The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1␣ (but not HIF-2␣) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxiainducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1␣ mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1␣ in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1␣ mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1␣ stabilization and activity. Based on these data and a mathematical model of HIF-1␣ suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1␣ activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1␣-dependent transcription.Tissue hypoxia is a common feature in a range of physiologic and pathophysiologic states, including exercise, development, cancer, and chronic inflammation. The hypoxia-inducible factor (HIF) is a ubiquitous hypoxia-responsive transcription factor that regulates the expression of a range of genes that promote adaptation to hypoxia (32, 57). The mechanism by which HIF is stabilized in hypoxia is well understood and is due to reduced activity of a family of oxygen-dependent HIFhydroxylases that target HIF␣ subunits for degradation and block transactivation in normoxia (5). Several studies (including the present one) have shown that the upregulation of HIF-1␣ that occurs in response to hypoxia is transient and involves a resolution phase even while the cells are maintained in hypoxia (23,26,59). However, the mechanism(s) underpinning the resolution of HIF-1␣ during prolonged hypoxia remains incompletely understood. Negative-feedback mechanisms involving HIF-dependent upregulation of PHD2 and PHD3 have been identified (5,26,47,59). In the present study, we aimed to expand our understanding of how the HIF response is resolved in prolonged hypoxia by investigating a possible role for hypoxiainduced microRNAs (miRNAs). miRNAs are endogenous small RNA molecules of approximately 22 nucleotides that regulate gene expression by destabilizing mRNA or repressing translation (4, 25). Approximately one-third of all genes in mammals have been predicted to be regulated by miRNAs (43,71), and the development of

Abstract-In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation. Key Words: vascular smooth muscle Ⅲ vein Ⅲ sphingosine-1-phosphate Ⅲ transient receptor potential Ⅲ calcium channel S phingosine-1-phosphate (S1P) has emerged as a major endogenous signaling phospholipid with diverse roles in yeast, plants, and mammals. 1 Proposed functions include the regulation of cell proliferation, migration, programmed death, and pathological processes including cancer, asthma, inflammation, and trauma. There has been particular interest in the role of S1P in the cardiovascular system, where it accumulates in atherosclerotic lesions and plays a role in ischemic preconditioning of the heart. 2-5 S1P is derived from the phosphorylation of sphingosine catalyzed by sphingosine kinase, sphingosine being from ceramide and ceramide from sphingomyelin, a constituent lipid of signaling microdomains of plasma membrane lipid rafts and caveolae. 3 S1P is detected in serum at almost 1 mol/L, although protein binding impacts on the available concentration and local concentrations may vary substantially. 6 S1P is quite unusual among signaling molecules in having separate intracellular and extracellular effects. 1,4,7,8 It affects vascular smooth muscle cell migration, 9,10 evokes contraction of rat mesenteric artery, 11 and slows pacemaker activity of the sino-atrial node of the heart. 12 The underlying mechanisms are only partially worked out, but vascular smooth muscle cells respond to S1P with transient followed by sustained elevation of the cytosolic Ca 2ϩ concentration, 10,11,13,14 whereas cardiac myocytes show activation of potassium current and S1P-evoked "Ca 2ϩ deregulation," depending on extracellular Ca 2ϩ . 12,15 Despite positive effects on Ca 2ϩ signaling, the molecular basis of a Ca 2ϩ channel stimulated by S1P is unknown. L-type voltage-gated Ca 2ϩ channels are inhibited by S1P. 12 The Drosophila transient receptor potential ...

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.