Fig 1: PPAR family Nuclear Receptors (NR) bind to the EGFR promoterA. DNA-binding capacities of increasing amounts of PPAR family nuclear receptors (PPARα, LXRα or RXRα) to a synthetic EGFR promoter DNA substrate (see Supplementary Figure S1). B. Negative control samples that measured the effect of NR (1:1 PPARα+LXRα) binding to the dsDNA substrate in the absence of dioleoyl-PA. Results expressed in terms of mean absorbance at 405 nm + SEM. Results are presented in terms of mean relative optical density at 405 nm + SEM. Experiments were performed in triplicate for n = 3. The (*) symbols above bars denote statistically significant (P < 0.05) ANOVA increases between samples and controls. C. Increasing dioleoyl-PA concentrations were pre-incubated with the PPARα+LXRα heterodimers and then subsequently were added to the EGFR promoter-containing DNA-BIND ELISA plate for completion of the assay. Results are expressed in terms of normalized relative DNA binding + SEM. Shown are images representative of n = 5 for each condition with similar results.
Fig 2: PA targets EGFR and also the nuclear receptors of the PPARα familyA. Western blot analysis showing the effects of 10 nM EGF or 300 nM PA or increasing concentrations of PA treatment on protein expression of EGFR and upstream nuclear receptor and transcription factor PPARα in the absence or presence of PLD2 overexpression. B.-C. QPCR analyses of samples detecting either EGFR (B) or PPARα (C) gene expression. D. QPCR analyses of samples detecting EGFR, PPAR or LXR gene expression with increasing amounts of PA treatment (0-1000 nM). The (*) symbols above bars denote statistically significant (P < 0.05) ANOVA increases between samples and controls. The (#) symbols above bars denote statistically significant (P < 0.05) ANOVA decreases between samples and controls.
Fig 3: Proposed model to explain results presented in this studyThe red circular arrow represents the PA “conveyor” transport system described in this study for the firt time that helps with the trafficking of the EGFR form the cell membrane to the nucleus and back (with newly-synthesized receptor). PA also targets PPARα that directly affects the EGFR promoter. The model attempts to explain the complex regulation of trafficking of the EGF receptor upon generation of PA and displacement of nuclear receptors from binding to the EGFR promoter. An increased loop of PA/EGFR trafficking is initiated by binding of EGF to its receptor. Both EGFR and PA are transferred to the nucleus. PA in the nucleus activates gene expression of several genes, as PA is a mitogen. However, it comes to a point in which there is an overabundance of PA in the nucleus, when this occurs, PA negatively shuts down the process. This is done through nuclear receptors (PPAR and LXR) as we show that PA is able to displace NR form DNA promoter of EGR. The trafficking and expression of new protein are shut down.
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