Publications by authors named "Tomokazu Hasegawa"

48 Publications

Prediction of treatment response from the microenvironment of tumor immunity in cervical cancer patients treated with chemoradiotherapy.

Med Mol Morphol 2021 May 8. Epub 2021 May 8.

Department of Radiology, Sapporo Medical University School of Medicine, S1 W16, Chuo-ku, Sapporo, Hokkaido, 060-8543, Japan.

To supplement clinical decision-making in the management of cervical cancer, various prognostic factors, including tumor immune microenvironments, were examined in patients with cervical cancer treated with definitive chemoradiotherapy. We retrospectively analyzed the expression of CD8, FoxP3, HLA-1, PD-L1, and XRCC4 in 100 cases of cervical cancer. The observed tumor immune microenvironments were also classified into three types: inflamed, excluded, and cold type. Less FoxP3+ T cells and cold-type tumor were found to be poor prognostic factors in addition to non-SCC, large pre-treatment tumor volume, and three or less cycles of concurrent chemotherapy based on multivariate analysis. Cold-type tumors had significantly worse prognoses than the other two types, whereas inflamed- and excluded-type tumors showed similar 5-year disease-specific survival (P < 0.001; 0% vs. 60.3% vs. 72.3%). Radiotherapy could overcome the inhibitory immune microenvironment that occurs in excluded type. Individualized combination therapy adapted to pre-treatment tumor immunity may be necessary to improve radiotherapy outcomes in cervical cancer.
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http://dx.doi.org/10.1007/s00795-021-00290-wDOI Listing
May 2021

Association between cancer immunity and treatment results in uterine cervical cancer patients treated with radiotherapy.

Jpn J Clin Oncol 2020 Oct;50(11):1290-1297

Department of Radiology, Sapporo Medical University School of Medicine, Sapporo, Japan.

Objective: To evaluate proteins related to tumor immune response and treatment outcome from radiotherapy for uterine cervical cancer patients.

Methods: We performed a retrospective immunohistochemical staining of 81 patients with uterine cervical cancer who underwent definitive radiotherapy. We examined the expression of programmed death ligand 1, human leukocyte antigen class I, tumor-infiltrating CD8+, and forkhead box P3+ (FoxP3+) T cells in tumor tissues.

Results: In biopsy specimen, patients with a higher number of CD8+ T cells and FoxP3+ T cells had a better disease-specific survival than patients with a lower number of CD8+ T cells and FoxP3+ cells (P = 0.018 and P = 0.009). Multivariate analysis showed that equivalent dose in 2 Gy fractions (EQD2) of the minimum dose to 90% of the high-risk clinical target volume, FoxP3+ T cells and expression of human leukocyte antigen class I were significant prognostic factors. When the EQD2 is 70 Gy or more, a higher local control rate is obtained regardless of the number of CD8- or FoxP3-positive cells. When EQD2 is <70 Gy, the number of CD8-positive cells has a significant impact on treatment outcome: the recurrence rate (local recurrence rate + distant metastasis rate) was 46.2% in the group with a CD8 value of 230 or higher, whereas the recurrence rate was 75.7% in the group with a CD8 value of less than 230.

Conclusion: The combination of CD8 or FoxP3 with EQD2 can be potentially useful to predict the treatment results of radiotherapy for cervical cancer, leading to individualized optimal selection of treatment for cervical cancer.
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http://dx.doi.org/10.1093/jjco/hyaa149DOI Listing
October 2020

Evaluation of the urethral α/β ratio and tissue repair half-time for iodine-125 prostate brachytherapy with or without supplemental external beam radiotherapy.

Brachytherapy 2020 May - Jun;19(3):290-297. Epub 2020 Apr 2.

Department of Radiology, Sapporo Medical University, Sapporo, Hokkaido, Japan. Electronic address:

Purpose: To assess the correlation between postimplant dosimetric quantifiers and the genitourinary (GU) toxicity of low-dose rate brachytherapy for prostate cancer.

Methods And Materials: The minimum urethral dose (UD10, 30, and 90) and the percent volume of the urethra receiving the prescription dose (V100, V150) were calculated from the postimplant dose-volume histograms of 182 patients. We then calculated various urethral biologically equivalent doses (uBEDs) using different values of the α/β ratio and tissue repair half-time (t1/2) and examined the correlations with GU toxicity.

Results: Common dosimetric quantifiers, such as UD90 (brachytherapy) + UD50 (external beam radiotherapy), showed no correlation with Grade ≥ 2 GU toxicity. There was a significant correlation between Grade ≥2 GU toxicity and uBED when the α/β value was 0.5 or 1 Gy and t1/2 was 0.5-2.5 h. An uBED (α/β = 1.0, t1/2 = 0.5) had the largest hazard ratio for GU toxicity, and it was also significantly correlated with Grade ≥ 2 GU toxicity according to multivariate analysis.

Conclusions: We observed a significant correlation of uBED with GU toxicity when α/β was 0.5 or 1.0 Gy and t1/2 was 0.5-2.5 h. As the simple formula we used has not been verified in basic experiments, more data are needed to validate our results.
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http://dx.doi.org/10.1016/j.brachy.2020.02.007DOI Listing
January 2021

Retrospective DVH analysis of point A based intracavitary brachytherapy for uterine cervical cancer.

J Radiat Res 2020 Mar;61(2):265-274

Department of Radiology, Sapporo Medical University School of Medicine, Chuo-ku, S1 W16, Sapporo, Hokkaido 060-8543, Japan.

