Fig 1.
TGF-β signaling pathways and induction of EMT-related genes.
Binding of TGF-β to the TGF-β receptor induces activation of SMAD2/3 and p38 MAPK via phosphorylation. The SMAD2/3 pathway directly activates EMT-related transcription factors including SNAIL1, whereas the p38 MAPK pathway activates these factors via AP-1. EMT related genes are then up- or downregulated.
Table 1.
Primers used in this study.
Fig 2.
Identification of AM251 as an EMT suppressing compound.
(A) HK-2 cells were incubated with 2 ng/ml TGF-β1 and individual compounds from the SCREEN-WELL® Bioactive lipid library (each at a 1:100 dilution of the original library) for 24 h. Cells were lysed and subjected to real-time RT-PCR to quantitate COL1A1 and PPIA mRNA levels as indicators of effects on EMT and cytotoxicity, respectively. Compounds causing ≥4-fold decreases in PPIA levels compared with control (no compound, +TGF-β1; gray circles) were eliminated from evaluation because of their toxicity (black circles). Compounds that decreased the ratio of expression levels of COL1A1 to PPIA to ≤50% compared with control (no compound, +TGF-β1; gray circles) were selected as hit compounds (red circles). (B–D) HK-2 (B) and RPTEC (C and D) cells were incubated with 2 ng/ml TGF-β1 and individual hit compounds (1-stearoyl-2-arachidonoyl-glycerol (SAG) at 20 μM; docosatrienoic acid (DTA) at 20 μM; carbaprostacyclin (CA) at 10 μM; ciglitazone (CI) at 10 μM; 24,25-dihydroxyvitamin D3 (DHVD3) at 20 μM; N-palmitoyl dopamine (PALDA) at 10 μM; N-arachidonoyl dopamine (NADA) at 10 μM; N-oleoyl dopamine (ODA) at 10 μM; or AM251 at 10 μM) for 24 h and then subjected to real-time RT-PCR to quantitate COL1A1 and PPIA mRNA levels. Values are means ± SD of the ratio of COL1A1 to PPIA mRNA levels, expressed relative to the ratio in the control (no treatment) (B and C) or PPIA mRNA levels relative to the control (no compound + TGF-β1) (D). Data were from three independent experiments. Statistically significant differences from the control (no compound +TGF-β1) are indicated (** P < 0.01, Student’s t-test). (E) Structure of AM251.
Fig 3.
AM251 reverses TGF-β1-dependent decreases in E-cadherin expression.
(A–C) RPTEC cells were incubated with 2 ng/ml TGF-β1 and/or 10 μM AM251, as indicated, for 24 h. (A) Total protein lysates were prepared and equal amounts of proteins (5 μg per sample) were separated by SDS-PAGE, followed by immunoblotting with an anti-E-cadherin antibody or an anti-GAPDH antibody as a loading control. (B) The results from (A) were quantified. Values are means ± SD of E-cadherin protein levels relative to those in cells with no treatment (TGF-β1(−) AM251(−)), from three independent experiments. (C) Total RNA was prepared and subjected to real-time RT-PCR to measure E-cadherin (CDH1) and PPIA mRNA levels. Values are means ± SD of the ratio of CDH1 to PPIA mRNA levels, expressed relative to the ratio in cells with no treatment (TGF-β1(−) AM251(−)), from three independent experiments. Statistically significant differences are indicated (* P < 0.05, ** P < 0.01, Student’s t-test).
Fig 4.
AM251 suppresses COL1A1 expression pre-induced by TGF-β1.
(A and B) RPTEC cells were cultured in REGM medium containing 2 ng/ml TGF-β1 and/or 10 μM AM251, as indicated, for 24, 48, 72, or 96 h. Total RNA was prepared and subjected to real-time RT-PCR to measure COL1A1 and PPIA mRNAs. Values are means ± SD of the ratio of COL1A1 to PPIA mRNA levels, expressed relative to the ratio in the control (no treatment) (A) or PPIA mRNA levels relative to the control (no treatment) (B), from three independent experiments. Statistically significant differences from the control (A, without AM251; B, no treatment) are indicated (** P < 0.01, Student’s t-test). (C and D) RPTEC cells were incubated with 2 ng/ml TGF-β1 for 96 h in REGM medium containing REGM Single Quots and incubated with 2 ng/ml TGF-β1 and/or 10 μM AM251, as indicated, for the following 24 h in REGM medium. Total RNA was prepared and subjected to real-time RT-PCR to measure COL1A1 and PPIA. Relative mRNA levels of COL1A1 (C) and PPIA (D) were determined as described for (A) and (B), respectively.
Fig 5.
CB1 is not involved in EMT suppression in RPTEC cells.
