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 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

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

A Comparative Analysis of Glomerulus Development in the Pronephros of Medaka and Zebrafish

<div><p>The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.</p> </div

Morphological types of pronephric glomeruli in teleost fishes.

*<p>Lebistes reticulatus was adopted as scientific name of guppy in Agarwal and John (1988)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045286#pone.0045286-Agarwal1" target="_blank">[⁴⁶]</a>.</p

Glomerulus development in medaka and zebrafish pronephros in comparison to mouse and rat metanephros.

<p>A1-B5: Pronephric glomerulus development in 3 to 6 dpf medaka (A1-A5) and 34 hpf to 4 dpf zebrafish (B1-B5) by JB4 cross sections stained by hematoxylin and eosin. The glomerular primordium of medaka exhibited a C-shaped epithelial layer (A2, A3), which is similar to the mouse and rat S-shaped body stage (C3, D3), unlike in zebrafish (B2). The C-shaped primordium contained a characteristic balloon-like or sinusoidal capillary. The paired pronephric glomerulus was fused at the midline to form a glomerulus in zebrafish (B3), but remained separated into two parts by the interposition of an interglomerular mesangium in medaka (arrowheads in A2-A5). C1-D5: Metanephric glomerulus development in rat epoxy resin sections stained by toluidine blue (C1-C5) and in mouse E18.5 kidney sections stained with H&E (D1-D5). Cross section of rat and mouse metanephros shows renal vesicle (C1, D1), comma-shaped body (C2, D2), S-shaped body at (C3, D3), capillary loop at (C4, D4), and maturing glomerulus (C5, D5). Rat vascular cleft in C2 (arrowhead) and primitive podocyte layer in C3 (asterisk). Mouse vascular cleft in D2 and D3 (arrowheads). Scale bars = 10 µm.</p

Transmission electron microscopy of podocytes development in medaka pronephric glomerulus.

<p>A-E: The ultrastructure of pronephric glomerulus cross sections. A: At 4.5 dpf, podocytes form a single columnar epithelium and are connected via intercellular junctions (arrowheads), which are located in the middle of the cell height. B: The intercellular junctions of podocytes are located in the vicinity of the GBM (arrows) at 5 dpf. Podocytes interdigitate at the cell periphery to form irregularly-shaped processes (arrowheads). C: The podocyte cell body is detached from the GBM to form a subpodocyte space (asterisks) by 6 dpf. The irregularly-shaped processes become flattened (arrowheads); however, foot processes with silt diaphragms have not formed yet. D: Foot processes connected by slit diaphragm are formed in some capillary walls (arrowheads) at 7 dpf. E: Foot processes are found in the most capillary walls (arrowheads) at 10 dpf. CL, capillary lumen; P, podocyte cell body: PE. Scale bars = 1 µm.</p