inhibitors. Furthermore, chromatin immunoprecipitation assay revealed functional binding of PPARγ2 within the cxcl3 promoter region. Taken together, these results have indicated that CXCL3 is a novel adipokine that facilitates adipogenesis in an autocrine and/or a paracrine manner through induction of c/ebpb and c/ebpd.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5036360PMC
http://dx.doi.org/10.1194/jlr.M067207DOI Listing
October 2016

Smoking, second-hand smoke exposure and smoking cessation in relation to leukocyte telomere length and mortality.

Oncotarget 2016 09;7(37):60419-60431

PILAR Research and Education, Cambridge, UK.

Objectives: To investigate the link between smoking exposure, telomere length and mortality, with emphasis on second-hand smoke (SHS) exposure and the duration of smoking cessation.

Results: A total of 1,018 participants died during follow-up (mean: 10.3 years). A 50 base-pair decrease in LTL was shown among cotinine-confirmed current versus never smokers. The 90th quantile of LTL decreased with increasing cotinine among never smokers, indicating a role of SHS. Longer telomeres with smoking cessation were indicated but limited to a 3-16 year period of abstaining smoking. When assessing mortality, we observed a lower risk of all-cause death for the second quintile compared to the first among never smokers (HR: 0.67, 95% CI: 0.52-0.87), and a higher risk was found among current smokers (HR: 1.89, 1.19-2.92).

Materials And Methods: We studied 6,456 nationally representative U.S. respondents with mortality follow-up through to 31 December 2011. Smoking status was assessed by interviews and cotinine levels. Relative leukocyte telomere length (LTL) was quantified by polymerase chain reaction (PCR). Multivariable linear regression was performed to examine LTL by smoking exposure, adjusted for age, sex, race/ethnicity, socioeconomic status, education, body mass index, alcohol consumption, and physical activity. We further estimated the association of LTL with cotinine levels using quantile regression, and with smoking cessation dynamics. Cox regression was used to estimate mortality by smoking status and LTL.

Conclusion: Our findings indicated a complex association between smoking, telomere length, and mortality. LTL alterations with SHS and smoking cessation warrant further investigation for translation to public health measures.
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http://dx.doi.org/10.18632/oncotarget.11051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312393PMC
September 2016

10. Low-Intensity Pulsed Ultrasound (LIPUS) Stimulation Helps to Maintain the Differentiation Potency of Mesenchymal Stem Cells by Induction in Nanog Protein Transcript Levels and Phosphorylation.

J Orthop Trauma 2016 Aug;30(8):S4-5

*Field of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Science; †Faculty of Dental, Kagoshima University; and ‡Department of Oral Biology and Tissue Engineering, Meikai University School of Dentistry.

Mesenchymal Stem Cells (MSCs) are pluripotent cells that can be differentiated as osteoblasts, adipocytes, myocytes or chondrocytes depending on the culture condition. However, MSCs are known to lose their differentiation potency after long-term culture. Development of a new cell culture method to maintain their stemness is required for successful application of MSCs. Here, we revealed that low-intensity pulsed ultrasound (LIPUS) stimulation was useful for maintaining the MSC stemness as LIPUS inhibited the loss of osteogenic differentiation potency of osteo-progenitor cells induced by serial subculture. LIPUS also increased the transcriptional and phosphorylation level of Nanog, a crucial stem cell marker gene in a MSC cell line. We also found that LIPUS induced the secretion of extracellular adenosine triphosphate (ATP) in MSC. The treatments of the conditioned medium from LIPUS-stimulated MSC and exogenous ATP promoted Nanog expression. Thus, LIPUS may maintain the long-term differentiation potency of MSCs and osteo-progenitor cells by induction in Nanog transcript level and phosphorylation.
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http://dx.doi.org/10.1097/01.bot.0000489983.17459.0bDOI Listing
August 2016

Automated Assay of Telomere Length Measurement and Informatics for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort.

Genetics 2015 Aug 19;200(4):1061-72. Epub 2015 Jun 19.

Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158-2517

The Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort includes DNA specimens extracted from saliva samples of 110,266 individuals. Because of its relationship to aging, telomere length measurement was considered an important biomarker to develop on these subjects. To assay relative telomere length (TL) on this large cohort over a short time period, we created a novel high throughput robotic system for TL analysis and informatics. Samples were run in triplicate, along with control samples, in a randomized design. As part of quality control, we determined the within-sample variability and employed thresholds for the elimination of outlying measurements. Of 106,902 samples assayed, 105,539 (98.7%) passed all quality control (QC) measures. As expected, TL in general showed a decline with age and a sex difference. While telomeres showed a negative correlation with age up to 75 years, in those older than 75 years, age positively correlated with longer telomeres, indicative of an association of longer telomeres with more years of survival in those older than 75. Furthermore, while females in general had longer telomeres than males, this difference was significant only for those older than age 50. An additional novel finding was that the variance of TL between individuals increased with age. This study establishes reliable assay and analysis methodologies for measurement of TL in large, population-based human studies. The GERA cohort represents the largest currently available such resource, linked to comprehensive electronic health and genotype data for analysis.
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http://dx.doi.org/10.1534/genetics.115.178624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574243PMC
August 2015

Induction of CXCL2 and CCL2 by pressure force requires IL-1β-MyD88 axis in osteoblasts.

