Hideki Takai

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

Insulin‐like growth factor‐I (IGF‐I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF‐I regulation of osteogenesis, we analyzed the effects of IGF‐I on the expression of BSP in osteoblast‐like Saos2 and in rat stromal bone marrow (RBMC‐D8) cells. IGF‐I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC‐D8 cells, IGF‐I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF‐I was observed at 12 h in pLUC3 construct that included the promoter sequence from −116 to +60. Effect of IGF‐I was abrogated by 2‐bp mutations in either the FGF2 response element (FRE) or homeodomain protein‐binding site (HOX). Gel shift analyses showed that IGF‐I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX‐protein complex was supershifted by Smad1 antibody, while the FRE‐protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE‐ and HOX‐protein complexes. The IGF‐I effects on the formation of FRE‐protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3‐kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF‐I effects on HOX‐protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF‐I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene. J. Cell. Physiol. 208: 326–335, 2006.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between -184LUC to -2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

Kaempferol stimulates bone sialoprotein gene transcription and new bone formation

Kaempferol is a typical flavonol‐type flavonoid that is present in a variety of vegetables and fruits, and has a protective effect on postmenopausal bone loss. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone and could be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by kaempferol in rat osteoblast‐like UMR106 cells, and the effect of kaempferol on new bone formation. Kaempferol (5 µM) increased BSP and Osterix mRNA levels at 12 h and up‐regulated Runx2 mRNA expression at 6 h. Kaempferol increased luciferase activity of the construct pLUC3, which including the promoter sequence between nucleotides −116 to +60. Transcriptional stimulation by kaempferol abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, and FRE elements. Gel shift analyses showed that kaempferol increased nuclear protein binding to CRE and FRE elements, whereas the CCAAT‐protein complex did not change after kaempferol stimulation. Twelve daily injections of 5 µM kaempferol directly into the periosteum of parietal bones of newborn rats increased new bone formation. These data suggest that kaempferol increased BSP gene transcription mediated through inverted CCAAT, CRE, and FRE elements in the rat BSP gene promoter, and could induce osteoblast activities in the early stage of bone formation. J. Cell. Biochem. 110: 1342–1355, 2010.

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

BackgroundClinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL).MethodsIn this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors.ResultsWe characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells.ConclusionsOur study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria.Clinical trialsUse of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC): Clinicaltrials.gov NCT01931007. Registered August 26, 2013.MSC for Occlusive Disease of the Kidney: Clinicaltrials.gov NCT01840540. Registered April 23, 2013.Mesenchymal Stem Cell Therapy in Multiple System Atrophy: Clinicaltrials.gov NCT02315027. Registered October 31, 2014.Efficacy and Safety of Adult Human Mesenchymal Stem Cells to Treat Steroid Refractory Acute Graft Versus Host Disease. Clinicaltrials.gov NCT00366145. Registered August 17, 2006.A Dose-escalation Safety Trial for Intrathecal Autologous Mesenchymal Stem Cell Therapy in Amyotrophic Lateral Sclerosis. Clinicaltrials.gov NCT01609283. Registered May 18, 2012.

Parathyroid hormone regulation of the human bone sialoprotein gene transcription is mediated through two cAMP response elements

Parathyroid hormone (PTH) regulates serum calcium and inorganic phosphate levels through its actions on kidney and bone. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation and bone metabolism. We here report that two cAMP response elements (CRE) in the human BSP gene promoter are target of PTH. In human osteoblast‐like Saos2 cells, PTH (human 1−34 PTH, 10 nM) increased BSP mRNA and protein levels at 3 h. From transient transfection assays, 2‐ to 2.5‐fold increase in transcription by PTH was observed at 3 and 6 h in −184, −211, −428, −868, and −927 luciferase constructs that included the human BSP gene promoter. Effect of PTH was abrogated by 2 bp mutations in either the CRE1 (−79 to −72) or CRE2 (−674 to −667). Luciferase activities induced by PTH were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel shift analyses showed that PTH increased binding of nuclear proteins to the CRE1 and CRE2 elements. The CRE1–protein and CRE2–protein complexes were disrupted by CRE binding protein 1 (CREB1) antibodies and supershifted by phospho‐CREB1 antibody. ChIP assays detected binding of CREB1 and phospho‐CREB1 to a chromatin fragment containing CRE1 and CRE2, and increased binding of phospho‐CREB1 to the both sites. These studies demonstrate that PTH stimulates human BSP gene transcription by targeting the two CREs in the promoter of the human BSP gene. J. Cell. Biochem. 106: 618–625, 2009.

