Dwayne G. Stupack

Get a ligand, get a life: integrins, signaling and cell survival

Programmed cell death is crucial for the development and maintenance of multicellular organisms. The decision to live, or to die, depends, at the cellular level, upon the cells interaction with extracellular cues that trigger cell signaling pathways promoting survival or death. The extracellular matrix (ECM) influences the execution of the apoptotic program through the actions of adhesion receptors. Among these, integrins initiate a variety of downstream signaling events in response to ECM ligation. Integrins directly activate survival pathways via the PI 3-kinase and MAPK pathways and act as essential cofactors for their stimulation by growth factors. Conversely, elevated integrin expression in the absence of appropriate ligands, or in the presence of natural or synthetic antagonists, can promote apoptosis under otherwise permissive growth conditions. Integrins thus act in a crucial biosensory role, coordinating survival or death responses as a function of ECM composition. This dual function provides an elegant mechanism through which tissue-remodeling events may regulate cell death or survival in a temporal, ECM-governed manner.

Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins.

Integrin-mediated adhesion promotes cell survival in vitro, whereas integrin antagonists induce apoptosis of adherent cells in vivo. Here, we demonstrate that cells adherent within a three-dimensional extracellular matrix undergo apoptosis due to expression of unligated integrins, the β subunit cytoplasmic domain, or its membrane proximal sequence KLLITIHDRKEF. Integrin-mediated death requires initiator, but not stress, caspase activity and is distinct from anoikis, which is caused by the loss of adhesion per se. Surprisingly, unligated integrin or β integrin tails recruit caspase-8 to the membrane, where it becomes activated in a death receptor–independent manner. Integrin ligation disrupts this integrin–caspase containing complex and increases survival, revealing an unexpected role for integrins in the regulation of apoptosis and tissue remodeling.

Decreased angiogenesis and arthritic disease in rabbits treated with an αvβ3 antagonist

Rheumatoid arthritis (RA) is an inflammatory disease associated with intense angiogenesis and vascular expression of integrin αvβ3. Intra-articular administration of a cyclic peptide antagonist of integrin αvβ3 to rabbits with antigen-induced arthritis early in disease resulted in inhibition of synovial angiogenesis and reduced synovial cell infiltrate, pannus formation, and cartilage erosions. These effects were not associated with lymphopenia or impairment of leukocyte function. Furthermore, when administered in chronic, preexisting disease, the αvβ3 antagonist effectively diminished arthritis severity and was associated with a quantitative increase in apoptosis of the angiogenic blood vessels. Therefore, angiogenesis appears to be a central factor in the initiation and persistence of arthritic disease, and antagonists of integrin αvβ3 may represent a novel therapeutic strategy for RA.

Src-mediated coupling of focal adhesion kinase to integrin αvβ5 in vascular endothelial growth factor signaling

Vascular endothelial growth factor (VEGF) promotes vascular permeability (VP) and neovascularization, and is required for development. We find that VEGF-stimulated Src activity in chick embryo blood vessels induces the coupling of focal adhesion kinase (FAK) to integrin αvβ5, a critical event in VEGF-mediated signaling and biological responsiveness. In contrast, FAK is constitutively associated with β1 and β3 integrins in the presence or absence of growth factors. In cultured endothelial cells, VEGF, but not basic fibroblast growth factor, promotes the Src-mediated phosphorylation of FAK on tyrosine 861, which contributes to the formation of a FAK/αvβ5 signaling complex. Moreover, formation of this FAK/αvβ5 complex is significantly reduced in pp60c-src-deficient mice. Supporting these results, mice deficient in either pp60c-src or integrin β5, but not integrin β3, have a reduced VP response to VEGF. This FAK/αvβ5 complex was also detected in epidermal growth factor-stimulated epithelial cells, suggesting a function for this complex outside the endothelium. Our findings indicate that Src can coordinate specific growth factor and extracellular matrix inputs by recruiting integrin αvβ5 into a FAK-containing signaling complex during growth factor–mediated biological responses.

Integrins and Angiogenesis

The growth of new blood vessels is a dynamic yet highly regulated process that depends on coordinated signaling by growth factor and cell adhesion receptors. As part of the molecular program regulating angiogenesis, endothelial cells acquire a proliferative and invasive phenotype but also show increased susceptibility to apoptotic stimuli. Integrins are the principle adhesion receptors used by endothelial cells to interact with their extracellular microenvironment, and integrin-mediated interactions play a critical role in regulating cell proliferation, migration, and survival. Alterations in the repertoire and?or activity of integrins, as well as the availability and structural property of their ligands, regulate the vascular cell during the growth or repair of blood vessels.