Combining external beam radiotherapy (EBRT) with intracavitary brachytherapy (ICBT) is important for definitive treatment of cervical cancer. In cervical cancer patients receiving radiotherapy, we evaluated treatment outcomes in relation to dose-volume histogram parameters, including the computed tomography (CT)-based high-risk clinical target volume (HR-CTV) for ICBT. Between 2010 and 2015, 89 consecutive cervical cancer patients were mostly treated with 40 Gy of EBRT in 20 fractions and 18 Gy of ICBT prescribed to point A in 3 fractions. CT scans were obtained during ICBT. The HR-CTV D90 was calculated and the total doses of ICBT and EBRT were converted to the equivalent dose in 2 Gy fractions (EQD2). When the patients were divided into four groups according to EQD2 of the HR-CTV D90, the 3-year local recurrence-free survival rates were 95.2, 78.4, 52.7 and 42.9% for patients receiving >80 , 70-80 , 60-70 and <60 Gy, respectively. There was a significant negative correlation between EQD2 of the HR-CTV D90 and the HR-CTV volume at first ICBT (r = -0.713). Local recurrence was more frequent when the HR-CTV volume was ≥22 cc and EQD2 of the HR-CTV D90 was <70 Gy. Multivariate analysis showed that EQD2 of the HR-CTV D90 and concurrent chemotherapy (≥4 cycles) were significant determinants of overall survival. HR-CTV D90 was an important prognostic indicator for local recurrence. HR-CTV D90 >70 Gy is required for the better local control, especially in patients with a larger HR-CTV (≥22 cc at initial ICBT).
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http://dx.doi.org/10.1093/jrr/rrz099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246069PMC
March 2020

Association between radiotherapy-induced alteration of programmed death ligand 1 and survival in patients with uterine cervical cancer undergoing preoperative radiotherapy.

Strahlenther Onkol 2020 Aug 17;196(8):725-735. Epub 2020 Jan 17.

Department of Radiology, Sapporo Medical University School of Medicine, S1W16, Chuo-Ku, 060-8543, Sapporo, Hokkaido, Japan.

Purpose: To evaluate radiotherapy-induced changes in the expression of programmed death ligand 1 (PD-L1), programmed death 1 (PD-1), and human leukocyte antigen class I (HLA-1) in patients with uterine cervical cancer, as well as infiltration of CD8+ and Forkhead box P3+ (FoxP3+) T lymphocytes into tumor tissue and the prognostic value of these parameters.

Materials And Methods: We performed immunohistochemical analysis of pre-radiotherapy biopsies and corresponding post-radiotherapy resected tissues in 104 uterine cervical cancer patients undergoing preoperative chemoradiotherapy or radiotherapy alone. We scored the expression of various proteins to distinguish positive from negative samples.

Results: PD-L1-expressing tumor cells (PD-L1 TC) increased significantly after chemoradiotherapy (p = 0.043). CD8+ T cell infiltration (p = 0.002) and FoxP3+ T cell infiltration (p = 0.003) decreased significantly after chemoradiotherapy. Expression of PD‑1, PD-L1-expressing immune cells (PD-L1 IC), and HLA‑1 did not change after chemoradiotherapy. In biopsy specimens obtained before chemoradiotherapy or radiotherapy, greater infiltration of CD8+ T cells (p = 0.001) and FoxP3+ T cells (p = 0.003) were significant predictors of better overall survival (OS). In surgical specimens obtained after chemoradiotherapy or radiotherapy, greater infiltration of PD-L1 TC was the only significant predictor of better OS (p < 0.001) and was related to a significantly lower probability of out-of-field recurrence (p = 0.005).

Conclusion: Chemoradiotherapy induced an immunologic shift that increased PD-L1 TC. Chemoradiotherapy has immunological effects that can influence the results of treatment for uterine cervical cancer.
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http://dx.doi.org/10.1007/s00066-019-01571-1DOI Listing
August 2020

Coordination of WNT signaling and ciliogenesis during odontogenesis by piezo type mechanosensitive ion channel component 1.

Sci Rep 2019 10 14;9(1):14762. Epub 2019 Oct 14.

Department of Pediatric Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 770-8504, Japan.

Signal transmission from the mechanical forces to the various intracellular activities is a fundamental process during tissue development. Despite their critical role, the mechanism of mechanical forces in the biological process is poorly understood. In this study, we demonstrated that in the response to hydrostatic pressure (HP), the piezo type mechanosensitive ion channel component 1 (PIEZO1) is a primary mechanosensing receptor for odontoblast differentiation through coordination of the WNT expression and ciliogenesis. In stem cells from human exfoliated deciduous teeth (SHED), HP significantly promoted calcium deposition as well as the expression of odontogenic marker genes, PANX3 and DSPP, and WNT related-genes including WNT5b and WNT16, whereas HP inhibited cell proliferation and enhanced primary cilia expression. WNT signaling inhibitor XAV939 and primary cilia inhibitor chloral hydrate blocked the HP-induced calcium deposition. The PIEZO1 activator Yoda1 inhibited cell proliferation but induced ciliogenesis and WNT16 expression. Interestingly, HP and Yoda1 promoted nuclear translocation of RUNX2, whereas siRNA-mediated silencing of PIEZO1 decreased HP-induced nuclear translocation of RUNX2. Taken together, these results suggest that PIEZO1 functions as a mechanotransducer that connects HP signal to the intracellular signalings during odontoblast differentiation.
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http://dx.doi.org/10.1038/s41598-019-51381-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6791893PMC
October 2019

Combination of ions promotes cell migration via extracellular signal‑regulated kinase 1/2 signaling pathway in human gingival fibroblasts.

Mol Med Rep 2019 Jun 8;19(6):5039-5045. Epub 2019 Apr 8.

Department of Pediatric Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770‑8504, Japan.

Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre‑reacted glass‑ionomer (S‑PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S‑PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S‑PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF‑1 was treated with various dilutions of an eluent solution of S‑PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF‑1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S‑PRG solution and revealed that it activated the phosphorylation of extracellular signal‑regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S‑PRG fillers can stimulate HGF‑1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing.
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http://dx.doi.org/10.3892/mmr.2019.10141DOI Listing
June 2019

Influence of XRCC4 expression by breast cancer cells on ipsilateral recurrence after breast-conserving therapy.

Strahlenther Onkol 2019 Jul 17;195(7):648-658. Epub 2019 Apr 17.

Department of Radiology, Sapporo Medical University School of Medicine, 060-8543, Chuo-ku, Sapporo, Hokkaido, Japan.

Background: We examined the expression of nonhomologous end-joining (NHEJ) proteins by breast cancer cells in patients with or without ipsilateral breast tumor recurrence (IBTR) after breast-conserving therapy. We also investigated whether there was a difference of NHEJ-related protein expression by tumor cells between two types of IBTR, i.e., true recurrence (TR) with regrowth from the tumor bed or development of a new primary tumor (NP).

