IADR Abstract Archives
Stanniocalcin 1 Enhances Osteogenic Differentiation of Human Periodontal Ligamental Fibroblasts
Objectives: Stanniocalcin 1 (STC1) is a mammalian homolog of STC gene that regulates calcium and phosphate levels in bony fish. A recent study presented that compression of periodontal ligamental fibroblasts (PDLFs) increases expression levels of STC1. Stc1-deficient mice did not exhibit any bony phenotype but overexpression of human STC1 gene in mice retarded growth and calvarial suture closure. However, the function of STC1 in PDLFs has not been elucidated. Therefore, in this study, we examined the regulatory role of STC1 in osteogenic differentiation of PDLFs.
Methods: Primary human PDLFs were maintained in growth medium (α-MEM+10% FBS) and osteogenic differentiation was induced by culturing cells in osteogenic medium (growth medium supplemented with β-glycerophosphate and ascorbic acid). Osteogenic differentiation was examined by performing quantitative RT-PCR of osteoblast marker genes (Runx2, osterix, alkaline phosphatase, osteocalcin), alkaline phosphatase activity assays and alizarin red S staining to quantify matrix mineralization. Knockdown of STC1 or VEGF gene was performed by transfecting cells with small interfering RNAs to the respective genes. Blocking the extracellular action of STC1 or VEGF was induced by using the respective neutralizing antibodies.
Results: Osteogenic stimuli increased mRNA and protein levels of STC1 in a time-dependent manner. Overexpression of STC1 gene or treating the cells with recombinant STC1 protein significantly enhanced osteogenic differentiation of PDLFs. Knockdown of STC1 or STC1 neutralizing antibody treatment blocked osteogenic differentiation of PDLFs. STC1 increased expression levels of VEGF. Treatment of PDLFs with VEGF significantly enhanced osteogenic differentiation. In addition, silencing of VEGF significantly downregulated STC1-induced osteogenic differentiation of PDLFs, which was rescued by addition of extracelluar VEGF.
Conclusions: These results suggest that STC1 is an endogenous regulatory protein that is involved in the progression of osteogenic differentiation of PDLFs and that STC1 enhances osteogenic differentiation through the induction of VEGF expression, at least in part.
Methods: Primary human PDLFs were maintained in growth medium (α-MEM+10% FBS) and osteogenic differentiation was induced by culturing cells in osteogenic medium (growth medium supplemented with β-glycerophosphate and ascorbic acid). Osteogenic differentiation was examined by performing quantitative RT-PCR of osteoblast marker genes (Runx2, osterix, alkaline phosphatase, osteocalcin), alkaline phosphatase activity assays and alizarin red S staining to quantify matrix mineralization. Knockdown of STC1 or VEGF gene was performed by transfecting cells with small interfering RNAs to the respective genes. Blocking the extracellular action of STC1 or VEGF was induced by using the respective neutralizing antibodies.
Results: Osteogenic stimuli increased mRNA and protein levels of STC1 in a time-dependent manner. Overexpression of STC1 gene or treating the cells with recombinant STC1 protein significantly enhanced osteogenic differentiation of PDLFs. Knockdown of STC1 or STC1 neutralizing antibody treatment blocked osteogenic differentiation of PDLFs. STC1 increased expression levels of VEGF. Treatment of PDLFs with VEGF significantly enhanced osteogenic differentiation. In addition, silencing of VEGF significantly downregulated STC1-induced osteogenic differentiation of PDLFs, which was rescued by addition of extracelluar VEGF.
Conclusions: These results suggest that STC1 is an endogenous regulatory protein that is involved in the progression of osteogenic differentiation of PDLFs and that STC1 enhances osteogenic differentiation through the induction of VEGF expression, at least in part.