title: Controlled Osteogenic Differentiation of Mouse Mesenchymal Stem Cells by Tetracycline-Controlled Transcriptional Activation of Amelogenin.
abstract: Regenerative dental therapies for bone tissues rely on efficient targeting of endogenous and transplanted mesenchymal stem cells (MSCs) to guide bone formation. Amelogenin is the primary component of Emdogain, which is used to regenerate periodontal defects; however, the mechanisms underlying the therapeutic effects on alveolar bone remain unclear. The tetracycline (Tet)-dependent transcriptional regulatory system is a good candidate to investigate distinct roles of genes of interest during stem cell differentiation. Here, we investigated amelogenin-dependent regulation of osteogenesis in MSCs by establishing a Tet-controlled transcriptional activation system. Clonal mouse bone marrow-derived MSCs were lentivirally transduced with the Tet repressor (TetR) expression vector followed by drug selection to obtain MSCs constitutively expressing TetR (MSCs-TetR). Expression vectors that contained the Tet operator and amelogenin-coding (Amelx) cDNA fragments were constructed using the Gateway system and lentivirally introduced into MSCs-TetR to generate a Tet regulation system in MSCs (MSCs-TetR/Amelx). MSCs-TetR/Amelx significantly overexpressed the Amelx gene and protein in the presence of the tetracycline derivative doxycycline. Concomitant expression of osterix, bone sialoprotein (BSP), osteopontin, and osteocalcin was modulated by addition or removal of doxycycline under osteogenic guidance. During osteogenic induction, MSCs-TetR/Amelx treated with doxycycline showed significantly increased gene expression of osterix, type I collagen, BSP, and osteocalcin in addition to increased alkaline phosphatase activity and mineralized nodule formation. Enhanced extracellular matrix calcification was observed when forced Amelx expression commenced at the early stage but not at the intermediate or late stages of osteogenesis. These results suggest that a Tet-controlled Amelx gene regulation system for mouse MSCs was successfully established, in which transcriptional activation of Amelx was associated with enhanced osteogenic differentiation, especially in the early stage of biomineralization.
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Wang
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.|Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Okawa
authors.affiliation: Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Kamano
authors.affiliation: Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, Japan.
authors.forename: Kunimichi
authors.initials: K
authors.lastname: Niibe
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Kayashima
authors.affiliation: Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Thanaphum
authors.initials: T
authors.lastname: Osathanon
authors.affiliation: Research Unit of Mineralized Tissue, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
authors.forename: Prasit
authors.initials: P
authors.lastname: Pavasant
authors.affiliation: Division of Dental Pharmacology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.
authors.forename: Makio
authors.initials: M
authors.lastname: Saeki
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yatani
authors.affiliation: Department of Fixed Prosthodontics, Osaka University Graduate School of Dentistry, Suita, Osaka, Japan.|Division of Molecular and Regenerative Prosthodontics, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Egusa
mesh_terms: D053523:Amelogenin / Q000235:genetics*; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002999:Clone Cells; D015870:Gene Expression / Q000187:drug effects; D016086:Lentivirus / Q000235:genetics; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics*; D013752:Tetracycline / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects
chemical_list: D053523:Amelogenin; C507200:Amelx protein, mouse; D013752:Tetracycline