Fumiko Itoh

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP‐Jκ/CBF‐1 and GC‐rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1‐induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.

The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/Smad signalling.

Abstract Background: Transforming growth factor‐β (TGF‐β) initiates intracellular signalling by inducing the formation of a heteromeric complex between TGF‐β type I (TβR‐I) and TGF‐β type II serine/threonine kinase receptors (TβR‐II). After the activation of TβR‐I kinase by TβR‐II kinase, specific receptor‐regulated Smads (R‐Smads) are phosphorylated by TβR‐I kinase. Smad anchor for receptor activation (SARA), which contains a FYVE finger domain, regulates the subcellular localization of R‐Smads and presents them to TβR‐I. However, it is unclear where SARA is localized in the cell and which phospholipid(s) interacts with its FYVE domain.

TMEPAI, a Transmembrane TGF-β-Inducible Protein, Sequesters Smad Proteins from Active Participation in TGF-β Signaling

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

Smad7 and protein phosphatase 1α are critical determinants in the duration of TGF-β/ALK1 signaling in endothelial cells

BackgroundIn endothelial cells (EC), transforming growth factor-β (TGF-β) can bind to and transduce signals through ALK1 and ALK5. The TGF-β/ALK5 and TGF-β/ALK1 pathways have opposite effects on EC behaviour. Besides differential receptor binding, the duration of TGF-β signaling is an important specificity determinant for signaling responses. TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs occurs transiently.ResultsThe temporal activation of TGF-β-induced Smad1/5 phosphorylation in ECs was found to be affected by de novo protein synthesis, and ALK1 and Smad5 expression levels determined signal strength of TGF-β/ALK1 signaling pathway. Smad7 and protein phosphatase 1α (PP1α) mRNA expression levels were found to be specifically upregulated by TGF-β/ALK1. Ectopic expression of Smad7 or PP1α potently inhibited TGF-β/ALK1-induced Smad1/5 phosphorylation in ECs. Conversely, siRNA-mediated knockdown of Smad7 or PP1α enhanced TGF-β/ALK1-induced signaling responses. PP1α interacted with ALK1 and this association was further potentiated by Smad7. Dephosphorylation of the ALK1, immunoprecipitated from cell lysates, was attenuated by a specific PP1 inhibitor.ConclusionOur results suggest that upon its induction by the TGF-β/ALK1 pathway, Smad7 may recruit PP1α to ALK1, and thereby control TGF-β/ALK1-induced Smad1/5 phosphorylation.

Preventive Effect of Dipeptidyl Peptidase-4 Inhibitor on Atherosclerosis Is Mainly Attributable to Incretin's Actions in Nondiabetic and Diabetic Apolipoprotein E-Null Mice

Aim Several recent reports have revealed that dipeptidyl peptidase (DPP)-4 inhibitors have suppressive effects on atherosclerosis in apolipoprotein E-null (Apoe −/−) mice. It remains to be seen, however, whether this effect stems from increased levels of the two active incretins, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Methods Nontreated Apoe −/− mice, streptozotocin-induced diabetic Apoe −/− mice, and db/db diabetic mice were administered the DPP-4 inhibitor vildagliptin in drinking water and co-infused with either saline, the GLP-1 receptor blocker, exendin(9–39), the GIP receptor blocker, (Pro3)GIP, or both via osmotic minipumps for 4 weeks. Aortic atherosclerosis and oxidized low-density lipoprotein-induced foam cell formation in exudate peritoneal macrophages were determined. Results Vildagliptin increased plasma GLP-1 and GIP levels without affecting food intake, body weight, blood pressure, or plasma lipid profile in any of the animals tested, though it reduced HbA1c in the diabetic mice. Diabetic Apoe −/− mice exhibited further-progressed atherosclerotic lesions and foam cell formation compared with nondiabetic counterparts. Nondiabetic and diabetic Apoe −/− mice showed a comparable response to vildagliptin, namely, remarkable suppression of atherosclerotic lesions with macrophage accumulation and foam cell formation in peritoneal macrophages. Exendin(9–39) or (Pro3)GIP partially attenuated the vildagliptin-induced suppression of atherosclerosis. The two blockers in combination abolished the anti-atherosclerotic effect of vildagliptin in nondiabetic mice but only partly attenuated it in diabetic mice. Vildagliptin suppressed macrophage foam cell formation in nondiabetic and diabetic mice, and this suppressive effect was abolished by infusions with exendin(9–39)+(Pro3)GIP. Incubation of DPP-4 or vildagliptin in vitro had no effect on macrophage foam cell formation. Conclusions Vildagliptin confers a substantial anti-atherosclerotic effect in both nondiabetic and diabetic mice, mainly via the action of the two incretins. However, the partial attenuation of atherosclerotic lesions by the dual incretin receptor antagonists in diabetic mice implies that vildagliptin confers a partial anti-atherogenic effect beyond that from the incretins.