Bone 2015 May 17;74:76-82. Epub 2015 Jan 17.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Electronic address:

Mechanical stresses including pressure force induce chemokine expressions in osteoblasts resulting in inflammatory reactions and bone remodeling. However, it has not been well elucidated how mechanical stresses induce inflammatory chemokine expressions in osteoblasts. IL-1β has been identified as an important pathogenic factor in bone loss diseases, such as inflammatory arthritis and periodontitis. Myeloid differentiation factor 88 (MyD88) is an essential downstream adaptor molecule of IL-1 receptor signaling. This study was to examine the gene expression profiles of inflammatory chemokines and the role of MyD88 in osteoblasts stimulated by pressure force. Pressure force (10g/cm(2)) induced significant mRNA increases of CXCL2, CCL2, and CCL5, as well as prompt phosphorylation of MAP kinases (ERK, p38 and JNK), in wild-type primary osteoblasts. The CXCL2 and CCL2 mRNA increases and MAP kinase phosphorylation were severely impaired in MyD88(-/-) osteoblasts. Constitutive low-level expression of IL-1β mRNA was similarly observed in both wild-type and MyD88(-/-) osteoblasts, which was not altered by pressure force stimulation. Notably, neutralization of IL-1β with a specific antibody significantly impaired pressure force-induced mRNA increases of CXCL2 and CCL2, as well as MAP kinase phosphorylation, in wild-type osteoblasts. Furthermore, pre-treatment with recombinant IL-1β significantly enhanced MAP kinase phosphorylation and mRNA increases of CXCL2 and CCL2 by pressure force in wild-type but not MyD88(-/-) osteoblasts. These results have suggested that the activation of MyD88 pathway by constitutive low-level IL-1β expression is essential for pressure force-induced CXCL2 and CCL2 expression in osteoblasts. Thus MyD88 signal in osteoblasts may be required for bone resorption by pressure force through chemokine induction.
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http://dx.doi.org/10.1016/j.bone.2015.01.007DOI Listing
May 2015

AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation.

Bone 2015 May 10;74:125-33. Epub 2014 Dec 10.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Electronic address:

Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity.
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http://dx.doi.org/10.1016/j.bone.2014.12.001DOI Listing
May 2015

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

PLoS Genet 2014 Jun 5;10(6):e1004299. Epub 2014 Jun 5.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America; Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore.

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.
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http://dx.doi.org/10.1371/journal.pgen.1004299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046920PMC
June 2014

MAP3K8 (TPL2/COT) affects obesity-induced adipose tissue inflammation without systemic effects in humans and in mice.

PLoS One 2014 24;9(2):e89615. Epub 2014 Feb 24.

Department of Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands ; Institute for Genomic and Metabolic Disease, Radboud University Medical Centre, Nijmegen, The Netherlands ; Department of Human Nutrition, Wageningen University and Research Centre, Wageningen, The Netherlands.

Chronic low-grade inflammation in adipose tissue often accompanies obesity, leading to insulin resistance and increasing the risk for metabolic diseases. MAP3K8 (TPL2/COT) is an important signal transductor and activator of pro-inflammatory pathways that has been linked to obesity-induced adipose tissue inflammation. We used human adipose tissue biopsies to study the relationship of MAP3K8 expression with markers of obesity and expression of pro-inflammatory cytokines (IL-1β, IL-6 and IL-8). Moreover, we evaluated obesity-induced adipose tissue inflammation and insulin resistance in mice lacking MAP3K8 and WT mice on a high-fat diet (HFD) for 16 weeks. Individuals with a BMI >30 displayed a higher mRNA expression of MAP3K8 in adipose tissue compared to individuals with a normal BMI. Additionally, high mRNA expression levels of IL-1β, IL-6 and IL-8, but not TNF -α, in human adipose tissue were associated with higher expression of MAP3K8. Moreover, high plasma SAA and CRP did not associate with increased MAP3K8 expression in adipose tissue. Similarly, no association was found for MAP3K8 expression with plasma insulin or glucose levels. Mice lacking MAP3K8 had similar bodyweight gain as WT mice, yet displayed lower mRNA expression levels of IL-1β, IL-6 and CXCL1 in adipose tissue in response to the HFD as compared to WT animals. However, MAP3K8 deficient mice were not protected against HFD-induced adipose tissue macrophage infiltration or the development of insulin resistance. Together, the data in both human and mouse show that MAP3K8 is involved in local adipose tissue inflammation, specifically for IL-1β and its responsive cytokines IL-6 and IL-8, but does not seem to have systemic effects on insulin resistance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089615PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3933658PMC
March 2015

Low intensity pulsed ultrasound (LIPUS) influences the multilineage differentiation of mesenchymal stem and progenitor cell lines through ROCK-Cot/Tpl2-MEK-ERK signaling pathway.

