title: Bone morphogenetic protein-induced cell differentiation involves Atg7 and Wnt16 sequentially in human stem cell-derived osteoblastic cells.
abstract: We established a differentiation method for homogeneous α7 integrin-positive human skeletal muscle stem cell (α7(+)hSMSC)-derived osteoblast-like cells with bone morphogenetic protein (BMP)-2. To explore the early signaling cascade for osteoblastic differentiation, we examined the upregulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling during BMP-2-mediated human osteoblastic differentiation. In a screening experiment, BMP-2 increased the mRNA and protein levels of Atg7, Wnt16, and Lrp5/Fzd2 (a Wnt receptor), but not microtubule-associated protein 1 light chain (LC3; a mammalian homolog of yeast Atg8), TFE3, Beclin1, Atg5, Atg12, Wnt3a, or Wnt5, together with the amounts of autophagosomes and autophagy fluxes. Treatment with siRNAs against Atg7 and Wnt16 individually suppressed the BMP-2-induced increase in osteoblastic differentiation. The osteoblastic phenotype, involving osteocalcin (BGLAP), osteopontin (SPP1), and osterix (SP7) expression, decreased when autophagy was inhibited by chloroquine (an autophagy inhibitor), but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have revealed a unique sequential cascade of BMP-2→Atg7→Wnt16→Lrp5/Fzd2→matrix metalloproteinase-13→osteoblastic differentiation. This cascade results in a potent increase in osteoblastic cell differentiation, indicating the unique involvement of Atg7, autophagy, and Wnt16 signaling in BMP-2-induced differentiation of α7(+)hSMSCs into osteoblast-like cells at a relatively early stage.
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan. Electronic address: ozeki@g.agu.ac.jp.
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Ozeki
authors.affiliation: Department of Integrative Education of Pharmacy, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, Aichi 464-8650, Japan.
authors.forename: Makio
authors.initials: M
authors.lastname: Mogi
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Hase
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Taiki
authors.initials: T
authors.lastname: Hiyama
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Yamaguchi
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Kawai
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Toru
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Department of Endodontics, School of Dentistry, Aichi Gakuin University, 2-11 Suemori-dori, Chikusa-ku, Nagoya, Aichi 464-8651, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Nakata
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D001343:Autophagy / Q000187:drug effects; D000071193:Autophagy-Related Protein 7 / Q000235:genetics / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology*; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002738:Chloroquine / Q000494:pharmacology; D020868:Gene Silencing / Q000187:drug effects; D006801:Humans; D039001:Integrin alpha Chains / Q000378:metabolism; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000166:cytology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D014212:Tretinoin / Q000494:pharmacology; D051153:Wnt Proteins / Q000378:metabolism*
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D039001:Integrin alpha Chains; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C417262:WNT16 protein, human; D051153:Wnt Proteins; C085725:integrin alpha7; D014212:Tretinoin; D002738:Chloroquine; C509349:ATG7 protein, human; D000071193:Autophagy-Related Protein 7; D020123:Sirolimus
keywords: Atg7; Cell differentiation; Matrix metalloproteinase-13; Osteoblast; Skeletal muscle stem cell; Wnt16