Coxsackievirus group B3 (CVB3) replication is influenced by host cell cycle status. However, the effect of CVB3 infection on cell cycle regulation and the mechanisms involved are not precisely defined. In this study, we examined cell cycle progression and regulation when the infection was initiated in late G 1 phase of the cell cycle. Analysis of cellular DNA synthesis in infected cells by thymidine incorporation assays showed a significant reduction in [ 3 H]thymidine uptake compared to that of sham-infected cells. To further clarify the effects of CVB3 on the host cell cycle, we examined the cell cycle regulatory proteins involved in G 1 progression and G 1 /S transition. Infection resulted in dephosphorylation of retinoblastoma protein and reduced G 1 cyclin-dependent kinase activities, accompanied by decreased levels of G 1 cyclin protein expression (cyclin D1 and cyclin E). We further investigated the mechanisms by which CVB3 infection down-regulates cyclin D1 expression. Northern blotting showed that cyclin D1 mRNA levels were modestly increased following CVB3 infection, suggesting that cyclin D1 regulation occurs by a posttranscriptional mechanism. Viral infection resulted in only a 20 to 30% inhibition of cyclin D1 protein synthesis 3 h postinfection. However, the proteasome inhibitors MG132 and lactacystin prevent CVB3-induced cyclin D1 reduction, indicating that CVB3-induced down-regulation of cyclin D1 is facilitated by ubiquitin-proteasome proteolysis. Finally, using GSK3␤ pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3␤ independent. Taken together, our results demonstrate that CVB3 infection disrupts host cell homeostasis by blocking the cell cycle at the G 1 /S boundary and induces cell cycle arrest in part through an increase in ubiquitin-dependent proteolysis of cyclin D1.Coxsackievirus group B3 (CVB3), an enterovirus of the family Picornaviridae, is a common human pathogen associated with various diseases, such as myocarditis, meningitis, and pancreatitis (7,30,48). It has been suggested that early virus and host cell interactions can determine the degree of viral replication and the progression of target organ injury. Like most viruses, CVB3 has evolved a variety of mechanisms to optimize cellular conditions to benefit its own replication. We have previously shown that the extracellular signal-regulated kinase (ERK) is activated during CVB3 infection and that inhibition of such activity blocks CVB3 replication. It has also been reported that CVB3 modifies host gene expression to optimize its replication (34, 44, 50). As part of such manipulation of host cells, CVB3 may modify the host cell cycle regulatory machinery to facilitate its replication. Indeed, during the preparation of this study, Feuer et al. (14) showed that coxsackievirus replication and persistence were affected by cell cycle status. Although it was proposed that virus-induced shutdown of host protein synthesis led to cell growth arrest (14), the effect of CVB3 infection on cell cycle control and the precis...

Abstract-Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P 2 )-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P 2 -null but not in wild-type arteries. This was accompanied with a significant increase in both medial and intimal smooth muscle cell (SMC) replication between days 4 to 28, with only minimal replication in wild-type arteries. S1P 2 -null SMCs showed a significant increase in migration when stimulated with S1P alone and together with platelet-derived growth factor, whereas both wild-type and null SMCs migrated equally well to platelet-derived growth factor. S1P increased Rho activation in wild-type but not in S1P 2 -null SMCs, and inhibition of Rho activity promoted S1P-induced SMC migration. Plasma S1P levels were similar and did not change after surgery. These results suggest that activation of S1P 2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development. Key Words: sphingosine 1-phosphate receptors Ⅲ smooth muscle cells Ⅲ neointima S phingosine 1-phosphate (S1P) is a bioactive sphingolipid formed by activation of sphingosine kinases. 1 It exerts pleiotropic effects on many cells by regulating cytoskeletal rearrangement, cell survival, cell migration, cell proliferation, angiogenesis, and vascular development. [2][3][4][5] Recently S1P has received attention as a regulator of the cardiovascular system. In part, this is because there are high levels of S1P in plasma, and a recent report showed that they correlate well with the reoccurrence of vascular events. 6 -10 Further platelets release S1P during their activation, and consequently S1P levels are likely to be high at sites of arterial injury. 11,12 S1P acts through 5 G protein-coupled receptors (S1P 1 to S1P 5 ), although arterial smooth muscle cells (SMCs) express only S1P 1 , S1P 2 , and S1P 3 . 4,13 Initially these receptors were called endothelial cell differentiation gene receptors. 14 Activation of S1P receptors induces coupling to a variety of G proteins, which in turn leads to activation of multiple pathways. In SMCs most work has concentrated on S1P 1 and S1P 2 because they have opposing actions. S1P 1 couples to Gi and leads to activation of extracellular signal-regulated kinase, phosphatidylinositol 3-kinase, and Rac. 13,15,16 Adult SMCs only weakly express S1P 1 , although it is more highly expressed in pup cells, and this has been linked to their increased ability to migrate and proliferate in response to S1P. 17 S1P 1 is also strongly expressed in SMCs from rat intimal lesions as well as in human atherosclerotic lesions. 17,18 These data have been used to suggest that activation of S1P 1 may induce events leading to restenosis and the formation of arterial lesions. S1P 2 is the main receptor expressed by most adult medial SMCs and couples to Gi, Gq, and G12/13, and its activation by S1P is associated with inhibition of SMC migration. 13,19 This is thought to occur via coupling...

