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Osteopontin (OPN) is expressed in various cell types including osteoblasts. OPN expression level is robustly increased during osteoblast differentiation. Although OPN was initially found as a secretory protein (sOPN), recent reports identified the intracellular isoform of OPN (iOPN). Distinct functions of each OPN isoform in osteoblasts, however, are not well established. Here, using the Tet-On inducible expression system, we examined the role of each OPN isoform during osteoblast differentiation. Induced overexpression of wild type OPN (wtOPN), which includes both sOPN and iOPN, significantly increased matrix mineralization and osteogenic marker gene expression during osteogenic differentiation induced by either ascorbic acid or bone morphogenetic protein (BMP) 9. In contrast, these osteogenic differentiation processes were significantly inhibited by the specific overexpression of iOPN. Furthermore, the addition of recombinant OPN or neutralizing anti-OPN antibody to the culture medium exerted promotive or inhibitory effect on osteoblast differentiation, respectively. These data strongly indicate that iOPN exerts inhibitory effects on osteoblast differentiation, whereas sOPN exerts positive effects. We also found that the secretion process of OPN is positively regulated by c-Jun N-terminal kinase (JNK) activity in osteoblasts., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Gingival epithelial cells (GECs) are physical and immunological barriers against outward pathogens while coping with a plethora of non-pathogenic commensal bacteria. GECs express several members of Toll-like receptors (TLRs) and control subsequent innate immune responses. TLR4 senses lipopolysaccharide (LPS) while TLR7/8 recognizes single-strand RNA (ssRNA) playing important roles against viral infection. However, their distinct roles in GECs have not been fully demonstrated. Here, we analyzed biological responses of GECs to  LPS and CL075, a TLR7/8 agonist. GE1, a mouse gingival epithelial cell line, constitutively express TLR4 and TLR7, but not TLR8, like primary skin keratinocytes. Stimulation of GE1 cells with CL075 induced cytokine, chemokine, and antimicrobial peptide  expressions, the pattern of which is rather different from that with LPS: higher mRNA levels of interferon (IFN) β, CXCL10, and β-defensin (BD) 14 (mouse homolog of human BD3); lower levels of tumor necrosis factor (TNF), CCL5, CCL11, CCL20, CXCL2, and CX3CL1. As for the intracellular signal transduction of GE1 cells, CL075 rapidly induced significant AKT phosphorylation but failed to activate IKKα/β-NFκB pathway, whereas LPS induced marked IKKα/β-NFκB activation without significant AKT phosphorylation. In contrast, both CL075 and LPS induced rapid IKKα/β-NFκB activation and AKT phosphorylation in a macrophage cell line. Furthermore, specific inhibition of AKT activity abrogated CL075-induced IFNβ, CXCL10, and BD14 mRNA expression in GE1 cells. Thus, TLR4/7 ligands appear to induce rather different host-defense responses of GECs through distinct intracellular signaling mechanisms., (© 2024 The Authors. Journal of Cellular Biochemistry published by Wiley Periodicals LLC.)

Bone morphogenic protein 9 (BMP9) is one of the most potent inducers of osteogenic differentiation among the 14 BMP members, but its mechanism of action has not been fully demonstrated. Hes1 is a transcriptional regulator with basic helix-loop-helix (bHLH) domain and is a well-known Notch effector. In this study, we investigated the functional roles of early induction of Hes1 by BMP9 in a mouse mesenchymal stem cell line, ST2. Hes1 mRNA was transiently and periodically induced by BMP9 in ST2, which was inhibited by BMP signal inhibitors but not by Notch inhibitor. Interestingly, Hes1 knockdown in ST2 by siRNA increased the expression of osteogenic differentiation markers such as Sp7 and Ibsp and matrix mineralization in comparison with control siRNA transfected ST2. In contrast, forced expression of Hes1 by using the Tet-On system suppressed the expression of osteogenic markers and matrix mineralization by BMP9. We also found that the early induction of Hes1 by BMP9 suppressed the expression of Alk1, an essential receptor for BMP9. In conclusion, BMP9 rapidly induces the expression of Hes1 via the SMAD pathway in ST2 cells, which plays a negative regulatory role in osteogenic differentiation of mesenchymal stem cells induced by BMP9., (© 2023 Wiley Periodicals LLC.)

