Defining the function of C/EBP-beta and its downstream target PSMB9 in atherosclerosis

PROJECT SUMMARY/ABSTRACT
Despite countless significant advances in the field of cardiovascular disease, it remains the number one
cause of death worldwide. Several systemic risk factors which increase disease risk and include hypertension,
high cholesterol, obesity, diabetes, smoking, and chronic inflammation. An additional local risk factor is disturbed
blood flow (d-flow), which activates the endothelium to permit of atherosclerosis, and is accelerated when other
risk factors are present. Despite our best efforts to treat these conditions to improve outcomes, cardiovascular
disease remains the number one killer, which suggests that there remain residual unaddressed risk factors.
We have identified through previous studies using the partial carotid ligation (PCL) model of d-flow
causing atherogenesis that CCAAT/Enhancer Binding Protein β (C/EBPβ), a protein previously identified to play
critical roles in development, cell differentiation, and inflammation, was upregulated at the mRNA level in d-flow
exposed inflamed endothelial cells in vivo. We found in orthogonal studies that C/EBPβ was upregulated at the
protein level in regions of d-flow, and that the compared to regions of stable flow, that the protein had localized
to the nucleus, suggested that it was playing a role in downstream signaling and transcription. Three isoforms
comprise C/EBPβ, which is translated from a single intron. The first isoform is LAP*, the full length protein, with
activating and repression domains, the second isoform is LAP, which is nearly identical to LAP* but lacks 23
amino acids (and one of the activating domains) at the N terminal region. The short isoform is LIP, which lacks
all activating domains and is thought to have a repressive role on the function of LAP* and LAP. We found that
flag-tagged LIP isoform co-immunoprecipitated with the Protein Subunit Beta type-9 immunoproteasome
component (PSMB9, alternatively named LMP2) in vitro, which notably, has also been implicated in
atherosclerosis. Finally, we found that in contrast to stable flow, d-flow induced PSMB9 activity in vitro.
Given these findings, we will set out to define the role of C/EBPβ and PSMB9 in atherosclerosis. We will
determine whether C/EBPβ and PSMB9 are required for endothelial cell inflammation caused by d-flow in vitro
using CRISPR-i and small molecule inhibition. We will employ a mouse model of endothelial-specific C/EBPβ or
PSMB9 deletion combined with PCL and hypercholesterolemia to determine if these proteins are required by
endothelial cells for the propagation of disturbed blood flow leading to endothelial inflammation and
atherosclerosis in vivo. Finally we will determine whether those two proteins are over-expressed in endothelial
cells from atherosclerotic regions compared to healthy control regions in human coronary arteries. We
hypothesize that C/EBPβ is a key protein required for atherosclerosis, and that it exerts its pro-atherogenic
effects by binding and activating the downstream PSMB9 immunoproteasome.
We hope that our findings will further elucidate unaddressed risk factors which contribute to
cardiovascular disease, and will lead to improved treatments for the number one cause of death.

Grant Number: 1F32HL182304-01
NIH Institute/Center: NIH
Principal Investigator: Jay Bowman-Kirigin