Patients And Methods: The original cohort comprised 560 breast cancer patients who received breast-conserving therapy between February 1995 and March 2006, including 520 patients without IBTR and 40 patients with IBTR. Propensity score matching was employed to select 40 trios (120 patients) consisting of 1 patient with IBTR and 2 patients without IBTR. Immunohistochemical examination of proteins related to NHEJ was performed in surgical specimens.

Results: The 40 patients with IBTR included 22 patients who developed TR and 18 who had NP. The 15-year overall survival rate was 85.9% for patients with NP and 95.5% for those with TR, while it was 96.5% for patients without IBTR. Patients with high XRCC4 expression in tumor cells had significantly higher IBTR rates than those with low XRCC4 expression (P < 0.001). The frequency of TR was significantly higher in patients with high expression of XRCC4 than in those with low XRCC4 expression (p < 0.001). XRCC4 expression by tumor cells was not significantly related to development of NP.

Conclusion: IBTR due to TR may be related to low radiosensitivity of tumor cells, possibly related to high XRCC4 expression.
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http://dx.doi.org/10.1007/s00066-019-01468-zDOI Listing
July 2019

Effect of hypoxia on the expression of CCAAT/enhancer-binding protein β and receptor activator of nuclear factor kappa-B ligand in periodontal ligament cells.

J Oral Sci 2018 ;60(4):544-551

Department of Pediatric Dentistry, Nihon University School of Dentistry.

Hypoxia after traumatic injuries to a tooth is one of the causes of subsequent root resorption. Inflammatory cytokines produced under hypoxic conditions are associated with root resorption, but the mechanism has not been fully understood. In this study, the role of hypoxia-inducible factor-1 (HIF-1) signaling in the regulation of CCAAT (cytosine-cytosine-adenosine-adenosine-thymidine)/enhancer-binding protein-β (C/EBPβ) and the receptor activator of nuclear factor kappa-B ligand (RANKL) expressions in immortalized human periodontal ligament (PDL) cells was investigated. PDL cells cultured under a hypoxic condition showed an increase in the expression of C/EBPβ and RANKL messenger RNAs (mRNAs), whereas the expression of osteoprotegerin and HIF-1α mRNAs was unaffected. Hypoxia had no effects on the secretion of interleukin (IL)-1β, IL-6, IL-8, IL-17A, tumor necrosis factor-alpha, macrophage migration inhibitory factor, monocyte chemoattractant protein-1, and macrophage colony-stimulating factor in the culture media. Treatment of the cells with dimethyloxaloylglycine, a competitive HIF prolyl hydroxylase inhibitor, significantly increased the expression of C/EBPβ and RANKL mRNAs. This suggested that the hypoxia-induced elevation of C/EBPβ and RANKL mRNAs was dependent on the HIF-1 activity. PDL cells transfected with a specific small interfering RNA designed to target the C/EBPβ gene showed a significant suppression of the RANKL mRNA. These findings indicated that C/EBPβ may play an important role in tooth root resorption via RANKL activation in hypoxia-exposed PDL cells.
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http://dx.doi.org/10.2334/josnusd.17-0436DOI Listing
May 2019

Hypoxia-induced upregulation of angiogenic factors in immortalized human periodontal ligament fibroblasts.

J Oral Sci 2018 ;60(4):519-525

Department of Pediatric Dentistry, Nihon University School of Dentistry.

Hypoxia induces complex cellular responses that are mediated by a key transcription factor, hypoxia-inducible factor-1 (HIF-1). HIF-1 promotes production of cytokines and angiogenic factors and contributes to recovery of injured tissues. In the present study, expressions of angiogenin (ANG) and vascular endothelial growth factor (VEGF), which are potent angiogenic factors in mammalian tissues, were examined in immortalized fibroblasts exposed to hypoxia. After 24 h of exposure to hypoxia, ANG and VEGF mRNAs expressions were significantly elevated in periodontal ligament (PDL) fibroblasts but not in embryonic fibroblasts. Hypoxia also increased productions of ANG and VEGF proteins in PDL fibroblasts. HIF-1α mRNA expression was not affected by hypoxia in either fibroblast, although HIF-1α protein expression was enhanced after exposure to hypoxia. Treatment of PDL fibroblasts with dimethyloxaloylglycine, a prolyl hydroxylase inhibitor that stabilizes the HIF-1α protein, significantly increased expressions of ANG and VEGF mRNAs under normoxia. This suggests that stabilization of HIF-1α is crucial for upregulation of ANG and VEGF in PDL fibroblasts. These results indicate that, under hypoxic conditions, HIF-1α upregulates synthesis of ANG and VEGF in PDL fibroblasts and promotes angiogenesis.
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http://dx.doi.org/10.2334/josnusd.17-0441DOI Listing
May 2019

Influence of PD-L1 expression in immune cells on the response to radiation therapy in patients with oropharyngeal squamous cell carcinoma.

Radiother Oncol 2018 11 21;129(2):409-414. Epub 2018 Sep 21.

Department of Radiology, Sapporo Medical University School of Medicine, Japan. Electronic address:

Background And Purpose: To investigate influences of proteins involved with tumor immunity on outcomes of radiotherapy for oropharyngeal squamous cell carcinoma (OPSCC).

Material And Methods: We performed immunohistochemical staining to examine expressions of p16 and proteins involved with tumor immunity in 92 OPSCC patients treated with radiotherapy.

Results: Patients with abundant infiltrating CD8-positive cells had the significantly better overall survival (OS) rate than patients with fewer CD8-positive cells (p = 0.026). Patients with higher PD-L1 expression in tumor cells (TC 1-3) had a better outcome than those with low PD-L1 expression in tumor cells (TC 0) for both OS (p = 0.019) and progression-free survival (PFS) rate (p = 0.032). Patients with high PD-L1 expression in infiltrating immune cells (IC 3) showed significantly better OS (p = 0.009) and PFS (p = 0.011) than those with low PD-L1 expression (IC 0-2). Patients with p16-negative and IC 3 showed similar OS to patients with p16-positive and IC 0-2. P16-positive tumors had a significantly higher CD8-positive cell infiltration and PD-L1 expression in tumor cells than p16-negative tumors.

Conclusions: In addition to tumor p16 expression, PD-L1 expression in TC and IC can be useful for predicting the response of OPSCC to radiotherapy.
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http://dx.doi.org/10.1016/j.radonc.2018.08.023DOI Listing
November 2018

Prediction of acute gastrointestinal and genitourinary radiation toxicity in prostate cancer patients using lymphocyte microRNA.

