title: Bone mesenchymal stem cell-conditioned medium induces the upregulation of Smad6, which inhibits the BMP-4/Smad1/5/8 signaling pathway.
abstract: OBJECTIVE
  In this study, we aimed to investigate the effect of bone mesenchymal stem cells (BMSCs) conditioned medium on BMP4-induced activation of the Smad signaling pathway during the process of neural stem cells differentiation.
  
  
  METHODS
  We designed four experimental groups: (A) control group, (B) BMP-4 treatment group, (C) BMSC-CM group, and (D) BMSC-CM (BMSC-conditioned medium) combined with BMP-4 group. The expression of microtube-associated protein-2 (MAP-2) detected by immunocytochemical staining and western blot. The expression of glial fibrillary acidic protein (GFAP) and BMP receptors (BMPRII and BMPRIa) was determined by western blot, while SMAD1/5/8 and SMAD6 mRNA expression was detected by quantitative real-time polymerase chain reaction (Q-RT-PCR).
  
  
  RESULTS
  We found that the expression of microtube-associated protein-2 (MAP-2) increased in group B compared to group A. In group C, the expression of GFAP was increased and the expression of MAP-2 was decreased compared to group A. This phenomenon was weaker in group D. We also demonstrated that BMSC-CM could inhibit the activation of BMP signaling by downregulating the expression of BMPRII and BMPRIa proteins. Moreover, BMSC-CM could upregulate the expression of Smad6 mRNA and inhibit the activation of Smad1/5/8 mRNA.
  
  
  CONCLUSIONS
  These observations suggest that BMSC-CM could neutralize the effect of BMP-4 on the differentiation of NSCs by upregulating the expression of Smad6 and decrease the expression of GFAP by inhibiting the BMP-4-SMAD1/5/8 signaling pathway, conversely increasing the generation of neurons.
authors.affiliation: a Department of Spinal Surgery , The First Affiliated Hospital of Anhui Medical University , Hefei , China.
authors.forename: Huang
authors.initials: H
authors.lastname: Fang
authors.affiliation: a Department of Spinal Surgery , The First Affiliated Hospital of Anhui Medical University , Hefei , China.
authors.forename: PeiWen
authors.initials: P
authors.lastname: Song
authors.affiliation: a Department of Spinal Surgery , The First Affiliated Hospital of Anhui Medical University , Hefei , China.
authors.forename: CaiLiang
authors.initials: C
authors.lastname: Shen
authors.affiliation: b School of Life Science, Anhui Medical University , Heifei , China.
authors.forename: XiaoYing
authors.initials: X
authors.lastname: Liu
authors.affiliation: a Department of Spinal Surgery , The First Affiliated Hospital of Anhui Medical University , Hefei , China.
authors.forename: HaiTai
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D055415:Bone Morphogenetic Protein 4 / Q000378:metabolism / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000737:chemistry*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D064231:Nestin / Q000378:metabolism; D009474:Neurons / Q000187:drug effects*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000235:genetics / Q000378:metabolism; D051905:Smad6 Protein / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*; D000099229:RE1-Silencing Transcription Factor
chemical_list: D055415:Bone Morphogenetic Protein 4; D017077:Culture Media, Conditioned; D005904:Glial Fibrillary Acidic Protein; D008869:Microtubule-Associated Proteins; D064231:Nestin; D012097:Repressor Proteins; D051785:Smad Proteins; D051905:Smad6 Protein; D000099229:RE1-Silencing Transcription Factor; C525809:BMP4 protein, human; C497803:Smad6 protein, mouse
keywords: BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; BMSC-CM, bone mesenchymal stem cell-conditioned medium; BMSCs, bone mesenchymal stem cells; Bone mesenchymal stem cell-conditioned medium; Bone morphogenetic protein-4; CNS, central nervous system; GFAP, glial fibrillary acidic protein; MAP-2, microtube-associated protein-2; NSC differentiation; NSCs, neural stem cells; Smad signaling pathway; Smad6