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are cell wall components of Gram-positive and Gram-negative bacteria, respectively. Notably, oral microflora consists of a variety of bacterial species, and osteomyelitis of the jaw caused by dental infection presents with symptoms of bone resorption and osteosclerosis. However, the effects of LTA and LPS on osteogenic differentiation have not yet been clarified. We examined the effects of LTA and LPS on osteoblasts and found that LTA alone promoted alizarin red staining at low concentrations and inhibited it at high concentrations. Additionally, gene expression of osteogenic markers (ALP, OCN, and OPG) were enhanced at low concentrations of LTA. High concentrations of LPS suppressed calcification potential, and the addition of low concentrations of LTA inhibited calcification suppression, restoring the gene expression levels of suppressed bone differentiation markers (ALP, BSP, and OCN). Moreover, the suppression of p38, a signaling pathway associated with bone differentiation, had opposing effects on gene-level expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), suggesting that mixed LTA and LPS infections have opposite effects on bone differentiation through concentration gradients, involving inflammatory markers (TNF-α and IL-6) and the p38 pathway.

Bone morphogenetic proteins (BMPs) are essential regulators of skeletal homeostasis, and BMP9 is the most potently osteogenic among them. Here, we found that BMP9 and BMP2 rapidly induced early growth response 1 (EGR1) protein expression in osteoblasts through MEK/ERK pathway-dependent transcriptional activation. Knock-down of EGR1 using siRNA significantly inhibited BMP9-induced matrix mineralization and osteogenic marker gene expression in osteoblasts. Knock-down of EGR1 significantly reduced SMAD1/5 phosphorylation and inhibited the expression of their transcriptional targets in osteoblasts stimulated by BMP9. In contrast, forced EGR1 overexpression in osteoblasts enhanced BMP9-mediated osteoblast differentiation and SMAD1/5 phosphorylation. An intracellular association between EGR1 and SMAD1/5 was identified using immunoprecipitation assays. These results indicated that EGR1 plays an important role in BMP9-stimulated osteoblast differentiation by enhancing SMAD1/5 phosphorylation., (© 2022 Federation of European Biochemical Societies.)

Objectives: The oral cavity is one of the main entry sites for SARS-CoV-2. Gingival keratinocytes express transmembrane serine protease 2 (TMPRSS2), responsible for priming the SARS-CoV-2 spike protein. We investigated whether periodontitis increased the expression of TMPRSS2., Methods: To investigate gene expression in periodontitis, we analyzed the expression of specific genes from (1) the Gene Expression Omnibus (GEO) dataset of 247 human gingival tissues and (2) an experimentally-induced periodontitis mouse model. Human gingival tissues with or without periodontitis were immunohistochemically stained using an anti-TMPRSS2 antibody. Analysis of the TMPRSS2 promoter was performed using a ChIP-Atlas dataset. TMPRSS2 expression was detected in cultured human keratinocytes using quantitative reverse transcription (qRT)-PCR and Western blot analysis., Results: GEO dataset analysis and an experimentally-induced periodontitis model revealed increased expression of TMPRSS2 in periodontitis gingiva. The keratinocyte cell membrane in periodontitis gingiva was strongly immunohistochemically stained for TMPRSS2. Using ChIP-Atlas and GEO datasets, we screened for transcription factors that bind to the TMPRSS2 promoter region. We found one candidate, estrogen receptor 1 (ESR1), highly expressed in periodontitis gingiva. Analysis of the GEO dataset revealed a correlation between ESR1 and TMPRSS2 expression in gingival tissues. An ESR1 ligand induced TMPRSS2 expression in cultured keratinocytes., Conclusions: Periodontitis increases TMPRSS2 expression in the cell membrane of gingival keratinocytes., (Copyright © 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Bone homeostasis is regulated by bone morphogenic proteins (BMPs), among which BMP9 is one of the most osteogenic. Here, we have found that BMP9 rapidly increases the protein expression of hypoxia-inducible factor-1α (HIF-1α) in osteoblasts under normoxic conditions more efficiently than BMP2 or BMP4. A combination of BMP9 and hypoxia further increased HIF-1α protein expression. HIF-1α protein induction by BMP9 is not accompanied by messenger RNA (mRNA) increase and is inhibited by the activation of prolyl hydroxylase domain (PHD)-containing protein, indicating that BMP9 induces HIF-1α protein expression by inhibiting PHD-mediated protein degradation. BMP9-induced HIF-1α protein increase was abrogated by inhibitors of phosphoinositide 3-kinase (PI3K) and protein kinase B (AKT) kinase, indicating that it is mediated by PI3K-AKT signaling pathway. BMP9 increased mRNA expression of pyruvate dehydrogenase kinase 1 (PDK1), a glycolytic enzyme, and vascular endothelial growth factor-A (VEGF-A), an angiogenic factor, in osteoblasts. Notably, BMP9-induced mRNA expression of PDK1, but not that of VEGF-A, was significantly inhibited by small interference RNA-mediated knockdown of Hif-1α. BMP9-induced matrix mineralization and osteogenic marker gene expressions were significantly inhibited by chemical inhibition and gene knockdown of either Hif-1α or Pdk-1, respectively. Since increased glycolysis is an essential feature of differentiated osteoblasts, our findings indicate that HIF-1α expression is important in BMP9-mediated osteoblast differentiation through the induction of PDK1., (© 2022 Wiley Periodicals LLC.)

