Original article
Sam68 impedes the recovery of arterial injury by augmenting inflammatory response

https://doi.org/10.1016/j.yjmcc.2019.10.003Get rights and content

Highlights

  • Deletion of Sam68 leads to accelerated re-endothelialization and attenuated neointima hyperplasia in injured carotid arteries.

  • Deletion of Sam68 results in a lowered expression of pro-inflammatory cytokines in injured arteries and inflamed macrophages.

  • In macrophages, Sam68 promotes TNF-α–induced NF-κB activation by interacting with TRAF2 and cytoskeleton protein Filamin A.

  • Upon TNF-α stimulation, Sam68 potentiate NF-κB activation to exacerbate inflammation, impede recovery, and worsen remodeling.

Abstract

Objective

The role of Src-associated-in-mitosis-68-kDa (Sam68) in cardiovascular biology has not been studied. A recent report suggests that Sam68 promotes TNF-α–induced NF-κB activation in fibroblasts. Here we sought to dissect the molecular mechanism by which Sam68 regulates NF-κB signaling and its functional significance in vascular injury.

Approach and results

The endothelial denudation injury was induced in the carotid artery of Sam68-null (Sam68−/−) and WT mice. Sam68−/− mice displayed an accelerated re-endothelialization and attenuated neointima hyperplasia, which was associated with a reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines in the injured vessels. Remarkably, the ameliorated vascular remodeling was recapitulated in WT mice after receiving transplantation of bone marrow (BM) from Sam68−/− mice, suggesting the effect was attributable to BM-derived inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-α–induced expression of TNF-α, IL-1β, and IL-6 and in the level of nuclear phospho-p65, indicating attenuated NF-κB activation; and these results were confirmed in peritoneal and BM-derived macrophages of Sam68−/− vs. WT mice. Furthermore, co-immunoprecipitation and mass-spectrometry identified Filamin A (FLNA) as a novel Sam68-interacting protein upon TNF-α treatment. Loss- and gain-of-function experiments suggest that Sam68 and FLNA are mutually dependent for NF-κB activation and pro-inflammatory cytokine expression, and that the N-terminus of Sam68 is required for TRAF2-FLNA interaction.

Conclusions

Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery by interacting with FLNA to stabilize TRAF2 on the cytoskeleton and consequently potentiate NF-κB signaling.

Introduction

Rapid expanding endovascular techniques, including angioplasty and stenting, are important options for patients with coronary and peripheral arterial disease. However, the procedure-associated injuries can result in adverse vascular remodeling characterized by impaired endothelial recovery, neointima hyperplasia, and loss of arterial lumen (i.e., restenosis); and currently restenosis remains one of the major barriers to the full success of these techniques [1]. Although the exact mechanisms of restenosis are not completely understood, experimental evidence suggests that vascular inflammatory response plays a critical role. Arterial injury first activates cytokine gene expression in monocytes, macrophages and/or vascular smooth muscle cells (VSMCs), evoking secondary, self-sustaining autocrine and paracrine growth factor and cytokine expression [[2], [3], [4]]. This cytokine-growth factor cascade appears to contribute to VSMC proliferation and migration, resulting in neointima hyperplasia [5]. Mononuclear phagocytes are important contributors in these processes, in part via secretion of pro-inflammatory cytokines and chemokines.

Among various pro-inflammatory cytokines, TNF-α is perhaps the most potent one. It represses post-injury re-endothelialization and promotes VSMC proliferation [6]. The pro-inflammatory activities of TNF-α are primarily mediated by TNF-α receptor 1 (TNFR1), which initiates a pro-survival pathway through the activation of the transcription factor NF-κB [7]. It is known that NF-κB plays a central role in the cellular response to pro-inflammatory stimuli and in the expression of pro-inflammatory cytokines. Repression of NF-κB activation has been suggested and tested an effective approach for restenosis prevention [8,9]. TNF-α stimulation of cells leads to formation of an early complex I in the membrane within minutes, which result in NF-κB activation [10]. The membrane complex I is composed of TNFR, TNFR1-associated DEATH domain protein (TRADD), receptor-interacting protein (RIP), TNFR-associated factor 2 (TRAF2), inhibitor of apoptosis proteins (cIAPs), and IkappaB kinases (IKKs). The TRAF2 is a prototypical member of the TRAF family and is crucial for TNF-α induction of NF-κB activation [11,12]. Notably, TNF-α mediated NF-κB signaling is critically dependent on cytoskeleton. For example, it has been shown that TRAF2 interacts with Filamin A (FLNA), an actin cross-linking protein; in the FLNA-deficient cells, TNF-α fails to activate NF-κB [13].

