Methods: Wild-type and mutant BMP4 expression constructs were generated, thus the expressed proteins would have a Flag tag on the prodomain or a HA tag on the mature BMP4. Various BMP4 expression constructs were examined in Cos-7 cells by both western-blot analysis and immunofluorescent staining using an antibody against the Flag or HA tag.
Results: In vitro functional analysis demonstrated that the Ala42Pro mutation did neither affect proBMP4 processing nor secretion when expressed in Cos-7 cells. However, immunofluorescent staining of Cos-7 cells transiently transfected with the wild-type BMP4 expression construct revealed a fibrillar distribution of the secreted BMP4 prodomains suggesting that BMP4 prodomains are instrumental in storing mature BMP4 dimers in the ECM. Although a similar fibrillar distribution was observed for the mutant BMP4, it is quite possible that the mutation alters the interaction of the prodomain with the fibrillar components in the ECM, thereby affecting availability/activity of mature BMP4.
Conclusion: Our work suggests that the BMP4 prodomain might play a role that is analogous to the TGF-β1 prodomain and is essential for regulating the storage of mature BMP4 in the ECM during tooth development. Studies are underway to further investigate how the mutation may affect such Bmp4/ECM interaction.
Keywords: Hypodontia, BMP4