Functional role of extracellular matrix proteins and their receptors in apoptosis and cell survival

• Main Author: Cardó i Vila, Marina
• Other Authors: Celada Cotarelo, Antonio
• Format: Doctoral Thesis
• Language: English
• Published: Universitat de Barcelona 2003
• Subjects: Ciències Experimentals i Matemàtiques; 576
• Online Access: http://hdl.handle.net/10803/3009; http://nbn-resolving.de/urn:isbn:8468850217

Macrophages require presence of M-CSF to survive and proliferate. Incubation of bone marrow macrophages with soluble factors (LPS or IFN-gamma) or insoluble factors such as extracellular matrix proteins (Decorin, FN) macrophage proliferation was blocked. Moreover, pretreatment of macrophages with IFN-gamma protects from apoptosis induced by several stimuli. Inhibition of p21Waf with antisense oligonucleotides or using null mice showed that the induction of p21Waf by IFN-gamma mediate this protection. Thus, IFN-gamma makes macrophages unresponsive to apoptotic stimuli by inducing p21Waf and arresting the cell cycle at the G1/S boundary. The insoluble factors, decorin and fibronectin, inhibit macrophage proliferation through p27kip1 expression and the modification of ERK activity. Decorin treated macrophages but not fibronectin protect from apoptosis mechanism that require p21Waf expression. Decorin enhances the IFNgamma-induced expression of IA-alpha and IAß MHC class II genes. Moreover, it increases the IFN-gamma or LPS-induced expression of inducible NO synthase, TNF-alpha, IL-1ß, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effect of decorin on macrophage activation is explained by its ability to block the binding of autocrine-produced TGFß on the surface of macrophages.These soluble and insoluble factors modulate the cell response through the interaction with surface receptors. Cell surface receptors of the integrin family are important regulators of the cell behavior. ß5 cytoplasmic domain has been reported to control cell migration and proliferation. Certain postadhesion are regulated through a pathway that requires both avß5 and PKC activity.Extensive data have been reported on the use of phage libraries to identify ligands. The large molecular diversity represented in phage peptide libraries facilitates the identification of motifs that map to protein interaction sites. Here we introduced an approach based on phage display technology to identify molecules that specifically interact with the cytoplasmic of the ß5 integrin subunit. We showed that a peptide that mimics annexin V binds to the cytoplasmic domain and triggers apoptosis. Annexin V is a cytosolic signaling protein known to inhibit PKC activity, and we demonstrated that annexin-V only binds to active form of PKC. Induction of apoptosis by this peptide is modulated by growth factors and by PKC antagonist. Caspase activity and the expression of ß5 integrin are also required.Caspases play an important role on apoptosis. XIAP functions as a caspase inhibitor and is a member of the inhibitors of apoptosis (IAP) family of proteins. All of the members of the IAP have been shown to inhibit programmed cell death. The human IAP family members bind to caspase 3 and caspase 7 with inhibitory constant values. We have selected peptides from a phage display library by using recombinant full-length human XIAP. A consensus motif was recovered from two independent screenings by using different libraries. Phage displaying variations of the consensus sequence bound specifically to the BIR2 domain of XIAP but not to other IAPs. Protein-protein interaction assays revealed that caspase-3 and -7 blocked the binding of the XIAP-binding phage to XIAP, indicating that this peptide targets a domain within XIAP that is related to the caspase-binding site. We also demonstrated that an internalizing version of the XIAP-binding peptide identified in our screenings could induce apoptosis in leukemia cells.Using a new approach for the screening by phage display technology we also characterize cells surface receptors in endothelial cell activation and proliferation. The molecular diversity in human blood vessels remains largely unexplored. We developed a selection method in which peptides that home to specific vascular beds are identified after administration of a peptide library. These data represents a step toward the construction of a molecular map of human vasculature and may have broad implications for development of targeted therapies.