By serving as the major conduits for communication between the interior of cells and the extracellular environment, integrin ?/ heterodimeric adhesion receptors are indispensable for many cellular responses including adhesion, migration, proliferation and survival. On vascular cells, optimal integrin function depends on thei ability to undergo activation, a transition from a low to a high affinity/avidity state for their cognate ligands. Such activation of integrins depends on the binding of proteins to the short cytoplasmic tail of the integrin subunit. Over the past 5 years, information has exploded to indicate that kindlins, a three member family of FERM domain proteins, are essential for appropriate integrin activation. This statement is supported by numerous in vitro studies, the phenotypes of mice in which the genes of each of the kindlins has been inactivated, and the pathologies associated with the deficiencies of the kindlins in humans. Humans with deficiencies of kindlin-3 (K3), the focus of this application, present severe bleeding, compromised immunity leading to increased susceptibility to infections, osteopetrosis and abnormal erythrocytes. Despite the clear significance of K3 in vascular biology and pathology, it is the kindlin whose structure- function relationships are least understood at a molecular level. Adding to the uncertainties regarding the roles of K3 is our recent findings that K3 is not restricted to hematopoietic cells but is also expressed in endothelial cells and breast cancer cells where it influences the properties of both cell types. The goal of this application is to provide new insights into the structure, function and biology K3 using molecular, cellular and in vivo approaches. Our driving hypothesis is that K3 has both integrin-dependent and integrin-independent functions, which allows it to mediate responses in vascular cells, including endothelial cells, erythrocytes and cancer cells. To test this hypothesis, unique mouse models (knock-in mice expressing an integrin defective K3 as well as the potential to knock-out K3 in specific tissues) and cell models where K3 can be expressed and support integrin activation. With these tools in hand, the following specific aims are proposed.
Aim 1 : Analysis of the role of specific subdomains of K3, its posttranslational modification, and its interaction with established as well as previously unconsidered binding partners, ADAP and actin, in controlling its function.
Aim 2 : Using our unique mouse strains, the role of K3 in endothelial cell responses, including its ability to mediate angiogenesis, and in controlling erythroid maturation will be dissected. In each case, it will be determined whether the roles of K3 are integrin-dependent and independent.
Aim 3 : With compelling preliminary data showing that K3 is a breast tumor promoter, the effects of K3 manipulation on tumor growth and metastasis will be evaluated in murine models, and its correlation with breast tumor grade and subtype will be assessed. Overall, these studies will address unresolved issues regarding the mechanism-of-action and identify previously unconsidered functions of K3.
Studies of humans have demonstrated that a deficiency of a single molecule, kindlin-3, leads to a variety of symptoms including bleeding, high susceptibility to infections, increase bone density and red cell abnormalities. This same molecule is also expressed at very high levels in human breast cancer tissue and furthermore increases the growth and metastasis of breast cancer in mouse models. We seek to determine how this single molecule exerts its broad range of functions. Our studies may identify new targets for the diagnosis, treatment and prevention of many diseases in which kindlin-3 exerts its effects ranging from cardiovascular disease to cancer.
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