Androgen receptor stimulates bone sialoprotein (BSP) gene transcription via cAMP response element and activator protein 1/glucocorticoid response elements.

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. The androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. To determine the molecular mechanism involved in the stimulation of bone formation, we have analyzed the effects of androgens and AR effects on BSP gene transcription. AR protein levels were increased after AR overexpression in ROS17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not changed by DHT (10−8 M, 24 h). Whereas luciferase (LUC) activities in all constructs, including a short construct (nts −116 to +60), were increased by AR overexpression, the basal and LUC activities enhanced by AR overexpression were not induced by DHT (10−8M, 24 h). The effect of AR overexpression was abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE‐protein complexes were supershifted by phospho‐CREB antibody, and CREB, c‐Fos, c‐Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE‐protein complexes were supershifted by c‐Fos antibody and c‐Jun, and AR antibodies disrupted the complexes formation. These studies demonstrate that AR stimulates BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene. J. Cell. Biochem. 102: 240–251, 2007.

Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals

In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well‐known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using αMEM‐based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198–1204, 2009.

Nicotine suppresses bone sialoprotein gene expression

BACKGROUND AND OBJECTIVE Tobacco smoking is a risk factor for periodontitis and osteoporosis. Nicotine is a major component of tobacco, and has been reported to inhibit proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. The purpose of this study was to determine the effects of nicotine on bone metabolism. MATERIAL AND METHODS We used rat osteobast-like UMR106 and ROS 17/2.8 cells and rat stromal bone marrow RBMC-D8 cells. To determine the molecular basis of the transcriptional regulation of the BSP gene by nicotine, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the BSP gene promoter linked to a luciferase reporter gene and gel mobility shift assays. RESULTS Nicotine (250 microg/mL) decreased the BSP mRNA levels at 12 and 24 h in UMR106 and ROS 17/2.8 cells. From transient transfection assays using various sized BSP promoter-luciferase constructs, nicotine decreased the luciferase activities of the construct, including the promoter sequence nucleotides -116 to +60, in UMR106 and RBMC-D8 cells. Nicotine decreased the nuclear protein binding to the cAMP response element (CRE), fibroblast growth factor 2 response element (FRE) and homeodomain protein-binding site (HOX) at 12 and 24 h. CONCLUSION This study indicates that nicotine suppresses BSP transcription mediated through CRE, FRE and HOX elements in the proximal promoter of the rat BSP gene.

Effects of initial periodontal therapy on interleukin-1β level in gingival crevicular fluid and clinical periodontal parameters.

Inflammatory cytokines may have important roles in periodontitis. We assessed the effects of initial periodontal therapy on clinical periodontal parameters and interleukin-1β (IL-1β) level in gingival crevicular fluid (GCF) from chronic periodontitis (CP) patients. After initial screening, baseline periodontal parameters such as probing pocket depth (PPD) and bleeding on probing (BOP) were measured. GCF samples were collected from 13 shallow (≤3 mm) and deep (≥5 mm) PPD sites from 13 CP patients, and GCF volume and IL-1β concentration were determined at baseline (before scaling and root planning) and at 2 and 4 months after initial therapy. Baseline BOP rate, GCF volume, and IL-1β level were significantly higher at deep PPD sites than at shallow PPD sites. Significant improvements in PPD and BOP were observed at 2 and 4 months after periodontal initial therapy in deep PPD sites only. In contrast, GCF volume and IL-1β concentration were lower at 2 and 4 months after initial therapy at all sites. These results suggest that GCF volume and IL-1β level in samples reflect disease severity and that these variables are better than PPD and BOP as markers of gingival inflammation.