AM251 Suppresses Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells

Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the causative mechanisms of kidney fibrosis. In our study, we screened lipophilic compounds using a lipid library including approximately 200 lipids to identify those that suppressed EMT induced by a transforming growth factor (TGF)-beta 1 stimulus. Initial screening was performed with the immortalized HK-2 renal tubule epithelial cell line. The most promising compounds were further tested in RPTEC primary renal tubule epithelial cells. We found that the synthetic lipid AM251 suppressed two hallmark events associated with EMT, the upregulation of collagen 1A1 (COL1A1) and downregulation of E-cadherin. Though AM251 is known to act as an antagonist for the cannabinoid receptor type 1 (CB1) and an agonist for the G protein-coupled receptor 55 (GRP55), the suppression of EMT by AM251 was not mediated through either receptor. Microarray analyses revealed that AM251 inhibited induction of several EMT transcription factors such as SNAIL1, which is the key inducer of EMT, and the AP-1 transcription factors FOSB and JUNB. Activation of SMAD2/3 and p38 mitogen-activated protein kinase (MAPK) was inhibited by AM251, with greater inhibition of the latter, indicating that AM251 acted upstream of SMAD/p38 MAPK in the TGF-beta signaling pathway. Our findings regarding the effects of AM251 on the TGF-beta signaling pathway may inform development of a novel therapeutic agent suppressing EMT, thus preventing kidney fibrosis

AM251 reverses TGF-β1-dependent decreases in E-cadherin expression.

<p>(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 (<i>CDH1</i>) and <i>PPIA</i> mRNA levels. Values are means ± SD of the ratio of <i>CDH1</i> to <i>PPIA</i> mRNA levels, expressed relative to the ratio in cells with no treatment (TGF-β1(−) AM251(−)), from three independent experiments. Statistically significant differences are indicated (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, Student’s <i>t</i>-test).</p

AM251 suppresses induction of <i>SNAILs</i>, <i>JUNB</i>, <i>FOSB</i>, <i>TGFR1</i>, and <i>TGFBs</i>.

<p>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 <i>PPIA</i> and <i>SNAIL1</i> (A), <i>SNAIL2</i> (B), <i>JUNB</i> (C), <i>FOSB</i> (D), <i>TGFBR1</i> (E), <i>TGFB2</i> (F), and <i>TGFB3</i> (G) mRNA levels. Values are means ± SD of the ratio of each gene to <i>PPIA</i> mRNA levels, expressed relative to the ratio in the control (no treatment), from three independent experiments. Statistically significant differences are indicated (* <i>P</i> < 0.05, ** <i>P</i> < 0.01, Student’s <i>t</i>-test).</p

AM251 reverses TGF-β1-induced changes in expression of EMT-related genes.

<p>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.</p

TGF-β signaling pathways and induction of EMT-related genes.

<p>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.</p

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

GPR55 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 <i>GRP55</i> and <i>PPIA</i> mRNA levels. Values are means ± SD of the ratio of <i>GRP55</i> to <i>PPIA</i> 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 <i>COL1A1</i> were determined as for (A). Statistically significant differences are indicated (* <i>P</i> < 0.05, ** <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