The establishment of new blood vessels, a process known as angiogenesis, promotes tumor progression by providing tumor cells the necessary nutrients and signals for survival. Our recent work has provided support for the notion that the a4 laminin subunit (alpha4LN), a component of the basement membrane secreted by endothelial cells, is crucial for blood vessel development through a novel interaction with alphavbeta3 integrin. There are three aims in the proposal.
In aim 1, integrin recognition motif(s) within the G domain of the alpha4LN subunit will be characterized by using recombinant proteins and classical biochemical analyses or by phase display technique. In the second aim, the molecular mechanisms regulating the affinity of alphavbeta3 integrin for the alpha4LN subunit will be evaluated.
This aim i s based on a series of preliminary data indicating 1) that there is crosstalk between (1containing integrins and the alphavbeta3 integrin heterodimer in endothelial cells 2) that such crosstalk regulates the affinity of alphavbeta3 integrin for the (4LN subunit and 3) a PKA inhibitor abrogates the crosstalk. Based on these data, we will determine whether specific serine/threonine phosphorylation events on the cytoplasmic tails of alphavbeta3 integrin or, possibly an alphavbeta3 integrin associated protein, negatively regulate the affinity of alphavbeta3 integrin for alpha4LN.
Aim 3 is based on studies which indicate that apoptosis plays a role in tube-like network assembly of endothelial cells in vitro on Matrigel and that such regulated apoptotic events can be inhibited by an excess of alpha4LN ligand. To further this analysis, the role of regulated apoptosis during tube-like network assembly by endothelial cells will be evaluated in vitro. To do so, endothelial cells will be stained with markers for apoptosis during their morphogenesis in vitro to assess where apoptosis occurs spatially during tube assembly. In addition, the ability of alphavbeta3-integrin interactions to regulate endothelial cell survival pathways during tube formation using blocking or activating ligand and integrin antibodies, peptide agonists and antagonists, and by molecular genetic approaches will be assessed. Together these results will provide new insight into the molecular mechanisms that regulate angiogenesis and, long term, may uncover useful reagents that can block tumor development by inhibiting their ability to develop a vasculature.
|Gonzalez, Annette M; Bhattacharya, Ramona; deHart, Gregory W et al. (2010) Transdominant regulation of integrin function: mechanisms of crosstalk. Cell Signal 22:578-83|
|Gonzalez, Annette M; Claiborne, Jessica; Jones, Jonathan C R (2008) Integrin cross-talk in endothelial cells is regulated by protein kinase A and protein phosphatase 1. J Biol Chem 283:31849-60|