THE GRADUATE COLLEGE OF THE
UNIVERSITY OF OKLAHOMA HEALTH SCIENCES CENTER
ANNOUNCES THE FINAL EXAMINATION OF
KYLE GRADY INGRAM
FOR THE DEFENSE OF THE DOCTOR OF PHILOSOPHY DEGREE
GRADUATE COLLEGE
DEPARTMENT OF CELL BIOLOGY
Monday, June 15, 2015, 2:00 P.M.
Room 109, Biomedical Research Center, OUHSC
The NuRD Chromatin-Remodeling Complex Regulates
Vascular Stability During Embryonic Development
COMMITTEE IN CHARGE: Courtney Griffin, Ph.D., Chair, Eric
Howard, Ph.D., Lorin Olson, Ph.D., Florea Lupu, Ph.D., Ralf Janknecht, Ph.D., Hong Chen,
Ph.D.
ABSTRACT: CHD4 is a catalytic subunit of the nucleosome remodeling and
histone deacetylase (NuRD) complex, which couples histone deacetylation and
chromatin-remodeling activities to repress or promote transcription of specific
target genes. To characterize the role of CHD4 and NuRD in murine vascular
development, we conditionally depleted Chd4 from vascular endothelium using a
Tie2-Cre transgenic line.
Chd4 mutant embryos died at embryonic day 11.5
(E11.5) due to vascular rupture of the dorsal aorta and cardinal veins. We found
two critical mediators of extracellular matrix (ECM) stability were misregulated in
mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR
or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated.
Misregulation of these genes resulted in increased activity of the extracellular
plasminogen activation pathway, causing increased ECM degradation. Genetic
reduction of the uPAR ligand urokinase (uPA; Plau) rescued ECM degradation and
vascular rupture of Chd4 mutant embryos. In addition to ECM degradation, we
also observed endothelial cell (EC) necrosis and upregulation of the receptorinteracting protein kinase (RIPK3) in our Chd4 mutant embryos. RIPK3 is the
most distinctive biochemical marker of programmed necrosis (necroptosis), a
recently discovered form of programmed necrosis induced by extrinsic and
intrinsic cell death signaling events. We found that Ripk3 expression is regulated
by an anchorage-dependent mechanism in the vascular endothelium. Loss of the
vascular extracellular matrix (ECM) by excessive proteolysis or impaired
deposition resulted in Ripk3 upregulation, endothelial cell necroptosis, and
embryonic lethality due to vascular rupture at midgestation. Genetic reduction of
Ripk3 partially rescued ECM-deficient mutants—including Chd4 mutants—from
vascular rupture. Matrix detachment resulted in increased Ripk3 transcription in
cultured human umbilical vein endothelial cells (HUVECs). Moreover, inhibition
of the matrix receptor
death that could be rescued with the necroptosis inhibitor necrosulfonamide,
indicating that necroptotic cell death prior to loss of cellular adhesion is integrindependent. Altogether, these studies demonstrate that CHD4/NuRD influences
endothelial cell-ECM interactions, which play an essential role in mediating
embryonic vascular integrity by regulating Ripk3 expression and preventing
endothelial cell necroptosis.
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