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...
. The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway.
An incomplete understanding of the molecular events that regulate the myometrial transition from the quiescent pregnant state to the active contractile state during labor has hindered the development of improved therapies for preterm labor. During myometrial activation, proteins that prime the smooth muscle for contraction are upregulated, allowing maximal responsiveness to contractile agonists and thereby producing strong phasic contractions. Upregulation of one such protein, COX-2, generates PGs that induce contractions. Intriguingly, the predominant myometrial PG produced just prior to labor is prostacyclin (PGI 2 ), a smooth muscle relaxant. However, here we have shown that activation of PGI 2 receptor (IP) upregulated the expression of several contractile proteins and the gap junction protein connexin 43 through cAMP/PKA signaling in human myometrial tissue in organ and cell culture. Functionally, these IP-dependent changes in gene expression promoted an enhanced contractile response to oxytocin in pregnant human myometrial tissue strips, which was inhibited by the IP antagonist RO3244794. Furthermore, contractile protein induction was dependent on the concentration and time of exposure to the PGI 2 analog iloprost and was blocked by both RO3244794 and PKA knockdown. We therefore propose that PGI 2 -mediated upregulation of contractile proteins and connexin 43 is a critical step in myometrial activation, allowing for a maximal contractile response. Our observations have important implications regarding activation of the myometrium prior to the onset of labor.
Objective: Levels of the smooth muscle relaxant prostacyclin increase prior to labor. We investigated the role of prostacyclin in myometrial activation.Methods: Myometrial tissue was obtained from term pregnant women during cesarean delivery and treated with vehicle or the prostacyclin receptor (IP) agonist iloprost in organ or cell culture models. siRNA was transfected using a Nucleofector. Contraction‐associated protein (CAP) and contractile protein expression were analyzed by immunoblotting or RT‐PCR.Results: Iloprost induced expression of the CAPs COX‐2 and connexin‐43, and the contractile proteins smooth muscle‐myosin heavy chain, h‐caldesmon, and calponin in organ culture and/or cell culture. COX‐2 activity increased as measured by PGF2αrelease. Iloprost activated cAMP/PKA, and siRNA against PKA reduced iloprost‐induced CAP and contractile protein expression, while 8‐Br‐cAMP mimicked the effect. siRNA against the transcription factors GATA‐6 or myocardin inhibited iloprost‐induced SM‐MHC and COX‐2.Conclusions: IP activation of cAMP/PKA induces CAPs and contractile proteins in human myometrium, perhaps by regulating GATA‐6 and myocardin. Prostacyclin may provide positive feedback, linking CAPs and contractile proteins, by increasing COX‐2 activity. These mechanisms may be involved in the conversion of the human uterus from quiescent to an actively contracting labor phenotype.This work was supported by grants from the NIH NHLBI and an NIH NCI training grant
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.