Role of endoplasmic reticulum stress in epithelial-mesenchymal transition of alveolar epithelial cells: effects of misfolded surfactant protein.
ABSTRACT Endoplasmic reticulum (ER) stress has been implicated in alveolar epithelial type II (AT2) cell apoptosis in idiopathic pulmonary fibrosis. We hypothesized that ER stress (either chemically induced or due to accumulation of misfolded proteins) is also associated with epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AECs). ER stress inducers, thapsigargin (TG) or tunicamycin (TN), increased expression of ER chaperone, Grp78, and spliced X-box binding protein 1, decreased epithelial markers, E-cadherin and zonula occludens-1 (ZO-1), increased the myofibroblast marker, α-smooth muscle actin (α-SMA), and induced fibroblast-like morphology in both primary AECs and the AT2 cell line, RLE-6TN, consistent with EMT. Overexpression of the surfactant protein (SP)-C BRICHOS mutant SP-C(ΔExon4) in A549 cells increased Grp78 and α-SMA and disrupted ZO-1 distribution, and, in primary AECs, SP-C(ΔExon4) induced fibroblastic-like morphology, decreased ZO-1 and E-cadherin and increased α-SMA, mechanistically linking ER stress associated with mutant SP to fibrosis through EMT. Whereas EMT was evident at lower concentrations of TG or TN, higher concentrations caused apoptosis. The Src inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4]pyramidine) (PP2), abrogated EMT associated with TN or TG in primary AECs, whereas overexpression of SP-C(ΔExon4) increased Src phosphorylation, suggesting a common mechanism. Furthermore, increased Grp78 immunoreactivity was observed in AT2 cells of mice after bleomycin injury, supporting a role for ER stress in epithelial abnormalities in fibrosis in vivo. These results demonstrate that ER stress induces EMT in AECs, at least in part through Src-dependent pathways, suggesting a novel role for ER stress in fibroblast accumulation in pulmonary fibrosis.
Article: Misfolded BRICHOS SP-C mutant proteins induce apoptosis via caspase-4- and cytochrome c-related mechanisms.[show abstract] [hide abstract]
ABSTRACT: Several mutations within the BRICHOS domain of surfactant protein C (SP-C) have been linked to interstitial lung disease. Recent studies have suggested that these mutations cause misfolding of the proprotein (proSP-C), which initiates the unfolded protein response to resolve improper folding or promote protein degradation. We have reported that in vitro expression of one of these proteins, the exon 4 deletion mutant (hSP-C(Deltaexon4)), causes endoplasmic reticulum (ER) stress, inhibits proteasome function, and activates caspase-3-mediated apoptosis. To further elucidate mechanisms and common pathways for cellular dysfunction, various assays were performed by transiently expressing two SP-C BRICHOS domain mutant (BRISPC) proteins (hSP-C(Deltaexon4), hSP-C(L188Q)) and control proteins in lung epithelium-derived A549 and kidney epithelium-derived (HEK-293) GFP(u)-1 cell lines. Compared with controls, cells expressing either BRICHOS mutant protein consistently exhibited increased formation of insoluble aggregates, enhanced promotion of inositol-requiring enzyme 1-dependent splicing of X-box binding protein-1 (XBP-1), significant inhibition of proteasome activity, enhanced induction of mitochondrial cytochrome c release, and increased activations of caspase-4 and caspase-3, leading to apoptosis. These results suggest common cellular responses, including initiation of cell-death signaling pathways, to these lung disease-associated BRISPC proteins.AJP Lung Cellular and Molecular Physiology 10/2007; 293(3):L720-9. · 3.66 Impact Factor
Article: Tamoxifen resistance in MCF7 cells promotes EMT-like behaviour and involves modulation of beta-catenin phosphorylation.[show abstract] [hide abstract]
ABSTRACT: We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.International Journal of Cancer 02/2006; 118(2):290-301. · 5.44 Impact Factor