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JNK‐mediated eIF4E phosphorylation and signaling in fibrotic functions of lung‐resident mesenchymal cells (MCs)
Author(s) -
Walker Natalie Maureen,
Lama Vibha N.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.651.14
Subject(s) - phosphorylation , eif4e , cancer research , signal transduction , western blot , kinase , blot , chemistry , medicine , microbiology and biotechnology , translation (biology) , biology , messenger rna , biochemistry , gene
INTRO We have recently demonstrated that cap‐dependent translation regulated through 4E‐BP1/eIF4E signaling plays a critical role in regulating fibrotic functions of activated mesenchymal cells, specifically collagen I expression. Fibrotic MC signaling to 4EBP1 is resistant to rapamycin, and resistance to TOR inhibitors has also been described in cancer. This underscores the need for better therapeutic options targeting aberrant translation, and further understanding of upstream signaling mechanisms regulating translational control will be key. Here we investigate mechanisms driving cap‐dependent translation upstream of eIF4E Serine 209 phosphorylation in lung MCs. Materials/methods Lung‐resident mesenchymal cells were isolated from Bronchoalveolar lavage fluid of lung transplant recipients with and without fibrosis by plastic adherence. MCs were treated for 2 hours with 10uM SP600125 (JNK inhibitor), U0126 (MEK inhibitor), SB202190 (p38inhibitor), or the MNK kinase inhibitors CGP57380 and eFT‐508. Constitutively active JNK1 (MKK7‐JNK1 fusion) was shuttled into plentiLOX‐IRES‐puro and packaged into lenti‐viral particles. Non‐fibrotic MCs were infected for 48 hours, lysed and protein expression was analyzed by western blot. RESULTS Fibrotic MCs isolated from patients with Bronchiolitis obliterans had significantly higher cap‐dependent translation compared to non‐fibrotic MCs when measured utilizing luciferase reporter assay (n=7; p<0.01). Fibrotic MCs had significantly higher phosphorylation of eIF4E at serine 209 compared to non‐fibrotic MCs when baseline expression was measured by western blotting (n=8; p<0.01), suggesting elevated translational activity. MCs treated with rapamycin (250 nM) for 2 hours demonstrated increased phosphorylation of eIF4E (S209), which was sensitive to MNK kinase inhibition. Treatment of fibrotic MCs with JNK MAPkinase inhibitor SP600125 or the MNK inhibitors eFT‐508 and CGP57380 led to a significant decrease in collagen I expression and eIF4E (S209) phosphorylation, while MEK and p38 inhibitors did not. Expression of constitutively active JNK led to a significant increase in phosphorylated eIF4E (S209) in non‐fibrotic MCs, implicating JNK activation as key to causing eIF4E phosphorylation and potentially increased cap‐dependent translation. Conclusion These data demonstrate that cap‐dependent translation downstream of 4E‐BP1/eIF4E is elevated in fibrotic MCs and that eIF4E phosphorylation at serine 209 is sensitive to JNK/MNK inhibition. This suggests that the JNK/MNK signaling axis upstream of 4E‐BP1/eIF4E and hence translation is key to fibrotic functions of MCs during pathogenesis of fibrotic lung diseases, and a viable target for treating fibrosis. Support or Funding Information This abstract is funded by NIH grants: RO1F035722 and RO1F037975. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .