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

Overview of the experimental design for induction of periodontitis by ligature (Experimental design 1).

<p>Mice received daily intraperitoneal injections of PBS, CSC or nicotine for 3 consecutive days (days −3 to −1). Mice were examined on day 7 after placement of the ligature.</p

Micro-CT analysis.

<p>(A) The maxillae were scanned by micro-computed tomography. (B) Measurement of total bone loss. Bar graphs represent the mean values ± SD (n = 3). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS-treated. Dashed line means baseline (unligated side in PBS treatment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155594#pone.0155594.g003" target="_blank">Fig 3B</a>).</p

Schematic illustration of the measurement of the distance between the cemento-enamel junction (CEJ) and the alveolar bone crest (ABC).

<p>Distance from the CEJ to the ABC was measured at four points indicated on the first molar to the third molar.</p

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

Overview of experimental design for alveolar bone repair after ligature removal (Experimental design 2).

<p>Mice were given daily intraperitoneal injections of PBS, CSC or nicotine for 3 consecutive days (days −3 to −1). Some of the mice were examined on day 7 after placement of the ligature, and some of the mice were examined 10 days after ligature removal. Three mice with nicotine-treated were given additional intraperitoneal injection of nicotine for 3 consecutive days (days 8–10) and examined 10 days after ligature removal.</p

TRAP staining.

<p>(A) Detection of osteoclasts in tissue sections by tartrate-resistance acid phosphate (TRAP) staining. (B) Quantification of TRAP-positive osteoclasts. The mean of number of positive cells was determined from five serial sections per mouse. Data are represented as mean ± SD (n = 7). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS-treated.</p

RANKL expression in submandibular lymph nodes.

<p><i>Rankl</i> mRNA expression in submandibular lymph nodes was assessed by real-time PCR. Quantitative mRNA values were normalized to the amount of <i>Gapdh</i>. Bar graphs represent the mean values ± SD (n = 7). * <i>p</i> < 0.01, vs. without ligation. ** <i>p</i> < 0.01, vs. PBS treated. Lower panels are images of submandibular lymph nodes. Representative lymph nodes are shown.</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