ATP binding without hydrolysis switches sulfonylurea receptor 1 (SUR1) to outward-facing conformations that activate KATP channels.

Neuroendocrine-type ATP-sensitive K+ (KATP) 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, KD , values for the conformation-sensitive channel antagonist [3H]glibenclamide ([3H]GBM), indicating that ATP can act as a pure agonist. Assembly with Kir6.2 reduced SUR1's KD for [3H]GBM. This reduction required the Kir N terminus (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 KD for [3H]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 KATP channels toward open states, consistent with SUR1 variants with lower KD values causing neonatal diabetes, whereas increased KD values cause congenital hyperinsulinism.

Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity.

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.

Sphingosine-1-phosphate receptor 3 promotes neointimal hyperplasia in mouse iliac-femoral arteries.

OBJECTIVE: The objective of this study was to define a role for sphingosine-1-phosphate receptor 3 (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 was higher in these arteries compared with carotid arteries. At 28 days after surgery, wild-type arteries formed significantly larger lesions than S1PR3-null arteries. Bromodeoxyuridine labeling experiments demonstrated that on injury, wild-type arteries exhibited higher medial as well as intimal proliferation than S1PR3-null arteries. Because S1PR3 expression in vitro was low, we expressed S1PR3 in S1PR3-null smooth muscle cells (SMCs) using retroviral-mediated gene transfer to study the effects of S1PR3 on cell functions and signaling. SMCs expressing S1PR3, but not vector-transfected controls, responded to sphingosine-1-phosphate stimulation with activation of Rac, Erk, and Akt. SMCs expressing S1PR3 also migrated more. CONCLUSIONS: In humans and mice, S1PR3 expression was higher in iliac-femoral arteries compared with carotid arteries. S1PR3 promoted neointimal hyperplasia on denudation of iliac-femoral arteries in mice, likely by stimulating cell migration and proliferation through activation of signaling pathways involving Erk, Akt, and Rac.

Regulation of arterial lesions in mice depends on differential smooth muscle cell migration: a role for sphingosine-1-phosphate receptors.

The response of mice arteries to injury varies significantly between strains. FVB mice develop large neointimas after injury, whereas very small lesions form in C57BL/6 mice. After injury, platelet interaction with the denuded artery and early smooth muscle (SMC) replication are identical in both strains; however, the migration of SMCs differs significantly. FVB cells readily move into the developing neointima, whereas only the occasional C57BL/6 cells migrate. Injured arteries showed no difference in matrix metalloproteinases (MMP-2 and MMP-9) and plasminogen activator activities. In vitro, sphingosine-1-phosphate (S1P) in combination with platelet-derived growth factor (PDGF) stimulates migration of FVB cells but inhibits migration of C57BL/6 SMCs. Both SMCs migrate equally well to PDGF alone. One explanation is that the SMCs express different S1P receptors. Real-time polymerase chain reaction shows that FVB cells express higher levels of S1P receptor-1 (S1P(1)) compared with C57BL/6 cells, which express higher levels of S1P receptor-2 (S1P(2)). In addition, the migration of C57BL/6 cells can be increased by inhibiting S1P(2), whereas inhibiting S1P(1) expression slows the migration of FVB cells. Taken together these studies suggest that expression of S1P receptors vary within inbred mouse strains and that S1P is critical for SMC migration and lesion formation after injury.

Sphingosine 1-phosphate receptor 2 negatively regulates neointimal formation in mouse arteries.

Neointimal lesion formation was induced in sphingosine 1-phosphate (S1P) receptor 2 (S1P2)-null and wild-type mice by ligation of the left carotid artery. After 28 days, large neointimal lesions developed in S1P2-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. S1P2-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 S1P2-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 S1P2 normally acts to suppress SMC growth in arteries and that S1P is a regulator of neointimal development.

Ubiquitin-dependent proteolysis of cyclin D1 is associated with coxsackievirus-induced cell growth arrest.

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 GSK3beta pathway inhibitors, we showed that the reduction of cyclin D1 is GSK3beta 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.