Fig 2: SRF is important for regulation of IL1B and CXCL8. (A–C) Human coronary SMCs were transduced with MRTF-A along with different titers of Ad-h-shSRF for knockdown of SRF. After harvesting the cells and isolation of RNA, SRF, IL1B, and CNN1 were assayed using RT-qPCR. In (A,B), IL1B and CNN1 were plotted vs. the relative SRF level in the respective samples. In (C), SRF, IL1B, and CNN1 were plotted vs. the titer (multiplicity of infection, MOI) of the short hairpin virus. (D) Shows the remainder of the inflammatory mediators in control vs. SRF-depleted cells (1000 MOI). (E) Is like (D), except that SRF silencing was done without simultaneous overexpression of MRTF-A. (F) Shows the gene locus for human CXCL8 and binding of RelA (light blue oval) 4 kb upstream of the transcription start site. The proximal promoter contained two DNA sequences with two deviations each from the classical SRF-binding sequence. When testing this sequence in a reporter assay, no suppression by MRTF-A was however, seen (G). In (H), Srf and Il1b were assayed in the aorta from smooth muscle specific and inducible Srf knockout mice. Because knockout was induced by tamoxifen, two control groups were included in addition to the knockout group. Vehicle controls (VC) are Cre-positive mice injected with sunflower oil, whereas tamoxifen controls (TC) are Cre-negative mice injected with tamoxifen. Knockouts (TKO) are Cre-positive mice injected with tamoxifen. All mice are homozygous for the floxed Srf allele. (I–L) Show the effect of overexpression of SRF in the absence and presence of MRTF-A. SRF was capable of suppressing CXCL8 on its own but was without effect on CCL2 and IL6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: EBV product-containing exosomes enhance fibrotic and inflammatory signalings in fibroblasts. A Analysis of signaling pathways in fibroblasts stimulated with HK1EBV cell-derived exosomes compared with those exposed to medium. The top-scoring signaling pathways were identified using Ingenuity Pathway Analysis (IPA) software (QIAGEN). The identified pathways were ranked by significance [−log (p-value)]. The number of genes that met cutoff criteria [|log2(FC)| > 0.5 and p value < 0.05] in a given signaling pathway was shown. Total RNAs were harvested 3 hours post HK1EBV cell-derived exosome (10 μg/ml) stimulation. B Analysis of KEGG pathways in fibroblasts stimulated with HK1EBV cell-derived exosomes compared with those exposed to medium. C Gene set enrichment analysis (GSEA) in fibroblasts treated with HK1EBV cell-derived exosomes. GSEA plots for gene sets involved in inflammatory response, TGFβ signaling, and epithelial-mesenchymal transition were identified. Expression values were normalized to that of control fibroblasts. Significance threshold set at FDR < 0.25. D Volcano plots of transcriptomic analyses revealed differentially expressed genes (DEGs). Cutoff values were defined as |log2(FC)| > 0.5 and a P-value < 0.05. Total RNA was harvested 3 hours and 6 hours post exosome stimulation (10 μg/ml). E Expression heatmap of select genes upregulated by EBV product-containing exosomes. Expression levels were measured by qRT-PCR and normalized with respect to peptidylprolyl isomerase A (PPIA), used as an internal control. Values are expressed as log2 (fold change). F Western blot analysis of IL8, IL6, and CCL2 in fibroblasts. Protein lysates were harvested after 2-hour exosomal stimulation plus 4-hr treatment with Brefeldin A. GAPDH was used as a loading control. G Quantification of secreted IL8, IL6, and CCL2 in cell-free culture supernatants collected 3 hours post exosomal stimulation of fibroblasts. Data are presented as means ± SD of triplicate experiments (*p < 0.05**, p < 0.01, ***p < 0.001; paired t test)

Fig 4: MRTF-A interacts with RelA and RelB. To explore if MRTF-A binds RelA and RelB, we performed co-immunoprecipitations followed by dot blotting (A). Lysates from cells where MRTF-A was overexpressed were incubated with control resin and with MRTF-A-antibody-conjugated resin. Flow through (FC: flow through control; FM: flow through MRTF-A) from both columns contained RelA, RelB, and MRTF-A. RelA, RelB and MRTF-A were moreover detectable in the eluate from the MRTF-A column (EM: eluate MRTF-A) but in the in the eluate from the control column (EC: eluate control). Western blotting (B) of the eluates showed the RelA band at the expected molecular weight. These findings support a model where MRTF binding to Rel proteins suppresses inflammation (C). (C) Also depicts the hypothesis that RelA suppression, by glucocorticoid receptor stimulation or silencing, should mitigate inflammatory suppression by MRTF-A. (D,E) Test this hypothesis using dexamethasone (glucocorticoid receptor agonist, 3 μM) or short hairpin silencing (200 MOI of shRELA). For the experiments in (D,E), the fold repression is given. Independent testing showed that the fold repression of CCL2 and IL1B by MRTF-A was reduced by dexamethasone. In contrast, fold suppression of CXCL8 and IL6 was unaffected. For RelA silencing, CXCL8 and IL6 suppression by MRTF-A were significantly reduced, but the overall effect, where MRTF-A suppression of inflammation was reduced by ≈25%, was also significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 5: Inhibition of YAP1 prevents EBV exosome-induced fibroblast activation. A Assessment of verteporfin- and saracatinib-mediated inhibition of HK1EBV exosome-induced fibroblast contractility. B YAP1 inhibition in fibroblasts decreases EBV exosome-promoted NPC cell proliferation. The culture medium of the fibroblasts treated with inhibitor or vehicle was replaced with fresh medium containing 1% FBS after 5 hours of drug treatment, followed by 3 hours of exosomal stimulation. Fibroblast supernatants were then collected and used to treat NPC cells. Data are presented as the mean and SD of triplicate experiments. C Immunofluorescence staining of active YAP1 (green) and FAPα (red) in the fibroblasts treated with HK1EBV cell-derived exosomes, alone or together with verteporfin or saracatinib. Cells were fixed 3 hours after exosome stimulation. Blue, nuclear staining. Scale bars, 20 μm. D Western blot analysis of active YAP and its downstream target proteins in the fibroblasts treated with HK1EBV-derived exosomes, alone or together with verteporfin (3 μM) or saracatinib. Protein lysates were harvested 3 hours after exosome treatment. GAPDH was used as a loading control. E–G Assessment of verteporfin-mediated suppression of the levels of IL8 (E), CCL2 (F), and IL6 (G) released from the fibroblasts treated under the indicated conditions. H–J, Assessment of saracatinib-mediated suppression of the production of IL8 (H), CCL2 (I), and IL6 (J) released from fibroblasts. Unless otherwise indicated, verteporfin (1 μM) or saracatinib (20 μM) was added to the growth medium of fibroblasts 2 hours prior to exosome (10 μg/ml) treatment. Data are presented as the mean and SD of triplicate experiments (*p < 0.05, **p < 0.01, ***p < 0.001; Bonferroni’s multiple comparison test)