Rapamycin promotes vascular smooth muscle cell differentiation through insulin receptor substrate-1/phosphatidylinositol 3-kinase/Akt2 feedback signaling

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.     

血清饥饿可诱导人血管平滑肌细胞再分化

体外培养的分化型血管平滑肌细胞 (vascularsmoothmusclecells ,VSMC)以特异性标志基因表达、长梭形外观及对兴奋剂刺激产生收缩反应为其表型特征 .以血清饥饿法培养处于超汇合 (overconfluence)状态的人VSMC ,观察其分化型标志基因表达活性及其与细胞形态特征和收缩反应性之间的关系 ,探讨细胞生存环境对VSMC基因表达及表型的影响 .研究显示 ,生长至超汇合的VSMC由含血清培养转为血清饥饿后 ,收缩蛋白如SMα肌动蛋白 (SMα actin)、SM2 2α、h1 calponin、肌球蛋白重链 (MHC)SM1和SM2亚型的表达活性明显上调 ,证实血清饥饿诱导的收缩蛋白基因表达和血清应答因子 (serumresponsefactor ,SRF)与CArG顺式元件结合活性的增强有关 .同时 ,血清饥饿还可激活参与VSMC分化调节的转录调控因子SmLIM、Gax和分化相关蛋白HRG 1基因的转录 .随着血清饥饿培养时间的延长 ,VSMC逐渐形成多层、束状、成极性排列的形式 ,对兴奋剂刺激产生的收缩反应明显增强 .结果表明 ,超汇合状态的去分化型VSMC脱离血清刺激后 ,可以再分化成熟并重新获得收缩能力     

The prostacyclin receptor induces human vascular smooth muscle cell differentiation via the protein kinase A pathway

Recent studies of cyclooxygenase-2 (COX-2) inhibitors suggest that the balance between thromboxane and prostacyclin is a critical factor in cardiovascular homeostasis. Disruption of prostacyclin signaling by genetic deletion of the receptor or by pharmacological inhibition of COX-2 is associated with increased atherosclerosis and restenosis after injury in animal models and adverse cardiovascular events in clinical trials (Vioxx). Human vascular smooth muscle cells (VSMC) in culture exhibit a dedifferentiated, migratory, proliferative phenotype, similar to what occurs after arterial injury. We report that the prostacyclin analog iloprost induces differentiation of VSMC from this synthetic, proliferative phenotype to a quiescent, contractile phenotype. Iloprost induced expression of smooth muscle (SM)-specific differentiation markers, including SM-myosin heavy chain, calponin, h-caldesmon, and SM alpha-actin, as determined by Western blotting and RT-PCR analysis. Iloprost activated cAMP/protein kinase A (PKA) signaling in human VSMC, and the cell-permeable cAMP analog 8-bromo-cAMP mimicked the iloprost-induced differentiation. Both myristoylated PKA inhibitor amide-(14-22) (PKI, specific PKA inhibitor), as well as ablation of the catalytic subunits of PKA by small interfering RNA, opposed the upregulation of contractile markers induced by iloprost. These data suggest that iloprost modulates VSMC phenotype via G(s) activation of the cAMP/PKA pathway. These studies reveal regulation of VSMC differentiation as a potential mechanism for the cardiovascular protective effects of prostacyclin. This provides important mechanistic insights into the induction of cardiovascular events with the use of selective COX-2 inhibitors.     

FGFR1 forms an FRS2-dependent complex with mTOR to regulate smooth muscle marker gene expression

Vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype in human cardiovascular disease such as atherosclerosis and restenosis after angioplasty. VSMCs show reduced expression of contractile proteins and are capable of responding to mitogens by increasing expression of growth factor receptors. Fibroblast growth factor receptor-1 (FGFR1) signaling is one of several signaling pathways involved in this VSMC phenotypic switching. The aim of this study was to examine the signaling pathway downstream of FGFR1 in the regulation of SM marker gene expression. We found that FGFR1 activated Akt/mTOR pathway and that the mTOR inhibitor rapamycin partially reversed FGFR1-mediated downregulation of SM marker gene expression. Furthermore, we showed that mTOR forms a multi-protein complex with FGFR1 in VSMCs. These findings provide novel information for future development of therapeutic strategies for the treatment of human cardiovascular disease.     

