Naoki Mochizuki

Spatio-temporal images of growth-factor-induced activation of Ras and Rap1

G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. Here we use FRET (fluorescent resonance energy transfer)-based sensors to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activated Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras was initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity was observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, we found that Ras at the neurites turned over rapidly; therefore, the sustained Ras activity at neurites was due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells.

Cyclic AMP Potentiates Vascular Endothelial Cadherin-Mediated Cell-Cell Contact To Enhance Endothelial Barrier Function through an Epac-Rap1 Signaling Pathway

ABSTRACT Cyclic AMP (cAMP) is a well-known intracellular signaling molecule improving barrier function in vascular endothelial cells. Here, we delineate a novel cAMP-triggered signal that regulates the barrier function. We found that cAMP-elevating reagents, prostacyclin and forskolin, decreased cell permeability and enhanced vascular endothelial (VE) cadherin-dependent cell adhesion. Although the decreased permeability and the increased VE-cadherin-mediated adhesion by prostacyclin and forskolin were insensitive to a specific inhibitor for cAMP-dependent protein kinase, these effects were mimicked by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′, 5′-cyclic monophosphate, a specific activator for Epac, which is a novel cAMP-dependent guanine nucleotide exchange factor for Rap1. Thus, we investigated the effect of Rap1 on permeability and the VE-cadherin-mediated cell adhesion by expressing either constitutive active Rap1 or Rap1GAPII. Activation of Rap1 resulted in a decrease in permeability and enhancement of VE-cadherin-dependent cell adhesion, whereas inactivation of Rap1 had the counter effect. Furthermore, prostacyclin and forskolin induced cortical actin rearrangement in a Rap1-dependent manner. In conclusion, cAMP-Epac-Rap1 signaling promotes decreased cell permeability by enhancing VE-cadherin-mediated adhesion lined by the rearranged cortical actin.

Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome

Background— The endoplasmic reticulum (ER) responds to various stresses by upregulation of ER chaperones, but prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear. Methods and Results— Coronary artery segments were obtained at autopsy from 71 patients, and atherectomy specimens were obtained from 40 patients. Smooth muscle cells and macrophages in the fibrous caps of thin-cap atheroma and ruptured plaques, but not in the fibrous caps of thick-cap atheroma and fibrous plaques, showed a marked increase of ER chaperone expression and apoptotic cells. ER chaperones also showed higher expression in atherectomy specimens from patients with unstable angina pectoris than in specimens from those with stable angina. Expression of 7-ketocholesterol was increased in the fibrous caps of thin-cap atheroma compared with thick-cap atheroma. Treatment of cultured coronary artery smooth muscle cells or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, whereas these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by small interfering RNA decreased ER stress-dependent death of cultured coronary artery smooth muscle cells and THP-1 cells. Conclusions— Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of smooth muscle cells and macrophages may contribute to plaque vulnerability.

Differential function of Tie2 at cell–cell contacts and cell–substratum contacts regulated by angiopoietin-1

Tie2 belongs to the receptor tyrosine kinase family and functions as a receptor for Angiopoietin-1 (Ang1). Gene-targeting analyses of either Ang1 or Tie2 in mice reveal a critical role of Ang1–Tie2 signalling in developmental vascular formation. It remains elusive how the Tie2 signalling pathway plays distinct roles in both vascular quiescence and angiogenesis. We demonstrate here that Ang1 bridges Tie2 at cell–cell contacts, resulting in trans-association of Tie2 in the presence of cell–cell contacts. In clear contrast, in isolated cells, extracellular matrix-bound Ang1 locates Tie2 at cell–substratum contacts. Furthermore, Tie2 activated at cell–cell or cell–substratum contacts leads to preferential activation of Akt and Erk, respectively. Microarray analyses and real-time PCR validation clearly show the differential gene expression profile in vascular endothelial cells upon Ang1 stimulation in the presence or absence of cell–cell contacts, implying downstream signalling is dependent upon the spatial localization of Tie2.

The sphingolipid transporter spns2 functions in migration of zebrafish myocardial precursors.

Sphingosine-1-phosphate (S1P) is a secreted lipid mediator that functions in vascular development; however, it remains unclear how S1P secretion is regulated during embryogenesis. We identified a zebrafish mutant, ko157, that displays cardia bifida (two hearts) resembling that in the S1P receptor-2 mutant. A migration defect of myocardial precursors in the ko157 mutant is due to a mutation in a multipass transmembrane protein, Spns2, and can be rescued by S1P injection. We show that the export of S1P from cells requires Spns2. spns2 is expressed in the extraembryonic tissue yolk syncytial layer (YSL), and the introduction of spns2 mRNA in the YSL restored the cardiac defect in the ko157 mutant. Thus, Spns2 in the YSL functions as a S1P transporter in S1P secretion, thereby regulating myocardial precursor migration.

Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms

The crescent‐shaped BAR (Bin/Amphiphysin/Rvs‐homology) domain dimer is a versatile protein module that senses and generates positive membrane curvature. The BAR domain dimer of human endophilin‐A1, solved at 3.1 Å, has a unique structure consisting of a pair of helix–loop appendages sprouting out from the crescent. The appendages short helices form a hydrophobic ridge, which runs across the concave surface at its center. Examining liposome binding and tubulation in vitro using purified BAR domain and its mutants indicated that the ridge penetrates into the membrane bilayer and enhances liposome tubulation. BAR domain‐expressing cells exhibited marked plasma membrane tubulation in vivo. Furthermore, a swinging‐arm mutant lost liposome tubulation activity yet retaining liposome binding. These data suggested that the rigid crescent dimer shape is crucial for the tubulation. We here propose that the BAR domain drives membrane curvature by coordinate action of the crescents scaffold mechanism and the ridges membrane insertion in addition to membrane binding via amino‐terminal amphipathic helix.

Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G|[alpha]|i

Many receptors for neuropeptides and hormones are coupled with the heterotrimeric Gi protein, which activates the p42/44 mitogen-activated protein kinase (ERK/MAPK) cascade through both the α- and βγ-subunits of Gi (refs 1–3). The βγ-subunit activates the ERK/MAPK cascade through tyrosine kinase. Constitutively active Gαi2 (gip2) isolated from adrenal and ovarian tumours, transforms Rat-1 fibroblasts and also activates the ERK/MAPK cascade by an unknown mechanism,. The ERK/MAPK pathway is activated by Ras, and is inhibited when the low-molecular-mass GTP-binding protein Rap1 antagonizes Ras function. Here we show that a novel isoform of Rap1 GTPase-activating protein, called rap1GAPII, binds specifically to the α-subunits of the Gi family of heterotrimeric G-proteins. Stimulation of the Gi-coupled m2-muscarinic receptor translocates rap1GAPII from the cytosol to the membrane and decreases the amount of GTP-bound Rap1. This decrease in GTP-bound Rap1 activates ERK/MAPK. Thus, the α-subunit of Gi activates the Ras-ERK/MAPK mitogenic pathway by membrane recruitment of rap1GAPII and reduction of GTP-bound Rap1.

Hybrid Cell–Gene Therapy for Pulmonary Hypertension Based on Phagocytosing Action of Endothelial Progenitor Cells

Background—Circulating endothelial progenitor cells (EPCs) migrate to injured vascular endothelium and differentiate into mature endothelial cells. We investigated whether transplantation of vasodilator gene-transduced EPCs ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Methods and Results—We obtained EPCs from cultured human umbilical cord blood mononuclear cells and constructed plasmid DNA of adrenomedullin (AM), a potent vasodilator peptide. We used cationic gelatin to produce ionically linked DNA-gelatin complexes. Interestingly, EPCs phagocytosed plasmid DNA-gelatin complexes, which allowed nonviral, highly efficient gene transfer into EPCs. Intravenously administered EPCs were incorporated into the pulmonary vasculature of immunodeficient nude rats given MCT. Transplantation of EPCs alone modestly attenuated MCT-induced pulmonary hypertension (16% decrease in pulmonary vascular resistance). Furthermore, transplantation of AM DNA-transduced EPCs markedly ameliorated pulmonary hypertension in MCT rats (39% decrease in pulmonary vascular resistance). MCT rats transplanted with AM-expressing EPCs had a significantly higher survival rate than those given culture medium or EPCs alone. Conclusions—Umbilical cord blood–derived EPCs had a phagocytosing action that allowed nonviral, highly efficient gene transfer into EPCs. Transplantation of AM gene-transduced EPCs caused significantly greater improvement in pulmonary hypertension in MCT rats than transplantation of EPCs alone. Thus, a novel hybrid cell–gene therapy based on the phagocytosing action of EPCs may be a new therapeutic strategy for the treatment of pulmonary hypertension.

Angiopoietin-like Protein 2 Promotes Chronic Adipose Tissue Inflammation and Obesity-Related Systemic Insulin Resistance

Recent studies of obesity have provided new insights into the mechanisms underlying insulin resistance and metabolic dysregulation. Numerous efforts have been made to identify key regulators of obesity-linked adipose tissue inflammation and insulin resistance. We found that angiopoietin-like protein 2 (Angptl2) was secreted by adipose tissue and that its circulating level was closely related to adiposity, systemic insulin resistance, and inflammation in both mice and humans. Angptl2 activated an inflammatory cascade in endothelial cells via integrin signaling and induced chemotaxis of monocytes/macrophages. Constitutive Angptl2 activation in vivo induced inflammation of the vasculature characterized by abundant attachment of leukocytes to the vessel walls and increased permeability. Angptl2 deletion ameliorated adipose tissue inflammation and systemic insulin resistance in diet-induced obese mice. Conversely, Angptl2 overexpression in adipose tissue caused local inflammation and systemic insulin resistance in nonobese mice. Thus, Angptl2 is a key adipocyte-derived inflammatory mediator that links obesity to systemic insulin resistance.

Requirement for C3G-dependent Rap1 activation for cell adhesion and embryogenesis

C3G is a guanine nucleotide exchange factor (GEF) for Rap1, and is activated via Crk adaptor protein. To understand the physiological role of C3G, we generated C3G knockout mice. C3G−/− homozygous mice died before embryonic day 7.5. The lethality was rescued by the expression of the human C3G transgene, which could be excised upon the expression of Cre recombinase. From the embryo of this mouse, we prepared fibroblast cell lines, MEF‐hC3G. Expression of Cre abolished the expression of C3G in MEF‐hC3G and inhibited cell adhesion‐induced activation of Rap1. The Cre‐expressing MEF‐hC3G showed impaired cell adhesion, delayed cell spreading and accelerated cell migration. The accelerated cell migration was suppressed by the expression of active Rap1, Rap2 and R‐Ras. Expression of Epac and CalDAG‐GEFI, GEFs for Rap1, also suppressed the accelerated migration of the C3G‐deficient cells. This observation indicated that Rap1 activation was sufficient to complement the C3G deficiency. In conclusion, C3G‐dependent activation of Rap1 is required for adhesion and spreading of embryonic fibroblasts and for the early embryogenesis of the mouse.