The role of nuclear actin dynamics in the regulation of vascular smooth muscle cell inflammation

Abstract

Atherosclerosis is a chronic inflammatory disease which has become a leading cause of mortality and morbidity. Numerous cell types contribute to atherosclerosis development and progression. Vascular smooth muscle cells (VSMC), typically a quiescent cell type, are known to contribute to disease progression through increased rates of proliferation and migration. More recently, VSMCs have been shown to contribute to the inflammatory environment that promotes atherosclerosis, through the activity of the pro-inflammatory transcription factor, NF-kB. The second messenger, cyclic-AMP (cAMP), has anti-inflammatory effects, many of which are attributed to the inhibition of NF-kB activity. However, the mechanisms mediating these effects remain unclear.
Here, I present findings which demonstrate that cAMP-induced elevation of nuclear actin monomer levels inhibits NF-kB activity. Elevation of cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-ActinR62D) suppressed TNFa-induced expression of NF-kB-dependent pro-inflammatory genes and NF-kB-dependent reporter gene activity. Preventing the cAMP-induced elevation of nuclear actin monomer levels, through expression of nuclear localised active mutant of the actin polymerising protein, mDIA, overexpression of the nuclear actin export complex, XPO6, and silencing components of the actin monomer nuclear import complex, IPO9 and CFL1, reversed the inhibitory action of cAMP signaling on NF-kB-dependent reporter gene activity. The inhibitory effects reported here are not mediated through the disruption of RelA/p65 interactions with potential transcriptional co-factors, ACTN4 and MKL1. I show that cAMP-induced elevation of nuclear actin monomer levels promotes accelerated degradation of RelA/p65, which can be reversed using the proteasome inhibitor, MG-132. Correspondingly, stimulation of cAMP signaling increased the association of RelA/p65 with ubiquitin affinity beads, indicative of increased RelA/p65 ubiquitination.
Cumulatively, the data presented in this thesis demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP signaling and highlight the importance of nuclear actin dynamics in the regulation of inflammation through the inhibition of NF-kB activity.

Date of Award	6 Dec 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Harry H Mellor (Supervisor) & Mark Bond (Supervisor)