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

Examination of the Relationship between Oral Health and Arterial Sclerosis without Genetic Confounding through the Study of Older Japanese Twins

<div><p>Objective</p><p>Although researchers have recently demonstrated a relationship between oral health and arterial sclerosis, the genetic contribution to this relationship has been ignored even though genetic factors are expected to have some effect on various diseases. The aim of this study was to evaluate oral health as a significant risk factor related to arterial sclerosis after eliminating genetic confounding through study of older Japanese twins.</p><p>Subjects and Methods</p><p>Medical and dental surveys were conducted individually for 106 Japanese twin pairs over the age of 50 years. Maximal carotid intima-media thickness (IMT-Cmax) was measured as a surrogate marker of arterial sclerosis. IMT-Cmax > 1.0 mm was diagnosed as arterial sclerosis. All of the twins were examined for the number of remaining teeth, masticatory performance, and periodontal status. We evaluated each measurement related with IMT-Cmax and arterial sclerosis using generalized estimating equations analysis adjusted for potential risk factors. For non-smoking monozygotic twins, a regression analysis using a “between within” model was conducted to evaluate the relationship between IMT-Cmax and the number of teeth as the environmental factor controlling genetic and familial confounding.</p><p>Results</p><p>We examined 91 monozygotic and 15 dizygotic twin pairs (males: 42, females: 64) with a mean (± standard deviation) age of 67.4 ± 10.0 years. Out of all of the oral health-related measurements collected, only the number of teeth was significantly related to arterial sclerosis (odds ratio: 0.72, 95% confidence interval: 0.52-0.99 per five teeth). Regression analysis showed a significant association between the IMT-Cmax and the number of teeth as an environmental factor (<i>p</i> = 0.037).</p><p>Conclusions</p><p>Analysis of monozygotic twins older than 50 years of age showed that having fewer teeth could be a significant environmental factor related to arterial sclerosis, even after controlling for genetic and familial confounding.</p></div

Histological analysis.

<p>Histological analysis of alveolar bone between the first molar and the second molar with or without a ligature. Decalcified periodontal tissue sections were prepared and stained with hematoxylin and eosin solution. Representative tissue sections are shown.</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

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 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

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