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

Kawahara T, Yamashita M, Ikegami K, Nakamura T, Yanagita M, Yamada S, Kitamura M, Murakami S - PLoS ONE (2015)

Bottom Line: SB431542 did not promote MPDL22 calcification without BMP-2 stimulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan.

ABSTRACT
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 alkaline phosphatase (ALPase) and Runt-related transcription factor (Runx) 2 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 Smurf1 and Smad6, 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.

No MeSH data available.


Related in: MedlinePlus

Effects of SB431542 on mineralized nodule formation by MPDL22 cells.(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 ALP, Runx2, and Osterix 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 GAPDH mRNA. **: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.
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pone.0125590.g003: Effects of SB431542 on mineralized nodule formation by MPDL22 cells.(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 ALP, Runx2, and Osterix 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 GAPDH mRNA. **: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.

Mentions: Next, we examined the effects of SB431542 on the hard tissue-forming ability of MPDL22 cells. We previously reported that MPDL22 cells differentiated into hard tissue-forming cells, such as osteoblasts and cementoblasts, during long-term culture with ascorbic acid (AA) and β-glycerophosphate (β-GP) [17]. As shown in Fig 3A, BMP-2 induced calcified nodule formation in MPDL22 cells at day 12. In contrast, stimulation with TGF-β or FGF-2 inhibited the formation of calcified nodules induced by the mineralization-inducing medium (AA + β-GP). Although SB431542 alone slightly induced mineralization in MPDL22 cells in this system, SB431542 dramatically enhanced BMP-2-induced calcified nodule formation. This suggested that the inhibition of endogenous TGF-β function accelerated the calcified nodule formation by BMP-2. To confirm this, we evaluated the effects of SB431542 at different concentrations on MPDL22 cell calcification in the presence of BMP-2. As shown in Fig 3C, the addition of SB431542 up to 10 μM increased the calcified nodule formation in a dose-dependent manner. Density analysis of scanned images of Alizarin staining showed that 10 μM SB431542 treatment increased the BMP-2-dependent mineralized nodule formation by over 4-fold (Fig 3B and 3C). RT-qPCR analysis revealed that the BMP-2 dependent expression of ossification-related genes ALP, Runx2 and Osterix were increased at days 6 and 12 in the presence of SB431542. These results suggested that SB431542 stimulated the BMP-2-induced osteoblastic differentiation of MPDL22 cells (Fig 3D).


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

Kawahara T, Yamashita M, Ikegami K, Nakamura T, Yanagita M, Yamada S, Kitamura M, Murakami S - PLoS ONE (2015)

Effects of SB431542 on mineralized nodule formation by MPDL22 cells.(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 ALP, Runx2, and Osterix 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 GAPDH mRNA. **: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.
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pone.0125590.g003: Effects of SB431542 on mineralized nodule formation by MPDL22 cells.(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 ALP, Runx2, and Osterix 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 GAPDH mRNA. **: p<0.01 vs BMP-2; *: p<0.05 vs BMP-2.
Mentions: Next, we examined the effects of SB431542 on the hard tissue-forming ability of MPDL22 cells. We previously reported that MPDL22 cells differentiated into hard tissue-forming cells, such as osteoblasts and cementoblasts, during long-term culture with ascorbic acid (AA) and β-glycerophosphate (β-GP) [17]. As shown in Fig 3A, BMP-2 induced calcified nodule formation in MPDL22 cells at day 12. In contrast, stimulation with TGF-β or FGF-2 inhibited the formation of calcified nodules induced by the mineralization-inducing medium (AA + β-GP). Although SB431542 alone slightly induced mineralization in MPDL22 cells in this system, SB431542 dramatically enhanced BMP-2-induced calcified nodule formation. This suggested that the inhibition of endogenous TGF-β function accelerated the calcified nodule formation by BMP-2. To confirm this, we evaluated the effects of SB431542 at different concentrations on MPDL22 cell calcification in the presence of BMP-2. As shown in Fig 3C, the addition of SB431542 up to 10 μM increased the calcified nodule formation in a dose-dependent manner. Density analysis of scanned images of Alizarin staining showed that 10 μM SB431542 treatment increased the BMP-2-dependent mineralized nodule formation by over 4-fold (Fig 3B and 3C). RT-qPCR analysis revealed that the BMP-2 dependent expression of ossification-related genes ALP, Runx2 and Osterix were increased at days 6 and 12 in the presence of SB431542. These results suggested that SB431542 stimulated the BMP-2-induced osteoblastic differentiation of MPDL22 cells (Fig 3D).

Bottom Line: SB431542 did not promote MPDL22 calcification without BMP-2 stimulation.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita-Osaka 565-0871, Japan.

ABSTRACT
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 alkaline phosphatase (ALPase) and Runt-related transcription factor (Runx) 2 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 Smurf1 and Smad6, 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.

No MeSH data available.


Related in: MedlinePlus