J Biol Chem 2014 Apr 18;289(15):10330-10344. Epub 2014 Feb 18.

Department of Biochemistry and Molecular Dentistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Electronic address:

Mesenchymal stem cells (MSCs) are pluripotent cells that can differentiate into multilineage cell types, including adipocytes and osteoblasts. Mechanical stimulus is one of the crucial factors in regulating MSC differentiation. However, it remains unknown how mechanical stimulus affects the balance between adipogenesis and osteogenesis. Low intensity pulsed ultrasound (LIPUS) therapy is a clinical application of mechanical stimulus and facilitates bone fracture healing. Here, we applied LIPUS to adipogenic progenitor cell and MSC lines to analyze how multilineage cell differentiation was affected. We found that LIPUS suppressed adipogenic differentiation of both cell types, represented by impaired lipid droplet appearance and decreased gene expression of peroxisome proliferator-activated receptor γ2 (Pparg2) and fatty acid-binding protein 4 (Fabp4). LIPUS also down-regulated the phosphorylation level of peroxisome proliferator-activated receptor γ2 protein, inhibiting its transcriptional activity. In contrast, LIPUS promoted osteogenic differentiation of the MSC line, characterized by increased cell calcification as well as inductions of runt-related transcription factor 2 (Runx2) and Osteocalcin mRNAs. LIPUS induced phosphorylation of cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, which was essential for the phosphorylation of mitogen-activated kinase kinase 1 (MEK1) and p44/p42 extracellular signal-regulated kinases (ERKs). Notably, effects of LIPUS on both adipogenesis and osteogenesis were prevented by a Cot/Tpl2-specific inhibitor. Furthermore, effects of LIPUS on MSC differentiation as well as Cot/Tpl2 phosphorylation were attenuated by the inhibition of Rho-associated kinase. Taken together, these results indicate that mechanical stimulus with LIPUS suppresses adipogenesis and promotes osteogenesis of MSCs through Rho-associated kinase-Cot/Tpl2-MEK-ERK signaling pathway.
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http://dx.doi.org/10.1074/jbc.M113.546382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036157PMC
April 2014

Long-time treatment by low-dose N-acetyl-L-cysteine enhances proinflammatory cytokine expressions in LPS-stimulated macrophages.

PLoS One 2014 4;9(2):e87229. Epub 2014 Feb 4.

Department of Oral Biochemistry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan.

N-acetyl-L-cysteine is known to act as a reactive oxygen species scavenger and used in clinical applications. Previous reports have shown that high-dose N-acetyl-L-cysteine treatment inhibits the expression of proinflammatory cytokines in activated macrophages. Here, we have found that long-time N-acetyl-L-cysteine treatment at low-concentration increases phosphorylation of extracellular signal-regulated kinase 1/2 and AKT, which are essential for the induction of proinflammatory cytokines including interleukin 1β and interleukin 6 in lipopolysaccharide-stimulated RAW264.7 cells. Furthermore, long-time N-acetyl-L-cysteine treatment decreases expressions of protein phosphatases, catalytic subunit of protein phosphatase-2A and dual specificity phosphatase 1. On the other hand, we have found that short-time N-acetyl-L-cysteine treatment at low dose increases p53 expression, which inhibits expressions of proinflammatory cytokines. These observations suggest that long-time low-dose N-acetyl-L-cysteine treatment increases expressions of proinflammatory cytokines through enhancement of kinase phosphorylation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0087229PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3913600PMC
December 2014

Low-intensity pulsed ultrasound (LIPUS) inhibits LPS-induced inflammatory responses of osteoblasts through TLR4-MyD88 dissociation.

Bone 2014 Jan 30;58:17-25. Epub 2013 Sep 30.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan.

