Rapamycin Promotes Vascular Smooth Muscle Cell Differentiation through Insulin Receptor Substrate-1/Phosphatidylinositol 3-Kinase/Akt2 Feedback Signaling

Abstract

The phenotypic plasticity of mature vascular smooth muscle cells (VSMCs) facilitates angiogenesis and wound healing, but VSCM dedifferentiation also contributes to vascular pathologies such as intimal hyperplasia. Insulin/insulin-like growth factor I (IGF-I) is unique among growth factors in promoting VSMC differentiation via preferential activation of phosphatidylinositol 3-kinase (PI3K) and Akt. We have previously reported that rapamycin promotes VSMC differentiation by inhibiting the mammalian target of rapamycin (mTOR) target S6K1. Here, we show that rapamycin activates Akt and induces contractile protein expression in human VSMC in an insulin-like growth factor I-dependent manner, by relieving S6K1-dependent negative regulation of insulin receptor substrate-1 (IRS-1). In skeletal muscle and adipocytes, rapamycin relieves mTOR/S6K1-dependent inhibitory phosphorylation of IRS-1, thus preventing IRS-1 degradation and enhancing PI3K activation. We report that this mechanism is functional in VSMCs and crucial for rapamycin-induced differentiation. Rapamycin inhibits S6K1-dependent IRS-1 serine phosphorylation, increases IRS-1 protein levels, and promotes association of tyrosine-phosphorylated IRS-1 with PI3K. A rapamycin-resistant S6K1 mutant prevents rapamycin-induced Akt activation and VSMC differentiation. Notably, we find that rapamycin selectively activates only the Akt2 isoform and that Akt2, but not Akt1, is sufficient to induce contractile protein expression. Akt2 is required for rapamycin-induced VSMC differentiation, whereas Akt1 appears to oppose contractile protein expression. The anti-restenotic effect of rapamycin in patients may be attributable to this unique pattern of PI3K effector regulation wherein anti-differentiation signals from S6K1 are inhibited, but pro-differentiation Akt2 activity is promoted through an IRS-1 feedback signaling mechanism. Vascular smooth muscle cells (VSMCs)3 maintain a phenotypic plasticity that is important in physiological processes such as arteriogenesis, and in pathological responses, including atherosclerosis, intimal hyperplasia, and restenosis. Mature VSMCs are quiescent and exhibit a differentiated, contractile phenotype. Differentiation status in vitro can be measured by expression of smooth muscle-specific contractile proteins, including calponin, caldesmon, and smooth muscle myosin heavy chain (SM-MHC) (1). In response to injury, or upon in vitro culture, VSMCs re-enter the cell cycle, proliferate, migrate toward attractants, down-regulate expression of contractile proteins, and up-regulate protein synthesis, particularly of the extracellular matrix. This de-differentiated phenotype is referred to as "synthetic" because of this property (1).VSMC de-differentiation and resultant intimal hyperplasia in response to vessel injury are common problems following vascular interventions such as angioplasty, stent placement, and bypass grafts. Since receiving FDA approval in 2003, the use of the mTOR inhibitor rapamycin on drug-eluting stents has had a profound impac...

Insulin-like Growth Factor-I-stimulated Insulin Receptor Substrate-1 Negatively Regulates Src Homology 2 Domain-containing Protein-tyrosine Phosphatase Substrate-1 Function in Vascular Smooth Muscle Cells

“…Because differentiated VSMCs in blood vessels are maintained in a non-proliferative state, expression of IRS-1 may be one of the important determinants that allow normal protein synthesis and hypertrophic responses to IGF-I and insulin while functioning simultaneously to inhibit cell replication. In contrast, transition to the dedifferentiated phenotype could possibly require a major decrease in IRS-1 in order to permit MAPK pathway activation (17,73). This conclusion is supported by the results of this study, wherein IRS-1 silencing increased basal IGF-IR phosphorylation, which in turn, in the absence of IRS-1 binding, had increased access to SHPS-1, thereby augmenting basal SHPS-1 phosphorylation.…”

“…IRS-1 has been linked to many of the insulinor IGF-I-mediated biological responses, including changes in cell size, mitogenesis, and cell migration (14 -16). IGF-Istimulated IGF-IR has been shown to bind to IRS-1 and phosphorylate it in several cell types, including vascular smooth muscle cells (17,18).…”

Pharmacodynamic and Antineoplastic Activity of BI 836845, a Fully Human IGF Ligand-Neutralizing Antibody, and Mechanistic Rationale for Combination with Rapamycin

“…S3). Interestingly, a similar effect was seen in a study using vascular smooth muscle cells (51). However, the precise definition of the mechanism by which rapamycin increases serum IGF bioactivity requires additional investigation and is an active area of research.…”

Effect of muscle‐derived stem cells on the restoration of corpora cavernosa smooth muscle and erectile function in the aged rat

“…1C). The MDSC conversion into SMCs was assessed for 4 weeks in DMEM-2.5% FBS and 20 n M rapamycin to enhance the contractile phenotype [38] by another SMC marker that is not expressed in myofibroblasts, namely calponin. In this case, the MDSCs were labelled with DAPI and calponin identified by immunodetection with Texas Red fluorescence, observing the positive cells in magenta colour in the overlay of the blue and red filters (Fig.…”