TGF-Beta Negatively Regulates the BMP2-Dependent Early Commitment of Periodontal Ligament Cells into Hard Tissue Forming Cells

<div><p>Transforming growth factor beta (TGF-β) is a multi-functional growth factor expressed in many tissues and organs. Genetic animal models have revealed the critical functions of TGF-β in craniofacial development, including the teeth and periodontal tissue. However, the physiological function of TGF-β in the periodontal ligament (PDL) has not been fully elucidated. In this study, we examined the roles of TGF-β in the cytodifferentiation of PDL cells using a TGF-β receptor kinase inhibitor, SB431542. Mouse PDL cell clones (MPDL22) were cultured in calcification-inducing medium with or without SB431542 in the presence or absence of various growth factors, such as bone morphogenetic protein (BMP)-2, TGF-β and fibroblast growth factor (FGF)-2. SB431542 dramatically enhanced the BMP-2-dependent calcification of MPDL22 cells and accelerated the expression of ossification genes <i>alkaline phosphatase</i> (<i>ALPase</i>) and <i>Runt-related transcription factor</i> (<i>Runx</i>) <i>2</i> during early osteoblastic differentiation. SB431542 did not promote MPDL22 calcification without BMP-2 stimulation. The cell growth rate and collagen synthesis during the late stage of MPDL22 culture were retarded by SB431542. Quantitative reverse transcription polymerase chain reaction analysis revealed that the expressions of <i>Smurf1</i> and <i>Smad6</i>, which are negative feedback components in the TGF-β/BMP signaling pathway, were downregulated in MPDL22 cells with SB431542 treatment. These results suggest that an endogenous signal from TGF-β negatively regulates the early commitment and cytodifferentiation of PDL cells into hard tissue-forming cells. A synthetic drug that regulates endogenous TGF-β signals may be efficacious for developing periodontal regenerative therapies.</p></div

SB431542 treatment on ALP activity and expression of osteoblastic differentiation-related genes in MPDL22 cells.

<p>(A) MPDL22 cells were cultured in mineralization-inducing medium in the presence or absence of BMP-2 (50 ng/mL), TGF-β (4 ng/mL) and SB431542 (10 μM). MPDL22 cells were harvested at the indicated time points. ALPase activity was determined as described in the methods section. Activity in U/mg protein for the cell lysates is shown. **: p<0.01 vs BMP-2. (-): control; B: BMP-2; T: TGF-β; SB: SB431542. (B) Relative quantification of <i>ALP</i>, <i>Runx2</i>, <i>Osterix</i> and <i>BSP</i> mRNA expression levels was performed after 4 and 6 days of MPDL22 cell culture in the mineralization inducing medium with or without BMP-2 (50 ng/mL) and SB431542 (10 μM). D: AA plus β-GP; B: BMP-2; SB: SB431542.**: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.</p

Effects of BMP-2 and SB431542 on collagen synthesis during osteoblastic differentiation of MPDL22 cells.

<p>(A) SB431542 (10 µM) was added to MPDL22 cells during osteogenic differentiation with or without BMP-2 (50 ng/mL) at different times as indicated in the left panel. The right panel shows the van Gieson staining, which stains collagen pink. (B) The relative quantification of <i>Col1A1</i> mRNA in BMP-2-induced MPDL22 cells was assessed during osteogenic differentiation in the presence or absence of SB431542 (10 μM). MPDL22 cells were harvested every 3 days and isolated mRNA was assessed by RT-qPCR. Quantitative mRNA values were normalized to the amount of <i>GAPDH</i> mRNA. B: BMP-2; SB: SB431542, **: p<0.01 vs BMP-2. (C) The protein synthesis of collagen I in MPDL22 cells was examined by western blotting. The culture supernatants were aspirated at the indicated time points from MPDL22 cells treated by BMP-2 (50 ng/mL) and TGF-β (4 ng/mL) in the presence or absence of SB431542 (10 μM) in long-term cultures. SB: SB431542.</p

Effects of SB431542 on mineralized nodule formation by MPDL22 cells.

