Title:ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders
Volume: 20
Issue: 18
Author(s): Faroogh Marofi , Jalal Choupani , Saeed Solali , Ghasem Vahedi , Ali Hassanzadeh , Tohid Gharibi and Majid F. Hagh*
Affiliation:
- Department of Immunology, Division of Hematology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz,Iran
Keywords:
DNA methylation, osteoblastic differentiation, mesenchymal stem cells, zoledronic acid, MS-qPCR, bone disorders.
Abstract:
Objective: Zoledronic Acid (ZA) is one of the common treatment choices used in various boneassociated
conditions. Also, many studies have investigated the effect of ZA on Osteoblastic-Differentiation
(OSD) of Mesenchymal Stem Cells (MSCs), but its clear molecular mechanism(s) has remained to be understood.
It seems that the methylation of the promoter region of key genes might be an important factor involved
in the regulation of genes responsible for OSD. The present study aimed to evaluate the changes in the mRNA
expression and promoter methylation of central Transcription Factors (TFs) during OSD of MSCs under treatment
with ZA.
Materials and Methods: MSCs were induced to be differentiated into the osteoblastic cell lineage using routine
protocols. MSCs received ZA during OSD and then the methylation and mRNA expression levels of target
genes were measured by Methylation Specific-quantitative Polymerase Chain Reaction (MS-qPCR) and
real-time PCR, respectively. The osteoblastic differentiation was confirmed by Alizarin Red Staining and the
related markers to this stage.
Results: Gene expression and promoter methylation level for DLX3, FRA1, ATF4, MSX2, C/EBPζ, and
C/EBPa were up or down-regulated in both ZA-treated and untreated cells during the osteodifferentiation process
on days 0 to 21. ATF4, DLX3, and FRA1 genes were significantly up-regulated during the OSD processes,
while the result for MSX2, C/EBPζ, and C/EBPa was reverse. On the other hand, ATF4 and DLX3 methylation
levels gradually reduced in both ZA-treated and untreated cells during the osteodifferentiation process on days 0
to 21, while the pattern was increasing for MSX2 and C/EBPa. The methylation pattern of C/EBPζ was upward
in untreated groups while it had a downward pattern in ZA-treated groups at the same scheduled time. The result
for FRA1 was not significant in both groups at the same scheduled time (days 0-21).
Conclusion: The results indicated that promoter-hypomethylation of ATF4, DLX3, and FRA1 genes might be
one of the mechanism(s) controlling their gene expression. Moreover, we found that promoter-hypermethylation
led to the down-regulation of MSX2, C/EBP-ζ and C/EBP-α. The results implicate that ATF4, DLX3 and FRA1
may act as inducers of OSD while MSX2, C/EBP-ζ and C/EBP-α could act as the inhibitor ones. We also determined
that promoter-methylation is an important process in the regulation of OSD. However, yet there was no
significant difference in the promoter-methylation level of selected TFs in ZA-treated and control cells, a methylation-
independent pathway might be involved in the regulation of target genes during OSD of MSCs.
