IGF-1 increases macrophage motility via PKC/p38-dependent αvβ3-integrin inside-out signaling
Introduction
Permanent infiltration of vascular lesions with activated macrophages and lymphocytes is a key feature in the initiation, progression and complications of atherosclerosis [1]. Motility of inflammatory mononuclear cells (MNCs) within the vessel wall depends on the action of chemokines/growth factors and their receptors, and also requires integrins and matrix metalloproteinases (MMPs). Heterodimeric α/β integrins serve as signaling bridges between the cytoskeleton and the extracellular matrix (ECM). Integrin ligation to ECM proteins (outside-in signaling) leads to changes in phosphorylation of integrin-linked non-receptor tyrosine kinases, such as focal adhesion kinase (FAK), resulting in adhesion and migration [2]. In contrast, inside-out integrin activation is initiated via growth factor receptor binding, activation of receptor tyrosine kinases and further downstream signaling pathways such as protein kinase C (PKC), leading to modulation in integrin affinity/avidity [3]. Integrins are assisted by MMPs, the major proteolytic enzymes within the interstitium, typically induced by chemokines/growth factors [4]. Integrins and MMPs collaborate in a number of ways, including MMP-binding to integrins, thereby coordinating ECM degradation and migration [5].
The insulin-like growth factor-1 (IGF-1) axis, comprised of IGF-1, its cognate receptor (IGF-1R) and binding proteins (IGFBPs), is highly expressed in human atherosclerotic lesions [6]. Increased levels of circulating IGF-1 and IGFBP-3 are found during vascular disease progression in patients [7]. Experimental in vivo studies demonstrated enhanced neointima formation upon overexpession of IGF-1 in vascular smooth muscle cells (VSMCs) [8] and lessening of neointima formation upon IGF-1 inhibition [9]. In vitro, IGF-1 has been shown to induce cancer cell migration, involving the activation of MMPs or integrins. Zhang et al. [10] demonstrated that IGF-1 upregulates the membrane-type MT1-MMP/MMP-2 activation cascade, whereas Marelli et al. found IGF-1 promoting migration of human androgen-independent prostate cancer cells primarily via integrin activation [11].
In light of enhanced IGF-1 production in vascular disease and the central pathological role of macrophage invasion in inflammatory atherosclerosis, we aimed at elucidating the impact of IGF-1 on macrophage migration, its involved signaling pathways, and at clarifying the contribution of integrins and/or MMPs in IGF-1 mediated macrophage motility.
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Materials and methods
Cell culture media and materials were form Gibco, IGF-1 was from PreproTech. Wortmannin, SB203580, LY303.511, LY294.002, the antibody to phospho-FAK and the antibody to the αv NH2-terminus (VNR139) were from Calbiochem. The furin inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) and integrin-blocking cyclic RGD and RGE hexapeptides were from Bachem. The MMP-inhibitor GM6001, rabbit polyclonal antibody against the hinge region of human MT1-MMP (AB815) and integrin αvβ3-blocking antibody
Facilitated macrophage migration by IGF-1 involves integrin activation
Differentiation of human monocytic THP-1 cells to macrophages by PMA (100 nmol/L; 48 h) was monitored by typical cell morphology and the expression of the macrophage marker protein vimentin (Fig. 1A and B) [13]. Immunoblotting demonstrated that monocyte → macrophage transformation is characterized by increases in IGF-1R expression (Fig. 1B). First, we investigated whether IGF-1 is chemoattractant to macrophages itself. Migration checker-box experiments revealed that IGF-1 (50 ng/mL) is strongly
Discussion
Here we demonstrate that IGF-1 is chemotactic to human THP-1 cell-derived macrophages. Enhanced macrophage migration induced by IGF-1 depends on αvβ3-integrin, whereas the activation of MMPs seems not to be required. IGF-1 facilitated macrophage migration relies on IGF-1 mediated integrin inside-out signaling activation. This involves compartmentalization of integrin adaptor proteins into plasma membrane-associated focal adhesion sites, resulting in increased cell motility. Thus, IGF-1
Acknowledgments
J.F. was supported by the Charité Universitätsmedizin-Berlin. P.S. and E.F. are supported by the Zukunftsfond Berlin/TSB Medici. K.K. is supported by the Deutsche Forschungsgemeinschaft (KA1820/4-1) and by the German Heart Foundation/German Foundation of Heart Research (F/04/08).
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