Rapamycin-mediated CD36 translational suppression contributes to alleviation of hepatic steatosis

Rapamycin, a mammalian target of rapamycin (mTOR)-specific inhibitor, has the effect of anti-lipid deposition on non-alcoholic fatty liver disease (NAFLD), but the mechanisms with which rapamycin alleviates hepatic steatosis are not fully disclosed. CD36 is known to facilitate long-chain fatty acid uptake and contribute to NAFLD progression. Hepatic CD36 expression is closely associated with hepatic steatosis, while mTOR pathway is involved in CD36 translational control. This study was undertaken to investigate whether rapamycin alleviates hepatic steatosis via the inhibition of mTOR pathway-dependent CD36 translation. Human hepatoblastoma HepG2 cells were treated with palmitate and C57BL/6J mice were fed with high fat diet (HFD) to induce hepatic steatosis. Hepatic CD36 protein expression was significantly increased with lipid accumulation in palmitate-treated HepG2 cells or HFD-fed C57BL/6J mice. Rapamycin reduced hepatic steatosis and CD36 protein expression, but it had no influence on CD36 mRNA expression. Rapamycin had no effect on CD36 protein stability, but it significantly decreased CD36 translational efficiency. We further confirmed that rapamycin inhibited the phosphorylation of mTOR and its downstream translational regulators including p70 ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). This study demonstrates that rapamycin inhibits hepatic CD36 translational efficiency through the mTOR pathway, resulting in reduction of CD36 protein expression and alleviation of hepatic steatosis.     

TGF-beta superfamily members do not promote smooth muscle-specific alternative splicing, a late marker of vascular smooth muscle cell differentiation

Smooth muscle (SM) specific alternate splicing of a number of genes is a late marker of the differentiated vascular smooth muscle cell (VSMC) phenotype and is one of the first differentiation characteristics to be lost during de-differentiation and in disease. An understanding of how this aspect of VSMC phenotype is regulated may provide insights into the earliest events of the atherosclerotic process. TGF-beta1 is a potent regulator of VSMC differentiation and can induce expression of SM-specific contractile proteins in both pluripotent stem cells and de-differentiated VSMCs. The purpose of this study was to test the hypothesis that members of the TGFbeta-superfamily can also effect SM-specific alternative splicing. Firstly, we established that SM-specific splicing of alpha-tropomyosin, vinculin and SM-myosin heavy chain (MHC) increases during rat fetal/neonatal development and is decreased in VSMCs following balloon-induced carotid injury in the rat. Treatment of cultured rat VSMCs with TGFbeta-superfamily members resulted in a significant reduction in the ratio of SM to non-muscle (NM) alpha-tropomyosin, but did not effect SM-specific alternative splicing of vinculin or SM-MHC. Treatment of pluripotent C3H10T1/2 cells with TGF-beta1, which increased SM differentiation marker expression, did not increase SM-specific alpha-tropomyosin splicing. Taken together, these results demonstrate differential regulation of SM-specific alternative splicing and indicate that although TGF-beta1 promotes VSMC differentiation marker expression, TGF-beta1 cannot act as the sole trigger of VSMC differentiation.     

Sustained activation of the JNK cascade and rapamycin-induced apoptosis are suppressed by p53/p21(Cip1)

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces apoptosis of cells lacking functional p53. Cells expressing wild-type p53 or p21(Cip1)arrest in G1 and remain viable. In cells lacking functional p53, rapamycin or amino acid deprivation induces rapid and sustained activation of apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase, and elevation of phosphorylated c-Jun that results in apoptosis. This stress response depends on expression of eukaryotic initiation factor 4E binding protein 1 and is suppressed by p21(Cip1) independent of cell cycle arrest. Rapamycin induces p21(Cip1) binding to ASK1, suppressing kinase activity and attenuating cellular stress. These results suggest that inhibition of mTOR triggers a potentially lethal response that is prevented only in cells expressing p21(Cip1).     

Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity

Under serum-free conditions, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), induces a cellular stress response characterized by rapid and sustained activation of the apoptosis signal-regulating kinase 1 (ASK1) signaling pathway and selective apoptosis of cells lacking functional p53. Here we have investigated how mTOR regulates ASK1 signaling using p53-mutant rhabdomyosarcoma cells. In Rh30 cells, ASK1 was found to physically interact with protein phosphatase 5 (PP5), previously identified as a negative regulator of ASK1. Rapamycin did not affect either protein level of PP5 or association of PP5 with ASK1. Instead, rapamycin caused rapid dissociation of the PP2A-B" regulatory subunit (PR72) from the PP5-ASK1 complex, which was associated with reduced phosphatase activity of PP5. This effect was dependent on expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Down-regulation of PP5 activity by rapamycin coordinately activated ASK1, leading to elevated phosphorylation of c-Jun. Amino acid deprivation, which like rapamycin inhibits mTOR signaling, also inhibited PP5 activity, caused rapid dissociation of PR72, and activated ASK1 signaling. Overexpression of PP5, but not the PP2A catalytic subunit, blocked rapamycin-induced phosphorylation of c-Jun, and protected cells from rapamycin-induced apoptosis. The results suggest that PP5 is downstream of mTOR, and positively regulated by the mTOR pathway. The findings suggest that in the absence of serum factors, mTOR signaling suppresses apoptosis through positive regulation of PP5 activity and suppression of cellular stress.     

Stretch-dependent growth and differentiation in vascular smooth muscle: role of the actin cytoskeleton

The smooth muscle cells in the vascular wall are constantly exposed to distending forces from the intraluminal pressure. A rise in blood pressure triggers growth of the vessel wall, which is characterized primarily by hypertrophy of smooth muscle cells with maintained differentiation in a contractile phenotype. Growth factor stimulation of dissociated smooth muscle cells, on the other hand, causes proliferative growth with loss of contractility. This type of response is also found in neointima development following angioplasty and in atherosclerotic lesions. An intact tissue environment is therefore critical for preserved differentiation. Recent advances point to a role of actin polymerization in the expression of smooth muscle differentiation marker genes, in concert with serum response factor (SRF) and cofactors, such as myocardin. Stretch of intact venous smooth muscle activates Rho and inhibits the actin filament severing factor cofilin, resulting in increased actin polymerization. Concomitantly, the rates of synthesis of SRF-regulated differentiation markers, such as SM22alpha, calponin, and alpha-actin, are increased. This increase in differentiation signals is parallel with activation of the mitogen-activated protein (MAP) kinase pathway. Thus stretch-induced growth in a maintained contractile phenotype occurs by dual activation of signal pathways regulating both growth and differentiation. A current challenge is to identify sites of crosstalk between these pathways in intact smooth muscle tissue.     

Rapamycin treatment augments both protein ubiquitination and Akt activation in pressure-overloaded rat myocardium

Ubiquitin-mediated protein degradation is necessary for both increased ventricular mass and survival signaling for compensated hypertrophy in pressure-overloaded (PO) myocardium. Another molecular keystone involved in the hypertrophic growth process is the mammalian target of rapamycin (mTOR), which forms two distinct functional complexes: mTORC1 that activates p70S6 kinase-1 to enhance protein synthesis and mTORC2 that activates Akt to promote cell survival. Independent studies in animal models show that rapamycin treatment that alters mTOR complexes also reduces hypertrophic growth and increases lifespan by an unknown mechanism. We tested whether the ubiquitin-mediated regulation of growth and survival in hypertrophic myocardium is linked to the mTOR pathway. For in vivo studies, right ventricle PO in rats was conducted by pulmonary artery banding; the normally loaded left ventricle served as an internal control. Rapamycin (0.75 mg/kg per day) or vehicle alone was administered intraperitoneally for 3 days or 2 wk. Immunoblot and immunofluorescence imaging showed that the level of ubiquitylated proteins in cardiomyocytes that increased following 48 h of PO was enhanced by rapamycin. Rapamycin pretreatment also significantly increased PO-induced Akt phosphorylation at S473, a finding confirmed in cardiomyocytes in vitro to be downstream of mTORC2. Analysis of prosurvival signaling in vivo showed that rapamycin increased PO-induced degradation of phosphorylated inhibitor of κB, enhanced expression of cellular inhibitor of apoptosis protein 1, and decreased active caspase-3. Long-term rapamycin treatment in 2-wk PO myocardium blunted hypertrophy, improved contractile function, and reduced caspase-3 and calpain activation. These data indicate potential cardioprotective benefits of rapamycin in PO hypertrophy.     

