ATF4, DLX3, FRA1, MSX2, C/EBP-ζ, and C/EBP-α Shape the Molecular Basis of Therapeutic Effects of Zoledronic Acid in Bone Disorders.

OBJECTIVE Zoledronic Acid (ZA) is one of the common treatment choices used in various bone-associated 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 reason involved in the regulation of genes responsible for OSD. The present study was 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 was 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 were falling 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 ZAtreated 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 leads to the downregulation 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 promotermethylation 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.