The present study describes, for the first time, the removal of the nuclear factor of activated T cells cytoplasmic 1 (NFATc1) by a decoy approach in human primary osteoblasts (hOBs). hOBs with different NFATc1 expression levels were used. The functionality of endogenous NFAT proteins in our experimental model was analyzed by monitoring the transcriptional activity on a luciferase reporter construct driven by three copies of an NFAT response element (pNFAT-TA-luc). Cell treatment with the decoy against NFATc1 resulted in a significant increase in the expression of osteoblastic markers, including ERα and ColXV. On the contrary, the expression of Runx2, which is known to not be transcriptionally regulated by NFATc1, was not altered, indicating the specificity of the decoy effect. To our knowledge, this is the first time that transcription factor decoy has been successful in hOBs to allow the investigation of the role of NFATc1 in an experimental model that, compared to the use of cell lines, more closely resembles an in vivo model. In addition, by using chromatin immunoprecipitation we found that in vivo NFATc1 is recruited on the ColXV gene promoter. The specific role of NFATc1 in osteoblast differentiation is not well understood, however, our findings reinforce the action of NFATc1 in the transcriptional program of osteoblasts, also supporting the therapeutic potential for the proper manipulation of NFATc1-mediated events in different bone disorders. At the same time, our data add important information on the regulation of the expression of ColXV, which only recently has been proposed as an osteoblastic marker.
- Chromatin immunoprecipitation
- Collagen type XV
- Human primary osteoblasts
- Nuclear factor of activated T cells
- Transcription factor decoy
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