This study examined intracellular signal events of arterial cells following balloon catheter injury to rat carotid artery. Within 30 minutes, a marked increase in extracellular signal-regulated kinase-1/2 (ERK1/2) activity was observed. This activity remained elevated for 12 hours but had decreased to control levels by day 1. No increase in ERK1/2 was detected at any later times. Injection of anti-fibroblast growth factor 2 antibody (60 mg i.v.) significantly inhibited the activation of ERK1/2 at 30 minutes after the injury. PD98059 (80 micromol/L), a selective inhibitor of mitogen-activated protein kinase/ERK kinase-1 (MEK1), decreased ERK1/2 activity in injured arteries and also reduced the medial cell replication. In contrast, PD98059 did not block the intimal cell replication at day 8. Mitogen-activated protein kinase phosphatase-1 (MKP-1) was expressed within hours after injury but only weakly at later times; MKP-1 was again expressed after 7 and 14 days. The expression of MKP-1 was associated with an activation of c-Jun amino-terminal kinase. Injury to the arterial wall also stimulated the activity of p70 S6 kinase from 30 minutes to 12 hours, suggesting an alternative pathway in mitogenic signaling of early cell replication. These findings demonstrate that fibroblast growth factor 2-induced ERK1/2 activation promotes medial cell replication after balloon injury; however, signaling of intimal cell replication may not be linked to the MEK1-dependent ERK pathway.

Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.

Background:Obesity is associated with reduced levels of circulating high-density lipoproteins (HDLs) and its major protein, apolipoprotein (apo) A-I. As a result of the role of HDL and apoA-I in cellular lipid transport, low HDL and apoA-I may contribute directly to establishing or maintaining the obese condition.Methods:To test this, male C57BL/6 wild-type (WT), apoA-I deficient (apoA-I−/−) and apoA-I transgenic (apoA-Itg/tg) mice were fed obesogenic diets (ODs) and monitored for several clinical parameters. We also performed cell culture studies.Results:ApoA-I−/− mice gained significantly more body weight and body fat than WT mice over 20 weeks despite their reduced food intake. During a caloric restriction regime imposed on OD-fed mice, apoA-I deficiency significantly inhibited the loss of body fat as compared with WT mice. Reduced body fat loss with caloric restriction in apoA-I−/− mice was associated with blunted stimulated adipose tissue lipolysis as verified by decreased levels of phosphorylated hormone-sensitive lipase (p-HSL) and lipolytic enzyme mRNA. In contrast to apoA-I−/− mice, apoA-Itg/tg mice gained relatively less weight than WT mice, consistent with other reports. ApoA-Itg/tg mice showed increased adipose tissue lipolysis, verified by increased levels of p-HSL and lipolytic enzyme mRNA. In cell culture studies, HDL and apoA-I specifically increased catecholamine-induced lipolysis possibly through modulating the adipocyte plasma membrane cholesterol content.Conclusions:Thus, apoA-I and HDL contribute to modulating body fat content by controlling the extent of lipolysis. ApoA-I and HDL are key components of lipid metabolism in adipose tissue and constitute new therapeutic targets in obesity.

