Molecular Mechanism in Thrombospondin 1 Mediated Antiangiogenesis

Gage-Heimark 225X190

Project Co-leaders

NAU

Matthew J. Gage, PhD, Assistant Professor

 

UACC 

William R. Montfort, PhD, Professor

 

 Abstract

Vascular endothelial growth factor A (VEGF-A) is key for developmental angiogenesis and a major drug target for inhibiting angiogenesis in cancer. Nitric oxide (NO) signaling is also required for angiogenesis, suggesting this pathway may also provide for an interventional opportunity. Thrombospondin 1 (TSP1) was the first identified endogenous inhibitor of angiogenesis, but the mechanism underlying this activity is still unclear. TSP1 was recently discovered to inhibit NO signaling through a mechanism involving binding to receptor CD47, which constitutively binds VEGFR, the VEGF-A receptor, in an interaction that is disrupted upon binding of TSP1, rendering VEGFR inactive. Thus, TSP1 represents an outstanding new target for intervention in angiogenesis-dependent cancer proliferation, as well as for cardiovascular disease and wound healing abnormalities, but little is known about how it functions. This is a proposal for a full project through the Partnership for Native American Cancer Prevention (NACP) that extends from a NACP-funded pilot project initiated 15 months ago. In our pilot study, we discovered a new arm of NO regulation, that of extracellular inhibition of sGC through a mechanism requiring calcium, thus linking two major signaling pathways, those requiring NO and those requiring calcium. We also found that TSP1 inhibition of NO signaling is by inducing an increase in cytosolic calcium upon binding to CD47. Here, we propose uncovering the binding surfaces between CD47, TSP1 and VEGFR, and the means by which binding leads to inhibition of VEGF and NO signaling. First, we intend use a combination of RNAi, mutagenesis, microarray analysis, quantitative PCR and cellular calcium imaging to determine how TSP1 binding to CD47 inhibits the NO receptor. Second, we intend to use FRET analysis and mutagenesis to map the interaction surfaces between VEGFR, CD47 and TSP1. Finally, we intend to use calcium, cGMP and NO imaging to uncover how the VEGFR, TSP1 and NO pathways are interrelated in endothelial cells. The answers to these questions will provide the background necessary for new drug discovery.

Specific Aims

  1. Discovering how TSP1 binding to CD47 inhibits the nitric oxide receptor. TSP1 is a trimericmatricellular protein of ~450 kDa. CD47 is transmembrane glycoprotein of ~50 kDa that interacts with integrins and may function as a non-canonical G-protein-coupled receptor (GPCR). Very little is known about signal transduction from TSP1 through CD47 into the cytosol, or how the resulting calcium pulse inhibits the nitric oxide receptor, soluble guanylyl cyclase (sGC). We will use a combination of RNAi, pharmacological intervention, mass spectrometry, microarray analysis, mutagenesis, quantitative PCR and cellular calcium imaging to uncover the details of the signaling pathway.
  2. Discovering VEGFR, CD47 and TSP1 binding interactions. The binding surface between CD47 and VEGFR is unknown but apparently required for VEGF-A signaling in endothelial cells. Binding of TSP1 releases and inactivates VEGFR. To uncover the details behind these events, we will employ fluorescent imaging, FRET analysis and mutagenesis.
  3. Uncovering the intersection of VEGFR, TSP1 and NO signaling pathways in endothelial cells. The interplay between VEGFR, calcium and autocrine NO signaling pathways is unclear at present but key for endothelial cell migration and angiogenesis. We will uncover how these pathways are integrated by developing cGMP, calcium and NO imaging tools in a single cell.