Jpn J Clin Oncol 2018 Feb;48(2):167-174

Department of Radiology, Sapporo Medical University School of Medicine, Hokkaido, Japan.

Background: To search for novel biomarkers that can predict acute radiation toxicity, we conducted microRNA expression analysis of peripheral blood lymphocytes (PBLs).

Methods: The discovery cohort was 69 patients with localized adenocarcinoma of the prostate who received intensity-modulated radiation therapy between October 2007 and October 2010. The validation cohort was 72 patients treated with low-dose-rate brachytherapy between May 2008 and March 2014. After13 microRNAs were selected by TaqMan® Array analysis in a preliminary experiment, expression of these microRNAs in all samples was analyzed by RT-PCR.

Results: In the discovery cohort, the average prostate volume, the rectal volume receiving 70 Gy, and expression of miR-410 and miR-221 were significant risk factors for Grade 1-2 gastrointestinal toxicity. Receiver operating characteristic analysis showed that the area under the curve (AUC) was 0.807. The maximum dose to the urinary bladder, prostate volume, pretreatment urinary function score, and miR-99a and miR-221 expression were risk factors for Grade 2 genitourinary toxicity. The corresponding AUC was 0.796. In the validation cohort, reproducibility of these markers was confirmed for gastrointestinal toxicity, but not for genitourinary toxicity.

Conclusion: Combining radiation dose parameters with microRNA expression in PBLs may be useful for predicting acute gastrointestinal toxicity of radiation therapy, thus contributing to personalized treatment of prostate cancer.
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http://dx.doi.org/10.1093/jjco/hyx181DOI Listing
February 2018

Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.

Sci Rep 2017 12 18;7(1):17696. Epub 2017 Dec 18.

Department of Pediatric Dentistry, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, 770-8504, Japan.

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.
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http://dx.doi.org/10.1038/s41598-017-18089-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735093PMC
December 2017

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro.

Mol Med Rep 2017 Jul 19;16(1):429-434. Epub 2017 May 19.

Department of Molecular Cell Pharmacology, Hokkaido University Graduate School of Dental Medicine, Kita‑ku, Sapporo, Hokkaido 060‑8586, Japan.

Bone is maintained by a balance between bone formation and resorption. This remodeling is controlled by a wide variety of systemic and local factors including hormones, cytokines and mechanical stresses. The present in vitro study examined the impact of medium volume, using 0.4, 0.6, 0.8, 1.0, 1.5 and 2.0 ml/well in a 24‑well plate, on the differentiation of osteoblasts and osteoclasts. There were no differences in the alkaline phosphatase activity of osteoblasts amongst the groups; however, the area of mineral deposition was decreased in a media volume‑dependent manner. A co‑culture of osteoblastic cells with bone marrow cells revealed a reduction in the total number of osteoclastic tartrate‑resistant acid phosphatase (TRAP)‑positive multinuclear cells (≥2 nuclei), whereas the formation of large osteoclastic TRAP‑positive multinuclear cells (≥8 nuclei) was increased, in a media volume‑dependent manner. There were also no differences in receptor activator of nuclear factor‑κB ligand mRNA and total osteoprotegerin (OPG) protein expression levels amongst the groups, however the concentration of OPG decreased in a media volume‑dependent manner. In conclusion, the present study demonstrated that the suppression of mineralization in osteoblastic cells and the stimulation of osteoclast fusion are dependent on the medium volume, indicating that media volume is an important factor in in vitro cell culture systems.
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http://dx.doi.org/10.3892/mmr.2017.6611DOI Listing
July 2017

Local tumor control and DNA-PK activity of peripheral blood lymphocytes in prostate cancer patients receiving radiotherapy.

J Radiat Res 2017 Mar;58(2):225-231

Department of Radiology, Sapporo Medical University School of Medicine, S1W16, Chuo-ku, Sapporo, Hokkaido, 060-8543, Japan.

Repair of DNA damage is critical for genomic stability, and DNA-dependent protein kinase (DNA-PK) has an important role in repairing double-strand breaks. We examined whether the DNA-PK activity of peripheral blood lymphocytes (PBLs) was related to biochemical (prostate-specific antigen: PSA) relapse and radiation toxicity in prostate cancer patients who have received radiotherapy. A total of 69 patients with localized adenocarcinoma of the prostate participated in this study. Peripheral blood was collected 2 years or later after radiotherapy and centrifuged, then DNA-PK activity was measured by a filter binding assay. The high DNA-PK activity group had a significantly higher PSA relapse-free survival rate than the low DNA-PK activity group. The 10-year PSA relapse-free survival was 87.0% in the high DNA-PK activity group, whereas it was 52.7% in the low DNA-PK activity group. Multivariate analysis showed the Gleason score and the level of DNA-PK activity were significant predictors of PSA relapse after radiotherapy. In addition, the low DNA-PK activity group tended to have a higher incidence of Grade 1-2 urinary toxicity than the high DNA-PK activity group. Prostate cancer patients with low DNA-PK activity had a higher rate of PSA relapse and a higher incidence of urinary toxicity. DNA-PK activity in PBLs might be a useful marker for predicting PSA relapse and urinary toxicity, possibly contributing to personalized treatment of prostate cancer.
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http://dx.doi.org/10.1093/jrr/rrw099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5571613PMC
March 2017

Crystal structure of the solute-binding protein BxlE from Streptomyces thermoviolaceus OPC-520 complexed with xylobiose.

J Biochem 2017 Jun;161(6):493-501

Department of Microbiology, Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka 569-1094, Japan.

