This proposal seeks to establish the role of kindlin-3 in physiological and pathophysiological responses. Kindlin-3 (FERMT3) is one of the three member family of FERM domain intracellular adapter proteins, which has >20 binding partners and thereby control numerous responses in numerous cells types. Our contributions to the kindlin field have included the demonstration of their essential role in integrin activation; delineation of the molecular basis for their interaction with integrin beta subunits; identification of a disease, LADIII, associated with severe bleeding, compromised immunity, increased susceptibility to infections and osteopetrosis arising from a deficiency of kindlin-3, demonstration of not only integrin dependent, but also integrin independent functions of kindlins (e.g. perturbed hemostasis and vascular permeability); and the implication of a central role of the kindlins in cancer biology. Adding to the importance of kindlin-3 is evidence that it is not restricted to hematopoietic cells, but is present in and contributes to the biological responses of endothelial cells and cancer cells, particularly to the progression and metastasis of breast cancer. Despite the clear significance of kindlin-3 in vascular biology and pathology, it is this kindlin that has been particularly resistant to analysis arising from the lack of cellular and in vivo models to understand the breadth of its biological functions. We have resolved some of these barriers and are now poised to resolve key questions regarding kindlin-3. We have developed assays and mouse strains in which the molecular basis of kindlin-3?s function can be dissected. Specifically, we have in hand mouse strains in which the integrin-dependent and independent responses can be distinguished (integrin binding site disabled) and tissue specific knockouts of kindlin-3 to delete it from monocyte/macrophages and erythroid cells. We have also developed CRISPR/cas9 technology which has allowed us to knockout or replace kindlin-3 in cancer cells. Importantly, we have identified a unique phosphorylation site in kindlin-3 that is not conserved in the other two kindlins, and have shown that this post-translational event is of functional significance in hematopoietic and breast cancer cells and distinguishes between kindlin-3 binding partners.
Our specific aims are: 1) identify the role of integrin- dependent and -independent functions in breast cancer progression and metastasis; 2) determine the importance of phosphorylation of kindlin-3 in vascular and cancer cells; and 3) determine how kindlin-3 influences erythropoiesis and erythroid cell shape. Overall, these studies will establish basic mechanisms by which kindlin-3 exerts its known functions, may identify previously unappreciated functions of this molecule and may establish if kindlin-3 can serve as a biomarker and as a therapeutic target.

Public Health Relevance

Patient studies have shown that deficiencies of a single molecule, kindlin-3, lead to a variety of symptoms including bleeding, high susceptibility to infections, increased bone density and red cell abnormalities. This same molecule is also expressed at very high levels in human breast cancer tissue and increases the growth and metastasis of breast cancer in mouse models and we seek to determine how this single protein exerts its broad range of effects. Our studies may establish fundamental mechanisms and identify new targets for the diagnosis, treatment and prevention of many diseases in which kindlin-3 exerts its broad effects in cardiovascular disease and in cancer.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL096062-10
Application #
9891081
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
2009-07-01
Project End
2023-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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Pluskota, Elzbieta; Bledzka, Kamila M; Bialkowska, Katarzyna et al. (2017) Kindlin-2 interacts with endothelial adherens junctions to support vascular barrier integrity. J Physiol 595:6443-6462
Bledzka, Kamila; Schiemann, Barbara; Schiemann, William P et al. (2017) The WAVE3-YB1 interaction regulates cancer stem cells activity in breast cancer. Oncotarget 8:104072-104089
Sossey-Alaoui, Khalid; Pluskota, Elzbieta; Bialkowska, Katarzyna et al. (2017) Kindlin-2 Regulates the Growth of Breast Cancer Tumors by Activating CSF-1-Mediated Macrophage Infiltration. Cancer Res 77:5129-5141
Wang, Yunmei; Gao, Huiyun; Shi, Can et al. (2017) Leukocyte integrin Mac-1 regulates thrombosis via interaction with platelet GPIb?. Nat Commun 8:15559
Meller, Julia; Chen, Zhihong; Dudiki, Tejasvi et al. (2017) Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages. JCI Insight 2:
Hirbawi, Jamila; Bialkowska, Katarzyna; Bledzka, Kamila M et al. (2017) The extreme C-terminal region of kindlin-2 is critical to its regulation of integrin activation. J Biol Chem 292:14258-14269

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