TGF-β₁-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.

Synergistic effect between EGF and TGF-β1 in inducing oncogenic properties of intestinal epithelial cells

“…Moreover, EGF and TGF-b1 synergistically induce the expression of a series of genes including MMPs involved in promoting invasion or AIG. Our finding of the cooperation between EGF and TGF-b1 in inducing EMT confirms similar previous observations in RIE and other cell systems (Stolz et al, 1997;Saha et al, 1999;Docherty et al, 2006). Our finding of the enhancing effect between TGF-b1 and EGF in promoting AIG, migration and invasion is in agreement with the recent reports that activation of TGF-b signaling promotes pulmonary metastasis of mammary tumors in MMTV/neu transgenic mice (Siegel et al, 2003) Figure 1a with or without addition of 25 mM PD98059 or 50 mM 6-gingerol in DMEM containing 10% FCS for 24 h. Two micrograms each was subjected to RT-PCR using genespecific primers (Supplementary Table 1).…”

Potential nephroprotective effects of the Chinese herbAngelica sinensisagainst cisplatin tubulotoxicity

“…Ki-67 is commonly used to assess the proliferative capacities of tumors. It has also been used in vitro and in vivo for the assessment of RPTEC regeneration (Docherty et al, 2006;Pozdzik et al, 2008).…”

Stem cell options for kidney disease

“…In muscle, slow-twitch fibres can be reprogrammed in vivo into the fast-twitch phenotype via forced expression of Eya1 and Six1 [98], and ectopic expression of Msx1 in mouse C2C12 myotubules results in dedifferentiation of a subset of cells to a pool of proliferating mononucleate cells with chondrogenic, osteogenic, myogenic and adipogenic potential [99]. In the kidney, a number of factors have been shown to cause epithelial-mesenchymal transdifferentiation (EMT) of adult tubular epithelial cells in vitro, although the particular mechanisms, for example TGFβ1 signalling, are mainly associated with fibrogenesis and disease [100][101][102].…”