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. 2016 Jun 21;7(25):38681-38692.
doi: 10.18632/oncotarget.9583.

EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells

Jin-Ling Ye  1 , Chun-Qi Gao  1 , Xiang-Guang Li  1 , Cheng-Long Jin  1 , Dan Wang  1 , Gang Shu  1 , Wen-Ce Wang  1 , Xiang-Feng Kong  2 , Kang Yao  2 , Hui-Chao Yan  1 , Xiu-Qi Wang  1
Affiliations

EAAT3 promotes amino acid transport and proliferation of porcine intestinal epithelial cells

Jin-Ling Ye et al. Oncotarget. .

Abstract

Excitatory amino acid transporter 3 (EAAT3, encoded by SLC1A1) is an epithelial type high-affinity anionic amino acid transporter, and glutamate is the major oxidative fuel for intestinal epithelial cells. This study investigated the effects of EAAT3 on amino acid transport and cell proliferation through activation of the mammalian target of the rapamycin (mTOR) pathway in porcine jejunal epithelial cells (IPEC-J2). Anionic amino acid and cystine (Cys) transport were increased (P<0.05) by EAAT3 overexpression and decreased (P<0.05) by EAAT3 knockdown rather than other amino acids. MTT and cell counting assays suggested that IPEC-J2 cell proliferation increased (P<0.05) with EAAT3 overexpression. Phosphorylation of mTOR (Ser2448), ribosomal protein S6 kinase-1 (S6K1, Thr389) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1, Thr70) was increased by EAAT3 overexpression and decreased by EAAT3 knockdown (P<0.05), as were levels of activating transcription factor 4 (ATF4) and cystine/glutamate antiporter (xCT) (P<0.05). Our results demonstrate for the first time that EAAT3 facilitates anionic amino acid transport and activates the mTOR pathway, promoting Cys transport and IPEC-J2 cell proliferation.

Keywords: amino acid; excitatory amino acid transporter 3; intestinal epithelial cells; mammalian target of rapamycin; proliferation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Construction of the recombinant plasmid, EAAT3-pcDNA3.1+
Cloning of the pig EAAT3 cDNA A. Identification of the recombinant plasmids EAAT3-pGEM-T B. and EAAT3-pcDNA3.1+ C. M: DNA Marker 2000; M1: DNA Marker 10000; Lanes 1, 2: PCR products of jejunal excitatory amino acid transporter 3 (EAAT3); Lanes 3, 4, 5: Double enzyme digestions of EAAT3-pGEM-T by EcoRI and XhoI; Lanes 6, 7: Single enzyme digestions of EAAT3-pcDNA3.1+ by EcoRI and XhoI, respectively; Lane 8: Double enzyme digestions of EAAT3-pcDNA3.1+ by EcoRI and XhoI.
Figure 2
Figure 2. EAAT3 overexpression in IPEC-J2 cells
Representative immunofluorescence images of Control and Overexpression cells at 48 h after seeding, labeled with DAPI (blue) and EAAT3 antibody (red) A. Scale bars: 100 μm. Control: control group; Overexpression: EAAT3 overexpression group. EAAT3 mRNA abundance (n=6) B. and protein level (n=3) C. Representative results of three independent experiments are shown as means ± SEM; *P<0.05.
Figure 3
Figure 3. siRNA Representative fluorescence images of IPEC-J2 cells 48 h post-transfection with the transfection control (Cy3)-siRNA
IPEC-J2 cells under normal light after transfection with the control (Cy3)-siRNA (100× A. and 200× C.). Corresponding fluorescence photos of IPEC-J2 cells after transfection with the control (Cy3)-siRNA (100× B. and 200× D.). Scale bars: 100 μm. Representative results of the three independent experiments are shown.
Figure 4
Figure 4. Screening for the optimal EAAT3 siRNA and interference time in IPEC-J2 cells
The effects of three siRNAs on EAAT3 mRNA abundance were measured by real-time PCR 48 h post-transfection A. Blank: blank control group; NC: negative control group; siRNA-001: EAAT3-siRNA-001 group; siRNA-002: EAAT3-siRNA-002 group; siRNA-003: EAAT3-siRNA-003 group. The effects of siRNA-003 transfection time on EAAT3 mRNA abundance were measured by real-time PCR B. Data are expressed as means ± SEM (n=6). Different letters indicate significant differences (P<0.05). siRNA-003 treatment reduced EAAT3 protein levels as compared to the Negative controls C. Negative control: negative control group; Knockdown: EAAT3-siRNA-003 group. Representative results of three independent experiments are shown. Data are expressed as means ± SEM (n=3); *P<0.05.
Figure 5
Figure 5. Effects of EAAT3 overexpression A. and knockdown B. on amino acid transport in IPEC-J2 cells
Control: control group; Overexpression: EAAT3 overexpression group. Negative control: negative control group; Knockdown: EAAT3-siRNA-003 group. Glu: glutamate; Asp: aspartate; Cys: cystine. Representative results of three independent experiments are shown. Data are expressed as means ± SEM (n=6); *P<0.05.
Figure 6
Figure 6. EAAT3 overexpression increased IPEC-J2 cell proliferation
The Overexpression group OD values and cell numbers were higher than those of the Control group at 72, 96, and 120 h after seeding, as assessed by MTT (n=20) A. and cell counting assay (n=6) B. respectively. Control: control group; Overexpression: EAAT3 overexpression group. Representative results of three independent experiments are shown. Data are expressed as means ± SEM; *P<0.05.
Figure 7
Figure 7. Effects of EAAT3 overexpression A–B. and knockdown C–D. on mTOR pathway related proteins in IPEC-J2 cells
Control: control group; Overexpression: EAAT3 overexpression group. Negative control: negative control group; Knockdown: EAAT3-siRNA-003 group. Representative results of three independent experiments are shown. Data are expressed as means ± SEM (n=3); *P<0.05.
Figure 8
Figure 8. xCT mRNA abundance (n=6) and protein level (n=3) and ATF4 protein level (n=3) after EAAT3 overexpression A–B. or knockdown C–D. as measured by real-time PCR and western blotting
Control: control group; Overexpression: EAAT3 overexpression group. Negative control: negative control group; Knockdown: EAAT3-siRNA-003 group. ATF4: activating transcription factor 4; xCT: cystine/glutamate antiporter. Representative results of three independent experiments are shown. Data are expressed as means ± SEM; *P<0.05.
Figure 9
Figure 9. mTOR pathway activation by EAAT3
EAAT3 facilitates Glu transport and activates the mTOR pathway. EAAT3 triggers mTOR pathway activation, which promotes ATF4-mediated xCT expression and might accelerate Cys transport. EAAT3: excitatory amino acid transporter 3; Glu: glutamate; Cys: cystine; mTOR: mammalian target of rapamycin; ATF4: activating transcription factor 4; xCT: cystine/glutamate antiporter.
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