Src-associated-in-mitosis-68-KD (Sam68) is an RNA binding protein and Src kinase substrate. It can act as an adaptor protein and involve in the regulation of cellular metabolism, nuclear export and stability of RNAs [14]. However, the role of Sam68 in cardiovascular biology has not been studied. Interestingly, emerging evidence suggests that Sam68 is a necessary component of the TNFR early complex I and contributes to TNF-α–induced NF-κB activation in MEF, T cells and fibroblast-like synoviocytes [[15], [16], [17]]. Specifically, Sam68 has been shown to interact with TRAF2, and its deficiency impairs the maintenance of the recruited TRAF2 at TNFR [15]. However, it is unknown whether Sam68 mediated regulation of TNF-α/NF-κB signaling involves cytoskeleton and whether it plays a role in vascular injury response.

In this study, we found that upon TNF-α stimulation, Sam68 interacts with FLNA to bridge TRAF2, thus TNFR complex I, with cytoskeleton to sustain NF-κB signaling in macrophages. Genetic deletion of Sam68 in mice significantly decreases NF-κB activation in macrophages, ameliorates inflammatory response and adverse remodeling of carotid artery from endothelial denudation injury.

Section snippets

Materials and methods

The authors declare that all supporting data are available within the article and its online Supplementary Materials files. The Major Resource Table is given as an online-only Supplementary Materials file.

Deletion of Sam68 leads to accelerated re-endothelialization and attenuated neointima hyperplasia and macrophage infiltration in the injured carotid artery

To understand the role of Sam68 in the vascular response to injuries, we induced wire-mediated endothelial denudation injury in the left carotid artery of Sam68−/− and WT mice. Re-endothelialization and neointima formation were evaluated at serial time points post-injury by Evan's Blue perfusion with en face photograph and by cross-section H.E. staining of neointima thickness, respectively. While the initial areas of injury were similar between the two groups, Sam68−/− mice demonstrated a

Discussion

In this report, we have provided compelling evidence that Sam68 is an essential component of the TNF-α/NF-κB signaling during vascular inflammatory response to denudation injury. Upon TNF-α stimulation in macrophages, Sam68 interacts with TRAF2 of the TNFR complex and the cytoskeleton protein FLNA to enhance NF-κB activation. Genetic deletion of Sam68 in mice or in the transplanted BM cells significantly reduces NF-κB signaling, inflammatory cytokine expression and macrophage infiltration in

Sources of funding

This work was supported by the National Institute of Health (R01 Grants# HL093439, HL113541, HL131110, HL138990 to G.Q.; HL142291 to H.Q & G.Q.); American Diabetes Association (Grant# 1-15-BS-148 to G.Q.); American Heart Association (Grant# 19TPA34910227 to G.Q.; 15POST25340008 to S.H.; 16POST29820001 to L.Y.; 18POST34070088 to S.X.; 18PRE34080358 to E.Z.; and 19CDA34630052 to A.Q.).

Author contributions

S.H., S.X., J.Z., and G.Q. conceived and designed the project. S.H., S.X., J.Z., A.Q, W.M., C.B., H.L, D.B., L.Y., and E.Z. acquired and analyzed data. S.R. provided key materials, Q.L., S.J., T.C.Z., P.K., C.Z., and J.Z. assisted data interpretation. S.H., S.X. and G.Q. wrote the manuscript.

Declaration of Competing Interest

The authors declare no competing financial interests.

Acknowledgement

We thank Dr. Chi Wai Eric So (The Institute of Cancer Research, Sutton, UK) for providing us with Myc-Sam68 plasmid.

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