Cytokines of the tumor necrosis factor (TNF) family regulate inflammation and immunity, and a subset of this family can also induce cell death in a context‐dependent manner. Although TNFα is cytotoxic to certain tumor cell lines, it induces apoptosis in normal cells only when NFκB signaling is blocked. Here we show that the matricellular protein CCN1/CYR61 can unmask the cytotoxic potential of TNFα without perturbation of NFκB signaling or de novo protein synthesis, leading to rapid apoptosis in the otherwise resistant primary human fibroblasts. CCN1 acts through binding to integrins αvβ5, α6β1, and syndecan‐4, triggering the generation of reactive oxygen species (ROS) through a Rac1‐dependent mechanism via 5‐lipoxygenase and the mitochondria, leading to the biphasic activation of JNK necessary for apoptosis. Mice with the genomic Ccn1 locus replaced with an apoptosis‐defective Ccn1 allele are substantially resistant to TNFα‐induced apoptosis in vivo. These results indicate that CCN1 may act as a physiologic regulator of TNFα cytotoxicity, providing the contextual cues from the extracellular matrix for TNFα‐mediated cell death.
Bladder cancer, the fourth most common noncutaneous malignancy in the United States, is characterized by high recurrence rate, with a subset of these cancers progressing to a deadly muscle invasive form of disease. Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA, thus potentially modulating signaling pathways in recipient cells. Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell–cell adhesion and gain migratory and invasive properties to become mesenchymal stem cells. EMT has been implicated in the initiation of metastasis for cancer progression. We investigated the ability of bladder cancer-shed exosomes to induce EMT in urothelial cells. Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media or from patient urine or bladder barbotage samples. Exosomes were then added to the urothelial cells and EMT was assessed. Urothelial cells treated with bladder cancer exosomes showed an increased expression in several mesenchymal markers, including α-smooth muscle actin, S100A4 and snail, as compared with phosphate-buffered saline (PBS)-treated cells. Moreover, treatment of urothelial cells with bladder cancer exosomes resulted in decreased expression of epithelial markers E-cadherin and β-catenin, as compared with the control, PBS-treated cells. Bladder cancer exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pretreatment. We further showed that exosomes isolated from patient urine and bladder barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. In conclusion, the research presented here represents both a new insight into the role of exosomes in transition of bladder cancer into invasive disease, as well as an introduction to a new platform for exosome research in urothelial cells.
Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin α6β1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type–specific manner and that integrin α6β1-HSPGs can function as receptors to induce p53-dependent apoptosis.
SummaryWe previously showed that the cell-cell junction protein plakoglobin (PG) not only suppresses motility of keratinocytes in contact with each other, but also, unexpectedly, of single cells. Here keratinocytes. PG -/-cells also exhibited Src-independent activation of the small GTPases Rac1 and RhoA. Both Src and RhoA inhibition attenuated PG -/-keratinocyte motility. We propose a novel role for PG in regulating cell motility through distinct ECM-Src and RhoGTPase-dependent pathways, influenced in part by PG-dependent regulation of FN mRNA stability. Key words: Armadillo protein, Desmosome, Extracellular matrix, KeratinocyteJournal of Cell Science (Caldelari et al., 2001) and adhesive strength (Acehan et al., 2008) in keratinocytes lacking PG. Moreover, PG-null mice and patients with pathogenic homozygous PG mutations have impaired tissue integrity associated with skin and heart defects (Aberle et al., 1995;Bierkamp et al., 1996;McKoy et al., 2000;Ruiz et al., 1996). PG is also found in the cytoplasm and nucleus (Green and Simpson, 2007;Schmidt and Koch, 2007), where it is able to act independently of its function in intercellular adhesion. Its adhesionindependent functions are still not well defined, but the data suggest that PG can regulate gene expression and protein stability (Aktary et al., 2010;Hakimelahi et al., 2000;Shimizu et al., 2008) in both a -catenin-dependent and -independent manner (Raurell et al., 2006;Teuliere et al., 2004;Yin and Green, 2004;Zhurinsky et al., 2000).Recently we demonstrated that PG not only inhibits motility of keratinocytes in contact, but also inhibits Src-dependent single cell motility (Yin et al., 2005b). The observed changes in motility and altered cell morphology of PG -/-keratinocytes suggested to us that PG could be regulating cell-substrate interactions by modulating components of the extracellular matrix (ECM), its integrin receptors and/or the molecules involved in ECM-triggered motility cues. Using a combination of live cell imaging and cross plating, we show here that PG expression has a potent impact on the organization of actin, its associated membrane protrusions, focal adhesions and Src-dependent motility, in large part through regulation of the expression levels of the underlying ECM components. In particular, the ability of PG to regulate fibronectin (FN; also know as Fn1) mRNA stability was identified as a novel mechanism contributing to PG-dependent suppression of keratinocyte motility. Further analysis indicated that PG-dependent alterations in activity of the small GTPases Rac1 and RhoA act in parallel with FN/Src-dependent regulation of cortical actin 3577 Plakoglobin regulates motility via ECM structures to fine tune the motile behavior of keratinocytes. Collectively, these results indicate that a desmosomal molecule, PG, is capable of regulating single cell motility through matrix deposition in concert with Rho GTPases, independently of its role as a cell-cell adhesion molecule. Results PG regulates keratinocyte cell polarity and single-cell ...
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