Abstract
Vascular smooth muscle cell (VSMC) phenotypic modulation is characterized by the downregulation of SMC actin cytoskeleton proteins. Our published study shows that depletion of SM22α (aka SM22, Transgelin, an actin cytoskeleton binding protein) promotes inflammation in SMCs by activating NF-κB signal pathways both in cultured VSMCs and in response to vascular injury. The goal of this study is to investigate the underlying molecular mechanisms whereby SM22 suppresses NF-κB signaling pathways under inflammatory condition. NF-κB inducing kinase (Nik, aka MAP3K14, activated by the LTβR) is a key upstream regulator of NF-κB signal pathways. Here, we show that SM22 overexpression suppresses the expression of NIK and its downstream NF-κB canonical and noncanonical signal pathways in a VSMC line treated with a LTβR agonist. SM22 regulates NIK expression at both transcriptional and the proteasome–mediated post-translational levels in VSMCs depending on the culture condition. By qPCR, chromatin immunoprecipitation and luciferase assays, we found that Nik is a transcription target of serum response factor (SRF). Although SM22 is known to be expressed in the cytoplasm, we found that SM22 is also expressed in the nucleus where SM22 interacts with SRF to inhibit the transcription of Nik and prototypical SRF regulated genes including c-fos and Egr3. Moreover, carotid injury increases NIK expression in Sm22-/- mice, which is partially relieved by adenovirally transduced SM22. These findings reveal for the first time that SM22 is expressed in the nucleus in addition to the cytoplasm of VSMCs to regulate the transcription of Nik and its downstream proinflammatory NF-kB signal pathways as a modulator of SRF during vascular inflammation.
Highlights
Vascular smooth muscle cell (VSMC) phenotypic modulation plays critical roles in the pathogenesis of vascular diseases such as atherosclerosis and aneurysms [1, 2]
Overexpression of SM22 suppresses the expression of proinflammatory genes in VSMCs under inflammatory condition
In LTβR-Fc treated PAC1 cells, we found that exogenous overexpression of SM22 reduced LTβR activation-induced transcription of proinflammatory genes Vcam1, Icam1, Ccl2 and Cx3cl1 by the Quantitative real time RT-PCR (qPCR) assay (Fig 1A) and the expression of ICAM1 and SDF-1 by western blotting (WB) assay (Fig 1B)
Summary
Vascular smooth muscle cell (VSMC) phenotypic modulation plays critical roles in the pathogenesis of vascular diseases such as atherosclerosis and aneurysms [1, 2]. SMC phenotypic modulation is accompanied by the down regulation of actin cytoskeleton proteins including smooth muscle α-actin (SMA), SM22α, Calponin and smooth muscle myosin heavy chain (SMMHC). Extensive studies have characterized the central role of SRF in SMC phenotypic switching from the differentiated state to a variety of dedifferentiated states involved in VSMC proliferation, migration, inflammation and calcification [1,2,3]. SM22 contains several distinct domains including a C-terminal actin binding domain (ABD) and the Calponin homology domain (CH) [6, 7]. In addition to actin binding, the ABD of SM22 facilitates the bundling of F-actin and the formation of cytoskeletal structures such as stress fibers [6]
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