Differential expressions of connexin32&43 in periodontal ligament-derived cells during mineralization

Introduction: Gap junctions (GJs) connect adjacent cells and facilitate intercellular communication by providing channels that permit small signaling molecules exchange. GJs comprise of connexin (Cx) tightly assembled in hexameric fashion. Several reports have suggested cell type specific expressions of Cxs and their functional-related specificity. Periodontal ligament cells (PDLs) are a group of heterogeneous cells that possess osteogenic phenotype. It has been shown that PDLs expressed Cx32 and 43, although specific functions have not yet been clarified.

Objective: To analyze the expression profiles of Cx32 and 43 during in PDLs induced to form mineralized nodules.

Methods: PDLs were derived from healthy third molars of 5 patients. Equal amount of cells numbers were seeded in 35 mm dishes and cultured in DMEM medium containing 10%FBS. At day7, medium was changed into DMEM supplemented with 10%FBS 50µg/ml ascorbic acid and 10mM ß-glycerophosphate (mineralizing medium) for another 21 days. At the end of each week, cell-matrix layer were stained with alizarin red S to visualize mineralization. Cells were lysed with lysis buffer and analyzed for Cx32 and 43 proteins levels using Western blot analysis (WB) with specific antibodies. Statistical analyses were performed using ANOVA.

Results: Mineralized nodules were observed during week3 of culture and were more extensive at week4. WB indicated that Cx32 was already expressed at the beginning of culture. However, the expression level decreased during mineralization. On the other hand, expression level of Cx43 appeared to increase when cells were induced to form mineralized nodules. At week4, when mineralized nodules were most abundant, Cx43 significantly increased and showed the highest level.

Conclusion: The results suggested that Cx32 and 43 might play a differential role in regulating osteoblastic phenotype of PDLs. Cx32 might be associated with maintenance of PDL characteristics, while Cx43 might regulate mineralization in these cells.