<p>(A) Osteogenic differentiation of MPDL22 cells was induced by culture in mineralization inducing medium with or without BMP-2 (50 ng/mL), FGF-2 (50 ng/mL) and PDGF-BB (20 ng/mL) in the presence or absence of TGF-β (4 ng/mL) and SB431542 (10 μM). Calcified nodule formation was determined at day 12 by Alizarin red staining. (B) Quantification of calcified nodule formation by MPDL22 cells induced by BMP-2 in the presence or absence of AA (50 mg/mL) plus β-GP (50 mM), BMP-2 (50 ng/mL) and SB431542 (10 μM). Densitometric analysis was applied to the scanned culture plate images at day 12. Positive scores were calculated by multiplying the stained area by its Alizarin red staining color density. B: BMP-2; SB: SB431542. **: p<0.01 vs BMP-2. (C) The effects of various concentrations of SB431542 on the mineralized nodule formation by MPDL22 cells. **: p<0.01 vs BMP-2. Quantification of the calcified nodule formation by BMP-2-stimulated MPDL22 cells in the presence of β-GP (50 mM) plus AA (50 mg/mL) with or without SB431542 (0.1, 1.0, and 10 μM). (D) The relative quantification of <i>ALP</i>, <i>Runx2</i>, and <i>Osterix</i> mRNAs during osteogenic differentiation of MPDL22 cells by BMP-2 (50 ng/mL) after treatment with or without SB431542 (10 μM) for 2 days. MPDL22 cells were harvested at days 6 and 12 and the isolated mRNA was assessed by RT-qPCR. Quantitative mRNA values were normalized to the amount of <i>GAPDH</i> mRNA. **: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.</p

Expression of TGF-β/BMP receptor and Smads in MPDL22 cells.

<p>(A) Semiquantitative RT-PCR analysis of the expression of TGF-β receptor genes <i>ALK-1</i>, <i>-4</i>, <i>-5</i>, <i>-7</i>, and <i>TβRII</i>, and BMP receptor genes <i>ALK-2</i>, <i>-3</i>, <i>-6</i>, <i>BMPR2</i>, and <i>Smad1–7</i>. Human glycerralaldehyde-3-phosphate dehydrogenase (<i>GAPDH</i>) was used as an internal control. (B) Western blotting analysis of TGF-β/BMP receptor induced by TGF-β (4 ng/mL) or BMP-2 (50 ng/mL) in the presence or absence of SB431542 (10 μM). Protein levels of TGF-β receptor I (TGF-βRI), TGF-β receptor II (TGF-βRII), BMP receptor I (BMPRI), and BMP receptor II (BMPRII) were measured. β-actin was used as a protein loading control. Quantitative analysis is shown as the relative ratios of TGF-β or BMP receptors I/II and β-actin by densitometric analysis. Values represent the mean ± SD of 3 independent assays. (-): control; B: BMP-2; T: TGF-β. (C) The relative quantification of <i>TGF-β1</i>, <i>TGF-β2</i>, and <i>TGF-β3</i> mRNAs in MPDL22 cells by RT-qPCR. Quantitative mRNA values were normalized to the amount of <i>GAPDH</i> mRNA. (D) TGF-β production from MPDL22 cells. Protein expression levels of TGF-β were examined by ELISA. Culture supernatants of MPDL22 cells were aspirated after 24 h of culture with or without BMP-2 (50 ng/mL) and SB431542 (10 μM). B: BMP-2; SB: SB431542. **: p<0.01 vs the BMP-2 stimulated group.</p

Effects of SB431542 on the TGF-β/Smad transcriptional responses in MPDL22 cells.

<p>(A) Activation of Smad3, Erk, and p38 induced by TGF-β (4 ng/mL) with or without pretreatment with SB431542 (10 μM). Phosphorylation levels and protein levels were determined by western blotting. (B) Promoter activity of TGF-β responsive gene <i>PAI-1</i>. MPDL22 cells were transfected with (<i>CAGA</i>)₁₂-Luc reporter plasmid as indicated. Twenty-four hours after transfection, cells were treated with TGF-β (4 ng/mL), SB431542 (10 μM) or both overnight. (-): control; B: BMP-2; T: TGF-β; SB: SB431542. **: p<0.01 vs the TGF-β stimulated group.</p