Gauchat, J.-F. | bioRxiv Science

bioRxiv2025

ARF6 controls VSMC cell phenotypic switching upon lipid stimulation to promote inflammatory signaling contributing to the progression of atherosclerosis

Vascular smooth muscle cells (VSMCs) play a pivotal role in the development and progression of atherosclerosis. Traditionally viewed as contractile cells that maintain vascular tone and structure, VSMCs undergo phenotypic switching in response to atherogenic stimuli, such as high circulating levels of LDL, thus adopting synthetic, osteogenic, or macrophage-like phenotypes. This plasticity contributes to plaque formation, extracellular matrix remodeling, and inflammatory signaling. We have previously shown that ADP-ribosylation factor 1 (ARF1), a small GTP-binding protein, regulates the expression and function of actin, which is important for maintaining the contractile phenotype of VSMCs. However, the role of ARF6 in phenotypic switching remains to be elucidated. Here, we demonstrate that ARF6 knockdown in human aortic smooth muscle cells (HASMCs) reduced lipid uptake through alterations in the expression of scavenger receptors (LOX-1, MSR1), cytokine production (IL-6) as well as modulation of inflammatory markers and pathways (adhesion molecules, PI3K, NFkB, p38). To confirm our findings in an in vivo setting, we engineered a novel conditional smooth muscle cell-specific ARF6 KO mouse in an atheroprone background (Acta2-Cre-ERT2+/-/ApoE-/-/Arf6f/f mice). Mice were fed a high-fat diet to accelerate plaque formation. ARF6 knockout resulted in a significant reduction of atherosclerotic lesions in the aortic arch, which was associated with a reduction of collagen and foam cells. Furthermore, we observed that ARF6 regulates the expression of inflammatory markers. These findings highlight the importance of ARF6 expression in VSMCs and its role in the pathogenesis of atherosclerosis.

biochemistry↗

bioRxiv2024

A proximity ligation screen identifies SNAT2 as a novel target of the MARCH1 E3 ubiquitin ligase

E3 ubiquitin ligases are part of various families of proteins and include hundreds of members, which play key roles in all aspects of cell biology. They generally regulate the half-life of other proteins but can also modulate their cellular localization and functions. The MARCH family of ubiquitin ligases is composed of 11 members and two closely related proteins, MARCH1 and MARCH8, share similar targets, while being active in different cell types. Although they appear to target principally immune cell components, such as MHC class II molecules and the co-stimulatory molecule CD86, the repertory of their targets remains to be fully documented. Here, to further define the MARCH1s interactome, we adapted a proximity-dependent biotin identification (BioID)-based screening approach in live HEK293 cells. We transfected a fusion protein consisting of mouse MARCH1 linked to YFP at its N-terminus and to the biotin ligase of Aquifex aeolicus at its C-terminus. Upon transient overexpression of this construct in the presence of exogenous biotin, we could recover biotinylated proteins that are presumably found within 10nm of MARCH1. To help in the identification of bona fide down-regulated specific targets, we compared MARCH1s interactome with the one obtained using a ubiquitination-deficient MARCH1 mutant (MARCH1W104A). CD98 and CD71, two previously described targets of MARCH1, were identified in this screen. Of 16 other biotinylated proteins identified by semi-quantitative mass spectrometry, 10 were tested directly by flow cytometry to monitor their expression in the presence or absence of transfected MARCH1. The protein levels of five of these endogenous targets, CD29, CD112, NKCC1, CD147 and SNAT2, confirmed their negative regulation by MARCH1 in this system. SNAT2 was particularly sensitive to the presence of MARCH1 and was found to be ubiquitinated on Western blots following immunoprecipitation. Thus, BioID2 is an effective mean of characterizing the interactome of MARCH1 and the identification of SNAT2 suggests a role of this ubiquitin ligase in cellular metabolism.

immunology↗