Periodontal ligament (PDL) cells are composed of several different kinds of cells including mesenchymal stem cells (MSC). It is well known that these MSC play an important role in the regeneration of periodontal ligament and alveolar bone. The heterodimeric bone morphogenetic protein (BMP-2/-7) is a very potent BMP. However, BMP and TGF-1 signaling pathways that induce and regulate osteogenesis of HPDL cells are not well understood. It was reported that phosphatidylinositol 3-kinase (PI3K), which is activated by insulin-like growth factor-1 (IGF-1) and subsequently promotes the Akt phosphorylation, might play an important role in BMP-2/-7 and TGF-1-induced osteogenesis. There are numerous signaling pathways located downstream of PI3K/Akt, including the mTOR/p70S6K pathways that was the focus of this study. Here we show that TGF-1 is a more potent inducer of osteogenesis of HPDL cells than BMP-2/-7. LY294002, a PI3K inhibitor, and rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), decreased both the Smad 3 phosphorylation and the osteogenic differentiation induced by TGF-1 treatment. In contrast, the phosphorylation of Smad1/5/ 8 by BMP-2/-7 treatment was affected by neither LY294002 nor rapamycin treatment and mTOR inhibition failed to reduce BMP-2/-7-induced osteogenesis of HPDL cells. Thus the PI3K -mTOR pathway is required for TGF-1-induced osteogenesis but is not essential for osteogenesis induced by BMP-2/-7. In conclusion, the PI3K/mTOR/p70S6K pathway plays different roles in the regulation of osteogenic differentiation by TGF-1 and BMP-2/-7.

Recent studies have suggested that a balance between Wnt and bone morphogenetic protein (BMP) output may be a key factor for regulatory networks involved in bone regeneration. Here we report that Wnt inhibitors coordinately regulate osteoblastic differentiation induced by BMPs of human periodontal ligament cell. Human periodontal ligament (HPDL) cells were stimulated with BMP2/7, Dexamethasone (Dex), or a combination of these reagents. After treatment, we analyzed the phosphorylated Smad1/5 and osteoblast differentiation markers. Specific antagonists such as Dickkopfs (Dkk1, Dkk2) , secreted Frizzled-related proteins (SFRP1,SFRP2), and sclerostin(SOST) which are believed to play important roles in osteoblast differentiations were also examined by quantitative RT-PCR. BMP2/7 treatment increased ALP activity modestly, and the combination of BMP2/7 with Dex greatly enhanced ALP activity. SOST expression increased sharply following BMP2/7 treatment; however, adding BMP2/7 and Dex drastically reversed this increase in SOST. BMP2/7 and Dex treatment synergistically decreased DKK2 and sFRP2 expression. In contrast, BMP2/7 and Dex had a significant inductive effect on DKK1 and sFRP1 expression. BMP2/7 treatment resulted in Smad1/5 phosphorylation, but adding Dex did not induce p-Smad1/5. However, BMP2/7-induced Smad1/5 phosphorylation was inhibited in the combined treatment group. These opposite trends support the previous reports showing mutual antagonism between DKK1 and DKK2 or sFRP1 and sFRP2. In conclusion, wellbalanced Wnt inhibitors may be crucial for bone tissue homeostasis.

Developmental studies indicate the Wnt/ -catenin pathway biphasically regulates tissue differentiation. Bone morphogenetic protein (BMP) and Wnt signaling reported function in osteoblast differentiation, but few studies have investigated these complex mechanisms. We hypothesized that Wnt/ -catenin signaling acts, either promoting or inhibiting osteogenesis of human periodontal ligament (HPDL) cells depending on times of treatment of Wnt3A. HPDL cells were treated with single or repeated administration (12-or 24-h intervals) of 10 ng/ml of Wnt3A and cultured for 3 days. Single Wnt3A administration increased alkaline phosphatase (ALP) activity. Conversely, repeated Wnt3A administration significantly decreased the expression of osteoblast marker genes. Single Wnt3A administration resulted in Smad1/5 and Akt phosphorylation; however, repeated Wnt3A administration inhibited it. BMP2/7 or IGF-1 treatment was sufficient to reverse the inhibitory effects of repeated Wnt3A treatment. Single administration of a glycogen synthase kinase 3 inhibitors, CHIR99021, increased ALP expression, but repeated CHIR99021 administration significantly decreased ALP expression and ALP activity compared with single administration. These findings suggest that repeated activation of Wnt signaling inhibits osteoblast differentiation by suppressing the BMP2/7-Smad1/5 and phosphatidylinositide 3-kinase (PI3K) /Akt pathways in HPDL cells. This indicated that crosstalk between the Wnt, Smad1/5, and PI3K/Akt pathways is important for osteoblastic differentiation of HPDL cells.