Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor

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Abstract

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p < 0.05 vs. controls), whereas it had no effect of PDGF-stimulated signaling pathways or cell growth. Both, IGF-1 and PDGF-BB, induced MT1-MMP expression, but only IGF-1-mediated MT1-MMP induction was inhibited by dec-CMK. Induction of MMP-2 by IGF-1 was inhibited by the PI3-kinase inhibitor wortmannin, but not by the MEK-inhibitor PD98059. Dec-CMK inhibited VSMC chemotaxis comparable to the effects of the MMP-inhibitor GM6001 (both p < 0.05 vs. controls), supporting that MMPs are involved. In conclusion, this study demonstrates that targeting furin-like PCs and thus inhibiting IGF-1R activation is a novel target to inhibit IGF-1-mediated signaling and cell functions, such as IGF-1-induced MT1-MMP/MMP-2 in VSMCs.

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Materials and methods

Materials. IGF-1 was from PreproTech and PDGF-BB was purchased from BioScource. Wortmannin and PD98059 were from Calbiochem. The furin-like proprotein convertase inhibitor decanoyl-RVKR-chloromethylketone (dec-CMK) was from Bachem and the broad spectrum hydroxamate class MMP-inhibitor GM6001 (Ilomastat) was from Chemicon. All other chemicals were from Sigma. Cell culture media and materials were from Gibco. The monoclonal antibody directed against the hemopexin-like domain of MT1-MMP (clone

Furin-like proprotein convertases activate the IGF-1R in VSMCs

The presence of furin (Fig. 1A) and PC5 (Fig. 1B) mRNAs and protein levels (Fig. 1C) in VSMC cultures was confirmed by PCR and immunoblotting. To investigate the requirement of furin-like PCs for IGF-1R activation, VSMCs were rendered quiescent by serum-starvation and treated with the furin-like PC-inhibitor dec-CMK (50 μM), followed by incubation of cells in 10% FCS (24 h) in the presence of the inhibitor. In controls, the anti-IGF-1R antibody directed against the C-terminus detected almost

Discussion

The present study demonstrates that IGF-1 induces MT1-MMP/MMP-2 expression and activity in VSMCs via IGF-1R-mediated PI3-kinase signaling. Furthermore, we found that furin-like PCs control IGF-induced MT1-MMP in VSMCs through endoproteolytic activation of the pro-IGF-1R.

MMPs are major contributors to tissue remodeling in arteriosclerosis [10]. MT1-MMP plays a unique role, because in addition to its ability to cleave a number of ECM proteins within the vessel wall [10], membrane-anchored,

Acknowledgments

This work was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF) (CAN02/005) to P.S., E.F., and K.G. C.M. was supported by the Deutscher Akademischer Austauschdienst (DAAD). M.C. and N.G.S. were supported by Canadian Institutes of Health Research (CIHR) Grants MOP-44362 and MGP-44363, respectively.

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