Osteogenetic oligodeoxynucleotide (osteoDN) osteoblast mineralization independently of TLR9

Osteogenetic oligodeoxynucleotide (osteoDN) osteoblast mineralization independently of TLR9

Yuma Nihashi1, Mana Miyoshi2, Koji Umezawa2,3, Takeshi Shimosato1,2,3, Tomohide Takaya1,2,3.

  1. Graduate School of Medicine, Science and Technology, Shinshu University.
  2. Faculty of Agriculture, Shinshu University.
  3. Institute for Biomedical Sciences, Shinshu University.

日本農芸化学会2021年度大会 (仙台), 2021/03/20 (口演).


Introduction: Osteoblasts are responsible for bone formation, and its differentiation is indispensable for bone homeostasis. Dysfunction of osteoblast differentiation is one of the causes of osteoporosis. We recently identified the oligodeoxynucleotide named "osteoDN" strongly promotes an early stage of osteoblast differentiation. However, the target of osteoDN is still unclear. Generally, the oligonucleotides having unmethylated CpG motifs (CG) are recognized by Toll-like receptor 9 (TLR9) and initiate immune responses. Since osteoDN has one CG sequence, in this study, we investigated the TLR9-dependency of osteoDN during osteoblast mineralization.

Methods: Mouse osteoblast cell line MC3T3-E1 was induced mineralization for 9-12 days in differentiation medium with 0.1-10 uM osteoDN in the presence or absence of a TLR9 antagonist, E6446. Osteoblast mineralization was evaluated by alizarin staining. Expression levels of osteogenic genes were quantified by qPCR.

Results: 10 uM osteoDN significantly induced transcriptions of osteogenic transcription factors Runx2 and Sp7 (osterix), and non-collagenous hormone Bglap2 (osteocalcin) at 48 h after the treatment. osteoDN significantly increased the alizarin-positive area at day 9 in a dose-dependent manner. These data indicate that osteoDN induces osteoblastic mineralization through activating gene expression. RT-PCR revealed that MC3T3-E1 cells scarcely express TLR9. Pre-treatment of E6446 from 3 h before osteoDN administration did not affect osteoblast mineralization induced by osteoDN. Furthermore, the mutant osteoDN which CG motif is substituted to GC (osteoDN-GC) promoted osteoblast mineralization as well as osteoDN did. It demonstrates that osteoDN facilitates osteoblast maturation in a TLR9-independent manner.

Conclusion: The 18-base single-strand osteoDN promoted osteogenic gene expression and osteoblast mineralization independently of TLR9. It is possible that osteoDN serve as an aptamer depending on its conformation. osteoDN accelerating osteoblast differentiation can be a nucleic acid drug for prevention and therapy for osteoporosis.

Keywords: osteoblast, osteogenic differentiation, myogenetic oligodeoxynucleotide