The GEF Cytohesin-2/ARNO Mediates Resistin induced Phenotypic Switching in Vascular Smooth Muscle Cells

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 PIP 3 , 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.Resistin is an adipokine, which is mainly expressed in peripheral blood mononuclear cells and macrophages 1,2 . Resistin levels were first found to be enhanced in serum of diabetic as well as obese mice 3 . In human, increased serum levels of resistin in diabetic and obese patients 4-6 was furthermore shown to correlate with the accumulation of metabolic syndrome factors in type 2 diabetes 7 as well as with other inflammatory diseases, such as atherosclerosis, rheumatic disease and sepsis 2,8 . As all these mentioned pathophysiological conditions are associated with vascular inflammation and thus an elevated risk of cardiovascular disease 9-11 , resistin has gained increasing interest as a potential biomarker 12 and target of therapeutic interventions 8 in the context of vascular inflammation and cardiovascular disease.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 . VSMC migration is a complex process involving remodelling of the cytoskeleton, integrins regulating adhesion by interaction with extracellular matrix (ECM) proteins ...

Cytohesin-2/ARNO: A Novel Bridge Between Cell Migration and Immunoregulation in Synovial Fibroblasts

“…Thus, we explored the role of ARNO in the IL-1β STAT3 signalling pathway, as this can play key roles in both acute and chronic inflammatory responses upon IL-1β stimulation in SFs ( 38 ). We also evaluated the p38 MAPK pathway, as it has been shown to be important for RA pathogenesis and IL-1β signalling ( 39 ) and also for ARNO signaling in relation to MMP expression ( 31 ). Reduction in ARNO expression by siRNA transfection resulted in inhibition of STAT3 activation (as evidenced by its phosphorylation and quantitated as pSTAT3/STAT3 expression) upon IL-1β stimulation, both in naïve ( Figure 4A ) and CIA SFs ( Figure 4B ).…”

“…To address the functional role of ARNO in SFs, we used small interfering RNA (siRNA) approaches which were highly effective at downregulating expression of both ARNO mRNA (78.3% ± 0.08, Figure 1C) and ARNO protein (79.1% ± 0.22, Figure 1D), relative to the negative control AllStars siRNA (Qiagen, siRNA with no homology to any known mammalian gene) in resting and IL-1b-stimulated SFs. ARNO activates ARF6 to compromise vascular permeability in arthritis and other inflammatory conditions (21,28,29) and it also triggers key effector mechanisms involved in cell migration (29)(30)(31)(32). Thus, to investigate whether the ARNO-ARF6 axis was also active in SFs, we knocked-down ARNO expression by siRNA and evaluated activation of ARF6 (GTP-bound) in response to IL-1b (Figure 1E).…”

LncRNA 220: A Novel Long Non-Coding RNA Regulates Autophagy and Apoptosis in Kupffer Cells via the miR-5101/PI3K/AKT/mTOR Axis in LPS-Induced Endotoxemic Liver Injury in Mice

“…The cells were then polarized into M0 macrophages by exposure to phorbol 12-myristate 13-acetate (PMA, Sigma, P8139-1MG, USA) at a concentration of 100 ng/mL in the final solution for 24 h [ 70 ]. Subsequently, the cells were recultivated in DMEM (Gibco, C11995500BT, USA) containing 10% FBS under the same conditions as before, with a concentration of 100 ng/mL in the final solution of intercalated resistin (Peprotech, 450-19-25, Rocky Hill, NJ, USA) according to the relevant literature [ 71 , 72 ] to induce polarization into M1 macrophages.…”

GEF Cytohesin-2/ARNO: a novel bridge between cell migration and immunoregulation in synovial fibroblasts

“…Thus, we explored the role of ARNO in the IL-1b STAT3 signalling pathway, as this can play key roles in both acute and chronic inflammatory responses upon IL-1b stimulation in SFs (38). We also evaluated the p38 MAPK pathway, as it has been shown to be important for RA pathogenesis and IL-1 signalling (39) and also for ARNO signaling in relation to MMP expression (31). Reduction in ARNO expression by siRNA transfection resulted in inhibition of STAT3 activation (as evidenced by its phosphorylation quantitated as pSTAT3/STAT3 expression) upon IL-1b stimulation, both in naïve (Figure 4A) and CIA SFs (Figure 4B).…”