(A) RPTEC cells were incubated with 2 ng/ml TGF-β1 for 24 h. Total RNA was prepared and subjected to real-time RT-PCR to measure CB1 (CNR1) and PPIA mRNA levels. Values are means ± SD of the ratio of CNR1 to PPIA mRNA levels, expressed relative to the ratio in the control, from three independent experiments. (B) RPTEC cells were incubated with 2 ng/ml TGF-β1 and the CB1 agonist anandamide (ANA) at the indicated concentrations for 24 h. Relative mRNA levels of COL1A1 were determined as for (A). (C) RPTEC cells were treated with control siRNA or a selective siRNA for CNR1 (siCB1-1 or siCB1-2) for 72 h. Relative mRNA levels of CNR1 were determined as for (A). (D) RPTEC cells were treated with control siRNA or selective siRNA for CNR1 (siCB1-1 or siCB1-2) for 48 h and then incubated with 2 ng/ml TGF-β1 and 10 μM AM251 for another 24 h. Relative mRNA levels of COL1A1 were determined as for (A). Statistically significant differences are indicated (** P < 0.01, Student’s t-test).
Fig 6.
GPR55 is not involved in EMT suppression in RPTEC cells.
(A) RPTEC cells were incubated with 2 ng/ml TGF-β1 for 24 h. Total RNA was prepared and subjected to real-time RT-PCR to measure GRP55 and PPIA mRNA levels. Values are means ± SD of the ratio of GRP55 to PPIA mRNA levels, expressed relative to the ratio in the control (no treatment), from three independent experiments. (B) RPTEC cells were incubated with 2 ng/ml TGF-β1 in the presence or absence of the GRP55 antagonist CID16020046 (CID; Tocris Bioscience, Minneapolis, MN, USA) at the indicated concentrations and 10 μM AM251 for 24 h. Relative mRNA levels of COL1A1 were determined as for (A). Statistically significant differences are indicated (* P < 0.05, ** P < 0.01, Student’s t-test).
Fig 7.
AM251 inhibits EMT with specificity.
(A–C) RPTEC cells were incubated with or without 2 ng/ml TGF-β1 (TGF) in the presence or absence of 10 μM AM251 (AM) for 24 h. Total RNA was isolated from three independent samples, pooled, and subjected to microarray analyses. (A) Comparison of the microarray data was conducted on the data from AM251(−) TGF-β1(−) versus those from AM251(−) TGF-β1(+) conditions (red circles); and from AM251(+) TGF-β1(−) versus those from AM251(+) TGF-β1(+) conditions (blue circles). Numbers of upregulated (≥2-fold; left panels) and downregulated (≥2-fold; right panels) genes are depicted as Venn diagrams. (B) Hierarchical clustering analyses were performed using genes upregulated by ≥2-fold upon treatment with TGF-β1 by the centroid distance method. Yellow bars indicate that the values are similar to the averages of four different conditions. Red and blue bars indicate that the values are higher and lower than the averages, respectively, with the color strengths representing the degrees of the values. (C) Enrichment pathway analyses were conducted using genes with ≥2-fold change upon treatment with TGF-β1 and the MetaCore gene regulatory network database. The top 10 pathways are shown. PI3K, phosphoinositide 3-kinase; ILK, integrin-linked kinase; IGF, insulin-like growth factor; TNFα, tumor necrosis factor α; MAG, myelin-associated glycoprotein.
Fig 8.
AM251 reverses TGF-β1-induced changes in expression of EMT-related genes.
RPTEC cells were incubated with or without 2 ng/ml TGF-β1 in the presence or absence of 10 μM AM251 for 24 h. Total RNA was isolated from three independent samples, pooled, and subjected to microarray analyses. Values represent gene expression changes upon treatment with TGF-β1. N.D., not detected.
Fig 9.
AM251 suppresses induction of SNAILs, JUNB, FOSB, TGFR1, and TGFBs.
RPTEC cells were incubated with or without 2 ng/ml TGF-β1 in the presence or absence of 10 μM AM251 for 24 h. Total RNA was prepared and subjected to real-time RT-PCR to measure PPIA and SNAIL1 (A), SNAIL2 (B), JUNB (C), FOSB (D), TGFBR1 (E), TGFB2 (F), and TGFB3 (G) mRNA levels. Values are means ± SD of the ratio of each gene to PPIA mRNA levels, expressed relative to the ratio in the control (no treatment), from three independent experiments. Statistically significant differences are indicated (* P < 0.05, ** P < 0.01, Student’s t-test).
Fig 10.
AM251 inhibits the SMAD2/3 and p38 MAPK signaling pathways.
(A) RPTEC cells were incubated with or without 2 ng/ml TGF-β1 in the presence or absence of 10 μM AM251 for 24 h. (A) Total RNA was isolated from three independent samples, pooled, and subjected to microarray analyses. Values for SMAD2, SMAD3, MAPK11 (p38β), MAPK12 (p38γ), MAPK13 (p38δ), and MAPK14 (p38) represent their gene expression changes in cells treated with TGF-β1. (B–D) RPTEC cells were incubated with or without 2 ng/ml TGF-β1 in the presence or absence of 10 μM AM251 for 1 h. (B) Total protein lysates were prepared, and equal amounts of protein (5 μg per sample) were separated by SDS-PAGE, followed by immunoblotting with anti-phopho-p38 (P-p38), anti-p38, anti-phospho-SMAD3 (P-SMAD3), or anti-SMAD3 antibodies. (C and D) The results from (B) were quantified. Values are means ± SD of phopho-p38 (C) or phospho-SMAD3 (D) levels relative to those in cells with no treatment (TGF-β1(−) AM251(−)), from three independent experiments. Statistically significant differences are indicated (** P < 0.01, Student’s t-test).