"Soluble" adenylyl cyclase-generated cyclic adenosine monophosphate promotes fast migration in PC12 cells.
ABSTRACT: In a model for neuronal movement, PC12 cells undergo fast migration in response to nerve growth factor (NGF) and phorbol ester (PMA). We previously showed that NGF increases intracellular cAMP via activation of soluble adenylyl cyclase (sAC). In this report, we demonstrate that sAC activation is an essential component of NGF- + PMA-induced fast migration in PC12 cells. Interestingly, PMA also raises intracellular cAMP but does so by stimulating transmembrane adenylyl cyclases (tmAC); however, this tmAC-generated cAMP does not contribute to fast migration. Therefore, cells must possess independent pools of cAMP capable of modulating distinct functions.
Project description:Nerve growth factor (NGF) and the ubiquitous second messenger cyclic AMP (cAMP) are both implicated in neuronal differentiation. Multiple studies indicate that NGF signals to at least a subset of its targets via cAMP, but the link between NGF and cAMP has remained elusive. Here, we have described the use of small molecule inhibitors to differentiate between the two known sources of cAMP in mammalian cells, bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC) and G protein-regulated transmembrane adenylyl cyclases. These inhibitors, along with sAC-specific small interfering RNA, reveal that sAC is uniquely responsible for the NGF-elicited rise in cAMP and is essential for the NGF-induced activation of the small G protein Rap1 in PC12 cells. In contrast and as expected, transmembrane adenylyl cyclase-generated cAMP is responsible for Rap1 activation by the G protein-coupled receptor ligand PACAP (pituitary adenylyl cyclase-activating peptide). These results identify sAC as a mediator of NGF signaling and reveal the existence of distinct pathways leading to cAMP-dependent signal transduction.
Project description:In beta cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein-responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.
Project description:Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.
Project description:Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.
Project description:Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.
Project description:The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.
Project description:cAMP is a critical second messenger mediating activity-dependent neuronal survival and neurite growth. We investigated the expression and function of the soluble adenylyl cyclase (sAC, ADCY10) in CNS retinal ganglion cells (RGCs). We found sAC protein expressed in multiple RGC compartments including the nucleus, cytoplasm and axons. sAC activation increased cAMP above the level seen with transmembrane adenylate cyclase (tmAC) activation. Electrical activity and bicarbonate, both physiologic sAC activators, significantly increased survival and axon growth, whereas pharmacologic or siRNA-mediated sAC inhibition dramatically decreased RGC survival and axon growth in vitro, and survival in vivo. Conversely, RGC survival and axon growth were unaltered in RGCs from AC1/AC8 double knock-out mice or after specifically inhibiting tmACs. These data identify a novel sAC-mediated cAMP signaling pathway regulating RGC survival and axon growth, and suggest new neuroprotective or regenerative strategies based on sAC modulation.
Project description:H2 O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion. This study investigated signalling downstream of the receptors activated by H2 O2 .Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 receptor agonist or ?2 -adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy.Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated anion currents. A similar Epac-mediated pathway was seen following ?2 -adrenoceptor or forskolin stimulation.H2 O2 initiated a complex signalling cascade that used direct stimulation of tmACs by G?s followed by Epac-mediated Ca(2+) crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.
Project description:Bidirectional promoters are the major source of gene activation-associated noncoding RNA (ncRNA). PC12 cells offer an interesting model for understanding the mechanism underlying bidirectional promoter-mediated cell cycle control. Nerve growth factor (NGF)-stimulated PC12 cells elongate neurites, and are in a reversible cell-cycle-arrested state. In contrast, these cells irreversibly differentiate and cannot re-enter the normal cell cycle after NGF plus cAMP treatment. In this study, using directional RNA-seq, we found that bidirectional promoters for protein-coding genes with promoter-associated ncRNA (pancRNA) were enriched for cAMP response element consensus sequences, and were preferred targets for transcriptional regulation by the transcription factors in the cAMP-dependent pathway. A spindle-formation-associated gene, Nusap1 and pancNusap1 were among the most strictly co-transcribed pancRNA-mRNA pairs. This pancRNA-mRNA pair was specifically repressed in irreversibly differentiated PC12 cells. Knockdown (KD) and overexpression experiments showed that pancNusap1 positively regulated the Nusap1 expression in a sequence-specific manner, which was accompanied by histone acetylation at the Nusap1 promoter. Furthermore, pancNusap1 KD recapitulated the effects of cAMP on cell cycle arrest. Thus, we conclude that pancRNA-mediated histone acetylation contributes to the establishment of the cAMP-induced transcription state of the Nusap1 locus and contributes to the irreversible cell cycle exit for terminal differentiation of PC12 cells.
Project description:Continuous NGF stimulation induces PC12 cell differentiation; however, it is unclear whether NGF is continuously required for differentiation. We found that discontinuous NGF stimulation, consisting of the first transient stimulation followed by an interval and the second sustained stimulation, similarly induces differentiation. The first stimulation did not induce neurite extension, whereas the second stimulation induced fast neurite extension; therefore, the first stimulation is required as prerequisite conditions. This indicates that the action of NGF can be divided into two processes: the first stimulation-driven latent process and the second stimulation-driven extension process. The latent process appears to require ERK and transcription, but not PI3K, activities, whereas the extension-process requires ERK and PI3K, but not transcription, activities. We also found that NGF in the first stimulation can be replaced by PACAP, but not by insulin, EGF, bFGF or forskolin, whereas NGF in the second stimulation cannot be replaced by any of these stimulants. These findings allowed us to identify potential genes specifically involved in the latent process, rather than in other processes. These results demonstrate that NGF induces differentiation of PC12 cells via mechanically distinct processes: the ERK-driven and transcription-dependent latent process; and the ERK- and PI3K-driven, and transcription-independent extension process. Overall design: Gene expression in PC12 cells was measured at 3 hours after following stimulations, complete medium (n = 4), continuous NGF (n = 2), 1 hour of NGF (n = 4), 1 hour of PACAP (n = 2), 1 hour of Insulin (n = 2), 1 hour of NGF in presence of U0126 (n = 2) or 1 hour of NGF in presence of LY294002 (n = 2).