title: Impact of zinc fingers and homeoboxes 3 on the regulation of mesenchymal stem cell osteogenic differentiation.
abstract: We propose zinc fingers and homeoboxes 3 (ZHX3) as new osteogenic markers for mesenchymal stem cells (MSC). ZHX3 mRNA expression was upregulated within 1-6 h after incubation of MSCs in the osteogenic induction medium, and reached maximum levels after 24 h of incubation. Two to 4 days later, ZHX3 mRNA levels had decreased sharply. Maximal mRNA levels were 3- to 5-fold higher than those in the undifferentiated state. In contrast, Runt-related transcription factor2 (RUNX2) mRNA expression was downregulated at 2-4 h after incubation, and levels were only enhanced 1.4-fold after 12 and 24 h of incubation. Further, Osterix mRNA levels increased only 1.6-fold after 4 and 24 h of incubation. Thus, ZHX3 expression may be a better marker of MSC osteogenic differentiation than RUNX2 or Osterix expression at the initial stage of differentiation. Knockdown of ZHX3 using 2 distinct small interfering RNA (siRNA) oligonucleotides had little effect on cell morphology or on MSC proliferation, regardless of the differentiation state of the cells. However, ZHX3 siRNAs suppressed Osterix, but not RUNX2 mRNA expression, within 1 h of osteogenic differentiation, and this suppression was sustained for at least 24 h. The 2 ZHX3 siRNAs also suppressed alkaline phosphatase induction and matrix mineralization (assessed using alizarin red staining), and, further, suppressed the calcium content of the cultures at a later stage of differentiation (days 6-21). The effects of ZHX3 siRNAs on the osteogenic differentiation were comparable to those of RUNX2 and Osterix siRNAs. These findings suggest that ZHX3 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and that ZHX3 may be useful as an early osteogenic differentiation marker.
authors.affiliation: Department of Prosthetic Dentistry, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Suehiro
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishimura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawamoto
authors.forename: Masami
authors.initials: M
authors.lastname: Kanawa
authors.forename: Yuu
authors.initials: Y
authors.lastname: Yoshizawa
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Murata
authors.forename: Yukio
authors.initials: Y
authors.lastname: Kato
mesh_terms: D050156:Adipogenesis; D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology; D002113:Calcification, Physiologic; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D020219:Chondrogenesis; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D010012:Osteogenesis; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D000943:Antigens, Differentiation; D050659:Core Binding Factor Alpha 1 Subunit; D018398:Homeodomain Proteins; C493709:RUNX2 protein, human; D012097:Repressor Proteins; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; C476878:ZHX3 protein, human; D000469:Alkaline Phosphatase