title: TGF-beta1-induced EMT can occur independently of its proapoptotic effects and is aided by EGF receptor activation.
abstract: Apoptosis and epithelial-mesenchymal transdifferentiation (EMT) occur in stressed tubular epithelial cells and contribute to renal fibrosis. Transforming growth factor (TGF)-beta(1) promotes these responses and we examined whether the processes were interdependent in vitro. Direct (caspase inhibition) and indirect [epidermal growth factor (EGF) receptor stimulation] strategies were used to block apoptosis during TGF-beta(1) stimulation, and the subsequent effect on EMT was assessed. HK-2 cells were exposed to TGF-beta(1) with or without preincubation with ZVAD-FMK (pan-caspase inhibitor) or concomitant treatment with EGF plus or minus preincubation with LY-294002 (PI3-kinase inhibitor). Cells were then assessed for apoptosis and proliferation by flow cytometry, crystal violet assay, and Western blotting. Markers of EMT were assessed by microscopy, immunofluorescence, real-time RT-PCR, Western blotting, PAI-1 reporter assay, and collagen gel contraction assay. TGF-beta(1) caused apoptosis and priming for staurosporine-induced apoptosis. This was blocked by ZVAD-FMK. However, ZVAD-FMK did not prevent EMT following TGF-beta(1) treatment. EGF inhibited apoptosis and facilitated TGF-beta(1) induction of EMT by increasing proliferation and accentuating E-cadherin loss. Additionally, EGF significantly enhanced TGF-beta(1)-induced collagen I gel contraction. EGF increased Akt phosphorylation during EMT, and the prosurvival effect of this was confirmed using LY-294002, which reduced EGF-induced Akt phosphorylation and reversed its antiapoptotic and proproliferatory effects. TGF-beta(1) induces EMT independently of its proapoptotic effects. TGF-beta(1) and EGF together lead to EMT. EGF increases proliferation and resistance to apoptosis during EMT in a PI3-K Akt-dependent manner. In vivo, EGF receptor activation may assist in the selective survival of a transdifferentiated, profibrotic cell type.
authors.affiliation: Conway Institute, Univ. College Dublin, Belfield, Dublin 4, Ireland.
authors.forename: Neil G
authors.initials: NG
authors.lastname: Docherty
authors.forename: Orfhlaith E
authors.initials: OE
authors.lastname: O'Sullivan
authors.forename: Declan A
authors.initials: DA
authors.lastname: Healy
authors.forename: Madeline
authors.initials: M
authors.lastname: Murphy
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: O'neill
authors.forename: John M
authors.initials: JM
authors.lastname: Fitzpatrick
authors.forename: R William G
authors.initials: RW
authors.lastname: Watson
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D061945:Caspase Inhibitors; D002454:Cell Differentiation; D002470:Cell Survival; D004815:Epidermal Growth Factor; D004848:Epithelium / Q000502:physiology; D066246:ErbB Receptors / Q000502:physiology; D005355:Fibrosis; D006801:Humans; D007668:Kidney / Q000473:pathology*; D007687:Kidney Tubules, Proximal / Q000502:physiology; D008648:Mesoderm / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000502:physiology*; D053773:Transforming Growth Factor beta1
chemical_list: D061945:Caspase Inhibitors; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors