Mitogen activated protein kinase phosphatase-1 (MKP-1) is an enzyme that dephosphorylates MAPK, which act as master regulators of cellular behavior. MKP-1 canonically acts as an off-switch for the MAPK;however, in angiogenesis, the formation of new blood vessels by endothelial cell (EC) sprouting, MKP-1 was found to act as an on-switch, enabling EC migration. Since the MAPK mediate pro-angiogenic processes such as migration and proliferation, these observations suggest that MKP-1 may contribute to angiogenesis independently of its canonical MAPK phosphatase activity. Previous reports from the applicant's laboratory identified histone H3, a protein that aids in packaging of DNA, as a novel substrate of MKP-1. Together, these data suggest that MKP-1 may facilitate angiogenesis by transcriptional control of angiogenic genes through histone modification. The overall objective of this project is therefore to elucidate the role of MKP-1 in angiogenesis by identifyin the mechanism of MKP-1-mediated angiogenesis in vitro and evaluate its effects in clinically relevant animal models of vascular injury, growth, and disease.
The first aim of this proposal is to characterize the role and mechanism of endothelial MKP-1 in vascular endothelial growth factor (VEGF)-induced angiogenesis in vitro by testing the hypothesis that MKP-1 positively mediates angiogenesis and regulates expression of pro-angiogenic genes such as fractalkine by modifying histones. The role of MKP-1 in both human and mouse EC migration, proliferation, and tube formation will be evaluated by MKP-1 depletion as well as exogenous reconstitution in human cells. Preliminary data demonstrated that MKP-1 immunoprecipitates with exonic DNA of the angiogenic gene fractalkine and is required for its induction by VEGF. This proposal will evaluate the mechanism by which MKP-1 interacts with fractalkine DNA by chromatin immunoprecipitation, and will identify other potential MKP-1-regulated pro-angiogenic genes by protein array screening.
The second aim of this proposal is to evaluate the role of MKP-1 in angiogenesis in vivo by testing the hypotheses that MKP-1 positively mediates recovery from ischemic injury, neovascular growth through angiogenesis, and pathological angiogenesis. These will be evaluated using MKP-1 knockout mouse models of hind limb ischemia, VEGF-induced subcutaneous Matrigel plug angiogenesis, and B16 melanoma tumor growth and angiogenesis. The MKP-1-dependence of fractalkine induction in vivo will be evaluated by immunohistochemistry and real-time quantitative PCR. Successful completion of these aims will enhance our fundamental knowledge of the mechanisms governing therapeutic and pathological angiogenesis, and will identify a novel role for MKP-1 in angiogenic gene regulation, which may lead to improved therapeutic strategies for stimulating or inhibiting angiogenesis.
The preliminary and proposed research contained in this proposal seek to enhance our fundamental knowledge of the basic physiological mechanisms that regulate the process of new blood vessel formation and aim to apply that knowledge to enhance health, lengthen life, and reduce the burdens of myriad angiogenesis- dependent conditions including metastatic and non-metastatic cancers, revascularization of ischemic tissues, inflammatory conditions, pulmonary hypertension, and blinding eye diseases. To date, these mechanisms remain incompletely understood and current clinical strategies are limited. Particularly, this work will elucidate the function and mechanism of the regulatory molecule, MKP-1, in the process of new blood vessel formation. !
|Boerckel, Joel D; Chandrasekharan, Unnikrishnan M; Waitkus, Matthew S et al. (2014) Mitogen-activated protein kinase phosphatase-1 promotes neovascularization and angiogenic gene expression. Arterioscler Thromb Vasc Biol 34:1020-31|