Edited by Norma M. AllewellNeuroendocrine-type ATP-sensitive K ؉ (K ATP ) channels are metabolite sensors coupling membrane potential with metabolism, thereby linking insulin secretion to plasma glucose levels. They are octameric complexes, (SUR1/Kir6.2) 4 , comprising sulfonylurea receptor 1 (SUR1 or ABCC8) and a K ؉ -selective inward rectifier (Kir6.2 or KCNJ11). Interactions between nucleotide-, agonist-, and antagonist-binding sites affect channel activity allosterically. Although it is hypothesized that opening these channels requires SUR1-mediated MgATP hydrolysis, we show here that ATP binding to SUR1, without hydrolysis, opens channels when nucleotide antagonism on Kir6.2 is minimized and SUR1 mutants with increased ATP affinities are used. We found that ATP binding is sufficient to switch SUR1 alone between inward-or outward-facing conformations with low or high dissociation constant, K D , values for the conformation-sensitive channel antagonist [ 3 H]glibenclamide ([ 3 H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's K D for [ 3 H]GBM.ThisreductionrequiredtheKirNterminus(KNtp), consistent with KNtp occupying a "transport cavity," thus positioning it to link ATP-induced SUR1 conformational changes to channel gating. Moreover, ATP/GBM site coupling was constrained in WT SUR1/WT Kir6.2 channels; ATP-bound channels had a lower K D for [ 3 H]GBM than ATP-bound SUR1. This constraint was largely eliminated by the Q1179R neonatal diabetes-associated mutation in helix 15, suggesting that a "swapped" helix pair, 15 and 16, is part of a structural pathway connecting the ATP/GBM sites. Our results suggest that ATP binding to SUR1 biases K ATP channels toward open states, consistent with SUR1 variants with lower K D values causing neonatal diabetes, whereas increased K D values cause congenital hyperinsulinism.Neuroendocrine-type ATP-sensitive K ϩ (K ATP ) 2 channels comprise an ATP-binding cassette (ABC) protein (1), ABCC8/ SUR1, and a K ϩ -selective inward rectifier (2), KCNJ11/Kir6.2, assembled as heterooctamers (3-5), (SUR1/Kir6.2) 4 . In pancreatic ␤-cells, these channels are metabolite sensors that couple cellular metabolism with membrane electrical activity to link insulin secretion with blood glucose levels. This coupling is critical for normal physiology; loss of channel function is a cause of congenital hyperinsulinism (Ref. 6; for reviews, see Refs. 7 and 8), whereas gain-of-function mutations in Kir6.2 (9) and SUR1 (10) cause neonatal diabetes (ND) (for reviews, see Refs. 11 and 12). Gain of function is one cause of mature-onset diabetes of the young (13), whereas a ABCC8/SUR1 polymorphism, the Ala amino acid allele at p.A1369S, is associated with an increased risk for type 2 diabetes (14).Channel activity is regulated positively by ATP and ADP binding to SUR1 and negatively by nucleotide binding to . Additionally, multiple metabolites, including phosphoinositides (21-23) and long-chain acyl-CoA esters (24 -26), and phosphorylation (27-29) positively modulate ch...

Objective The objective of this study is to define a role for S1PR3 in intimal hyperplasia. Methods and Results A denudation model of the iliac-femoral artery in wild-type and S1PR3-null mice was used to define a role for S1PR3 in the arterial injury response because we found in humans and mice that expression of S1PR3 is higher in these arteries when compared to carotid arteries. At 28 days after surgery, wild-type arteries form significantly larger lesions than S1PR3-null arteries. BrdU labeling experiments demonstrate that upon injury, wild-type arteries exhibit higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro is low, we expressed S1PR3 in S1PR3-null SMCs using retroviral-mediated gene transfer to study S1PR3 effects on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, respond to S1P stimulation with activation of Rac, Erk and Akt. SMCs expressing S1PR3 also grow migrate more. Conclusion In humans and mice, S1PR3 expression is higher in iliac-femoral arteries compared to carotid arteries. S1PR3 promotes neointimal hyperplasia upon denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt and Rac.

Numerous physiological agents and conditions modulate cellular insulin sensitivity by downregulating or upregulating total cellular insulin receptors. In this study, we examined the effects of replacing complete medium in the absence or presence of insulin on the regulation of insulin-receptor gene expression in cultured human hepatoma cells (HepG2). Failure to replace complete medium resulted in growth arrest of HepG2 cells and a six- to sevenfold increase in insulin-receptor mRNA due to the prolongation of insulin-receptor mRNA half-life. Northern analysis revealed multiple insulin-receptor mRNA species; the largest species (11 kilobases) was disproportionately increased in growth-arrested cells. High concentrations of insulin (500 ng/ml) induced a 33.8% decrease in the abundance of insulin-receptor mRNA (n = 14). At lower concentrations, a trend of inhibition was observed but was not statistically significant. Insulin (500 ng/ml) did not affect insulin-receptor mRNA stability. The effect of conditioned media, insulin, and dexamethasone on insulin-receptor promoter activity was also examined. Various constructs of the 5'-flanking region of the insulin-receptor gene were attached immediately upstream to a chloramphenicol acetyltransferase (CAT) reporter gene and transiently transfected into HepG2 cells via a pBR322-derived plasmid (pCAT). In cells replaced with complete medium, 12 and 118% of the promoter activity was contained within 578 and 877 base pairs, respectively, from the major translational initiation site. Conditioned media from growth-arrested cells in culture for 7 days increased promoter activity approximately twofold in 48 h. However, this increase failed to localize to any specific region on the insulin-receptor promoter.(ABSTRACT TRUNCATED AT 250 WORDS)