S100A4: a novel negative regulator of mineralization and osteoblast differentiation.
S100A4 is an intracellular calcium-binding protein expressed by osteoblastic cells. However, its roles in bone physiology are unknown. Because before matrix mineralization, its expression is markedly diminished, we hypothesized that S100A4 negatively regulates the mineralization process. In this study, we investigated the effects of the inhibition of S100A4 synthesis on osteoblast differentiation and in vitro mineralized nodule formation. Inhibition of S100A4 synthesis was achieved by an antisense approach in the mouse osteoblastic cell line MC3T3-E1. Cell clones that synthesized low levels of S100A4 (AS clones) produced markedly increased number of mineralized nodules at much earlier stages in comparison with controls as demonstrated by Alizarin red S and von Kossa staining. The expression of type I collagen (COLI) and osteopontin (OPN) increased in AS clones compared with controls. Bone sialoprotein (BSP) and osteocalcin (OCN), molecules associated with mineralization and markers for mature osteoblastic phenotype, were expressed in AS clones before their detection in controls. Because S100A4 was not localized in the nucleus of MC3T3-E1 cells and AS clones, it is unlikely that S100A4 directly regulates the expression of these genes. Moreover, the expression of Cbfal/Osf-2 and Osx, transcription factors necessary for the expression of osteoblast-associated genes, remained unchanged in AS clones, indicating that S100A4 may be downstream to these transcription factors. These findings indicate that S100A4 is a novel negative regulator of matrix mineralization likely by modulating the process of osteoblast differentiation.
Duarte
Wagner R
WR
Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-745, USA. wagner_duarte@dentistry.unc.edu
Shibata
Tatsuya
T
Takenaga
Keizo
K
Takahashi
Etsuko
E
Kubota
Kaori
K
Ohya
Keiichi
K
Ishikawa
Isao
I
Yamauchi
Mitsuo
M
Kasugai
Shohei
S
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers; D015153:Blotting, Western; D002113:Calcification, Physiologic / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers; D005455:Fluorescent Antibody Technique; D051379:Mice; D010006:Osteoblasts / Q000166:cytology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000235:genetics / Q000502:physiology*
D015415:Biomarkers; D017931:DNA Primers; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C470944:S100a4 protein, mouse