Objectives: The colonic self-expandable metallic stent (C-SEMS) with a 9-French (Fr) delivery system allows for a small-caliber endoscope (SCE) to be used to treat malignant colonic obstruction. Despite the lack of evidence, the SCE has become popular because it is considered easier to insert than the large-caliber endoscope (LCE). We aimed to determine whether the SCE is more suitable than the LCE for C-SEMS placement., Methods: Between July 2018 and November 2019, 50 consecutive patients who were scheduled to undergo C-SEMS for colon obstruction were recruited in this study. Patients were randomized to the SCE or LCE group. The SCE and LCE were used with 9-Fr and 10-Fr delivery systems, respectively. The primary outcome was the total procedure time. Secondary outcomes were the technical success rate, complication rate, clinical success rate, insertion time, guidewire-passage time, stent-deployment time, and colonic obstruction-scoring-system score., Results: Forty-five patients (SCE group, n = 22; LCE group, n = 23) were analyzed. The procedure time in the LCE group (median, 20.5 min) was significantly ( p = 0.024) shorter than that in the SCE group (median, 25.1 min). The insertion time in the LCE group (median, 2.0 min) was significantly ( p = 0.0049) shorter than that in the SCE group (median, 6.0 min). A sub-analysis of the procedure difficulties showed that the insertion time in the LCE group (median, 5.0 min) was significantly shorter than that in the SCE group (median, 8.5 min)., Conclusion: Both LCE and SCE can be used for C-SEMS; however, LCE is more suitable than SCE as it achieved a faster and equally efficacious C-SEMS placement as that of SCE., Clinical Trial Registration Number: University Hospital Medical Information Network Clinical Trials Registry (UMIN 32748)., Competing Interests: Conflict of interest statement: The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: EI belongs to an endowed course supported by the companies Ono Pharmaceutical Co., Ltd, Miyarisan Pharmaceutical Co. Ltd, Sanwa Kagaku Kenkyusho Co., Ltd, Otsuka Pharmaceutical Factory, Inc., Fujifilm Medical Co., Ltd, Terumo Corporation, FANCL Corporation, Ohga Pharmacy, and Abbott Japan, LLC. EI receives lecture honorarium from Takeda Pharmaceutical Company. YO conducts collaborative research with Fujifilm Medical Co., Ltd and FANCL Corporation. The authors declare no other conflicts of interest in association with this study., (© The Author(s), 2022.)

Objectives: Traditionally, evaluation is considered a measurement process that can be performed independently of the cultural context. However, more recently the importance of considering raters' sense-making, that is, the process by which raters assign meaning to their collective experiences, is being recognised. Thus far, the majority of the discussion on this topic has originated from Western perspectives. Little is known about the potential influence of an Asian culture on raters' sense-making. This study explored residents' sense-making associated with evaluating their clinical teachers within an Asian setting to better understand contextual dependency of validity., Design: A qualitative study using constructivist grounded theory., Setting: The Japanese Ministry of Health, Labour and Welfare has implemented a system to monitor the quality of clinical teaching within its 2-year postgraduate training programme. An evaluation instrument was developed specifically for the Japanese setting through which residents can evaluate their clinical teachers., Participants: 30 residents from 10 Japanese teaching hospitals with experience in evaluating their clinical teachers were sampled purposively and theoretically., Methods: We conducted in-depth semistructured individual interviews. Sensitising concepts derived from Confucianism and principles of response process informed open, axial and selective coding., Results: Two themes and four subthemes were constructed. Japanese residents emphasised the awareness of their relationship with their clinical teachers (1). This awareness was fuelled by their sense of hierarchy (1a) and being part of the collective society (1b). Residents described how the meaning of evaluation (2) was coloured by their perceived role as senior (2a) and their experienced responsibility for future generations (2b)., Conclusions: Japanese residents' sense-making while evaluating their clinical teachers appears to be situated and affected by Japanese cultural values. These findings contribute to a better understanding of a culture's influence on residents' sense-making of evaluation instruments and the validity argument of evaluation., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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 2 O 2 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., (© 2021 Wiley Periodicals LLC.)