Roles of TGF-β family signals in the fate determination of pluripotent stem cells.

Members of the transforming growth factor-β (TGF-β) family have been implicated in embryogenesis as well as in the determination of the cell fates of mouse and human embryonic stem (ES) cells, which are characterized by their self-renewal and pluripotency. The cellular responses to TGF-β family signals are divergent depending on the cellular context and local environment. TGF-β family signals play critical roles both in the maintenance of the pluripotent state of ES cells by inducing the expression of Nanog, Oct4, and Sox2, and in their differentiation into various cell types by regulating the expression of master regulatory genes. Moreover, multiple lines of evidence have suggested the importance of TGF-β family signals in establishing induced pluripotent stem (iPS) cells. Since ES and iPS cells have great potential for applications in regenerative medicine, it is critical to figure out the mechanisms underlying their self-renewal, pluripotency, and differentiation. Here, we discuss the roles of TGF-β family ligands and their downstream signaling molecules, Smad proteins, in the maintenance of the pluripotency and lineage specification of mouse and human ES and iPS cells.

Smad2/Smad3 in endothelium is indispensable for vascular stability via S1PR1 and N-cadherin expressions.

Transforming growth factor-β (TGF-β) is involved in vascular formation through activin receptor-like kinase (ALK)1 and ALK5. ALK5, which is expressed ubiquitously, phosphorylates Smad2 and Smad3, whereas endothelial cell (EC)-specific ALK1 activates Smad1 and Smad5. Because ALK5 kinase activity is required for ALK1 to transduce TGF-β signaling via Smad1/5 in ECs, ALK5 knockout (KO) mice were not able to give us the precise mechanisms by which TGF-β/ALK5/Smad2/3 signaling is implicated in angiogenesis. To delineate the role of Smad2/3 signaling in endothelium, the Smad2 gene in Smad3 KO mice was selectively deleted in ECs using Tie2-Cre transgenic mice, termed EC-specific Smad2/3 double KO (EC-Smad2/3KO) mice. EC-Smad2/3KO embryos revealed hemorrhage leading to embryonic lethality around E12.5. EC-Smad2/3KO embryos exhibited no abnormality of vasculogenesis and angiogenesis in both the yolk sac and the whole embryo, whereas vascular maturation was incomplete because of inadequate assembly of mural cells in the vasculature. Wide gaps between ECs and mural cells could be observed in the vasculature of EC-Smad2/3KO mice because of reduced expression of N-cadherin and sphingosine-1-phosphate receptor-1 (S1PR1) in ECs from those mice. These results indicated that Smad2/3 signaling in ECs is indispensable for maintenance of vascular integrity via the fine-tuning of N-cadherin, VE-cadherin, and S1PR1 expressions in the vasculature.

