Proteomic characterization of epicardial-myocardial signaling reveals novel regulatory networks including a role for NF-κB in epicardial EMT

Figures

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  Fig 1. Preparation of EHE-CM for mass spectrometry. Representative images of chicken heart explant at 0 hour (A), EHE co-culture at 24 hours (B) and 36 hours (C). Scale bar: 80 μm in (A); 40 μm in (B-C). (D) Silver-stained SDS-PAGE gel of 3 independent preparations, CM1-3. (E) Western blot of TGFβ2 and TGFβ3 in CM preparations. CM, conditioned medium; H, heart explant; HL, heart lysate; A, atria; V, ventricle.

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  Fig 2. The NF-κB regulatory network. Ingenuity Pathway Analysis was utilized to generate network connections of the EHE secretome. In the depicted network NF-κB was identified as a central node. Nodes are depicted as molecule names over shapes. Shaded shapes represent focus molecules and open shapes represent molecules were not present in our dataset but placed by IPA due to their connections to the focus molecules. Single lines represent protein—protein interactions and arrows indicate regulatory relationship(s) between connected molecules. Solid lines denote a direct relationship between gene products while dotted lines indicate an indirect relationship. TGFβ (circle) and NF-κB (square) connectivity nodes are marked.

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  Fig 3. NF-κB p65 subcellular localization in primary mouse epicardial cells after TGFβ2 and PDGFBB treatment. Immunofluorescent detection of p65 in untreated (UTD) cells (A-D), cells treated with TGFβ2/PDGFBB for 8 hours (E-H), and cells co-treated with TGFβ2/PDGFBB and BMS345541 (I-L). Arrows point to untreated cells with elevated nuclear p65 staining as compared to cytoplasmic staining levels. Scale bar: 20 μm. Box-and-Whisker plots (Min to Max method, Graphpad Prism) of p65 nuclear:cytoplasmic ratio (M). *p < 0.0001 (Mann—Whitney test).

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  Fig 4. NF-κB p65 subcellular localization in primary chicken epicardial cells after TGFβ2 and PDGFBB treatment. Immunofluorescent detection of p65 in untreated (UTD) cells (A-D), cells treated with TGFβ2/PDGFBB for 8 hours (E-H), and cells co-treated with TGFβ2/PDGFBB and BMS345541 (I-L). Nuclei in the DAPI channel were outlined to enhance the visibility of nuclear p65 (D, H, L). Scale bar: 20 μm. Box-and-Whisker plots (Min to Max method, Graphpad Prism) of p65 nuclear:cytoplasmic ratio (M). *p < 0.05, **p < 0.01 (Mann—Whitney test).

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  Fig 5. Inhibition of NF-κB blocks TGFβ2- and PDGFBB-induced EMT in primary chicken epicardial cells. Primary chicken epicardial cells cultured in the absence or presence of TGFβ2/PDGFBB and/or BMS345541for 48 hours. Determination of cell morphology using phase contrast (A-D). F-actin stress fiber formation (E-H) and β-catenin localization in the same cells (E’-H’) were visualized by immunofluorescence. Scale bar: 80 μm in (A-D); 40 μm in (E-H, E’-H’). qRT-PCR analysis of WT1, MMP2 and DCN in untreated cells versus cells treated with TGFβ2/PDGFBB and/or BMS345541 (I). Error bars represent the means ± s.e.m. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001 (student’s t Test).

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  Fig 6. Inhibition of NF-κB blocks TGFβ2- and PDGFBB-induced EMT in the mouse epicardial cell line MEC1. MEC1 cells cultured in the absence or presence of TGFβ2/PDGFBB and/or BMS345541 for 48 hours. Immunodetection of F-actin (A-D) and β-catenin localization (A’-D’) in the same cells. In addition, expression of ZO1 (E-H) and ACTA2-positive stress fiber formation (E’-H’) was determined in the same cells. Scale bar: 40 μm.

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  Fig 7. NF-κB p65 is expressed in mouse embryonic epicardium. Immunofluorescence imaging of mouse E11.5 (A-B) and E13.5 (C-D) sections using antibodies directed against p65 (red) and cTnT (green). Nuclei identified by DAPI (blue) staining. p65 proteins were found enriched in the epicardium and endocardium. Nuclear accumulation was observed in a subset of epicardial cells (arrows). Dotted lines delineate the border between the myocardium and the epicardium. Scale bars: 20 μm.

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  Fig 8. NF-κB p65 is expressed in chicken embryonic epicardium. Cryosections from HH22 (A, A’, B, B’), HH26 (C, C’, D, D’) and HH30 (E, E’, F, F’) chick embryos were processed for immunohistochemistry using antibodies specific for p65 (red) and anti-sarcomeric myosin (MF20). The cardiomyocyte marker MF20 (green) labels the myocardium. Nuclei are detected by DAPI (blue). Rectangles mark the regions that are magnified in the lower panel for each stage. Scale bar: 50 μm for (A’, B’, C’, D’, E’, F’); 315 μm for (A, B, C, D, E, F).

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  Fig 9. Proposed model for the function of NF-κB signaling in epicardial EMT. TGFβ2 and PDGFBB induce the activation of epicardial NF-κB and consequently upregulation of mesenchymal genes MMP2 and DCN as well as downregulation of epicardial cell marker gene WT1.