Calcium and osteoprotegerin regulate IGF1R expression to inhibit vascular calcification

Abstract

Osteoprotegerin (OPG) inhibits vascular calcification in vitro, and OPG(-/-) mice develop vascular calcification. Insulin-like growth factor-1 (IGF1) signalling has been implicated in vascular smooth muscle cell (VSMC) survival; however, the role of IGF1-receptor (IGF1R) expression in calcification is unclear. We sought to determine whether the protective effects of OPG in vascular calcification were mediated by IGF1R. Calcium-induced mineralization of VSMCs was blocked in cells expressing the IGF1R and by treatment with OPG. OPG induced IGF1R mRNA, protein, and transcription optimally at 1 ng/mL. Calcium also positively regulated both OPG and IGF1R, and siRNA targeting of OPG inhibited calcium-inducible IGF1R mRNA. Addition of calcium to VSMCs reduced camptothecin-stimulated apoptosis and increased expression of survival genes Bcl2 and nuclear factor-kappa B without altering levels of proliferation. Calcium's induction of IGF1R and OPG was dose and time dependent but was blunted at higher calcium doses. Calcium- and OPG-inducible IGF1R transcription occurred between -446 and -188 bp of the IGF1R promoter, and inducible-IGF1R expression was blocked by specificity protein-1 (Sp1) silencing studies. Furthermore, elevated IGF1R and OPG protein levels were present in calcified atherosclerotic tissue. We have shown for the first time that IGF1R expression and activity via OPG can modulate VSMC calcification in vitro. We suggest a feedback mechanism: moderate calcium levels increase OPG, which then increases IGF1R to enhance VSMC survival and block calcification induced by calcium. In contrast, high calcium leads to inhibition of IGF1R expression and activity to stimulate VSMC calcification further.