Inhibition of tumor cell surface ATP synthesis by pigment epithelium-derived factor: implications for antitumor activity.
ABSTRACT Recently, we have shown that the antiangiogenic pigment epithelium-derived factor (PEDF) can bind the catalytic β-subunit of F1-ATP synthase and inhibit endothelial cell surface ATP synthase activity. This factor can additionally restrict tumor growth, invasion and metastasis, and can directly induce death on several tumor cell types. Active cell surface ATP synthase is also present in certain tumor cells and its ATP product is considered a stimulus for tumor growth. The present study aimed to elucidate the biological implications of the interactions between the extracellular PEDF and tumor cell surface ATP synthase. Incubation of T24 human urinary bladder carcinoma cells in media containing human recombinant PEDF protein for 48-96 h dramatically decreased cell viability in a concentration-dependent fashion as monitored by real-time cell impedance with a microelectronic system, microscopic imaging and biomarkers of live cells. Intact tumor cells exhibited cell surface ATP synthesis activity, which was inhibited by piceatannol, a specific inhibitor of F1/F0-ATP synthase. Immunoblotting revealed that the β subunit of F1-ATP synthase was present in plasma membrane fractions of these cells. Interestingly, pre-incubation of tumor cells with PEDF inhibited the activity of cell surface ATP synthase in a concentration-dependent fashion. The PEDF-derived peptide 34-mer decreased tumor cell viability and inhibited extracellular ATP synthesis to the same extent as full-length PEDF. Moreover, ATP additions attenuated both the PEDF-mediated decrease in tumor cell viability and the inhibition of endothelial cell tube formation. The results lead to conclude that PEDF is a novel inhibitor of tumor cell surface ATP synthase activity that exhibits a cytotoxic effect on tumor cells, and that the structural determinants for these properties are within the peptide region 34-mer of the PEDF polypeptide. The data strongly suggest a role for the interaction between the 34-mer region of PEDF and tumor cell-surface ATP synthase in promoting tumor cell death.
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ABSTRACT: The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell-surfaces. We have previously reported that PEDF binds and stimulates PEDF-R, a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (L159-M325) with affinity similar to the full-length PEDF-R (M1-L504). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (I193-L232) and P1 (T210-L249) peptides. Recombinant C-terminal truncated PEDF-R4 (M1-L232), and internally truncated PEDF-R and PEDF-R4 (ΔH203-L232) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the H203-L232 region lost the PEDF affinity that stimulated their enzymatic activity. Cell-surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knockdown PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF/PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell-surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells, and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.Journal of Biological Chemistry 07/2013; · 4.60 Impact Factor
Article: Revisiting the matricellular concept[Show abstract] [Hide abstract]
ABSTRACT: The concept of a matricellular protein was first proposed by Paul Bornstein in the mid-1990s to account for the non-lethal phenotypes of mice with inactivated genes encoding thrombospondin-1, tenascin-C, or SPARC. It was also recognized that these extracellular matrix proteins were primarily counter or de-adhesive. This review reappraises the matricellular concept after nearly two decades of continuous investigation. The expanded matricellular family as well as the diverse and often unexpected functions, cellular location, and interacting partners/receptors of matricellular proteins are considered. Development of therapeutic strategies that target matricellular proteins are discussed in the context of pathology and regenerative medicine.Matrix Biology 07/2014; · 3.65 Impact Factor
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ABSTRACT: Endothelin-1 acts on endothelial cells to enhance mechanical stimulation-induced release of ATP, which in turn can act on sensory neurons innervating blood vessels to contribute to vascular pain, a phenomenon we have referred to as stimulus-dependent hyperalgesia (SDH). In the present study we evaluated the role of the major classes of ATP release mechanisms to SDH: vesicular exocytosis, plasma membrane associated ATP synthase, ATP-Binding Cassette (ABC) transporters, and ion channels. Inhibitors of vesicular exocytosis (i.e., monensin, brefeldin A and bafilomycin), plasma membrane associated ATPase (i.e., oligomycin and pigment epithelium-derived factor-derived peptide 34-mer) and connexin ion channels (carbenoxolone and flufenamic acid), but not ABC transporters (i.e., dipyridamole, nicardipine or CFTRinh-172) attenuated stimulus-dependent hyperalgesia. These studies support a role of ATP in SDH, and suggest novel targets for the treatment of vascular pain syndromes. Endothelin-1 acts on endothelial cells to produce mechanical stimulation-induced hyperalgesia. Inhibitors of three different ATP release mechanisms attenuated this stimulus-dependent hyperalgesia. These studies provide support for a role of ATP in stimulus-dependent hyperalgesia, and suggest novel targets for the treatment of vascular pain syndromes.The journal of pain: official journal of the American Pain Society 04/2014; · 4.22 Impact Factor