NFκB-Mediated Expression of Phosphoinositide 3-Kinase δ Is Critical for Mesenchymal Transition in Retinal Pigment Epithelial Cells

Abstract

Epithelial mesenchymal transition (EMT) plays a vital role in a variety of human diseases including proliferative vitreoretinopathy (PVR), in which retinal pigment epithelial (RPE) cells play a key part. Transcriptomic analysis showed that the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was up-regulated in human RPE cells upon treatment with transforming growth factor (TGF)-β2, a multifunctional cytokine associated with clinical PVR. Stimulation of human RPE cells with TGF-β2 induced expression of p110δ (the catalytic subunit of PI3Kδ) and activation of NFκB/p65. CRISPR-Cas9-mediated depletion of p110δ or NFκB/p65 suppressed TGF-β2-induced fibronectin expression and activation of Akt as well as migration of these cells. Intriguingly, abrogating expression of NFκB/p65 also blocked TGF-β2-induced expression of p110δ, and luciferase reporter assay indicated that TGF-β2 induced NFκB/p65 binding to the promoter of the PIK3CD that encodes p110δ. These data reveal that NFκB/p65-mediated expression of PI3Kδ is essential in human RPE cells for TGF-β2-induced EMT, uncovering hindrance of TGF-β2-induced expression of p110δ as a novel approach to inhibit PVR.

Keywords: CRISPR/Cas9; EMT; NFκB/p65; PI3Kδ; PVR; RPE; TGF-β2.

Figure 1

RNA sequencing analysis of TGF-β2-induced major changes in human RPE cells. (A). Number of significantly altered genes in TGF-β2-treated ARPE-19 cells. Volcano plot: X-axis: indication of the protein difference multiple (take log2); Y-axis: corresponding −log10 (p value). Red points: significantly up-regulated proteins; green points: significantly down-regulated proteins; gray points: proteins without significant changes. Untreated cells were used as controls. (B). GO analysis of biological processes, cellular components and molecular functions of genes altered by TGF-β2 treatment. (C). KEGG enrichment of signaling pathways in TGF-β2-treated cells. Top 20 of up-regulated pathways.

Figure 2

TGF-β2-induced activation of Akt, expression of p110δ, nuclear translocation of NFκB/p65 as well as EMT. (A). ARPE-19 cells were treated with TGF-β2 (10 ng/mL) for the indicated time points, followed by immunoblotting analyses. (B). ARPE-19 cells treated with TGF-β2 (10 ng/mL) for 48 h were immunofluorescence stained with antibodies to NFκB/p65 (green). Blue: DAPI. Red line: the location of the co-localization. Scale bar: 10 μm. (C,D). Co-localization analysis of NFκB with nucleus was performed by Image J [40]. (E–I). Quantitation of Western blotting band intensity in A. The graphs are mean ± standard deviation (SD) of three independent experiments. ** p < 0.01, *** p < 0.001. (J–L). mRNA from ARPE-19 cells treated with TGF-β2 (10 ng/mL) for 48 h was extracted and subjected to reverse qPCR. The graphs are mean ± SD of 4 independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. ** p < 0.01, *** p < 0.001.

