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This study investigated the effect of various treatments with TGF-β1 in osteogenic differentiation of human periodontal ligament (HPDL) cells. HPDL cells were treated with single 1 ng/ml, 100 ng/ml or multiple (12-h intervals) 1 ng/ml TGF-β1. Alkaline phosphatase (ALP) activity was detected by ALP activity staining at 4 days. Phospho-Smad3 was detected by western blotting and transcription of osteogenic differentiation markers (RUNX2, ALPL, IBSP: bone sialoprotein, BGLAP: osteocalcin) were determined by real-time PCR at various culture periods. Cells were pretreated with or without SB505124, a TGF-β/Smad pathway inhibitor, for 1 h before 1 ng/ml TGF-β1 treatment at 12-h intervals. ALP activity and phosphorylated Smad3 were detected. Single treatment with 1 ng/ml TGF-β1 significantly induced Smad3 phosphorylation, subsequent ALP upregulation and transcription of osteogenic differentiation markers, compared to these inductions with 100 ng/ml TGF-β1 treatment. In multiple treatments with 1 ng/ml TGF-β1 at 12-h intervals, only the first treatment significantly induced Smad3 phosphorylation but not the succeeding treatments. Inductive effects of single 1 ng/ml TGF-β1 in osteogenic differentiation were almost completely abolished by multiple treatments. Furthermore, multiple treatments with 1 ng/ml TGF-β1 did not induce ALP activity although the TGF-β/Smad pathway was inhibited by SB505124. Single treatment with low dose TGF-β1 significantly induced osteogenic differentiation. Multiple treatments inhibited osteogenic differentiation despite inhibition of TGF-β/Smad pathway. These findings suggest that TGF-β1 both positively and negatively regulates osteogenic differentiation and the inhibitory effect may be mediated by the non-Smad signaling pathway in HPDL cells.

Dual Nature of TGF-β1 in Osteoblastic Differentiation of Human Periodontal Ligament Cells