AM251 suppresses <i>COL1A1</i> expression pre-induced by TGF-β1.

<p>(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 <i>COL1A1</i> and <i>PPIA</i> mRNAs. Values are means ± SD of the ratio of <i>COL1A1</i> to <i>PPIA</i> mRNA levels, expressed relative to the ratio in the control (no treatment) (A) or <i>PPIA</i> 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 (** <i>P</i> < 0.01, Student’s <i>t</i>-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 <i>COL1A1</i> and <i>PPIA</i>. Relative mRNA levels of <i>COL1A1</i> (C) and <i>PPIA</i> (D) were determined as described for (A) and (B), respectively.</p

AM251 inhibits the SMAD2/3 and p38 MAPK signaling pathways.

<p>(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 <i>SMAD2</i>, <i>SMAD3</i>, <i>MAPK11</i> (p38β), <i>MAPK12</i> (p38γ), <i>MAPK13</i> (p38δ), and <i>MAPK14</i> (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 (** <i>P</i> < 0.01, Student’s <i>t</i>-test).</p

CB1 is not involved in EMT suppression in RPTEC cells.

<p>(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 (<i>CNR1</i>) and <i>PPIA</i> mRNA levels. Values are means ± SD of the ratio of <i>CNR1</i> to <i>PPIA</i> 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 <i>COL1A1</i> were determined as for (A). (C) RPTEC cells were treated with control siRNA or a selective siRNA for <i>CNR1</i> (siCB1-1 or siCB1-2) for 72 h. Relative mRNA levels of <i>CNR1</i> were determined as for (A). (D) RPTEC cells were treated with control siRNA or selective siRNA for <i>CNR1</i> (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 <i>COL1A1</i> were determined as for (A). Statistically significant differences are indicated (** <i>P</i> < 0.01, Student’s <i>t</i>-test).</p

AM251 inhibits EMT with specificity.

<p>(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.</p

Generation of Novel Anti-MUC1 Monoclonal Antibodies with Designed Carbohydrate Specificities Using MUC1 Glycopeptide Library

Numerous anti-mucin 1 (anti-MUC1) antibodies that recognize <i>O</i>-glycan core structures have already been developed. However, most of them show low specificities toward <i>O</i>-glycan structures and/or low affinity toward a monovalent epitope. In this study, using an MUC1 glycopeptide library, we established two novel anti-MUC1 monoclonal antibodies (1B2 and 12D10) with designed carbohydrate specificities. Compared with previously reported anti-MUC1 antibodies, 1B2 and 12D10 showed quite different features regarding their specificities, affinities, and reactivity profiles to various cell lines. Both antibodies recognized specific <i>O</i>-glycan structures at the PDT*R motif (the asterisk represents an <i>O</i>-glycosylation site). 1B2 recognized <i>O</i>-glycans with an unsubstituted <i>O</i>-6 position of the GalNAc residue (Tn, T, and 23ST), whereas 12D10 recognized Neu5Ac at the same position (STn, 26ST, and dST). Neither of them bound to glycopeptides with core 2 <i>O</i>-glycans that have GlcNAc at the <i>O</i>-6 position of the GalNAc residue. Furthermore, 1B2 and 12D10 showed a strong binding to not only native MUC1 but also 20-mer glycopeptide with a monovalent epitope. These anti-MUC1 antibodies should thus become powerful tools for biological studies on MUC1 <i>O</i>-glycan structures. Furthermore, the strategy of using glycopeptide libraries should enable the development of novel antibodies with predesigned <i>O</i>-glycan specificities