Methods: RT-PCR, immunofluorescent staining and Western-blotting analyses were used to determine the expression and subcellular localization of DMP1 in various cell lines, including odontoblast-like (17IIA11), preosteoblast (MC3T3-E1), mesenchymal cells (C3H10T1/2), and human dental pulp stem cells (DPSC). In addition, a haemagglutinin (HA) tagged DMP1 expression construct was generated and examined for its subcellular localization in kidney fibroblast cells (COS-7).
Results: Western-blot analysis showed the presence of DMP1 in the cytoplasmic and nuclear extracts of MC3T3-E1, 17IIA11, and C3H10T1/2 cells. Dmp1 transcripts were consistently detected in all three cell lines by RT-PCR analysis. However, immunofluorescent staining revealed the presence of two distinct subpopulations of cells in these cell lines with nuclear or cytoplasmic staining, when using a DMP1 monoclonal antibody. Nuclear and cytoplasmic DMP1 was confirmed in MC3T3-E1 cells by immuofluorescent staining using a rabbit polyclonal antibody; and the staining was inhibited when the antibody was preincubated with the synthetic peptide used to generate the antibody, confirming the specificity of the antibody. Moreover, nearly half DPSC cells had nuclear staining and the other half showed cytoplasmic staining. Nuclear and cytoplasmic localization was also observed in COS-7 cells transfected with HA-tagged DMP1 expression construct when detected with an antibody against the HA tag.
Conclusion: These findings suggest that, apart from its role as a constituent of dentin/bone matrix, DMP1 might play a regulatory or structural role in the nucleus that is not unique to the odontoblast/osteoblast cells.
Keywords: Bone, DMP1, Dentin, Odontoblasts and Osteoblasts/osteoclasts
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