TNFα Modulates Fibroblast Growth Factor Receptor 2 Gene Expression through the pRB/E2F1 Pathway: Identification of a Non-Canonical E2F Binding Motif

Abstract

Interactions between epithelium and mesenchyme during wound healing are not fully understood, but Fibroblast Growth Factors (FGFs) and their receptors FGFRs are recognized as key elements. FGFR2 gene encodes for two splicing transcript variants, FGFR2-IIIb or Keratinocyte Growth Factor Receptor (KGFR) and FGFR2-IIIc, which differ for tissue localization and ligand specificity. Proinflammatory cytokines play an essential role in the regulation of epithelial-mesenchymal interactions, and have been indicated to stimulate FGFs production. Here we demonstrated that upregulation of FGFR2 mRNA and protein expression is induced by the proinflammatory cytokines Tumor Necrosis Factor-α, Interleukin-1β and Interleukin 2. Furthermore, we found that TNFα determines FGFR2 transcriptional induction through activation of pRb, mediated by Raf and/or p38 pathways, and subsequent release of the transcription factor E2F1. Experiments based on FGFR2 promoter serial deletions and site-directed mutagenesis allowed us to identify a minimal responsive element that retains the capacity to be activated by E2F1. Computational analysis indicated that this element is a non-canonical E2F responsive motif. Thus far, the molecular mechanisms of FGFR2 upregulation during wound healing or in pathological events are not known. Our data suggest that FGFR2 expression can be modulated by local recruitment of inflammatory cytokines. Furthermore, since alterations in FGFR2 expression have been linked to the pathogenesis of certain human cancers, these findings could also provide elements for diagnosis and potential targets for novel therapeutic approaches.

Autophagy activation is required for myofibroblast differentiation during healing of oral mucosa

“…Total RNA from biopsies or cell cultures was extracted using TRIzol reagent (Invitrogen by Life Technologies, Carlsbad, CA, USA) following the manufacturer's instructions. Quantitative real‐time PCR (qRT‐PCR) was performed as previously described (D'Amici et al., ). Briefly, cDNA obtained were used for amplification of αSMA and vimentin using the appropriate TaqMan gene expression assay kits (Applied Biosystems by Life Technologies).…”

“…Cells were lysed in RIPA buffer and processed for Western blot analysis as previously described (D'Amici et al., ). Membranes were incubated overnight at 4°C with the following antibodies: anti‐Vimentin (1:500 dilution; Dako), anti‐αSMA (1:1,000 dilution; Sigma), Col1a1 (1:3,000 dilution; Merk Millipore), LC‐3 (1:2,000 dilution; Sigma), p62 (1:1,000 dilution; BD Biosciences), p‐mTOR (1:1,000 dilution; Cell Signaling Technology), p‐AKT (1:1,000 dilution; Cell Signaling Technology), followed by the appropriate horseradish peroxidase (HRP)‐conjugated secondary antibody (Santa Cruz Biotechnology).…”

“…Cells were lysed in RIPA buffer and processed for Western blot analysis as previously described (D'Amici et al, 2013). Membranes were incubated overnight at 4°C with the following antibodies: anti-Vimentin (1:500 dilution; Dako), anti-αSMA (1:1,000 dilution; Sigma), Col1a1…”

Synergetic Effect of a-Lipoic Acid with Keratinocyte Growth Factor on Protecting Alveolar Epithelial Type II Cells of Rat Fetus from Hyperoxia -Induced Injury

“…This behavior is consistent with ATIICs being less sensitive to KGF. Similarly, Marchese et al demonstrated that KGFR gene and protein expression could be modulated by proinflammatory cytokines and oxidative stress [39,40,41], and excessive oxidative stress induced by UVB could result in the internalization of KGFR after temporary UVB exposure [40]. Down-regulation was observed 6 h to 24 h after UVB exposure [41,42].…”

In Vitro Biophysical and Biological Characterization of Lipid Nanoparticles Co-Encapsulating Oncosuppressors miR-199b-5p and miR-204-5p as Potentiators of Target Therapy in Metastatic Melanoma

“…Melanoma cells were lysed with RIPA buffer purchased by Sigma-Aldrich (St. Louis, MO, USA) and total proteins were run using Invitrogen Bolt Bis-Tris 4-12% Plus gels precast polyacrylamide gels [43]. Proteins were then transferred using the iBlot ® Transfer Stacks in nitrocellulose membranes through the fast iBlot®Dry Blotting System (ThermoFisher Scientific, Foster City, CA, USA).…”