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Macrophage migration is a key aspect in the initiation and progression of atherosclerosis. Insulin-like growth factor (IGF)-1 is highly expressed in macrophages in human atheroma. Its function in macrophage motility, however, remains to be elucidated. The aim of this study was to investigate the impact of IGF-1 on macrophage migration, its signaling pathways and the involvement of integrins and/or matrix metalloproteinases (MMPs). Results Migration checker-box experiments demonstrated that IGF-1 induced chemotaxis in human THP-1/macrophages. IGF-1 induced migration was inhibited by RGD-containing peptides and the αvβ3-blocking antibody LM609, but was unaffected by the MMP-inhibitor GM6001. Immunoblotting demonstrated that IGF-1 did not affect the activation of MMPs or TIMPs, nor did it increase αv-integrin protein levels. However, IGF-1 induced recruitment of αvβ3, as well as trans-location of the integrin adaptor protein phospho-paxillin to focal adhesion sites. Pharmacological blocking experiments with specific inhibitors of Akt, PKC and p38 MAP-kinase revealed that IGF-1-dependent activation of focal adhesion kinase (FAK) and paxillin, and consecutively IGF-1 facilitated migration, required IGF-1/IGF-1R-mediated PI3-kinase/PKC/p38-dependent integrin inside-out signaling. Conclusion IGF-1 plays a vital role in macrophage migration critically implicated in tissue inflammation. This involves activation of integrins and focal adhesion formation via inside-out PI3-kinase/PKC/p38-dependent signaling, but does not require MMP activation.

Vascular smooth muscle cell (VSMC) invasion is a key element in atherogenesis and restenosis, requiring integrins for adhesion/de-adhesion as well as matrix metalloproteinases (MMPs) for focalized proteolysis. Among the MMP family, pro-MMP-2 is unique in its activation, depending on the formation of a multiprotein complex with MT1-MMP/TIMP-2 at the cell surface, in which integrin alphavbeta3 participates. Integrin alphav and MT1-MMP are synthesized from precursors via furin-dependent cleavage of their pro-peptide. Furin is the prototypical proprotein convertase highly expressed in VSMCs and human atherosclerotic lesions. Its precise role in the tight network involving MMPs/integrins and their coordination and cooperation required for VSMC invasion is unknown. We demonstrate that furin-inhibition with decanoyl-RVKR-chloromethylketone inhibits VSMC invasion in a comparable degree to MMP inhibitors, which reduce the MT1-MMP-MMP-2 proteolytic cascade. Furin-inhibition did not prevent MT1-MMP/MMP-2 maturation. In contrast, it strongly reduced pro-alphav cleavage, but did not lessen its cell membrane expression. However, inhibition of pro-alphav processing via furin-inhibition strongly reduced pro-MMP-2 binding to the cell surface, thereby lessening its full maturation and diminishing the cell surface in situ proteolysis required for invasion. Thus, our data demonstrate a novel mechanism of furin-dependent alphav cleavage that enhances pro-MMP-2 binding and activation at the cell membrane in cooperation with MT1-MMP in primary VSMCs. Processing of alphav by furin contributes to the recruitment of enzymatic energy to the cell surface, thereby providing focalized proteolysis associated with VSMC invasion.

SummaryIntegrins link the cytoskeleton to the extracellular matrix, providing outside-in/inside-out signalling essential for vascular smooth muscle cell (VSMC) migration in atherosclerosis. The integrin αv subunit is synthesised from its precursor via furin-dependent endoproteolytic cleavage. Furin is a proprotein convertase (PC) highly expressed in VSMCs and in human atherosclerotic lesions. Inhibition of αv processing inhibits binding to vitronectin and migration. However, the precise role of furin-dependent αv cleavage on integrin bidirectional signalling and subsequent VSMC functions is unknown. Our present study demonstrates that the furin-like PC inhibitor decanoyl-RVKR-chloromethylke-tone (dec-CMK) inhibited αv cleavage. This reduced vitronectin-induced (outside-in) focal adhesion kinase (FAK)- and paxillin-phosphorylation, and VSMC motility. Inside-out-stimulated, integrin-mediated VSMC adhesion/migration relied on integrin-adaptor protein activation following protein kinase C (PKC) and ERK1/2 phosphorylation. In contrast to outside-in signalling, PKC-dependent phosphorylation of FAK and paxillin was unaffected by the status of integrin cleavage. Still, cytoskeleton and focal adhesion site rearrangements were modulated by the inhibition of furin-dependent integrin cleavage, thereby lessening inside-out dependent migration. Hence, we find that integrin bidirectional signalling is critically controlled by furin. Furin-dependent integrin processing modulates rapid adaptive integrin/cytoskeleton changes, essential to VSMC motility, which represents a crucial component in atherosclerosis and restenosis.