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., (© 2020 Federation of European Biochemical Societies.)

Background: Endoscopic submucosal dissection (ESD) is a standard treatment for tumors of the gastrointestinal tract. We developed a self-completion method of ESD using Endosaber to eliminate the need for an additional device or human assistance during the procedure. The aim of this study was to evaluate the technical feasibility and outcomes of this method in an ex vivo porcine training model., Methods: This was a pilot study, and the procedures were performed by 4 experts. Mock lesions measuring 15 mm in diameter were prepared at the posterior wall in the middle or lower esophagus obtained from domestic pigs. Each operator performed ESD on the mock lesions in 3 models. The primary outcome was ESD completion rate using the self-completion method. The secondary outcomes were procedure time, en bloc resection rate, perforation rate, and number of injections during the procedure., Results: All 12 ESDs were completed using the self-completion method. The median procedure time (interquartile range) was 483.5 (399-619.3) s (median incision time: 240.4 [168.3-332.5] s; median dissection time: 222 [182.8-257] s). En bloc resection rate was 100%. No perforation was noted during any of the procedures. The median number of injections was 10.5 (9-14.3). The procedure time decreased significantly with increase in experience (p = 0.020)., Conclusions: The self-completion ESD method using one Endosaber without any assistance achieved a 100% en bloc resection rate without any perforation. The need for an additional device or assistance was successfully eliminated. This method may prove to be a simple and cost-effective ESD procedure for lesions in humans., (© 2019 S. Karger AG, Basel.)

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., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background/aims: It is important to appropriately manage patients with procedure-related artificial mucosal ulcers or procedure-related complications. Many endoscopic closure techniques have been reported; however, they often require the use of special devices. We developed a single-channel endoscopic closure technique (SCCT) that can be performed with conventional devices. In the present study, we describe the technique and evaluate its efficacy., Methods: Twenty-five consecutive patients who underwent endoscopic treatment and whose artificial ulcer was closed using the SCCT were enrolled in this study. The technical success rate, number of clips for closure, procedure time, complication rate on the day of the procedure, clinical success rates on days 1 and 5, and incidence of severe stenosis of the gastrointestinal (GI) tract at 2 months after the procedure were evaluated., Results: The median ulcer diameter was 20 mm. The tumor locations were the stomach ( n = 19), jejunum ( n = 1), and colon ( n = 5). The technical success rate was 100% (25/25), and the rate of incomplete closure was 0% (0/25). Eight clips were needed on average. The median procedure time was 18 min (range 5-49 min). The complication rate was 0% (25/25). The clinical success rates on days 1 and 5 were 100% (19/19) and 100% (9/9), respectively. No patients presented stenosis as a late complication at 2 months after the procedure (0/25)., Conclusion: The SCCT could be applied in the treatment of artificial ulcers in several parts of the GI tract with a high clinical success rate and no complications. The SCCT appears to be a good option for closing artificial mucosal ulcers., Competing Interests: The authors have no conflicts of interest to declare., (Copyright © 2020 by S. Karger AG, Basel.)

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., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2019. Published by Elsevier Inc.)

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., (© 2019 Wiley Periodicals, Inc.)

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 Porphyromonas gingivalis 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).

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., (© 2017 Wiley Periodicals, Inc.)

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- jun N-terminal kinases (JNKs) in adipogenic differentiation using in vitro 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 Adiponectin , fatty acid protein 4 ( Fabp4 ), Pparg2 , and C/ebpa 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 C/ebpd , but not C/ebpb , during the initial stage of adipogenic differentiation. JNK activation increased C/ebpd mRNA and protein expression through the induction and phosphorylation of activating transcription factor 2 (ATF2) that binds to a responsive element within the C/ebpd gene promoter region. Taken together, these data indicate that constitutive JNK activity is specifically required for the initial stage differentiation events of adipocytes., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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.