Requirement of TCF7L2 for TGF-β-dependent transcriptional activation of the TMEPAI gene

The TGF-β and Wnt pathways are involved in cell fate and tumorigenicity. A recent report indicated that a TGF-β target gene, TMEPAI (transmembrane prostate androgen-induced RNA), is possibly also a downstream target of Wnt signaling. Although TMEPAI was believed to be involved in tumorigenicity because of its blockage of TGF-β signaling, how TGF-β and Wnt signals affect the activation of the TMEPAI gene is not well understood. Herein, we show that the TMEPAI promoter is regulated synergistically by TGF-β/Smad and Wnt/β-catenin/T cell factor (TCF) 7L2. The critical cis-element for dual signals, termed TGF-β-responsive TCF7L2-binding element (TTE), is located in intron 1 of the TMEPAI gene. TCF7L2, but not Smad proteins, bound to TTE, whereas the disruption of TTE by mutagenesis remarkably counteracted both TGF-β and TCF7L2 responses. The introduction of mutations in critical Smad-binding elements blocked the activation of the TMEPAI promoter by TCF7L2. Furthermore, our DNA-protein interaction experiments revealed the indirect binding of TCF7L2 to Smad-binding elements via Smad3 upon TGF-β stimulation as well as its TGF-β-dependent association with TTE. We demonstrate that the Wnt/β-catenin/TCF7L2 pathway is preferentially able to alter the transcriptional regulation of the TGF-β-target gene, TMEPAI.

Inhibition of endothelial cell activation by bHLH protein E2-2 and its impairment of angiogenesis

E2-2 belongs to the basic helix-loop-helix (bHLH) family of transcription factors. E2-2 associates with inhibitor of DNA binding (Id) 1, which is involved in angiogenesis. In this paper, we demonstrate that E2-2 interacts with Id1 and provide evidence that this interaction potentiates angiogenesis. Mutational analysis revealed that the HLH domain of E2-2 is required for the interaction with Id1 and vice versa. In addition, Id1 interfered with E2-2-mediated effects on luciferase reporter activities. Interestingly, injection of E2-2-expressing adenoviruses into Matrigel plugs implanted under the skin blocked in vivo angiogenesis. In contrast, the injection of Id1-expressing adenoviruses rescued E2-2-mediated inhibition of in vivo angiogenic reaction. Consistent with the results of the Matrigel plug assay, E2-2 could inhibit endothelial cell (EC) migration, network formation, and proliferation. On the other hand, knockdown of E2-2 in ECs increased EC migration. The blockade of EC migration by E2-2 was relieved by exogenous expression of Id1. We also demonstrated that E2-2 can perturb VEGFR2 expression via inhibition of VEGFR2 promoter activity. This study suggests that E2-2 can maintain EC quiescence and that Id1 can counter this effect.

Salusins: Potential Use as a Biomarker for Atherosclerotic Cardiovascular Diseases

Human salusin-α and salusin-β are related peptides produced from prosalusin. Bolus injection of salusin-β into rats induces more profound hypotension and bradycardia than salusin-α. Central administration of salusin-β increases blood pressure via release of norepinephrine and arginine-vasopressin. Circulating levels of salusin-α and salusin-β are lower in patients with essential hypertension. Salusin-β exerts more potent mitogenic effects on human vascular smooth muscle cells (VSMCs) and fibroblasts than salusin-α. Salusin-β accelerates inflammatory responses in human endothelial cells and monocyte-endothelial adhesion. Human macrophage foam cell formation is stimulated by salusin-β but suppressed by salusin-α. Chronic salusin-β infusion into apolipoprotein E-deficient mice enhances atherosclerotic lesions; salusin-α infusion reduces lesions. Salusin-β is expressed in proliferative neointimal lesions of porcine coronary arteries after stenting. Salusin-α and salusin-β immunoreactivity have been detected in human coronary atherosclerotic plaques, with dominance of salusin-β in macrophage foam cells, VSMCs, and fibroblasts. Circulating salusin-β levels increase and salusin-α levels decrease in patients with coronary artery disease. These findings suggest that salusin-β and salusin-α may contribute to proatherogenesis and antiatherogenesis, respectively. Increased salusin-β and/or decreased salusin-α levels in circulating blood and vascular tissue are closely linked with atherosclerosis. Salusin-α and salusin-β could be candidate biomarkers and therapeutic targets for atherosclerotic cardiovascular diseases.