Analysis of the Interaction between Mitf and miR-148a in the regulation of osteoclast differentiation

• Main Author: Fu, Qi
• Other Authors: Ostrowski, Michael, Sharma, Sudarshana
• Format: Thesis
• Language: English
• Published: The Ohio State University 2011
• Subjects: MicroRNAs; Bone development; Signaling pathways; Iceland
• Online Access: http://hdl.handle.net/1811/48904

Osteoclast differentiation plays an important role in maintaining a healthy bone density. Mitf is a transcription factor that plays a significant role in regulating osteoclast differentiation. Recently, microRNAs (miRs) have been shown to be heavily involved in development. I decided to investigate the interaction between microRNAs and Mitf during osteoclast differentiation. Preliminary results from a miR microarray comparing Mitf+/+ mice and Mitfmi/mi mice showed that many miRs were differentially regulated during osteoclast differentiation, but only a few miRs were significantly regulated. MiR-148a was one of the miRs that were significantly down regulated by 0.27 fold during osteoclast differentiation. A recent publication from the University of Iceland using a luciferase assay has shown that miR-148a translation is inhibited upon over-expression of Mitf. This led me to my hypothesis that Mitf and miR-148a interacted in a negative feedback-loop mechanism to regulate osteoclast differentiation. The down regulation of miR-148a in Mitf mutant mice was confirmed using Real-time PCR by comparing miR-148a expression between Mitf+/+ and Mitfvga/mi mice and miR-148a expression between Mitfce/+ and Mitfce/ce mice. Target analysis shows that Mitf has four putative binding sites on the miR-148a promoter. ChIP analysis showed an increase in miR-148a abundance on the Mitf protein complex, but not on the Pu.1 protein complex during osteoclast differentiation, suggesting that miR-148a directly interacts with Mitf alone during osteoclast differentiation. Mitfce/ce mice are another type of Mitf mutant mice that haven’t been well-studied. X-ray scans showed a decrease in bone density and MicroCT analysis showed a decrease in bone volume fraction in Mitfce/ce pups that was not present in Mitfce/ce adults, suggesting that Mitfce/ce mice exhibit a slight osteopetrotic phenotype that is resolved with age. TRAP Staining shows that Mitfce/ce mice exhibit defects in osteoclast differentiation that are not resolved with age. Chromatin Immunoprecipitation (ChIP) analysis and Real-time PCR show that Mitf recruitment and osteoclast-specific gene expression is decreased in Mitfce/ce mice. This shows that the defects in osteoclast differentiation are at least partially due to a dysregulation of Mitf. These results provide us with a better understanding of the bone biology of Mitfce/ce mice and how Mitf and miR-148a interact to regulate osteoclast differentiation. No embargo