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 ( Nos1 ) and Nos2 was increased by low-intensity pulsed ultrasound (LIPUS) in MC3T3-E1 cells and primary osteoblasts. The increase of Nos1/2 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 ( Vdr ) 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., (© 2017 Kusuyama et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

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 trend  = 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 2 ), and those with low CRP. However, a significant interaction was only found between total PSA and race/ethnicity (P interaction  = 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., (© 2016 Wiley Periodicals, Inc.)

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., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

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., Competing Interests: There are no conflicts of interest for all the contributors.

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.

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.

Telomerase core enzyme minimally consists of the telomerase reverse transcriptase domain-containing protein (Est2 in budding yeast S. cerevisiae) and telomerase RNA, which contains the template specifying the telomeric repeat sequence synthesized. Here we report that in vivo, a fraction of S. cerevisiae telomerase RNA (TLC1) molecules form complexes containing at least two molecules of TLC1, via two separable modes: one requiring a sequence in the 3' region of the immature TLC1 precursor and the other requiring Ku and Sir4. Such physical TLC1-TLC1 association peaked in G1 phase and did not require telomere silencing, telomere tethering to the nuclear periphery, telomerase holoenzyme assembly, or detectable Est2-Est2 protein association. These data indicate that TLC1-TLC1 associations reflect processes occurring during telomerase biogenesis; we propose that TLC1-TLC1 associations and subsequent reorganization may be regulatory steps in telomerase enzymatic activation.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 by the Genetics Society of America.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

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.

Short leukocyte telomere length (LTL) has been associated with atherosclerosis in cross-sectional studies, but the prospective relationship between telomere shortening and risk of developing carotid atherosclerosis has not been well-established. This study examines whether LTL at baseline predicts incidence and progression of carotid atherosclerosis in American Indians in the Strong Heart Study. The analysis included 2,819 participants who were free of overt cardiovascular disease at baseline (2001-2003) and were followed through the end of 2006-2009 (average 5.5-yr follow-up). Discrete atherosclerotic plaque was defined as focal protrusion with an arterial wall thickness ≥50% the surrounding wall. Carotid progression was defined as having a higher plaque score at the end of study follow-up compared to baseline. Associations of LTL with incidence and progression of carotid plaque were examined using Cox proportional hazard regression, adjusting for standard coronary risk factors. Compared to participants in the highest LTL tertile, those in the lowest tertile had significantly elevated risk for both incident plaque (HR, 1.49; 95% CI, 1.09-2.03) and plaque progression (HR, 1.61; 95% CI, 1.26-2.07). Our results provide initial evidence for a potential prognostic utility of LTL in risk prediction for atherosclerosis.

Shorter leukocyte telomere length (LTL) has been associated with a wide range of age-related disorders including cardiovascular disease (CVD) and diabetes. Obesity is an important risk factor for CVD and diabetes. The association of LTL with obesity is not well understood. This study for the first time examines the association of LTL with obesity indices including body mass index, waist circumference, percent body fat, waist-to-hip ratio, and waist-to-height ratio in 3,256 American Indians (14-93 years old, 60% women) participating in the Strong Heart Family Study. Association of LTL with each adiposity index was examined using multivariate generalized linear mixed model, adjusting for chronological age, sex, study center, education, lifestyle (smoking, alcohol consumption, and total energy intake), high-sensitivity C-reactive protein, hypertension and diabetes. Results show that obese participants had significantly shorter LTL than non-obese individuals (age-adjusted P=0.0002). Multivariate analyses demonstrate that LTL was significantly and inversely associated with all of the studied obesity parameters. Our results may shed light on the potential role of biological aging in pathogenesis of obesity and its comorbidities.

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.

