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

Abstract

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.

The Comparison between Single vs Repeated Administration of Wnt3A of HPDL Cells

“…1B and C), similar to repeated TGF-β1 administration, as we previously reported 4) . Expressions of other osteoblast markers and differentiation-related genes such as BSP, IGF-1, RUNX2, and MSX2 are also decreased following repeated Wnt3A administration ( Fig.…”

“…First, repeated Wnt3A treatment downregulates ALP expression and early-phase osteoblast differentiation of HPDL cells. Previously, we reported that repeated TGF-β1 treatment to HPDL cells downregulates ALP expression 4) . We observed subsequent decreased IGF-1 expression and inhibition of PI3K/Akt signaling 3) .…”

“…We previously reported an experimental model that allows the investigation of inhibitory effects of transforming growth factor-1 (TGF-1) on osteoblast differentiation and found that repeated or high-dose administration of TGF-1 inhibited osteoblast differentiation of HPDL cells by decreasing insulin-like growth factor-1 (IGF-1) expression and subsequently downregulating phosphatidylinositide 3-kinase (PI3K)/Akt signaling 3,4) . However, many aspects of the mechanisms by which HPDL cells differentiate into osteoblasts remain unclear.…”

Phosphoinositide 3-Kinase (PI3K) Activation is Differentially Regulated during Osteogenesis induced by TGF-β1 and BMP-2/BMP-7

“…Exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-1 administration. Thus, IGF-1 plays a central role of osteoblast differentiation 13) . BMP-2, an agent that enhances osteoblast differentiation and function, increases IGF-1 synthesis in osteoblasts 26) .…”

“…TGF-1 increases mRNA levels of osteoblast differentiation markers and alkaline phosphatase (ALP) activity in murine bone marrow stromal cells 11) . However, TGF-1 also blocks osteogenesis by various mechanisms depending on its concentration, the cell density, and differentiation stage of the cells 13) . Some studies reported that TGF-1 has biphasic and concentration-dependent effects on osteoblast differentiation [14][15][16] .…”