BxlE from Streptomyces thermoviolaceus OPC-520 is a xylo-oligosaccharide (mainly xylobiose)-binding protein that serves as the initial receptor for the bacterial ABC-type xylo-oligosaccharide transport system. To determine the ligand-binding mechanism of BxlE, X-ray structures of ligand-free (open form) and ligand (xylobiose)-bound (closed form) BxlE were determined at 1.85 Å resolution. BxlE consists of two globular domains that are linked by two β-strands, with the cleft at the interface of the two domains creating the ligand-binding pocket. In the ligand-free open form, this pocket consists of a U-shaped and negatively charged groove located between the two domains. In the xylobiose-bound closed form of BxlE, both the N and C domains move to fold the ligand without conformational changes in either domain. Xylobiose is buried in the groove and wrapped by the N-domain mainly via hydrogen bond interactions and by the C-domain primarily via non-polar interactions with Trp side chains. In addition to the concave shape matching the binding of xylobiose, an inter-domain salt bridge between Asp-47 and Lys-294 limits the space in the ligand-binding site. This domain-stabilized mechanism of ligand binding to BxlE is a unique feature that is not observed with other solute-binding proteins.
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http://dx.doi.org/10.1093/jb/mvw097DOI Listing
June 2017

Involvement of interleukin‑23 induced by Porphyromonas endodontalis lipopolysaccharide in osteoclastogenesis.

Mol Med Rep 2017 Feb 14;15(2):559-566. Epub 2016 Dec 14.

Department of Histology and Oral Histology, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima 770‑8504, Japan.

Periapical lesions are characterized by the destruction of periapical bone, and occur as a result of local inflammatory responses to root canal infection by microorganisms including Porphyromonas endodontalis (P. endodontalis). P. endodontalis and its primary virulence factor, lipopolysaccharide (LPS), are associated with the development of periapical lesions and alveolar bone loss. Interleukin‑23 (IL‑23) is critical in the initiation and progression of periodontal disease via effects on peripheral bone metabolism. The present study investigated the expression of IL‑23 in tissue where a periapical lesion was present, and the effect of P. endodontalis LPS on the expression of IL‑23 in periodontal ligament (PDL) cells. Reverse transcription‑ quantitative polymerase chain reaction and immunohistochemistry revealed increased levels of IL‑23 expression in tissue with periapical lesions compared with healthy PDL tissue. Treatment with P. endodontalis LPS increased the expression of IL‑23 in the SH‑9 human PDL cell line. BAY11‑7082, a nuclear factor κB inhibitor, suppressed P. endodontalis LPS‑induced IL‑23 expression in SH‑9 cells. Treatment of RAW264.7 cells with conditioned medium from P. endodontalis LPS‑treated SH‑9 cells promoted osteoclastogenesis. By contrast, RAW264.7 cells treated with conditioned medium from IL‑23‑knockdown SH‑9 cells underwent reduced levels of osteoclastogenesis. The results of the present study indicated that the expression of IL‑23 in PDL cells induced by P. endodontalis LPS treatment may be involved in the progression of periapical lesions via stimulation of the osteoclastogenesis process.
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http://dx.doi.org/10.3892/mmr.2016.6041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364876PMC
February 2017

Release from optimal compressive force suppresses osteoclast differentiation.

Mol Med Rep 2016 Nov 5;14(5):4699-4705. Epub 2016 Oct 5.

Department of Orthodontics, Hokkaido University Graduate School of Dental Medicine, Sapporo, Hokkaido 060‑8586, Japan.

Bone remodeling is an important factor in orthodontic tooth movement. During orthodontic treatment, osteoclasts are subjected to various mechanical stimuli, and this promotes or inhibits osteoclast differentiation and fusion. It has been previously reported that the release from tensile force induces osteoclast differentiation. However, little is known about how release from compressive force affects osteoclasts. The present study investigated the effects of release from compressive force on osteoclasts. The number of tartrate‑resistant acid phosphatase (TRAP)‑positive multinucleated osteoclasts derived from RAW264.7 cells was counted, and gene expression associated with osteoclast differentiation and fusion in response to release from compressive force was evaluated by reverse transcription‑quantitative polymerase chain reaction. Osteoclast number was increased by optimal compressive force application. On release from this force, osteoclast differentiation and fusion were suppressed. mRNA expression of NFATc1 was inhibited for 6 h subsequent to release from compressive force. mRNA expression of the other osteoclast‑specific genes, TRAP, RANK, matrix metalloproteinase‑9, cathepsin‑K, chloride channel 7, ATPase H+ transporting vacuolar proton pump member I, dendritic cell‑specific transmembrane protein and osteoclast stimulatory transmembrane protein (OC‑STAMP) was significantly inhibited at 3 h following release from compressive force compared with control cells. These findings suggest that release from optimal compressive force suppresses osteoclast differentiation and fusion, which may be important for developing orthodontic treatments.
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http://dx.doi.org/10.3892/mmr.2016.5801DOI Listing
November 2016

Expression of Ku70 predicts results of radiotherapy in prostate cancer.

Strahlenther Onkol 2017 Jan 27;193(1):29-37. Epub 2016 Jul 27.

Department of Radiology, Sapporo Medical University School of medicine, 060-8543, Chuo-ku, Sapporo, Hokkaido, Japan.

Background And Purpose: Therapeutic strategy for prostate cancer is decided according to T stage, Gleason score, and prostate-specific antigen (PSA) level. These clinical factors are not accurate enough to predict individual risk of local failure of prostate cancer after radiotherapy. Parameters involved with radiosensitivity are required to improve the predictive capability for local relapse.

Patients And Methods: We analyzed 58 patients with localized adenocarcinoma of the prostate between August 2007 and October 2010 treated with 76 Gy of intensity-modulated radiotherapy (IMRT) as a discovery cohort and 42 patients between March 2001 and May 2007 treated with three-dimensional conformal radiotherapy (3D-CRT) as a validation cohort. Immunohistochemical examination for proteins involved in nonhomologous end-joining was performed using biopsy specimens.

Results: Ku70 expression was not correlated with various clinical parameters, such as the Gleason score and D'amico risk classification, indicating that Ku70 expression was an independent prognostic factor. The predictive value for PSA relapse was markedly improved after the combination of Gleason score and Ku70 expression, as compared with Gleason score alone. In patients treated with radiotherapy and androgen deprivation therapy (ADT), no relapses were observed in patients with Gleason score ≤7 or low Ku70 expression. In contrast, patients with Gleason score ≥8 and high Ku70 expression had high PSA relapse rates. In the validation cohort, similar results were obtained.

Conclusion: Treatment with 76 Gy and ADT can be effective for patients with Gleason score ≤7 or low Ku70 expression, but is not enough for patients with Gleason score ≥8 and high Ku70 expression and, thus, require other treatment approaches.
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http://dx.doi.org/10.1007/s00066-016-1023-7DOI Listing
January 2017

Influence of XRCC4 expression in esophageal cancer cells on the response to radiotherapy.

