NFIc Regulates the Expression of Growth Factors FGF5 and CSF

Transcription factor NFI-C has shown to be essential for root development.  Knockout mice for Nfic do not form roots and these mice end up losing their teeth. Histological studies have shown morphological abnormalities in the dental papilla mesenchyme (DPM)-derived odontoblast cells in these mice, as well as the formation of a sparse and irregular dentin layer. Using an established DPM cell-line lacking Nfic (silenced using small hairpin RNA (shRNA) we can determine changes in gene expression as compared to control cells expressing Nfic. This cell line can be used to determine down-stream genes affected by Nfic during dentinogenesis. Objective: Compare gene expression in DPM cells maintained in vitro where Nfi-C has been silenced with shRNA and DPM cells where Nfic is expressed normally and confirm changes at the protein level. Methods: Immortalized DPM cells maintained in vitro were stably transfected with Nfic-shRNA. The presence of Nfic in these cells was reduced by 94%. Cells were grown in culture; RNA or proteins were extracted and mRNA was converted to cDNA for DNA microarray analysis between the treated and control cells. RT² Profiler™ PCR Arrays for Growth factors (SuperArrays, Bioscience Corp) were used. Proteins were used for immunostaining.  Results: Our data shows statistically significant changes in more than 35 different growth factors when DPM cells depleted of Nfic were compared to the control DPM cells grown under the same conditions. The most significant changes was a 100 fold increase in fibroblast growth factor 5 (fgf5), in DPM cells lacking Nfic. There was also considerable reduction (17 fold) in the amount of Colony stimulation factor 1 (Csf1) in this cells. Analysis at the protein level using western blot immunostaining confirmed these results. Conclusions: These results suggest that one mechanism that Nfic uses to regulate root formation is by controlling expression of FGF5.