Expression Of FHL2Protein During Mouse Molar Development And Preliminary Study Of Its Mechnism

Objective:Tooth morphogenesis is a complex multifactorial process that involves sequential and reciprocal interactions between dental epithelium and the neural crest-derived mesenchyme. During tooth development, multiple differentially expressed signaling molecules are involved that interact with each other in regulating cell differentiation and tooth morphogenesis. However, the precise mechanism of tooth development and dentin formation is still not well-known, and more potential molecules might be involved in this process.Four and a half LIM domains2(FHL2) is a LIM-only protein of279amino acids and consists of a half LIM domain at the N-terminal followed by four full LIM domains, which acts as transcriptional co-regulator. Through the LIM domains, FHL2can bind many proteins such as kinase, receptor and transcription factors to regulate gene expression, cell differentiation, morphogenesis and hard tissues morphogenesis. Depending on the protein partners involved, FHL2functions either as an activator or as a repressor to participate in cell differentiation and plays important roles in these processes. Recent studies demonstrated that FHL2could play an important role in osteoblasts differentiation and bone formation via interacting with many transcription factors.Bone and dentin are all hard tissues. In addition, osteoblasts and odontoblasts are all differentiated from mesenchymal cells and share several similarities including the expression of similar genes. It is logically reasonable that we hypothesize FHL2might have a potential role in tooth development and dentin formation.During early tooth development, Runx2can increase the transcriptional capability of the other signaling molecules and plays an important role in regulating cell differentiation and hard tissues morphogenesis. During osteoblasts differentiation and bone formation, FHL2functions as an activator to increase the transcription capability of Runx2directly or via Wnt/β-catenin pathways. It is logically reasonable that we hypothesize FHL2might function as a co-activator to interact with the transcription factor Runx2during tooth development, odontoblasts differentiation and dentin formation.Methods, Results and Conclusions:Experiment1:Immunohistochemistry was used to investigate the expression patterns of FHL2at different stages of mouse molar development. Immunohistochemical staining showed that at the bud and cap stage, FHL2was expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2appeared in the inner and outer enamel epithelium, and stratum intermedium. Positive staining gradually converged at the cusps of dental papilla. At the late bell stage, FHL2was expressed in the terminal differentiated ameloblasts and odontoblasts and stratum intermedium. At the postnatal day, FHL2was detected in the secretory and mature ameloblasts and odontoblasts and mature enamel, and gradually appeared at Hertwig’s epithelial root sheath (HERS) and periodontal tissues. The spatial-temporal expression patterns of FHL2from the bud stage to the postnatal day (28) suggested that during tooth development, FHL2might play an important role in tooth development, cell differentiation, morphogenesis, secretion and mineralization of hard tissue matrix.Experiment2:Immunofluorescence was used to investigate the expression patterns of FHL2and Runx2at the early stages of mouse molar development. Immunofluorescence staining showed that the spatial and temporal expression of FHL2and Runx2was found in the early developing tooth germ. The expression locations were similar at the tooth germ. At the bud and cap stage, FHL2and Runx2were expressed both in enamel organ and the underlying mesenchyme. At the early bell stage, FHL2and Runx2appeared in the inner and outer enamel epithelium, and stratum intermedium. Positive staining gradually converged at the cusps of dental papilla.Experiment3:Western Blot was used to investigate the expression of FHL2and Runx2at the protein level during dental pulp stem cells differentiation into odontoblasts. And immunoprecipitation was used to determine the relationship between FHL2and Runx2. Western Blot showed on inducing day0、7、14, FHL2and Runx2were detected in all three inducing days. FHL2was gradually increased, but the highest protein level of Runx2on inducing day7was showed. Immunoprecipitation showed the direct interaction between FHL2and Runx2.Experiment2and3demonstrated that during tooth morphogenesis and odontoblasts differentiation, FHL2might functions as co-activator to increase the transcription capability of Runx2.