Med Mol Morphol 2017 Mar 23;50(1):25-33. Epub 2016 Jun 23.

Department of Radiology, Sapporo Medical University Schoolo of Medicine, S1W16, Chuo-Ku, Sapporo, 060-8543, Hokkaido, Japan.

DNA double-strand break (DSB) is one of the most serious forms of damage induced by ionizing irradiation and is mainly repaired by the non-homologous end joining (NHEJ) repair. Immunohistochemical analysis of proteins involved in NHEJ, such as XRCC4 (X-ray repair cross-complementing protein 4), Ku86 and DNA-PKcs (DNA-dependent protein kinase, catalytic subunits), may be useful for predicting tumor radiosensitivity. We examined 92 patients with esophageal squamous cell carcinoma (ECSS) who were treated by radiotherapy between 1999 and 2008. Immunohistochemical examination of tumor tissue for Ki-67 and DSB-related proteins, including XRCC4, Ku86, and DNA-PKcs, was performed using pretreatment biopsy specimens. Low expression of XRCC4 was detected in 31 of 92 examined samples (33.7 %). The 5-year overall survival (OS) rate was 67.7 % in the low expression group and 31.0 % in the high expression group (P = 0.00). Multivariate analysis confirmed that advanced T-stage (HR 3.24, P = 0.01), radiation dose less than 66 Gy (HR 2.23, P = 0.02), absence of systemic chemotherapy (HR 2.59, P = 0.05), and high expression of XRCC4 (HR 12.0, P = 0.02) were independent prognostic factors for predicting poor OS. Other DSB-related proteins and Ki-67 were not predictive factors. XRCC4 expression might have an influence on results of radiotherapy for patients with ESCC.
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http://dx.doi.org/10.1007/s00795-016-0144-5DOI Listing
March 2017

Optimal compressive force accelerates osteoclastogenesis in RAW264.7 cells.

Mol Med Rep 2015 Oct 29;12(4):5879-85. Epub 2015 Jul 29.

Department of Orthodontics, Hokkaido University Graduate School of Dental Medicine, Sapporo, Hokkaido 060‑8586, Japan.

Mechanical stress produced by orthodontic forces is a factor in the remodeling of periodontal ligaments (PDLs) and alveolar bone. It has been reported that the expression of a number of cytokines associated with osteoclastogenesis is upregulated when compressive forces act on osteoblasts and PDL cells. The present study investigated the effects of compressive forces on the formation of osteoclasts from the macrophage cell line RAW264.7. Compressive forces on osteoclasts were exerted using layers of 3, 5, 7, 9 or 14 glass cover slips on the 4th day of culture for 24 h. The number of osteoclasts was determined by counting the number of cells positive for tartrate-resistant acid phosphatase staining. Osteoclastogenesis advanced rapidly on days four and five. The number of osteoclasts with >8 nuclei peaked when the force of 7 slips was applied, which was therefore regarded as the optimal compressive force. Alterations in the expression of osteoclast-associated genes are associated with changes in the differentiation and fusion of macrophages in response to compressive forces; therefore, osteoclast-associated genes were assessed by reverse transcription quantitative polymerase chain reaction in the present study. The mRNA expression of osteoclast‑associated genes increased significantly after 3 h of optimal compression, whereas mRNA expression increased after 24 h in the control group. These findings suggested that osteoclastogenesis of macrophages was accelerated when an optimal compressive force was applied.
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http://dx.doi.org/10.3892/mmr.2015.4141DOI Listing
October 2015

Recruitment of mesenchymal stem cells by stromal cell-derived factor 1α in pulp cells from deciduous teeth.

Int J Mol Med 2015 Aug 16;36(2):442-8. Epub 2015 Jun 16.

Department of Pediatric Dentistry, Tokushima University Hospital, Tokushima 770‑8504, Japan.

Dental pulp cells (DPCs), including dental pulp (DP) stem cells, play a role in dentine repair under certain conditions caused by bacterial infections associated with caries, tooth fracture and injury. Mesenchymal stem cells (MSCs) have also been shown to be involved in this process of repair. However, the mechanisms through which MSCs are recruited to the DP have not yet been elucidated. Therefore, the aim of the present in vitro study was to investigate whether stromal cell-derived factor 1α (SDF1)-C-X-C chemokine receptor type 4 (CXCR4) signaling is involved in tissue repair in the DP of deciduous teeth. A single-cell clone from DPCs (SDP11) and UE7T-13 cells were used as pulp cells and MSCs, respectively. The MG-63 and HuO9 cells, two osteosarcoma cell lines, were used as positive control cells. Reverse transcription polymerase chain reaction (RT-PCR) revealed that all cell lines (SDP11, UE7T-13 MG-63 and HuO9) were positive for both SDF1 and CXCR4 mRNA expression. Moreover, immunocytochemical analysis indicated that SDF1 and CXCR4 proteins were expressed in the SDP11 and UE7T-13 cells. SDF1 was also detected in the cell lysates (CLs) and conditioned medium (CM) collected from the SDP11 and UE7T-13 cells, and AMD3100, a specific antagonist of CXCR4, inhibited the migration of the UE7T-13 cells; this migration was induced by treatment with CM, which was collected from the SDP11 cells. In addition, real-time PCR showed that the expression of SDF1 in the SDP11 cells was inhibited by treatment with 20 ng/ml fibroblast growth factor (FGF)-2, and exposure to AZD4547, an inhibitor of the FGF receptor, blocked this inhibition. Collectively, these data suggest that SDF1 produced by DP plays an important role in homeostasis, repair and regeneration via the recruitment of MSCs.
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http://dx.doi.org/10.3892/ijmm.2015.2247DOI Listing
August 2015

Novel SCRG1/BST1 axis regulates self-renewal, migration, and osteogenic differentiation potential in mesenchymal stem cells.

Sci Rep 2014 Jan 13;4:3652. Epub 2014 Jan 13.

Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

Human mesenchymal stem cells (hMSCs) remodel or regenerate various tissues through several mechanisms. Here, we identified the hMSC-secreted protein SCRG1 and its receptor BST1 as a positive regulator of self-renewal, migration, and osteogenic differentiation. SCRG1 and BST1 gene expression decreased during osteogenic differentiation of hMSCs. Intriguingly, SCRG1 maintained stem cell marker expression (Oct-4 and CD271/LNGFR) and the potentials of self-renewal, migration, and osteogenic differentiation, even at high passage numbers. Thus, the novel SCRG1/BST1 axis determines the fate of hMSCs by regulating their kinetic and differentiation potentials. Our findings provide a new perspective on methods for ex vivo expansion of hMSCs that maintain native stem cell potentials for bone-forming cell therapy.
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http://dx.doi.org/10.1038/srep03652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3888969PMC
January 2014

PDGF-induced PI3K-mediated signaling enhances the TGF-β-induced osteogenic differentiation of human mesenchymal stem cells in a TGF-β-activated MEK-dependent manner.

Int J Mol Med 2014 Mar 27;33(3):534-42. Epub 2013 Dec 27.

Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, Yahaba, Iwate 028‑3694, Japan.

Transforming growth factor-β (TGF-β) is a critical regulator of osteogenic differentiation and the platelet-derived growth factor (PDGF) is a chemoattractant or mitogen of osteogenic mesenchymal cells. However, the combined effects of these regulators on the osteogenic differentiation of mesenchymal cells remains unknown. In this study, we investigated the effects of TGF-β and/or PDGF on the osteogenic differentiation of human mesenchymal stem cells (hMSCs). The TGF-β-induced osteogenic differentiation of UE7T-13 cells, a bone marrow-derived hMSC line, was markedly enhanced by PDGF, although PDGF alone did not induce differentiation. TGF-β induced extracellular signal-regulated kinase (ERK) phosphorylation and PDGF induced Akt phosphorylation. In addition, the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, suppressed the osteogenic differentiation induced by TGF-β alone. Moreover, U0126 completely suppressed the osteogenic differentiation synergistically induced by TGF-β and PDGF, whereas the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002, only partially suppressed this effect. These results suggest that the enhancement of TGF-β-induced osteogenic differentiation by PDGF-induced PI3K/Akt-mediated signaling depends on TGF-β-induced MEK activity. Thus, PDGF positively modulates the TGF-β-induced osteogenic differentiation of hMSCs through synergistic crosstalk between MEK- and PI3K/Akt-mediated signaling.
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http://dx.doi.org/10.3892/ijmm.2013.1606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926498PMC
March 2014

Forced mastication increases survival of adult neural stem cells in the hippocampal dentate gyrus.

Int J Mol Med 2013 Feb 18;31(2):307-14. Epub 2012 Dec 18.

Department of Pediatric Dentistry, Tokushima University Hospital, Tokushima 770‑8504, Japan.

In this study, we examined the effect of forced mastication on neurogenesis in the hippocampal dentate gyrus (DG) of adult mice. Six-week-old mice were subjected to either a hard or normal diet for 13 weeks. They received a daily injection of bromodeoxyuridine (BrdU) for 12 consecutive days beginning at 14 weeks of age. The number of BrdU-positive cells in the DG was counted 1 day after and 5 weeks after the final BrdU injection. The number of BrdU-positive cells 1 day after injection did not differ between the 2 diet groups. However, the number of BrdU-positive cells in the group fed the hard diet was significantly increased 5 weeks after BrdU injection compared to the group fed the normal diet. The results of the Morris water maze test showed that mice fed a hard diet required significantly less time to reach the platform than the control mice when tested at 10 days. Moreover, mice in the group fed the hard diet spent significantly more time in the former platform area than the group fed the normal diet, indicating that hard diet feeding improved spatial memory compared to normal diet feeding. Real-time PCR analysis showed that the expression of glutamate receptor 1 mRNA was significantly increased in the group fed the hard diet compared with the group fed the normal diet. These results suggest that mastication increases the survival of adult neural stem cells in the hippocampal DG.
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http://dx.doi.org/10.3892/ijmm.2012.1217DOI Listing
February 2013

Stage-dependent suppression of the formation of dentin-resorbing multinuclear cells with migration inhibitory factor in vitro.

Exp Ther Med 2012 Jan 7;3(1):37-43. Epub 2011 Oct 7.

Departments of Dentistry for Children and Disabled Person, and.

The macrophage migration inhibitory factor (MIF) is a crucial mediator of immune responses and is known to play a pivotal role in cell proliferation and differentiation. In this study, we assessed whether MIF exerts regulatory effects on osteoclast formation in bone marrow cells and, if so, by what mechanism. Bone marrow cells were either co-cultured with MC3T3-E1 cells or cultured with macrophage-colony stimulating factor (M-CSF) and the soluble form of the receptor activator of the nuclear factor-κB ligand (RANKL). Under the influence of MIF, the formation of osteoclastic multinuclear cells was examined. The number of multinuclear TRAP-positive cells formed in the co-culture was significantly reduced when MIF (≥0.1 μg/ml) was exogenously applied during the third and fourth days of the 6-day cultivation period. MIF affected neither the number of mononuclear TRAP-positive cells induced with M-CSF and RANKL, nor the expression of RANKL and osteoprotegerin in MC3T3-E1 cells. TRAP-positive cells cultured on dentin slices with MIF showed lower dentin-resorbing activity than those cultured without MIF. These results suggest that MIF has no regulatory roles in the differentiation of bone marrow cells to mononuclear TRAP-positive cells, but has inhibitory effects on the formation of mature osteoclasts by preventing cell fusion, which may eventually interfere with the osteoclast-mediated dentin resorption.
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http://dx.doi.org/10.3892/etm.2011.362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3438539PMC
January 2012

TGF-β-operated growth inhibition and translineage commitment into smooth muscle cells of periodontal ligament-derived endothelial progenitor cells through Smad- and p38 MAPK-dependent signals.

Int J Biol Sci 2012 22;8(7):1062-74. Epub 2012 Aug 22.

Division of Cellular Biosignal Sciences, Department of Biochemistry, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 028-3694, Japan.