Potentiation of neuroblastoma metastasis by loss of caspase-8.

Neuroblastoma, the most common paediatric solid tumour, arises from defective neural crest cells. Genetic alterations occur frequently in the most aggressive neuroblastomas. In particular, deletion or suppression of the proapoptotic enzyme caspase-8 is common in malignant, disseminated disease, although the effect of this loss on disease progression is unclear. Here we show that suppression of caspase-8 expression occurs during the establishment of neuroblastoma metastases in vivo, and that reconstitution of caspase-8 expression in deficient neuroblastoma cells suppressed their metastases. Caspase-8 status was not a predictor of primary tumour growth; rather, caspase-8 selectively potentiated apoptosis in neuroblastoma cells invading the collagenous stroma at the tumour margin. Apoptosis was initiated by unligated integrins by means of a process known as integrin-mediated death. Loss of caspase-8 or integrin rendered these cells refractory to integrin-mediated death, allowed cellular survival in the stromal microenvironment, and promoted metastases. These findings define caspase-8 as a metastasis suppressor gene that, together with integrins, regulates the survival and invasive capacity of neuroblastoma cells.

A homing mechanism for bone marrow–derived progenitor cell recruitment to the neovasculature

CD34+ bone marrow-derived progenitor cells contribute to tissue repair by differentiating into endothelial cells, vascular smooth muscle cells, hematopoietic cells, and possibly other cell types. However, the mechanisms by which circulating progenitor cells home to remodeling tissues remain unclear. Here we show that integrin alpha4beta1 (VLA-4) promotes the homing of circulating progenitor cells to the alpha4beta1 ligands VCAM and cellular fibronectin, which are expressed on actively remodeling neovasculature. Progenitor cells, which express integrin alpha4beta1, homed to sites of active tumor neovascularization but not to normal nonimmune tissues. Antagonists of integrin alpha4beta1, but not other integrins, blocked the adhesion of these cells to endothelia in vitro and in vivo as well as their homing to neovasculature and outgrowth into differentiated cell types. These studies describe an adhesion event that facilitates the homing of progenitor cells to the neovasculature.

Integrin αvβ1 Is an Adenovirus Coreceptor

ABSTRACT The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor αvβ3 or αvβ5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin αvβ1. Function-blocking antibodies directed against αv or β1, but not β3, β5, or α5, integrin subunits block Ad infection and viral endocytosis. Therefore, αvβ1 serves as a coreceptor for Ad infection, and the lack of β3 and/or β5 but the relatively high expression of αvβ1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

Integrin αVβ3mediates diverse responses in vascular cells, ranging from cell adhesion, migration, and proliferation to uptake of adenoviruses. However, the extent to which αVβ3 is regulated by changes in receptor conformation (affinity), receptor diffusion/clustering (avidity), or post-receptor events is unknown. Affinity regulation of the related integrin, αIIbβ3, has been established using a monovalent ligand-mimetic antibody, PAC1 Fab. To determine the role of affinity modulation of αVβ3, a novel monovalent ligand-mimetic antibody (WOW-1) was created by replacing the heavy chain hypervariable region 3 of PAC1 Fab with a single αV integrin-binding domain from multivalent adenovirus penton base. Both WOW-1 Fab and penton base bound selectively to activated αVβ3, but not to αIIbβ3, in receptor and cell binding assays. αVβ3 affinity varied with the cell type. Unstimulated B-lymphoblastoid cells bound WOW-1 Fab poorly (apparent K d = 2.4 μm), but acute stimulation with phorbol 12-myristate 13-acetate increased receptor affinity >30-fold (K d = 80 nm), with no change in receptor number. In contrast, αVβ3 in melanoma cells was constitutively active, but ligand binding could be suppressed by overexpression of β3 cytoplasmic tails. Up-regulation of αVβ3 affinity had functional consequences in that it increased cell adhesion and spreading and promoted adenovirus-mediated gene transfer. These studies establish that αVβ3 is subject to rapid regulated changes in affinity that influence the biological functions of this integrin.

ECM Remodeling Regulates Angiogenesis: Endothelial Integrins Look for New Ligands

The process of angiogenesis is a dynamic one. Vascular endothelial cells are changing at the same time the extracellular matrix is being remodeled. Stupack and Cheresh discuss how remodeling of the extracellular matrix (ECM) and changes in the endothelial cell protein production and integrin expression contribute to the complex process of new blood vessel growth from an existing vascular bed.