Previous reports have shown that osteoblasts are mechano-sensitive. Low-intensity pulsed ultrasound (LIPUS) induces osteoblast differentiation and is an established therapy for bone fracture. Here we have examined how LIPUS affects inflammatory responses of osteoblasts to LPS. LPS rapidly induced mRNA expression of several chemokines including CCL2, CXCL1, and CXCL10 in both mouse osteoblast cell line and calvaria-derived osteoblasts. Simultaneous treatment by LIPUS significantly inhibited mRNA induction of CXCL1 and CXCL10 by LPS. LPS-induced phosphorylation of ERKs, p38 kinases, MEK1/2, MKK3/6, IKKs, TBK1, and Akt was decreased in LIPUS-treated osteoblasts. Furthermore, LIPUS inhibited the transcriptional activation of NF-κB responsive element and Interferon-sensitive response element (ISRE) by LPS. In a transient transfection experiment, LIPUS significantly inhibited TLR4-MyD88 complex formation. Thus LIPUS exerts anti-inflammatory effects on LPS-stimulated osteoblasts by inhibiting TLR4 signal transduction.
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http://dx.doi.org/10.1016/j.bone.2013.09.018DOI Listing
January 2014

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages.

FEBS Lett 2012 May 21;586(10):1540-6. Epub 2012 Apr 21.

Department of Oral Biochemistry, Field of Developmental Medicine, Kagoshima University, Graduate School of Medical and Dental Sciences, Sakuragaoka, Kagoshima, Japan.

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-?B, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88(-/-) and cot/tpl2(-/-) macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages.
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http://dx.doi.org/10.1016/j.febslet.2012.04.018DOI Listing
May 2012

Bleomycin-induced lung injury in mice investigated by MRI: model assessment for target analysis.

Magn Reson Med 2012 Feb 7;67(2):499-509. Epub 2011 Jun 7.

Global Imaging Group, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Magnetic resonance imaging (MRI) has been used to follow the course of bleomycin-induced lung injury in mice and to investigate two knockout mouse lines with the aim of providing potential therapeutic targets. Bleomycin (0.25 mg/kg) was administered intranasally six times, once a day. MRI was carried out on spontaneously breathing animals up to day 70 after bleomycin. Neither cardiac nor respiratory gating was applied during image acquisition. A long lasting response following bleomycin has been detected by MRI in the lungs of male C57BL/6 mice. Histology showed that, from day 14-70 after bleomycin, fibrosis was the predominant component of the injury. Female C57BL/6 mice displayed a smaller response than males. Bleomycin-induced injury was significantly more pronounced in C57BL/6 than in Balb/C mice. MRI and histology demonstrated a protection against bleomycin insult in female heterozygous and male homozygous cancer Osaka thyroid kinase knockout animals. In contrast, no protection was seen in cadherin-11 knockout animals. In summary, MRI can quantify, in spontaneously breathing mice, bleomycin-induced lung injury. With the ability for repetitive measurements in the same animal, the technique is attractive for in vivo target analysis and compound profiling in this murine model.
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http://dx.doi.org/10.1002/mrm.23009DOI Listing
February 2012

Functional involvement of dual specificity phosphatase 16 (DUSP16), a c-Jun N-terminal kinase-specific phosphatase, in the regulation of T helper cell differentiation.

J Biol Chem 2011 Jul 25;286(28):24896-905. Epub 2011 May 25.

Department of Oral Biochemistry, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan.

Naïve CD4(+) T helper (Th) cells differentiate into distinct subsets of effector cells (Th1, Th2, Th17, and induced regulatory T cells (iTreg)) expressing different sets of cytokines upon encounter with presented foreign antigens. It has been well established that Th1/Th2 balance is critical for the nature of the following immune responses. Previous reports have demonstrated important roles of c-Jun N-terminal kinase (JNK) in Th1/Th2 balance, whereas the regulatory mechanisms of JNK activity in Th cells have not been elucidated. Here, we show that dual specificity phosphatase 16 (DUSP16, also referred to as MKP-M or MKP-7), which preferentially inactivates JNK, is selectively expressed in Th2 cells. In the in vitro differentiation assay of naïve CD4(+) cells, DUSP16 expression is up-regulated during Th2 differentiation and down-regulated during Th1 differentiation. Chromatin immunoprecipitation revealed the increased acetylation of histone H3/H4 at the dusp16 gene promoter in CD4(+) T cells under the Th2 condition. Adenoviral transduction of naïve CD4(+) T cells with DUSP16 resulted in increased mRNA expression of IL-4 and GATA-3 in Th2 and decreased expression of IFNγ and T-bet in Th1 differentiation. In contrast, transduction of a dominant negative form of DUSP16 had the reverse effects. Furthermore, upon immunization, T cell-specific dusp16 transgenic mice produced antigen-specific IgG2a at lower amounts, whereas DN dusp16 transgenic mice produced higher amounts of antigen-specific IgG2a accompanied by decreased amounts of antigen-specific IgG1 and IgE than those of control mice. Together, these data suggest the functional role of DUSP16 in Th1/Th2 balance.
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http://dx.doi.org/10.1074/jbc.M111.245019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3137064PMC
July 2011
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