Abstract-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 -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.
Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-beta or PLC-gamma isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled beta2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-epsilon isoform.
We have recently reported that two typical G s -coupled receptors, the  2 -adrenergic receptor and the receptor for prostaglandin E 1 , stimulate phospholipase C-⑀ (PLC- Activation of mitogen-activated protein (MAP) 1 kinases plays a prominent role in many cellular responses to a large variety of membrane receptors. Initially identified as signal transducers of growth factor receptors with intrinsic tyrosine kinase activity, activation of MAP kinases, specifically of the extracellular signal-regulated kinases 1 and 2 (ERK1/2), is now also recognized as a major signal transduction pathway of many receptors coupled to heterotrimeric G proteins. ERK activation by these second messenger-generating receptors is apparently accomplished by diverse molecular mechanisms, depending on the cell types studied as well as the receptor and the heterotrimeric G protein involved (1-5).⑀The cAMP-producing G s -coupled receptors play a rather unique role in ERK activation. These receptors inhibit ERK activation by growth factor receptors in several cell types while stimulating this cellular response in others, most notably in neuronal and endocrine cells. Several models have been proposed to explain these diverse actions of cAMP and G s -coupled receptors on ERK activation, both apparently involving in most cases the principal cAMP target, the cAMP-activated protein kinase (PKA) (6, 7). In particular, PKA-dependent ERK activation in HEK-293 cells by the  2 -adrenergic receptor ( 2 -AR), a prototypical G s -coupled receptor, has been extensively studied. Lefkowitz and coworkers (8) reported that cAMP-activated PKA phosphorylates the  2 -AR and thereby alters the coupling specificity of the receptor from G s to G i proteins. By this G protein "switching," the  2 -AR then apparently induces ERK activation by releasing G␥ dimers from the pertussis toxin (PTX)-sensitive G i proteins, followed by activation of the cytosolic tyrosine kinase c-Src, the GTPase Ras, and the MAP kinase kinase kinase, Raf-1. Meanwhile, it has been reported that the switching of  2 -AR from G s to G i proteins is controlled by -arrestin, which recruits the cAMP-degrading phosphodiesterase 4 to the plasma membrane and thereby alters the activity state of PKA (9). In contrast, Schmitt and Stork (10) report that ERK activation by the  2 -AR in HEK-293 cells, although also PKA-and c-Src-dependent, is PTX-insensitive and thus apparently does not require  2 -AR switching to G i proteins, a finding recently confirmed by others using a PKAinsensitive  2 -AR mutant (11). Furthermore, Schmitt and Stork (10) show that ERK activation by  2 -AR is not mediated by Ras but by the related GTPase Rap1, activating the MAP kinase kinase kinase B-Raf, but not Raf-1. Interestingly, activation of the  2 -AR also resulted in Ras activation, but this receptor action was apparently independent of cAMP and PKA (10).
e 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 ␣v and 3 integrin expression and that the down-modulation of ␣v3 rescues the angiogenic phenotype in FOSL1-silenced HUVEC, while the ectopic expression of ␣v3 alone reproduces the phenotypic alterations induced by FOSL1 knockdown. FOSL1 represses the transcription of both ␣v and 3 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 ␣v3 expression on endothelial cells is required for endothelial assembly into vessel structures. V asculogenesis and angiogenesis are complex processes that, in response to angiogenic stimuli initiated by growth factors, result in a highly organized sequence of events, including cellular proliferation, migration, and formation of primitive endothelial tubes. During these processes endothelial cells (ECs) must proliferate, migrate, and establish highly dynamic cell-cell contacts and interactions with the extracellular matrix (ECM).Adhesion of endothelial cells with the ECM is mediated by integrins, which have been shown to be required during the vasculogenic and angiogenic processes (1). Mice null for ␣v die in utero showing vasculature abnormalities in the placenta (2), and neutralizing antibodies to integrin ␣v3 lead to abnormal vessel structures (3). The interaction of endothelial cells with the ECM is essential for endothelial cell proliferation, migration, and survival (4) and is required for tissue organization and differentiation. Moreover, upon interaction with the ECM, integrins form complexes with angiogenic receptors, contributing to their activation (5-10).Fosl1 (Fos-like 1; also named Fra1) knockout mice die between embryonic day 10.0 (E10.0) and E10.5 showing abnormal yolk sacs with placentas that are largely avascular (11).Fosl1 is an early gene that belongs to the activator protein 1 (AP-1) family of dimeric transcription factor genes (12). Fosl1 regulation is mediated by an intronic enhancer, which contains an AP-1 consensus and an E-box element next to each other (13-15).Fos proteins, including Fosl1, bind to the DNA, forming heterodimers with Jun proteins although they cannot homodimerize or heterodimerize with ATF proteins. Fosl1 lacks a transactivation domain. Therefore, its contribution to AP-1-dependent transcription dep...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.