title: Akt activation is required for TGF-β1-induced osteoblast differentiation of MC3T3-E1 pre-osteoblasts.
abstract: BACKGROUND
  We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation.
  
  
  RESULTS
  MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation.
  
  
  CONCLUSIONS
  TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells.
authors.affiliation: Department of Periodontology, Tokyo Dental College, Tokyo, Japan; Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.
authors.forename: Eiichi
authors.initials: E
authors.lastname: Suzuki
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ochiai-Shino
authors.affiliation: Department of Periodontology, Tokyo Dental College, Tokyo, Japan.
authors.forename: Hideto
authors.initials: H
authors.lastname: Aoki
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Onodera
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Saito
authors.affiliation: Department of Periodontology, Tokyo Dental College, Tokyo, Japan; Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Saito
authors.affiliation: Department of Biochemistry, Tokyo Dental College, Tokyo, Japan; Oral Health Science Center, Tokyo Dental College, Tokyo, Japan.
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Azuma
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D005786:Gene Expression Regulation / Q000187:drug effects; D005819:Genetic Markers; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010012:Osteogenesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C480020:Atrnl1 protein, mouse; D005819:Genetic Markers; D008565:Membrane Proteins; D053773:Transforming Growth Factor beta1; C514862:Akt1 protein, mouse; D051057:Proto-Oncogene Proteins c-akt