title: ZBTB16 induces osteogenic differentiation marker genes in dental follicle cells independent from RUNX2.
abstract: BACKGROUND
  Dental follicle cells (DFCs) are neural crest cell-derived cells and the genuine precursor cells of cementoblast and alveolar osteoblasts. After osteogenic differentiation, expression levels of the transcription factor zinc factor and BTB domain containing 16 (ZBTB16) were significantly increased. ZBTB16 is associated with the process of osteogenic differentiation in bone marrow-derived mesenchymal stem cells and crucial for the expression of the osteogenic transcription factor runt-related transcription factor 2 (RUNX2). It is proposed that ZBTB16 plays also a crucial role for the differentiation of DFCs into osteoblasts.
  
  
  METHODS
  In this study, the differentiation of DFCs by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and electron-dispersive x-ray spectrometry (EDX) analysis is investigated. The expression of ZBTB16 during osteogenic differentiation and the expression of osteogenic differentiation markers were assessed by real-time reverse transcription polymerase chain reaction. Glucocorticoid stimulation was inhibited using RU486 (11β-[p-(Dimethylamino)phenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), and ZBTB16 was overexpressed via transient transfection of an expression vector.
  
  
  RESULTS
  After the initiation of osteogenic differentiation, ZBTB16 levels were increased highly in DFCs, whereas RUNX2 was expressed constitutively only. An EDX analysis verified the differentiation of DFCs into osteoblast-like cells because clusters of mineralization consisted of hydroxyapatite. ZBTB16 induced the expression of nuclear receptor subfamily 4, group A, member 3; osteocalcin; and stanniocalcin 1 (STC1) but not of RUNX2 and ALP in DFCs. STC1 was upregulated in DFCs downstream of ZBTB16 and after the osteogenic differentiation. The overexpression of STC1 in DFCs increased the expression of ZBTB16 and specific markers for biomineralization.
  
  
  CONCLUSIONS
  The present study shows that ZBTB16 induced the expression of osteogenic differentiation markers independently of RUNX2. Moreover, STC1 is a new candidate for the evaluation of late mechanisms of osteogenic differentiation downstream of ZBTB16.
authors.affiliation: Department of Cranio- and Maxillofacial Surgery, University Hospital Regensburg, Regensburg, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Felthaus
authors.forename: Martin
authors.initials: M
authors.lastname: Gosau
authors.forename: Christian
authors.initials: C
authors.lastname: Morsczeck
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D000880:Anthraquinones; D015415:Biomarkers / Q000032:analysis; D002113:Calcification, Physiologic / Q000235:genetics; D002118:Calcium / Q000032:analysis; D002454:Cell Differentiation / Q000235:genetics; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics; D003795:Dental Sac / Q000166:cytology* / Q000378:metabolism; D017886:Durapatite / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D005938:Glucocorticoids / Q000037:antagonists & inhibitors; D006023:Glycoproteins / Q000235:genetics; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000502:physiology*; D015735:Mifepristone / Q000494:pharmacology; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics; D010012:Osteogenesis / Q000235:genetics*; D010758:Phosphorus / Q000032:analysis; D000075722:Promyelocytic Leukemia Zinc Finger Protein; D060888:Real-Time Polymerase Chain Reaction; D011987:Receptors, Steroid / Q000235:genetics; D011988:Receptors, Thyroid Hormone / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013052:Spectrometry, X-Ray Emission; D013234:Stem Cells / Q000502:physiology*; D014162:Transfection
chemical_list: D000880:Anthraquinones; D015415:Biomarkers; D004396:Coloring Agents; D050659:Core Binding Factor Alpha 1 Subunit; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D006023:Glycoproteins; D006727:Hormone Antagonists; D051741:Kruppel-Like Transcription Factors; C490412:NR4A3 protein, human; D000075722:Promyelocytic Leukemia Zinc Finger Protein; C493709:RUNX2 protein, human; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D015675:Osteocalcin; C092037:ZBTB16 protein, human; D010758:Phosphorus; D015735:Mifepristone; C010078:alizarin; C051795:teleocalcin; D017886:Durapatite; D000469:Alkaline Phosphatase; D002118:Calcium