Figure 3

Depletion of NFκB/p65 attenuates TGF-β2-induced Akt activation, expression of p110δ as well as EMT. (A). Sanger DNA sequencing identification of CRISPR-Cas9-mediated genome editing. (B). Western blotting analysis identification of CRISPR-Cas9-mediated protein depletion using the indicated antibodies. Shown is a representative of at least 3 independent experiments. (C). Quantitation of Western blotting band intensity. The graphs are mean ± SD of three independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. (D,G). Serum-starved cells with LacZ or NF-κB/p65 sgRNA were treated with TGF-β2 (10 ng/mL) for 24 h or 48 h. Cell lysates were subjected to immunoblotting analysis using the indicated antibodies. Shown is a representative of at least 3 independent experiments. (E,F,H,I). Quantitation of Western blotting band intensity in (D,G). The bar graphs are mean ± SD of 3 independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. (J). ARPE-19 cells expressing SpCas9 with LacZ or RELA (encoding NF-κB/p65) sgRNA treated with TGF-β2 (10 ng/mL) for 48 h were stained with antibodies to fibronectin (rabbit) and pan-keratin (mouse), followed by incubation with fluorescently labeled secondary antibodies. The slides were mounted in mounting medium containing DAPI (blue). Red signals indicate protein expressions. Shown is a representative of three independent experiments. Scale bar: 20 μm. (K,L). Integrated density of the fluorescence was analyzed by Image J. The graphs are mean ± SD of 3 independent experiments. The data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Depletion of NFκB/p65 attenuates TGF-β2-induced Akt activation, p110δ expression and EMT. (A,B). ARPE-19 cells with lacZ-sgRNA/SpCas9 or PK2-sgRNA/SpCas9 were examined by Western blotting using indicated antibodies. Shown is a representative of at least 3 independent experiments (A); Quantitation of Western blotting band intensity in A (B). (C–G). Serum-starved ARPE-19 expressing SpCas9 with LacZ or PK2 sgRNA were treated with TGF-β2 (10 ng/mL) for 48 h. Their lysates were subjected to Western blot analyses using indicated antibodies. Shown is representative of at least 3 independent experiments (C,F). Quantitation of Western blotting band intensity in (C–G). (H). ARPE-19 cells expressing SpCas9 with LacZ or PK2 sgRNA treated with TGF-β2 (10 ng/mL) for 48 h were first stained with antibodies against fibronectin (rabbit), pan-keratin (mouse), N-cadherin (rabbit) and β-catenin (rabbit), followed by incubation with fluorescently labeled secondary antibodies and mounting of the slides in mounting medium containing DAPI (blue). Red signals indicate proteins expression. Shown is a representative of 3 independent experiments. Scale bar: 20 μm. (I–L). Integrated density of the fluorescence was analyzed by Image J. The bar graphs are mean ± SD of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

NFκB/p65 binding to the PIK3CD-2e promoter is required for TGF-β2-induced expression of p110δ in ARPE-19 cells. (A,B). After cells had attached to a 96-well plate, they were transfected overnight with p-GL3-R1 (or p-GL3 control) or pRL-RK using lipofectamine 3000. Cells were then treated with TGF-β2 (10 ng/mL) for the next 24 h. Firefly luciferase activity and Renilla luciferase activity were read in a TD-20/20 luminometer. Data were calculated as firefly luciferase activity/Renilla luciferase activity. The mean ± SD of 3 independent experiments is shown. Data were analyzed using one-way ANOVA followed by the Tukey HSD post hoc test. * Denotes p < 0.05. (C). NFκB/p65 binding to the PIK3CD-2e promoter determined by a ChIP assay. DNA from ChIP was subjected to PCR and gel analysis. Non-immune IgG served as a negative control.

Figure 6

Depletion of PI3Kδ blocks TGF-β2-induced migration of ARPE-19 cells. (A). Wells of confluence cells were scratched using 200 μL pipet tips, washed and photographed at the initial width, followed by treatment with TGF-β2 (10 ng/mL) for 24 h. Cell images were then taken, and wound areas were analyzed by image J and Adobe Photoshop CS4 software. The data of bar graphs are the mean ± SD of 3 independent experiments. Representative raw data of 3 independent experiments are shown underneath the bar graphs. (B). *** denotes p < 0.001 using one-way ANOVA followed by the Tukey HSD post hoc test. (C). Diagram of a loop pathway of PI3Kδ/Akt/NFκB/PI3Kδ induced by TGF-β2. Growth factors (GFs) and cytokines (CKs) in the vitreous [24,45] activate the PI3K/Akt signaling pathway, resulting in Mdm2 phosphorylation and a decline in p53 levels [28,44]. Receptor-regulated PI3Ks consist of PI3Kα, PI3Kβ and PI3Kδ [31]. In particular, TGF-β2 in the vitreous [46] triggers heightened expression of Mdm2 [47], resulting in elevated levels of NFκB/p65 [48] and p110δ [30], the catalytic subunit of PI3Kδ [31]. These biochemical events (e.g., a decrease in p53 [49,50,51] and an increase in NFκB/p65 [52,53]) consequently promote cellular responses (e.g., proliferation, EMT, migration and contraction), driving PVR pathogenesis.