Abstract
The pro-inflammatory adipokine resistin induces a phenotypic switch of vascular smooth muscle cells (VSMC), a process decisive for atherosclerosis, including morphological changes, increased synthetic activity, proliferation and migration. The guanine-exchange factor ARNO (Cytohesin-2) has been shown to be important for morphological changes and migration of other cell types. In this study we dissected the role of ARNO in resistin induced VSMC phenotypic switching and signalling. Firstly, treatment with the cytohesin inhibitor Secin H3 prevented the resistin mediated induction of morphological changes in VSMC. Secondly, Secin H3 treatment as well as expression of an inactive ARNO (EK) reduced resistin induced VSMC synthetic activity, as assessed by matrix metalloproteinase 2 (MMP-2) expression, as well as the migration into a wound in vitro compared to ARNO WT expression. Thirdly, we found ARNO to influence MMP-2 expression and migration via activation of p38 MAPK and the JNK/AP-1 pathway. Interestingly, these processes were shown to be dependent on the binding of PIP3, as mutation of the ARNO PH-domain inhibited VSMC migration, MMP-2 expression as well as p38 MAPK and JNK signalling. Thus, we demonstrate that ARNO is an important link in resistin dependent cell signalling leading to morphological changes, MMP-2 production and migration of VSMC.
Highlights
Upon stimulation with inflammatory cytokines or adipokines, vascular smooth muscle cells (VSMC) undergo a phenotypic switch from a quiescent differentiated contractile phenotype to a proliferative, migratory and synthetic dedifferentiated one[13]
We analysed the influence of resistin on vascular smooth muscle cell (VSMC) morphology by stimulating cells for different times with a high concentration of resistin (100 ng/ml), as this has been shown to induce an inflammatory response in VSMC and endothelial cells[28,29,30], and compared it to PDGF-B stimulation, a known VSMC activator
Quantitative real-time PCR measurements revealed that cytohesin-1, -2 and -3 are expressed in VSMC with cytohesin-2 (ARNO), being the predominant form (Fig. 1d)
Summary
Upon stimulation with inflammatory cytokines or adipokines, vascular smooth muscle cells (VSMC) undergo a phenotypic switch from a quiescent differentiated contractile phenotype to a proliferative, migratory and synthetic dedifferentiated one[13]. The phenotypic changes of VSMC contribute to the development of atherosclerosis[14] During this process VSMC undergo morphological changes and downregulate proteins determining the contractile phenotype, such as smooth muscle (SM) α-Actin or SM myosin heavy chain, and instead upregulate genes such as metalloproteinases (MMP), characteristic for an increased synthetic activity, important for the invasive process, i.e. proliferation and migration[13]. Not much is known about which signalling intermediates are involved in resistin induced VSMC migration and MMP production. ARNO has been demonstrated to bind to phosphatidylinositol-3,4,5-triphosphate (PIP3), a membrane associated product generated by the phosphatidylinositol 3-kinase (PI3-K)[21,22,23] and PI3-K has been shown to be important for activation of ARNO to induce cell migration[24]. ARNO GEF activity as well as its PH domain were necessary for these processes
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