Shear Stress Induces Phenotypic Modulation of Vascular Smooth Muscle Cells via AMPK/mTOR/ULK1-Mediated Autophagy

Phenotypic modulation of vascular smooth muscle cells (VSMCs) is involved in the pathophysiological processes of the intracranial aneurysms (IAs). Although shear stress has been implicated in the proliferation, migration, and phenotypic conversion of VSMCs, the molecular mechanisms underlying these events are currently unknown. In this study, we investigated whether shear stress(SS)-induced VSMC phenotypic modulation was mediated by autophagy involved in adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR)/Unc-51-like kinase 1 (ULK1) pathway. The results show that shear stress could inhibit the expression of key VSMC contractile genes and induce pro-inflammatory/matrix-remodeling genes levels, contributing to VSMCs phenotypic switching from a contractile to a synthetic phenotype. More importantly, Shear stress also markedly increased the levels of the autophagy marker microtubule-associated protein light chain 3-II (LC3II), Beclin-1, and p62 degradation. The autophagy inhibitor 3-methyladenine (3-MA) significantly blocked shear-induced phenotypic modulation of VSMCs. To further explore the molecular mechanism involved in shear-induced autophagy, we found that shear stress could activate AMPK/mTOR/ULK1 signaling pathway in VSMCs. Compound C, a pharmacological inhibitor of AMPK, significantly reduced the levels of p-AMPK and p-ULK, enhanced p-mTOR level, and finally decreased LC3II and Beclin-1 level, which suggested that activated AMPK/mTOR/ULK1 signaling was related to shear-mediated autophagy. These results indicate that shear stress promotes VSMC phenotypic modulation through the induction of autophagy involved in activating the AMPK/mTOR/ULK1 pathway.     

Differing effects of rapamycin and mTOR kinase inhibitors on protein synthesis

mTOR (mammalian target of rapamycin) forms two distinct types of complex, mTORC (mTOR complex) 1 and 2. Rapamycin inhibits some of the functions of mTORC1, whereas newly developed mTOR kinase inhibitors interfere with the actions of both types of complex. We have explored the effects of rapamycin and mTOR kinase inhibitors on general protein synthesis and, using a new stable isotope-labelling method, the synthesis of specific proteins. In HeLa cells, rapamycin only had a modest effect on total protein synthesis, whereas mTOR kinase inhibitors decreased protein synthesis by approx. 30%. This does not seem to be due to the ability of mTOR kinase inhibitors to block the binding of eIFs (eukaryotic initiation factors) eIF4G and eIF4E. Analysis of the effects of the inhibitors on the synthesis of specific proteins showed a spectrum of behaviours. As expected, synthesis of proteins encoded by mRNAs that contain a 5'-TOP (5'-terminal oligopyrimidine tract) was impaired by rapamycin, but more strongly by mTOR kinase inhibition. Several proteins not known to be encoded by 5'-TOP mRNAs also showed similar behaviour. Synthesis of proteins encoded by 'non-TOP' mRNAs was less inhibited by mTOR kinase inhibitors and especially by rapamycin. The implications of our findings are discussed.     

Dual role of PKA in phenotypic modulation of vascular smooth muscle cells by extracellular ATP

Extracellular ATP is released from activated platelets and endothelial cells and stimulates proliferation of vascular smooth muscle cells (VSMC). We found that ATP stimulates a profound but transient activation of protein kinase A (PKA) via purinergic P2Y receptors. The specific inhibition of PKA by adenovirus-mediated transduction of the PKA inhibitor (PKI) attenuates VSMC proliferation in response to ATP, suggesting a positive role for transient PKA activation in VSMC proliferation. By contrast, isoproterenol and forskolin, which stimulate a more sustained PKA activation, inhibit VSMC growth as expected. On the other hand, the activity of serum response factor (SRF) and the SRF-dependent expression of smooth muscle (SM) genes, such as SM--actin and SM22, are extremely sensitive to regulation by PKA, and even transient PKA activation by ATP is sufficient for their downregulation. Analysis of the dose responses of PKA activation, VSMC proliferation, SRF activity, and SM gene expression to ATP, with or without PKI overexpression, suggests the following model for the phenotypic modulation of VSMC by ATP, in which the transient PKA activation plays a critical role. At low micromolar doses, ATP elicits a negligible effect on DNA synthesis but induces profound SRF activity and SM gene expression, thus promoting the contractile VSMC phenotype. At high micromolar doses, ATP inhibits SRF activity and SM gene expression and promotes VSMC growth in a manner dependent on transient PKA activation. Transformation of VSMC by high doses of ATP can be prevented and even reversed by inhibition of PKA activity. adenosine triphosphate; purinergic receptors; protein kinase A; serum response factor; proliferation; -actin; SM22