Differentiation-inducing factor-1 alters canonical Wnt signaling and suppresses alkaline phosphatase expression in osteoblast-like cell lines.
Because DIF-1 has been shown to affect Wnt/beta-catenin signaling pathway, the effects of DIF-1 on osteoblast-like cell lines, SaOS-2 and MC3T3-E1, were examined. We found that DIF-1 inhibited this pathway, resulting in the suppression of ALP promoter activity through the TCF/LEF binding site.


BACKGROUND
Differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium, inhibits cell proliferation and induces cell differentiation in several mammalian cells. Previous studies showed that DIF-1 activated glycogen synthase kinase-3beta, suggesting that this chemical could affect the Wnt/beta-catenin signaling pathway. This pathway has been shown to be involved in bone biology.


METHODS
We studied the effects of DIF-1 on SaOS-2 and MC3T3-E1, osteosarcoma cell lines widely used as a model system for ostoblastic cells and murine osteoblast-like cell line, respectively. Reporter gene assays were also carried out to examine the effect of DIF-1 on the Wnt/beta-catenin signaling pathway.


RESULTS
DIF-1 inhibited SaOS-2 proliferation and reduced alkaline phosphatase (ALP) activity in a concentration- and a time-dependent manner. The expression of ALP was markedly suppressed by DIF-1-treatment in protein and mRNA levels. DIF-1 also suppressed the expression of other osteoblast differentiation markers, including core binding factor alpha1, type I collagen, and osteocalcin, in protein and mRNA levels and inhibited osteoblast-mediated mineralization. Subsequently, we examined the effect of DIF-1 on the Wnt/beta-catenin signaling pathway. We found that DIF-1 suppressed the expression of beta-catenin protein and the activity of the reporter gene containing T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) consensus binding sites. We examined the effect of DIF-1 on a reporter gene driven by the human ALP promoter and found that DIF-1 significantly reduced the ALP reporter gene activity through the TCF/LEF binding site (-1023/-1017 bp). Furthermore, the effect of DIF-1 on MC3T3-E1, a murine osteoblast-like cell line, was examined, and it was found that DIF-1 suppressed ALP mRNA expression by the reduction of the ALP reporter gene activity through the TCF/LEF binding site.


CONCLUSIONS
Our data suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of ALP promoter activity. To our knowledge, this is the first report to analyze the role of the TCF/LEF binding site (-1023/-1017 bp) of the ALP gene promoter in osteoblast-like cell lines.
Matsuzaki
Etsuko
E
Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.
Takahashi-Yanaga
Fumi
F
Miwa
Yoshikazu
Y
Hirata
Masato
M
Watanabe
Yutaka
Y
Sato
Noriharu
N
Morimoto
Sachio
S
Hirofuji
Takao
T
Maeda
Katsumasa
K
Sasaguri
Toshiyuki
T
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015415:Biomarkers / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006588:Hexanones / Q000494:pharmacology*; D006801:Humans; D006843:Hydrocarbons, Chlorinated / Q000494:pharmacology*; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051822:T Cell Transcription Factor 1 / Q000378:metabolism; D051153:Wnt Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051176:beta Catenin / Q000037:antagonists & inhibitors* / Q000378:metabolism
D015415:Biomarkers; C490570:Differentiation-inducing factor-1, Dictyostelium; D006588:Hexanones; D006843:Hydrocarbons, Chlorinated; D051821:Lymphoid Enhancer-Binding Factor 1; D012333:RNA, Messenger; D051822:T Cell Transcription Factor 1; D051153:Wnt Proteins; D051176:beta Catenin; D000469:Alkaline Phosphatase