Hyaluronan (ha)-Cd44 Interactions With Ankyrin And Rho Kinase (rok) Regulate Ip3 Receptor-Mediated Calcium Ion Signaling And Endothelial Cell Function
Date of Award
Doctor of Philosophy (Ph.D.)
Molecular Cell and Developmental Biology
First Committee Member
Theodore Lampidis, Committee Chair
Hyaluronan ((HA)---a major extracellular matrix component) and its membrane binding protein, CD44, are important for aortic endothelium-related vascular integrity, barrier function and angiogenesis. HA binding to CD44 promotes signal transduction and the recruitment of various intracellular signaling molecules including the cytoskeletal protein ankyrin and rho kinase leading to a variety of cellular functions. Aortic endothelial cells express an alternatively spliced form of CD44 called CD44v10. Phospholipase C (PLC) activity hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) to diacylglycerol DAG) and inositol-1,4,5-triphosphate (IP3). IP3 then binds to the IP3 receptor (an intracellular IP3-gated Ca2+ channel) and induces Ca 2+ release from intracellular Ca2+ storage sites. Using PLC and IP3 receptor inhibitors, we have determined that HA binding to CD44v10 induces a PLC- and IP3 receptor-dependent increase in intracellular Ca2+ concentration in endothelial cells.In addition, we have demonstrated that HA treatment of aortic endothelial cells recruits CD44v10, the cytoskeletal protein, ankyrin, and the IP 3 receptor into cholesterolcontaining plasma membrane microdomains (called lipid rafts or caveolae). In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for binding to both the cytoplasmic domain of CD44 and the IP3 receptor leading to ankyrin/IP3 receptor membrane colocalization and Ca2+ mobilization. In addition, overexpression of ARD or disruption of lipid rafts by a cholesterol-depletion drug inhibits HA-induced recruitment of ankyrin and IP3 receptor into CD44v10-containing lipid rafts. The loss of ankyrin/IP3 receptor membrane colocalization impairs HA-stimulated Ca2+ mobilization and Ca2+-dependent endothelial cell function [i.e. nitric oxide (NO) production, adhesion and proliferation].Furthermore, we have found that HA treatment of endothelial cells induces Rho signaling and Rho kinase (ROK) activity in a CD44v10-specific manner. Our results indicate that ROK phosphorylates the IP3 receptor; and the phosphorylated IP3 receptor induces Ca2+ mobilization. Overexpression of a dominant-negative form of ROK inhibits HA-induced IP3 receptor phosphorylation, Ca2+ signaling and subsequent endothelial cell migration. These observations support the notion that CD44v10-ROK interaction is involved in regulating IP3 receptor-mediated Ca2+ signaling required for HA-mediated endothelial cell migration. Taken together, our findings suggest that CD44v10 interaction with important signaling molecules (i.e. the cytoskeletal protein ankyrin and ROK) plays a pivotal role in regulating IP3 receptor-mediated Ca2+ signaling and Ca2+-dependent endothelial cell functions. We believe that this information has provided new insights toward a better understanding of endothelial biology underlying the clinical behaviors of HA/CD44 and IP3 receptor/Ca2+ signaling-related cardiovascular disease and pathological angiogenesis.
Biology, Molecular; Biology, Cell
Singleton, Patrick A., "Hyaluronan (ha)-Cd44 Interactions With Ankyrin And Rho Kinase (rok) Regulate Ip3 Receptor-Mediated Calcium Ion Signaling And Endothelial Cell Function" (2003). Dissertations from ProQuest. 2037.