The periodontal ligament (PDL) is a fibrous connective tissue that attaches the tooth to the alveolar bone. We previously demonstrated the ability of PDL fibroblast-like cells to construct an endothelial cell (EC) marker-positive blood vessel-like structure, indicating the potential of fibroblastic lineage cells in PDL tissue as precursors of endothelial progenitor cells (EPCs) to facilitate the construction of a vascular system around damaged PDL tissue. A vascular regeneration around PDL tissue needs proliferation of vascular progenitor cells and the subsequent differentiation of the cells. Transforming growth factor-β (TGF-β) is known as an inducer of endothelial-mesenchymal transition (EndMT), however, it remains to be clarified what kinds of TGF-β signals affect growth and mesenchymal differentiation of PDL-derived EPC-like fibroblastic cells. Here, we demonstrated that TGF-β1 not only suppressed the proliferation of the PDL-derived EPC-like fibroblastic cells, but also induced smooth muscle cell (SMC) markers expression in the cells. On the other hand, TGF-β1 stimulation suppressed EC marker expression. Intriguingly, overexpression of Smad7, an inhibitor for TGF-β-induced Smad-dependent signaling, suppressed the TGF-β1-induced growth inhibition and SMC markers expression, but did not the TGF-β1-induced downregulation of EC marker expression. In contrast, p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 suppressed the TGF-β1-induced downregulation of EC marker expression. In addition, the TGF-β1-induced SMC markers expression of the PDL-derived cells was reversed upon stimulation with fibroblast growth factor (FGF), suggesting that the TGF-β1 might not induce terminal SMC differentiation of the EPC-like fibroblastic cells. Thus, TGF-β1 not only negatively controls the growth of PDL-derived EPC-like fibroblastic cells via a Smad-dependent manner but also positively controls the SMC-differentiation of the cells possibly at the early stage of the translineage commitment via Smad- and p38 MAPK-dependent manners.
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http://dx.doi.org/10.7150/ijbs.4488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3432854PMC
November 2012

Differential effects of TGF-β1 and FGF-2 on SDF-1α expression in human periodontal ligament cells derived from deciduous teeth in vitro.

Int J Mol Med 2012 Jul 2;30(1):35-40. Epub 2012 Apr 2.

Department of Pediatric Dentistry, Tokushima University Hospital, Tokushima 770-8504, Japan.

Stromal cell-derived factor (SDF)-1α has been reported to play a crucial role in stem cell homing and recruitment to injured sites. However, no information is available about its role in periodontal tissues. The aim of this in vitro study was to investigate the effects of basic fibroblast growth factor (FGF-2) and transforming growth factor (TGF)-β1 on SDF-1α expression in immortalized periodontal ligament (PDL) cells derived from deciduous teeth (SH9 cells). Real-time PCR and western blot analyses showed that SDF-1α mRNA expression in SH9 cells was markedly inhibited by FGF-2 treatment for 48 h. SU5402, which directly interacts with the catalytic domain of the FGF receptor 1 (FGFR1) and suppresses its phosphorylation, inhibited the FGF-2-related decrease in SDF-1α expression. These results suggest that FGF-2 signaling via the FGFR1 pathway inhibits SDF-1α expression. Conversely, SDF-1α expression in SH9 cells was increased by TGF-β1 treatment for 12 h. Western blot analysis showed that this treatment induced Smad2/3 phosphorylation. A time-course experiment showed that SDF-1α expression levels reached a maximum 12 h after the TGF-β1 treatment and returned to basal levels by 48 h. Real-time PCR analysis showed that Smad7 mRNA expression peaked by 6 h after TGF-β1 treatment. Since Smad7 siRNA downregulated Smad7 expression by approximately 2.5-fold compared with the negative control siRNA, the induction of SDF-1α expression was prolonged. Furthermore, treatment of SH9 cells with TGF-β1 for 12 h induced transwell migration of UE7T-13 cells, which are mesenchymal stem cells derived from human bone marrow. Therefore, SDF-1α may play an important role in stem and progenitor cell recruitment and homing to injured sites in the periodontal ligament, and regulation of SDF-1α expression may be a useful tool in cell-based therapy for periodontal tissue regeneration.
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http://dx.doi.org/10.3892/ijmm.2012.957DOI Listing
July 2012

Effects of bisphosphonates on osteoclastogenesis in RAW264.7 cells.

Int J Mol Med 2012 Jun 23;29(6):1007-15. Epub 2012 Mar 23.

Department of Oral Diagnosis and Oral Medicine, Hokkaido University Graduate School of Dental Medicine, Kita-ku, Sapporo 060-8586, Japan.

Bisphosphonates are used as therapeutic agents for the management of osteoporosis and other bone diseases. However, the precise effects and mechanisms of bisphosphonates on osteoclastogenesis are unclear, as previous studies have reported contradictory findings and no studies have circumstantially assessed the effects of bisphosphonates on osteoclastogenesis. Therefore, the aim of this study was to determine the effects of bisphosphonates on osteoclastogenesis in RAW264.7 (RAW) cells. To examine the direct effects of bisphosphonates on osteoclast differentiation via receptor activator of nuclear factor-κB (RANK) ligand (RANKL), RAW cells were cultured with bisphosphonates. Addition of bisphosphonates to RAW cells led to a significant decrease in the number of osteoclasts and large osteoclasts (≥ 8 nuclei) in a bisphosphonate concentration-dependent and time-dependent manner. The cytotoxicity of non-nitrogen-containing bisphosphonates was specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells. Resorption activity was significantly diminished by treatment with bisphosphonates, thus confirming that bisphosphonates impair the absorptive activity of osteoclasts. We also investigated the effects of bisphosphonates on the mRNA expression of genes associated with osteoclastogenesis, osteoclast-specific markers and apoptosis-related genes using quantitative real-time PCR. The results suggest that bisphosphonates suppress osteoclast differentiation and infusion, and induce osteoclast apoptosis. With regard to osteoclast apoptosis induced by bisphosphonates, we further investigated the detection of DNA fragmentation and Caspase-Glo 3/7 assay. DNA fragmentation was confirmed after treatment with bisphosphonates, while caspase-3/7 activity increased significantly when compared with controls. In conclusion, bisphosphonates directly inhibited RANKL-stimulated osteoclast differentiation and fusion in RAW cells. It was confirmed that bisphosphonates impair osteoclast resorption activity and induce apoptosis. The effects of non-nitrogen-containing bisphosphonates were also specific to osteoclasts, while nitrogen-containing bisphosphonates were cytotoxic and induced cell death in both osteoclasts and RAW cells.
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http://dx.doi.org/10.3892/ijmm.2012.952DOI Listing
June 2012