Short telomere length, a marker of biological aging, has been associated with age-related metabolic disorders. Telomere attrition induces profound metabolic dysfunction in animal models, but no study has examined the metabolome of telomeric aging in human. Here we studied 423 apparently healthy American Indians participating in the Strong Family Heart Study. Leukocyte telomere length (LTL) was measured by qPCR. Metabolites in fasting plasma were detected by untargeted LC/MS. Associations of LTL with each metabolite and their combined effects were examined using generalized estimating equation adjusting for chronological age and other aging-related factors. Multiple testing was corrected using the q-value method (q<0.05). Of the 1,364 distinct m/z features detected, nineteen metabolites in the classes of glycerophosphoethanolamines, glycerophosphocholines, glycerolipids, bile acids, isoprenoids, fatty amides, or L-carnitine ester were significantly associated with LTL, independent of chronological age and other aging-related factors. Participants with longer (top tertile) and shorter (bottom tertile) LTL were clearly separated into distinct groups using a multi-marker score comprising of all these metabolites, suggesting that these newly detected metabolites could be novel metabolic markers of biological aging. This is the first study to interrogate the human metabolome of telomeric aging. Our results provide initial evidence for a metabolic control of LTL and may reveal previously undescribed new roles of various lipids in the aging process.

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.

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.

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., (© 2013. Published by Elsevier Inc. All rights reserved.)

Telomeres play a central role in cellular aging, and shorter telomere length has been associated with age-related disorders including diabetes. However, a causal link between telomere shortening and diabetes risk has not been established. In a well-characterized longitudinal cohort of American Indians participating in the Strong Heart Family Study, we examined whether leukocyte telomere length (LTL) at baseline predicts incident diabetes independent of known diabetes risk factors. Among 2,328 participants free of diabetes at baseline, 292 subjects developed diabetes during an average 5.5 years of follow-up. Compared with subjects in the highest quartile (longest) of LTL, those in the lowest quartile (shortest) had an almost twofold increased risk of incident diabetes (hazard ratio [HR] 1.83 [95% CI 1.26-2.66]), whereas the risk for those in the second (HR 0.87 [95% CI 0.59-1.29]) and the third (HR 0.95 [95% CI 0.65-1.38]) quartiles was statistically nonsignificant. These findings suggest a nonlinear association between LTL and incident diabetes and indicate that LTL could serve as a predictive marker for diabetes development in American Indians, who suffer from disproportionately high rates of diabetes.

Telomeres play a central role in cellular senescence and are associated with a variety of age-related disorders such as dementia, Alzheimer's disease and atherosclerosis. Telomere length varies greatly among individuals of the same age, and is heritable. Here we performed a genome-wide linkage scan to identify quantitative trait loci (QTL) influencing leukocyte telomere length (LTL) measured by quantitative PCR in 3,665 American Indians (aged 14-93 years) from 94 large, multi-generational families. All participants were recruited by the Strong Heart Family Study (SHFS), a prospective study to identify genetic factors for cardiovascular disease and its risk factors in American Indians residing in Oklahoma, Arizona and Dakota. LTL heritability was estimated to be between 51% and 62%, suggesting a strong genetic predisposition to interindividual variation of LTL in this population. Significant QTLs were localized to chromosome 13 (Logarithm of odds score (LOD)=3.9) at 13q12.11, to 18q22.2 (LOD=3.2) and to 3p14.1 (LOD=3.0) for Oklahoma. This is the first study to identify susceptibility loci influencing leukocyte telomere variation in American Indians, a minority group suffering from a disproportionately high rate of type 2 diabetes and other age-related disorders.

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., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Wiley Periodicals, Inc.)

Mast cells are best known as central effector cells in IgE-mediated type I allergic diseases including asthma and hay fever. An increasing amount of evidence, however, has demonstrated that mast cells are sentinel cells playing a critical role in host defense against invading microbes. Mast cells are located immediately beneath the epithelial surfaces exposed to the outer environment, such as genitourinary and gastrointestinal tracts, skin, and airways. This review discusses recent studies on the critical roles of mast cells in host defense against Gram-negative bacterial infection. Mast cells are equipped with multiple receptors detecting the invading Gram-negative bacteria in both direct (opsonin-independent) and indirect (opsonin-dependent) mechanisms. The former includes Toll-like receptors (TLRs), CD48, and nucleotide-binding oligomerization (NOD) proteins, while the latter includes Fcγ receptors (FcγRs) and complement receptors. In addition to the detecting systems, mast cells are also armed with versatile tools to combat and kill Gram-negative bacteria. In response to the recognition of the Gram-negative bacterial infection, mast cells secrete various types of mediators which either regulate host immune system or directly attack the bacteria. Mast cells can also phagocytize and subsequently display the bacterial antigens on their cell surfaces. Moreover, recent findings have revealed the formation of extra-cellular traps by mast cells. Finally this review will especially focus on recent findings on LPS signaling in mast cells, both the functional outcome and the molecular mechanisms.

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.