FOSL1 controls the assembly of endothelial cells into capillary tubes by direct repression of αv and ß3 integrin transcription.

To form three-dimensional capillary tubes, endothelial cells must establish contacts with the extracellular matrix that provides signals for their proliferation, migration, and differentiation. The transcription factor Fosl1 plays a key role in the vasculogenic and angiogenic processes as Fosl1 knockout embryos die with vascular defects in extraembryonic tissues. Here, we show that Fosl1(-/-) embryonic stem cells differentiate into endothelial cells but fail to correctly assemble into primitive capillaries and to form tube-like structures. FOSL1 silencing affects in vitro angiogenesis, increases cell adhesion, and decreases cell mobility of primary human endothelial cells (HUVEC). We further show that FOSL1 is a repressor of αv and ß3 integrin expression and that the down-modulation of αvß3 rescues the angiogenic phenotype in FOSL1-silenced HUVEC, while the ectopic expression of αvß3 alone reproduces the phenotypic alterations induced by FOSL1 knockdown. FOSL1 represses the transcription of both αv and ß3 integrin genes by binding together with JunD to their proximal promoter via the transcription factor SP1. These data suggest that FOSL1-dependent negative regulation of αvß3 expression on endothelial cells is required for endothelial assembly into vessel structures.

Study of cyclic adenosine monophosphate microdomains in cells.

Cyclic adenosine monophosphate (cAMP) controls the physiological response to many diverse extracellular stimuli. To maintain signal specificity, cAMP-mediated signaling is finely tuned by means of a complex array of proteins that control the spatial and temporal dynamics of the second messenger within the cell. To unravel the way a cell encodes cAMP signals, new biosensors have recently been introduced that allow imaging of the second messenger in living cells with high spatial resolution. The more recent generation of such biosensors exploits the phenomenon of fluorescence resonance energy transfer between the green fluorescent protein- tagged subunits of a chimeric protein kinase A, as the way to visualize and measure the dynamic fluctuations of cAMP. This chapter describes the molecular basis on which such a genetically encoded cAMP sensor relies and the tools and methods required to perform cAMP measurements in living samples.

Epac- and Ca2+ -controlled activation of Ras and extracellular signal-regulated kinases by Gs-coupled receptors.

We have recently reported that two typical Gs-coupled receptors, the beta2-adrenergic receptor and the receptor for prostaglandin E1, stimulate phospholipase C-epsilon (PLC-epsilon) and increase intracellular Ca2+ concentration ([Ca2+]i) in HEK-293 cells and N1E-115 neuroblastoma cells, respectively, by a pathway involving Epac1, a cAMP-activated and Rap-specific guanine nucleotide exchange factor (GEF), and the GTPase Rap2B. Here we have demonstrated that these Gs-coupled receptors use this pathway to activate H-Ras and the extracellular signal-regulated kinases 1 and 2 (ERK1/2). Specifically, agonist activation of the receptors resulted in activation of H-Ras and ERK1/2. The latter action was suppressed by dominant negative H-Ras, but not Rap1A. The receptor actions were independent of protein kinase A but fully mimicked by an Epac-specific cAMP analog as well as by a constitutively active Rap2B mutant. On the other hand, a cAMP-binding-deficient Epac1 mutant, the Rap GTPase-activating proteinII, and a dominant negative Rap2B mutant suppressed receptor- and Epac-mediated activation of H-Ras and ERK1/2. Finally, we have demonstrated that activation of H-Ras and ERK1/2 requires the lipase activity of PLC-epsilon and the subsequent [Ca2+]i increase, suggesting that H-Ras activation is mediated by a Ca2+ -activated GEF. In line with this hypothesis, receptor-mediated activation of H-Ras and ERK1/2 was strongly enhanced by expression of RasGRP1, a Ca2+ -regulated Ras-GEF. Collectively, our data indicated that Gs-coupled receptors can activate H-Ras and subsequently the mitogen-activated protein kinases ERK1/2 by a Ca2+ -activated Ras-GEF, possibly RasGRP1, mediated by cAMP-activated Epac proteins, which then lead via Rap2B and PLC-epsilon stimulation to [Ca2+]i increase.

Measuring dynamic changes in cAMP using fluorescence resonance energy transfer.

cAMP is a ubiquitous second messenger that controls numerous cellular events including movement, growth, metabolism, contraction, and synaptic plasticity. With the emerging concept of compartmentalization of cAMP-dependent signaling, a detailed study of the spatio-temporal intracellular dynamics of cAMP is required. Here we describe a new methodology for monitoring fluctuations of cAMP in living cells, based on the use of a genetically encoded biosensor. The regulatory and catalytic subunits of the main cAMP effector, the protein kinase A (PKA), fused with two suitable green fluorescent protein (GFP) mutants is used for measuring changes in fluorescence resonance energy transfer (FRET) that correlate with changes in intracellular cAMP levels. This method allows the study of cAMP fluctuations in living cells with high resolution both in time and in space.

Fluorescence resonance energy transfer-based analysis of cAMP dynamics in live neonatal rat cardiac myocytes reveals distinct functions of compartmentalized phosphodiesterases.

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a beta-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3.

Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.

Role of Rho kinase signalling in healthy and varicose human saphenous veins.

1. The present study was performed to determine the role of Rho-Rho kinase signalling pathway in smooth muscle cells from both healthy and varicose human saphenous vein. 2. The Rho kinase inhibitor Y-27632 inhibited the noradrenaline (NA)-induced contraction in human saphenous veins with IC(50) corresponding to 0.5 microM and 10.9 microM in control and varicose veins, respectively. The maximal amplitude of the NA-induced contraction was smaller in varicose vein compared to control (1263+/-172 mg versus 1974+/-245 mg, P<0.05). 3. In beta-escin permeabilized strips, GTPgammaS induced a rise in tension that was inhibited by Y-27632. The amplitude of the GTPgammaS-induced contraction was smaller in varicose compared to control veins (23.1+/-2.4% versus 41.3+/-2.2%, P<0.002). 4. In smooth muscle cells, Y-27632 induced disassembly of both actin cytoskeleton and extracellular fibronectin matrix. In comparison to control cells, varicose vein smooth muscle cells show decreased actin cytoskeleton organization and reduction of fibronectin matrix deposition. 5. The Rho proteins Rnd1 and RhoA, and Rho kinase 1 are expressed in human saphenous veins. A 2.6 fold reduction of Rho kinase expression was found in varicose veins. 6. These results indicate that RhoA-Rho kinase mediated Ca(2+) sensitization of the contraction and regulated actin cytoskeleton and extracellular fibronectin matrix assembly in human saphenous smooth muscle. The decrease of Rho kinase expression and Rho kinase-dependent functions detected in smooth muscle from varicose veins supports a role of this signalling pathway in the functional alterations of the vein wall occurring in the course of the disease.

Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B.

Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.